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backgroundadjuvant radiation therapy is commonly administered to breast cancer patients who received breast - conserving surgery . however , lengthy treatment times of standard radiotherapy pose certain challenges . here , we performed a prospective controlled study comparing standard radiation to hypofractionated radiotherapy in terms of efficacy and outcome.material/methodseighty breast cancer patients ( tumor stage pt1 - 2n0 - 1m0 ) who had undergone breast - conservation surgery were randomly divided into 2 groups ( 40 patients / group ) . the experimental group received 43.2 gy to the whole breast in 18 fractions for 24 days with a concomitant boost ( 50.4 gy ) to the tumor bed . the control group received 45 gy to the whole breast in 25 fractions for 44 days with a boost to the tumor bed of 59 gy . survival , locoregional recurrence , adverse effects , and aesthetic results were all considered for analysis.resultsthe following criteria were included as part of study follow - up : local control , survival , adverse skin reactions , cosmetic outcome , and hematological toxicity . at a median follow - up of 27 months ( follow - up rate 100% ) , there were no statistical differences in any of the categories between the 2 groups . the 2-year survival rate of both groups was 100% without any locoregional recurrence . although there was some skin toxicity , these instances were not severe and they cleared on their own within 6 weeks . the most common problems encountered by patients were breast fibrosis and altered pigmentation.conclusionsa shortened whole - breast hypofractionated irradiation schedule with a concomitant boost is as effective as standard radiation and may be a reasonable alternative following breast conservation surgery .

breast cancer is one of the most common cancers affecting women worldwide and a primary cause of cancer - related death in women . despite high morbidity and mortality , therapeutic options for breast cancer include both targeted ( e.g. , anti - estrogens and her2 antagonists ) and non - targeted therapies ( e.g. , radiation therapy ) . if diagnosed at an early stage , breast conservation surgery can often be performed ; this involves surgical removal of the cancer cells with minimal disturbance of normal breast tissue . , patients often receive adjuvant radiation therapy , which helps kill any remaining cancerous cells that may not have been removed during the operation . to date , an optimal fractionation schedule for breast irradiation has not been universally accepted , and many studies have examined the benefits and drawbacks of various treatment regimens [ 58 ] . previous studies have shown that breast - conserving surgery in conjunction with irradiation has a similar outcome as a radical operations such as a full mastectomy . the current standard for radiation treatment involves whole - breast tangential irradiation with a subsequent boost to the tumor bed ; this has been proven to decrease locoregional recurrence . however , this standard regimen occurs over a long period of time and can potentially interfere with post - operative chemotherapy treatment . radiation treatment following breast - conserving surgery for early - stage disease typically occurs over the course of 67 weeks . previous work has shown that breast cancer has a low / ratio ( 34 gy ) and that hypofractionated radiation therapy ( > 2 gy / fraction ) may be effective without significantly increasing adverse effects . interestingly , a study showed that implementation of a cost - minimization strategy was effective on a patient - by - patient basis and significantly reduced medical expenses . here , we further examine the influence of radiotherapy scheduling on patients undergoing breast conservation surgery . specifically , we examined whether there were any outcome differences between early - stage breast cancer patients receiving standard radiotherapy compared to those receiving whole - breast hypofractionated irradiation with a concomitant boost . patients met the inclusion criteria were enrolled the study in the department of radiation oncology , tianjin medical university cancer institute and hospital between january 2011 and december 2011 . patients were randomly divided into an experimental group ( 24-day group ) and a control group ( 44-day group ) using a random number table . written informed consent was obtained from all patients and the ethics committee of tianjin medical university cancer institute and hospital approved the study protocol . patients were enrolled in the study if they met the following inclusion criteria : patients were diagnosed at an early disease stage ( pt1 - 2n0 - 1m0 ) and underwent breast - conserving surgery . all patients were 18 years of age or older , with a karnofsky performance status ( kps ) score greater than or equal to 70 . silver clips were used to denote tumor volume and help localize radiation , which had to be administered within 1 month of surgery . patients who met any of the following exclusion criteria were not enrolled in the study : any patient who had received radiation therapy in the ipsilateral breast , chest wall , lung , or lymph nodes was excluded from the study . additionally , women diagnosed with either inflammatory breast cancer or bilateral breast cancer or who had received prior breast - conserving surgery or breast reconstruction surgery were excluded . finally , those who experienced breast cancer recurrence were prevented from enrolling in the study . patients had undergone various treatment modalities based on their presentation and tumor characteristics . here , we outline the primary treatment regimens used for all patients in the study . breast - conserving surgery was performed by surgical excision of the primary tumor , with a 23 cm margin of macroscopically normal tissue and an axillary dissection . when the sentinel node biopsy was positive , the axillary lymph nodes were dissected ; in all other cases , no dissection of axillary lymph nodes was performed . the dissection of axillary lymph nodes was conducted at either a level i ii or a level i iii , depending on the patient s risk level . during surgery , patients received postoperative chemotherapy if they were axillary lymph node positive or if they were axillary lymph node negative but at a high risk of recurrence ( i.e. < 35 years old with a tumor diameter 2 cm , a histological grade of ii iii , signs of vascular invasion , her-2 positive , er / pr negative ) . a 2-drug chemotherapy regimen was used in this study either pharmorubicin combined with cyclophosphamide or pharmorubicin combined with paclitaxel . there was no difference in the proportion of different chemotherapy regimens between the 2 groups . radiotherapy was started within 1 month of surgery and 3 weeks after the first cycle of chemotherapy . patients in experimental group underwent 24 days of whole - breast 2-field tangent radiotherapy of 43.2 gy in 18 fractions ( dose / fraction=2.4 gy ) with a concomitant boost to the tumor bed of 50.4 gy in 18 fractions ( dose / fraction=0.4 gy ) . those in control group underwent 44 days of whole - breast 2-field tangent radiotherapy of 45 gy in 25 fractions with a dose / fraction of 1.8 gy and a subsequent boost to the tumor bed of 59 gy in 7 fractions ( dose / fraction=2 gy ) . irradiation was administered to the entire breast tissue and to lymph nodes between the pectoral muscles and lymphatic drainage area of the pectoral wall under the breast . we used 6 mv of x - ray , which was 95% of the prescribed isodose x - ray . the irradiated range of the tumor bed was 12 cm outside of the silver clip . the photon beam energy was set between 6 and 9 mv based on tumor bed depth . patients with tumors that were hormone receptor - positive ( er & pr ) received endocrine therapy after chemotherapy . patients were followed until september 2013 . specifically , follow - up was performed immediately at the end of radiation and 6 weeks , 6 months , 12 months , and 24 months after this time . follow - up observations included a review of patient medical history , physical examination , bilateral breast anteroposterior x - ray examination , chest radiography , mammography , and breast ultrasound . secondary endpoints included acute skin reactions , advanced skin reactions , aesthetic outcome , and hematological toxicity . locoregional recurrence was defined as the area of the breast and the supraclavicular lymph drainage area within the radiation field ; definite diagnosis was confirmed by both clinical and imaging examinations . skin adverse reactions ( levels l4 ) were assessed according to american acute and late radiation tumor tissue radiation injury grading standards . aesthetic results , including breast edema , skin sag , fibrosis , telangiectasia , scarring , pigmentation , breast size , nipple level , and bilateral symmetry were graded as excellent , good , fair , or poor according to guidelines established by the joint center for radiation therapy ( jcrt ) and as previously described . data for comparability , adverse reactions , and aesthetic outcomes in the 2 groups were compared and analyzed by chi - square test . all statistical analyses were performed using spss version 16.0 ( spss , inc . , chicago , il ) . patients met the inclusion criteria were enrolled the study in the department of radiation oncology , tianjin medical university cancer institute and hospital between january 2011 and december 2011 . patients were randomly divided into an experimental group ( 24-day group ) and a control group ( 44-day group ) using a random number table . written informed consent was obtained from all patients and the ethics committee of tianjin medical university cancer institute and hospital approved the study protocol . patients were enrolled in the study if they met the following inclusion criteria : patients were diagnosed at an early disease stage ( pt1 - 2n0 - 1m0 ) and underwent breast - conserving surgery . all patients were 18 years of age or older , with a karnofsky performance status ( kps ) score greater than or equal to 70 . silver clips were used to denote tumor volume and help localize radiation , which had to be administered within 1 month of surgery . patients who met any of the following exclusion criteria were not enrolled in the study : any patient who had received radiation therapy in the ipsilateral breast , chest wall , lung , or lymph nodes was excluded from the study . additionally , women diagnosed with either inflammatory breast cancer or bilateral breast cancer or who had received prior breast - conserving surgery or breast reconstruction surgery were excluded . finally , those who experienced breast cancer recurrence were prevented from enrolling in the study . patients had undergone various treatment modalities based on their presentation and tumor characteristics . here , we outline the primary treatment regimens used for all patients in the study . breast - conserving surgery was performed by surgical excision of the primary tumor , with a 23 cm margin of macroscopically normal tissue and an axillary dissection . when the sentinel node biopsy was positive , the axillary lymph nodes were dissected ; in all other cases , no dissection of axillary lymph nodes was performed . the dissection of axillary lymph nodes was conducted at either a level i ii or a level i iii , depending on the patient s risk level . during surgery patients received postoperative chemotherapy if they were axillary lymph node positive or if they were axillary lymph node negative but at a high risk of recurrence ( i.e. < 35 years old with a tumor diameter 2 cm , a histological grade of ii iii , signs of vascular invasion , her-2 positive , er / pr negative ) . a 2-drug chemotherapy regimen was used in this study either pharmorubicin combined with cyclophosphamide or pharmorubicin combined with paclitaxel . there was no difference in the proportion of different chemotherapy regimens between the 2 groups . radiotherapy was started within 1 month of surgery and 3 weeks after the first cycle of chemotherapy . patients in experimental group underwent 24 days of whole - breast 2-field tangent radiotherapy of 43.2 gy in 18 fractions ( dose / fraction=2.4 gy ) with a concomitant boost to the tumor bed of 50.4 gy in 18 fractions ( dose / fraction=0.4 gy ) . those in control group underwent 44 days of whole - breast 2-field tangent radiotherapy of 45 gy in 25 fractions with a dose / fraction of 1.8 gy and a subsequent boost to the tumor bed of 59 gy in 7 fractions ( dose / fraction=2 gy ) . irradiation was administered to the entire breast tissue and to lymph nodes between the pectoral muscles and lymphatic drainage area of the pectoral wall under the breast . we used 6 mv of x - ray , which was 95% of the prescribed isodose x - ray . the irradiated range of the tumor bed was 12 cm outside of the silver clip . the photon beam energy was set between 6 and 9 mv based on tumor bed depth . patients with tumors that were hormone receptor - positive ( er & pr ) received endocrine therapy after chemotherapy . breast - conserving surgery was performed by surgical excision of the primary tumor , with a 23 cm margin of macroscopically normal tissue and an axillary dissection . when the sentinel node biopsy was positive , the axillary lymph nodes were dissected ; in all other cases , no dissection of axillary lymph nodes was performed . the dissection of axillary lymph nodes was conducted at either a level i ii or a level i iii , depending on the patient s risk level . during surgery , patients received postoperative chemotherapy if they were axillary lymph node positive or if they were axillary lymph node negative but at a high risk of recurrence ( i.e. < 35 years old with a tumor diameter 2 cm , a histological grade of ii iii , signs of vascular invasion , her-2 positive , er / pr negative ) . a 2-drug chemotherapy regimen was used in this study either pharmorubicin combined with cyclophosphamide or pharmorubicin combined with paclitaxel . there was no difference in the proportion of different chemotherapy regimens between the 2 groups . radiotherapy was started within 1 month of surgery and 3 weeks after the first cycle of chemotherapy . patients in experimental group underwent 24 days of whole - breast 2-field tangent radiotherapy of 43.2 gy in 18 fractions ( dose / fraction=2.4 gy ) with a concomitant boost to the tumor bed of 50.4 gy in 18 fractions ( dose / fraction=0.4 gy ) . those in control group underwent 44 days of whole - breast 2-field tangent radiotherapy of 45 gy in 25 fractions with a dose / fraction of 1.8 gy and a subsequent boost to the tumor bed of 59 gy in 7 fractions ( dose / fraction=2 gy ) . irradiation was administered to the entire breast tissue and to lymph nodes between the pectoral muscles and lymphatic drainage area of the pectoral wall under the breast . we used 6 mv of x - ray , which was 95% of the prescribed isodose x - ray . the irradiated range of the tumor bed was 12 cm outside of the silver clip . the photon beam energy was set between 6 and 9 mv based on tumor bed depth . patients with tumors that were hormone receptor - positive ( er & pr ) received endocrine therapy after chemotherapy . patients were followed until september 2013 . specifically , follow - up was performed immediately at the end of radiation and 6 weeks , 6 months , 12 months , and 24 months after this time . follow - up observations included a review of patient medical history , physical examination , bilateral breast anteroposterior x - ray examination , chest radiography , mammography , and breast ultrasound . secondary endpoints included acute skin reactions , advanced skin reactions , aesthetic outcome , and hematological toxicity . locoregional recurrence was defined as the area of the breast and the supraclavicular lymph drainage area within the radiation field ; definite diagnosis was confirmed by both clinical and imaging examinations . skin adverse reactions ( levels l4 ) were assessed according to american acute and late radiation tumor tissue radiation injury grading standards . aesthetic results , including breast edema , skin sag , fibrosis , telangiectasia , scarring , pigmentation , breast size , nipple level , and bilateral symmetry were graded as excellent , good , fair , or poor according to guidelines established by the joint center for radiation therapy ( jcrt ) and as previously described . data for comparability , adverse reactions , and aesthetic outcomes in the 2 groups were compared and analyzed by chi - square test . all statistical analyses were performed using spss version 16.0 ( spss , inc . , chicago , il ) . in total , 80 patients met the inclusion criteria and were enrolled in the study ( experimental group , n=40 ; control group , n=40 ) ( figure 1 ) . the clinical characteristics between the 2 groups were similar , and detailed information is provided in table 1 . at a median follow - up of 27 months ( range : 2032 months ) the 2-year survival rate of both groups was also 100% , and there was no locoregional recurrence . adverse skin reactions ( levels 12 ) experienced by patients in the 2 groups were similar . no skin toxicities higher than grade 3 were detected in any patient during the follow - up period . patients in both groups experienced overall good aesthetic results ; the only problems encountered were breast fibrosis and alteration in pigmentation in both groups ( 3 cases in experimental group and 2 cases in control group for breast fibrosis , 7 cases in both of the 2 groups for alteration in pigmentation ) . finally , we did not detect any significant differences in hematological toxicity ( i.e. , neutropenia , levels 12 or platelet decline , level 1 ) . overall , there were no statistically significant differences in any of the examined categories between the 2 groups ( table 2 ) . in this study , we examined whether there were any outcome differences between early - stage breast cancer patients receiving standard radiotherapy compared to those receiving whole - breast hypofractionated irradiation with a concomitant boost . we found that there were no significant differences between the 2 regimens in any of the examined outcomes . thus , a shortened whole - breast hypofractionated irradiation schedule with a concomitant boost is as effective as standard radiation and may be a reasonable alternative following breast conservation surgery an earlier study by whelan et al . showed that a significant portion of early - stage breast cancer patients could benefit from accelerated , hypofractionated whole - breast irradiation ( ah - wbi ) . they found that ah - wbi ( 42.5 gy in 16 fractions over 22 days ) was not inferior to standard radiation ( 50 gy in 25 fractions over 35 days ) in terms of acute skin reactions , local recurrence within 10 years , or cosmetic outcome . however , subgroup analysis showed that , compared with standard radiation treatment , ah - wbi had a lower efficiency in high - histological grade patients ; the locoregional recurrence over 10 years in patients undergoing ah - wbi compared to those receiving standard radiation treatment was 15.6% and 4.7% , respectively ( p=0.01 ) . in 2012 , the british columbia cancer center observed a total of 1335 early breast cancer patients with grade 3 disease ( t1t2 , n0 , m0 ) and compared the local relapse rates between hypofractionated radiotherapy and conventionally fractionated schedules ; 252 patients underwent conventional fractionation of 4550 gy in 25 fractions , and 1083 patients received a hypofractionated schedule of 42.544 gy in 16 fractions . the 10-year cumulative incidence of local relapse was 6.9% in the hypofractionated group and 6.2% in the conventionally fractionated group ( p=0.99 ) . the data show that the hypofractionated schedule was no worse than conventional fractionation , even for histologic grade 3 breast tumors . importantly , these results are not consistent with the findings of the whelan study , previously described . another group compared the 5-year local relapse rates between hypofractionated radiotherapy and conventionally fractionated schedules [ 1921 ] . results from this study showed that hypofractionated radiotherapy did not increase the local relapse rate . overall , both treatment regimens showed good efficacy and aesthetic outcomes . additionally , adverse reactions were acceptable , and the cost of treatment was significantly reduced . taken together , these studies show that hypofractionated radiotherapy following breast - conserving surgery is a feasible and worthwhile option . did not include a concomitant boost to the tumor bed , which may prevent direct comparison to our study . there was a statistically significant difference ( p<.0001 ) in local recurrence at 10-year follow - up ( 10.2% versus 6.2% ) . there was also a significant increase in the amount of fibrosis detected in the boost group . in contrast to these 2 outcomes , there was no difference in overall survival between the 2 groups . another study reported that patients receiving a concomitant boost of 14gy to the tumor bed had an increased risk of breast hardening and telangiectasia . however , there were no reported differences in breast appearance , aesthetic outcomes , breast contractures , deformation , edema , or swelling between the 2 groups . recently , raza et al . compared accelerated intensity - modulated radiation therapy ( imrt ) with a concomitant boost to the tumor bed delivered over 3 or 5 weeks against standard 6-week accelerated radiotherapy with a sequential electron boost . acute complications such as breast pain , fatigue , and dermatitis were significantly less in the 3-week regimen compared to the other treatment schedules ( p<0.05 ) . results from studies such as these have made whole - breast radiotherapy with a concomitant boost the standard of treatment in china . while our study supports a shorter radiation schedule , it has certain limitations , including the short follow - up time and the limited number of patients enrolled . thus , additional studies with larger sample sizes must be performed to understand the long - term effects of hypofractionated radiation . in conclusion , our results show that abbreviated hypofractionated radiation is well - tolerated and comparable to longer standard treatment regimens . thus , shortened whole - breast hypofractionated irradiation schedule with a concomitant boost is as effective as standard radiation and may be a reasonable alternative following breast conservation surgery .

proinflammatory cytokines have been implicated in the pathophysiology of both type 1 diabetes ( t1d ) and type 2 diabetes ( t2d ) . t1d is an autoimmune disease involving the adaptive immune system responding to pancreatic beta - cells as antigen - presenting cells . this attracts immune cells that surround pancreatic islets ( insulitis ) and secrete cytokines , such as il-1beta , ifn - gamma , and tnf - alpha , in close proximity to pancreatic beta - cells . in contrast , there is little evidence for such a focused autoimmune response in t2d . instead , the innate immune system , which responds to cellular damage and pathogens , appears to play a key role . there are three major sources of proinflammatory cytokines that may impact islet / beta - cell function in t2d : ( 1 ) from islet cells , ( 2 ) from increased numbers of intraislet macrophages / immune cells , and ( 3 ) from increased circulating levels of proinflammatory cytokines due to obesity , presumably coming from inflamed adipose tissue . these differences between t1d and t2d are reflected by significant differences in the cytokine concentration , duration , and milieu . this review focuses on chronic versus acute cytokine action , cytokine concentrations , and cytokine milieu from the perspective of the pancreatic islet in t2d . we conclude that new cytokine models may be needed to reflect the pathophysiology of t2d more effectively than what are currently employed .

nearly a century has passed since insulin codiscoverers frederick banting and charles best observed the first patient to receive insulin therapy in 1922 , instantly transforming the prognosis of type 1 diabetes ( t1d ) from a death sentence to a manageable disease . however , the root cause of t1d is still not known , but the processes that lead to the destruction of insulin - producing pancreatic beta - cells have been fairly well elucidated . t1d is now considered an autoimmune disease that causes the gradual , systematic destruction of pancreatic beta - cells within the islets of langerhans . proinflammatory cytokines play a prominent role in the pathophysiology of t1d [ 16 ] , but increasing evidence also suggests a significant role for cytokines in islet dysfunction in t2d as well [ 68 ] . in this review , i address the substantial differences in the inflammatory environment of the pancreatic islet in t1d versus t2d and then consider alternative models of cytokine exposure that may more accurately reflect the pancreatic environment in t2d , with particular emphasis on the islet . there are notable similarities and differences in the action of cytokines in the development of t1d versus t2d [ 9 , 10 ] , as shown in figure 1 and described below . in t1d , beta - cells are the direct target of an autoimmune invasion beginning with peri - insulitis and ending in beta - cell death [ 1012 ] . in t2d , metabolic stress is thought to activate the innate immune system , resulting in a chronic inflammatory state marked by increased cytokines , increased islet - associated macrophages , and beta - cell apoptosis [ 10 , 13 , 14 ] . in t1d , autoimmunity is the prime effector of beta - cell destruction . in t2d , a number of metabolic stress factors related to excess nutrients are thought to contribute to beta - cell decline and destruction including glucotoxicity [ 1517 ] , lipotoxicity [ 1517 ] , oxidative stress [ 16 , 18 ] , endoplasmic reticulum ( er ) stress [ 19 , 20 ] , amyloid deposition , and inflammation [ 8 , 13 ] . some evidence suggests that all of these factors may even be connected through inflammasome activation [ 2224 ] . to bring this discussion full circle , emerging research suggests that some of the putative factors causing beta - cell dysfunction in t2d , most notably er stress and/or oxidative stress , may play a role in antigen presentation to trigger the autoimmune response in t1d [ 2527 ] . the key proinflammatory cytokines in t1d are interleukin- ( il- ) 1beta , tumor necrosis factor alpha ( tnf - alpha ) , and interferon - gamma ( ifn - gamma ) . il-1beta is produced by monocytes ( leukocytes ) , macrophages , and other immune cells . tnf - alpha is produced by macrophages , lymphoid , stromal cells , and many other cell types and can exist in membrane - bound as well as in soluble forms [ 28 , 29 ] . ifn - gamma is primarily produced by natural killer cells and certain types of t cells , although macrophages can also produce ifn - gamma under certain conditions , such as exposure to il-12 and il-18 [ 31 , 32 ] . infiltrating immune cells are thought to secrete these three cytokines in close proximity to the beta - cell at high concentrations in t1d , and these cytokines interact synergistically to inflict cytotoxic effects [ 3 , 3336 ] . although rodent islets may succumb to high concentrations of any of these cytokines , classic studies demonstrated that combinations of these cytokines produced more consistent cytotoxic effects on human islets [ 3 , 4 , 34 , 37 , 38 ] . in t2d , the metabolic stress of obesity is thought to elevate cytokine production in adipose tissue [ 3943 ] and other organs to a lesser extent , resulting in an overall chronic low - grade inflammatory state . in the next section , key parameters will be examined to define a model of islet exposure to cytokines that may resemble the pancreatic environment more closely during the development of t2d . a number of proinflammatory cytokines are elevated in the general circulation of obese individuals compared to lean individuals , and these increased levels are associated with an increased risk of developing t2d [ 4553 ] . this low - grade systemic inflammation may also play a direct role in triggering beta - cell dysfunction , particularly in t2d . we have previously reported that cytokines can directly affect aspects of islet function in rodent islets at circulating concentrations in vitro [ 5456 ] . however , these effects , which are primarily related to intracellular calcium handling , do not appear to impact cell death rate or insulin secretion in normal islets . as shown in figure 2 , the t2d - like cytokines , in the range of circulating levels in the blood , do not increase cell death or decrease insulin secretion significantly . the t1d - like concentrations of cytokines necessary to induce cell death are at least 50-fold higher than what is observed in low - grade systemic inflammation in t2d ( see also similar published data in ) . the 5 ng / ml concentration of il-1beta commonly found in the literature is 10 times greater than the t1d - like concentration and 500 times greater than the t2d - like concentration shown in figure 2 . this suggests that the use of high concentrations of death - inducing cytokines is not necessarily appropriate for recreating an islet environment related to t2d . to build an appropriate in vitro model of chronic low - grade inflammation that can reasonably mimic the islet environment in t2d in vivo , several questions must be addressed : what cytokines and chemokines are elevated in the systemic circulation in obesity and t2d that can negatively impact islet function?what immune cells are found in or near islets that can secrete cytokines in close proximity?what cytokines are secreted by islet cells themselves in response to damage or stress?what concentration and duration of exposure mimic chronic low - grade inflammation?each of these considerations is discussed in sections 3.13.4 . what cytokines and chemokines are elevated in the systemic circulation in obesity and t2d that can negatively impact islet function ? what immune cells are found in or near islets that can secrete cytokines in close proximity ? circulating levels of several cytokines and chemokines have been identified as potential risk factors for developing t2d , including tnf - alpha , c - reactive protein ( crp ) , monocyte chemoattractant protein-1 ( mcp-1 ) , il-1beta , and il-6 [ 4553 ] . il-1beta at high enough concentrations can destroy beta - cells [ 58 , 59 ] . antagonizing the actions of il-1beta has been shown to have clinical value in partially mitigating the symptoms of t2d [ 6063 ] , suggesting that il-1beta plays a role in the pathophysiology of the disease . numerous studies have also associated increased levels of il-6 with the development of type 2 diabetes [ 6467 ] . however , il-6 has also been associated with beneficial effects of exercise ( reviewed in ) and with improving islet function [ 69 , 70 ] , resulting in a complex and evolving view over the role(s ) of il-6 in t2d [ 7173 ] . a prospective study focused primarily on links between nutrition and cancer generated an intriguing report that identified the combination of both il-1beta and il-6 as key cocontributors to the development of t2d . we reported that serum levels of il-1beta and il-6 in diabetes - prone mice at an age before hyperglycemia developed were 2 - 3 times higher than for age - matched heterozygous control mice , suggesting that low - grade systemic inflammation develops early in the disease process . in addition , many other cytokines and chemokines are increased in obesity including leptin , resistin , il-7 , il-8 , retinol binding protein- ( rbp- ) 4 , plasminogen activator inhibitor- ( pai- ) 1 , chemokine- ( c - x - c motif- ) ligand 5 ( cxcl5 ) , visfatin , chemerin , and vaspin [ 75 , 76 ] . other cytokines are decreased with increased obesity including adiponectin , il-10 , and omentin [ 75 , 76 ] . we conducted a 32-plex cytokine detection array of mouse blood serum from leptin - receptor - deficient male bks.cg - dock7m+/+ leprdb / j ( db / db ) mice and heterozygous controls at different ages to identify additional cytokines and chemokines . many of the 32 cytokines tested were above or below the sensitivity for appropriate detection or were highly variable . however , cxcl1 and cxcl5 were increased significantly in serum at the onset of obesity and t2d . we further showed that exposure to circulating concentrations of these chemokines synergistically produced mild inhibition of islet function and that expression of cxcl1 and cxcl5 increased markedly in islets in response to low - grade inflammation . regarding the classic trio of t1d cytokines , il-1beta appears to also play a role in t2d , but tnf - alpha and ifn - gamma may or may not be as prominent . in our studies , we did not observe elevated serum levels of tnf - alpha in prediabetic mice ( ifn - gamma was not examined ) , and tnf - alpha does not appear to have the same degree of impact as il-1beta and il-6 on the development of t2d in humans . ifn - gamma appears to be elevated in obesity and may play a role in islet dysfunction in t2d [ 78 , 79 ] . however , at present there is not sufficient evidence that circulating levels of ifn - gamma impact islet function or that ifn - producing immune cells are localized within islets in models of t2d . additional studies are needed to determine the full milieu of obesity - associated cytokines and the extent to which this milieu of circulating cytokines can impact islet function . in addition to the chronic inflammatory state marked by increased circulating cytokines , signs of increased inflammation in t2d have also been observed within the pancreas itself . specifically , increased numbers of islet - associated macrophages have been reported in rodent models of t2d and in humans with t2d [ 80 , 81 ] . in the db / db mouse model of diabetes , a 4-fold increase in m1 type macrophages was reported as early as 8 weeks of age . macrophages are involved in adaptation and cellular repair as well as in the discrete removal of dead and dying cells . thus , increased islet presence of macrophages could merely be an indicator of more dying cells that must be cleared away . however , macrophages could also be active instigators of destruction by secreting various proinflammatory cytokines in close proximity to islet cells . the potential of macrophage instigation of islet dysfunction has been confirmed by studies showing that palmitate causes macrophage accumulation , increased cytokine / chemokine production within islets , and islet dysfunction , but islet dysfunction is prevented when macrophage accumulation is blocked [ 83 , 84 ] . these findings suggest that macrophages play an important role in inflammation caused by metabolic stress . in addition to macrophage involvement , recent work also suggests that clusters of differentiation- ( cd- ) 45-positive leukocytes are also increased in islets from t2d donors , suggesting a possible role of the adaptive immune system in t2d [ 85 , 86 ] . chronic high levels of glucose , for example , can stimulate il-1beta production in beta - cells . for example , treatment with ifn - gamma + tnf - alpha increases islet expression of il-15 , interferon - gamma - induced protein- ( ip- ) 10 , cxcl9 , cxcl11 , and chemokine ( c - c motif ) ligand- ( ccl- ) 20 . exposure to free fatty acids , palmitate in particular , can induce islet expression of numerous cytokines and chemokines , including il-1beta , tnf - alpha , il-6 , il-8 , cxcl1 , and mcp-1 [ 83 , 84 , 90 ] . the importance of intraislet cytokine production is that cytokine concentrations within islets may be orders of magnitude higher when cytokines are produced by islet cells and/or resident immune cells within the islet in comparison to circulating levels . it should be noted that the increased cytokine expression in many of these studies could not be attributed to any specific cell type(s ) within the islet . thus , it is possible that immune cells within the islet could be responsible for most or all of the increased expression of these cytokines , rather than insulin - producing beta - cells , glucagon - producing alpha cell , or other endocrine cell types in the islet . thus , intraislet secretion of cytokines , regardless of the source , may markedly increase the potential concentration of cytokine exposure in islet cells . the net effect of cytokine exposure on tissue depends markedly on factors such as cytokine concentration , duration of cytokine exposure , and the combination of cytokines involved ( also called the cytokine milieu [ 91 , 92 ] ) . il-1beta , for example , causes islet dysfunction or cell death in a number of studies of chronic exposure [ 37 , 58 ] , and blockade of the il-1 receptor mitigates many of these effects [ 60 , 93 , 94 ] . however , il-1beta can also enhance insulin secretion [ 9597 ] , and recent work even suggests il-1beta may play a key role in islet compensation for nutrient overload in the early stages of metabolic disorders . in one early series of studies , both positive and negative impacts of il-1beta were described based on differences and dose and duration of exposure [ 99101 ] . in the context of t2d , the duration of low - grade inflammation may be a key factor , as cytokines could have compensatory / protective effects initially that become deleterious under chronic conditions . thus , the dose and duration of cytokine exposure [ 98 , 101 , 102 ] , species being tested [ 103105 ] , and milieu of interacting cytokines [ 3 , 4 , 95 , 105 , 106 ] are all important factors that contribute to both positive or negative effects on the beta - cell . in addition , synergistic activity among multiple cytokines can alter or amplify signaling pathways [ 3 , 4 , 107 , 108 ] , adding an additional layer of complexity to cytokine action . our recent work has focused on elucidating possible differences in response to chronic low - grade inflammation between islets from diabetes - prone and normal ( nondiabetes - prone ) environments . male db / db mice and heterozygous controls were used at 4 - 5 weeks of age , an age at which the capacity for glucose - stimulated insulin secretion and intracellular calcium responses are similar for both mouse strains . the combination of il-1beta and il-6 significantly impaired islet function in ways that either alone could not . further , islets isolated from prediabetic db / db mice and exposed in vitro to il-1beta + il-6 overnight at approximately circulating levels ( in mice ) caused a significant decrease in glucose - stimulated insulin secretion , a significant increase in er stress markers ( nitric oxide synthase 2 ( nos2 ) , 78-kda glucose - regulated protein ( grp78 ) , activating transcription factor 4 ( atf4 ) , and dna damage inducible transcript 3 ( ddit3 ) , also known as c / ebp homologous protein ( chop ) ) , and increased cell death ; these cytokines produced no such effect in heterozygous control islets . further , when nondiabetic control mice were implanted with subcutaneous osmotic mini - pumps containing il-1beta + il-6 to mimic the serum increases found in prediabetic db / db mice , we were able to produce 2 - 3-fold increases in circulating cytokines , thus reproducing cytokine levels observed in low - grade inflammation in obesity . the increased circulating levels of il-1beta + il-6 were insufficient to impact physiology in these normal mice . when compared with saline - implanted controls , however , isolated islets from cytokine - pump mice showed deficiencies in calcium handling and insulin secretion that were similar to cytokine effects on islets in vitro . these results in mice suggest that mild increases in circulating cytokines may be sufficient to trigger islet dysfunction leading to islet failure in genetically susceptible individuals . the cytokine signaling pathways may also differ in conditions of low - grade inflammation when compared to classic cytokine cocktails associated with t1d . a microarray study of islet gene expression in response to low concentrations 10 pg / ml il-1beta + 20 pg / ml il-6 produced a large number of gene hits that were apparently not associated with canonical signaling pathways for il-1beta and il-6 . several highly cytokine - induced genes related to proteins involved with iron regulation , including steap4 ( six - transmembrane epithelial antigen of prostate 4 ) , lipocalin-2 ( lcn-2 ) , and hepcidin antimicrobial peptide ( hamp ) . moreover , seven cytokine - sensitive genes were identified for single nucleotide polymorphisms related to the acute insulin response to glucose ( airg , a test of islet function ) in a genome - wide association scan of a population with a high prevalence for t2d . the seven genes were arap3 ( arfgap with rhogap domain , ankyrin repeat , and ph domain 3 ) , f13a1 ( coagulation factor xiii , a1 polypeptide ) , klhl6 ( kelch - like protein 6 ) , nid1 ( nidogen 1 ) , pamr1 ( peptidase domain - containing protein associated with muscle regeneration 1 ) , ripk2 ( receptor - interacting protein kinase 2 ) , and steap4 . the microenvironment of the pancreatic islet during the development of t2d has yet to be fully elucidated . with regard to inflammatory - mediated processes , several sources of proinflammatory cytokines have been suggested throughout this review : ( 1 ) immune - cell - derived cytokines from macrophages and lymphocytes , ( 2 ) cytokines derived from inflamed fat tissues or other distal sources that increase cytokine concentration in the general circulation , and ( 3 ) cytokines / chemokines derived from peptide - producing islet cells such as alpha - cells or beta - cells ( see figure 3 ) . creating accurate in vitro models of the concentration , duration , and combination of key cytokines involved with each of these sources will contribute greatly toward a better understanding of how islets and other organs such as the liver , muscle , fat , and kidney respond to low - grade inflammation . we have made a first attempt at a model of the circulating levels of proinflammatory cytokines utilizing il-1beta and il-6 . although the inflammatory environment is dynamic , we focused on cytokines involved prior to the onset of hyperglycemia ( the prediabetic stage ) . several observations served as our rationale for developing this model of circulating cytokines in t2d . first , the combination of these two cytokines is sufficient to significantly increase the risk of developing t2d in humans . second , increased levels of il-1beta and il-6 were observed in db / db mice prior to hyperglycemia or substantial differences in weight ( < 10% ) compared to control mice . third , exposure to combinations of these cytokines at circulating levels affected islets in unique ways that treatment with individual cytokines could not replicate [ 55 , 74 ] . these studies collectively point to the combination of 510 pg / ml il-1beta and 1020 pg / ml il-6 as being sufficient to promote islet dysfunction in t2d . to produce these levels of low - grade systemic inflammation in vivo osmotic mini - pumps are ideally designed to produce the small and precise changes in the circulating levels of cytokines that define chronic inflammation . we induced low - grade inflammation in vivo in normal healthy mice loading alzet osmotic mini - pumps ( model 1007d , rate 0.5 l / h for 7 days ) with 32 g / ml for il-1b plus 4 g / ml for il-6 in saline or saline only controls . we inserted each pump subcutaneously into an incision at the nape of the neck to cause low - grade inflammation in mice without significantly impacting blood glucose or insulin levels . elisa assays confirmed that serum levels of il-1b and il-6 were approximately doubled in mice treated with cytokine mini - pumps versus saline control pumps . islets isolated from cytokine - pump - treated mice showed impaired function compared to saline - pump controls . these findings are detailed in and provide a method for elevating specific circulating cytokines related to chronic low - grade inflammation in t2d , independent of obesity . differences in susceptibility to cytokine - induced damage between diabetes - prone and nondiabetes - prone islets suggest that certain genetic predispositions may render some individuals much more susceptible to the negative impact of proinflammatory cytokines than others . the relatively small increases in circulating cytokines caused by obesity or other chronic inflammatory conditions could conceivably contribute to a plethora of conditions including asthma [ 110 , 111 ] , rheumatoid arthritis , heart disease , various cancers [ 114116 ] , polycystic ovarian syndrome [ 117 , 118 ] , and even mood disorders . thus , the combination of the genetic predisposition of the individual and the dose , duration , and milieu of cytokine exposure may significantly contribute to numerous chronic diseases .

backgroundthere has been an increase in the use of fine needle aspiration cytology ( fnac ) for the diagnosis of parathyroid lesions ( pls ) . differentiation between a thyroid lesion and a pl is not easy because of their similar features . we reviewed parathyroid aspirates in our institution and aimed to uncover trends in diagnostic criteria.methodswe selected 25 parathyroid aspirates ( from 6 men and 19 women ) confirmed surgically or immunohistochemically from 2006 to 2011.resultsmajor architectural findings of pls include scattered naked nuclei , loose clusters , a papillary pattern with a fibrovascular core , tight clusters , and a follicular pattern . these architectures were commonly admixed with one another . cytological features included anisokaryosis , stippled chromatin , a well - defined cell border , and oxyphilic cytoplasm . eighteen of the 25 patients were diagnosed with pl using fnac . seven patients had been misdiagnosed with atypical cells ( n=2 ) , benign follicular cells ( n=2 ) , adenomatous goiter ( n=2 ) and metastatic carcinoma ( n=1 ) in fnac . using clinicoradiologic data , the sensitivity of the cytological diagnosis was 86.7% . the cytological sensitivity decreased to 50% without this information.conclusionsfnac of pl is easily confused with thyroid lesions . a combination of cytological parameters and clinical data will be required to improve the diagnostic sensitivity of pls .

we evaluated 25 fnac specimens from 25 patients with well - documented cases of pl according to serum parathyroid hormone ( pth ) level , immunohistochemical staining , ultrasonography , and pathology results at the korea cancer center hospital from january 2006 to may 2011 . cases that presented as a single mass were included , and those that were not confirmed by operation or immunohistochemistry were excluded . clinicoradiologic information , such as signs or symptoms , serum pth level , ultrasonography , and operation records , were obtained from patients ' medical records . the aspirated material was smeared onto four glass slides that were immediately fixed in 95% alcohol and subjected to papanicolaou staining . thyroid transcription factor-1 ( ttf-1 ) , pth , and chromogranin a were selectively used for immunohistochemistry on cell blocks . diagnostic terminology of " parathyroid lesion " ( pl ) was used if the cytological findings or immunohistochemistry results indicated a parathyroid origin . twenty patients underwent surgery , and five patients did not . in those patients who underwent surgery , all sections of the resection specimens the five patients who did not receive surgery , pl was confirmed by immunohistochemical staining of cell blocks . all the slides were reviewed with a multi - headed microscope by two separate pathologists . the cytological features were categorized as follows : architectural features ( scattered naked nuclei , loose clusters , a papillary pattern with a fibrovascular core , tight clusters , and a follicular pattern ) , nuclear features ( anisokaryosis , stippled chromatin , prominent nucleoli , intranuclear pseudoinclusion , and eccentric nuclei ) and other features ( a well - defined cell border , oxyphilic cytoplasm , colloid - like material , and macrophages ) . the patient population included six males and 19 females with an age range of 39 to 71 years ( mean age , 51 years ) ( table 1 ) . of the 25 patients , 22 were evaluated for serum pth before the cytological examination . the pl was found at the posterior surface of the thyroid gland in nine patients ( 36% ) , inferior to the thyroid gland in seven patients ( 28% ) , in the thyroid parenchyma in six patients ( 24% ) , and in the paratracheal area in three patients ( 12% ) . the clinicoradiologic impression consisted of 11 incidentalomas ( 44% ) , eight phpts ( 32% ) , four pls ( 16% ) , and two metastatic papillary thyroid carcinomas ( ptc ) ( 8% ) . eighteen of the 25 cases ( 72% ) were cytologically diagnosed as pl ; the remaining seven patients were diagnosed as follows : two with benign follicular cells ( 8% ) , two with adenomatous goiter ( 8% ) , two with atypical cells ( 8% ) , and one with metastatic carcinoma ( 4% ) . when the clinicoradiologic impression suggested pl , the sensitivity of the cytological diagnosis was 86.7% ( 13/15 ) . in all other cases , a minimally invasive parathyroidectomy was performed in 17 of the 20 patients who underwent surgery , and the remaining three patients received thyroid surgery or neck dissection . eighteen of the 20 cases who received surgery were diagnosed as parathyroid adenoma , and the remaining two were determined to have parathyroid carcinoma . in the aspirates from the five patients who did not receive surgery , pth was expressed in two , and the remaining three aspirates demonstrated positivity for chromogranin a. ttf-1 was negative in all four cases who were examined ( fig . the most impressive feature of the parathyroid aspirates was the diversity of the architectural features . most of the smears showed scattered naked nuclei ( 25/25 , 100% ) and loose clusters ( 22/25 , 88% ) in varying degrees . other architectural features included a papillary pattern with a fibrovascular core ( 17/25 , 68% ) , tight clusters ( 14/25 , 56% ) , and a follicular pattern ( 11/25 , 44% ) ( table 2 , fig . each aspirate showed a combination of foregoing architectures in varying degrees . in three aspirates , the entire specimen consisted of scattered naked nuclei and closely resembled thyroiditis ( 3/25 , 12% ) . almost all nuclei were centrally located , round to oval and similar in size to red blood cells with a regular nuclear membrane . a small proportion of parathyroid cells showed mild to moderate anisokaryosis in each aspirate ( 24/25 , 96% ) . prominent nucleoli and intranuclear pseudoinclusions were only detected in one aspirate ( 1/25 , 4% ) ( table 2 , fig . 3 ) . many parathyroid cells had finely granular cytoplasm , and a small portion of cells revealed a well - defined cell border ( 9/25 , 36% ) and oxyphilic cytoplasm ( 9/25 , 36% ) . fragments of hyaline colloid - like material were noted in three aspirates ( 12% ) . another two cases ( patients nos . 3 and 4 ) were diagnosed as adenomatous goiter ; these patients underwent minimally invasive parathyroidectomy because pl was diagnosed using an intraoperative frozen biopsy . clinical impression and cytological diagnosis were metastatic carcinoma , so neck dissection was performed . the dominant cytological architecture in this case consisted of tight clusters , although other cytological features of pl were present in small proportions . a papillary pattern with a fibrovascular core , anisokaryosis and stippled chromatin were also found . although pl was diagnosed using intraoperative frozen biopsy , the remaining lobe was removed by lobectomy . in patient no . 7 , who had simultaneous ptc , the paratracheal mass was clinically thought to be metastatic ptc . the dominant architecture was loose clusters , and other cytological findings were a papillary pattern with scattered naked nuclei , tight clusters , a follicular pattern , anisokaryosis , and stippled chromatin . the patient population included six males and 19 females with an age range of 39 to 71 years ( mean age , 51 years ) ( table 1 ) . of the 25 patients , 22 were evaluated for serum pth before the cytological examination . the pl was found at the posterior surface of the thyroid gland in nine patients ( 36% ) , inferior to the thyroid gland in seven patients ( 28% ) , in the thyroid parenchyma in six patients ( 24% ) , and in the paratracheal area in three patients ( 12% ) . the clinicoradiologic impression consisted of 11 incidentalomas ( 44% ) , eight phpts ( 32% ) , four pls ( 16% ) , and two metastatic papillary thyroid carcinomas ( ptc ) ( 8% ) . eighteen of the 25 cases ( 72% ) were cytologically diagnosed as pl ; the remaining seven patients were diagnosed as follows : two with benign follicular cells ( 8% ) , two with adenomatous goiter ( 8% ) , two with atypical cells ( 8% ) , and one with metastatic carcinoma ( 4% ) . when the clinicoradiologic impression suggested pl , the sensitivity of the cytological diagnosis was 86.7% ( 13/15 ) . in all other cases , a minimally invasive parathyroidectomy was performed in 17 of the 20 patients who underwent surgery , and the remaining three patients received thyroid surgery or neck dissection . eighteen of the 20 cases who received surgery were diagnosed as parathyroid adenoma , and the remaining two were determined to have parathyroid carcinoma . in the aspirates from the five patients who did not receive surgery , pth was expressed in two , and the remaining three aspirates demonstrated positivity for chromogranin a. ttf-1 was negative in all four cases who were examined ( fig . the most impressive feature of the parathyroid aspirates was the diversity of the architectural features . most of the smears showed scattered naked nuclei ( 25/25 , 100% ) and loose clusters ( 22/25 , 88% ) in varying degrees . other architectural features included a papillary pattern with a fibrovascular core ( 17/25 , 68% ) , tight clusters ( 14/25 , 56% ) , and a follicular pattern ( 11/25 , 44% ) ( table 2 , fig . each aspirate showed a combination of foregoing architectures in varying degrees . in three aspirates , the entire specimen consisted of scattered naked nuclei and closely resembled thyroiditis ( 3/25 , 12% ) . almost all nuclei were centrally located , round to oval and similar in size to red blood cells with a regular nuclear membrane . a small proportion of parathyroid cells showed mild to moderate anisokaryosis in each aspirate ( 24/25 , 96% ) . prominent nucleoli and intranuclear pseudoinclusions were only detected in one aspirate ( 1/25 , 4% ) ( table 2 , fig . many parathyroid cells had finely granular cytoplasm , and a small portion of cells revealed a well - defined cell border ( 9/25 , 36% ) and oxyphilic cytoplasm ( 9/25 , 36% ) . fragments of hyaline colloid - like material were noted in three aspirates ( 12% ) . another two cases ( patients nos . 3 and 4 ) were diagnosed as adenomatous goiter ; these patients underwent minimally invasive parathyroidectomy because pl was diagnosed using an intraoperative frozen biopsy . clinical impression and cytological diagnosis were metastatic carcinoma , so neck dissection was performed . the dominant cytological architecture in this case consisted of tight clusters , a papillary pattern with a fibrovascular core , anisokaryosis and stippled chromatin were also found . although pl was diagnosed using intraoperative frozen biopsy , the remaining lobe was removed by lobectomy . in patient no . 7 , who had simultaneous ptc , the paratracheal mass was clinically thought to be metastatic ptc . the dominant architecture was loose clusters , and other cytological findings were a papillary pattern with scattered naked nuclei , tight clusters , a follicular pattern , anisokaryosis , and stippled chromatin . the ability to distinguish between pl and thyroid lesions using fnac has important clinicoradiologic significance for the treatment and quality of life of patients.6 in thyroid neoplasm , a more extensive operation , such as a lobectomy , total thyroidectomy , or neck dissection , is required , whereas pl can be selectively removed with minimal incisions.4 therefore , pathologists need to note the cytomorphologic features of pl to ensure accurate diagnoses . in our study , the variable architectural and cytological features of pl were well presented as follows : scattered naked nuclei , loose clusters , a papillary pattern with a fibrovascular core , tight clusters , a follicular pattern , anisokaryosis , stippled chromatin , a well - defined cell border , and oxyphilic cytoplasm . although a few cases showed absolute predominance of one type of architecture , most cases had variable cytological features , each being mixed in varying degrees . absher et al.2 reported that the presence of a pl may be suspected when recognizing a combination of diverse cytological features rather than a single distinct factor . a few attempts were made to distinguish between pl and thyroid lesions by using fnac . absher et al.2 recommended determining certain characteristic features of pl , including cohesive cellular fragments admixed with numerous single naked nuclei , absent or inconspicuous nucleoli , round to oval uniform nuclei , smaller nuclei than thyroid nuclei , anisokaryosis , and hyperchromatic chromatin rather than finely granular chromatin . dimashkieh and krishnamurthy6 documented that smaller nuclei than thyroid follicular cells , bare nuclei , stippled chromatin , and a prominent vascular network with epithelial cells indicated parathyroid origin . the papillary pattern with a fibrovascular core presented in our study corresponded well with the prominent vascular network and the attached epithelial cells as well as the specific " spot of fire " hypervascularity pattern of the parathyroid gland observed on the color doppler sonogram.4 past studies have stated that colloid - like substances and macrophage accumulation are distinguishing cytological features of thyroid lesions from pl.1,7,8 however , recent studies have revealed that they also can be seen in pl.2,3,6 similarly , colloid - like substances were observed in three of our cases ( 12% ) , even though macrophages were not detected . there was one case with intranuclear pseudoinclusion , which postoperatively proved to be a parathyroid carcinoma . however , intranuclear pseudoinclusion has also been described in parathyroid tumors,9,10 and it was noted in just a few cells in this case . that particular patient had a history of total thyroidectomy due to ptc , and there was no evidence of ptc recurrence . clinicoradiologic features and ancillary tests , such as immunohistochemical testing , can contribute to the correct diagnosis of pl.2,3,6 the sensitivity of cytological diagnosis varied substantially depending upon available clinicoradiologic information , such as serum pth , ultrasonography , and location . an unusual location and other radiologic findings of pl can increase diagnostic difficulty . in our series , six of 25 pls ( 24% ) this number is higher than the previously reported incidence of 53 of 4,868 ( 1% ) parathyroid adenomas.11 in general , there are no distinguishing cytological features between parathyroid adenoma and hyperplasia.2,6 therefore , it is recommended that pl is used in the routine cytological diagnosis if an aspirate suggests a parathyroid origin.2 however , a few studies have indicated that there might be distinguishing cytological features between parathyroid adenoma and hyperplasia.1,12,13 in conclusion , fnac is a very useful method for the diagnosis of pl , although a few reports have mentioned the limitations of parathyroid fnac , such as its low sensitivity , smear inadequacy , and contamination with thyroid tissue.3,8,13 on the basis of the findings from previous reports as well as our study , common cytological architectures of pl are scattered naked nuclei , loose clusters , prominent vasculature with epithelial cells resembling a papillary pattern , tight clusters , and a follicular pattern . common cellular features included anisokaryosis , stippled chromatin , a well - defined cell border , oxyphilic cytoplasm , and smaller nuclei than thyroid follicular cells . minor features include intranuclear pseudoinclusions , colloid - like material , prominent nuclei , and eccentric nuclei . fnac of the parathyroid can easily be confused with that of the thyroid because of their overlap in cytological features . although no single cytomorphologic characteristic can be used as a diagnostic tool,2 a combination of cytological parameters and clinicoradiologic findings should be used to improve the diagnostic sensitivity of pl .

keratoconus ( kc ) is a progressive degenerative inflammatory - related disease of the human cornea leading to decreased visual function . the pathogenesis of kc remains to be understood . recent genetic studies indicate that gene variants of an inflammation - related molecule , hepatocyte growth factor ( hgf ) , are associated with an increased susceptibility for developing kc . however hgf protein expression in kc has not been explored . in this initial study , we investigated late - stage kc and control corneas for the expression of hgf and its receptor mesenchymal - epithelial transition factor ( c - met / met ) . kc buttons ( ~8 mm diameter ) ( n = 10 ) and whole control corneas ( n = 6 ) were fixed in 10% formalin or 2% paraformaldehyde , paraffin embedded and sectioned . sections were immunolabelled with hgf and c - met antibodies , visualised using immunofluorescence , and examined with scanning laser confocal microscopy . semiquantitative grading was used to compare hgf and c - met immunostaining in kc and control corneas . overall , kc corneas showed increased hgf and c - met immunostaining compared to controls . kc corneal epithelium displayed heterogeneous moderate - to - strong immunoreactivity for hgf and c - met , particularly in the basal epithelium adjacent to the cone area . taken together with the recent genetic studies , our results further support a possible role for hgf / c - met in the pathogenesis of kc .

keratoconus ( kc ) is the most common primary human degenerative corneal disease with a prevalence of around 1 in 2000 worldwide . it is bilateral , asymmetric , and progressive , leading to corneal thinning and irregularity . onset primarily occurs in the 2nd decade of life and is associated with significant decreasing visual function and morbidity . kc is the main indication recorded for corneal grafts in australia , and currently its progression can only be halted through surgical interventions including collagen cross - linking that stiffens the cornea using riboflavin and uva . more recently a surgical procedure was developed transplanting isolated bowman 's layer from donor corneas to kc eyes as a further late - stage intervention . the histopathology of kc is well described and includes epithelial and stromal thinning within the apical cone region , breaks in the bowman 's layer , focal fibrosis , and anterior stromal keratocyte apoptosis [ 2 , 7 ] . recent evidence indicates a role for inflammation in the disease , with increased recruitment of inflammatory cells ( e.g. , macrophages , lymphocytes , and antigen presenting cells ) and inflammatory markers such as interleukin-1 ( il-1 ) and transforming growth factor - beta ( tgf- ) observed in kc corneal tissue sections . increased expression of inflammatory markers such as interleukin-6 ( il-6 ) , tumour necrosis factor alpha ( tnf- ) , and matrix metalloproteinase 9 ( mmp-9 ) has also been found in tears collected from kc patients compared to controls . furthermore , a recent review examining the biochemical changes in kc proposed a two - hit hypothesis with a genetic predisposition to the corneal disease and a second hit that may induce abnormalities of inflammatory components . single nucleotide polymorphism ( snp ) refers to a change in a single nucleotide within a dna sequence and is the most common type of genetic variation observed in the human genome . two parallel genome - wide association studies identifying potential snps associated with kc , using independent sample cohorts , reported a significant association between kc and the hepatocyte growth factor ( hgf ) gene , identifying two single nucleotide polymorphisms ( snps ; rs3735520 and rs17501108 ) in the promoter region . ( 2011 ) also examined hgf protein abundance in the serum of controls correlating to the rs3735520 genotype and found a significant increase in hgf serum protein associated with the minor allele t . hgf is a pleiotropic growth factor that activates the hgf / c - met pathway after binding to its receptor , mesenchymal - epithelial transition factor ( c - met / met ) . once activated , downstream pathways such as mitogen - activated protein kinase ( mapk ) cascades , pi3k - akt axis or janus kinase / signal transducers , and activators of transcription ( jak / stat ) pathways may be activated . for example , together with mmp-1 , hgf is reported to initiate human corneal epithelial cell migration in vitro , and exogenous hgf has been found to promote the proliferation of both corneal epithelial and endothelial cells . in injured rabbit corneas , wilson et al . ( 1999 ) reported an obvious increase of hgf mrna expression in keratocytes and c - met mrna expression in epithelial cells compared to unwounded corneas , suggesting that the hgf / c - met pathway plays a role in corneal wound healing . studying bovine corneal wound healing in organ culture models , carrington and boulton ( 2005 ) showed that hgf delayed epithelial layer formation , together with increased differentiation of keratocytes to myofibroblasts , compared with untreated and keratinocyte growth factor ( kgf ) treated corneas . the expression of hgf and c - met proteins in human kc corneas has not been investigated to date . one study has reported increased serum hgf expression for at least the minor allele of hgf snp rs3735520 , associated with increased potential for developing kc . as a first step in assessing the role of hgf protein and its receptor ( c - met ) in kc , we used corneal buttons from patients with severe kc and control human corneas to compare and examine the distribution and expression of these proteins . kc corneal tissue buttons ( vision eye institute , chatswood , nsw australia ) and donor corneas ( lions new south wales ( nsw ) eye bank ) were obtained with consent and approval from the sydney eye hospital human research ethics committee ( hrec 2013/1041 ) . normal donor corneas were obtained from the lions nsw eye bank following appropriate consent and hrec approval . ten corneal buttons were collected from kc patients ( age range from 18 to 32 years ) undergoing corneal transplantation at vision eye institute . all kc patients were diagnosed on the basis of clinical signs and corneal topography and were classified as kc grade 4 ( most severe stage ) ( table 1 ) . six normal donor corneas ( age range 53 to 83 years ) were obtained from the lions nsw eye bank ( table 2 ) . kc corneal buttons ( ~8 mm diameter ) , with the cone apex location marked , were fixed in 10% neutral buffered formalin ( nbf ) . all specimens were paraffin embedded and cut at 6 m . sections were collected on super - frost plus slides ( menzel - glaser , saarbruckener , germany ) and dried before use . , sections were incubated in 0.01 m citrate buffer ( ph 6 ) at 85c for 10 minutes , cooled to 40c , and rinsed in tris - buffered saline ( tbs , ph 7.4 ) with 0.1% tween-20 ( tbst ) . sections were incubated at room temperature ( rt ) in 5% bovine serum albumin ( bsa ) in tbst for 30 minutes , followed by incubation overnight at 4c in hgf or c - met primary antibodies , or appropriate negative controls ( mouse or rabbit igg ) ( table 3 ) . after overnight incubation , sections were washed in tbst and incubated in either goat anti - mouse alexa fluor 488 or donkey anti - rabbit alexa fluor 488 . for co - immunolabelling experiments , a separate series of slides were prepared and hgf visualised with alexa fluor 488 and c - met visualised with alexa fluor 594 , combined with nuclear hoechst stain ( table 3 ) . immunolabelling was repeated at least twice per specimen for each antibody , and appropriate ig controls were included for each experiment . all slides were mounted in 20% glycerol / pbs , coverslipped , sealed with nail varnish , and viewed using a zeiss lsm700 scanning laser confocal microscope and image software ( zen 2011 , carl zeiss microimaging ghbh , jena , germany ) . where more than one colour was to be detected , multichannel excitation bleed - through was minimized using fluorochromes with separated peak excitations . emission bleed - through was minimized by multitracking , where signal crosstalk between neighbouring channels was corrected by performing a sequential image capture routine . semiquantitative grading of single - immunolabelled sections was used to assess the intensity and distribution of hgf and c - met immunoreactivity in the corneal epithelium , stroma , and endothelium . grading for kc buttons was made in the region adjacent to the cone ( adj ) and for control corneas in a similar central corneal region . immunolabelling of the thinned cone area of kc buttons was examined in each specimen but was not graded for comparison with controls because of the obviously altered morphology ( only 1 - 2 epithelial layers present ) . the grading scale used was based on the intensity of the immunofluorescence ( 0 = no staining , 0.5 = very weak , 1 = weak , 2 = moderate , and 3 = strong ) and the percentage ( % ) area immunolabelled ( 0 = 0% , 1 = 1% to 10% , 2 = 11% to 50% , and 3 > 50% ) as described previously . a final grade of 0 to 6 ( intensity + % area immunolabelled ) was then given for each specimen and this data is used to generate frequency histograms for hgf and c - met immunostaining in kc and control specimens , respectively . all kc corneas showed a thickened epithelium adjacent to the more central cone region ( figures 1(a ) and 2(a ) ) , compared to similar regions in the control corneas ( figures 1(c ) and 2(c ) ) , as we previously reported . only one to two layers of flattened epithelium were observed within the cone region ( figures 1(b ) and 2(b ) ) . weak and more evenly distributed cytoplasmic immunostaining for hgf and c - met was detected in the epithelium of control corneas ( figures 1(c ) and 2(c ) ) . in kc corneas , moderate - to - strong cytoplasmic immunostaining was seen within the basal and wing cell epithelial layers adjacent to the cone region ( green fluorescence , figures 1(a ) and 2(a ) ) . only weak - to - moderate immunostaining of hgf and c - met was detected in the kc cone region ( figures 1(b ) and 2(b ) ) . co - immunolabelling for hgf and c - met showed that c - met ( red ) was primarily localised in the epithelium of both control and kc corneas ( figure 3 ) . hgf ( green ) was primarily localised within the stroma ( both keratocytes and extracellular matrix ) of control and kc corneas ( figure 3 ) . weak and relatively uniform immunostaining was observed in similar regions from control corneas for hgf and c - met ( figure 3(a ) ) . however , for kc corneas , increased hgf staining ( green ) was detected in the basal epithelium adjacent to the cone region and colocalised with increased c - met staining ( red ) ( figure 3(d ) ) . limbal areas of control corneas showed slightly stronger stromal immunostaining of hgf compared to the central region of control corneas ( figure 3(b ) ) . semiquantitative grading of hgf and c - met immunostaining in kc samples was increased overall compared to the control corneas for similar regions , as indicated by the skewed distribution of the frequency histograms ( figures 4(a ) and 4(b ) ) . overall , a greater proportion of kc corneas showed > grade 3 immunostaining for both hgf and c - met ( kc : 70% and 90% , resp . , versus control : 16% and 66% resp . ) ( figures 4(a ) and 4(b ) ) . despite the importance of the hgf / c - met pathway in regulating a number of key cellular activities , little is known about its potential role in normal or kc human corneas . as a first step to understand the possible role(s ) of this pathway , we examined patterns of hgf and c - met protein expression in both control and kc corneas . higher levels of hgf and c - met immunostaining were detected in the basal epithelium adjacent to the kc cone , compared to the weaker and more uniform epithelial staining pattern seen in control corneas . in control corneas , we found hgf expression was more intense in the stroma compared to the epithelium . this is consistent with previous studies showing low level hgf mrna expression in the human corneal epithelium compared to keratocytes and endothelium [ 16 , 20 ] . the surface of normal rabbit corneal epithelium was reported to show only low level hgf protein . our immunostaining experiments detected stronger c - met ( hgf receptor ) staining in epithelium compared to keratocytes in control corneas . this is consistent with c - met mrna findings , which showed that human corneal epithelium , keratocytes , and endothelium all expressed c - met , but with highest mrna expression in epithelium [ 16 , 20 , 21 ] . these observations suggest that secreted keratocyte hgf may preferentially bind to the epithelium that expresses higher levels of c - met ( hgf receptor ) , compared to keratocytes that express low levels of c - met , thus potentially regulating key epithelial cellular activities such as cell proliferation . hgf is reported to be involved in two major processes , cell proliferation and migration , and inflammatory - related signalling cascades . hgf is a potent enhancer for corneal epithelial cell proliferation and migration in vivo , and increased hgf and c - met mrna expressions have been detected during corneal wound healing . increased hgf protein expression has been detected in the whole corneal epithelium , keratocytes , and tears of wounded compared to unwounded rabbit corneas . hgf mrna expression was also upregulated in the lacrimal gland when studying injured compared to unwounded rabbit corneas . no difference in c - met immunostaining was detected between wounded and unwounded rabbit corneas . in secondary this was suggested to be most likely due to normal tissue repair pathways already being damaged or compromised , implying involvement of hgf in tissue regeneration . these studies also suggested that lacrimal gland and keratocyte - derived hgf are closely associated with corneal tissue repair [ 17 , 24 ] . in kc corneal epithelium , we observed increased hgf in the region adjacent to the cone , as well as increased c - met ; however it remains to be determined whether the increased expressions of hgf and c - met observed are directly involved in kc pathogenesis or are secondary responses . poorly regulated and overexpressed hgf may be detrimental to tissues , related to the involvement of hgf in inflammation . for example , delayed formation of the epithelium layer and increased formation of stromal myofibroblasts have been detected during rabbit corneal wound healing , for corneas treated with recombinant hgf . applying recombinant hgf to corneas of mice with pseudomonas aeruginosa keratitis also worsened the disease progression , with a significantly higher grade of corneal opacity and thinning compared to pbs - treated corneas . in vitro treatment of corneal stromal keratocytes with proinflammatory interleukin- ( il- ) elevated corneal hgf expression also enhanced proinflammatory cytokines and decreased anti - inflammatory cytokines in pseudomonas aeruginosa keratitis in mice , most likely under the control of inflammatory cytokines and cell growth kinases . , emerging evidence clearly indicates that inflammation - related processes within the epithelium and stroma are involved in the pathogenesis of kc . significant increases in proinflammatory molecules such as il-6 , il-4 , il-5 , il-8 , and il-12 [ 2729 ] , mmp-1 , mmp-3 , mmp-7 , and mmp-13 , and chemokine c - c motif ligand 5 ( ccl5 ) and significantly decreased levels of lactoferrin , serum albumin , and secreted iga ( siga ) have been reported in kc compared to control tears . keratocytes in kc are reported to express more il-1 receptors than controls , and one group has proposed that keratocyte apoptosis observed in kc may be induced by the binding of il-1 receptors secondary to increased levels of il-1 associated with epithelial trauma , for example , due to eye - rubbing . most recently , increased mrna expression of mmp-9 and its inducer proteins il-6 and tnf- were detected in kc corneal epithelium in an indian cohort ( > 100 patients ) compared to healthy controls ( n = 20 ) , and tear protein levels of mmp-9 and il-6 were also increased in kc . treatment with topical cyclosporine a ( an immunosuppressant ) in a small group of kc patients ( n = 20 ) reduced the tear levels of mmp-9 and appeared to halt the progression of kc , consistent with the involvement of inflammation in kc pathogenesis . the link between hgf variants and kc susceptibility was first reported by burdon et al . , ( 2011 ) and then confirmed in an independent european cohort which replicated the association of snp rs3735520 and detected a new hgf snp rs2956540 . in addition , an independent study of an australian population of european descent ( n = 830 ) focused on the hgf locus and detected a different snp ( rs4954218 ) significantly associated with kc . however , a study of rs3735520 implicated a possible regulatory effect of this snp for hgf protein expression in serum from kc patients . as the snps identified to date are all located in the noncoding region of the gene ( rs3735520 , rs17501108 , and rs1014091 in the upstream of hgf and rs2286194 in the intron between exon 8 and exon 9 ) , it is likely that they will affect gene expression through mechanisms such as rna splicing , transcription factor binding , and mirna regulation . our results provide evidence of increased hgf protein in kc epithelium compared to control corneal epithelium ; however the role of the reported hgf snps in the increased protein expression is unclear and will be further investigated . previous studies showed an independent , repeatable association between certain snps and hgf in kc [ 13 , 34 , 35 ] . the snp variations detected were located in the noncoding region of hgf consistent with a role for these snps in the regulating hgf expression , and increased serum hgf expression associated with the minor snp allele has been reported . in the current study , we showed increased hgf protein expression within kc corneal epithelium . taken together previous snp studies , these observations indicate that the hgf / c - met pathway may be involved in the pathogenesis of kc . further studies investigating how the hgf / c - met pathway may be altered in kc , including the associations between snps and protein expression and the role of inflammation requires further investigation . studies on the downstream signalling pathways regulated by hgf / c - met , such as mapk cascades , the pi3k - akt axis , and the jak / stat pathway may help to identify the potential role of this pathway in kc and in normal corneal homeostasis .

backgroundsmoking is the principle risk factor for development of chronic obstructive pulmonary disease ( copd ) . multidrug resistance - associated protein 1 ( mrp1 ) is known to protect against toxic compounds and oxidative stress , and might play a role in protection against smoke - induced disease progression . we questioned whether mrp1-mediated transport is influenced by pulmonary drugs that are commonly prescribed in copd.methodsthe immortalized human bronchial epithelial cell line 16hbe14o was used to analyze direct in vitro effects of budesonide , formoterol , ipratropium bromide and n - acetylcysteine ( nac ) on mrp1-mediated transport . carboxyfluorescein ( cf ) was used as a model mrp1 substrate and was measured with functional flow cytometry.resultsformoterol had a minor effect , whereas budesonide concentration - dependently decreased cf transport by mrp1 . remarkably , addition of formoterol to the highest concentration of budesonide increased cf transport . ipratropium bromide inhibited cf transport at low concentrations and tended to increase cf transport at higher levels . nac increased cf transport by mrp1 in a concentration - dependent manner.conclusionsour data suggest that , besides their positive effects on respiratory symptoms , budesonide , formoterol , ipratropium bromide , and nac modulate mrp1 activity in bronchial epithelial cells . further studies are required to assess whether stimulation of mrp1 activity is beneficial for long - term treatment of copd .

smoking generates oxidative stress in the lungs and is the principal risk factor for development of lung cancer and chronic obstructive pulmonary disease ( copd ) . detoxification and elimination processes of noxious substances present in cigarette smoke are important for both disease prevention and progression , yet little is known on these processes in the lung so far . proteins of the atp - binding cassette ( abc ) superfamily such as the multidrug resistance - associated protein 1 ( mrp1 ) may play a role , since they protect against oxidative stress and other xenobiotics ( cole et al 1992 ) . substrates for mrp1 are glutathione , glucuronate , and sulfate conjugates and unconjugated compounds in presence of glutathione , eg , tobacco - specific nitrosamines ( leslie et al 2001 ) . interestingly , the lung and trachea and other tissues with a barrier function highly express several abc transporters ( langmann et al 2003 ) . especially mrp1 is expressed at high levels in human lung tissue ( van der deen et al 2005 ) , mainly at the basolateral side of bronchial epithelium ( brechot et al 1998 ; scheffer et al 2002 ) . we observed that mrp1 expression is diminished in bronchial epithelial cells of copd patients ( van der deen et al 2006 ) supporting the hypothesis that lower functional mrp1 activity is related to copd development . so far , copd is recognized as a relentlessly progressive disease in which only smoking cessation reduces the accelerated lung function decline . there is no cure for copd , yet recently it has been shown that some drugs are beneficial in the disease management . inhaled corticosteroids and long - acting beta - agonists such as budesonide and formoterol reduce the number of exacerbations in copd ( alsaeedi et al 2002 ; sutherland et al 2003 ) and their combination has been shown to be very effective ( calverley et al 2003 ) . treatment of copd patients with the anticholinergic drug ipratropium bromide results in a small improvement of lung function , yet does not influence the long - term decline in mild copd ( anthonisen et al 1994 ) . studies on oral use of the anti - mucolytic drug n - acetylcysteine ( nac ) have provided contradictory results ( stey et al 2000 ; decramer et al 2005 ) . in contrast to the extensive knowledge on chemotherapeutic drugs as substrates for mrp1 , limited data are available on the effect of pulmonary drugs on the functional expression of mrp1 ( hamilton et al 2002 ) . nac induces higher cellular glutathione levels and in this way can protect against oxidative stress that may indirectly affect mrp1 function ( akan et al 2005 ) but no information is available about budesonide , formoterol , and ipratropium bromide in this respect . in the present study , we questioned whether medications commonly prescribed to copd patients affect mrp1-mediated transport . therefore , we analyzed the direct in vitro effects of budesonide , formoterol , ipratropium bromide , and nac on mrp1 by means of functional flow cytometry in immortalized human bronchial epithelial cells . bovine serum albumin ( bsa , fraction v ) , minimal essential medium ( mem , supplemented with earle s salts and l - glutamine ) and rpmi 1640 medium ( supplemented with 25 mm hepes and l - glutamine ) were purchased from invitrogen life technologies ( breda , the netherlands ) . carboxyfluorescein diacetate ( cfda ) , ipratropium bromide , nac and propidium iodide ( pi ) were obtained from sigma - aldrich bv ( zwijndrecht , the netherlands ) . budesonide ( pulmicort ) was obtained from astrazeneca bv ( zoetermeer , the netherlands ) , formoterol fumarate dihydrate from astra draco ab ( lund , sweden ) , ethylenedinitrilo tetraacetic acid disodiumsalt dihydrate ( edta ) from merck ( darmstadt , germany ) , fetal calf serum ( fcs ) from bodinco bv ( alkmaar , the netherlands ) , vitrogen from nutacon ( leimuiden , the netherlands ) , and mk571 from omnilabo ( breda , the netherlands ) . the human bronchial epithelial cell line 16hbe14o , immortalized with psvori plasmid transfection , dc gruenert ( california pacific medical center research institute ; san francisco , ca ) ( cozens et al 1994 ) . , cells were washed twice with phosphate buffered saline ( pbs : 6.4 mm na2hpo4 , 1.5 mm kh2po4 , 0.14 mm nacl , 2.7 mm kcl , ph = 7.4 ) with 0.5 mm edta . 16hbe14o cells were grown on tissue culture plastics coated with vitrogen ( 30 g / ml ) and bsa ( 10 g / ml ) . to determine mrp1-mediated transport , cells were incubated with 0.1 m cfda as described previously ( van der kolk et al 1998 ) with slight modifications . cfda is intracellularly converted to carboxyfluorescein ( cf ) which is a fluorescent mrp1 substrate . to establish the effect of copd drugs on mrp1-mediated activity , 1 10 cells were incubated in 0.5 ml rpmi 1640 medium without fcs ( 37 c , 5% co2 ) with cfda and with or without the addition of drugs for 1 hour . mk571 ( 20 m ) was used as a positive control for inhibition of mrp1 activity ( van der kolk et al 1998 ) and always showed strong inhibition ( more than 10-fold ) of mrp1-mediated efflux of cf . budesonide and formoterol were added in concentrations of 10 m to 10 m. these drugs were dissolved in 96% ethanol and dimethyl sulfoxide ( dmso ) , respectively . in part of the experiments , cells were simultaneously incubated with budesonide and formoterol . when budesonide and formoterol are combined in one inhaler ( eg , symbicort , astrazeneca ) , the budesonide concentration is ~18- to 35-fold higher than the concentration of formoterol ; therefore we added concentrations of either drug with a comparable proportion . ipratropium bromide was added in concentrations from 10 m to 2 10 m and nac from 10 m to 1.6 10 m and these drugs were dissolved in pbs and water respectively . cells were pelleted for 15 seconds at 12,000 g and resuspended in ice - cold rpmi medium without fcs . drug or mk571 was added a second time in appropriate concentrations for 1 hour , this time without substrate cfda ( 37 c , 5% co2 ) . after pelleting , cells were put on ice to stop efflux of substrate and were resuspended in 350 l rpmi medium with 0.1 g / ml pi to distinguish dead from living cells . fluorescence of cf was analyzed with a facscalibur flow cytometer ( becton dickinson medical systems ; franklin lakes , ny , usa ) . ; topsham , me , usa ) was used to calculate mean fluorescence intensity ( mfi ) values . all measurements were corrected for the negative control ( medium alone ) and for incubation with the solvents 96% ethanol and dmso if appropriate . the percentage of dead cells ( pi positive population ) did not increase with all tested drug concentrations . none of the tested drugs caused autofluorescence ( fluorescence without cfda and pi incubation ) . data are expressed as the mean standard error of the mean ( sem ) . the paired student s t - test ( two - tailed ) was used to calculate modulating effects of the drug under study compared to control . bovine serum albumin ( bsa , fraction v ) , minimal essential medium ( mem , supplemented with earle s salts and l - glutamine ) and rpmi 1640 medium ( supplemented with 25 mm hepes and l - glutamine ) were purchased from invitrogen life technologies ( breda , the netherlands ) . carboxyfluorescein diacetate ( cfda ) , ipratropium bromide , nac and propidium iodide ( pi ) were obtained from sigma - aldrich bv ( zwijndrecht , the netherlands ) . budesonide ( pulmicort ) was obtained from astrazeneca bv ( zoetermeer , the netherlands ) , formoterol fumarate dihydrate from astra draco ab ( lund , sweden ) , ethylenedinitrilo tetraacetic acid disodiumsalt dihydrate ( edta ) from merck ( darmstadt , germany ) , fetal calf serum ( fcs ) from bodinco bv ( alkmaar , the netherlands ) , vitrogen from nutacon ( leimuiden , the netherlands ) , and mk571 from omnilabo ( breda , the netherlands ) . the human bronchial epithelial cell line 16hbe14o , immortalized with psvori plasmid transfection , was kindly provided by dr . dc gruenert ( california pacific medical center research institute ; san francisco , ca ) ( cozens et al 1994 ) . cells were cultured in mem supplemented with 10% heat inactivated fcs . before trypsinization , cells were washed twice with phosphate buffered saline ( pbs : 6.4 mm na2hpo4 , 1.5 mm kh2po4 , 0.14 mm nacl , 2.7 mm kcl , ph = 7.4 ) with 0.5 mm edta . 16hbe14o cells were grown on tissue culture plastics coated with vitrogen ( 30 g / ml ) and bsa ( 10 g / ml ) . to determine mrp1-mediated transport , cells were incubated with 0.1 m cfda as described previously ( van der kolk et al 1998 ) with slight modifications . cfda is intracellularly converted to carboxyfluorescein ( cf ) which is a fluorescent mrp1 substrate . to establish the effect of copd drugs on mrp1-mediated activity , 1 10 cells were incubated in 0.5 ml rpmi 1640 medium without fcs ( 37 c , 5% co2 ) with cfda and with or without the addition of drugs for 1 hour . mk571 ( 20 m ) was used as a positive control for inhibition of mrp1 activity ( van der kolk et al 1998 ) and always showed strong inhibition ( more than 10-fold ) of mrp1-mediated efflux of cf . budesonide and formoterol were added in concentrations of 10 m to 10 m. these drugs were dissolved in 96% ethanol and dimethyl sulfoxide ( dmso ) , respectively . in part of the experiments , cells were simultaneously incubated with budesonide and formoterol . when budesonide and formoterol are combined in one inhaler ( eg , symbicort , astrazeneca ) , the budesonide concentration is ~18- to 35-fold higher than the concentration of formoterol ; therefore we added concentrations of either drug with a comparable proportion . ipratropium bromide was added in concentrations from 10 m to 2 10 m and nac from 10 m to 1.6 10 m and these drugs were dissolved in pbs and water respectively . cells were pelleted for 15 seconds at 12,000 g and resuspended in ice - cold rpmi medium without fcs . drug or mk571 was added a second time in appropriate concentrations for 1 hour , this time without substrate cfda ( 37 c , 5% co2 ) . after pelleting , cells were put on ice to stop efflux of substrate and were resuspended in 350 l rpmi medium with 0.1 g / ml pi to distinguish dead from living cells . fluorescence of cf was analyzed with a facscalibur flow cytometer ( becton dickinson medical systems ; franklin lakes , ny , usa ) . the winlist 5.1 program ( verity software house inc . ; topsham , me , usa ) was used to calculate mean fluorescence intensity ( mfi ) values . all measurements were corrected for the negative control ( medium alone ) and for incubation with the solvents 96% ethanol and dmso if appropriate . the percentage of dead cells ( pi positive population ) did not increase with all tested drug concentrations . none of the tested drugs caused autofluorescence ( fluorescence without cfda and pi incubation ) . data are expressed as the mean standard error of the mean ( sem ) . the paired student s t - test ( two - tailed ) was used to calculate modulating effects of the drug under study compared to control . budesonide increased the accumulation of cf at concentrations of 10 m and 10 m with 26% and 97% , respectively . formoterol had a small yet significant effect on the accumulation of cf at 10 m , but at higher concentrations this effect was not significant ( figure 1b ) . copd patients are often treated with a combined therapy of budesonide and formoterol . to address the question whether the effect of budesonide is altered by addition of formoterol , we incubated the highest concentration of budesonide ( 10 m ) with increasing concentrations of formoterol . with this approach , the budesonide - induced cf accumulation decreased in a dose - dependent manner ; with 10 m , 10 m , and 10 m formoterol , respectively , 198% , 124% , and 58% compared with the control ( figure 2 ) . ipratropium bromide increased cf accumulation at relatively low concentrations ( 10 m to 10 m ) with a maximum of 24% at 10 m ( figure 3a ) . increasing the concentration of ipratropium bromide to 2 10 m potentiated the cf efflux ( 56% compared with control ) , suggesting that mrp1-mediated activity is stimulated rather than inhibited with ipratropium bromide . similar results were obtained with nac , ie , cf accumulation was reduced in a concentration - dependent manner ( 57% at 8 10 m ) ( figure 3b ) . increasing the nac concentrations to 1.6 budesonide increased the accumulation of cf at concentrations of 10 m and 10 m with 26% and 97% , respectively . formoterol had a small yet significant effect on the accumulation of cf at 10 m , but at higher concentrations this effect was not significant ( figure 1b ) . copd patients are often treated with a combined therapy of budesonide and formoterol . to address the question whether the effect of budesonide is altered by addition of formoterol , we incubated the highest concentration of budesonide ( 10 m ) with increasing concentrations of formoterol . with this approach , the budesonide - induced cf accumulation decreased in a dose - dependent manner ; with 10 m , 10 m , and 10 m formoterol , respectively , 198% , 124% , and 58% compared with the control ( figure 2 ) . ipratropium bromide increased cf accumulation at relatively low concentrations ( 10 m to 10 m ) with a maximum of 24% at 10 m ( figure 3a ) . increasing the concentration of ipratropium bromide to 2 10 m potentiated the cf efflux ( 56% compared with control ) , suggesting that mrp1-mediated activity is stimulated rather than inhibited with ipratropium bromide . similar results were obtained with nac , ie , cf accumulation was reduced in a concentration - dependent manner ( 57% at 8 10 m ) ( figure 3b ) . increasing the nac concentrations to 1.6 the membrane protein mrp1 is highly expressed in human lung tissue and may protect the airways against damage induced by cigarette smoking ( scheffer et al 2002 ) . this study shows for the first time the direct influence of pulmonary drugs on mrp1 functional activity , drugs that have already proven their effectiveness in the clinical management of copd . since mrp1 function is most likely cytoprotective in lung cells , we argued that modulation of its function with pulmonary drugs might induce either a positive or negative effect in copd with long - term treatment . this implies either that budesonide is an mrp1 substrate or that it acts on mrp1 function in another way . budesonide is intracellularly esterified to a fatty acid which is thought to be the mechanism of its prolonged activity after a single dose ( oconnell 2003 ) . mrp1 has been shown to be capable of rather slow outward transport of phospholipids , phophatidylcholine and sphingomyelin , and phospholipid analogues in the presence of oxidized glutathione and atp in human erythrocytes and epithelial cells ( dekkers et al 2000 ) . it is tempting to speculate that esterified budesonide could behave like a phospholipid analogue with a polar head and two fatty acid tails and is then outwards transported by mrp1 . as such , budesonide can compete with other mrp1 ( physiological ) substrates and in that way affect the functional activity of mrp1 . in addition , budesonide is also able to affect the transcriptional regulation of mrp1 , as shown by diminished expression of mrp1 in the lung epithelial cell line calu-1 after long - term incubation with budesonide ( 10 m ) ( bandi and kompella 2002 ) . taken together , the data suggest that treatment with budesonide could potentially have a negative effect in copd , given the cell protective properties of mrp1 . our observation does not distract from the well known beneficial effects of budesonide in diseases like asthma , since steroids suppress virtually every step of the inflammatory cascade . however , our findings may contribute an alternative hypothesis why inhaled steroids are not capable of altering the long - term course of copd , particularly in smokers with copd ( alsaeedi et al 2002 ) . in combination with formoterol , the direct inhibitory effect of budesonide on cf accumulation of epithelial cells was reduced , whereas formoterol as such had only minor effects on mrp1-mediated transport . this indicates that budesonide or its esterified derivatives is less available as an mrp1 substrate / inhibitor when combined with formoterol or that formoterol interferes with the interaction between budesonide ( esters ) and mrp1 . although formoterol is moderately lipophilic , it has a high affinity for cellular membrane lipid bilayers ( anderson et al 1994 ) . high concentrations of formoterol might interfere with the function of membrane proteins as this depends on the lipid environment of the protein . the effect with formoterol may also indirectly run via adenosine 3,5-cyclic monophosphate ( camp ) since formoterol stimulates cells via the beta(2)-adrenergic receptor ( johnson and rennard 2001 ) resulting in higher intracellular levels of camp . however , it is not clear why this occurs in the presence of budesonide while formoterol alone had no effect . ipratropium bromide incubation resulted in lower cf retentions with increasing concentrations in a concentration - dependent manner . ipratropium bromide competitively inhibits acetylcholine binding to the muscarinic receptor resulting in decreased guanosine 3,5-cyclic monophosphate ( cgmp ) levels which lowers broncho - constriction . there are no reports in the literature on its mechanism of action with respect to mrp1 , but it can be speculated that cgmp might play a role in this process since cgmp is a substrate of other members of the mrp family , mrp4 and mrp5 ( wielinga et al 2003 ) . effects of nac were similar to those observed with ipratropium bromide , ie , mrp1-mediated transport was stimulated with increasing concentrations of nac . glutathione and many glutathione conjugates are substrates for mrp1 , and therefore , intracellular glutathione levels are of major importance for mrp1 function in antioxidant defense . it is known that transport of anionic species such as cf can be stimulated with glutathione ( akan et al 2005 ; bagrij et al 2001 ) . it was observed that the compounds sulfinpyrazone and indomethacin stimulate glutathione transport by mrp2 at low concentrations , whereas relatively high concentrations inhibit gsh transport into the medium ( evers et al 2000 ) . our results with nac and ipratropium bromide might be explained by a similar co - transport of these substances with cf . it may occur that certain ( at present undefined ) toxic inhaled substances can be transported by mrp1 more effectively with increasing glutathione levels as well . for example , the tobacco - derived nnal - o - glucuronide was identified as an mrp1 substrate that requires glutathione for transport ( leslie et al 2001 ) . the pulmonary drugs that we tested are , apart from nac , clinically administered by inhalation . these drugs act directly on epithelial cells or pass the epithelial layer and act on eg smooth muscle cells or inflammatory cells . the intracellular levels that can be reached with treatment highly depend on the biochemical features of the substance , action of drug metabolism proteins ( phase ii conjugation enzymes such as cytochrome p450 isoforms ) , drug efflux pumps ( phase iii systems ) , as well as the route of administration . we have chosen the ranges of the applied drug concentrations based on published data ( gustafsson and persson 1991 ; anderson et al 1994 ; gillissen et al 1997 ; nagy et al 1997 ; bandi and kompella 2002 ; borchard et al 2002 ) and theoretical models , ie , ranges that will be clinically effective . mrp1 is located in bronchial epithelium , the first cells that drugs have to encounter to reach the underlying tissue to act on eg smooth muscle cells , fibroblasts and inflammatory cells . thus , local high cellular concentrations of pulmonary drugs are very likely to affect mrp1 activity in bronchial epithelium which was indeed confirmed in vitro in the present study . in conclusion , our studies show that drugs that are clinically used in copd affect mrp1 function , a transporter that protects against oxidative stress and toxic compounds . budesonide inhibits this mrp1-mediated transport and addition of formoterol on its turn reduces this inhibitory effect whereas ipratropium bromide and nac stimulate mrp1 activity in a concentration - dependent manner . further studies are required to investigate whether stimulation of mrp1 activity is beneficial for long - term treatment of copd with regard to the protective properties of mrp1 .

the task of gene identification frequently confronting researchers working with both novel and well studied genomes can be conveniently and reliably solved with the help of the genemark web software ( ) . the website provides interfaces to the genemark family of programs designed and tuned for gene prediction in prokaryotic , eukaryotic and viral genomic sequences . currently , the server allows the analysis of nearly 200 prokaryotic and > 10 eukaryotic genomes using species - specific versions of the software and pre - computed gene models . in addition , genes in prokaryotic sequences from novel genomes can be identified using models derived on the spot upon sequence submission , either by a relatively simple heuristic approach or by the full - fledged self - training program genemarks . a database of reannotations of > 1000 viral genomes by the genemarks program is also available from the web site . the genemark website is frequently updated to provide the latest versions of the software and gene models .

intrinsic , or ab initio , gene finders make no explicit use of information about dnas or proteins outside the sequence being studied . extrinsic gene finders utilize sequence similarity search methods to identify the locations of protein - coding regions . it is common for gene finders of both types to be used in concert in a gene finding project , owing to their complementary nature . such programs are the only means to identify genes with no homologues in current databases . as these genes make up a sizeable percentage of the whole gene complement for particular species , the importance of ab initio programs will not diminish in the foreseeable future . the genemark web software includes two major programs , called genemark ( 1 ) and genemark.hmm ( 2 ) . both programs employ inhomogeneous ( three - periodic ) markov chain models describing protein - coding dna and homogeneous markov chain models describing non - coding dna . genemark uses a bayesian formalism to calculate the a posteriori probability of the presence of the genetic code ( in at least one of six possible frames ) in a short dna sequence fragment , thus being a local approach . the genemark.hmm program uses a hidden markov model ( hmm ) framework and the generalized viterbi algorithm to determine the most likely sequence of hidden states ( which are actually labels designating the coding or non - coding function ) based on the whole observed dna sequence . additional details about the genemark and genemark.hmm algorithms can be found in refs ( 13 ) . the architecture of the hmm itself can be altered to fit the organization of a particular type of genome under study in a better way . for example , the prokaryotic version of genemark.hmm contains hidden states for the characteristic features of genes in prokaryotes , including ribosomal binding sites ( rbs ) , uninterrupted genes and gene overlaps . the eukaryotic version utilizes an extended hmm architecture , including states for splice sites , translation initiation ( kozak ) sites and interrupted genes ( exons and introns ) . the web software programs have been extensively used currently > 21 000 nucleotide sequence and 329 000 protein sequence records in genbank ( 4 ) contain references to the genemark programs . as many of the model parameters are species - specific , the accuracy of an ab initio gene finder is highly dependent on the selection of adequate training data as well as on the use of sound methods to create the models . the models available at the genemark website were constructed using our recently developed self - training methods ( 3 ) and were tested locally before being released . both genemark ( 1 ) and genemark.hmm ( 2 ) can be used via the genemark website for the analysis of prokaryotic dna , with 175 pre - computed species - specific statistical models available . analysis of dna from any prokaryotic species is supported by ( i ) a special version of genemark.hmm using a heuristic model calculated from the nucleotide frequencies of an input sequence at least 400 nt long ( 5 ) and ( ii ) a self - training program , genemarks ( 3 ) , which can be used for longer sequences on the order of 1 mb in length . thus , the dna of any prokaryote can be analysed , via either a pre - computed species - specific model or a model created on the fly . as many of the programs at the genemark website share similar interfaces , we use here the prokaryotic genemark.hmm program as an exemplar and discuss program - specific differences below , where appropriate . the genemark.hmm web interface accepts as input a single dna sequence as an uploaded file or as text pasted into a textbox . if a fasta description line begins the sequence , all text on the line following the greater than symbol ( > ) is used as the title . in the remainder of the submission , digits and white space characters are ignored and letters other than t , c , a and g ( assumed to appear rarely ) are converted to n. the interface requires selection of the species name . selection of a model for the rbs ( in the form of a position - specific weight matrix and a spacer length distribution ) is optional . in certain cases , such as the crenarchaeote pyrobaculum aerophilum , the rbs model is replaced by a promoter model , which is the dominant regulatory motif located upstream to gene starts in this species ( 6 ) . the interface also includes the option of using other types of genetic codes such as the mycoplasma genetic code . genemark.hmm reports all predicted genes in a format that includes the strand the gene resides on , its boundaries , length in nucleotides and gene class ( table 1 ) . class indicates which of the two markov chain models used in genemark.hmm , typical or atypical gene model , provided the higher likelihood for the gene sequence . genes of the typical class exhibit codon usage patterns specific to the majority of genes in the given species , while atypical class genes may not follow such patterns and frequently contain significant numbers of laterally transferred genes ( 7,8 ) . the nucleotide sequences of predicted genes and translated protein sequences are available as an output to facilitate further analysis , such as blast searching ( 9 ) . an option to generate genemark predictions in parallel with the genemark.hmm analysis provides important additional information . in this case , genemark is set up to use models derived from the same training data as models for the current run of genemark.hmm . it is worth noting that the genemark.hmm and genemark algorithms are complementary to each other in the same way as the viterbi algorithm and the posterior decoding algorithm are . therefore , though the two algorithms are distinct , they are supposed to generate predictions largely corroborating and validating each other . differences frequently indicate sequence errors and deviations in gene organization , very short genes , gene fragments , gene overlaps , etc . graphical output of the analysis is available in pdf or postscript format . a fragment of this output , illustrating the predictions of both genemark and genemark.hmm , is shown in figure 1 . the graphical output clearly depicts the advantage of using multiple markov chain models representing different classes of genes . here , the coding potential graph obtained using the typical gene model , derived by genemarks , is denoted by a solid black line , and the coding potential graph obtained using the atypical gene model ( derived by a heuristic approach ) is denoted by a dotted line . whereas the first and last genes in figure 1 could be detected using either of the two models , as both of them produced high enough coding potentials , the gene located in positions from 846 to 1112 was detected only by the atypical model . further , figure 1 demonstrates the ability of the genemark programs to detect genes of both the typical and atypical gene classes ( 7 ) . the genemark graph also includes indications of frameshift positions ( also listed in the text report ) , which are often sequencing errors but in rare cases are natural and biologically very interesting . for the genemark program , the window size and step size parameters ( 96 nt and 12 nt , respectively , by default ) define the size of the sliding window and how far this window is moved along the sequence in one step . the threshold parameter determines the minimal average coding potential for an open reading frame ( orf ) to be predicted as a gene . there are several options which allow fine - tuning of the genemark graphical output . in addition , there are options supporting the analysis of eukaryotic dna sequences by genemark including the ability to provide lists of putative splice sites and protein translations of predicted exons . as might be expected , genemark ( the posterior decoding algorithm ) does not produce high enough resolution for the precise prediction of exon intron borders . thus , genemark.hmm ( the generalized viterbi algorithm ) in its eukaryotic version is the major tool for the identification of exon intron structures in eukaryotic dna sequences . the output of the genemark program consists of a list of orfs predicted as genes , i.e. those with average coding potential above the selected threshold . although each predicted gene can have more than one potential start , additional data is provided to help the researcher annotate one of the alternatives as the true one . the start probability ( abbreviated start prob ) is derived from the sequences in the windows immediately upstream and downstream of each potential start . rbs information is provided in the form of a probability score along with the position and sequence of the potential rbs ( abbreviated rbs prob , in addition to the list of predicted genes , genemark provides a list of regions of interest , spans of significant length between in - frame stop codons where spikes of coding potential are wide enough and may warrant further analysis even if no genes are predicted therein based on automatic comparison with the threshold . analysis of prokaryotic dna sequences for which there is no pre - computed species - specific model can be carried out using a program version which heuristically derives a model for any input sequence > 400 nt ( 5 ) . this approach has also proven useful for the analysis of inhomogeneous genomes , particularly regions too divergent from the bulk of the genome , such as pathogenicity islands ( 10,11 ) . if models ( including rbs models ) have to be computed de novo for an anonymous dna sequence with length of the order of 1 mb or longer , the genemarks program can be used ( 3 ) . this program needs significantly more computational resources ; thus , its output is provided via email . a modified version of genemarks tuned for the analysis of viruses of eukaryotic hosts creates a model for the kozak consensus sequence instead of a two - component rbs model . the eukaryotic version of genemark.hmm is currently available for the analysis of 11 eukaryotic genomes : homo sapiens , arabidopsis thaliana , caenorhabditis elegans , chlamydomonas reinhardtii , drosophila melanogaster , gallus gallus , hordeum vulgare , mus musculus , oryza sativa , triticum aestivum and zea mays . from the prediction accuracy tables given at the website ( ) , it follows that the latest versions of genemark.hmm produce remarkably accurate gene predictions for plant genomes such as rice and arabidopsis . this fact has not escaped the attention of plant genome sequencing consortiums , which have used the program intensively ( 12,13 ) . the analysis of cdna and est sequences from eukaryotes , which typically contain no introns , is facilitated by a special version of genemark called genemark.spl . interestingly , eukaryotic genomes with rare introns present difficulty in terms of collecting enough statistics for the intron and internal exon related models , the important components of a full - fledged eukaryotic gene finder . for this reason , currently , this interface employs versions of prokaryotic genemark and genemark.hmm augmented with kozak start site models instead of the prokaryotic rbs model . the eukaryotic species - specific models are represented by several variants built for distinct g + c% ranges covering the whole scale of g + c inhomogeneity observed in a particular genome . genemark.hmm automatically selects the model variant which fits the g + c% of the input sequence . note that , in the eukaryotic case , the repeatmasker program ( a. m. a. smit , r. hubley , and p. green , ) , which is frequently used for pre - processing , can introduce a significant number of these characters do not influence the selection of the markov chain model used in prediction . a sample text output produced by the eukaryotic version of genemark.hmm is shown in table 2 . in the graphical output of the eukaryotic version of genemark.hmm , the thick horizontal bars ( which represent whole genes in the prokaryotic case ) vertical ticks on these bars show the starts and ends of predicted initial and terminal exons , respectively . for the analysis of virus and phage dna , the heuristic ( for short genomes ) and genemarks ( for long genomes ) options , mentioned above , are recommended . in addition , a database called violin containing pre - computed reannotations of > 1000 virus genomes is available ( 14 ) . future directions for genemark web software development include detection of several genomic elements currently not predicted by either genemark or genemark.hmm , such as rrna and trna genes ( which can be mis - predicted as protein - coding genes in low g+c% species ) and improving the detection of gene 5 ends . currently , the server supports the analysis of sequences masked by trnascan ( 15 ) or similar programs . n characters ) ; thus , the predictions will be compatible with this extrinsic information . the detection of exact gene starts remains a challenging problem in gene finding , as many genes have relatively weak patterns indicating sites of translation and transcription initiation . this problem is made especially difficult by the lack of available data sets containing verified gene start locations to be used for training and evaluation . refinements in the rbs and kozak models and the potential inclusion of hidden states representing upstream promoter sequences are currently being explored to address this issue . graphical output from the combination of genemark and genemark.hmm for a fragment of the escherichia coli k12 genome . the solid black and dashed traces indicate the coding potential calculated by the genemark program using the typical and atypical markov chain models of coding dna , respectively . only the three reading frames in the direct strand are shown as there are no genes ( either predicted or annotated ) on the reverse strand in this section of the genome . the thick grey horizontal bars indicate regions of interest provided by the genemark program . the thin black horizontal lines indicate ( longest ) orfs observed in each reading frame ; ticks extending above and below this line indicate potential start and stop codons , respectively . gene predictions made by the prokaryotic version of genemark.hmm for a fragment of the escherichia coli k12 genome in the class column , 1 and 2 indicate typical and atypical , respectively . direct and reverse complement strands are indicated by + and , respectively . prediction of a single gene ( with seven exons ) made by the eukaryotic version of genemark.hmm for a fragment of the arabidopsis thaliana genome the gene that an exon belongs to and its strand are necessarily the same for all exons in a gene . the start frame and end frame indicate the position of the codon ( first , second or third ) that the exon begins and ends with , respectively . notably , all complete gene structures begin in codon position 1 and end in codon position 3 .

erbb2 ( v - erb - b2 avian erythroblastic leukemia viral oncogene homolog 2 ) is an oncogenic tyrosine kinase receptor that is overexpressed in breast cancer . antibodies and inhibitors targeting erbb2 are currently available , although therapeutic failures remain frequent . we discuss here recent data showing that the scaffold protein erbb2ip ( erbb2 interacting protein , best known as erbin ) regulates erbb2 stability and may represent a future therapeutic target .

v - erb - b2 avian erythroblastic leukemia viral oncogene homolog 2 erbb2 interacting protein human epidermal growth factor receptor 2 heat shock protein 90 mouse mammary tumor virus - neu psd95/discs large / zo-1 promyelocytic leukemia - retinoic acid receptor a polyomavirus middle t antigen the mode of action of approved targeted therapies for cancer treatment mostly consists of inhibiting the function of the oncogenic target . although effective , these drugs are not totally efficient , in part due to remaining active oncoprotein in the cancer cell . the perfect drug would ideally degrade the bad guy , definitively eliminating the roots of the disease . such a drug , arsenic trixoxyde , has been very successfully developed to treat acute promyelocytic leukemia by eradicating the promyelocytic leukemia retinoic acid receptor a ( pml - rara ) fusion product through sumo - dependent proteasomal degradation . it is thus anticipated that a better knowledge of the mechanisms of oncoprotein degradation would benefit the development of innovative and efficient drugs . v - erb - b2 avian erythroblastic leukemia viral oncogene homolog 2 ( erbb2 ; also named human epidermal growth factor receptor 2 or her2 ) is a tyrosine kinase receptor whose gene amplification and protein overexpression leads to a poor prognosis in breast cancer . this genetic lesion is retrieved in 1520% of cases of breast cancer and is associated with metastatic development . for almost 10 years , so - called erbb2-positive patients have been treated with potent monoclonal antibodies ( trastuzumab , pertuzumab ) , and more recently with tyrosine kinase inhibitors ( lapatinib ) . although there is no doubt that these drugs have improved patient care , relapse is frequent . erbb2 induces a strong mitogenic pathway in cancer cells by activating classic tyrosine kinase downstream molecules such as ras , mapk , and pi3k . erbb2 protein levels are controlled by heat shock protein 90 ( hsp90 ) , a chaperon protein that binds the erbb2 intracellular domain and protects the protein from proteosomal degradation . hsp90 inhibitors such as geldanamycin that induce erbb2 degradation have been developed and have entered into clinical trials . more than a decade ago , our laboratories identified erbb2 interacting protein ( erbb2ip , best known as erbin ) , which has a psd95/discs large / zo-1 ( pdz ) domain that interacts with erbb2 . erbb2 interaction is highly specific as erbin has no affinity for other members of the erbb2/her family ( egfr , erbb3/her3 , and erbb4/her4 ) . erbin stabilizes erbb2 at the plasma membrane through its pdz interaction , and is necessary for the function of the receptor in the myelination of axons in the peripheral nervous system . erbin knock - out mice ( erbin ) are viable and fertile , despite their neurological defects . erbin mutation leads to decreased amounts of erbb2 at the plasma membrane of mammary epithelial cells . these data obtained in a physiological context enabled investigation into the role of erbin in tumor growth promoted by erbb2 . transgenic mouse mammary tumor virus ( mmtv)-neu ( neu ) mice express oncogenic neu , the rat homolog of erbb2 , in the mammary gland and form tumors similar to erbb2-positive breast cancer . remarkably , erbinneu mice exhibit a very significant delayed appearance of mammary tumor development compared to animals bearing oncogenic neu in a wild - type erbin background . tumor growth was diminished in the absence of erbin and correlated with lower amounts of total and active erbb2 . no effect was observed on protein levels of other erbin interactors such as integrin-4 , smad2 , and smad3 . moreover , a lower rate of cell proliferation was detected in neu - driven tumors and in 2d and 3d cultures of human cancer cells deficient for erbin expression . this led to the conclusion that erbin stabilizes erbb2 and is a component of the oncogenic program of this tyrosine kinase receptor . complementary results were obtained with 2 other mouse models developed to answer the following important questions . the answer is clearly no , as erbin deficiency had no effect on the development of mammary tumors induced by mmtv - pyvt ( polyomavirus middle t antigen is another strong oncoprotein ) . the answer to this is yes , as deletion of the erbin pdz domain in the mouse genome led to a similar decrease in neu - driven tumoral development as complete erbin deficiency . so how does erbin function to stabilize erbb2 ? obviously not by transcriptional regulation , as erbin deficiency did not affect erbb2 mrna levels . in fact , the answer to this question came from the discovery that erbin promotes the formation of a complex containing itself , erbb2 , and hsp90 , another stabilizing molecule for the receptor . disruption of this complex by a competitor peptide for the erbin pdz - erbb2 interaction or by geldanamycin condemned erbb2 to proteasomal degradation . this mechanism highlighted the importance of the very c - terminal sequence of erbb2 in protein stability , as previously shown and the role of erbin in maintaining receptor levels in collaboration with a chaperon ( fig . 1 ) . it remains to be determined how erbin participates in this stabilizing process at the molecular level , and whether other parts of the protein outside of the pdz domain can perform the job , either alone or with associated partners . of interest , erbin was found to be overexpressed in erbb2-positive breast cancer , suggesting that it may represent a suitable target for drug development . even though development of protein protein interaction inhibitors mimicking the erbb2 pdz binding site is no easy task , these data form the basis for strategies aiming to target the erbin pdz erbb2 interface with the objective of promoting degradation of the oncoprotein . on the left , erbb2 forms a complex with erbin and hsp90 at the plasma membrane . this interaction stabilizes erbb2 and allows a potent mitogenic signal . on the right , disruption of the erbb2erbin complex with a peptide mimicking the c - terminal sequence of erbb2 erbb2 , v - erb - b2 avian erythroblastic leukemia viral oncogene homolog 2 ; erbin , erbb2 interacting protein ; hsp90 , heat shock protein 90 : pdz , psd95/discs large / zo-1 . disruption of the erbb2erbin complex decreases erbb2 levels and limits oncogenicity . on the left , erbb2 forms a complex with erbin and hsp90 at the plasma membrane . this interaction stabilizes erbb2 and allows a potent mitogenic signal . on the right , disruption of the erbb2erbin complex with a peptide mimicking the c - terminal sequence of erbb2 induces degradation of erbb2 leading to lower amounts of erbb2 and decreased mitogenicity . erbb2 , v - erb - b2 avian erythroblastic leukemia viral oncogene homolog 2 ; erbin , erbb2 interacting protein ; hsp90 , heat shock protein 90 : pdz , psd95/discs large / zo-1 . the laboratory of jpb is supported in part by grants from la ligue nationale contre le cancer ( label ligue 2013 ) , institut paoli - calmettes , cancrople paca and by siric ( inca - dgos - inserm 6038 ) .

two new cyclic depsipeptides , companeramides a ( 1 ) and b ( 2 ) , have been isolated from the phylogenetically characterized cyanobacterial collection that yielded the previously reported cancer cell toxin coibamide a ( collected from coiba island , panama ) . the planar structures of the companeramides , which contain 3-amino-2-methyl-7-octynoic acid ( amoya ) , hydroxy isovaleric acid ( hiva ) , and eight -amino acid units , were established by nmr spectroscopy and mass spectrometry . the absolute configuration of each companeramide was assigned using a combination of marfey s methodology and chiral - phase hplc analysis of complete and partial hydrolysis products compared to commercial and synthesized standards . companeramides a ( 1 ) and b ( 2 ) showed high nanomolar in vitro antiplasmodial activity but were not overtly cytotoxic to four human cancer cell lines at the doses tested .

a marine cyanobacterial assemblage comprising a small filament leptolyngbya species , based on morphological analysis , was collected in 2004 by hand using scuba from a reef pinnacle in coiba national park , panama . the alcohol - preserved tissue was extracted with ch2cl2meoh , and the extract fractionated by normal - phase silica gel vacuum liquid chromatography ( np - vlc ) . in preliminary biological activity profiling , the 100% etoac fraction was cytotoxic to nci - h460 human lung tumor cells with an ic50 of 300 ng / ml and also showed preliminary activity in the icbg panel of antiparasitic assays : malaria ( ic50 6 g / ml ) , american trypanosomiasis ( ic50 35 g / ml ) , and leishmaniasis ( ic50 > 50 g / ml ) . rp18 solid - phase extraction ( spe ) , hplc , and further cytotoxicity testing of this fraction resulted in the discovery of the potent antiproliferative metabolite coibamide a , to which was attributed the preliminary antiparasitic activities observed for the parent fraction . however , h nmr analysis of the rp18-spe subfractions indicated the presence of additional , but unrelated , peptidic metabolites in the 70% meoh hplc purification of this more polar spe fraction has afforded two new cyclic depsipeptides , companeramides a ( 1 ) and b ( 2 ) , subsequently found to be responsible for the observed antiparasitic activity of the parent fraction . laboratory culture of the 2010 field - collected cyanobacterial assemblage yielded predominantly companeramides a ( 1 ) and b ( 2 ) , with a relatively low yield of coibamide a during the first six months of culture . microscopic examination of these cultures also revealed that a small - filament cyanobacterium was the dominant organism in culture , over its companion large - filament cyanobacterium . beyond six months in laboratory culture , this assemblage no longer produced coibamide a , but continued to produce the companeramides , and microscopic examination revealed the presence of almost exclusively small filaments , with only sporadic large filaments in the cultured material ( figure s21 , supporting information ) . subsequently a monoculture of this small - filament cyanobacterium was achieved that could be associated with companeramide production ( pac-10 - 3 csf1 , genbank km882611 ) . phylogenetically , this organism is most closely related to a taxonomically unassigned filamentous cyanobacterium associated with black band disease ( flk9 , genbank eu196364 , see supporting information figures s19 and s20 ) . companeramide a ( 1 ) yielded a prominent [ m + h ] ion by hrtofms for a molecular formula of c57h97n9o11 , which was supported by nmr spectroscopic analysis . the h nmr spectrum for 1 exhibited signals typical for a relatively hydrophobic cyanobacterial peptide metabolite , including four n - methyl singlets ( h 3.14 , 3.06 , 2.92 , 2.69 ) , four nh doublets ( h 8.86 , 6.92 , 6.91 , 6.75 ) , nine -h multiplets ( h 4.585.26 ) , and numerous overlapped methyl doublets ( h 0.771.32 , table 1 ) . the c nmr spectrum for 1 displayed nine distinguishable signals for ester / amide - type carbonyls ( c 169.4174.4 ) , one signal for an oxygenated sp - hybridized carbon ( c 75.4 ) , and two signals at c 83.8 and 68.6 , diagnostic of a terminal acetylenic moiety ( table 1 ) . the hmbc spectrum displayed prominent three - bond hmbc correlations from the four n - methyl singlets , permitting n - methyls h3 - 15 , h3 - 25 , h3 - 42 , and h3 - 52 to be associated with corresponding -carbons c-11 , c-20 , c-38 , and c-50 , respectively . the side chain spin systems of these four n - methylated amino acid residues were delineated by multiplicity - edited hsqc and hsqc - tocsy experiments as two n - methyl valine ( n - me - val ) , one n - methyl leucine ( n - me - leu ) , and one n - methyl alanine ( n - me - ala ) residue . further analysis of the cosy , hsqc , hsqc - tocsy , and hmbc experiments identified the regular amino acids alanine ( ala ) , proline ( pro ) , and two isoleucines ( ile ) , as well as hydroxyisovaleric acid ( hiva ) ( table s1 , supporting information ) . cosy and tocsy nmr experiments were used to identify a spin system in which a methine doublet of quartets ( h 2.51 , h-2 ) was correlated to both a methyl doublet ( h 1.31 , h3 - 9 ) and a second downfield methine multiplet ( h 3.97 , h-3 ) . this h-3 multiplet was in turn relay - coupled to the signals for three contiguous methylenes ( h2 - 4 to h2 - 6 ) and also to an nh doublet at h 6.75 ( nh-3 ; figure 1 ) . hmbc correlations from the distal methylene h signal ( h2 - 6 , h 2.17 ) to nonprotonated sp - hybridized c-7 ( c 83.8 ) and methine c-8 ( c 68.6 ) signals completed the hydrocarbon chain with a terminal acetylene . finally , three - bond hmbc correlations from the h3 - 9 doublet to methine c-3 ( c 51.2 ) and the c-1 carbonyl signal ( c 174.3 ) aided in the establishment of this -methyl--amino acid as 3-amino-2-methyl-7-octynoic acid ( amoya ) . key 2d nmr correlations for ( a ) companeramide a ( 1 ) and ( b ) companeramide b ( 2 ) . despite several closely overlapping -proton signals ( h-17 , h-32 , h-38 , h-44 ) correlated to overlapped carbonyl signals ( c-10 , c-31 , and c-49 ) in the hmbc spectrum for companeramide a ( 1 ) , all nine amino and one hydroxy acid subunits were sequenced from hmbc and roesy data collected at 700 mhz . two obvious fragments consisting of n - me - val-1ala n - me - leu pro ( fragment 1 ) and ile-1n - me - val-2ile-2n - me - ala hiva ( fragment 2 ) were assembled by hmbc correlations between n - methyl , amide , and/or -h and the carbonyl c signals for neighboring residues ( figure 1 and table s1 , supporting information ) . at the n - terminus of fragment 2 , the ester linkage between hiva and the amoya residue was defined by hmbc correlations from the hiva -h ( h-54 , h 4.80 ) , amoya -h ( h-2 , h 2.51 ) , and amoya methyl ( h3 - 9 , h 1.31 ) to the amoya carbonyl c-1 signal ( c 174.3 ) . roesy correlations between the pro h2 - 30 multiplets ( h 3.84 and 3.76 ) and the ile-1 -h-32 doublet of doublets ( h 4.74 ) corroborated an hmbc correlation from the pro -h-27 doublet of doublets ( h 4.92 ) to the overlapped ile carbonyl c-31 signal ( c 170.0 ) . although hmbc experiments with varying delay times failed to show correlations from the amoya nh-3 ( h 6.75 ) or -h to any carbonyl c nmr signals , a roesy correlation between the nh-3 signal and h 4.58 ( -h-11 ) was used to connect the amoya and n - me - val-1 residues to complete the planar macrocyclic structure of companeramide a ( 1 ) ( figure 1a ) . ms / ms data for 1 were consistent with the proposed amino acid sequence ( figure s15 , supporting information ) . companeramide b ( 2 ) gave a prominent hresims peak at m / z 1056.7013 , which was 28 mass units less than the major [ m + h ] ion for companeramide a ( 1 ) and indicated a molecular formula of c55h93n9o11 for 2 . the h nmr spectrum for 2 displayed four n - methyl amide singlets ( h 3.23 , 3.06 , 2.89 , 2.74 ) , four amide proton doublets ( h 8.84 , 7.48 , 7.44 , 6.80 ) , and midfield multiplets integrating to 10 -h ( h 3.815.40 ) . in the c nmr spectrum for 2 there were two signals indicative of a terminal acetylene ( c 83.4 and 69.4 ) , as for 1 , and eight distinct signals for ester / amide carbonyls , one of which was noticeably broad and of relatively higher intensity . analysis of the 2d nmr data for companeramide b ( 2 ) , including the cosy , hsqc , hsqc - tocsy , and hmbc spectra , revealed a similar structural composition to 1 , with the presence of amoya , two n - me - val , pro , ile , and hiva residues ( tables 1 and s2 , supporting information ) . the hsqc - tocsy spectrum for 2 additionally defined two val spin systems , each comprising an amide ( nh-17 or nh-43 ) , two coupled methines , and two methyl h nmr signals . two remaining sets of coupled -methine and methyl doublet h nmr signals could be attributed to two n - me - ala residues on the basis of hmbc correlations from the n - methyl singlets at h 2.74 ( h3 - 24 ) and 2.89 ( h3 - 50 ) to their respective coupled -methine signals ( h 4.96 , h-22 ; 5.40 , h-48 ) . as in the case of 1 , the sequential assembly of amino and hydroxy acids in 2 was complicated due to a number of closely overlapping -h chemical shifts ( h-11 and h-31 , h-11 and h-43 , h-31 and h-52 ) correlated to closely overlapped carbonyl signals ( c-10 , c-30 , and c-47 ) in the hmbc spectrum . in this case , hmbc and roesy correlations supported a fragment 1 sequence of n - me - val-1val-1n - me - ala-1pro ( figure 1b ) . although the order of val-1 and n - me - ala-1 in 2 was originally in question , when compared to the corresponding second and third residues ( ala and n - me - leu , respectively ) in 1 , key hmbc correlations were present from the n - ch3 - 15 singlet ( h 3.23 , n - me - val-1 ) to the c-16 carbonyl signal ( c 173.4 , val-1 ) , from the -h-17 triplet ( h 4.58 , val-1 ) to carbonyl c-21 ( c 169.8 n - me - ala-1 ) , and from the n - ch3 - 24 singlet ( h 2.74 , n - me - ala-1 ) to the c-25 carbonyl signal ( c 171.8 , pro ) . in addition , ms / ms fragments of m / z 183.11 ( b2 ) , 282.18 ( b3 ) , and 395.26 ( b4 ) ( figure s16 , supporting information ) supported the assigned fragment 1 sequence for 2 . this consistent pattern of n - methylation for 1 and 2 is biosynthetically logical . hmbc and roesy correlations supported an extended fragment 2 for 2 , in which the ile present in 1 was substituted for a val , providing a sequence of ile-1n - me - val-2val-2n - me - ala hiva . again , the two fragments could be connected by a roesy correlation between the pro methylene ( h2 - 29 ) signal and ile -h-31 signal in combination with a hmbc correlation from the pro -h-26 signal ( h 4.83 ) to the overlapped ile carbonyl c-30 signal ( c 170.2 ) . finally , the macrocycle could be closed by a roesy correlation between the signals at h 7.44 ( amoya nh-3 ) and 4.72 ( -h-11 ) together with an hmbc correlation from the nh-3 doublet to the c-10 carbonyl signal ( c 170.2 ) of n - me - val-1 . the resulting planar structure of companeramide b ( 2 , figure 1b ) was again supported by ms / ms data ( figure s16 , supporting information ) . the absolute configurations of the amino and hydroxy acid units in companeramides a ( 1 ) and b ( 2 ) were determined by both marfey s methodology and direct chiral - phase hplc analyses of acid hydrolysates ( 0.5 mg , 6 n hcl , 110 c , overnight ) . for each natural product , a portion ( ca . 0.25 mg ) of the acid hydrolysate was derivatized with n--(2,4-dinitro-5-fluorophenyl)-l - alaninamide ( l - fdaa , marfey s reagent ) and analyzed by comparative rp18 hplc with fdaa - derivatized d- and l - amino acid standards . the additional 0.25 mg of each natural product acid hydrolysate was reconstituted with h2o , analyzed by chiral - phase hplc , and compared with the retention times of authentic r- and s - hiva standards . for companeramide a ( 1 ) , an l - configuration for ala , n - me - ala , pro , and both ile , n - me - leu , and n - me - val residues , as well as s - hiva , was established . the hydrolysate from companeramide b ( 2 ) contained l - pro , two l - n - me - val , two l - val , l - ile , and both d- and l - n - me - ala , as well as s - hiva . in order to correctly assign the relative position of the d- and l - n - me - ala residues in companeramide b ( 2 ) , a partial hydrolysis and purification of resulting fragments containing n - me - ala was attempted , for subsequent complete hydrolysis and marfey s analyses . to this end , 2 ( 4 mg ) was partially hydrolyzed with 3 n hcl , and the product mixture analyzed by lc - ms to identify fragments containing a single n - me - ala residue . a tetrapeptide fragment ( m / z 413.9 ) identified as n - me - val val n - me - ala pro from ms / ms data ( figure s17 , supporting information ) was purified from the product mixture and subjected to complete hydrolysis and derivatization with n--(2,4-dinitro-5-fluorophenyl)-l - leucinamide ( l - fdla , advanced marfey s reagent ) for analysis . the retention times for the derivatized amino acids from the latter ( tetrapeptide fragment 1 of 2 ) corresponded to fdla - derivatized standards for n - me - l - val , l - val , n - me - d - ala , and l - pro . no other partial hydrolysis fragments could be isolated in reasonable amount for marfey s analysis ; however , assignment of the n - me - d - ala in the northern hemisphere ( fragment 1 ) of 2 led n - me - l - ala to be assigned in the southern hemisphere ( fragment 2 ) of 2 . the -amino acid amoya present in companeramides a ( 1 ) and b ( 2 ) was first identified in the molluscan metabolite onchidin , but has since been reported as a component of several marine cyanobacterial metabolites including ulongapeptin , guineamide c , and malevamide c. while the absolute configurations of the amoya residues in guineamide c and malevamide c have not been determined , those in ulongapeptin were assigned as 2s , 3s by marfey s analysis using comparison with a synthetic mixture of c-2 diastereomers ( 2s , 3r and 2r , 3r ) derivatized with both d- and l - fdla enantiomers . the ( 2s,3s)-configuration was also assigned to the amoya unit in onchidin based on noe studies and h nmr coupling constant analysis ; however , the total synthesis of onchidin suggested that a revision of the structure was necessary . therefore , it was planned to use marfey s analysis for assignment of the amoya unit in companeramides a and b , facilitated by the availability of the synthetic 3(r)-amino-2(r , s)-methyloctanoate ( amo ) standards . two portions of the synthetic material were derivatized separately with d- or l - fdla to provide hplc retention times for all four possible amo diastereomers , with retention times of l - fdla-(2r,3s)-amo and l - fdla-(2s,3s)-amo being inferred from the retention times of the enantiomeric d - fdla-(2s,3r)-amo and d - fdla-(2r,3r)-amo standards , respectively . hydrogenation of each companeramide to reduce the terminal alkyne was followed by acid hydrolysis to release amo for separate derivatization with d- and l - fdla . reported retention times for the four amo diastereomers using similar hplc conditions were used to assign the order of elution of the pairs of standards generated in our protocol . for each companeramide , the d - fdla - amo product coeluted with the d - fdla - derivatized ( 2s,3r)-amo standard , and similarly the l - fdla - amo product coeluted with the l - fdla - derivatized ( 2s,3r)-amo , supporting a 2s,3r - amoya unit in companeramides a ( 1 ) and b ( 2 ) . companeramides a ( 1 ) and b ( 2 ) showed no significant cytotoxicity at 1 m against four human cancer cell lines ( nci - h460 non - small - cell lung carcinoma , mda - mb-231 breast adenocarcinoma , sf-295 glioblastoma , and sk - ov3 ovarian carcinoma cells ) . instead preliminary antiparasitic activity of the parent fractions led to testing of the two pure compounds against three strains of the malaria parasite plasmodium falciparum in a fluorescence - based assay . neither compound was as active as the chloroquine control against the chloroquine - sensitive d6 or chloroquine - insensitive dd2 and 7g8 strains ( table 2 , figure s18 , supporting information ) . however , the differential activity between the two companeramides across all strains is noteworthy considering their structural similarity . the chloroquine - sensitive d6 strain was approximately twice as sensitive to companeramide a ( 1 ) as the chloroquine - resistant dd2 and 7g8 strains . in contrast , companeramide b ( 2 ) showed comparable activity against the chloroquine - sensitive d6 and chloroquine - resistant strain dd2 , but was about three times less active against the chloroquine - resistant 7g8 strain . the companeramides add to the growing repertoire of cyanobacterial depsipeptides that show some degree of antiplasmodial activity , including dolastatin 10 , the venturamides , carmabins and dragonamides , and symplostatin 4 ( first reported as gallinamide ) . while some of these compounds ( e.g. , dolastatin 10 ) are potently toxic to mammalian cells as well as to the malarial parasite , others such as the venturamides and carmabin a show relatively little toxicity to mammalian cells . at the concentrations tested , it is not possible to distinguish any selective antiplasmodial activity for companeramide a ( 1 ) , although the results for companeramide b ( 2 ) suggest potential antiplasmodial selectivity . the requisite testing of 2 for mammalian cell toxicity at higher micromolar concentrations was not pursued in the interest of saving material for other assays in which the target activity may be more pronounced than the moderate antiplasmodial activity presented here . it is interesting to speculate that related large cyclic alkynoic depsipeptides that are reported to be nontoxic , such as the malevamides , may possess antiparasitic activity . noteworthy also is that despite the structural relationship between the companeramides and ulongapeptin , only the heptameric ulongapeptin displays nanomolar cytotoxicity . uv and ir data were obtained on a hitachi u-2000 spectrophotometer and a nicolet ir100 ft - ir instrument , respectively . nmr spectra were acquired on bruker avance 700 mhz and bruker avance drx 600 mhz spectrometers with the residual chcl3 solvent used as an internal standard ( c 77.23 , h 7.26 ) . lr fabms and hrtofms ( es ) mass spectra were recorded on absciex 3200 qtrap and waters micromass mass spectrometers . the isolation of compounds 1 and 2 was performed using a waters hplc system consisting of two waters 515 pumps , a rheodyne 7725i injector , and a waters 996 photodiode array detector . marfey s and chiral - phase hplc analyses were conducted on a shimadzu hplc system equipped with two lc-20ad pumps and an spd - m20a photodiode array detector . the marine cyanobacterial assemblage ( mcphail laboratory voucher number pac-6/25/04 - 2 ) was first collected in june 2004 by hand using scuba from a reef pinnacle off the west coast of coiba national park . the material collected for chemical extraction ( 1 l ) was stored in 50% etoh for transport and then stored at 20 c until extraction . for phylogenetic analyses , subsamples of the field collection ( 0.5 ml of cyanobacteria in 5 ml of rna later solution ) were stored at room temperature ( rt ) and then 4 c before dna extraction , and live cultures were also initiated . additional field collections were made subsequently in 2010 ( voucher number pac-10 - 03 ) and 2012 ( voucher number pac-7/10/12 - 1 ) , for chemical and dna extraction , as well as laboratory culture . dna extraction of the 2010 rna later samples and comparison of the genomic 16s rdna ( rdna ) sequence with the ncbi blast database led to the identification of the cf . symploca strain , which will be assigned to a new genus , hyalidium . monoculture of the small - filament cyanobacterium ( pac-10 - 3 csf1 ) associated with companeramide production in the 2010 collection provided material for genomic dna isolation ( genbank accession number km882611 ) , and this organism was found to be 99% identical to that of filamentous cyanobacteria associated with black band disease ( see supporting information , s19 and s20 ) . approximately 95.6 g dry weight of the 2004 cyanobacterial collection was extracted repeatedly with ch2cl2meoh ( 2:1 ) to produce 5.75 g of an organic extract . the extract was fractionated by normal - phase vlc on si gel with a stepwise gradient solvent system from hexanes to etoac to meoh , yielding nine fractions . the fraction eluting with 100% etoac was cytotoxic ( ic50 300 ng / ml ) to nci - h460 lung cancer cells and further chromatographed by rp18 spe using a stepwise solvent gradient ( meoh h2o , 6:4 , 7:3 , 8:2 , 9:1 , 100% meoh and ch2cl2 ) . the noncytotoxic spe fraction eluting with 70% meoh possessed interesting peptide - like h nmr spectroscopic parameters and was purified further using rp18 hplc ( 85:15 meoh h2o , phenomenex synergi fusion 4 m , 10 250 mm , 2 ml / min , uv detection at 216 nm ) to yield companeramide a ( 1 , 2.3 mg , tr 30.5 min ) and companeramide b ( 2 , 5.3 mg , tr 21.4 min ) . colorless oil ; [ ]d 140 ( c 0.6 , meoh ) ; uv ( meoh ) max ( log ) 215 ( 3.45 ) nm ; ir max ( neat ) 3311 , 2963 , 2930 , 1720 , 1628 , 1517 , 1462 , 1231 , 1098 cm ; h and c nmr data , see tables 1 and s1 ( supporting information ) ; hrtofms ( es+ ) m / z 1084.7395 [ m + h ] ( calcd for c57h98n9o11 , 1084.7386 ) . colorless oil ; [ ]d 78 ( c 0.5 , meoh ) ; uv ( meoh ) max ( log ) 225 ( 4.06 ) nm ; ir max ( neat ) 3311 , 2963 , 2930 , 1720 , 1628 , 1517 , 1462 , 1231 , 1098 cm ; h and c nmr data , see tables 1 and s2 ( supporting information ) ; hrtofms ( es+ ) m / z 1056.7115 [ m + h ] ( calcd for c55h94n9o11 , 1056.7073 ) , m / z 1078.6865 [ m + na ] ( calcd for c55h93n9o11na , 1078.6892 ) . the absolute configurations of the amino acid residues in companeramides a ( 1 ) and b ( 2 ) were determined by marfey s methodology . approximately 0.5 mg of each of companeramides a and b were separately hydrolyzed ( 6 n hcl at 110 c for 16 h ) , evaporated to dryness , and reconstituted in h2o ( 50 l ) . fdaa solutions in acetone ( 0.1% , 100 l ) and 1 n nahco3 ( 50 l ) were added to each hydrolysate and heated to 37 c for 1 h. the solutions were allowed to cool to rt , neutralized with 2 n hcl ( 25 l ) , and evaporated to dryness . the residues were resuspended in dmso h2o ( 1:1 , 100 l ) and analyzed by rp18 hplc ( waters symmetry shield c18 column , 3.9 150 mm , 1 ml / min , uv detection at 340 nm ) using a linear gradient of 9:1 40 mm ammonium acetate buffer ( ph 5.2)ch3cn to 1:1 40 mm ammonium acetate buffer the absolute configurations of the amino acids in the hydrolysates of 1 and 2 were determined by direct comparison with the retention times ( tr , min ) for marfey s derivatives of authentic standards . the retention times ( min ) of the fdaa - derivatized -amino acids in the hydrolysate of 1 matched those of l - pro ( 12.8 ; d - pro , 13.5 ) , n - me - l - val ( 23.2 ; d - n - me - val , 27.5 ) , l - allo - ile ( 21.4 ; l - ile , 21.2 , d - ile , 28.3 , d - allo - ile , 28.8 ) , l - ala ( 12.3 ; d - ala , 16.9 ) , n - me - l - ala ( 14.3 ; n - me - d - ala , 16.6 ) , and n - me - l - leu ( 26.6 ; n - me - d - leu , 30.3 ) . the retention times ( min ) of the derivatized amino acids in the hydrolysate of 2 matched l - pro ( 12.8 ; d - pro , 13.5 ) , n - me - l - val ( 23.2 ; n - me - d - val , 27.5 ) , l - val ( 17.3 ; d - val , 24.0 ) , l - allo - ile ( 21.4 ; l - ile , 21.2 , d - ile , 28.3 and d - allo - ile , 28.8 ) , and both n - me - l - ala ( 14.3 ) and n - me - d - ala ( 16.6 ) . chiral - phase hplc analysis was used to determine the absolute configurations of the hiva residues in the two depsipeptides . a portion of the acid hydrolysate of 1 ( 0.25 mg ) and 2 ( 0.25 mg ) was reconstituted in h2o prior to chiral - phase hplc analysis ( 85:15 2 mm cuso4ch3cn ; column phenomenex chirex 3126 ( d ) , 4.6 250 mm , flow 1.0 ml / min , uv detection at 254 nm ) . the hiva residue in the hydrolysates of 1 and 2 eluted with the same retention time ( tr , min ) as the s - hiva standard ( 40.0 ) but not that of r - hiva ( 61.7 ) . to assign the position of n - me - l - ala versus n - me - d - ala in companeramide b ( 2 ) , 4 mg of 2 was partially hydrolyzed in 3 n hcl ( 2 ml , constant stirring , 100 c ) ; the reaction was monitored by lc - ms at 30 min intervals and was halted after 3 h by cooling to 20 c , after which the acid was removed under high vacuum . h2o ( 100 l ) for lc - ms / ms analysis ( es+ ) to identify fragments containing single n - me - ala residues . tetrapeptide n - me - val - val - n - me - ala - pro ( m / z 413.9 , 0.1 mg ) was purified from the total hydrolysate by rp18 hlc using a linear gradient of 5100% ch3cn tfa over 60 min ( phenomenex synergi hydro column , 10 250 mm , 2.5 ml / min , 340 nm ) . subsequent complete hydrolysis of the tetrapeptide ( 6 n hcl , microwave irradiation , 1000 w , 50 s ) was followed by marfey s derivatization of the resulting hydrolysate ( with l - fdla ) and commercial amino acid standards ( with l - fdla and d - fdla ) . in each case , 1 m nahco3 ( 20 l ) was added to a 50 mm amino acid solution in h2o , followed by fdla ( 1% w / v in acetone , 44 l ) , and the reaction mixture heated to 40 c ( 1 h ) . the reactions were quenched by cooling to rt and acidification ( 20 l of 1 n hcl ) , before drying under high vacuum and resuspension in 100 l of dmso for lc - ms analysis ( thermo aquasil c18 column , 3 150 mm , 2555% linear gradient of ch3cn tfa over 45 min , 1 ml / min , 340 nm , es+ ) . retention times ( m / z [ m + na ] , tr min ) for derivatized residues of the tetrapeptide corresponded to standards for n - me - l - val ( 426.2 , 23.55 ; n - me - d - val , 28.00 ) , l - val ( 412.2 , 19.06 ; d - val , 28.40 ) , n - me - d - ala ( 398.2 , 18.31 ; n - me - l - ala , 17.84 ) , and l - pro ( ion not identified , 16.34 , d - pro , 19.50 ) . assignment of the amoya residues relied on hydrogenation of the amoya unit in 1 and 2 to 3-amino-2-methyloctanoic acid ( amo ) , peptide hydrolysis , marfey s derivatization with fdla , and comparison with fdla - derivatized synthetic amo standards . in each case , approximately 0.5 mg of cyclic depsipeptide was dissolved in dry meoh and stirred over 5% pd / c under an atmosphere of hydrogen ( rt , 18 h ) . after filtration over celite , washing with etoh , and concentration in vacuo , the products ( m / z 1088 , tetrahydro-1 , and 1060 , tetrahydro-2 ) were resuspended in 6 n hcl and stirred overnight at 100 c . the acid hydrolysate of tetrahydro-1 was derivatized with l - fdla , as described above , while that of tetrahydro-2 was divided into two portions and derivatized with d- and l - fdla . h2o ) was derivatized with each of d- and l - fdla for rp18 hplc - ms analysis ( 50% ch3cn h2o ; phenomenex synergi hydro column , 4.6 250 mm , flow 0.2 ml / min , uv detection at 340 nm ) . observed elution times of the standards were 9.4 min ( d - fdla-2s,3r - amo = l - fdla-2r,3s - amo ) , 10.0 min ( d - fdla-2r,3r - amo = l - fdla-2s,3s - amo ) , 17.4 min ( l - fdla-2r,3r - amo ) , and 20.9 min ( l - fdla-2s,3r - amo ) , as assigned by comparison to reported elution times . the l - fdla - hydrolysate of tetrahydro-1 provided a small peak at 20.9 min for l - fdla-2s,3r - amo , which was supported by retention times of 20.9 min ( l - fdla-2s,3r - amo ) and 9.4 min ( d - fdla-2s,3r - amo ) for the two derivatized portions of tetrahydro-2 . plasmodium falciparum strains d6 , dd2 , and 7g8 were cultured in human erythrocytes at 2% hematocrit in rpmi 1640 containing 0.5% albumax , 45 g / l hypoxanthine , and 50 g / l gentamicin , as previously described . in vitro antimalarial activity was determined by the malaria sybr green i - based fluorescence ( msf ) assay described previously with slight modification . stock solutions of each test sample were prepared in sterile distilled h2o at a concentration of 10 mm . the sample solutions were serially diluted with culture medium and distributed to asynchronous parasite cultures on 96-well plates in quadruplicate in a total volume of 100 l to achieve 0.2% parasitemia with a 2% hematocrit . automated pipetting and dilution were carried out with a programmable precision 2000 robotic station ( bio - tek ) . after incubation , 100 l of lysis buffer with 0.2 l / ml sybr green i was added to each well . the plates were incubated at rt for 1 h in the dark and then placed in a 96-well fluorescence plate reader ( spectramax gemini - em ; molecular diagnostics ) with excitation and emission wavelengths at 497 and 520 nm , respectively , for measurement of fluorescence . the 50% inhibitory concentration ( ic50 ) was determined by nonlinear regression analysis of logistic dose cell viability was assessed with a standard mtt assay as described previously with the viability of vehicle - treated cells defined as 100% and coibamide a ( 30 nm ) included as a control cancer cell toxin . all compounds were reconstituted in 100% dmso and stored at 20 c until the day of treatment ; final concentrations of dmso never exceeded 0.1% . human h460 lung and sf-295 glioblastoma cells were from the national cancer institute cell line repository , and mda - mb-231 breast and sk - ov3 ovarian carcinoma cells were from the american type culture collection ( atcc ) . all cells were maintained under standard growth conditions and seeded into 96-well plates at a density of 3000 cells per well the day before treatment .

we report a case of tourette 's disease ( td ) with impulse control disorder which is rare;these type of patients are prone to rage attack and explosive outbursts in the childhood and adolescence which can be detrimental . hence , a case is reported to understand the phenomenology of its co - morbidity in td .

the literature concerning this rare syndrome alternatively knows it as maladie des tics , which was mainly descriptive since it was first mentioned by itard ( 1825 ) and later by gilles de la tourette 's syndrome in 1885 . the prevalence of tourette 's disorder was 4.9 per 10,000 males and 3.1 per 10,000 females . the most common co - occurring disorders with tourette 's disorder are attention deficit hyperkinetic disorder ( adhd ) 50 - 60% ; obsessive compulsive disorder ( ocd ) 30 - 70% , but there is no report regarding prevalence of impulse control disorder ( icd).however , icd is described as a rare one . the children with tourette 's disorder are prone to rage attacks due to underlying brain dysfunction . hence , the clinician should be aware of the possibility of co - morbidity of tourette 's disease ( td ) with icd . in tourette 's disorder , there is triad of multiple motor tics , coprolalia 2 - 6% and childhood / adolescent onset . shapiro and shapiro ( 1992 ) described impulsion , a manifestation of rage , which is interchangeable with intermittent explosive disorder . ( 1993 ) confirmed a relationship between levels of 5-hydroxyindoleacetic acid ( 5-hiaa ) in cerebrospinal fluid ( csf ) and impulsive or aggressive behavior . there is a defect in the serotonergic system , which acts as an inhibitor of motor activity ( staner , 1998 ) and positron emission tomography ( pet ) scan shows decrease in glucose metabolism in the prefrontal and frontal cortex of patients with impulsive acts ( raine et al 1994 ) . to our knowledge , this is the first case report from india which delineates the co - morbidity of td with icd . x , a 17-year - old , unmarried male and a 11th class student , reported in the outpatient clinic / department of psychiatry , government medical college , patiala . history dates back to 1 year when the patient had symptoms of sudden irregular involuntary body movements of head turn and shoulder shrug with isolated episodes of vocalization of obscene phrase bloody kill him . the symptoms were of waxing and waning in nature , and there was no period of remission for more than 3 months during the consecutive 12 months of illness . for the last 3 months , the patient complained of irresistible urge to throw stones without any provocation or any other motive upon his neighbors and destroyed their property . he derived pleasure after acting out and did not try to escape from the scene or resisted his arrest . when he tried to control his rage of throwing stones , there was mounting of tension and increased restlessness within him . anatomically , he felt intense discomforts on his palms , shoulders , midline abdomen and throat . the body movements started from the face , eye blinking , frowning and grimacing of the mouth , further progressing down to involve neck with shoulder shrug and wringing of hands . these movements were exaggerated by excitement , emotional stress or anxiety and attenuated by focused attention or relaxation . for the last 2 months , the patient had an irresistible urge to stab his brother with a knife . realizing his urge getting out of control , he asked for help . though he did not complete or act upon the urge , yet found it irresistible , followed by ejaculatory words of coprolalia ( bloody kill him ! ! ) . neither was it a persistent idea , thought or an image nor he tried to substitute it with any other subsidiary thought or engaged in any ritual of undoing it . in the family history , his father was aged 45 years and illiterate . he persistently nagged the patient and was strict , rigid and demanding with obsessive traits . his mother was aged 40 years , illiterate , housewife with no abnormal pre - morbid personality traits . in personal history , the patient was full - term , wanted child , delivered through cesarean section and third in birth order . he was breast fed , had mild physiological jaundice on 7th day and passed his developmental milestones normally . he was fully immunized and his schooling started at the age of 5 years , and had a normal peer group relationship . he was educated in government school till the age of 15 years and left it on financial grounds . after leaving the school , as assessed on wechsler adult intelligent scale ( wais ) , his iq was 95 . he described his relationship with his brother as loving and caring and denied any conflict with him . there was no pre - morbid personality traits of borderline , antisocial , histrionic , narcissistic and anankastic ( obsessive ) personality disorder as assessed on international personality disorders examination ( icd-10 : ipde ) . there was no history of mood disorder , anxiety disorder , adhd , ocd , conduct disorder or learning disorder . there was no history of substance abuse or medical history of head injury , seizure , athetosis , restless leg syndrome , huntington 's chorea , syndehams chorea , dystonia , meig 's syndrome , blepharospasm or family history of involuntary movement disorders . the patient was assessed using dsm - iv - tr diagnostic criteria and diagnosed as a case of tourette 's disorder with icd , specifier : intermittent explosive disorder . the global functional impairment as assessed on global assessment of functioning scale score ( gaf score ) was 61 . he was treated with sertraline 50 mg / day and gradually titrated upward within 4 weeks to the target level of 150 mg / day in divided doses . low dose of haloperidol was also given ( less than 4.5 mg / day ) and the dose of clonidine ranged from 0.1 to 0.3 mg / day in divided doses . as assessed on yale global tic severity scale ( ygtss ) , there was 50% improvement . the cardinal features of tourette 's disorder and other tic disorders are motor and vocal tics and childhood onset.[7914]in simple motor tics , there is a brief movement of individual muscle group like eye blinking , head shaking and shoulder shrugging , whereas in complex motor tics there is involvement of multiple muscle groups . in our case study , the onset of motor tics was at the age of 15 years , which confirms the findings of sandor et al . ( 1993 ) , i.e. , the onset of disease between the age of 1 and 21 years . it started as simple motor tics involving eye blinking and frowning , which gradually became complex with head turn , shoulder shrug , neck cracking and trunk bending . simple vocal tics include sniffing , grunting , throat clearing , snorting , and sucking , puffing , squeaking , barking and other meaningless sounds . complex vocal tics may present as stereotyped words or phrases , palilalia ( repeating one 's own words ) , echolalia or coprolalia . though coprolalia is often incorrectly considered essential for the diagnosis of tourette 's disorder , it is an uncommon symptom as it is seen in only 2 - 6% of the cases . in our case , the development of vocal tics ( coprolalia ) follows the usual pattern of development , i.e. , motor tics . coprolalia has , indeed , been regarded as hostile reaction toward the authority figures , especially within the family ( tobin and reinhart , 1961 as cited in enoch and ball ) . the patient does not have sufficient outlets for his hostility ; being afraid of punishment , his pent up feelings of hostility toward his father build up to the point where it is released in crescendo of explosive tics and obscene utterances . this view is supported by the fact that the father of the patient was strict , rigid and demanding with obsessive traits . tics are exacerbated by excitement or emotional stress and can be attenuated during periods of focused productive activity , attention and sleep . tics are involuntary ; yet because they are briefly suppressible and can be triggered by environmental stimuli , they may appear as volitional acts . in our case , it was triggered by emotional stress . other movement disorders such as chorea and dystonia are continuous , whereas tics are intermittent . mannerisms are often not impairing ; stereotypy tends to occur exclusively in children and adults with developmental disabilities and mental retardation . hence , it supports the diagnosis of tourette 's disorder as assessed using dsm - iv - tr diagnostic criteria , i.e. , both multiple motor and vocal tics ; tics occurring many times a day for more than 12 months with no period of remission for more than 3 months ; significant functional impairment as assessed on global assessment of functioning scale ( gaf score 61 ) ; onset before 18 years of age and absence of substance abuse or medical / neurological cause of disease . the complex motor tics of tourette 's disorder may be difficult to distinguish from the ocd compulsions . both tics and compulsion are preceded by an intrusive urge followed by feeling of relief . however , ocd compulsion is preceded by fear and obsessional concern with the mental urge to do something repeatedly until it is felt just right , whereas in tourette 's disorder there is a physical urge to perform a motor tic and not preceded by fear . an impulsion is an action performed until a sense of rightness is achieved , rather than compulsion which is designed to reduce anxiety brought by obsession . there is inability to resist the impulse , rather than the rapid transduction of thought to action as in ocd . there is considerable evidence that there is a broad range of co - occurring clinical problems like mood disorder , ocd , impulse control , anxiety , adhd and learning / conduct disorder . up to 50% of clinically ascertained children and adolescents with tourette 's disorder may be affected with problems of attention , concentration activity level or impulse control.in some individuals , tics are preceded or provoked by a thought or physical sensation referred to as premonitory urges . budman ( 1998 ) suggested that the clinical phenomenon of rage attack in children with tourette 's disorder resembles intermittent explosive disorder which may reflect specific neurobiological disturbances . these rage attacks in tourette 's disorder may be related to the presence of co - morbid disorder . the lifetime prevalence of intermittent explosive disorder is 6.3% ; the age of onset is in teens and the incidence is more in males as compared to females . in our case , the patient described the aggressive episodes of throwing stones or an urge to stab his brother as spell or attack . it was preceded by the sense of tension or arousal and was followed immediately by a sense of relief or release of tension . the obsession in tourette 's disorder has multiple concerns such as symmetry , violent , sexual images , urges , worries or loss of control , whereas in intermittent explosive disorder , the behavior has no gratifying element . in addition , fear is the underlying drive in ocd that leads to compulsion which in turn decreases anxiety . in icd , the aggressive acts of throwing stones toward neighbors and destruction of their property and an urge to kill his brother were without any premeditation . these impulsive acts were performed in response to instinctive , or involuntary , irresistible impulses . there was failure to resist the aggressive impulse and the aggressive acts were not accounted by any another mental illness , personality disorder , psychotic , mood , conduct , adhd and are not due to direct physiological effect of a substance or general medical condition ( head trauma , seizure , alzheimer 's disease ) . the essential feature of intermittent explosive disorder was the occurrence of urge for destruction of property . therefore , more epidemiological research is necessary to extend these intriguing findings to provide estimates for a range of presentations that reflect the clinical reality of the tourette 's disorder . it is important to know how often people with tourette 's disorder have tics specific impairment or co - morbidity and are in need of medication .

in the absence of whole genome sequences for many organisms , the use of expressed sequence tags ( est ) offers an affordable approach for researchers conducting phylogenetic analyses to gain insight about the evolutionary history of organisms . reliable alignments for phylogenomic analyses are based on orthologous gene sequences from different taxa . so far , researchers have not sufficiently tackled the problem of the completely automated construction of such datasets . existing software tools are either semi - automated , covering only part of the necessary data processing , or implemented as a pipeline , requiring the installation and configuration of a cascade of external tools , which may be time - consuming and hard to manage . to simplify data set construction for phylogenomic studies , we set up a web server that uses our recently developed orthoselect approach . to the best of our knowledge , our web server is the first web - based est analysis pipeline that allows the detection of orthologous gene sequences in est libraries and outputs orthologous gene alignments . additionally , orthoselect provides the user with an extensive results section that lists and visualizes all important results , such as annotations , data matrices for each gene / taxon and orthologous gene alignments . the web server is available at http://orthoselect.gobics.de .

the rapid development of genome - sequencing techniques has led to the generation of complete genome sequences for > 600 species . most of these sequences belong to model organisms , covering only small portions of the tree of life . the generation of massive numbers of expressed sequence tag ( est ) libraries that can now be sequenced inexpensively by third - generation sequencing is a cheap alternative to whole genome sequencing , and has also provided a wealth of phylogenetically relevant data . several recent phylogenomic studies have used est sequences to generate large data matrices ( 14 ) . these studies generated and assembled est sequences , which were screened for orthologous sequence regions to build useful orthologous gene alignments . orthologous sequences result from a speciation event , and are likely to have a conserved function , whereas paralogous sequences evolve through a gene duplication event within a species , and are less likely to maintain their original function , due to processes such as neo-/or sub - functionalization ( 5 ) . orthologous and paralogous together are called homologues ( 6 ) . since the prime goal of building reliable phylogenetic trees is to decipher the evolutionary relationships among organisms based on their shared common ancestry a reliable protocol is needed to build sets of orthologous sequences from est libraries for successive phylogenomic analyses . we recently proposed such a protocol , which we called orthoselect ( schreiber et al . the main idea is to keep user interaction simple , by simultaneously using state - of - the - art methods for orthology assignment , est translation and elimination of paralogues , as well as construction and automated refinement of multiple sequence alignments . orthoselect has been extensively tested and proven to be a useful tool for managing this complex task . figure 1.the main workflow of orthoselect : each est ( a ) is assigned to a pre - defined orthologous group ( og ) by the kog database , and translated ( b ) . after all ests have been assigned to ogs , a subset of the ogs can be selected which will be further processed to exclude all redundant sequences , compute a sequence alignment and refine it in the last step ( c ) . the main workflow of orthoselect : each est ( a ) is assigned to a pre - defined orthologous group ( og ) by the kog database , and translated ( b ) . after all ests have been assigned to ogs , a subset of the ogs can be selected which will be further processed to exclude all redundant sequences , compute a sequence alignment and refine it in the last step ( c ) . here , we present a web interface to orthoselect , the first web - based est analysis pipeline for constructing orthologous gene alignments from est libraries . the user simply uploads est libraries and chooses parameters ( or uses default settings ) to conduct the analysis . orthoselect then provides the user with a dataset useful for subsequent phylogenetic analysis , as well as numerous helpful data and statistics , such as annotations , a data matrix showing est assignments to the orthologous groups ( ogs ) and visualizations of the orthologous gene alignments . the main purpose of our web server is the construction of orthologous gene alignments from assembled est libraries or other nucleotide sequences . after the user uploads pools of est sequences , ests are assigned to ogs and the sequences most likely to be orthologous in case there were multiple sequences per species are used to compute an alignment that is post - processed in a final step . the workflow of the pipeline is depicted in figure 1 , and is described in more detail in the next section . using the ogs defined by the eukaryotic orthologous groups ( kog ) database ( 7 ) , each est is assigned to the closest og . the assignment is done using a reimplementation of blasto ( 8) that clusters hits from a similarity search of the est against the kog database . the similarity between a query sequence and an og is defined as the mean e - value between the query and the sequences from the og . we then translate the ests using the tools estscan ( 9 ) and genewise , ( 10 ) to account for frame shift errors . considering the best blast hit of the est as a reference sequence , our program selects the translated sequence that is most similar to the reference sequence . only ogs with at least three taxa are further considered . at this stage of the analysis , it is possible to preselect individual or groups of taxa . the set of ogs is then further reduced to contain only ogs containing all preselected taxa . redundant ( e.g. paralogous ) sequences are removed from each og . this is done by considering only the sequence from each species that maximizes a global alignment score as being most likely orthologous . all sequences from each orthologous group are then aligned using either muscle ( 11 ) , t - coffee ( 12 ) or dialign - tx ( 13 ) . these alignments are used to build hidden markov models ( 14 ) that will be used to search the est libraries for additional hits . est sequences may only partially cover genes , there is an option to exclude sequences from the alignment that are too short . this procedure outputs gene alignments whose member sequences are the ones most likely to be orthologous , given the dataset . our web server allows the use of orthoselect with default or adapted parameter values , e.g. the e - value for similarity searches using blast ( 16 ) or methods for computing multiple sequence alignments . orthoselect accepts nucleotide sequences in fasta format . in the absence of a standard format for sequence identifiers in fasta headers , sequence identifiers have to be adapted at some stage of a phylogenetic analysis to allow viewing taxa . orthoselect requires the fasta header to be in a certain format ( the first word up to the first whitespace is taken as an accession number ) , and uploaded files have to match that format , or can be adapted using a converter supplied on the web page . several syntax checks for the uploaded est sequences have been implemented to ensure optimal performance from orthoselect . our web interface offers the possibility to set up sequence identifiers ( e.g. abbreviated taxon names ) that will be used throughout the analysis . our web server will then return a list of those ogs to which the submitted ests have been assigned , as well as a subset of those ogs containing the preselected taxa . the maximum number of input sequences is 30 000 , and the maximum number of est libraries is 10 . an email address has to be supplied , since notification about the results will be sent via email . having generated est libraries for the species under study , one of the main questions that arise is what genes those est libraries have in common . these set of common genes can be used as a base for subsequent phylogenetic analysis . our web server outputs those genes present in all est libraries , but also provides additional information that will help the user to interpret the data and to decide which data are useful as input for phylogeny programs . the web interface offers a wide range of diagrams , charts , tables , etc . to supply the user with useful information ( figure 2 ) . the most important part is the graphical representation of individual ogs with all assigned and translated est sequences , and an overview of its taxonomical composition . single sequences can be viewed along with their translation , as well as the computed multiple sequence alignment prior and subsequent to the final post - processing step in which the program gblocks or aliscore are used . the web server outputs an overview of the ests functional classifications and og assignments as a data matrix with presence / absence information for each gene and species in the study , and annotations for each species . the data matrix shows how many sequences from which taxa have been assigned to an og . this way , the user can easily select ogs with all or a certain percentage of taxa present . figure 2.the output of the orthoselect web server . besides a general overview page of the results ( a ) , our web server reports information about functional annotations ( b ) , a gene / taxa presence / absence matrix ( c , d ) , annotations for each taxon ( e ) , as well as an overview of the orthologous groups ( f ) . additionally , for each orthologous group the resulting alignments are visualized using the jalview ( 17 ) applet ( g ) . the output of the orthoselect web server . besides a general overview page of the results ( a ) , our web server reports information about functional annotations ( b ) , a gene / taxa presence / absence matrix ( c , d ) , annotations for each taxon ( e ) , as well as an overview of the orthologous groups ( f ) . additionally , for each orthologous group the resulting alignments are visualized using the jalview ( 17 ) applet ( g ) . besides an overview of all ogs with sequences assigned ( all orthologous groups ) , orthoselect automatically builds a subset of ogs ( best orthologous groups ) that have either at least three different taxa or the pre - defined taxa present . the all orthologous groups contain all orthologous groups to which sequences have been assigned , whereas the best orthologous groups only contain one sequence per taxon ( see methods section ) . the results page is intended to give the user an elaborate overview and useful information , but also provides all results to be downloaded for further examination and use in phylogenetic studies . the orthoselect server consists of a web interface , a mysql database management system ( dbms ) , and the core program orthoselect . the web interface for orthoselect has been constructed using grails , which is a web application framework that uses the groovy scripting language on the java platform to help standardize the development of web interfaces ( http://www.grails.org ) . grails follows the idea of keeping data and web pages separate with a controller functioning as a mediator between them . all jobs are split into equal chunks to be computed in parallel on our computer cluster , and all data is stored in the dbms . german research foundation ( dfg , project wo896/6 - 1,2 ) within dfg priority programme spp 1174 deep metazoan phylogeny. funding for open access charge : deptartment of bioinformatics , georg - august - universitt gttingen . conflict of interest statement .

key clinical messagemixed phenotype acute leukemia with t(9;22 ) is a rare disease with poor prognosis , and information on optimal treatment is limited . we describe a case where our patient experienced positive outcome after nonacute myeloid leukemiatype intensive induction therapy followed by postremission therapy with stem cell transplant .

the philadelphia chromosome ( ph+ ) is derived from t(9;22)(q34 ; q11 ) or variants and carries the chimeric bcr / abl1 fusion gene which is classically associated with chronic myeloid leukemia ( cml ) 1 . chimeric bcr / abl1 fusion transcript e13a2(b2a2)/e14a2(b3a2 ) encodes for a 210kda protein ( p210 ) , e1a2 encodes for a 190kda protein ( p190 ) , and less commonly e19a2 encodes for a 230kda protein ( p230 ) resulting in constitutively active tyrosine kinase that leads to oncogenic transformation of cell to leukemia 2 . ph+ is found in 1530% of adults and 25% of children with acute lymphoblastic leukemia ( all ) , and on rare occasions , < 3% , in acute myeloid leukemia ( aml ) 3 . ph+ lymphoma and myeloma have been described in the literature 4 , 5 . among mixed phenotype leukemia with abnormal cytogenetics , acute leukemia presenting with ph+ blasts can mean cml in myeloid blast crisis ( cml / mbc ) , ph+ all , ph+ aml , or ph+ mixed phenotype acute leukemia . certain features such as the presence of basophilia , often seen in cml / mbc , or the absence of basophilia commonly seen in ph+ aml can help distinguish one from the other 7 . however , it can be very challenging to delineate these separate entities even in expert hands . efforts are being made to characterize the genetic differences between the malignancies to better manage treatment as these patients typically have poor prognosis 3 . mixed phenotype acute leukemia patients with t(9;22 ) or 11q23 cytogenetic abnormalities have very adverse prognosis 8 . central nerves system ( cns ) involving leukemia add to the disease complexity as extramedullary aml with marrow involvement is considered independent adverse prognostic factor with 5year survival rate ranging from 20% to 30% 9 . here , we present a challenging case of ph+ aml with cns involvement treated with intensive chemotherapy , tyrosine kinase inhibitor ( tki ) , cns directed therapy , and achieved lasting remission after allogeneic hematopoietic stem cell transplantation ( allohsct ) . patient is in complete remission for over 18 months despite its poor prognosis and remains on maintenance tki for relapse prevention . a previously healthy 61yearold man presented with easy bruising , slow wound healing sustained during routine household chores , and general malaise for the past 3 months . physical examination revealed petechiae on lower extremity , mild splenomegaly , and axillary adenopathy . complete blood count ( cbc ) showed significant leukocytosis with white blood count of 150,100/l , normocytic anemia with hemoglobin of 6.7 g / dl , and thrombocytopenia with platelet count of 37,000/l . initial laboratory workup bone marrow biopsy ( bmb ) showed diffuse infiltration by numerous blast cells , which effaced the normal marrow architecture ( fig . 1a ) . cytogenetics and fluorescence in situ hybridization ( fish ) analysis revealed the loss of chromosome 7 and the presence of philadelphia chromosome : 45,xy,7 , t(9;22)(q34;q11.2)/46,xy . morphologically these cells were atypical with immature expression of cd34 and coexpression of cd13 and hladr favoring an early myeloid differentiation rather than lymphoid origin . flow cytometry on the marrow aspirate showed an abnormal population of cells expressing dim cd45 , moderate cd19 , dim cd13 , bright cd34 , hladr , with a subset expressing dim cd117 , dim mpo , and dim tdt is identified , comprising 72% of total events . these blasts were negative for surface kappa and surface lambda light chain immunoglobulins , as well as cd10 , cd11b , cd14 , cd16 , cd64 , cd56 . molecular studies showed bcr / abl p190 mrna pcr of 33.8% on international scale and negative p210 . ( a ) core biopsy of the marrow showed diffuse infiltration by numerous blast cells , which effaced the normal marrow architecture ( h&e , x400 ) . ( b ) a large number of blast cells are also evident in the marrow aspirate ( wirght giemsa , x1000 ) . on admission , patient was started on hydroxyurea for cytoreduction along with routine tumor lysis prophylaxis with hydration and allopurinol . dasatinib was started after 2 days once initial reports became available showing positive philadelphia chromosome . patient was then started on hypercvad a few days later after extensive discussions about therapy choice . five days after admission , patient developed headache , blurry vision , and floaters bilaterally . a computerized tomography ( ct ) of head was performed , and it did not show any acute intracranial bleed , mass , or midline shift . ophthalmology was consulted , and they noted the following on fundus examination : right eye has preretinal hemorrhage near fovea and left eye with intraretinal and preretinal hemorrhage . their final impression was preretinal hemorrhages on both eyes and intraretinal hemorrhages on left eye secondary to thrombocytopenia . he underwent lumbar puncture ( lp ) , and eight blasts were found in cerebral spinal fluid ( csf ) on smear consistent with cns involvement with leukemia . patient was placed on hypercvad ( cyclophosphamide , vincristine , doxorubicin , dexamethasone ) with dasatinib 100 mg daily . biweekly intrathecal chemotherapy including intrathecal cytarabine ( arac ) was initiated with subsequent lp showing clearance of blast cells in csf . dasatinib was continued throughout his treatment course , and no significant adverse effect was noted . bmb performed 3 months after diagnosis showed mildly hypocellular bone marrow without morphologic or immunophenotypic evidence of aml . patient achieved complete cytogenetic response ( ccyr ) and major molecular response ( mmr ) at this point . due to the high risk of relapse in ph+ aml , patient subsequently received allohsct . he had 10/10 hlamatched haploidentical allohsct from his brother ( a total of 4.31 10e6 cd34 + cells were transfused ) with conditioning regimen consists of cyclophosphamide 60 mg / kg and total body irradiation ( tbi ) 1200 centigray ( cgy ) . his course was complicated by chronic graftversushost disease ( gvhd ) , but overall patient had been doing well for more than 18 months . patient continued on dasatinib with plan to stop after 2 years from the date of allohsct . fish study for t(9;22 ) was negative , and monthly bcr / abl p190 mrna pcr level remained negative . by all tests , patient is in complete remission and minimal residual disease ( mrd ) negative . philadelphia chromosomepositive de novo aml is a rare condition with estimated incidence rate of 0.53% 1 , 6 , 10 . the 2016 revision of the world health organization ( who ) classification has recognized mixed phenotype aml with t(9;22 ) as a separate entity 11 . distinction between unrecognized cml / mbc and de novo aml is possible on many grounds . compare to cml / mbc , patients with ph+ aml lack a history of cml or chronic phase / accelerated phase cml after aml induction chemotherapy . coexistence of normal metaphases along with ph+ metaphases , absence of basophilia , and lack of moderate / massive splenomegaly at the time of diagnosis favors ph+ aml over cml / mbc 3 , 12 . normal karyotype after induction therapy is suggestive of ph+ acute leukemia , and in cml / mbc , cytogenetic abnormality often persist 13 . in addition to the t(9;22 ) , cml / mbc is associated with trisomy 8 , trisomy 19 , and isochromosome 17q , whereas monosomy ( 7 ) is associated with ph+ aml 1 , 3 , 14 . the p190 bcrabl fusion protein is common in ph+ aml but only rarely present in cml / mbc 1 . on the molecular level , konoplev et al . found 22% of its patients with ph+ aml had npm1 mutation and none in patients with cml / mbc 2 . while none of the aforementioned features alone is diagnostic of ph+ aml , the summation provides a good indicator of the correct diagnosis , which is important because of its potential treatment implications . outcome of ph+ aml is generally poor with some studies showed median survival of 918 months 1 , 14 . the mainstay of treatment includes imatinib and allohsct , but the optimal treatment is not clear because of the limited number of cases and short followup reported in literature . with the presence of ph chromosome , complete response is thought to be unlikely with imatinib alone 1 . current recommendation according to the national comprehensive cancer network ( nccn ) clinical practice guidelines in oncology for aml is to treat ph+ aml as cml / mbc with tki ( alone or with induction chemotherapy ) followed by allohsct 15 . a recent study by chantepie et al . showed a promising 68% 2year overall survival in patients with ph+ aml after receiving allohsct 10 . there are also reports that showed benefits with using imatinib as interim therapy prior to allohsct and even imatinib or later generations of tki alone 16 , 17 , 18 . reboursiere et al . reported 21 cases of patients with ph+ aml in the literature treated with tki therapy 1 . eight of the patients received allohsct and three of them continued tki as maintenance therapy afterward . it appears tki after allohsct provides survival benefits , but it is far from conclusive at this point given the limited number of cases reported . more studies will be needed to elucidate the best treatment in this rare subtype of aml . any type of aml with cns involvement is uncommon with incidence rate range from 3.1% to 3.6% 19 . incidence rate is even lower at 0.37% when cns involvement is found at diagnosis according to a study conducted by shihadeh et al . these patients tend to be younger , have a higher white blood cell ( wbc ) count , lactate dehydrogenase ( ldh ) , and peripheral blood blasts at diagnosis 19 , 20 . one study found 80% of patients with cns involvement had ldh levels > 700 iu / l 19 . other predictive factors include flt3itd , african american ethnicity , and cytogenetic abnormalities of chromosome 11 , inv(16 ) , and trisomy(8 ) 20 . unlike acute all where cns involvement is more common in which routine intrathecal prophylaxis chemotherapy at the time of diagnosis has shown benefits , such prophylactic measure is not recommended for aml due to the lack of supporting data . different treatment options include intrathecal chemotherapy , systemic chemotherapy , and brain / craniospinal radiation 20 . it is important to note that aml with cns involvement confers a worse diseasefree and overall survival ( os ) compared to those without cns involvement , so any clinical suspicion should prompt timely diagnostic workup such as lumbar puncture and/or head imaging 19 . our patient presented with features that are suggestive of primary ph+ aml including no history of chronic phase , a lack of basophilia , monosomy ( 7 ) , and positive p190 . his course was complicated by cns involvement at diagnosis , and he had a number of predictive factors such as high wbc , ldh , blasts , and chromosome 11 abnormalities . patients with biphenotypic acute leukemia treated with allbased induction regimen have reported 87% complete remission ( cr ) while only 20% of patients treated with an amlbased regimen achieved cr 21 . based on strongly favorable data of intrathecal component to treat cns disease he was successfully treated and later received allohsct who is now in complete remission for more than 18 months . philadelphia chromosomepositive mixed phenotype de novo aml is rare and can be challenging to distinguish from cml / mbc . features associated with ph+ aml include lack of chronic phase , absence of basophilia or splenomegaly , monosomy ( 7 ) , npm1 mutation , and presence of p190 . nonamltype intensive induction therapy followed by postremission therapy by allohsct in suitable patients can potentially overcome adverse prognostic factors , achieve complete remission with longterm survival , and be cured . the authors declare that there is no conflict of interest regarding the publication of this article .

proper regulation of the phosphoinositide 3-kinase akt pathway is critical for the prevention of both insulin resistance and tumorigenesis . many recent studies have characterized a negative feedback loop in which components of one downstream branch of this pathway , composed of the mammalian target of rapamycin and ribosomal s6 kinase , block further activation of the pathway through inhibition of insulin receptor substrate function . these findings form a novel basis for improved understanding of the pathophysiology of metabolic diseases ( e.g. , diabetes and obesity ) , tumor syndromes ( e.g. , tuberous sclerosis complex and peutz - jegher 's syndrome ) , and human cancers .

tor proteins are ser / thr kinases with highly conserved homologues that are found in all eukaryotes ( schmelzle and hall , 2000 ) . in mammals , the two best - characterized targets of mtor are the ribosomal s6 kinases ( s6k1 and s6k2 ) and the eukaryotic initiation factor 4e ( eif4e)binding protein 1 ( 4e - bp1 ) . mtor activity leads to s6k1/2 phosphorylation and activation and to 4e - bp1 phosphorylation and release from the cap - dependent translation initiation factor eif4e . these two events , likely combined with other mtor targets , lead to an increase in ribosomal biogenesis and the selective translation of specific mrna populations . this ability to increase the protein synthesis capacity of the cell is responsible , at least in part , for the ability of tor proteins to drive cell growth and proliferation ( for review see fingar and blenis , 2004 ) . full activation of the mtor pathway requires signals from both nutrients ( e.g. , amino acids and glucose ) and growth factors . although the mechanism by which amino acids are sensed by tor proteins is unknown , their presence is essential for basal activation of mtor signaling , as well as for further stimulation by growth factors ( hara et al . , 1998 ) . interestingly , amino acids have been shown to modulate insulin signaling through mtor - dependent effects on irs-1 ( takano et al . it has been demonstrated that , via mtor regulation , excess amino acids attenuate and amino acid starvation stimulates akt activation and subsequent glucose uptake in response to insulin . these effects are concomitant with changes in the state of irs-1 phosphorylation , localization , and/or degradation . therefore , the levels of circulating amino acids and other nutrients may have a profound effect on the insulin sensitivity of peripheral tissues , such as fat and skeletal muscle , through mtor - mediated regulation of irs-1 function . the primary pathway by which most growth factors and cytokines activate mtor and its downstream targets appears to be the pi3k akt pathway ( for review see fingar and blenis , 2004 ) . as with amino acids , several research groups have found that pi3k akt - mediated activation of mtor also leads to attenuation of insulin signaling . their studies have demonstrated that activation of the pi3k akt mtor pathway in various cell lines by pdgf ( li et al . , 1999 ) , tnf- ( ozes et al . , 2001 ) , or insulin itself ( haruta et al . , 2000 carlson et al . , 2004 ) leads to irs-1 and/or irs-2 serine phosphorylation and down - regulation . although all of these studies found that irs protein phosphorylation is sensitive to the mtor - specific inhibitor rapamycin , the serine residues that are proposed to be involved vary . pdgf - mediated inhibition of irs-1 required the proline - directed sites s632 , s662 , and s731 ( li et al . , 1999 ) , tnf- treatment correlated with s632 and s635 phosphorylation ( ozes et al . , 2001 ) , , 2001 ; greene et al . , 2003 ; carlson et al . , 2004 ; all numbering is for mouse irs-1 ) . however , s307 ( s312 in humans ) is the only site demonstrated to be required for insulin - stimulated irs-1 down - regulation ( greene et al . , 2003 ) . although this site was independently found to be phosphorylated by jnk in response to tnf- ( aguirre et al . 2002 ) , jnk does not appear to be responsible for s307 phosphorylation in response to insulin ( rui et al . , 2001 ; greene et al . , 2003 ) , and the identity of the mtor - dependent kinase that directly phosphorylates this site remains unknown . tuberous sclerosis complex ( tsc ) is a syndrome characterized by a pleiotropic array of benign tumors , collectively referred to as hamartomas , often containing abnormally large cells ( for a recent review see kwiatkowski , 2003 ) . although these tumors rarely become malignant , their presence in various tissues gives rise to severe clinical manifestations , including neurological disorders , skin lesions , cardiac dysfunction , and kidney and lung failure . the disease has been mapped to mutations in either of the two tumor suppressor genes tsc1 , which encodes the hamartin protein , and tsc2 , which encodes the tuberin protein . the hamartin and tuberin proteins form a complex that has recently been found to act within the pathway leading from pi3k akt activation to mtor signaling ( for review see manning and cantley , 2003 ) . hamartin complex potently inhibits tor - dependent signaling in both flies and mammals ( gao et al . , 2002 ; , 2002 ; tee et al . , 2002 ) , and this inhibition is relieved by akt - directed phosphorylation of tuberin ( inoki et al . , 2002 ; manning et al . , 2002 ; potter et al . , 2002 ) therefore , in response to growth factors such as insulin , the pi3k akt pathway activates mtor signaling through phosphorylation and inhibition of tuberin ( fig . interestingly , the responsiveness of tor proteins to other signals , such as nutrient and energy availability , also appears to be dependent on the tsc genes ( gao et al . , 2002 ; inoki et al . , 2003 , shaw et al . , 2004 ) . therefore , homozygous loss of tsc1 or tsc2 leads to high constitutive activation of mtor signaling , as detected in mouse embryonic fibroblasts ( mefs ; jaeschke et al . , 2002 ; kwiatkowski et al . , 2002 ; zhang et al . , 2003 ) , in the tumors of rodent models of tsc ( kenerson et al . , 2002 ; el - hashemite et al . , 2003a ) , and in human tsc cells and tumors ( goncharova et al model of the pi3k akt tsc tor pathway and feedback regulation of insulin / igf - i signaling . ( a ) under normal conditions , insulin / igf - i engagement of the insulin or igf - i receptor ( ir ) leads to tyr phosphorylation of irs proteins and subsequent recruitment and activation of pi3k . pi3k activity leads to activation of akt , which phosphorylates and inhibits many downstream substrates , including bad , foxo transcription factors , gsk3 , and tuberin ( tsc2 ) . through these and other targets , akt activity stimulates glucose uptake and cell growth and proliferation , and inhibits apoptosis . akt - directed phosphorylation of tuberin relieves its inhibition of tor , via activation of the small g protein rheb ( not depicted ) . tor activation by this pathway requires the presence of nutrients and results in activation of s6k and inhibition of 4e - bp1 . s6k phosphorylates the ribosomal s6 protein ( not depicted ) and can feedback and inhibit irs proteins ( see s6k1 : closing the loop section for details ) . ( b ) under atypical conditions of constitutive tor activation , arising from chronic insulin / igf - i exposure , excess nutrients ( e.g. , amino acids [ aas ] and free fatty acids [ ffas ] ) , inflammatory cytokines , or genetic loss of specific tumor suppressor genes , such as tsc2 ( encoding tuberin ) , aberrantly high s6k activity shuts down insulin / igf - i signaling . therefore , although this unregulated tor activity leads to constitutive eif4e activation , it leads to insulin / igf - i resistance through down - regulation of irs protein function . this feedback inhibition prevents akt - mediated glucose uptake and regulation of its downstream substrates . the study of tsc1 and tsc2 mefs has uncovered a striking inability of serum , pdgf , and , especially , insulin and igf - i to activate the pi3k akt pathway in these cells ( jaeschke et al . , 2002 ; kwiatkowski et al . , 2002 ; zhang et al . , 2003 ) . this strong attenuation of akt activation is also seen in drosophila melanogaster tsc1 cells and larvae ( radimerski et al . , 2002 ) . the insensitivity of tsc - deficient mefs to pdgf is due to a loss of expression of both pdgf receptor and pdgf receptor ( zhang et al . , 2003 ) . however , two groups have now characterized the mechanism of insulin / igf - i resistance in these mefs ( harrington et al . , 2004 ; they found that the resistance is a consequence of constitutive down - regulation of irs-1 and irs-2 that is due to sustained and unregulated mtor signaling . ( 2004 ) found that in the absence of tsc2 , both irs-1 and irs-2 shift to a slower mobility during sds - page , which is generally indicative of an increase in ser / thr phosphorylation , and this shift is blocked by short ( 1 h ) treatments with rapamycin . this group found that the mrna and protein levels of irs-1 alone are decreased in tsc - deficient mefs , whereas shah et al . ( 2004 ) found that the levels and stability of both the irs-1 and irs-2 proteins are decreased in these cells . importantly , both studies found that long - term ( 24 h ) treatment of tsc - deficient mefs with rapamycin completely restores irs-1 protein levels and the insulin / igf - i responsiveness of the pi3k akt pathway . together , these studies demonstrate that high levels of constitutive mtor activity strongly down - regulate insulin / igf - i signaling through combined effects on irs-1 expression and irs-1 and irs-2 serine phosphorylation and protein stability . it is now well established that mtor signaling is intimately involved in many pathways that lead to insulin resistance through irs-1/2 down - regulation . the finding in drosophila that larvae doubly mutant for dtsc1 and ds6k have normal levels of akt activity , relative to the attenuated state of dtsc1 larvae , was the first suggestion that this feedback mechanism downstream of mtor is likely to be mediated by s6k ( radimerski et al . , 2002 ) . ( 2004 ) have now found that small interfering rna knockdown of s6k1 or s6k2 can restore the expression of irs-1 mrna in tsc2 mefs . 2004 ) found that a kinase - dead version of s6k1 can block this negative feedback loop and activate akt . although the mechanism of transcriptional regulation of irs-1 by s6k1/2 remains unknown , it appears that the effect of s6k1 on irs-1 serine phosphorylation is direct . in in vitro kinase assays , s6k1 directly phosphorylates s302 of mouse irs-1 ( harrington et al . , 2004 ) , a site matching its preferred substrate recognition motif ( alessi et al . , 1996 ) . furthermore , s302 is constitutively phosphorylated in tsc2 mefs , and this phosphorylation is inhibited by treatment with either rapamycin or s6k1 small interfering rnas . the results from in vitro binding assays suggest that phosphorylation of this site by s6k hinders the ability of irs-1 to associate with the insulin receptor ( harrington et al . , 2004 ) . a recent independent study also found that insulin and igf - i stimulate mtor - dependent s302 phosphorylation in various cell lines , as well as in skeletal muscle ( giraud et al . , 2004 ) . however , the results of overexpression experiments led this group to conclude that s302 phosphorylation enhances , rather than inhibits , insulin signaling . although additional experiments are necessary to resolve this discrepancy , two other research groups have recently provided further evidence that s302 phosphorylation inhibits signaling from irs-1 ( greene et al . a critical in vivo role for s6k1 in desensitizing tissues to insulin was demonstrated in a recent study on s6k1-deficient mice ( um et al . , 2004 ) . the researchers found that these mice are hypersensitive to insulin and impervious to the obesity - induced insulin resistance detected in wild - type mice . in the adipose tissue of wild - type mice , a dramatic increase in s6k1 activation is observed in response to a high fat diet or in genetic models of obesity , and this is accompanied by an increase in irs-1 phosphorylation on s307 , s632 , and s635 and by attenuation of insulin - induced akt activation . however , under these same conditions in s6k1 mice , these sites on irs-1 are not phosphorylated , and akt remains responsive to insulin . although this study did not examine s302 , an increase in phosphorylation of this site has recently been demonstrated in mouse models of obesity and insulin resistance ( werner et al . , 2004 ) . these results demonstrate that s6k1 is essential to multiple pathways that render cells unresponsive to insulin , including those originating from chronic exposure to insulin , ffas , and perhaps tnf-. interestingly , mice lacking either s6k1 or jnk1 are similarly protected from obesity - induced insulin resistance and display loss of irs-1 phosphorylation on s307 ( hirosumi et al . , 2002 ; um et al . , 2004 ) , a site found to be a direct target of jnk ( aguirre et al . , 2000 ) . it appears that phosphorylation of s307 and other serines on irs-1 might , therefore , exhibit cooperation with one another in response to some stimuli . because mtor signaling appears to be required for the effects of several pathways on irs-1 , it seems likely that there is an initial s6k1-mediated phosphorylation event , perhaps on s302 , that is required for other serine kinases to subsequently phosphorylate irs-1 . if this model is correct , this mechanism would prevent any pathway from blocking insulin signaling when mtor activity is low , such as during glucose or amino acid starvation . this would allow cells , and mtor , to remain maximally sensitive to the insulin pi3k akt pathway , which is involved in nutrient uptake . as a downstream target , s6k is poised to sense the degree of activation of mtor , which is stimulated by both nutrients and growth factors ( fig . 1 a ) . as a critical component of the negative feedback loop , s6k then has the ability to modify accordingly the level of input from some growth factors , such as insulin and igf - i . however , under nutritional conditions in which mtor is chronically activated , such as in the presence of excess amino acids or during high fat diets , s6k will constitutively shut down the responsiveness of certain cells to insulin ( fig . this suggests that rapamycin ( or , more specifically , an inhibitor of s6k ) might be an effective treatment for certain metabolic disorders , such as type 2 diabetes , characterized by insulin resistance ( um et al . , 2004 ) . chronic activation of mtor signaling can also result from genetic loss of certain tumor suppressor genes , including pten , tsc1/2 , and lkb1 ( neshat et al . , 2001 ; kwiatkowski , 2003 ; shaw et al . , 2004 ) . interestingly , mutations in these genes are associated with the specific tumor syndromes cowden 's disease ( pten ) , tsc ( tsc1/2 ) , and peutz - jegher 's syndrome ( lkb1 ) , which are all characterized by the development of benign tumors classified as hamartomas . although aberrant mtor activation appears to be a common biochemical link between cells lacking these tumor suppressors , the activation state of the pi3k akt pathway varies . loss of pten ( phosphatase and tensin homologue deleted on chromosome 10 ) , which counters the activity of pi3k , leads to constitutive activation of akt ( for review see cantley and neel , 1999 ) . however , as discussed above for tsc1/2 , mefs lacking lkb1 also exhibit some attenuation of akt activation ( shaw et al . , 2004 ) . this discrepancy might have profound effects on the nature of the tumors that arise in patients with these different syndromes . the overall development of a hamartoma might be the result of constitutive mtor activity , but the severity and malignancy potential of the tumor might depend on the ability to overcome the feedback inhibition of akt . the attenuation of akt is strongest in the absence of the tsc genes , and the inability of akt to phosphorylate and inhibit its downstream substrates other than tuberin , such as specific members of the foxo family of transcription factors , gsk3 , and bad , could limit the proliferation and survival capacity of tsc hamartomas ( fig . consistent with this idea , malignant tumors are very rare in tsc patients ( kwiatkowski , 2003 ) , whereas in cowden 's disease , where loss of pten activates akt independently of growth factor signaling , the development of malignant tumors is much more common ( cantley and neel , 1999 ) . because rapamycin relieves this feedback inhibition of akt ( harrington et al . , 2004 ; shah et al . , 2004 ) , a difficult question in the field is whether this mtor inhibitor would be beneficial or detrimental to patients with hamartomas exhibiting aberrant mtor activation . consistent with mtor activation driving initial tumor development in these related syndromes , rapamycin has been proven to be effective at blocking the formation of tumors in rodent models of tsc ( kenerson et al . , 2002 ) . however , it is less clear whether rapamycin would be a useful treatment once the tumor has already been established , as is the case in patients diagnosed with tsc . in fact , studies of tsc1/2- and lkb1-deficient mefs have demonstrated that under some growth conditions rapamycin actually enhances survival of cells devoid of these tumor suppressors ( inoki et al . , 2003 ; , 2004 ; shaw et al . , 2004 ) , and this is likely due to the restoration of critical survival pathways controlled by akt . however , these studies are done on a short - term basis and under very specific conditions . it seems likely that , although rapamycin 's inhibition of s6k activation would eliminate the negative feedback mechanism , it would also shut down the aberrant activation of eif4e and cap - dependent translation . based on many studies suggesting that eif4e is a potent oncogene ( e.g. , for review see mamane et al . , 2004 ; ruggero et al . , 2004 ; wendel et al . , 2004 ) , it would appear that the mtor - dependent activation of eif4e , through 4e - bp1 phosphorylation , may be at the heart of tumorigenesis in syndromes exhibiting constitutive mtor signaling . if this is true , then long - term treatment with rapamycin would block the primary cellular process driving tumor growth , irrespective of akt activity . furthermore , it is currently unknown whether the down - regulation of pdgf receptors observed in tsc - deficient mefs is sensitive to rapamycin ( zhang et al . , 2003 ) . it remains to be established whether the state of this mtor - dependent feedback mechanism is what delineates the severity of some tumors and metabolic diseases . however , studies on this pathway , and the diseases to which it contributes , underscore the importance of elucidating the complex wiring of seemingly basic signal transduction pathways .

asians and pacific islanders ' ( apis ) leading cause of death is cancer . we compared apis ' age - adjusted cancer mortality rates to other racial / ethnic groups and by api subgroup ( i.e. , chinese , koreans , asian indians , and filipinos ) using new york city ( nyc ) mortality data and census bureau population estimates for 20012010 . while other racial / ethnic groups ' overall cancer mortality rates declined in nyc during the last decade , apis remained stable . apis overall had the lowest mortality rates for more common cancer types ( i.e. , lung , colorectal , breast , and prostate ) , but the highest mortality rates for certain less common cancers ( i.e. , nasopharyngeal , stomach , and liver ) . chinese new yorkers ' lung cancer death rates were very high compared to other apis and comparable to non - hispanic whites ( 47.1/100,000 versus 49.5/100,000 , resp . ) . chinese men had much higher nasopharyngeal cancer mortality rates ( 4.5/100,000 versus 0.3/100,000 for non - hispanic whites ) . korean men had the highest liver and stomach cancer mortality rates ( 25.3/100,000 and 27.7/100,000 , resp . , versus 7.9/100,000 and 6.0/100,000 for non - hispanic whites ) . analysis of cancer rates by api subgroup provides the detailed information needed to plan cancer prevention efforts . these findings warrant consideration of targeted cancer mortality prevention efforts for affected subgroups , including hepatitis vaccination , screening , and treatment ; smoking cessation ; and cancer screening .

asians and pacific islanders ( apis ) constitute 5% of the united states ( us ) population and 13% of the new york city ( nyc ) population . from 2000 through 2010 , the api population experienced the fastest rate of growth than any other racial / ethnic groups both nationally ( 43% increase ) and in nyc ( 32% increase ) . the city 's api population is the largest among us cities , 1.1 million persons , according to the 2010 census . while cardiovascular disease is the leading cause of mortality among non - hispanic whites , non - hispanic blacks , and hispanics , cancer is the leading cause of death among apis , both nationally in 2007 ( 106.7 per 100,000 ) and in nyc in 2010 ( 105.9 per 100,000 ) [ 3 , 4 ] . apis experience lower death rates for certain common cancers ( i.e. , lung , colorectal , breast , and prostate ) than other racial / ethnic groups in the united states . however , apis experience the highest death rates from some less common cancers , particularly those associated with infectious agents , such as nasopharynx , liver , and stomach cancer . published analyses on cancer mortality among apis have largely focused on california [ 7 , 8 ] and new york city [ 9 , 10 ] , except one study utilizing data from selected sites throughout the us , but all these studies are based on data that is more than a decade old . in california , mccracken et al . examined cancer mortality among asian americans overall from 2000 to 2002 , and kwong et al . described cancer mortality by asian subgroups from 1997 through 2001 . in nyc , freeman et al . examined cancer mortality by overall racial / ethnic group between 1990 and 2000 . miller et al . examined cancer incidence and mortality by asian subgroups for 19982002 from selected state and regional cancer registries covering 54% of the us api population . cancer mortality among api subgroups has not been studied in nyc , aside from a 20-year - old nyc study ( 1988 through 1992 ) examining site - specific cancer mortality rates among the chinese only . thus , to our knowledge , no recent analyses of api or api subgroup cancer mortality rates within a us population have been conducted . we aim to characterize site - specific cancer mortality rates among apis in nyc , comparing them to other racial / ethnic groups for the 10-year period from 2001 through 2010 . we will also examine site - specific cancer rates among different api subgroups ( i.e. , chinese , korean , asian indian , and filipino ) to explore heterogeneity within the diverse api group . this analysis was based on new york city annual population estimates obtained from the us census bureau , the american community survey public use microdata samples ( 2005 through 2009 ) , and mortality data for the 10-year period ( 2001 through 2010 ) from the new york city department of health and mental hygiene 's ( nyc dohmh ) bureau of vital statistics . variables included sex , age , race / ethnicity , ancestry , birthplace , education , marital status , and underlying cause of death . underlying causes of death were coded according to the 10th revision of the international statistical classification of diseases and related health problems ( icd-10 ) . common site - specific cancers across all racial / ethnic groups ( i.e. , lung , colon and rectum , esophagus , prostate , breast , ovary , and cervix ) and those specifically common in apis ( i.e. , nasopharynx , liver , and stomach ) were selected for study . api subgroups were determined by the races reported on death certificates , which is provided by funeral directors in nyc . we did not differentiate between foreign - born versus us - born apis because the proportion of us - born was only 2.1% of deaths which is too small to merit analysis . this is likely because of the relatively young age of new york city 's us - born asian population ; 81% are less than 45 years of age based on us census bureau 's 20052009 american community survey microdata sample . cancer mortality rates and 95% confidence intervals were calculated for major racial / ethnic groups using nyc dohmh mortality data and us census population estimates . cancer mortality rates and 95% confidence intervals for chinese , korean , asian indian , and filipino subgroups were calculated using nyc dohmh mortality records and the 20052009 american community survey public use microdata sample . the 2010 us census reports information on 6 asian subgroups : chinese , korean , asian indian , filipino , japanese , and vietnamese . new york city 's japanese and vietnamese populations were too small to allow for inclusion in this analysis . in 2010 , 13,333 ( 25.4% ) new york city decedents died from a malignant neoplasm . among api decedents , 943 ( 29.9% ) died from a malignant neoplasm . apis have the overall lowest cancer mortality rates across all racial / ethnic groups . however , between 2001 and 2010 , cancer mortality rates declined in all racial / ethnic groups in nyc , except apis . the api cancer mortality rate has remained stable from 2001 through 2010 ( see figure 1 ) . from 2001 through 2010 , a total of 82,608 decedents died in new york city from the 10 selected cancers ( table 4 ) ; 5,548 ( 6.7% ) of these decedents were apis ( 3,200 males and 2,348 females ) ( table 1 ) . compared with non - hispanic whites , api decedents were more likely to be male , married , and having less than a high school education . on average , api decedents died 5 years earlier from cancer than did non - hispanic whites . the age - adjusted nasopharyngeal cancer mortality rate among api men ( 2.6 per 100,000 ) was the highest across all racial / ethnic groups and nearly 10 times higher than the rate for non - hispanic white men ( 0.29 per 100,000 ; see table 2 ) . the mortality rates of liver and stomach cancer were twice as high in api men as in non - hispanic white men ( liver cancer , 15.9 versus 7.9 per 100,000 and stomach cancer , 11.8 versus 6.0 per 1000,000 , resp . ) . the mortality rate for nasopharyngeal cancer among api women ( 0.7 per 100,000 ) was the highest of all racial / ethnic groups , 7 times as high as in non - hispanic white women ( 0.1 per 100,000 ) . the liver cancer mortality rate was almost 2 times higher in api women ( 5.4 per 100,000 ) than in non - hispanic white women ( 3.0 per 100,000 ) , and api women had the highest stomach cancer mortality rate ( 5.6 per 100,000 ) of all racial / ethnic groups . among both men and women , apis have the lowest colorectal cancer mortality rates of all racial / ethnic groups . api men and women have age - adjusted lung cancer mortality rates ( 38.2 and 16.7 per 100,000 , resp . ) that surpass those of hispanic men and women ( 33.5 and 14.7 per 100,000 , resp . ) but not non - hispanic whites or blacks . for all studied sites , cancer death rates were higher in api men than api women . from 2001 through 2010 , api males experienced higher age - adjusted liver cancer mortality rates than non - hispanic white males ( 15.6 to 14.0 per 100,000 versus 8.3 to 10.3 per 100,000 , resp . ; , api females experienced a slight increase in age - adjusted liver cancer mortality rates , while this rate was stable among non - hispanic white females ( 5.3 to 6.1 per 100,000 versus 3.1 to 3.0 per 100,000 , resp . , api males did not experience the dramatic decline in age - adjusted lung cancer mortality rates that non - hispanic white males did ( 38.6 to 38.2 per 100,000 versus 62.2 to 47.0 per 100,000 , resp . ; see figure 4 ) . age - adjusted lung cancer mortality rates among api females increased during 2001 through 2010 , while non - hispanic white females experienced a decrease ( 17.4 to 19.7 per 100,000 versus 39.8 to 34.5 per 100,000 , resp . ; see figure 5 ) . overall , 5,548 cancer deaths occurred among apis in new york city from 2001 through 2010 ( see table 3 ) . chinese comprise the majority ( 66.7% ) of api cancer decedents . compared to other api subgroups , chinese decedents were more likely to be male , have less than a high school education , and die at an older age on average . compared to other api subgroups , asian indian cancer decedents were more likely to be married and die at a younger age . filipino decedents were more likely to be female and have more than a high school education . chinese men and women had the highest lung cancer mortality rates ( 47.1 and 20.0 per 100,000 , resp . ) as compared to other api subgroups . lung cancer mortality rates in the chinese have increased from 2001 to 2010 ( 40.0 to 51.5 per 100,000 in men and 16.2 to 25.8 per 100,000 in women ) , while they have decreased in non - hispanic whites ( 62.2 to 47.0 per 100,000 in men and 39.8 to 34.5 per 100,000 in women ) . among apis , chinese women had the highest colon cancer mortality rate ( 10.6 per 100,000 ) among women and chinese men had the second highest colon cancer mortality rate ( 16.1 per 100,000 ) among men . chinese men and women had the highest nasopharyngeal cancer mortality rates ( 4.5 and 1.2 per 100,000 , resp . ) across all racial / ethnic groups , and these rates were statistically significantly higher than in the other racial / ethnic groups . korean men had the highest liver and colon cancer rates ( 25.3 and 17.6 per 100,000 , resp . ) compared to other api subgroups . korean women had the highest liver cancer mortality rate , which was statistically significantly higher than other api subgroups ( 7.5 per 100,000 versus 6.0 per 100,000 for chinese women , 2.9 per 100,000 for asian indian women and 2.6 per 100,000 for filipino women ) . korean men and women had the highest stomach cancer rates , which were statistically significantly higher than other api subgroups ( 27.7 and 11.0 per 100,000 , resp . ) . korean men and women had the second highest age - adjusted lung cancer mortality rates ( 38.9 and 12.9 per 100,000 , resp . ) . among api subgroups , filipino men had the highest prostate mortality rate ( 9.5 per 100,000 ) . filipino women had the highest breast and ovarian cancer mortality rates ( 13.4 and 6.4 per 100,000 , resp . ) . we investigated site - specific cancer mortality rates among apis and api subgroups , given that cancer is the leading cause of death among apis . while apis in nyc have the lowest overall cancer mortality rate of the 4 major racial / ethnic groups , certain site - specific cancer rates are much higher among apis than other racial / ethnic groups and among api subgroups . examining rates for apis as a group masks the true heterogeneity of their cancer burden . varying environmental and behavioral influences , including tobacco use , exposure to infectious agents , and diet , are likely driving these observed patterns of api cancer mortality . these findings warrant consideration of targeted cancer mortality prevention efforts for affected subgroups , including hepatitis vaccination , screening , and treatment ; smoking cessation ; and cancer screening . lung cancer death rates in chinese new yorkers are high and have increased from 2001 through 2010 for both men and women . it is known that tobacco smoking , primarily cigarettes , is the major cause of lung cancer . in nyc , the 2010 community health survey , a nyc dohmh population - based telephone survey , found a smoking prevalence of 20% among chinese men and less than 1% among chinese women . the increasing lung cancer death rate among chinese women in the setting of low prevalence of smoking is worrisome , and more research is needed to determine factors contributing to the increased lung cancer death rates in chinese women . second - hand smoke exposure at home and work and exposure to cooking oil from high - temperature frying may contribute to the increased risk of lung cancer in chinese women . in addition , the smoking prevalence among the chinese population overall has remained the same over 8 years ( 9.2% in 2002 and 10.2% in 2010 ) , whereas the overall smoking prevalence in nyc dropped significantly from 21.5% in 2002 to 14% in 2010 . given that nyc mounted an aggressive tobacco control program during this period , the increasing lung cancer death rates and the stable smoking prevalence rates among the chinese suggest that we may not be reaching this subgroup . global data suggest that lung cancer mortality rates in china are lower than in nyc . it is not known why this is true , but two possibilities would be increased immigration to nyc from areas of china with higher rates of lung cancer and decreased accuracy of cause of death information . in 2008 , according to the international agency for research on cancer , lung cancer mortality rates were 39.6 per 100,000 in chinese men and 18.3 per 100,000 in chinese women versus 47.1 and 20.0 per 100,000 for chinese men and women in nyc , respectively , per our analyses . one reason that could explain these persistently high rates of lung cancer mortality among the chinese in nyc is the culturally accepted nature of tobacco smoking in certain asian countries , particularly among men . according to the global adult tobacco survey , 63% of men in china currently smoked in 2010 . in addition , giving cigarettes as gifts during special occasions is a cultural norm , and immigrants have access to low - cost cigarettes from taiwan and china . since 2005 , the nyc mayor 's office and nyc dohmh have launched several culturally relevant media campaigns focusing on the chinese , including translated posters and print advertisements to promote tobacco cessation . the nyc dohmh has a long - standing nicotine patch and gum give - away program , and in 2010 , online registration was made available in chinese along with translated nicotine patch and gum kit contents . dohmh also has a 311 hotline with chinese language capability to help interested callers quit smoking . in 2012 , a dedicated chinese hotline was created to attract more participants to the nicotine patch and gum give - away program . despite these efforts , more research is needed to determine the barriers to quitting smoking in the asian population . closely following the chinese , koreans have the second highest lung cancer mortality rates ( 38.9 per 100,000 for men and 12.9 per 100,000 for women ) among apis . in south korea from 2008 to 2010 , 42.3% of korean men have been reported to smoke . in nyc , for 20062008 , korean 's self - reported rate of smoking was 16.9% based on the nyc community health survey , although small numbers limit the precision of that estimate . nyc has not focused on special tobacco cessation efforts in this group due to its small population size ( only 1.0% of nyc population ) . nasopharyngeal cancer death rates in nyc are higher among apis than other racial / ethnic groups , and this cancer disproportionately affects the chinese . the nasopharyngeal cancer mortality rate in chinese men ( 4.5 per 100,000 ) was 15 times that of non - hispanic whites ( 0.3 per 100,000 ) . in chinese women , the nasopharyngeal cancer mortality rate ( 1.2 per 100,000 ) was 12 times as high as in non - hispanic white women ( 0.1 per 100,000 ) . according to the world health organization , nasopharyngeal cancer mortality rates in 2008 in china for males and females were 1.9 and 0.8 per 100,000 , respectively , based on data from the international agency for research on cancer . most immigrants from china to the us come from guangdong province , where the incidence of nasopharyngeal cancer is very high ( 25 cases per 100,000 ) . several epidemiologic studies have proposed that the epstein - barr virus ( ebv ) , smoked fish products , and genetic predisposition are associated with high nasopharyngeal cancer rates among the chinese [ 22 , 23 ] . in certain parts of southern china , particularly guangdong province where nasopharyngeal cancer is common , at - risk individuals are being screened by ebv testing , followed by periodic examination of the nasopharynx and neck if positive . earlier detection of nasopharyngeal carcinoma allows for treatment with improved 5-year survival . in the united states , no nasopharyngeal cancer screening guidelines exist . in cities like nyc where a large proportion of chinese immigrants are from southern china , a high index of clinical suspicion for this type of cancer should be maintained by clinicians caring for these immigrants . further research is indicated to evaluate whether the screening approach used in southern china is effective at preventing mortality and to determine whether screening is appropriate for chinese new yorkers . in nyc , the liver cancer death rate is higher among apis , particularly koreans and chinese , than in other racial / ethnic groups . hepatitis b virus ( hbv ) infection is highly prevalent in asian countries , including south korea and china , and is the main risk factor for developing liver cancer in countries endemic with hbv . other contributing risk factors include cirrhosis ( associated with infection , obesity , and alcohol ) and aflatoxin exposure . nyc dohmh surveillance for chronic hbv infection from june 2008 to november 2009 identified 156 cases of infection , of whom 87 ( 56% ) were in chinese born persons and few ( <3 ) were in persons originating from korea . however , a 2008 prevalence study found that koreans represented 4.6% of nyc 's chronic hbv infections , while they constituted only 1.0% of the nyc population , confirming their higher risk for chronic infection compared to other new yorkers . in one 2005 nyc study of a clinic - based screening program , chinese had the highest prevalence ( 21% ) of chronic hbv infection among the patients screened , but koreans also had a high prevalence ( 5% ) of infection . given elevated rates of liver cancer among the chinese and koreans , nyc clinicians should be aware that the centers for disease control and prevention and dohmh currently recommend screening individuals at risk for hbv infection , including persons from hbv - endemic countries such as korea and china , treatment of chronic hbv infection , and periodic liver cancer screening in those with chronic hbv infection , which may help to prevent progression and death from liver cancer . given that current evidence suggests that koreans have a lower chronic hbv infection rate compared with chinese , other factors may be playing a role in elevating their liver cancer death rate above that of the chinese . when liver cancer was listed as the underlying cause of death , the use of alcohol as a contributing cause was mentioned in more korean decedents ( 1.8% ) than chinese decedents ( 0.2% ) , although its mention was infrequent for both . in nyc 's community health survey for the period 2002 to 2008 , individuals of korean descent reported binge drinking almost 4 times more frequently than individuals of chinese descent ( 23.5% versus 5.8% , resp . ) and heavy drinking was reported more frequently as well ( 3.4% versus 1.3% ) , although small numbers limit the precision of these later estimates . synergism between alcohol consumption and chronic hbv infection may possibly increase the risk of cirrhosis and thus liver cancer deaths in koreans in nyc . further research is needed to determine the contribution of alcohol use to liver cancer deaths among koreans . in addition , stomach cancer has also been associated with eating preserved food and foods rich in nitrites and/or nitrates . no routine stomach cancer screening is recommended in the us , but screening programs have been implemented in japan . the risk of stomach cancer deaths in the japanese living in japan ( 20.5 per 100,000 in men ) is comparable to mortality rates in nyc chinese ( 12.9 per 100,000 in men ) and koreans ( 27.7 per 100,000 in men ) . an improving trend in the stomach cancer survival rate has been observed in japan and can possibly be attributed to early cancer detection . a formal evaluation of the risk and benefits of using the japanese screening approach in high - risk groups should be considered for nyc and other urban areas with a sizeable api population . breast cancer is the leading cause of cancer death in nyc asian indian and filipina women . multiple factors contribute to breast cancer mortality , including genetic predisposition and socioeconomic and cultural circumstances , but late - stage diagnosis is strongly associated with higher mortality rates . in the nyc community health survey , women of asian descent aged 40 years and older reported a lower rate of mammography screening ( 67.5% ) than nyc women overall ( 75.3% ) , and specifically , asian indian women in nyc had a substantially lower rate of screening ( 58.9% ) . the rate for filipina women was a few percent below the citywide rate ( 72.1% ) based on their self - reported responses . however , a hawaiian study found that filipina women are less compliant with mammography guidelines and are diagnosed at a more advanced stage of cancer at time of diagnosis . efforts to target specific racial / ethnic groups to increase breast cancer screening may be needed . more research is also needed in nyc to determine the reasons for lower rates of mammography screening in asian indian and possibly filipina women , which may contribute to higher breast cancer mortality rates . the limitations of this study include potential underascertainment of api population size by the 2010 census or american community survey , which would result in an overestimation of mortality rates in the population . in particular , undocumented immigrants may not be captured in the census because of their reluctance to be counted , contributing to underestimation of the population [ 37 , 38 ] . underestimation of deaths among apis may also occur if persons who spend most of their working life here in the us return to their home country for retirement and death . asians are the fastest growing racial / ethnic group in the us , and they have a unique cancer burden that requires special attention . the leading cause of death in apis nationally and in nyc is malignant neoplasms . apis are disproportionately affected by some less common cancers , including nasopharyngeal , liver , and stomach cancers . hepatitis b screening has been recommended by cdc for high - risk groups , including persons from china and korea . culturally tailored tobacco control policies and programs have been implemented over the last decade , and further evaluation is needed to determine their impact . in those populations that have reduced smoking rates , the impact may become more apparent in the next decade as fewer people develop smoking - related cancer , given that there is a long lag time between smoking exposure and cancer development . potential population - based screening approaches to detect and treat early nasopharyngeal and stomach cancers in apis require more evaluation before implementation but should be considered . the substantial variation in cancer - specific mortality rates across asian racial subgroups indicates that api subgroup analyses such as these are necessary to plan public health interventions .

background : prevalence of metabolic syndrome ( mes ) is high among asians , including indians and is rising , particularly with the adoption of modernized lifestyle . various studies have reported a significant relationship between periodontal status and mes . the objective of this study is to investigate the association between periodontitis and mes.materials and methods : the study included 259 subjects ( 130 cases with chronic periodontitis , 129 controls without chronic periodontitis ) who underwent medical and periodontal checkup . five components ( obesity , high blood pressure , low- and high - density lipoproteins , cholesterol , hypertriglyceridemia , and high plasma glucose ) of mes were evaluated , and individuals with 3 positive components were defined as having mes . the periodontal parameter was clinical attachment level ( cal ) on the basis of which cases were selected with moderate ( cal loss 34 mm ) and severe ( cal loss 5 mm ) generalized chronic periodontitis . the association between chronic periodontitis and mes components was investigated using odds ratios ( ors ) and 95% confidence intervals ( cis).results : the association of mes and chronic periodontitis was strong and significant with or : 2.64 , 95% ci : 1.365.18 , and p < 0.003 . comparison of mean values of components of mes between cases and controls reveals that the mean waist circumference ( mean difference : 4.8 [ 95% ci : 7.751.84 ] , p < 0.002 ) and mean triglycerides level ( mean difference : 25.75 [ 95% ci : 49.222.28 ] , p < 0.032 ) were significantly higher in cases than in control groups . although mean systolic blood pressure , diastolic blood pressure , and fasting blood sugar level were higher in cases ( 125.77 , 82.99 and 86.38 , respectively ) compared with control ( 122.81 , 81.3 and 83.68 , respectively ) , it was statistically insignificant.conclusion:the results of this study suggest that there is a strong association between chronic periodontitis and mes . the association was independent of the various potential confounding risk factors affecting the chronic periodontitis such as age , sex , residential background , and tobacco consumption .

periodontitis is a chronic infectious disease of the supporting tissues of the teeth , with multiple related factors . in recent years , interest has grown regarding the relationship with other systemic conditions to identify some new aspects to improve diagnostic tools and treatment outcomes . metabolic syndrome ( mes ) consists of a combination of impaired glucose regulation , abdominal obesity , dyslipidemia , and high blood pressure . it is estimated that approximately one - fourth of the world 's adult population is affected by mes . it is generally accepted that the origin is a pro - inflammatory state derived from excessive calorie intake and over nutrition and other chronic inflammatory diseases . oxidative stress has been proposed as a potential common link to explain relationship among each component of mes and periodontitis . a cross - sectional study conducted by shimazaki et al . , in 2007 , showed that individuals exhibiting more components of mes had a higher odds ratio ( or ) for a greater periodontal probing depth ( pd ) and clinical attachment level ( cal ) . in addition , other cross - sectional studies revealed that individuals with mes had a higher risk for poor periodontal status , and those individuals with poor periodontal status had a higher risk for mes . elevated blood levels of inflammatory markers , such as c - reactive protein ( crp ) and interleukin ( il)-6 , were reported in patients with periodontal disease , suggesting that periodontal disease is a mild chronic inflammatory disease affecting the systemic conditions . aggravation of glucose tolerance in people with deep periodontal pockets was also shown epidemiologically , suggesting that infection with periodontal disease pathogens enhance tumor necrosis factor - alpha ( tnf- ) production , induces the prediabetic condition , and leads to abnormal glucose tolerance . furthermore , negative influences by lipopolysaccharide and cytokines produced by inflammation , such as tnf- and il-1 on lipid metabolism , were reported , suggesting that gram - negative anaerobe - induced periodontal disease has some influence on lipid metabolism . considering these findings , it is possible that periodontal disease increases the risk of developing mes as a gram - negative anaerobe - induced mild chronic inflammatory disease . prevalence of mes is high among asians including indians and is rising , particularly with the adoption of modernized lifestyle . however , little information is available on the possible association between periodontitis and mes as a sole clinical entity in indian population ; this study was carried out to assess the association between mes and chronic periodontitis . the total percentage of rural population in himachal pradesh 90.20% of the total population residing in 17,495 inhabited villages . the rural population has limited access to health - care facility as compared to the urban population . himachal pradesh has the highest percentage of rural population among all the states of the country with poor socioeconomic status as compared to those residing in urban areas . the cases and controls were screened on the basis of cal according to the american academy of periodontology classification system . subjects meeting the inclusion / exclusion criteria were invited to take part in the study and were provided with the subject information sheet and consent form . three hundred and seventy - eight subjects were enrolled for the study of duration 22 months . among all the consecutive patients population referred for care to the outpatient department of periodontology and services of h.p . government dental college and hospital , shimla , india , 119 ( out of 378 subjects ) patients did not complete the study . therefore , among the 259 subjects who completed the study , 130 individuals diagnosed with moderate / severe periodontitis were enrolled in the periodontal clinic , whereas 129 patients without periodontitis were invited to participate while attending the oral surgery or restorative clinics and served as nonaffected controls . thus , patients with moderate / severe chronic periodontitis formed the study population and those without periodontitis on clinical evaluation formed the control group . however , screening of cases , i.e. , individuals with moderate / severe periodontitis , was based on mean clinical attachment loss measured at four sites per tooth ( mesiobuccal , buccal , distobuccal , and lingual ) of the entire dentition except for third molars . inclusion criteria for the cases were diagnosis of moderate / severe periodontitis based on the 1999 consensus classification of periodontal disease by armitage and thomas f. flemmig ( 1999 ) . inclusion criteria for cases with moderate / severe chronic periodontitis : ( 1 ) patients with at least 20 teeth and more than 30% of sites exhibiting 34 mm of mean clinical attachment loss for moderate chronic periodontitis and more than 30% of sites 5 mm of mean clinical attachment loss for severe chronic periodontitis , ( 2 ) minimum age of 20 years , and ( 3 ) patients consenting to participate . inclusion criteria for controls : ( 1 ) the absence of the clinical and radiographic manifestations of periodontal disease , ( 2 ) at least , 20 teeth present in the oral cavity and no history of periodontal treatment in the past , ( 3 ) minimum age of 20 years , and ( 4 ) patients consenting to participate . exclusion criteria : ( 1 ) patients not willing to participate in the study , ( 2 ) pregnant patients , ( 3 ) patients of rheumatic heart disease , congenital heart disease and/or with prosthetic valves who were at high risk for endocarditis , and ( 4 ) all patients medically unfit to participate in the study . mes was diagnosed based on the presence of three of the five following components using modified adult treatment panel iii criteria : ( 1 ) abdominal / central obesity ( as indicated by increased waist circumference or markedly increased body mass index [ bmi ] and defined as waist circumference with ethnicity - specific values ) : waist circumference 90 cm in men and 80 cm in women ( based on a chinese , malay , and asian indian population ) , ( 2 ) hypertriglyceridemia : 150 mg / dl ( 1.69 mmol / l ) , ( 3 ) low- and high - density lipoprotein cholesterol : < 40 mg / dl ( 1.04 mmol / l ) in men ; < 50 mg / dl ( 1.29 mmol / l ) in women , ( 4 ) high blood pressure : 130/85 mmhg , and ( 5 ) high fasting glucose : 100 mg / dl ( 6.1 mmol / l ) . spss package version 16 ( spss inc . , chicago , il , usa ) was used . the significance of difference in mean values of continuous variables between study and control group was estimated by student 's t - test . the significance of the difference in the distribution of discrete variables between the groups was estimated with chi - square test . association between chronic periodontitis and mes was estimated by calculating or with 95% confidence interval ( ci ) . the cases and controls were screened on the basis of cal according to the american academy of periodontology classification system . subjects meeting the inclusion / exclusion criteria were invited to take part in the study and were provided with the subject information sheet and consent form . three hundred and seventy - eight subjects were enrolled for the study of duration 22 months . among all the consecutive patients population referred for care to the outpatient department of periodontology and services of h.p . government dental college and hospital , shimla , india , 119 ( out of 378 subjects ) patients did not complete the study . therefore , among the 259 subjects who completed the study , 130 individuals diagnosed with moderate / severe periodontitis were enrolled in the periodontal clinic , whereas 129 patients without periodontitis were invited to participate while attending the oral surgery or restorative clinics and served as nonaffected controls . thus , patients with moderate / severe chronic periodontitis formed the study population and those without periodontitis on clinical evaluation formed the control group . however , screening of cases , i.e. , individuals with moderate / severe periodontitis , was based on mean clinical attachment loss measured at four sites per tooth ( mesiobuccal , buccal , distobuccal , and lingual ) of the entire dentition except for third molars . inclusion criteria for the cases were diagnosis of moderate / severe periodontitis based on the 1999 consensus classification of periodontal disease by armitage and thomas f. flemmig ( 1999 ) . inclusion criteria for cases with moderate / severe chronic periodontitis : ( 1 ) patients with at least 20 teeth and more than 30% of sites exhibiting 34 mm of mean clinical attachment loss for moderate chronic periodontitis and more than 30% of sites 5 mm of mean clinical attachment loss for severe chronic periodontitis , ( 2 ) minimum age of 20 years , and ( 3 ) patients consenting to participate . inclusion criteria for controls : ( 1 ) the absence of the clinical and radiographic manifestations of periodontal disease , ( 2 ) at least , 20 teeth present in the oral cavity and no history of periodontal treatment in the past , ( 3 ) minimum age of 20 years , and ( 4 ) patients consenting to participate . exclusion criteria : ( 1 ) patients not willing to participate in the study , ( 2 ) pregnant patients , ( 3 ) patients of rheumatic heart disease , congenital heart disease and/or with prosthetic valves who were at high risk for endocarditis , and ( 4 ) all patients medically unfit to participate in the study . mes was diagnosed based on the presence of three of the five following components using modified adult treatment panel iii criteria : ( 1 ) abdominal / central obesity ( as indicated by increased waist circumference or markedly increased body mass index [ bmi ] and defined as waist circumference with ethnicity - specific values ) : waist circumference 90 cm in men and 80 cm in women ( based on a chinese , malay , and asian indian population ) , ( 2 ) hypertriglyceridemia : 150 mg / dl ( 1.69 mmol / l ) , ( 3 ) low- and high - density lipoprotein cholesterol : < 40 mg / dl ( 1.04 mmol / l ) in men ; < 50 mg / dl ( 1.29 mmol / l ) in women , ( 4 ) high blood pressure : 130/85 mmhg , and ( 5 ) high fasting glucose : 100 mg / dl ( 6.1 spss package version 16 ( spss inc . , chicago , il , usa ) was used . the significance of difference in mean values of continuous variables between study and control group was estimated by student 's t - test . the significance of the difference in the distribution of discrete variables between the groups was estimated with chi - square test . association between chronic periodontitis and mes was estimated by calculating or with 95% confidence interval ( ci ) . the study is attempted to evaluate the association between mes and chronic periodontitis in case control study design . the cases and controls were well matched with insignificant difference for age ( mean age : 39.06 vs. 37.9 , p < 0.475 ) , sex ( male : 46.9% vs. 58.9% , p < 0.070 ) , residential background ( rural : 60.8% vs. 55.8% , p < 0.494 ) , educational status ( illiterate : 6.2% vs. 3.9% , p < 0.655 ) , and tobacco consumption status ( current tobacco consumers : 14.6% vs. 11.6% , p < 0.597 ) . the prevalence of diabetes ( 3.1% vs. 0.8% , p < 0.37 ) , hypertension ( 15.4% vs. 13.2% , p < 0.741 ) , and coronary artery disease / myocardial infarction ( 0.8% vs. 1.6% , p < . clinical characteristics of the study population clinical characteristics of the study population risk factors and atherovascular disease distribution among the study population however , the association of mes and chronic periodontitis was strong and significant with or : 2.64 , 95% ci : 1.365.18 , and p < 0.003 [ table 3 ] . comparison of mean values of components of mes and lipid parameters between cases and controls reveals that the mean waist circumference ( mean difference : 4.8 [ 95% ci : 7.751.84 ] , p < 0.002 ) , mean total cholesterol level ( mean difference : 9.43 [ 95% ci : 18.720.14 ] , p < 0.047 ) , mean triglycerides level ( mean difference : 25.75 [ 95% ci : 49.222.28 ] , p < 0.032 ) , and mean ldl level ( mean difference : 7.6 [ 14.20.99 ] , p there was no significant difference in the mean systolic blood pressure ( 2.9 [ 95% ci : 6.70.83 ] , p < 0.126 ) , diastolic blood pressure ( 1.69 [ 95% ci : 3.870.48 ] , p < 0.127 ) , and hdls levels ( 0.05% [ 95% ci : 2.432.54 ] , p < 0.963 ) in the two groups . although mean systolic blood pressure , diastolic blood pressure , and fasting blood sugar level were higher in cases ( 125.77 , 82.99 , and 86.38 , respectively ) compared with control ( 122.81 , 81.3 , and 83.68 , respectively ) , it was statistically insignificant [ table 4 ] . the observed strong association between chronic periodontitis and mes is unlikely to be influenced by the tobacco consumption pattern in the study and control groups as comparison of the distribution of tobacco consumption index in two groups was statistically insignificant [ table 5 ] . frequency and distribution of biological variables among the study population frequency and distribution of tobacco consumption index of current tobacco consumers among the subjects with or without metabolic syndrome the results of this study suggest that there is a strong association of mes and chronic periodontitis was strong and significant with or : 2.64 , 95% ci : 1.365.18 , and p < 0.003 . the association was independent of the various potential confounding risk factors affecting the chronic periodontitis such as age , gender , residential background , and tobacco consumption . chronic periodontitis has been associated with systemic alteration such as low - grade inflammation , dyslipidemia , a procoagulant state , glucose intolerance , and endothelial dysfunction . it leads to the state of the chronic systemic inflammatory state through the release of various inflammatory cytokines such as il-6 , il-1 , tnf- , and crp , which may worsen the insulin resistance . further , the production of oxidative stress - enhancing reactive oxygen species in affected periodontal tissues is a potential factor which could increase insulin resistance that may result in mes . mes is characterized by state of constellation of metabolic and biological risk factors presumed to be the result of insulin resistance which is thought to be the outcome of the complex interaction of acquired environmental factors related to lifestyle and genetic predisposition . the studies also suggested the association between mes and chronic periodontitis but the exact mechanism is not known . mes could also be a predisposing factor for chronic periodontitis and it can influence the periodontal disease process . mes is known to affect normal vascular function and it may increase the vulnerability for infection because of its predisposition to glucodysregulation . individual risk factors of mes such as obesity can affect the levels of plasminogen activator inhibitor-1 , alterations in t - cells and monocytes for macrophage and increased cytokine production , abnormally elevated cytokines il-6 and -1 and tnf- , all of which contribute to periodontitis . adipose tissue can produce cytokines and prostaglandins , which play an important role in tissue destruction during periodontal disease . in obese , hypertensive rats , hyperplasia and hypertrophy of the walls of blood vessels supplying the periodontium was observed . thus , all the individual risk factors for mes either affect the normal periodontium or the periodontal disease process . conversely , as an infectious process with a prominent inflammatory component , periodontal disease can adversely affect mes . the underlying cause - and - effect relationship needs to be explored in future interventional studies that would have important implications in management and preventive strategies of both these conditions more effectively . the finding from this study is in agreement with the findings by shimazaki et al . , which reported that mes among 584 japanese women was associated with an increased risk for periodontitis . showed that the severity of periodontal disease , as measured by average pd and average cal , and the extent of periodontal disease , as measured by the percentage of sites with cal 3 mm and the percentage of sites with pd 3 mm , were significantly greater among patients with mes compared to those without mes . it is generally accepted that the origin of metabolic disorders is in pro - inflammatory state derived from excessive caloric intake , over nutrition , and other chronic inflammatory conditions . the pro - inflammatory status also leads to an increase in oxidative stress , which has potential to impair several crucial biological mechanisms . a study has demonstrated an increase in the products of oxidative damage in peripheral blood from persons with periodontitis , which emphasized the influence of periodontitis on serum and/or plasma oxidative markers . reduced antioxidant capacity pro / anti - oxidant capacity between periodontitis and mes could add the evidence for elucidating the mechanism of the link between periodontitis and mes . by removing all confounding potential risk factors for periodontitis and mes , the strong association between the two diseases in our study was not by chance , thus disproving the null hypothesis which was also substantiated by the other previous studies . the design of the study is such that the causal relationship between periodontal diseases and mes can not be identified . attrition of 119 subjects from the study can lead to important loss of the information of the study . multiple logistic regression and multivariate analysis were not done which would have helped in finding out the correlation of the different variables in the study . other confounding variables , for example , in between snacking , lack of exercise , genetics , and hereditary were not included in the study . as the study is conducted in the individuals with more than 20 teeth , the relation between periodontal disease and mes with < 20 teeth and among edentulous remains unknown . in future , the design of the study is such that the causal relationship between periodontal diseases and mes can not be identified . attrition of 119 subjects from the study can lead to important loss of the information of the study . multiple logistic regression and multivariate analysis were not done which would have helped in finding out the correlation of the different variables in the study . other confounding variables , for example , in between snacking , lack of exercise , genetics , and hereditary were not included in the study . as the study is conducted in the individuals with more than 20 teeth , the relation between periodontal disease and mes with < 20 teeth and among edentulous remains unknown . in future , the association was independent of the various potential confounding risk factors affecting the chronic periodontitis such as age , gender , residential background , and tobacco consumption . keeping in mind this association , dental professionals in general and periodontists in particular should take mes into consideration when evaluating periodontitis .

mammary ductal carcinoma in - situ ( dcis ) , a malignant appearing lesion on cytological and histological grounds , is in fact a non - obligate precancer . dcis is difficult to manage and is sometimes treated more aggressively than invasive carcinoma . although most dcis classifications take into account the architectural growth pattern , when it comes to architecture , the literature is full of contradictory information . we examined 289 breast cancers and found dcis in 265 of the cases . the majority of the dcis cases were seen in the setting of invasive cancer and only 9% of the cases represented pure dcis with no invasive cancer . the dcis commonly displayed a mixed pattern with micropapillary , cribriform and solid components with the micropapillary type being the rarest , occurring seldom on its own . a continuum of growth with a micropapillary pattern evolving into a cribriform type could be seen in some of the cases . this may explain some of the conflicting information , in the literature , regarding the different architectural types of dcis . the comedo - pattern of necrosis could be seen in all types of dcis . we therefore conclude that the study of the determinants of growth pattern in dcis would be the key to unravelling the diverse , often non - concordant evidence one encounters in the literature .

classifying and managing dcis has always been a thorny issue , often dividing various groups of pathologists around the world . amongst dcis features , the architectural pattern , its prognostic value , and role in grading dcis the current literature on the subject accepts the existence of 3 major architectural patterns of dcis , namely , the solid , cribriform , and micropapillary patterns . it has been variably considered as an early micropapillary dcis or even a subvariant of the atypical ductal hyperplasia . other special types of dcis , such as the apocrine , the endocrine ( argyrophilic ) , and signet ring dcis , are all defined on histological criteria , rather than architectural pattern and they actually belong to the solid pattern of growth . with respect to grading , it is universally accepted that the nuclear grade is the essential feature , recurring in all classification systems previously proposed and currently in use . an association , albeit inconsistent , exists between the nuclear grade and the architectural growth pattern . it is generally accepted that most micropapillary and cribriform in situ carcinomas are of low nuclear grade and relatively indolent . however , in a recent publication by fisher and colleagues , micropapillary dcis was found to be associated with both ipsilateral and contralateral recurrence of malignancy in a statistically significant number of cases . as this result appears somewhat puzzling in the light of our present knowledge and understanding of dcis , we decided to have a new , fresh look at all cases of dcis reported during the past approximately 10 years at the department of pathology of the royal darwin hospital , nt , australia and report our findings . all cases of dcis reported at the royal darwin hospital , northern territory , australia between january 2001 and september 2010 , representing 60% of all breast cancers in the nt were retrospectively reviewed . in order to capture all the cases , including those that may have been incorrectly coded , we verified all breast tissue reports and then selected for active review all cases of dcis . the architectural and cytological aspects of dcis were assessed . the presence or absence of necrosis the 3 main types of dcis , according to the architectural growth pattern ( micropapillary , cribriform , and solid ) were assessed . 90% of the in situ tumour displayed one architectural pattern , and mixed when the dominant pattern constituted < 90% of the in situ carcinoma . nuclear grading is based on the size of malignant cells nuclei in comparison to normal ductal epithelial cells . grade 1 is applied when the nuclei of the malignant cell are between 1.5 and 2 times that of normal ductal epithelial cell . grade 2 is applied when the nuclei of the malignant cell are between 2 and 2.5 times that of normal ductal epithelial cell . grade 3 is applied when the nuclei of the malignant cell are greater than 2.5 times that of normal ductal epithelial cell . the term comedonecrosis , which is poorly defined in the literature , was applied when significant necrosis , creating an appearance similar the comedos , seen in cutaneous acne , was noted in ducts with dcis . a total of 289 breast carcinomas had been received at the royal darwin hospital during the period of the study . these consisted of 265 invasive and 24 pure in situ cancers . these cancers consisted of 231 infiltrating duct carcinomas of no special type , 24 infiltrating lobular carcinomas and 10 carcinomas of special type . of these special breast cancers , 5 were pure mucinous carcinomas , 2 were invasive papillary carcinomas , 2 were tubular carcinomas , and 1 was medullary carcinoma . the proportion of invasive ductal carcinomas containing dcis varied from year to year , ranging from 16/26 ( 62% ) in 2002 to 8/26 ( 31% ) in 2007 . table 1 also shows the frequency of pure dcis and dcis associated with invasive cancer . of the 157 cases of dcis ( pure dcis and invasive cancers with dcis ) , 91 ( 58% ) displayed a single growth pattern and 66 ( 42% ) showed a mixed growth pattern ( table 1 ) . of the 91 cases of dcis with a single growth pattern , 4 ( 5% ) were micropapillary , 23 ( 25% ) were cribriform , 61 ( 67% ) were solid , and 3 ( 3% ) were encysted papillary ( table 1 ) . of the 66 mixed type dcis cases , all 3 growth patterns ( micropapillary , cribriform , and solid ) were noted in 11 ( 17% ) of the cases . micropapillary and cribriform was present in 14 ( 21% ) , micropapillary and solid was seen in 11 ( 17% ) , and cribriform and solid was identified in 30 ( 45% ) of the cases ( table 1 ) . most of the pure in situ carcinomas were of the mixed type and there were no cases of single pattern micropapillary dcis ( table 2 ) . overall , a micropapillary pattern was seen in 40/157 ( 25% ) , a cribriform component in 78/157 ( 50% ) , a solid component in 113/157 ( 72% ) , and macropapillary ( encysted papillary carcinoma ) in 3 ( 1.9% ) of the dcis cases ( table 2 ) . comedonecrosis was present in 18/40 ( 45% ) of the micropapillary , 67/113 ( 59% ) of the solid , and 20/78 ( 26% ) of the cribriform cases of dcis . these findings show that comedonecrosis is more likely to be seen in micropapillary and solid types dcis than in the cribriform type . this association is statistically significant with p values of 0.033 and 0.00004 , respectively . at the same time , no statistically significant difference in comedonecrosis occurrence was noted between the micropapillary and solid dcis ( p = 0.117 ) . of the 157 cases of dcis high nuclear grade was recognised in 70 ( 45% ) , intermediate nuclear grade in 65 ( 41% ) , and low nuclear grade in 22 ( 14% ) of the cases ( table 3 ) . of the 40 cases of micropapillary dcis , 2 ( 5% ) were low grade , 18 ( 45% ) were intermediate grade , and 20 ( 50% ) were high grade ( table 3 ) . of the 78 cases of cribriform dcis , 17 ( 22% ) were low grade , 39 ( 50% ) were intermediate grade , and 22 ( 28% ) were high grade ( table 3 ) . of the 113 cases of solid dcis , 8 ( 7% ) were low grade , 47 ( 42% ) were intermediate grade , and 58 ( 51% ) were high grade ( table 3 ) . these results show a statistically significant difference between the presence of high nuclear grade ( grade 3 ) in the solid and micropapillary types of dcis compared to the cribriform type ( p = 0.0008 , and p = 0.019 resp . ) . on the other hand , no statistically significant nuclear grade differences were noted between the micropapillary and solid types ( p = 0.884 ) . all three cases of macropapillary ( encysted papillary carcinoma ) were low grade ( table 3 ) . overall , we identified 40 cases with a micropapillary dcis component ( table 2 ) . thirty - six of these were mixed with other growth patterns and 4 had only micropapillary growth pattern . in the cases of dcis with no invasive cancer , there was a micropapillary component in 9 cases ; all mixed with other growth patterns . of these , 4 had only a micropapillary growth and in 27 the micropapillary pattern was mixed with other growth patterns . of all the 91 cases of dcis with a single growth pattern , 4 ( 4% ) had only micropapillary growth ( table 1 ) . of the 24 cases of pure dcis , none was only micropapillary but 9 had a micropapillary component , mixed with other architectural patterns . of these , 6 cases were high grade , 2 cases were intermediate grade , and 1 case was low grade ( table 3 ) . the age of patients who had a micropapillary component ranged from 38 years to 88 years with a median of 50 years . the age range for the low grade was 52 years to 63 years , for the intermediate grade was 38 years to 88 years , and for the high grade was 40 years to 80 years . the invasive component of cases that included a micropapillary type dcis was grade 3 in 22.6% ( 7/31 ) , grade 2 in 38.7% ( 12/31 ) , and grade 1 in 38.7% ( 12/31 ) of the cases . of the 4 cases with micropapillary dcis not associated with any other type of dcis ( pure micropapillary type ) , 3 were intermediate grade and 1 was low grade . the micropapillary dcis represented only a minor component ( < 25% ) in 9/36 of the cases in which it appeared in combination with the other types of dcis . the cases had been reported by 6 pathologists , of which three were responsible for reporting 94% of cases . the overall concordance for all cases of dcis , in respect to nuclear grade , regardless of architectural pattern , was very good , with a kappa score of 0.87 . in cases with micropapillary component there was a good concordance between our evaluation and the initial report with a kappa score of 0.61 . the nonconcordant cases , which differed by 1 grade , were all between low to intermediate grade ; none involved a high grade . the concordance rate for cases not including a micropapillary pattern was also very good with a kappa score of 0.88 . it is beyond the scope of this study to chronologically recapitulate all aspects of this complex histopathological entity . regarding the micropapillary dcis , our results are surprising , but may explain partly the increased correlation with breast carcinoma recurrence identified by fisher et al . . fisher and colleagues reviewing dcis from 1456 patients enrolled in the national surgical adjuvant breast and bowel project ( nsabp ) protocol b-24 to determine predictors for ipsilateral breast tumour recurrences and contralateral breast cancers , after a median follow - up time of 10.5 years , found ductal comedonecrosis , micropapillary histological tumour type to be independent high risk factors for ipsilateral breast tumour recurrence and for contralateral breast cancers . in a recent article , castellano et al . have shown the nuclear grade to be crucial in determining the biology of micropapillary dcis . they also showed that high nuclear grade micropapillary dcis more frequently overexpressed her2 , showed a higher proliferation index , and displayed necrosis and microinvasion . logistic regression analysis confirmed high nuclear grade ( odds ratio , 6.86 ; confidence interval , 1.4033.57 ) as the only parameter associated with elevated risk of local recurrence after breast - conserving surgery . however , the recurrence rate of 19 micropapillary dcis , which were part of a cohort of 338 consecutive dcis , was significantly higher ( log - rank test , p = 0.019 ) than that of nonmicropapillary , independent of nuclear grade . the authors concluded that although nuclear grade may significantly influence the biological behaviour of micropapillary ductal carcinoma in situ , micropapillary growth pattern represents a risk factor for local recurrence after breast - conserving surgery . while the number of cases in which the micropapillary growth pattern constitutes the only pattern was very small in our study , it may not be a coincidence that none of these cases was high nuclear grade and none was associated with necrosis . this finding would be consistent with the dogma defining micropapillary dcis as a predominantly low grade dcis . the frequent association between micropapillary dcis and necrosis may explain why authors , considering comedonecrosis a separate pattern of dcis , have been discarding all those cases also displaying comedonecrosis from the micropapillary group . another notion on which all publications seem to agree is the fact that the solid variant tends to be associated with high grade dcis , whereas the micropapillary and cribriform variants most often form a low grade dcis . we believe comedocarcinoma is not a separate type of dcis and should not be used as such . in other words , each dcis should be given an architectural pattern label and the presence or absence of necrosis should be described separately . in all the cases of dcis with a pure micropapillary growth pattern , the nuclear grade was low or intermediate . this may mean that those cases of low grade dcis that may have a certain genetic makeup will maintain an indolent course , whereas the more aggressive ones will undergo the changes described above and assume a mixed growth pattern with comedonecrosis . because of this supposed evolution , the micropapillary component may represent a minor proportion of the entire dcis at the time the tissue is removed for histological evaluation . if comedocarcinoma is considered a separate type of dcis in which the architectural pattern is neglected and not reported , then our results , with only 4 cases of micropapillary dcis , are all in keeping with the old dogma that micropapillary dcis is a low or intermediate grade with no necrosis . we agree with fisher and colleagues in considering comedocarcinoma a separate feature , rather than a histological type . pinder and o'malley hit the nail on the head and explain that comedonecrosis may be seen in association with any dcis architectural type , and that it is not a type itself , as it is neither a grade nor an architectur . so they clearly recommend that the term comedo - type dcis should not be used as a characterisation of growth pattern . also in keeping with our view is the fact that the rare , special type dcis , namely , the signet ring , endocrine , and other types , also have a growth pattern that is , the solid one , even if the cells show specific our results show that over 40% ( 66/157 ) of the dcis cases are not of pure architectural type , but in fact they are mixed . approximately half of the solid ( 51% ) and micropapillary ( 50% ) cases in our series are high grade . these values are statistically significant when compared to the occurrence of high grade cribriform dcis , of which only 28% are high grade . just under half ( 45% ) of the micropapillary and nearly two - thirds ( 67% ) of the solid cases of dcis are associated with comedonecrosis , while only a quarter ( 26% ) of the cribriform cases of dcis are associated with comedonecrosis . the micropapillary type is the rarest one , occurring seldom as a pure form with less than 3% of all cases of dcis and less than 5% of dcis cases with a single pattern being pure micropapillary ones . slightly more than half ( 54% ) of the solid and almost a third ( 30% ) of the cribriform cases occur on their own , whereas in only 10% of the cases low grade micropapillary and solid dcis were very rare in our series ( 5% and 7% , resp . ) . our results can be translated into the newly accepted din system by replacing the nuclear grade value with the equivalent din . after carefully observing and analysing our data , we draw the conclusion that in many cases the pattern of growth may be a continuum , starting as micropapillary , with papillae then either joining one another to form arches resembling the cribriform pattern or even continuing to proliferate until a solid sheet of cells fills the entire lumen . as necrosis occurs toward the tips or on the sides of the micropapillary structures , the lesion becomes intermediate grade . with progression , nuclear pleomorphism and a comedo - type necrosis appear , and the lesion then qualifies as a high grade dcis . we therefore believe the study of the determinants of growth pattern in dcis would be the key to unravelling the diverse , often nonconcordant evidence one encounters in the literature .

this minireview provides current summaries of beta - blocker use in the management of hypertension and/or chronic kidney disease . accumulated evidence suggests that atenolol is not sufficiently effective as a primary tool to treat hypertension . the less - than - adequate effect of beta - blockers in lowering the blood pressure and on vascular protection , and the unfavorable effects of these drugs , as compared to other antihypertensive agents , on the metabolic profile have been pointed out . on the other hand , in patients with chronic kidney disease , renin - angiotensin system blockers are the drugs of first choice for achieving the goal of renal protection . recent studies have reported that vasodilatory beta - blockers have adequate antihypertensive efficacy and less harmful effects on the metabolic profile , and also exert beneficial effects on endothelial function and renal protection . however , there is still not sufficient evidence on the beneficial effects of the new beta - blockers .

several recent meta - analyses have questioned the usefulness of beta - blockers as the primary tools to treat hypertension [ 13 ] . this has led to hesitation in the proper use of beta - blockers in the management of hypertension and chronic kidney disease ( ckd ) . this review describes the opinion base promoting the use of beta - blockers for the treatment of hypertension and ckd . prichard classified beta - blockers into three types according to their beta1-selectivity and vasodilatory potential . an additional classification is lipophilic or hydrophilic beta - blockers [ 5 , 6 ] . atenolol is a beta1-selective agent , and it has been widely used as the control drug in large randomized prospective controlled trials of newer antihypertensive agents such as calcium channel blockers and renin - angiotensin ( ra ) system blockers . table 1 summarizes the plausible reasons why beta - blockers are considered to be relatively ineffective for the prevention of cardiovascular events [ 79 ] . in the anglo - scandinavian cardiac outcomes trial - blood pressure lowering arm ( ascot - bpla ) study , blood pressure values were lower in those allocated to the calcium channel blocker - based regimen as compared to those allocated to the beta - blocker - based regimen throughout the trial period . recently , webb et al . reported a meta - analysis in which they described visit - to - visit blood pressure instability in patients receiving beta - blocker treatment , and also that this instability was associated with an increased risk of stroke . atenolol was used in the ascot - bpla study , and not only the analysis conducted by webb et al . but some studies demonstrated that once - daily atenolol does not provide adequate blood pressure control during the night - time and early morning periods because of its pharmacokinetic profile and half - life [ 13 , 14 ] . these drug profiles of atenolol may be the cause for its relatively weak blood pressure - lowering effect and the blood pressure instability . on the other hand , metoprolol or bisoprolol have been shown to be more effective in sustaining 24-hour and early morning bp reductions as compared with atenolol [ 15 , 16 ] . in the arterial tree , the arteries branch and taper as they reach peripheral sites , associated with the increase of the arterial resistance . reflected pulse wave ( from the periphery to the heart ) occurs at sites of abrupt increase of the arterial resistance , such as at sites of arterial branching . interaction between the incident pulse wave ( from the heart to the periphery ) and reflected pulse wave ( from the periphery to the central region ) is observed in the arterial tree ( figure 1 ) ; therefore , the blood pressure values differ between central and peripheral sites of the arterial tree . central ( aortic and carotid ) blood pressure is pathophysiologically more relevant than the peripheral pressure in the pathogenesis of cardiovascular disease . augmentation index ( ai ) , a marker of the interaction of incident pressure wave and reflected pressure wave , was significantly and inversely related to heart rate due to an alteration in the relative timing of the reflected pressure wave . beta - blockers reduce the heart rate and decrease ai , which reduces their efficacy in reducing the central blood pressure as compared to other antihypertensive agents . in their meta - analysis , fagard et al . reported that beta - blockers exert a relatively weak effect in causing regression of the left ventricular mass . in fagard et al . 's review , atenolol was used in about 70% of the study subjects prescribed beta - blockers , and no study involving the use of vasodilatory beta - blockers was included . recently , the advantages of nebivolol , a vasodilatory beta - blocker , over conventional -blockers in reducing the central blood pressure and inducing regression of the left ventricular mass have been reported . compared with atenolol , nebivolol exerts a more favorable effect on 24-hour blood pressure profile . furthermore , nebivolol and telmisartan , an angiotensin ii receptor blocker , decreased the left ventricular mass to a similar degree . reported that angiotensin - converting enzyme inhibitors improve endothelial function and are superior antihypertensive agents as compared to calcium channel blockers and beta - blockers . however , in all of the studies cited in their meta - analysis , atenolol had been used as the beta - blocker . in contrast to atenolol , carvedilol and nebivolol also improve the endothelial function [ 26 , 27 ] . while the meta - analysis conducted by messerli et al . reported the unfavorable effects of beta - blockers on the metabolic profiles , this analysis did not include studies in which vasodilatory beta - blockers had been used . more recent studies have reported the relatively less harmful effects of vasodilatory beta - blockers on the metabolic profiles and also on weight gain [ 29 , 30 ] . as described above , new generations of beta - blockers , such as the long - acting and/or vasodilatory beta - blockers may overcome the relatively weak effect of beta - blockers in preventing cardiovascular events . , which was a representative analysis to evaluate the usefulness of beta - blockers in the management of hypertension , suggested that first - line beta - blocker use was not as good as other classes of antihypertensive drugs to decrease the mortality or morbidity [ 1 , 2 ] . in his review , 6070% of the subjects were receiving atenolol . as mentioned above , there is some doubt about the suitability of atenolol as a first - line antihypertensive drug , because of its low lipophilic profile and relatively weak effect on cardiovascular protection . the maphy study demonstrated the significantly lower risk for coronary events in patients on metoprolol , a lipophilic beta - blocker , as compared to those on diuretics . the usefulness of lipophilic beta - blockers for the prevention of cardiovascular events is still under debate [ 5 , 6 ] . ( total number of analyzed subjects , 91561 ) reported the higher risk for cardiovascular events in patients on beta - blockers as compared to those on diuretics . however , the number of study subjects prescribed metoprolol included in their meta - analysis was 7663 ( 8.4% ) . on the other hand , the meta - analysis conducted by turnbull et al . ( total number of study subjects for the comparison of the outcomes of major cardiovascular events ( angiotensin converting enzyme inhibitor or calcium antagonist versus beta - blocker ) was 14583 , which was calculated from figure 4 in ) demonstrated no evidence of any difference in the effect between beta - blockers and other classes of antihypertensive agents in preventing major cardiovascular events . this meta - analysis included two studies in which metoprolol alone was used in the beta - blocker arm and two other studies in which metoprolol was used as one of the beta - blockers in the beta - blocker arm . the number of study subjects prescribed metoprolol included in this meta - analysis was 10062 ( 13.5% ) . thus , the meta - analysis conducted by turnbull et al . might have included a lower number of subjects prescribed atenolol and higher number of study subjects prescribed metoprolol , as compared to the meta - analysis conducted by wiysonge et al . . then , recently , turnbull et al . suggested that lipophilic beta - blockers may be preferable to hydrophilic beta - blockers for reducing the mortality in patients with coronary artery disease , though lipophilic -blockers are associated with an increased risk of depressive symptoms . reported that the differential effects between nonatenolol beta - blockers and other antihypertensive drugs on the risk of major cardiovascular events could not be fully evaluated because of the small number of studies including subjects prescribed nonatenolol beta - blockers . anyhow , atenolol is one of the most widely used beta - blockers , and more than 50% of the data in previous meta - analyses were derived from subjects prescribed atenolol . a meta - analysis to examine the effects of lipophilic and/or vasodilatory beta - blockers on the risk of major cardiovascular events the meta - analysis conducted by bangalore et al . demonstrated an inverse relationship between beta - blocker - induced heart rate lowering and the reduction in the risk of future cardiovascular events in patients with hypertension . this heart rate lowering causes a pseudoantihypertensive effect ; that is , the central aortic pressure becomes less than the brachial pressure [ 7 , 17 , 18 ] . this phenomenon is thought to be one of mechanisms underlying the lower cardiovascular - protective effects of beta - blockers . on the other hand , the risk seems to increase as the heart rate begins to exceed 70 bpm . the aforementioned meta - analysis conducted by bangalore et al . demonstrated an inverse relationship between heart rate and cardiovascular events in subjects with heart rates under 70 bpm , and no study until the current date has examined the relationship between the heart rate and the risk for cardiovascular events in cases receiving beta - blocker treatment with heart rates over 70 bpm . furthermore , atenolol was used in more than 80% of the study subjects of the meta - analysis conducted by bangalore et al . . thus , no study has examined the association between the reduction in heart rate and the increased risk of cardiovascular events by the treatment with beta - blockers other than atenolol . our previous prospective study identified high heart rate as an independent risk factor for vascular damage ( increase in arterial stiffening ) . therefore , it has not been concluded that beta - blocker - induced heart rate lowering increases the risk of cardiovascular events in cases with heart rate over 70 bpm . in the case of patients with hypertension and a high heart rate , we have sometimes experienced good efficacy of beta - blockers for the control of both the blood pressure and the heart rate ( figure 2 ) . in addition to the prevention of cardiovascular events , renal protection is crucial in the management of ckd . recent guidelines recommended ra system blockers as the agents of first choice for the management of hypertension in patients with ckd , because of the significant renal - protective effects of this class of drugs [ 38 , 39 ] . on the other hand , the sympathetic nervous system is activated in ckd [ 40 , 41 ] , which acts as a key player in the progression of renal dysfunction and may also contribute to the onset / progression of cardiovascular disease [ 40 , 41 ] . however , reduction of the cardiac output and the consequent impairment of renal perfusion caused by beta - blockers are thought to be harmful in patients with ckd . actually , recent studies have demonstrated that only 2030% of patients with ckd are prescribed beta - blockers [ 43 , 44 ] . the kidney disease outcomes quality initiative ( k / doqi ) guideline recommend that beta - blockers be used as the third - line antihypertensive agents in patients with proteinuria . however , as mentioned above , while meta - analyses have suggested the demerits of atenolol in the management of hypertension [ 13 ] , controversial results of the renal - protective effects of atenolol as compared to those of other antihypertensive agents were reported in subjects with hypertension accompanying ckd [ 46 , 47 ] . concerning other beta - blockers , the african american study of kidney disease and hypertension study ( aask trials ) demonstrated the absence of any significant differences in the clinical composite outcomes ( renal function decline , onset of end - stage renal disease , and/or death ) between patients treated with metoprolol and those treated with amlodipine . two multicenter studies reported the renal protective effects of carvedilol , a vasodilatatory beta - blocker [ 29 , 49 ] . thus , it has not yet been concluded whether beta - blockers ( especially vasodilatory beta - blockers ) may simply represent third - line of antihypertensive agents for the management of hypertension in patients with ckd . further study is proposed to clarify whether calcium channel blockers ( especially nondihydropyridines , which reduce proteinuria ) or vasodilatory beta - blockers may be more suitable for renal protection and controlling blood pressure in patients with ckd . traditional risk factors such as hypertension , diabetes mellitus , and dyslipidemia , and nontraditional risk factors such as inflammation , oxidative stress , abnormal mineral metabolisms , hyperparathyroidism , homocysteinemia , and anemia are known to be associated with cardiovascular events in patients with ckd . the reported rate of sudden cardiac death in patients with end - stage renal disease is 50-fold higher than that in the general population . in addition to the prevalence of coronary artery disease and/or heart failure , left ventricular hypertrophy , electrolyte abnormalities such as hyperkalemia , and vascular calcification might also be associated with sudden cardiac death in patients with ckd . 's meta - analysis demonstrated that beta - blockers reduce the risk of all - cause and cardiovascular mortality in patients with ckd and systolic heart failure . basically , in their review , studies involving the use of beta - blockers other than atenolol were included , and ace inhibitors were concomitantly prescribed for most of the patients in these studies . however , their meta - analysis did not evaluate the effect of beta - blockers on sudden cardiac death . the hemodialysis ( hemo ) study suggested a trend towards the benefit of beta - blockers for the prevention of sudden cardiac death in patients with ckd and coronary heart disease , but not in ckd patients without coronary artery disease ( this study did not describe the details about the kind of beta - blockers used , e.g. , atenolol ) . this review does not support the use of beta - blockers as the primary tool to treat hypertension . however , it does propose proper use of beta - blockers , especially in cases with high heart rate and/or resistant hypertension , considering their long - acting , vasodilatory , and/or lipophilic profiles . beta - blockers are recommended as second - line agents after ra system blockers for controlling hypertension in patients with ckd and systolic heart failure . as compared to other antihypertensive agents , except ra system blockers , it has been confirmed that there are no demerits to using beta - blockers for renal protection . in addition , vasodilatory beta - blockers may also have beneficial renal - protective effects . even in patients with ckd , control of blood pressure is crucial for the prevention of cardiovascular events . while the combination of ra system blockers with diuretics is effective for reducing the blood pressure , in ckd often three or more different antihypertensive drugs are required to control blood pressure level . then , there is no evidence that beta - blockers , especially vasodilatory beta blockers , are inferior to diuretics or calcium channel blockers as second- or third - line agents for renal protection and control of blood pressure in patients with ckd .

the mutual influence of the slow rearrangements of secondary structure and fast collapse of the long single - stranded rna ( ssrna ) in approximation of coarse - grained model is studied with analytic calculations . it is assumed that the characteristic time of the secondary structure rearrangement is much longer than that for the formation of the tertiary structure . a nonequilibrium phase transition of the 2nd order has been observed .

in addition to transmitting genetic information from dna to proteins , rna molecules participate actively in a variety of cellular processes . examples are translation ( rrna , trna , and tmrna ) , editing of mrna , intracellular protein targeting , nuclear splicing of pre - mrna , and x - chromosome inactivation . the rna molecules involved in these processes do not code for proteins but act themselves as functional products . in addition , some rna molecules prepared in vitro can bind to specific molecules such as atp . in all these cases , the information encoded in the sequence of nucleotide bases of each rna molecule determines its functional tertiary structure . the forces which stabilize the secondary structure of rna are stronger than interactions responsible for the tertiary structure and hence these two structures are characterized by two different energy scales . according to one of the currently accepted concepts of rna folding the secondary structure elements , such as helices and loops hairpins , are formed first and then stack together to form a three - dimensional tertiary structure . however , some experiments show that the folding rate of large rna is lower than that predicted by the hierarchical mechanism . the landscape of the energy function of large rna is extremely rugged and contains multiple deep minima which act as kinetic traps in the folding pathways . the molecule can remain trapped in the states distinct from the native structure for time periods even longer than the average lifetime of rna in a living cell [ 3 , 4 ] . it should be noted that excellent models for describing the rna secondary structure formation have been developed as well [ 510 ] . what remains relatively poorly understood is the full path of formation of the tertiary structure and , in particular , the mutual interplay between the secondary and tertiary structures [ 1115 ] . it has been shown that the characteristic pattern of the secondary structure of rna is a tree - like structure , formed by relatively short double - stranded helices . the hierarchical folding scenario has been studied , for example , in , where the folding of rna with fixed secondary structure is described by the model of tree - like polymer with quenched random branching . in the concept of annealed randomly branched polymer has been applied to study the equilibrium characteristics of rna . the completely annealed branching patterns describe the ensemble of secondary structures , wherein the tertiary structure is being formed as a result of substantial rearrangements of secondary structure elements . this scenario is typical for large rnas , to which the hierarchical folding mechanism is most probably not applicable . while the model of a polymer with randomly annealed branching can be applied successfully for studying the equilibrium features of rna folding , it can not describe the folding kinetics efficiently . at the same time , the kinetic effects in the folding process are viewed to be of great importance due to the existence of long - living intermediates , as mentioned above . in the present study we focus on the thermodynamic behaviour of the rna molecule in the steady nonequilibrium state that occurs in case of well - defined separation between the relaxation timescales of secondary and tertiary structures . we introduce a reasonable coarse - grained model of rna and study its behavior through analytical equations . the obtained results provide evidence for the existence of a nonequilibrium phase transition of the second order between the glassy phase and the ensemble of freely fluctuating spatial structures . the ssrna molecule is dissolved initially in the solvent at temperature t which satisfies inequalities < t < tm , where tm is the melting temperature and is the flory temperature . under these conditions next , transfer a very small amount of our rna containing solution into the same kind of solvent but with the temperature t , such that t < . in the beginning , the secondary and spatial structures in this state still correspond to the temperature t but they start to relax to the new temperature t. in the end of the process , the rna will arrive at a compact globular state with some secondary structure pattern . the tertiary structure of rna is stabilized by interactions between different elements of secondary structure : helical stems , hairpins , internal loops , mismatches , and so forth ( figure 1 ) . interactions between helical stems can be considered as homogeneous since the nitrogen bases are located inside the double helix . at the same time , interactions between single - stranded regions are heterogeneous because of the interactions between nitrogen bases of different types . to describe the conformation of rna in a coarse - grained approximation , n. the center of mass of monomer i is placed at the point with coordinates xi . secondary structure is described in terms of the randomly branched polymer ( figure 2 ) as the matrix b^=bij , where bij = 1 if the ith and jth monomers are linked by helical stems and bij = 0 , otherwise [ 14 , 16 ] . . then we introduce the following hamiltonian of the model:(1)h = hii+i < jvijxixj+vconf , where the term hiixi , b^=dt/22i < jbijxi - xj2 mimics the helical spring elasticity between the ith and jth effective monomers and is the equilibrium distance between neighbouring monomers which here coincides with the mean length of the helical stem . thus , the helical spring elasticity constant is assumed to be equal to dt. here vij is the second virial coefficient of interaction between the ith and jth effective monomers , which refer to the tertiary contacts between nonpaired regions ( loops ) . the interactions between nonpaired regions ( loops ) i and j are governed by their size and nucleotide sequences ( figure 3 ) . since many nucleotides contribute to the interaction of these effective monomers i and j , it is reasonable to consider coefficients vij as statistically independent random gaussian variables with distribution(2)pvijexpvij222,where is the variance of virial coefficients vij . collapse of the ssrna molecule is driven mostly by electrostatic interactions and has been investigated experimentally [ 17 , 18 ] and theoretically . their impact is present implicitly in the confinement potential vconfxi = tv0i < jxi - xj+g0i < j < kxi - xjxk - xj , which ensures existence of globular state of the rna molecule . here v0 < 0 and g0 > 0 are the second and third virial coefficients of interactions between helical stems , correspondingly . two types of conformational rearrangements in rna are possible : rearrangement of the secondary structure with characteristic time scale 2 and tertiary structure fluctuations with characteristic time scale 3 . thus as shown in the collapse of 400-nucleotide - long rna takes about 3 - 4 ms while the two - order shorter 21-nucleotide sequence of rna folds into a hairpin in about 10 ms . the reason for this is not only the higher stability of base pairs as compared with the tertiary contacts . formation of the base pairs requires twisting of two single - stranded subchains into a double helix , which is kinetically hampered to unwind . on the timescale , such that 3 2 , secondary structure and spatial arrangement of the effective monomers ( nonpaired regions ) are not in thermal equilibrium , and this stationary nonequilibrium steady state can be described in terms of the effective partition function ( 3)z = zb^,v^nb^v^,where zb^,v^ is the partition function of a branched molecule with the given branching pattern b^ and interaction matrix v^=vij . b^ means the average over all possible branching patterns , v^ is the average over intermonomer interactions , and n = t / t. t and t are the effective temperatures of coarse - grained spatial and secondary structures correspondingly . the effective partition function ( 3 ) is calculated by using the replica technique developed for systems with quenched disorder ( see , e.g. , [ 21 , 22 ] ) . in our case the limit n 0 corresponds to the quenched disorder , n = 1 describes the completely annealed disorder , and 0 < n < 1 for the partially annealed disorder . following the method described in we rewrite the partition function ( 3 ) in the form(4)z=def,where =1/t , x=i=1na=1nxia - xa , x=x1, ,xn , and f{ } = e{ } ts{ } is the n - replica free energy with ( 5)e=avconfca24abdx dyqab2x,ysl42dxx2x. here cax=dxxxa - x is the one - replica density of monomers , qabx,y=dxxxa - xxb - y is the two - replica overlapping parameter , and is the laplace operator in the nd - dimensional space . further in all equations we set kb = 1 . the energy term in ( 5 ) is obtained by averaging the nth power of the partition function over variables vij , and the entropy term includes averaging over all possible branching patterns corresponding to the rooted tree with coordination number equal to three . for function the confinement potential is written as a virial expansion vconf(c ) = nt((1/2)v0c + ( 1/3!)g0c ) . in compactly packed chain the density of monomers c0 and vconf(c0 ) nt(v0/4 ) . like in we parameterize the offdiagonal entries of the matrix k by function k(u ) , where u [ n , 1 ] , and the diagonal entries as kaa = k. the inverse matrix m^=k^-1 is parameterized by m(u ) , u [ n , 1 ] , and maa = m~=2n2/d . by introducing notations [ k](u ) = uk(u ) ndvk(v ) and k = n(du / u)[k](u ) , the free energy functional takes a form(6)f = nvconfc0+44nn2d22dn1dum~2mu2d/2tl42dnnk2+n1duu2ku2k~2 . variation of the free energy ( 6 ) over k~ and [ k](u ) as independent variables gives that the free energy per monomer has two branches:(7)f = f0nn = tv04ld+f<,nu0f>,n > u0,where the disordered free energy is(8)f<=f<0na1tl4dz02,where n u0 , = ( 3d + 4)/(4 d ) , and a1(d , , , ) ( ) . f < = f<(t , d , , ) is the free energy of ssrna with frozen secondary structure at n u0 . at the same time , for n u0 the disordered free energy can be written as(9)f>=f>0na1u01+tl4dz02u0+11n2,where f > = f>(t , d , , ) . here the following notations are introduced : z0(d , , , ) ( ) , (d ) = ( 4 d)/8 , (d ) = ( d + 4)/(4 d ) , and u0(d ) = ( 3/2)((3(d/2 + 1)+(d + 1)(d/2 1))/(d + 2 ) ) 0.6 if d = 3 . thus , at n < u0 a glassy phase with replica - symmetry breaking is observed while at values n > u0 a replica symmetric phase is obtained . the second derivatives of the free energy branches ( 8) and ( 9 ) with respect to variable n at the point of transition n = u0 satisfy equations ( 10)2f<n2n = u0=1u02a1tl4dz022f>n2n = u0=2u02tl4dz02while the first derivatives are as follows : ( f</n)|n = u0 = ( f>/n)|n = u0 = u0(a1 tdz0 ) . n < 1 the model exhibits nonequilibrium phase transition of the second order . the temperature of transition is(11)tc=tu0 . in the framework of the proposed model the value of the parameter 1 n serves a measure of the distance from the equilibrium state , corresponding to the n = 1 . if rna molecule is far enough from the equilibrium ( n < u0 ) , then the glassy phase is realized which is dominated by a few long - lived intermediates , which were observed experimentally in . the interplay between the secondary structure formation and fast collapse of the single - stranded rna is addressed in terms of the model with interaction between heterogeneous nonpaired and homogeneous double - stranded regions . thus , the nucleotide sequence heterogeneity is approximately described in terms of statistical - mechanical model with disorder . the memory effects in rna compact structure formation are governed by slow rearrangements of the secondary structure with subsequent fast relaxation of the spatial degrees of freedom . under these conditions , monomers rapidly attain their equilibrium at the temperature t and thus , because of the wide timescale gap , their equilibrium free energy acts as a driving force pushing the slow dynamics of the elements of secondary structure to reach a nonequilibrium stationary state at long times . this scheme is known generally as the adiabatic elimination of fast variables [ 2426 ] . the observed experimentally long - lived intermediates are obtained if rna molecule is far enough away from the state of thermodynamic equilibrium .

technological advances in computing , imaging and genomics have created new opportunities for exploring relationships between histology , molecular events and clinical outcomes using quantitative methods . slide scanning devices are now capable of rapidly producing massive digital image archives that capture histological details in high - resolution . commensurate advances in computing and image analysis algorithms enable mining of archives to extract descriptions of histology , ranging from basic human annotations to automatic and precisely quantitative morphometric characterization of hundreds of millions of cells . these imaging capabilities represent a new dimension in tissue - based studies , and when combined with genomic and clinical endpoints , can be used to explore biologic characteristics of the tumor microenvironment and to discover new morphologic biomarkers of genetic alterations and patient outcomes . in this paper we review developments in quantitative imaging technology and illustrate how image features can be integrated with clinical and genomic data to investigate fundamental problems in cancer . using motivating examples from the study of glioblastomas ( gbms ) , we demonstrate how public data from the cancer genome atlas ( tcga ) can serve as an open platform to conduct in silico tissue based studies that integrate existing data resources . we show how these approaches can be used to explore the relation of the tumor microenvironment to genomic alterations and gene expression patterns and to define nuclear morphometric features that are predictive of genetic alterations and clinical outcomes . challenges , limitations and emerging opportunities in the area of quantitative imaging and integrative analyses are also discussed .

visual information embedded in histologic specimens carries prognostic value and reflects the underlying molecular traits of disease . human evaluation of histology is a time - honored practice and serves as the basis of modern pathology , yet is highly subjective and known for its inter- and intra - observer variations ( 1 ) . human observers are also limited by scale and the need to reduce information into summary categorical descriptions . diagnostic evaluation of histologic specimens is often performed over a prescribed number of high - power fields , and reasonable reporting can not possibly capture detailed descriptions of the tissue heterogeneities observed in many diseases . the digitization of pathologic specimens has advanced with improvements in charge - coupled device ( ccd ) sensors , storage and network performance . early versions of slide scanning hardware suffered from slow image acquisition , and their practical use was limited by the expense of storage and network limitations that made file transfer and remote viewing difficult . contemporary slide scanning devices are now capable of digitizing a single slide at 40x objective magnification in two minutes or less , and can produce hundreds of whole - slide images ( wsis ) in a single day . with each image occupying hundreds of megabytes to several gigabytes , the recent precipitous decline in storage costs in the past decade has made generation and analysis of large wsi archives more practical . faster networks and improved software have enabled users to fluidly view and interact with large wsi archives at their desktop by streaming imaging data directly from remote servers . currently , no universal standards exists for file format or image compression within a wsi , despite some work by the dicom working - group 26 , creating significant challenges in the interoperability of various hardware and software platforms from different wsi vendors . improvements in computing performance and image analysis algorithms enable large wsi archives to be mined to extract quantitative and objective imaging features that describe the visual characteristics of tissue architecture and microanatomy ( 2 , 3 ) . advances in the theory of image analysis algorithms make it possible to reliably delineate objects across biological scales from cell nuclei and membranes ( where stained ) to complex multicellular structures and tissue interfaces ( 416 ) . with these objects delineated , a set of descriptive features can be calculated to describe their appearance including shape , texture , and spatial relation to one another . new computing hardware like multi - core processors and graphics cards enable these techniques to be scaled to wsi archives that can contain billions of such objects . a collection of algorithms has even emerged to mitigate technical effects introduced by the physical processing of tissues , allowing the automatic detection of artifacts , and the correction of color differences caused by variations in section thickness and staining ( 1722 ) . these procedures improve the robustness of image segmentation processes and result in uniform features that reflect biological properties , while reducing noise introduced by technical artifacts . the size in bytes of features extracted from an image can rival that of the image itself , and the management and standardization of image features and their provenance is not trivial . image analysis algorithms that precisely describe microscopic features within pathologic specimens provide tremendous opportunities for integration with genomic analyses and a new platform for advancing genotype - phenotype comparisons . contemporary genomic platforms have generated a new view of the genetic , transcriptional and epigenetic events that are embedded within tissue samples . deep molecular characterizations of biospecimens are increasingly available and gaining clinical relevance and the complementary nature of genomic and quantitative imaging descriptions creates new opportunities for their integrated analysis . genomics provide extremely high molecular resolution but poor spatial resolution , and the genomic signature of a specimen therefore represents an aggregate measure of heterogeneous molecular profiles within distinct components of the tissue analyzed . laser capture microdissection provides a way to increase the purity of genomic measurements , but is labor - intensive and difficult to carry out on large cohorts , although image analysis has been used to reduce this burden ( 23 ) . an alternative approach is the integration of genomic and imaging features through computational means to deconvolve distinct profiles from the aggregate profile , with the goal of recovering information that is lost when tissue is homogenized for genomic analysis . histology is also a manifestation of underlying molecular profiles within tissues , so quantitative imaging features can be expected to contain predictive power as biomarkers of genetic alterations and gene expression patterns . by integrating imaging and genomic features into risk models , the availability of large de - identified data - sets from the cancer genome atlas ( tcga ) has greatly facilitated integrated analyses that use imaging , genomic and clinical data . this well - characterized and comprehensive data set would be difficult to duplicate at a single institution due to prohibitive cost , privacy concerns , and patient volumes . tcga is a large public resource that provides comprehensive molecular characterizations of more than 22 cancers types . although intended primarily as a genomic resource , tcga contains over 22,000 whole - slide images from more than 10,000 tumors , in addition to detailed clinical descriptions , and serves as an open platform to perform studies that integrate quantitative histology with molecular and clinical data . the use of these existing resources to conduct in silico scientific investigations has enabled researchers in this area to focus effort on developing analysis methods rather than data production , and to scale studies to a number of samples that would be otherwise difficult to achieve ( fig 1 ) . while tcga is an exceptional resource at this point in time , such multifaceted descriptions of tissues will likely become more commonplace within academic research institutions with increasing clinical adoption of genomics and digital pathology , and as the information management systems that manage these data improve . in this paper we present a review of developments in the area of quantitative histology , using examples from glioblastoma to illustrate how imaging features can be integrated with genomic and clinical data to improve understanding . the first example explores issues of tumor microenvironment ( tme ) , and how imaging features can illuminate the impact of the tme on the genomic signatures and molecular classifications . in the second example we present a pipeline for the morphometric characterization of nuclei that is capable of extracting quantitative descriptions of billions of cell nuclei in digital wsi archives . we show how this pipeline can be used along with statistical and statistical learning techniques to define imaging biomarkers of genetic alterations and epigenetic and transcriptional patterns , as well as clinical outcomes . we finish by describing near - term potential opportunities for quantitative imaging and integrated studies , and discuss the limitations and challenges associated with these approaches . the cancer genome atlas ( tcga ) was established in 2005 to improve understanding of the molecular basis of human cancers through large - scale genomic analysis . with a goal of accruing 500 tumors for each cancer selected for study , tcga has expanded beyond initial pilot projects in glioblastoma , lung and ovarian carcinoma to now span more than 22 tumor types . this effort relies on a pipeline of participating institutions that submit frozen tissues and clinical data to a central repository , a set of de - centralized genomic analysis centers that produce messenger rna , micro rna , dna exome sequencing , dna copy number , dna methylation , and protein expression profiles , and an electronic clearinghouse that then makes this data available to the public ( https://tcga-data.nci.nih.gov ) . an important , yet underappreciated aspect of acquiring clinical data from tissue source sites includes the collection of digitized whole - slide images of submitted tumors . frozen sections are produced from the top and bottom of tissue samples that are submitted for genomic analysis , and are used for quality control to evaluate the percentage of tumor , the presence of necrosis and other factors that will influence the quality of genomic results . these images are a valuable resource since they are immediately adjacent to tissues used for genomics , and provide the most faithful representation of genomic - annotated tissues . the higher quality of these images and lack of freezing artifacts makes them more suitable for algorithmic analysis , particularly at high magnification . expert pathology committees that are selected by disease area review these permanent sections to ensure correct diagnosis and to evaluate the presence of important pathologic criteria . examples from the gbm project include the categorical scoring ( 0 , 1 + , 2 + ) of qualities like microvascular proliferation , pseudopalisading necrosis and lymphocytic infiltration . all permanent and frozen sections are digitized at 20x or 40x objective magnification and made publicly available for download . one of the main outcomes of the tcga analyses has been the development of genomic sub - classifications of many cancers . using clustering analysis of gene expression and other molecular platforms , the goal of these analyses is to define cohesive sub - classes of tumors with distinct molecular signatures that may benefit from class - specific targeted therapies . in glioblastoma , two studies using tcga data have identified tumor sub - classifications based on gene expression and dna methylation ( 24 , 25 ) . the initial tcga analysis of gbms identified four gene expression classes ( gess ) : proneural , neural , classic and mesenchymal . these classes exhibit clear and distinct patterns of gene expression , and are highly correlated with genetic alterations in egfr , idh1 , nf1 , pdgfra , and tp53 . a subsequent analysis of dna methylation data revealed that pro - neural gbms are further subdivided into two groups - those with idh mutations that have significant hypermethylation of cpg islands ( gcimp ) and are typically secondary gbms afflicting younger patients , and idh wildtype tumors that do not exhibit dna hypermethylation patterns . one of the first goals of our in silico research was to investigate the relationships between gene expression classifications and the tumor microenvironment in gbms ( 26 , 27 ) . most tissue - based transcriptional classification studies of tumors are subjective in that neoplasms are highly heterogeneous , and gene expression measurements can vary significantly among different samples from the same tumor . glioblastomas are no exception , being spatially complex tumors that harbor a variety of non - neoplastic cell types and microenvironmental elements that can significantly impact gene expression measurements . pseudopalisading necrosis and micro - vascular proliferation are perhaps the most notable elements , being part of the diagnostic criteria that distinguish glioblastomas from lower - grade gliomas , and indicators of poor prognosis ( 28 ) . the development of necrosis and microvascular proliferation is can be focal at first , but then expands , and signals severe underlying hypoxia with resultant profound transcriptional changes . having access to both frozen sections and comprehensive molecular profiles from adjacent tissues from tcga , we sought to measure the impact of necrosis and angiogenesis on gene expression patterns used to classify gbms . we hypothesized the extent of necrosis and angiogenesis in a histologic section are tightly associated with the presence of hypoxia , which could play an important role in establishing ges signatures by activation of hypoxia - inducible transcription factors . with the degree of hypoxia varying spatially throughout a tumor , multiple ges classes could possibly co - exist within the same tumor , and so classification by gene expression could be subject to random effects in tissue sampling . intra - tumoral variations in ges classification would have significant implications in using these classes as platforms for the development of targeted therapies . using a human - computer interface , we annotated 177 digitized frozen section images to define the boundaries of necrosis and angiogenesis for 99 tumors . the sections in these images are immediately adjacent to those used for genomic analysis , and these annotations therefore provide the most faithful representation of the microenvironmental conditions in genomically analyzed tissues ( fig 2a ) . the extent of necrosis and angiogenesis was calculated as a percentage of the total tissue area , and these quantitative features were linked to gene expression and other genomic measurements from the same tissue ( fig 2b ) . we first examined the abundance of necrosis and angiogenesis in tumors organized by tcga transcriptional class . tumors with a mesenchymal ges were clearly enriched with higher amounts of necrosis ( one - way anova p = 8.7e-4 , see fig 2c ) , suggesting a strong association between the mesenchymal gene - expression signatures , necrosis , and hypoxia . there were only 4 outliers with much higher levels of angiogensis and 3 were from the mesenchymal ges and 1 was from the proneural ges . while we would expect the presence of angiogenesis to influence gene expression , the ability to detect this feature may in part be limited by the relatively small contribution these regions make to the total amount of dna / rna extracted for analysis . next we performed a genome - wide analysis of transcriptional data to discover the impact of necrosis on gene expression . a normalized linear regression coefficient was calculated for each transcript to measure the strength of relationship between extent of necrosis and gene expression for more than 22,000 transcripts measured with affymetrix arrays . significance analysis of microarrays ( sam ) correction was applied to obtain multiple - test corrected p - values for each gene ( 29 ) . this analysis identified 2422 genes that are significantly correlated with extent of necrosis at 5% false - discovery rate or below , suggesting that necrosis has tremendous influence on gene expression in gbms . among the genes most significantly correlated with necrosis were a set of transcription factors known as mesenchymal master regulators : cebpb , fosl2 , cebpd , stat3 , bhlhe40 ( ranked 4 , 10 , 60 , 213 and 221 respectively ) . these transcription factors have been shown to form a small module regulating a much broader gene expression network that is responsible for mesenchymal tumor phenotype in glioblastomas ( 30 ) . at the top of this regulatory module are the transcription factors ce - bpb / cebpd and stat3 , whose coexpression is necessary and sufficient for activating the mesenchymal expression network . to explore the expression of these regulators in tissues , we performed immunohistochemistry on archived surgically resected glioblastomas from our own institution . we observed that cebpb / cebpd expression was strongly and specifically expressed in the hypoxic pseudopalisading cells surrounding areas of necrosis ( fig 1d ) . cebpb was strongly expressed in nuclei of the first 25 cell layers immediately surrounding necrosis , and cebpd expression was found in both nuclear and cytoplasmic regions of perinecrotic cells but extending slightly farther beyond cebpd . in regions between foci of necrosis , gene expression classifications are made by measuring the distances in gene expression space between a tumor s expression profile and a set of points or centroids that represent each class . the tumor is assigned to the class with the nearest centroid , which can be measured using a variety of metrics including simple euclidean distance . while a given tissue could potentially contain individual cells / regions with diverging gene expression profiles , these classifications force a selection of a single gene expression class that best defines the entire sample . to explore how the formation of necrosis influences the expression patterns of non - mesenchymal gbms , we examined the relationship between extent of necrosis and distance to the mesenchymal expression centroid in this cohort . we observed a clear trend the more necrosis that a sample contains , the more its expression profile resembles the mesenchymal centroid ( fig 1e ) . this finding further suggests that mesenchymal gene expression is strongly impacted by hypoxia and that expression signatures are strongly impacted by regionally varying elements of the microenvironment . the morphologic characteristics of cell nuclei convey important clinical information in many types of neoplasms . besides determining histologic classification and subtype , nuclear qualities including shape , texture and spatial arrangement can be indicative of more specific molecular alterations and patient prognosis . gains , losses and rearrangements of dna along with epigenetic modifications affecting chromatin structure can manifest in observable changes within nuclei of neoplastic cells . in the diffuse gliomas , nuclear features are of particular importance , as their classification of oligodendroglioma or astocytoma is based in large part on nuclear morphology . however , histopathologic classification based on human review is subjective and prone to substantial interobserver variation . understanding the relationships between nuclear morphology , tumor genetics and clinical outcomes will provide a better understanding of tumor biology and further improve the precision of clinical predictions . our studies of tumor microenvironment used human markups and annotations to generate quantitative features from whole - slide images . the limitations of human annotations are apparent when dealing with nuclear morphology - nuclei can number in the hundreds of millions in even a modestly sized set of images , and qualities of interest like nuclear texture are difficult to accurately characterize objectively by human observers . to address these challenges we have developed a computational system for the study of nuclear morphometry in large archives of whole - slide images ( fig 3a ) . this system uses image analysis algorithms to delineate individual cell nuclei , and to calculate a set of objective nuclear features for each nucleus to describe its shape and texture . high performance and parallel computing approaches are used to scale this approach to hundreds of millions of cells . this system presents opportunities to define quantitative morphologic biomarkers of molecular and clinical endpoints by enabling the extraction of objective , repeatable measurements from wsi archives . our initial morphometric study focused on the quantitative characterization of oligodendroglial differentiation in glioblastomas ( 31 ) . although gbm is defined as a grade iv astrocytoma , an important subset exhibits varying degrees of oligodendroglial differentiation in addition to the dominant astrocytic component ( 28 , 3234 ) . neoplasms with pure oligodendroglial differentiation typically have slower growth and better survivals when compared with astrocytomas of the same grades . the morphologic characteristics of oligodendrogliomas distinguish them from astrocytomas : oligodendroglial nuclei tend to be smaller , round and hyperchromatic with a lack of detailed texture , in contrast with astrocytoma nuclei that are larger , irregularly shaped , typically elongated and unevenly textured . in most instances , gbms contain a heterogeneous mixture of neoplastic cells with wide variations in nuclear characteristics , many of which are not clearly astrocytic or oligodendroglial . the volume and heterogeneity of cells present in gbms combined with subtle differences in morphologic diversity make them an ideal candidate for computational morphometric approaches . using our computational pipeline , we analyzed 200 million nuclei from digitized images of diagnostic slides corresponding to 117 tcga gbms . twenty - three quantitative features from four categories ( shape , intensity , texture and gradient ) were calculated to describe each nucleus . to represent the differentiation of each nucleus along the oligodendroglial / astrocytic spectrum , we built a regression model that uses the nuclear feature values to calculate a score for each nucleus representing its degree of oligodendroglial appearance ( fig 3b ) . combining the 200 million scores obtained from our pipeline with gene expression , copy number , dna sequence and methylation data from the same tcga tumors , we were able to clearly separate a set of tumor enriched with oligodendroglial - like cells that had strong associations with pdgfra amplification , proneural transcriptional class , and expression of the oligodendrocyte signature genes mbp , hoxd1 , plp1 , mobp and pdgfra . these results provide molecular validation that the quantitative features extracted by our software pipeline can capture the morphologic variations of nuclei encountered in gliomas . our differentiation study used a supervised approach to build a quantitative model of the oligodendroglial / astrocytic spectrum in gliomas . model - based approaches are a powerful way to incorporate prior knowledge into morphologic analyses , and to use quantitative measures of recognized morphologic patterns to explore their molecular correlates . because model - based approaches are built on prior knowledge , their ability to reveal previously unrecognized or unknown morphologic patterns is limited . to address this limitation , we have developed several unsupervised or model - free approaches that do not impose established constructs in the morphological analysis of wsi data . instead , these approaches let data speak for itself , using clustering analysis and other statistical learning techniques to reveal natural structure within the feature data in a bottom - up fashion . our first study with unsupervised methods investigated patient clustering of gbms into morphologically defined subtypes ( 35 , 36 ) . using nuclear features , we sought to determine if there are clear and distinct groups of tumors that emerge from clustering analysis , similar to gene expression studies where transcriptional profiles are clustered to reveal molecular tumor subtypes . taking the nuclear features from the tcga cohort , a morphologic signature was calculated for each tumor to represent the morphologic properties of its average nuclei . these signatures were analyzed using a consensus - clustering algorithm to find natural groups within the data and to measure their robustness . three clear clusters emerged from this analysis and we named them for themes observed in their molecular correlates : cell cycle ( cc ) , protein biosynthesis ( pb ) and chromatin modification ( cm ) . we observed that these clusters had significant differences in patient survival ( logrank p=1.4e-3 ) , with the pb cluster containing patients with relatively better outcomes and the cm cluster relatively worse . these clusters were also observed in an independent dataset of 84 gbms where the relative differences in outcomes between the clusters were also confirmed . to explore the meaning of these clusters we used the various genomic platforms made available by tcga including gene expression , dna methylation , copy number and dna sequencing . a pathway analysis found that the clusters varied in the extent of tp53 wnt , and nfkb signaling , and had variations in the extent of total dna methylation . an analysis of the pathologic features using categorical human annotations ( 0,1+,2 + ) found that tumors in the cm cluster had enriched presence of lymphocytes , and that pb cluster tumors exhibited a conspicuous lack of inflammation . to further explore model - free associations of nuclear morphometry in gbm and clinical and genomic endpoints , we took a more direct approach of correlating raw nuclear features with genomic and clinical endpoints ( 37 ) . for each patient , we calculated the mean and standard deviation of each feature as metrics and correlating them directly with patient survival using cox proportional hazards analysis using sam . notably , the mean circularity was significantly associated with longer patient survival , an observation consistent with prolonged clinical outcomes in gliomas with oligodendroglial differentiation . other features that were significantly associated with outcome include major axis length , with longer nuclei associated with a shorter survival , and min nuclear pixel intensity , with higher values associated with longer survival . the fact that these features emerged from a more data - driven approach provides some level of confidence in our analysis workflow . to correlate these features with genomic measurements we performed a one - way anova for each feature metric across transcriptional classifications , somatic mutations and dna copy number alterations . features distinguishing transcriptional classes include nuclear eccentricity ( p = 3.81e-4 ) , minor axis length ( p = 8.87e-3 ) and nuclear extent ( p = 3.2e-2 ) . those hypermethylated ( gcimp ) tumors within the proneural group had greater variation of pixel intensities within their nuclei ( nuclear energy , p = 2.28e-5 ) , and greater variation in nuclear size . genetic events having significant differences in nuclear morphometry included pten and tp53 mutations , and pdg - fra amplification . pten and tp53 mutant tumors were both associated with less circular nuclei ( p = 9.68e-3 , 3.77e-2 respectively ) . pdgfra amplified tumors were associated with greater circularity ( p = 2.31e-2 ) , consistent with pdgfra amplifications being associated with oligodendroglial differentiation . other genetic alterations with significant associations included egfr amplification , which was associated with greater nuclear eccentricity and canny , and mdm2 amplifications , which were associated with greater minor axis length , area and circularity . the cancer genome atlas ( tcga ) was established in 2005 to improve understanding of the molecular basis of human cancers through large - scale genomic analysis . with a goal of accruing 500 tumors for each cancer selected for study , tcga has expanded beyond initial pilot projects in glioblastoma , lung and ovarian carcinoma to now span more than 22 tumor types . this effort relies on a pipeline of participating institutions that submit frozen tissues and clinical data to a central repository , a set of de - centralized genomic analysis centers that produce messenger rna , micro rna , dna exome sequencing , dna copy number , dna methylation , and protein expression profiles , and an electronic clearinghouse that then makes this data available to the public ( https://tcga-data.nci.nih.gov ) . an important , yet underappreciated aspect of acquiring clinical data from tissue source sites includes the collection of digitized whole - slide images of submitted tumors . frozen sections are produced from the top and bottom of tissue samples that are submitted for genomic analysis , and are used for quality control to evaluate the percentage of tumor , the presence of necrosis and other factors that will influence the quality of genomic results . these images are a valuable resource since they are immediately adjacent to tissues used for genomics , and provide the most faithful representation of genomic - annotated tissues . the higher quality of these images and lack of freezing artifacts makes them more suitable for algorithmic analysis , particularly at high magnification . expert pathology committees that are selected by disease area review these permanent sections to ensure correct diagnosis and to evaluate the presence of important pathologic criteria . examples from the gbm project include the categorical scoring ( 0 , 1 + , 2 + ) of qualities like microvascular proliferation , pseudopalisading necrosis and lymphocytic infiltration . all permanent and frozen sections are digitized at 20x or 40x objective magnification and made publicly available for download . one of the main outcomes of the tcga analyses has been the development of genomic sub - classifications of many cancers . using clustering analysis of gene expression and other molecular platforms , the goal of these analyses is to define cohesive sub - classes of tumors with distinct molecular signatures that may benefit from class - specific targeted therapies . in glioblastoma , two studies using tcga data have identified tumor sub - classifications based on gene expression and dna methylation ( 24 , 25 ) . the initial tcga analysis of gbms identified four gene expression classes ( gess ) : proneural , neural , classic and mesenchymal . these classes exhibit clear and distinct patterns of gene expression , and are highly correlated with genetic alterations in egfr , idh1 , nf1 , pdgfra , and tp53 . a subsequent analysis of dna methylation data revealed that pro - neural gbms are further subdivided into two groups - those with idh mutations that have significant hypermethylation of cpg islands ( gcimp ) and are typically secondary gbms afflicting younger patients , and idh wildtype tumors that do not exhibit dna hypermethylation patterns . one of the first goals of our in silico research was to investigate the relationships between gene expression classifications and the tumor microenvironment in gbms ( 26 , 27 ) . most tissue - based transcriptional classification studies of tumors are subjective in that neoplasms are highly heterogeneous , and gene expression measurements can vary significantly among different samples from the same tumor . glioblastomas are no exception , being spatially complex tumors that harbor a variety of non - neoplastic cell types and microenvironmental elements that can significantly impact gene expression measurements . pseudopalisading necrosis and micro - vascular proliferation are perhaps the most notable elements , being part of the diagnostic criteria that distinguish glioblastomas from lower - grade gliomas , and indicators of poor prognosis ( 28 ) . the development of necrosis and microvascular proliferation is can be focal at first , but then expands , and signals severe underlying hypoxia with resultant profound transcriptional changes . having access to both frozen sections and comprehensive molecular profiles from adjacent tissues from tcga , we sought to measure the impact of necrosis and angiogenesis on gene expression patterns used to classify gbms . we hypothesized the extent of necrosis and angiogenesis in a histologic section are tightly associated with the presence of hypoxia , which could play an important role in establishing ges signatures by activation of hypoxia - inducible transcription factors . with the degree of hypoxia varying spatially throughout a tumor , multiple ges classes could possibly co - exist within the same tumor , and so classification by gene expression could be subject to random effects in tissue sampling . intra - tumoral variations in ges classification would have significant implications in using these classes as platforms for the development of targeted therapies . using a human - computer interface , we annotated 177 digitized frozen section images to define the boundaries of necrosis and angiogenesis for 99 tumors . the sections in these images are immediately adjacent to those used for genomic analysis , and these annotations therefore provide the most faithful representation of the microenvironmental conditions in genomically analyzed tissues ( fig 2a ) . the extent of necrosis and angiogenesis was calculated as a percentage of the total tissue area , and these quantitative features were linked to gene expression and other genomic measurements from the same tissue ( fig 2b ) . we first examined the abundance of necrosis and angiogenesis in tumors organized by tcga transcriptional class . tumors with a mesenchymal ges were clearly enriched with higher amounts of necrosis ( one - way anova p = 8.7e-4 , see fig 2c ) , suggesting a strong association between the mesenchymal gene - expression signatures , necrosis , and hypoxia . there were only 4 outliers with much higher levels of angiogensis and 3 were from the mesenchymal ges and 1 was from the proneural ges . while we would expect the presence of angiogenesis to influence gene expression , the ability to detect this feature may in part be limited by the relatively small contribution these regions make to the total amount of dna / rna extracted for analysis . next we performed a genome - wide analysis of transcriptional data to discover the impact of necrosis on gene expression . a normalized linear regression coefficient was calculated for each transcript to measure the strength of relationship between extent of necrosis and gene expression for more than 22,000 transcripts measured with affymetrix arrays . significance analysis of microarrays ( sam ) correction was applied to obtain multiple - test corrected p - values for each gene ( 29 ) . this analysis identified 2422 genes that are significantly correlated with extent of necrosis at 5% false - discovery rate or below , suggesting that necrosis has tremendous influence on gene expression in gbms . among the genes most significantly correlated with necrosis were a set of transcription factors known as mesenchymal master regulators : cebpb , fosl2 , cebpd , stat3 , bhlhe40 ( ranked 4 , 10 , 60 , 213 and 221 respectively ) . these transcription factors have been shown to form a small module regulating a much broader gene expression network that is responsible for mesenchymal tumor phenotype in glioblastomas ( 30 ) . at the top of this regulatory module are the transcription factors ce - bpb / cebpd and stat3 , whose coexpression is necessary and sufficient for activating the mesenchymal expression network . to explore the expression of these regulators in tissues , we performed immunohistochemistry on archived surgically resected glioblastomas from our own institution . we observed that cebpb / cebpd expression was strongly and specifically expressed in the hypoxic pseudopalisading cells surrounding areas of necrosis ( fig 1d ) . cebpb was strongly expressed in nuclei of the first 25 cell layers immediately surrounding necrosis , and cebpd expression was found in both nuclear and cytoplasmic regions of perinecrotic cells but extending slightly farther beyond cebpd . in regions between foci of necrosis , gene expression classifications are made by measuring the distances in gene expression space between a tumor s expression profile and a set of points or centroids that represent each class . the tumor is assigned to the class with the nearest centroid , which can be measured using a variety of metrics including simple euclidean distance . while a given tissue could potentially contain individual cells / regions with diverging gene expression profiles , these classifications force a selection of a single gene expression class that best defines the entire sample . to explore how the formation of necrosis influences the expression patterns of non - mesenchymal gbms , we examined the relationship between extent of necrosis and distance to the mesenchymal expression centroid in this cohort . we observed a clear trend the more necrosis that a sample contains , the more its expression profile resembles the mesenchymal centroid ( fig 1e ) . this finding further suggests that mesenchymal gene expression is strongly impacted by hypoxia and that expression signatures are strongly impacted by regionally varying elements of the microenvironment . the morphologic characteristics of cell nuclei convey important clinical information in many types of neoplasms . besides determining histologic classification and subtype , nuclear qualities including shape , texture and spatial arrangement can be indicative of more specific molecular alterations and patient prognosis . gains , losses and rearrangements of dna along with epigenetic modifications affecting chromatin structure can manifest in observable changes within nuclei of neoplastic cells . in the diffuse gliomas , nuclear features are of particular importance , as their classification of oligodendroglioma or astocytoma is based in large part on nuclear morphology . however , histopathologic classification based on human review is subjective and prone to substantial interobserver variation . understanding the relationships between nuclear morphology , tumor genetics and clinical outcomes will provide a better understanding of tumor biology and further improve the precision of clinical predictions . our studies of tumor microenvironment used human markups and annotations to generate quantitative features from whole - slide images . the limitations of human annotations are apparent when dealing with nuclear morphology - nuclei can number in the hundreds of millions in even a modestly sized set of images , and qualities of interest like nuclear texture are difficult to accurately characterize objectively by human observers . to address these challenges we have developed a computational system for the study of nuclear morphometry in large archives of whole - slide images ( fig 3a ) . this system uses image analysis algorithms to delineate individual cell nuclei , and to calculate a set of objective nuclear features for each nucleus to describe its shape and texture . high performance and parallel computing approaches are used to scale this approach to hundreds of millions of cells . this system presents opportunities to define quantitative morphologic biomarkers of molecular and clinical endpoints by enabling the extraction of objective , repeatable measurements from wsi archives . our initial morphometric study focused on the quantitative characterization of oligodendroglial differentiation in glioblastomas ( 31 ) . although gbm is defined as a grade iv astrocytoma , an important subset exhibits varying degrees of oligodendroglial differentiation in addition to the dominant astrocytic component ( 28 , 3234 ) . neoplasms with pure oligodendroglial differentiation typically have slower growth and better survivals when compared with astrocytomas of the same grades . the morphologic characteristics of oligodendrogliomas distinguish them from astrocytomas : oligodendroglial nuclei tend to be smaller , round and hyperchromatic with a lack of detailed texture , in contrast with astrocytoma nuclei that are larger , irregularly shaped , typically elongated and unevenly textured . in most instances , gbms contain a heterogeneous mixture of neoplastic cells with wide variations in nuclear characteristics , many of which are not clearly astrocytic or oligodendroglial . the volume and heterogeneity of cells present in gbms combined with subtle differences in morphologic diversity make them an ideal candidate for computational morphometric approaches . using our computational pipeline , we analyzed 200 million nuclei from digitized images of diagnostic slides corresponding to 117 tcga gbms . twenty - three quantitative features from four categories ( shape , intensity , texture and gradient ) were calculated to describe each nucleus . to represent the differentiation of each nucleus along the oligodendroglial / astrocytic spectrum , we built a regression model that uses the nuclear feature values to calculate a score for each nucleus representing its degree of oligodendroglial appearance ( fig 3b ) . combining the 200 million scores obtained from our pipeline with gene expression , copy number , dna sequence and methylation data from the same tcga tumors , we were able to clearly separate a set of tumor enriched with oligodendroglial - like cells that had strong associations with pdgfra amplification , proneural transcriptional class , and expression of the oligodendrocyte signature genes mbp , hoxd1 , plp1 , mobp and pdgfra . these results provide molecular validation that the quantitative features extracted by our software pipeline can capture the morphologic variations of nuclei encountered in gliomas . our differentiation study used a supervised approach to build a quantitative model of the oligodendroglial / astrocytic spectrum in gliomas . model - based approaches are a powerful way to incorporate prior knowledge into morphologic analyses , and to use quantitative measures of recognized morphologic patterns to explore their molecular correlates . because model - based approaches are built on prior knowledge , their ability to reveal previously unrecognized or unknown morphologic patterns is limited . to address this limitation , we have developed several unsupervised or model - free approaches that do not impose established constructs in the morphological analysis of wsi data . instead , these approaches let data speak for itself , using clustering analysis and other statistical learning techniques to reveal natural structure within the feature data in a bottom - up fashion . our first study with unsupervised methods investigated patient clustering of gbms into morphologically defined subtypes ( 35 , 36 ) . using nuclear features , we sought to determine if there are clear and distinct groups of tumors that emerge from clustering analysis , similar to gene expression studies where transcriptional profiles are clustered to reveal molecular tumor subtypes . taking the nuclear features from the tcga cohort , a morphologic signature was calculated for each tumor to represent the morphologic properties of its average nuclei . these signatures were analyzed using a consensus - clustering algorithm to find natural groups within the data and to measure their robustness . three clear clusters emerged from this analysis and we named them for themes observed in their molecular correlates : cell cycle ( cc ) , protein biosynthesis ( pb ) and chromatin modification ( cm ) . we observed that these clusters had significant differences in patient survival ( logrank p=1.4e-3 ) , with the pb cluster containing patients with relatively better outcomes and the cm cluster relatively worse . these clusters were also observed in an independent dataset of 84 gbms where the relative differences in outcomes between the clusters were also confirmed . to explore the meaning of these clusters we used the various genomic platforms made available by tcga including gene expression , dna methylation , copy number and dna sequencing . a pathway analysis found that the clusters varied in the extent of tp53 wnt , and nfkb signaling , and had variations in the extent of total dna methylation . an analysis of the pathologic features using categorical human annotations ( 0,1+,2 + ) found that tumors in the cm cluster had enriched presence of lymphocytes , and that pb cluster tumors exhibited a conspicuous lack of inflammation . to further explore model - free associations of nuclear morphometry in gbm and clinical and genomic endpoints , we took a more direct approach of correlating raw nuclear features with genomic and clinical endpoints ( 37 ) . for each patient , we calculated the mean and standard deviation of each feature as metrics and correlating them directly with patient survival using cox proportional hazards analysis using sam . notably , the mean circularity was significantly associated with longer patient survival , an observation consistent with prolonged clinical outcomes in gliomas with oligodendroglial differentiation . other features that were significantly associated with outcome include major axis length , with longer nuclei associated with a shorter survival , and min nuclear pixel intensity , with higher values associated with longer survival . the fact that these features emerged from a more data - driven approach provides some level of confidence in our analysis workflow . to correlate these features with genomic measurements we performed a one - way anova for each feature metric across transcriptional classifications , somatic mutations and dna copy number alterations . features distinguishing transcriptional classes include nuclear eccentricity ( p = 3.81e-4 ) , minor axis length ( p = 8.87e-3 ) and nuclear extent ( p = 3.2e-2 ) . those hypermethylated ( gcimp ) tumors within the proneural group had greater variation of pixel intensities within their nuclei ( nuclear energy , p = 2.28e-5 ) , and greater variation in nuclear size . genetic events having significant differences in nuclear morphometry included pten and tp53 mutations , and pdg - fra amplification . pten and tp53 mutant tumors were both associated with less circular nuclei ( p = 9.68e-3 , 3.77e-2 respectively ) . pdgfra amplified tumors were associated with greater circularity ( p = 2.31e-2 ) , consistent with pdgfra amplifications being associated with oligodendroglial differentiation . other genetic alterations with significant associations included egfr amplification , which was associated with greater nuclear eccentricity and canny , and mdm2 amplifications , which were associated with greater minor axis length , area and circularity . advances in whole - slide imaging and computing hardware have made it possible to approach increasingly difficult image analysis problems in pathology . at the same time , the increasing availability of rich genomic data have made pathology image analysis studies more interesting by allowing linkage of histologic features with comprehensive molecular measurements . within the last decade , the goals of pathology image analysis have shifted from attempts to implement computer - aided diagnostic procedures , to more creative analyses that explore complex genotype - phenotype associations and define novel prognostication methods . the emerging goal is to go beyond computational replication of pathologists and to develop novel techniques to unmask latent content within image sets that has as yet unrecognized clinical and scientific value . this convergence of image analysis and bioinformatics has produced some exciting results in several different areas . in glioblastoma , morphometry - driven tumor subtypes were identified on the basis of nuclear morphometry and cellularity and found to be predictive of clinical outcomes and pathway activation ( 6 , 38 ) . in breast cancer , morphologic features describing stromal / tumor interface were found to be predictive of overall survival independent of other clinical , pathological and molecular features in two independent cohorts ( 39 ) . features of cellularity derived from images were found to improve the estimation of copy number variations , and a prognostic model that combines image measurements with gene expression features was developed and validated in independent cohorts ( 40 ) . focusing specifically on triple - negative breast cancer , a prognostic model based on morphologic features was also developed and validated in an independent dataset of triple negative breast cancers containing histology images ( 41 ) . a gene - expression signature derived from this prognostic model was then developed and used to further validate the prognostic value of this model in gene expression datasets where histology images were not available . one barrier to progress in this area is the dissemination of algorithms and image features beyond image analysis experts to the broader research community . making software and feature data publicly accessible will facilitate advances in this field by more fully engaging the pathology community and providing opportunities for comparative studies . establishing the computational resources needed to execute image analysis algorithms on the primary images is difficult , and the sharing of derived feature data is limited by a lack of standardization . to begin to address these issues , we have developed web - based interfaces and data standards to support the visualization , federation and analysis of pathology image data . the cancer digital slide archive ( cdsa , http://cancer.digitalslidearchive.net/ ) is a web - based resource that was originally developed to facilitate the visualization and analysis of pathology imaging and clinical data from tcga ( 42 ) . the cdsa currently hosts over 22,000 images and associated clinical data from over 22 different cancers represented in tcga . this interface provides access to pathology and clinical data through a simple web - browser interface . although currently focused on serving primary images for visualization , the cdsa and other similar resources could naturally serve as clearinghouses that allow a broader set of users to interact with image analysis algorithms and feature sets . cloud - based services could be established that enable primary image data , derived features and computational tools to reside in a common computing environment , avoiding the need for costly transfer of massive amounts of image and feature data . users could then perform end - to - end integrated analyses of pathology imaging online without the need to establish local computing resources or shepherding primary image or feature data between systems across the internet . in cases where feature data and algorithms are exchanged , we have also developed the pathology analytic imaging standards ( pais ) to support the standardization of image analysis algorithms and image features ( 43 , 44 ) . pais provides data standards that enable users to capture software and algorithm parameter provenance , and a common file format that enables results to be stored in a database for search and exchange . this issue is particularly important in studies identifying image biomarkers of clinical outcomes or genomic features . one of the risks in using image features to predict outcome or genomic measurements is overfitting are we are learning meaningful relationships that will generalize to new unseen datasets , or simply generating predictions that are specific to the noise and artifacts of the dataset that were analyzed ? validating findings in external datasets , when available , or by proper cross - validation of a single dataset is important for distinguishing true findings from artifacts . sometimes the morphologic features that are predictive can be visualized , while other times their predictive power is clearly measurable but not apparent to the human eye . some features are calculated ( e.g. standard deviation of canny ) are difficult to correlate with concrete histologic findings that can be visualized . in the cases where visualization is not possible or does not produce any obvious visible distinction , it is difficult to interpret the meaning of predictive features or to link them to existing knowledge about the histology of that disease . greater availability of benchmark datasets containing whole - slide images , and genomic and clinical data could help to establish reproducibility and improve confidence in the relationships defined through computational analysis . another area for growth is the integration of radiology imaging with pathology , genomics and clinical data . image analysis methods for radiology data are more mature than for pathology , and are able to extract meaningful features from mr , pet , ct and other medical imaging modalities . the global perspective of tumor provided by medical imaging is complementary to the tissue and molecular scale measurements provided by pathology and genomics , and a number of studies have already explored the relationships between quantitative radiology imaging features , genomic profiles and clinical outcomes ( 4548 ) . integrating complementary features across biological scales into prognostic models is a promising avenue to improve the precision of clinical predictions and risk stratification of patients . advances in whole - slide imaging and computing hardware have made it possible to approach increasingly difficult image analysis problems in pathology . at the same time , the increasing availability of rich genomic data have made pathology image analysis studies more interesting by allowing linkage of histologic features with comprehensive molecular measurements . within the last decade , the goals of pathology image analysis have shifted from attempts to implement computer - aided diagnostic procedures , to more creative analyses that explore complex genotype - phenotype associations and define novel prognostication methods . the emerging goal is to go beyond computational replication of pathologists and to develop novel techniques to unmask latent content within image sets that has as yet unrecognized clinical and scientific value . this convergence of image analysis and bioinformatics has produced some exciting results in several different areas . in glioblastoma , morphometry - driven tumor subtypes were identified on the basis of nuclear morphometry and cellularity and found to be predictive of clinical outcomes and pathway activation ( 6 , 38 ) . in breast cancer , morphologic features describing stromal / tumor interface were found to be predictive of overall survival independent of other clinical , pathological and molecular features in two independent cohorts ( 39 ) . features of cellularity derived from images were found to improve the estimation of copy number variations , and a prognostic model that combines image measurements with gene expression features was developed and validated in independent cohorts ( 40 ) . focusing specifically on triple - negative breast cancer , a prognostic model based on morphologic features was also developed and validated in an independent dataset of triple negative breast cancers containing histology images ( 41 ) . a gene - expression signature derived from this prognostic model was then developed and used to further validate the prognostic value of this model in gene expression datasets where histology images were not available . one barrier to progress in this area is the dissemination of algorithms and image features beyond image analysis experts to the broader research community . making software and feature data publicly accessible will facilitate advances in this field by more fully engaging the pathology community and providing opportunities for comparative studies . establishing the computational resources needed to execute image analysis algorithms on the primary images is difficult , and the sharing of derived feature data is limited by a lack of standardization . to begin to address these issues , we have developed web - based interfaces and data standards to support the visualization , federation and analysis of pathology image data . the cancer digital slide archive ( cdsa , http://cancer.digitalslidearchive.net/ ) is a web - based resource that was originally developed to facilitate the visualization and analysis of pathology imaging and clinical data from tcga ( 42 ) . the cdsa currently hosts over 22,000 images and associated clinical data from over 22 different cancers represented in tcga . this interface provides access to pathology and clinical data through a simple web - browser interface . although currently focused on serving primary images for visualization , the cdsa and other similar resources could naturally serve as clearinghouses that allow a broader set of users to interact with image analysis algorithms and feature sets . cloud - based services could be established that enable primary image data , derived features and computational tools to reside in a common computing environment , avoiding the need for costly transfer of massive amounts of image and feature data . users could then perform end - to - end integrated analyses of pathology imaging online without the need to establish local computing resources or shepherding primary image or feature data between systems across the internet . in cases where feature data and algorithms are exchanged , we have also developed the pathology analytic imaging standards ( pais ) to support the standardization of image analysis algorithms and image features ( 43 , 44 ) . pais provides data standards that enable users to capture software and algorithm parameter provenance , and a common file format that enables results to be stored in a database for search and exchange . this issue is particularly important in studies identifying image biomarkers of clinical outcomes or genomic features . one of the risks in using image features to predict outcome or genomic measurements is overfitting are we are learning meaningful relationships that will generalize to new unseen datasets , or simply generating predictions that are specific to the noise and artifacts of the dataset that were analyzed ? validating findings in external datasets , when available , or by proper cross - validation of a single dataset is important for distinguishing true findings from artifacts . sometimes the morphologic features that are predictive can be visualized , while other times their predictive power is clearly measurable but not apparent to the human eye . some features are calculated ( e.g. standard deviation of canny ) are difficult to correlate with concrete histologic findings that can be visualized . in the cases where visualization is not possible or does not produce any obvious visible distinction , it is difficult to interpret the meaning of predictive features or to link them to existing knowledge about the histology of that disease . greater availability of benchmark datasets containing whole - slide images , and genomic and clinical data could help to establish reproducibility and improve confidence in the relationships defined through computational analysis . another area for growth is the integration of radiology imaging with pathology , genomics and clinical data . image analysis methods for radiology data are more mature than for pathology , and are able to extract meaningful features from mr , pet , ct and other medical imaging modalities . the global perspective of tumor provided by medical imaging is complementary to the tissue and molecular scale measurements provided by pathology and genomics , and a number of studies have already explored the relationships between quantitative radiology imaging features , genomic profiles and clinical outcomes ( 4548 ) . integrating complementary features across biological scales into prognostic models is a promising avenue to improve the precision of clinical predictions and risk stratification of patients .

the two - component systems ( tcs ) , or histidine - to - aspartate phosphorelays , are evolutionarily conserved common signal transduction mechanisms that are implicated in a wide variety of cellular responses to environmental stimuli in both prokaryotes and eukaryotes including plants . among higher plants , legumes including lotus japonicus have a unique ability to engage in beneficial symbiosis with nitrogen - fixing bacteria . we previously presented a genome - wide compiled list of tcs - associated components of mesorhizobium loti , which is a symbiont specific to l. japonicus ( hagiwara et al . 2004 , dna res . , 11 , 5765 ) . to gain both general and specific insights into tcs of this currently attractive model legume , here we compiled tcs - associated components as many as possible from a genome - wide viewpoint by taking advantage that the efforts of whole genome sequencing of l. japonicus are almost at final stage . in the current database ( http://www.kazusa.or.jp/lotus/index.html ) , it was found that l. japonicus has , at least , 14 genes each encoding a histidine kinase , 7 histidine - containing phosphotransmitter - related genes , 7 type - a response regulator ( rr)-related genes , 11 type - b rr - related genes , and also 5 circadian clock - associated pseudo - rr genes . these results suggested that most of the l. japonicus tcs - associated genes have already been uncovered in this genome - wide analysis , if not all . here , characteristics of these tcs - associated components of l. japonicus were inspected , one by one , in comparison with those of arabidopsis thaliana . in addition , some critical experiments were also done to gain further insights into the functions of l. japonicus tcs - associated genes with special reference to cytokinin - mediated signal transduction and circadian clock .

this study was supported by a grant - in - aid for scientific research on priority areas ( 19039013 to t.m . and 20061016 to t.y . ) and a grant - in - aid for the gcoe programs ( systems biology ) of the ministry of education , culture , sports , science , and technology of japan .

purposethis study was designed to evaluate the risk on development and persistence of constipation after hysterectomy.methodswe conducted a prospective , observational , multicenter study with three - year follow - up in 13 teaching and nonteaching hospitals in the netherlands . a total of 413 females who underwent hysterectomy for benign disease other than symptomatic uterine prolapse were included . all patients underwent vaginal hysterectomy , subtotal abdominal hysterectomy , or total abdominal hysterectomy . a validated disease - specific quality - of - life questionnaire was completed before and three years after surgery to assess the presence of constipation.resultsof the 413 included patients , 344 ( 83 percent ) responded at three - year follow - up . constipation had developed in 7 of 309 patients ( 2 percent ) without constipation before surgery and persisted in 16 of 35 patients ( 46 percent ) with constipation before surgery . preservation of the cervix seemed to be associated with an increased risk of the development of constipation ( relative risk , 6.6 ; 95 percent confidence interval , 1.333.3 ; p = 0.02 ) . statistically significant risk factors for the persistence of constipation could not be identified.conclusionshysterectomy does not seem to cause constipation . in nearly half of the patients reporting constipation before hysterectomy , this symptom will disappear .

the design of the study has been described previously.9 in short , a prospective , observational study was performed among females undergoing hysterectomy for benign disease in 13 teaching and nonteaching hospitals in the netherlands . the study was approved by all local ethical committees , and written informed consent was obtained from all patients . we recruited consecutive females who had been offered hysterectomy between january 1999 and july 2000 . exclusion criteria were known endometriosis and symptomatic descensus of the uterus as indication for hysterectomy . before hysterectomy , the gynecologist who had set the indication completed a standardized form to score age , obstetric history , history of abdominal surgery , indication for hysterectomy , duration of complaints , postmenopausal status , maximal diameter of the uterus as assessed by ultrasound , indication for vaginal or abdominal hysterectomy , and indication for removal or preservation of the cervix . after surgery , the gynecologist who had performed the hysterectomy completed a standardized form to score the following variables : duration of surgery in minutes , amount of blood loss in ml , and complications during surgery . at the day of discharge , this form was completed by documenting the duration of hospital stay and complications during hospital stay . all patients completed before , and at three years after , surgery the defecation distress inventory ( ddi).10 the ddi is a dutch validated questionnaire that was developed identical to the urogenital distress inventory11,12 by our research group and is used to assess the presence and experienced discomfort of defecation symptoms . the 15 questions were developed after studying the literature and international definitions , interviewing patients who presented with constipation or fecal incontinence , and by interviewing three experts in the field from the department of surgery and department of obstetrics and gynecology from the university medical centre utrecht , the netherlands . eventually , a structured interview of the 15 selected items was held with 20 female patients . for this study , we used the response to two questions of the ddi : do you have less than three bowel movements per week ? and the question : do you have to strain > 25 percent of the time to have a bowel movement ? according to the definition of drosmann et al . , constipation was considered to be present if the patient responded positive to both of these questions.13 this statistical analysis was designed to calculate the risk of constipation after hysterectomy and to identify which patient characteristics are prognostic factors for the development or persistence of constipation . the prevalence of constipation that persisted or had developed after hysterectomy was compared for the presence or absence of different patient characteristics and tested for statistical significance by using fisher s exact test . the risk of constipation after hysterectomy was expressed by the relative risk ( rr ) and 95 percent confidence interval ( ci ) . the rr expresses the risk of a patient in whom a condition is present compared with a patient in whom this condition is not present . variables that were tested for their statistical significance were age , body mass index , parity , history of abdominal surgery , presence of comorbidity , indication for hysterectomy , presence of fibroma , maximal diameter of the uterus , vaginal or abdominal approach , and removal of the cervix . the statistical package spss 11.5 was used to perform our analysis ( spss , inc . , this statistical analysis was designed to calculate the risk of constipation after hysterectomy and to identify which patient characteristics are prognostic factors for the development or persistence of constipation . the prevalence of constipation that persisted or had developed after hysterectomy was compared for the presence or absence of different patient characteristics and tested for statistical significance by using fisher s exact test . the risk of constipation after hysterectomy was expressed by the relative risk ( rr ) and 95 percent confidence interval ( ci ) . the rr expresses the risk of a patient in whom a condition is present compared with a patient in whom this condition is not present . variables that were tested for their statistical significance were age , body mass index , parity , history of abdominal surgery , presence of comorbidity , indication for hysterectomy , presence of fibroma , maximal diameter of the uterus , vaginal or abdominal approach , and removal of the cervix . the statistical package spss 11.5 was used to perform our analysis ( spss , inc . , of the 413 included patients , 344 ( 83 percent ) responded at three - year follow - up . if abdominal hysterectomy was performed , the cervix was preserved in one of three patients . table 1patient characteristics ( n = 334)age ( yr)44(6)parous ( n)281(84)body mass index ( kg / m)25(4)history of abdominal surgery ( n)126(38)comorbidity ( n)235(70)duration of symptoms ( mo)34(32)indication for hysterectomy ( n ) menorrhagia231(69 ) metrorrhagia104(31 ) abdominal pain144(43 ) dysmenorroe76(23)fibroma present on ultrasound ( n)228(68)maximal diameter of uterus ( cm)10.3(3.5)descensus of uterus ( cm)-5.4(2.9)values are means with standard deviations in parentheses or numbers with percentages in parentheses.not mutually exclusive.measured under anesthesia by pulling down the cervix with a forceps.table 2surgical procedures and complicationsno . of patients 334surgical procedures total abdominal hysterectomy158(47 ) subtotal abdominal hysterectomy91(27 ) vaginal hysterectomy85(25)surgery time ( min)62(20)blood loss ( ml)275(210)complications during surgery18(5 ) bleeding requiring transfusion14(4 ) bleeding requiring reoperation2(1 ) bladder lesion2(1)complications during hospital admission14(4 ) vault abscess2(1 ) vault hematoma1(0 ) cystitis3(1 ) bladder retention4(1 ) fever of unknown origin2(1 ) pulmonary embolism2(1)values are means with standard deviations in parentheses or numbers with percentages in parentheses.some patients had more than one complication . patient characteristics ( n = 334 ) values are means with standard deviations in parentheses or numbers with percentages in parentheses . measured under anesthesia by pulling down the cervix with a forceps . surgical procedures and complications values constipation had developed in 7 of 309 patients ( 2 percent ) without constipation before surgery . table 3 shows the risk on development of constipation according to the different patient characteristics and surgical parameters . it seemed that preservation of the cervix was associated with a higher risk to develop constipation after hysterectomy . we reviewed the medical files of the seven patients who developed constipation and found that in three of these patients total hysterectomy was planned ; however , during surgery because of difficult surgical conditions , such as adhesions and/or fibroma extending into the cervix , it was decided to preserve the cervix . all patients who developed constipation had undergone abdominal hysterectomy , had fibroma on ultrasound , had not undergone previous abdominal surgery , and did not have metrorragia as indication for hysterectomy . table 3risk of development or persistence of constipation three years after hysterectomy for different patient characteristics and surgical parameterspatient characteristic / surgical parameterrisk of development of constipationrisk of persistence of constipationn(%)rr95 percent cip valuen(%)rr95 percent cip valueage ( yr)<403/70(4)2.3(0.221.2)0.635/10(50)**140503/179(2)0.9(0.18.4)19/21(43)**0.45>501/53(2)11/1(100)1body mass index ( kg / m)<220/51(0)**0.335/10(50)0.9(0.42)0.8722252/99(2)0.6(0.13)0.71/5(20)0.4(0.12.3)0.32>255/145(3)18/15(53)1parousyes4/252(2)114/29(48)1no3/50(6)3.8(0.916.4)0.091/3(33)0.7(0.13.6)1history of abdominal surgeryyes0/116(0)14/10(40)1no7/186(4)**0.0611/22(50)1.3(0.53)0.71comorbidityyes3/85(4)14/11(36)1no4/208(2)0.5(0.12.4)0.4210/20(50)1.4(0.63.4)0.71menorrhagia**yes5/206(2)110/25(40)1no2/96(2)0.9(0.24.3)15/7(71)1.8(0.93.5)0.21metrorrhagia**yes0/95(0)15/9(56)1no7/207(3)**0.110/23(44)0.8(0.41.7)0.7abdominal pain**yes4/133(3)16/11(55)1no3/169(2)0.6(0.12.6)0.79/21(43)0.8(0.41.6)0.71dysmenorroe**yes1/67(1)15/9(56)1no6/235(2)1.7(0.214)110/23(44)0.8(0.41.7)0.7fibroma present on ultrasoundyes7/206(3)17/19(37)1no0/92(0)**0.18/13(62)1.7(0.83.5)0.28maximal diameter of uterus ( cm)<80/52(0)**0.183/6(50)1(0.32.8)18102/142(1)0.3(0.11.5)0.247/16(44)0.9(0.42)1>105/108(5)15/10(50)1surgical approachvaginal0/74(0)16/11(55)1abdominal7/228(3)**0.29/21(43)0.8(0.41.6)0.71removal of the cervixtotal2/219(1)111/23(48)1subtotal5/83(6)6.6(1.333.3)0.024/9(44)0.9(0.42.2)1rr = relative risk ; ci = confidence interval.*cannot be calculated.**indication for hysterectomy.calculated by fisher s exact test . risk of development or persistence of constipation three years after hysterectomy for different patient characteristics and surgical parameters rr = relative risk ; ci = confidence interval . constipation persisted in 16 of 35 patients ( 46 percent ) with constipation before surgery . table 3 shows the risk on persistence of constipation according to the different patient characteristics and surgical parameters . at three years after surgery , constipation had developed in 7 of 309 patients ( 2 percent ) without constipation before surgery . table 3 shows the risk on development of constipation according to the different patient characteristics and surgical parameters . it seemed that preservation of the cervix was associated with a higher risk to develop constipation after hysterectomy . we reviewed the medical files of the seven patients who developed constipation and found that in three of these patients total hysterectomy was planned ; however , during surgery because of difficult surgical conditions , such as adhesions and/or fibroma extending into the cervix , it was decided to preserve the cervix . all patients who developed constipation had undergone abdominal hysterectomy , had fibroma on ultrasound , had not undergone previous abdominal surgery , and did not have metrorragia as indication for hysterectomy . table 3risk of development or persistence of constipation three years after hysterectomy for different patient characteristics and surgical parameterspatient characteristic / surgical parameterrisk of development of constipationrisk of persistence of constipationn(%)rr95 percent cip valuen(%)rr95 percent cip valueage ( yr)<403/70(4)2.3(0.221.2)0.635/10(50)**140503/179(2)0.9(0.18.4)19/21(43)**0.45>501/53(2)11/1(100)1body mass index ( kg / m)<220/51(0)**0.335/10(50)0.9(0.42)0.8722252/99(2)0.6(0.13)0.71/5(20)0.4(0.12.3)0.32>255/145(3)18/15(53)1parousyes4/252(2)114/29(48)1no3/50(6)3.8(0.916.4)0.091/3(33)0.7(0.13.6)1history of abdominal surgeryyes0/116(0)14/10(40)1no7/186(4)**0.0611/22(50)1.3(0.53)0.71comorbidityyes3/85(4)14/11(36)1no4/208(2)0.5(0.12.4)0.4210/20(50)1.4(0.63.4)0.71menorrhagia**yes5/206(2)110/25(40)1no2/96(2)0.9(0.24.3)15/7(71)1.8(0.93.5)0.21metrorrhagia**yes0/95(0)15/9(56)1no7/207(3)**0.110/23(44)0.8(0.41.7)0.7abdominal pain**yes4/133(3)16/11(55)1no3/169(2)0.6(0.12.6)0.79/21(43)0.8(0.41.6)0.71dysmenorroe**yes1/67(1)15/9(56)1no6/235(2)1.7(0.214)110/23(44)0.8(0.41.7)0.7fibroma present on ultrasoundyes7/206(3)17/19(37)1no0/92(0)**0.18/13(62)1.7(0.83.5)0.28maximal diameter of uterus ( cm)<80/52(0)**0.183/6(50)1(0.32.8)18102/142(1)0.3(0.11.5)0.247/16(44)0.9(0.42)1>105/108(5)15/10(50)1surgical approachvaginal0/74(0)16/11(55)1abdominal7/228(3)**0.29/21(43)0.8(0.41.6)0.71removal of the cervixtotal2/219(1)111/23(48)1subtotal5/83(6)6.6(1.333.3)0.024/9(44)0.9(0.42.2)1rr = relative risk ; ci = confidence interval.*cannot be calculated.**indication for hysterectomy.calculated by fisher s exact test . risk of development or persistence of constipation three years after hysterectomy for different patient characteristics and surgical parameters rr = relative risk ; ci = confidence interval . constipation persisted in 16 of 35 patients ( 46 percent ) with constipation before surgery . table 3 shows the risk on persistence of constipation according to the different patient characteristics and surgical parameters . the purpose of our study was to investigate whether hysterectomy causes constipation and to identify prognostic factors for the development or persistence of constipation . preservation of the cervix seemed to be associated with an increased occurrence of constipation , but the small numbers make the relevance of this finding questionable . in nearly half of the patients reporting constipation before hysterectomy , the present study was based on a multicenter cohort study of 413 females undergoing vaginal , subtotal abdominal , or total abdominal hysterectomy . data were prospectively collected , potential confounders were accurately documented , and a validated questionnaire was used to asses the occurrence of constipation . first , the number of patients in the stratum with constipation present before surgery was relatively small . second , the number of patients that developed constipation was so low that it is questionable whether one should attempt to identify risk factors for this occurrence . one of the strengths of our study is that we managed to assess complete follow - up of more than 80 percent of our cohort at three years after surgery . there is a widespread but poorly quantified belief that hysterectomy is associated with bowel problems , especially constipation . taylor and coauthors6 compared females with bowel symptoms after hysterectomy with age - matched healthy control subjects . posthysterectomy females reported more commonly infrequent bowel movement , use of laxatives , and consulted more often a doctor because of constipation . van dam et al.7 compared bowel function in 593 females who had undergone hysterectomy to a control group consisting of 100 women who had undergone laparoscopic cholecystectomy and found that bowel dysfunction was significantly more common after hysterectomy . heaton and coauthors5 also observed that constipation was more common after hysterectomy than after laparoscopic cholecystectomy . because of the retrospective design of these studies , it seems reasonable to assume that the operation precedes the onset of bowel dysfunction . furthermore , retrospective studies may be biased by recall bias , in specific , patients may have forgotten the exact timing of the onset of constipation . similar to our findings , one other study prospectively evaluating the effects of hysterectomy on constipation did not observe an increased incidence of constipation postoperatively.14 in a randomized trial comparing the effects of total and subtotal abdominal hysterectomy on pelvic floor function , the prevalence of constipation after surgery was lower than before surgery . the authors did not present the data stratified for presence or absence of constipation before hysterectomy . the reported prevalence of constipation in the community ranges from 2 to 28 percent and depends on the used definition.15 the definition for constipation we use has been described by drossman et al.13 and includes both frequency of defecation and the necessity to strain . in most studies , constipation is defined as a bowel frequency of less than three times per week.15 the use of our stricter definition may explain the low prevalence of constipation both before and after hysterectomy . however , it has been shown that adding the necessity to strain to the definition significantly increases the sensitivity of the symptoms to identify individuals with constipation.16 studies initiated to identify prognostic factors for the development or persistence of constipation have , as far as known , not been published . in this study , the small number of patients that developed constipation ( 7/309 ) and the small number with constipation before surgery ( 35/344 ) limited the identification of prognostic variables for the development and persistence of constipation . however , it was an interesting observation that preservation of the cervix seemed to be associated with an increased incidence of constipation after hysterectomy . in a randomized trial comparing the effects of total and subtotal abdominal hysterectomy on pelvic floor function , preservation of the cervix and prevalence of constipation after surgery were not related to each other.17 as mentioned in the results section , review of the medical files of the seven patients who developed constipation showed that in three of these patients during surgery the cervix could not be removed because of difficult surgical conditions , such as adhesions and/or fibroma extending into the cervix . as a consequence , the prognostic value of the variable preservation of the cervix seems to be confounded by the technical difficulty of the procedure . even while preserving the cervix , it is likely that these surgical procedures have caused more autonomic nerve damage with the development of constipation as a result . because the prevalence of constipation increases with age , the observed development in our study might reflect the natural course of this symptom.18 therefore , we conclude that hysterectomy does not affect the occurrence of constipation . preservation of the cervix was associated with an increased risk on the development of constipation after hysterectomy , but this was largely explained by more difficult and extensive surgery . if present before hysterectomy , constipation had disappeared at three years after surgery in almost half of the patients . to the editor hysterectomy is the most common major gynecologic operation and the procedure usually has a low risk for postoperative morbidity . sometimes patients report unwanted effects on bowel function after hysterectomy , but there are a limited number of studies 18 on this subject and most of them are retrospective in nature . roovers et al . should be commended for conducting a prospective large - scale study to determine the development of constipation after hysterectomy . the 83 percent response rate after three years is impressive . in a recent prospective study , 9 we found similar results as roovers et al . we evaluated the influence of hysterectomy on bowel function in 120 consecutive patients , and we found a trend toward more anal incontinence symptoms but no deterioration of constipation . roovers et al . have defined constipation as the presence of less than three bowel movements per week and straining more than 25 percent of the time to have a bowel movement . we do not agree with this restrictive definition of constipation , and there is ample support from our standpoint in the literature . it is correct that koch et al.10 found that straining is a sensitive criterion for constipation . in their conclusion , however , they recommend physiologic testing in the assessment of constipated patients rather than using the definition used in the present study . the authors reference a study by drossman et al . 11 for their definition of constipation . this study is 15 years old and is primarily focused on describing the range of bowel patterns in the general population and not to define constipation . drossman is otherwise one of the authorities behind the rome criteria for functional bowel disorders , including constipation . 12 in the latest communication from the rome initiative , constipation is defined as persistently difficult , infrequent , or seemingly incomplete defecation , which do not meet ibs criteria . 13 the american college of gastroenterology states that constipation is characterized by unsatisfactory defecation that results from infrequent stools , difficult stool passage , or both . 14 the restrictive definition of constipation in the present study limits the number of constipated patients , which may have influenced the results and conclusions . it would be interesting to see whether the outcome would change with a more liberal , and more commonly used , definition of constipation . we are still lacking large prospective studies focused on evaluating the influence of hysterectomy on different aspects of bowel function . this is an important topic for future studies , given the high incidence of this operation in the female population . to the editor colleagues mellgren and altman do not agree with the restrictive definition of constipation . in our study , constipation was considered to be present if the patient responded both positive to the questions : do you have less than three bowel movements per week ? and do you have to strain > 25 percent of the time to have a bowel movement ? the ddi questionnaire does not only question whether a symptom is present but also the experienced amount of bother by that symptom . we reanalyzed our data and found that 2.4 percent of the asymptomatic patients developed infrequent bowel movement , whereas 17.6 percent of the asymptomatic patients developed the need to frequently strain . we also analyzed how bothered the patients were by these symptoms and found that three years after surgery 38 percent of the patients were severely bothered by infrequent bowel movements , whereas only 11 percent of the patients were severely bothered by the need to frequently strain . we concluded that frequency of bowel movement and the need to strain are represented in the definition of constipation according to the rome criteria and the american college of gastroenterology . based on our own data , we state that quality of life related to constipation is mainly determined by the frequency of bowel movement . in our study , 2.4 percent of the preoperative asymptomatic patients had developed this symptom at three years after surgery .

objectiveto evaluate the effects of ultrasonic instrumentation with different scaler - tip angulations on the shear bond strength ( sbs ) and bond failure mode of metallic orthodontic brackets.methodsadhesive pre - coated metallic brackets were bonded to 72 extracted human premolars embedded in autopolymerizing acrylic resin . the teeth were randomly divided into 3 groups ( n = 24 each ) to undergo no treatment ( control group ) or ultrasonic instrumentation with a scaler - tip angulation of 45 ( 45-angulation group ) or 0 ( 0-angulation group ) . sbs was tested in a universal testing machine , and adhesive remnant index ( ari ) scores were recorded . the kruskal - wallis test and mann - whitney u - test were used for statistical analysis.resultsthe control group had a significantly higher mean sbs value than the treated groups , which showed no significant differences in their mean sbs values . the ari scores were not significantly different among the groups.conclusionsultrasonic instrumentation around the bracket base reduces the sbs of metallic orthodontic brackets , emphasizing the need for caution during professional oral hygiene procedures in orthodontic patients . the scaler - tip angulation does not influence the sbs reduction and bond failure mode of such brackets .

the placement of fixed orthodontic appliances increases the difficulty in mechanical plaque removal and thus predisposes orthodontic patients to plaque accumulation and enamel demineralization.1 - 3 fixed orthodontic treatment is also associated with increased probing pocket depths , elevated bacterial count in plaque , and a shift in the healthy microbial composition of subgingival plaque to a periodontal pathogenic one.4 - 6 therefore , optimal oral home care and professional prophylactic programs are of paramount importance in patients undergoing fixed orthodontic treatment.7,8 during professional oral hygiene procedures , ultrasonic scalers are used around the bracket base , a critical site of plaque accumulation , and can affect the bracket - enamel interface.9,10 prolonged ultrasonic vibration at maximum power is used to facilitate the removal of posts , crowns , or bridges and could similarly debond orthodontic brackets.11 - 15 this action is attributable to the propagation of vibrations from the ultrasonic device to the object that is to be removed , as well as to the biophysical action of ultrasound within the coolant ( i.e. , cavitational activity and acoustic microstreaming).16 however , to date , no study of the effects of ultrasonic instrumentation for oral hygiene maintenance on the shear bond strength ( sbs ) of orthodontic brackets has been conducted . the aim of this study was to evaluate the effects of ultrasonic instrumentation with different scaler - tip angulations on the sbs and bond failure mode of metallic orthodontic brackets . seventy - two premolars extracted for orthodontic reasons from patients aged 12 - 18 years were collected after receiving adequate informed consent . the teeth did not have cracks visible under 4 magnification , hypoplasia , white spots , caries , or labial restorations.17 they were washed in water to remove contamination and stored in distilled water in a refrigerator ( i.e. , nominal 4 ) for 1 - 6 months , in accordance with the iso / ts 11405 standard.18 in strict accordance with the protocol described by alessandri bonetti et al.,19 the labial enamel surfaces were cleaned , polished , and etched . a thin uniform coat of primer ( transbond xt ; 3 m unitek , monrovia , ca , usa ) was applied by using a microbrush . adhesive pre - coated metallic brackets ( apc ii adhesive coating on victory series brackets ; 3 m unitek ) were then placed on the teeth , adjusted to their final positions , and pressed firmly . after removing excess resin from the periphery of the bracket base with a dental probe , the adhesive was cured by using a light - emitting diode light source ( ortholux luminous curing light ; 3 m unitek ) for 6 s ( 3 s mesially and 3 s distally ) , according to the manufacturer 's instruction . after storage in distilled water at 37 for 24 h ( iso / ts 1140518 ; test type 1 : short - term test ) , the teeth were embedded in autopolymerizing acrylic resin ( orthocryl ; dentaurum , bologna , italy ) in polyvinyl chloride molds ( 15-mm diameter and 20-mm height ) so that the roots were fully embedded in acrylic resin and the bonding surface of the brackets remained perpendicular to the horizontal plane and parallel to the direction of the force to be applied , in an effort to minimize peel and maximize shear during testing . to ensure the fulfillment of these requirements , the roots of each tooth were initially inserted in a wax pedestal , which was molded until the correct position was achieved and verified with the aid of a parallelometer ( paraline ; dentaurum ) . the teeth were randomly divided into 3 groups of 24 specimens each : control group , no treatment ; 45-angulation group , ultrasonic instrumentation with a scaler - tip angulation of 45 ( figure 1 ) ; 0-angulation group , ultrasonic instrumentation with a scaler - tip angulation of 0 ( figure 2 ) . to maintain the desired angulation in the 45-angulation group , an external support with a component forming an angle of 45 to the scaler tip was fastened to the ultrasonic device and the component was maintained parallel to the tooth surface , thus achieving an angle of 45 between the scaler tip and the tooth surface . a piezoelectric ultrasonic scaler ( piezosteril 5 ; castellini , bologna , italy ) was used with insert " c1 " ( recommended by the manufacturer for supragingival scaling ) at a power setting of 7.5 w. tap water , delivered directly from the dental unit , was used as the coolant . ultrasonic instrumentation was performed for 1 min , with 20 s each on the mesial , distal , and occlusal sides of the bracket base . the gingival side of the bracket base was omitted because the roots were embedded in acrylic resin up to the cementoenamel junction , preventing the access of the scaler tip to that surface . the bracket slot was not instrumented because the bracket wings did not allow the chosen scaler - tip angulations to be consistently achieved along that surface . after storage in distilled water at room temperature for 24 h , the specimens were tested according to the iso / ts 1140518 standard in a universal testing machine ( instron , milan , italy ) . each acrylic block was placed in the machine so that the bracket base was parallel to the direction of the force to be applied and a chisel - shaped blade was placed on the occlusal side of the ligature groove between the bracket base and the wings . a shear debonding force was applied in the occlusogingival direction at a crosshead speed of 1 mm / min,18 and the amount of shear force required to debond each bracket was recorded in newtons . stress values were calculated as the ratio of the debonding load ( in newtons ) to the area of the bracket base ( 9.79 mm ) and measured in megapascals ( mpa).20 to evaluate the mode of bond failure , the specimens were examined at 20 magnification with an optical microscope.21 by using open - source image - analysis software ( imagej ; national institutes of health , bethesda , md , usa ) , the amount of adhesive remaining on the tooth was expressed as a percentage of the total bonded area.19 the analysis was performed by an examiner trained to use the software and blinded to the groups . the evaluations were repeated by the same examiner 1 week later ; if any discrepancies arose , a third assessment was performed to determine the final score . an adhesive remnant index ( ari ) score was assigned to each specimen : 0 , no adhesive left on the tooth ; 1 , less than half of the adhesive remained on the tooth ; 2 , more than half of the adhesive remained on the tooth ; 3 , all the adhesive was left on the tooth , with a distinct impression of the bracket mesh.22,23 statistical analyses were performed by using statistical software ( pasw statistics for windows , version 18.0 ; ibm co. , armonk , ny , usa ) . the significance level was set at 0.05 . a pilot study estimated the expected difference in the mean sbs values between the untreated and the treated specimens as 2.3 mpa and the within - group standard deviation as 2.4 mpa . given the level of 0.0167 ( bonferroni adjustment for multiple comparisons ) with a power of 80% the shapiro - wilk test showed that the sbs data were consistent with a gaussian distribution in the control and 0-angulation groups , but they were not normally distributed in the 45-angulation group . pairwise comparisons were performed by using the mann - whitney u - test with bonferroni correction ( adjusted level = 0.0167 ) . the kruskal - wallis test was also used to assess the significance of the differences in the ari scores among the groups . seventy - two premolars extracted for orthodontic reasons from patients aged 12 - 18 years were collected after receiving adequate informed consent . the teeth did not have cracks visible under 4 magnification , hypoplasia , white spots , caries , or labial restorations.17 they were washed in water to remove contamination and stored in distilled water in a refrigerator ( i.e. , nominal 4 ) for 1 - 6 months , in accordance with the iso / ts 11405 standard.18 in strict accordance with the protocol described by alessandri bonetti et al.,19 the labial enamel surfaces were cleaned , polished , and etched . a thin uniform coat of primer ( transbond xt ; 3 m unitek , monrovia , ca , usa ) was applied by using a microbrush . adhesive pre - coated metallic brackets ( apc ii adhesive coating on victory series brackets ; 3 m unitek ) were then placed on the teeth , adjusted to their final positions , and pressed firmly . after removing excess resin from the periphery of the bracket base with a dental probe , the adhesive was cured by using a light - emitting diode light source ( ortholux luminous curing light ; 3 m unitek ) for 6 s ( 3 s mesially and 3 s distally ) , according to the manufacturer 's instruction . after storage in distilled water at 37 for 24 h ( iso / ts 1140518 ; test type 1 : short - term test ) , the teeth were embedded in autopolymerizing acrylic resin ( orthocryl ; dentaurum , bologna , italy ) in polyvinyl chloride molds ( 15-mm diameter and 20-mm height ) so that the roots were fully embedded in acrylic resin and the bonding surface of the brackets remained perpendicular to the horizontal plane and parallel to the direction of the force to be applied , in an effort to minimize peel and maximize shear during testing . to ensure the fulfillment of these requirements , the roots of each tooth were initially inserted in a wax pedestal , which was molded until the correct position was achieved and verified with the aid of a parallelometer ( paraline ; dentaurum ) . the teeth were randomly divided into 3 groups of 24 specimens each : control group , no treatment ; 45-angulation group , ultrasonic instrumentation with a scaler - tip angulation of 45 ( figure 1 ) ; 0-angulation group , ultrasonic instrumentation with a scaler - tip angulation of 0 ( figure 2 ) . to maintain the desired angulation in the 45-angulation group , an external support with a component forming an angle of 45 to the scaler tip was fastened to the ultrasonic device and the component was maintained parallel to the tooth surface , thus achieving an angle of 45 between the scaler tip and the tooth surface . a piezoelectric ultrasonic scaler ( piezosteril 5 ; castellini , bologna , italy ) was used with insert " c1 " ( recommended by the manufacturer for supragingival scaling ) at a power setting of 7.5 w. tap water , delivered directly from the dental unit , was used as the coolant . ultrasonic instrumentation was performed for 1 min , with 20 s each on the mesial , distal , and occlusal sides of the bracket base . the gingival side of the bracket base was omitted because the roots were embedded in acrylic resin up to the cementoenamel junction , preventing the access of the scaler tip to that surface . the bracket slot was not instrumented because the bracket wings did not allow the chosen scaler - tip angulations to be consistently achieved along that surface . a single experienced and trained operator performed all these procedures . after storage in distilled water at room temperature for 24 h , the specimens were tested according to the iso / ts 1140518 standard in a universal testing machine ( instron , milan , italy ) . each acrylic block was placed in the machine so that the bracket base was parallel to the direction of the force to be applied and a chisel - shaped blade was placed on the occlusal side of the ligature groove between the bracket base and the wings . a shear debonding force was applied in the occlusogingival direction at a crosshead speed of 1 mm / min,18 and the amount of shear force required to debond each bracket was recorded in newtons . stress values were calculated as the ratio of the debonding load ( in newtons ) to the area of the bracket base ( 9.79 mm ) and measured in megapascals ( mpa).20 to evaluate the mode of bond failure , the specimens were examined at 20 magnification with an optical microscope.21 by using open - source image - analysis software ( imagej ; national institutes of health , bethesda , md , usa ) , the amount of adhesive remaining on the tooth was expressed as a percentage of the total bonded area.19 the analysis was performed by an examiner trained to use the software and blinded to the groups . the evaluations were repeated by the same examiner 1 week later ; if any discrepancies arose , a third assessment was performed to determine the final score . an adhesive remnant index ( ari ) score was assigned to each specimen : 0 , no adhesive left on the tooth ; 1 , less than half of the adhesive remained on the tooth ; 2 , more than half of the adhesive remained on the tooth ; 3 , all the adhesive was left on the tooth , with a distinct impression of the bracket mesh.22,23 statistical analyses were performed by using statistical software ( pasw statistics for windows , version 18.0 ; ibm co. , armonk , ny , usa ) . the significance level was set at 0.05 . a pilot study estimated the expected difference in the mean sbs values between the untreated and the treated specimens as 2.3 mpa and the within - group standard deviation as 2.4 mpa . given the level of 0.0167 ( bonferroni adjustment for multiple comparisons ) with a power of 80% the shapiro - wilk test showed that the sbs data were consistent with a gaussian distribution in the control and 0-angulation groups , but they were not normally distributed in the 45-angulation group . pairwise comparisons were performed by using the mann - whitney u - test with bonferroni correction ( adjusted level = 0.0167 ) . the kruskal - wallis test was also used to assess the significance of the differences in the ari scores among the groups . three specimens of the 0-angulation group could not be tested because debonding of the brackets occurred during ultrasonic instrumentation . in these cases , an sbs value of 0 mpa was attributed because it was considered more representative of the clinical situation than a value obtained by interpolation or considering the loss of the specimen . a significant difference in the mean sbs values was noted among the groups ( p = 0.004 ) . the mean sbs value of the control group ( 9.92 mpa ) was significantly higher than that of the 45-angulation ( 7.77 mpa ; p = 0.004 ) and 0-angulation ( 7.16 mpa ; p = 0.006 ) groups . the kruskal - wallis test showed no significant differences among the groups . in many specimens ( 62.5% of the 45-angulation group and 66.67% of the control and 0-angulation groups ) , most of the adhesive remained on the enamel ( ari scores of 2 or 3 ) . proper professional oral hygiene maintenance is of paramount importance in patients undergoing fixed orthodontic treatment to reduce the risk of periodontal diseases and enamel demineralization.1 - 3 ultrasonic instrumentation is usually carried out around the bracket base , which is a critical site of plaque accumulation,9,10 but no studies have been performed yet to assess whether there is any influence on the sbs and mode of bond failure of metallic orthodontic brackets . to simulate extreme conditions and highlight the most detrimental effect of ultrasonic instrumentation on the sbs of metallic orthodontic brackets , the time of instrumentation was overrated when compared with a routine clinical procedure , in which the scaler tip probably contacts the bracket - enamel interface for only a short period . therefore , 1 minute was chosen as the total application time.24 further , because manufacturers generally recommend a power setting of medium to high for supragingival scaling and chapple et al.25 demonstrated that a half power setting is as effective as a full one , a power setting of 7.5 w ( 50% of the maximum power [ 15 w ] of the device ) was selected in this study . finally , the experienced operator who performed all the ultrasonic procedures had been trained to maintain the force of scaler - tip application as low as possible while contacting the bracket in a consistent manner , similarly to the routine clinical setting . however , as this method is not a standardized way of force application , it may limit the reproducibility of the study . in this study , ultrasonic instrumentation around the bracket base decreased the sbs of the metallic orthodontic brackets compared with the control specimens . the mean sbs values of both the treated groups were still beyond 6 mpa , which is reportedly adequate for orthodontic purposes.26 however , such a reduction is undesirable because it increases the risk of bracket failure after professional oral hygiene procedures , consequently impeding treatment progress and prolonging treatment time.27 the angulation of the scaler tip has been investigated in relation to the potential damage to the tooth surface but not to the impairment of the sbs of metallic orthodontic brackets . the extent of damage to the tooth surface is reportedly the highest with a tip angulation of 45 when using piezoelectric ultrasonic scalers.28,29 in this study , no significant differences in the sbs were found between the 0-angulation and the 45-angulation groups , suggesting that the tested angulations did not influence to bracket bond failure after professional oral hygiene procedures . however , the scaler tip should preferably be parallel to the tooth surface ( i.e. , 0 angulation ) , or at an angle less than 15 , to orient the pattern of ultrasonic vibration parallel to the tooth surface and decrease the risk of damage.28,29 interestingly , 3 brackets detached in the 0-angulation group before the sbs testing ; the reason for their failure is not fully understood . finally , the ari scores were similar among the groups , indicating that ultrasonic instrumentation did not affect the mode of bracket bond failure . in all the groups , most of the adhesive remained on the enamel , indicating a low risk of enamel damage after debonding . further studies are required to evaluate the influence of other parameters ( e.g. , power setting , load , instrumentation time ) pertaining to ultrasonic instrumentation for oral hygienic purposes on the sbs of orthodontic brackets . under the conditions of this in vitro study , 1 minute ultrasonic instrumentation around the bracket base at medium power reduced the sbs of metallic orthodontic brackets , indicating a higher risk of bracket bond failure after professional oral hygiene procedures . therefore , clinicians should avoid prolonged ultrasonic instrumentation around the bracket base during plaque removal . the scaler - tip angulation does not influence the sbs reduction and failure mode of metallic orthodontic brackets .

backgroundhigh red blood cell distribution width ( rdw ) is related to impairment of erythropoiesis , reflecting chronic inflammation and increased levels of oxidative stress , both of which are telltale signs of type 2 diabetics . the aim of this study was to evaluate the relationship between the rdw and fasting blood sugar / blood pressure , and compare the results from diabetics with nondiabetic controls.methodsthis was an unmatched case - control study involving 200 participants consisting of 100 diabetics and 100 nondiabetic controls . blood ( 4.5 ml ) was collected from all of the diabetics and nondiabetic controls , and placed into edta anticoagulant tubes . a full blood count was performed using the sysmex kx-21n , a three - part auto analyzer able to run 19 parameters per sample , including rdw . blood pressure was measured during sample collection and in a sitting position.resultsthe mean fasting blood sugar level was 95.2030.10 mg / dl in the controls , and 147.8572.54 mg / dl in the diabetics . the mean blood pressures for diabetics was 138/90 mmhg and for non - diabetics 120/80 mmhg . the mean rdw - sd ( rdw standard deviation ) was 46.444.64 fl in the controls , and 46.843.18 in the diabetics . the mean rdw - cv ( rdw coefficient of variation ) was 14.74%1.94% in controls , and 14.800.71 for diabetics . no statistically significant correlation was found between the rdw - sd and fasting blood sugar / blood pressure in the diabetics . a statistically significant positive correlation was found between the rdw - cv and blood pressure in the diabetics.conclusiona positive correlation between the rdw - cv and blood pressure was established in the diabetics in this study .

diabetes mellitus ( dm ) is a chronic metabolic disorder characterized by hyperglycemia , and resulting from defects in insulin secretion , insulin action , or both.1 the chronic hyperglycemia occurring in dm is associated with long - term damage , as well as dysfunction and failure of different organs , especially in the eyes , kidneys , nerves , heart and blood vessels.1 the world health organization ( who ) estimates that about 347 million people worldwide are presently living with dm.2 of this figure , approximately 80% are from low and medium income countries.3 who also projects that by 2030 , dm will be the seventh leading cause of death worldwide.4 there are two main types of dm : type 1 which usually develops in childhood and adolescence , and is insulin dependent ; and type 2 which develops in adulthood and represents more than 90% of cases worldwide . risk factors for type 2 dm include sedentary lifestyle , obesity , and old age . diagnosis of dm can be made with a simple fasting plasma glucose test with values 126 mg / dl ( 7.0 mmol / l ) being diagnostic of dm ( fasting is defined as no caloric intake for at least 8 hours).5 in the presence of symptoms of hyperglycemia ( polyuria , polydipsia , polyphagia , weight loss ) , a casual plasma glucose level of > 200 mg / dl ( 11.1 mmol / l ) is diagnostic.5 recently , various researchers have proposed that type 2 dm is connected to a state of subclinical chronic inflammation.6,7 it may be that abnormal levels of chemokines released by the expanded adipose tissue in obesity activates monocytes , and increases the secretion of proinflammatory adipokines . such cytokines in turn enhance insulin resistance in adipose and other tissues , thereby increasing the risk of type 2 dm.8,9 the red blood cell distribution width ( rdw ) is a measure of variation in size of the circulating erythrocytes ( anisocytosis)10 which is routinely obtained from a standard automated complete blood count . high rdw indicates the presence of anisocytosis which is related to impairment of erythropoiesis and degradation of erythrocytes,10 reflecting chronic inflammation and increased levels of oxidative stress , both of which are telltale signs in type 2 diabetics , and this may significantly contribute to development of atherosclerotic diseases.11,12 many recent studies have investigated changes in rdw in association with cardiac and noncardiac related deaths.1319 most of these studies report a positive correlation of rdw with the erythrocyte sedimentation rate ( esr ) and c - reactive protein ( crp ) levels an increase in the rdw during inflammation , similar to that seen in other inflammatory parameters . malandrino et al20 recently reported a positive correlation between a high rdw and increased incidence of both macro- and microvascular complications in dm patients without marked vascular complications . the aim of this study was to evaluate the relationship between the rdw and fasting blood sugar / blood pressure , and compare with nondiabetic controls . the research was approved by the ethics review committee of lagos state university teaching hospital ( lasuth ) . this was a case - control study consisting of 100 type 2 dm patients receiving treatment , attending the diabetic clinic of the lasuth , and 100 nondiabetic controls consisting of medical students , nurses and doctors in the same institution . during the study period between june to september 2013 , all patients who gave informed consent and satisfied the study inclusion criteria were recruited into the study . they were asked to fill structured questionnaires including demographic information , height , weight , last fasting blood sugar , blood pressure , drug history , and family history of diabetes . all the diabetics were on oral hypoglycemic and antiplatelet drugs , like clopidogrel and vasoprin tablets , and some were on antihypertensive and lipid lowering drugs . information on family history of diabetes was also obtained from the controls and they were subjected to fasting blood sugar testing before enlistment . blood was withdrawn with minimal stasis from the antecubital vein using a dry sterile disposable syringe and needle . the edta samples were kept at room temperature until processed within 4 hours of collection . a full blood count was performed using the sysmexkx-21n ( sysmex corporation , kobe , japan ) , a three - part auto analyzer able to run 19 parameters per sample , including hemoglobin concentration , packed cell volume , red blood cell concentration , mean corpuscular hemoglobin , mean cell volume , mean corpuscular hemoglobin concentration , white blood cell and platelet count , and mean platelet volume . standardization , calibration of the instrument , and processing of the samples were carried out according to the manufacturer s instructions . well - mixed blood samples were aspirated , by leaving the equipment sampling probe in the blood sample and then pressing the start button . the research was approved by the ethics review committee of lagos state university teaching hospital ( lasuth ) . this was a case - control study consisting of 100 type 2 dm patients receiving treatment , attending the diabetic clinic of the lasuth , and 100 nondiabetic controls consisting of medical students , nurses and doctors in the same institution . during the study period between june to september 2013 , all patients who gave informed consent and satisfied the study inclusion criteria were recruited into the study . they were asked to fill structured questionnaires including demographic information , height , weight , last fasting blood sugar , blood pressure , drug history , and family history of diabetes . all the diabetics were on oral hypoglycemic and antiplatelet drugs , like clopidogrel and vasoprin tablets , and some were on antihypertensive and lipid lowering drugs . information on family history of diabetes was also obtained from the controls and they were subjected to fasting blood sugar testing before enlistment . blood was withdrawn with minimal stasis from the antecubital vein using a dry sterile disposable syringe and needle . the edta samples were kept at room temperature until processed within 4 hours of collection . a full blood count was performed using the sysmexkx-21n ( sysmex corporation , kobe , japan ) , a three - part auto analyzer able to run 19 parameters per sample , including hemoglobin concentration , packed cell volume , red blood cell concentration , mean corpuscular hemoglobin , mean cell volume , mean corpuscular hemoglobin concentration , white blood cell and platelet count , and mean platelet volume . standardization , calibration of the instrument , and processing of the samples were carried out according to the manufacturer s instructions . well - mixed blood samples were aspirated , by leaving the equipment sampling probe in the blood sample and then pressing the start button . data were analyzed using spss version 16.0 ( spss inc . , chicago , il , usa ) . a total of 200 participants were enrolled into the study consisting of 100 diabetics and 100 nondiabetic controls . the mean age of the control group was 32.386.44 years , with a minimum age of 17 years , and a maximum age of 70 years . the mean age of the type 2 dm group was 62.359.84 years , the minimum was 34 years old , and the maximum 90 years old . the overall female : male ratio was 68% to 32% . in the type 2 dm participants , 73% were female , and 27% were male . in the nondiabetic participants , 63% were female , and in the diabetic group , the mean body mass index was 32.104.85 kg / m , and the mean fasting blood sugar level was 147.8572.54 mg / dl . in the nondiabetic group , the mean body mass index was 255.23 kg / m , and the mean fasting blood sugar was 95.2030.10 mg / dl . the minimum blood pressure of the diabetics was 100/90 mmhg , maximum was 200/90 mmhg , and the mean was 138/90 mmhg . amongst the diabetics , a total of 45 out of 100 ( 45% ) gave a positive family history of type 2 diabetes , while 55% had no family history of diabetes . the rdw - cv ( rdw coefficient of variation ) in the control group was 14.741.94 , and 14.800.71 in the type 2 dm group . the rdw - sd ( rdw standard deviation ) was 46.444.64 in the control group , and 46.843.18 in the type 2 dm group ( table 2 ) . both the rdw - sd and the rdw - cv were not statistically significantly correlated with the fasting blood sugar level in the type 2 dm group ; p - values were 0.10 and 0.55 respectively ; spearman s rho values were 0.61 and 0.12 respectively . the rdw - sd was not statistically significantly correlated with blood pressure ; p=0.99 ; spearman s rho of 0.11 . a statistically significant correlation was achieved between the rdw - cv and the blood pressure of type 2 dm patients ; p=0.02 , spearman s rho of 0.02 . rdw - sd and the rdw - cv values were not statistically significantly correlated with the duration of diagnosis of diabetes in the patients ; p - values were 0.8 and 0.38 respectively ; and spearman s rho of 0.096 and 0.035 respectively . tukey s post hoc analysis could not be performed between the rdw - cv and blood pressure because at least one group had fewer than two cases , the significant level of 0.29 was obtained with test of homogeneity of variance . dm affects a sizeable proportion of the working population , and has economic consequences for both the individual and the society as a whole . the rdw , a widely available and inexpensive test conducted as part of the complete blood cell count , measures the degree of anisocytosis . it is calculated as follows : rdw - cv=(standard deviation of red blood cell volumemean cell volume)100 the normal range for the rdw - cv is 11.5%14.5% , and higher values indicate greater variations in cell sizes.21 high rdw indicates a high degree of anisocytosis which is associated with distortion and degradation of erythropoiesis,21 reflecting chronic inflammation and an increased level of oxidative stress.22 dm increases vascular inflammation and oxidative stress while vascular inflammation affects erythropoiesis and deformability of red blood cells thus elevating rdw levels.23 rdw is strongly associated with chronic inflammation and is a strong indicator of risk of cardiovascular mortality in people with cardiovascular diseases , dm , as well as in the general population.13,14,18,24 increased rdw may also arise as a result of anemia . thus , causes like iron deficiency , and megaloblastic anemia with associated micro- or macrocytosis are potential confounders because our participants were not screened for iron , vitamin 12 , nor folic acid . however , the effect of such confounders is negligible as all the participants selected for the study were healthy individuals ( control group ) and diabetics without complications or obvious comorbidities as at the time of the study . the effect of other causes of chronic inflammation such as tuberculosis , cancers , and connective tissue disorders as confounders is similarly negligible . the mean age of diagnosis of type 2 dm among adults aged 1879 years in the us between 1997 and 2011 was 54 years,25 and is similar to the 62.359.84 years obtained in our study ; however this value doubles the mean age of our controls of 32.386.44 years . all controls used for this study were relatively young men and women working in our institution including nurses , doctors , and medical students , which are not representative of the general population ; this could impact on results obtained and is a possible limitation of the study . however , the sex distribution in the type 2 dm group and the control group was similar , with approximately 2:1 female : male in both cases . our type 2 dm study population had relatively well controlled diabetes , hence rdw - cv and rdw - sd of the type 2 dm group ( 14.800.71 and 46.843.18 ) was almost the same with control values ( 14.741.94 and 46.444.64 ) . also , the mean fasting blood sugar of 147.8572.54 mg / dl in diabetics compared well with the nondiabetic controls of 95.2030.10 mg / dl . these closely related results in our study may be accounted for by the fact that the majority of diabetics enrolled had long been on treatment before this study , and their medications performed well . this finding is in contrast with the findings of studies by lee and partley,26 malandrino et al20 and heba et al27 who found significant associations between the rdw and macrovascular complications of dm , suggesting that rdw may be a predictor of the onset of diabetic macrovascular complications . also in contrast to the studies of malandrino et al20 and sherif et al,27 our results showed a statistically insignificant negative correlation between rdw and fasting blood sugar levels in diabetics . while in keeping with many other studies,2831 a statistically significant positive correlation was achieved between rdw and blood pressure in our patients . elevated rdw had been reported as a prognostic marker reflecting an underlying inflammatory state.28 high rdw was strongly associated with poor clinical outcomes in patients with heart failure,28 coronary artery disease,29,30 pulmonary hypertension31 and peripheral arterial disease.32 increased rdw was also associated with increased mortality in diabetic patients with coronary artery disease treated with percutaneous coronary intervention.33 we could only establish a statistically significant correlation between rdw and blood pressure , not between rdw and fasting blood sugar . we appreciate the efforts of mr isa usman who assisted in blood collection from the participants , and mr phillip o oluwamuhuru who carried out the full blood count on the samples .

backgroundthe purpose of this study was to report outcomes of congenital hallux varus deformity after surgical treatment.methodswe evaluated ten feet of eight patients with a congenital hallux varus deformity , including four feet combined with a longitudinal epiphyseal bracket ( leb ) . there were seven male patients and one female patient with a mean age of 33 months ( range , 7 to 103 months ) at the time of surgery . two patients were bilaterally involved . the mean duration of follow - up was 5.9 years ( range , 2.3 to 13.8 years ) . clinical outcomes were assessed according to the criteria of phelps and grogan . surgical procedures included the farmer procedure , the mcelvenny procedure or an osteotomy at the first metatarsal or proximal phalanx.resultsthe clinical results were excellent in two feet , good in six and poor in two feet . the leb was associated with hallux varus in four feet and were treated by osteotomy alone or in conjunction with soft tissue procedure.conclusionscongenital hallux varus was successfully corrected by surgery with overall favorable outcome . preoperatively , a leb should be considered as a possible cause of the deformity in order to prevent recurrent or residual varus after surgery .

totally ten feet of eight patients with a congenital hallux varus deformity were surgically treated at our institute between 1993 and 2008 by the senior author . indications for surgery were to improve appearance and to enable the wearing of normal shoes.5 ) there were seven male and one female with a mean age at the time of surgery of 33 months ( range , 7 to 103 months ) . mean duration of the follow - up was 5.9 years after index operation ( range , 2.3 to 13.8 years ) . six of the patients had hallux varus as an isolated congenital anomaly , while two patients had other anomalies ; one had a congenital anterolateral bowing of the tibia on the ipsilateral side , and the other patient had a congenital anterolateral bowing of the tibia and hypoplasia of the second finger on the ipsilateral side . a preoperative , simple radiograph was used to evaluate the angular deformity , the presence of supernumerary bones or toes and abnormal appearances of the first metatarsal or phalanges , including a leb . based on a preoperative radiograph and operative findings , pathological structures in patients reported to be associated with congenital hallux varus deformity by mcelvenny11 ) were described as follows : ( 1 ) short , thick metatarsal , ( 2 ) accessory bones or toes , ( 3 ) varus deformity of one or more of the four metatarsals , and ( 4 ) a firm fibrous band extending from the medial side of the great toe to the base of the first metatarsal . residual varus deformity or recurrence was evaluated by postoperative , simple radiographs and clinical photographs in all patients at follow - up . clinical outcomes were assessed according to the criteria of phelps and grogan.7 ) pain , calluses , residual deformities and scar cosmesis were examined in all patients and the final results were graded as excellent , good or poor . the final result was rated as the most unsatisfactory grade out of the four categories . furthermore , the patients were asked if they experienced any difficulty in wearing shoes and if they felt that the cosmetic results were satisfactory . all of the deformities in our patients were associated with one or more of the four components described by mcelvenny11 ) ( table 1 ) . five feet of three patients had been operated to remove polydactyly approximately 12 months after birth at another institute ( patients 5 , 7 , and 8) . all remaining five patients had preaxial polydactyly of incompletely duplicated phalanges ( type 4 according to venn - watson classification7 ) ) , although most of the phalanges were poorly formed with little bony contact and arose from the medial border of the foot at the level of the metatarsophalangeal joint.5 ) these were removed at surgery . a firm fibrous band that extended from the medial side of the great toe to the base of the first metatarsal was also common , present in nine of the ten feet . a combined leb was identified in four feet and involved the first metatarsal in three feet ( right foot of patient 5 and both feet of patient 7 ) and the proximal phalanx of the great toe in one foot ( patient 8) . interestingly , varus deformities were still present even though all of them had a previous history of surgical removal of polydactyly . surgical procedures varied according to the severity of the deformity and the anatomic configuration of the first metatarsal or proximal phalanx ( table 1 ) . decisions regarding surgical options were individualized , mainly depending on the pathology and severity of the deformity as well as on the age of the patient . a soft tissue procedure , as described by farmer1 ) or mcelvenny,11 ) was in isolation performed for three feet ( patients 1 , 3 , and 4 ) and in conjunction with osteotomy of the first metatarsal or proximal phalanx in four feet . specifically , a medial open - wedge osteotomy of the first metatarsal was performed for the three feet with a combined leb of the first metatarsal ( right foot of patient 5 and both feet of patient 7 ) , followed by an interposition of an appropriately sized allogenous strut graft ( fig . an osteotomy was performed at the proximal phalanx for the foot with hallux varus combined with a leb of the proximal phalanx . the farmer technique was initially performed for patient 6 with a severe deformity , but the varus alignment was not sufficiently corrected . a lateral closing - wedge osteotomy of the first metatarsal was performed for further correction because the first metatarsal was not shortened . after the farmer technique was performed , the varus deformity was still present in patient 2 with an interphalangeal joint and a severe varus preoperative angulation of 60. further correction with a medial open - wedge osteotomy at the proximal phalanx was necessary to achieve a neutral alignment ( fig . the clinical results were excellent in two feet , good in six and poor in two feet ( table 1 ) . gross appearances were satisfactory compared with preoperative appearances , although none of the feet could be described as cosmetically normal.5 ) an unsatisfactory rating was generally related to scar cosmesis . one patient with poor results ( patient 5 ) who had been bilaterally treated was not satisfied due to scar cosmesis of both feet , even though he did not experience pain or footwear problems . the mean varus angulation of 29.1 at the metatarsophalangeal joint was improved to a mean angle of 4.0 of valgus at follow - up ( p < 0.05 ) ( table 1 ) . overcorrection was seen in one patient , who had 30 of valgus angulation at follow - up , although he did not complain of pain or difficulties wearing footwear . one patient ( patient 1 ) with a previous surgery using the farmer technique complained of a painful bump on the medial aspect of the great toe and persistent widening of the first web space at weight - bearing at year 6 postoperative . soft tissue procedures were performed to resect the medial skin bump and to narrow the first web space . the patients ' outcome was rated as good at the final follow - up . the other patient ( patient 3 ) experienced footwear problems due to shortening of the first ray with mild , residual varus after the farmer procedure . this patient required a medial open - wedge osteotomy of the first metatarsal and insertion of a strut allograft for further correction 7.5 years postoperative . all of the deformities in our patients were associated with one or more of the four components described by mcelvenny11 ) ( table 1 ) . five feet of three patients had been operated to remove polydactyly approximately 12 months after birth at another institute ( patients 5 , 7 , and 8) . all remaining five patients had preaxial polydactyly of incompletely duplicated phalanges ( type 4 according to venn - watson classification7 ) ) , although most of the phalanges were poorly formed with little bony contact and arose from the medial border of the foot at the level of the metatarsophalangeal joint.5 ) these were removed at surgery . a firm fibrous band that extended from the medial side of the great toe to the base of the first metatarsal was also common , present in nine of the ten feet . a combined leb was identified in four feet and involved the first metatarsal in three feet ( right foot of patient 5 and both feet of patient 7 ) and the proximal phalanx of the great toe in one foot ( patient 8) . interestingly , varus deformities were still present even though all of them had a previous history of surgical removal of polydactyly . surgical procedures varied according to the severity of the deformity and the anatomic configuration of the first metatarsal or proximal phalanx ( table 1 ) . decisions regarding surgical options were individualized , mainly depending on the pathology and severity of the deformity as well as on the age of the patient . a soft tissue procedure , as described by farmer1 ) or mcelvenny,11 ) was in isolation performed for three feet ( patients 1 , 3 , and 4 ) and in conjunction with osteotomy of the first metatarsal or proximal phalanx in four feet . specifically , a medial open - wedge osteotomy of the first metatarsal was performed for the three feet with a combined leb of the first metatarsal ( right foot of patient 5 and both feet of patient 7 ) , followed by an interposition of an appropriately sized allogenous strut graft ( fig . an osteotomy was performed at the proximal phalanx for the foot with hallux varus combined with a leb of the proximal phalanx . the farmer technique was initially performed for patient 6 with a severe deformity , but the varus alignment was not sufficiently corrected . a lateral closing - wedge osteotomy of the first metatarsal was performed for further correction because the first metatarsal was not shortened . after the farmer technique was performed , the varus deformity was still present in patient 2 with an interphalangeal joint and a severe varus preoperative angulation of 60. further correction with a medial open - wedge osteotomy at the proximal phalanx was necessary to achieve a neutral alignment ( fig . the clinical results were excellent in two feet , good in six and poor in two feet ( table 1 ) . gross appearances were satisfactory compared with preoperative appearances , although none of the feet could be described as cosmetically normal.5 ) an unsatisfactory rating was generally related to scar cosmesis . one patient with poor results ( patient 5 ) who had been bilaterally treated was not satisfied due to scar cosmesis of both feet , even though he did not experience pain or footwear problems . the mean varus angulation of 29.1 at the metatarsophalangeal joint was improved to a mean angle of 4.0 of valgus at follow - up ( p < 0.05 ) ( table 1 ) . overcorrection was seen in one patient , who had 30 of valgus angulation at follow - up , although he did not complain of pain or difficulties wearing footwear . one patient ( patient 1 ) with a previous surgery using the farmer technique complained of a painful bump on the medial aspect of the great toe and persistent widening of the first web space at weight - bearing at year 6 postoperative . soft tissue procedures were performed to resect the medial skin bump and to narrow the first web space . the patients ' outcome was rated as good at the final follow - up . the other patient ( patient 3 ) experienced footwear problems due to shortening of the first ray with mild , residual varus after the farmer procedure . this patient required a medial open - wedge osteotomy of the first metatarsal and insertion of a strut allograft for further correction 7.5 years postoperative . in the current study , congenital hallux varus deformities were treated with various surgical techniques on a case - to - case basis depending on the severity and anatomic characteristics of the deformity . overall , these techniques yielded favorable results in terms of pain , ability to wear footwear and recurrence of the deformity . several studies have reported surgical outcomes for congenital hallux varus ( table 2).1,4,5,8,10,12 ) these studies evaluated patients with mixed diseases or were based on only a few case reports . no uniform method was used to assess clinical outcome and also various surgical procedures were used . this heterogeneity in previous studies , as well as in ours , is most likely not only due to the rarity of congenital hallux varus , but also to the lack of an established , clear definition of the deformity , its underlying pathomechanisms and the surgical treatment of choice for correction . general guidelines recommend that the issues to be considered for surgery are the correction of a polydactyly if present ; correction of the soft tissue tether on the medial side of the foot and the enlarged web space between the great and second toe ; correction of a metatarsal - phalangeal incongruity and the correction of a metatarsal or bracket epiphyseal deformity.18 ) soft tissue procedure by mcelvenny11 ) or farmer1 ) is recommended if the first metatarsal is normal . we followed this guideline in our series and decisions regarding surgical options were based on the pathology and severity of deformity and were individualized for each patient . mills and menelaus5 ) reported surgical outcomes of 20 feet of 12 patients followed - up for an average of 12.7 years . surgical procedures varied , including mcelvenny technique for nine feet , farmer technique for four , metatarsal osteotomy for two and arthrodesis of the first metatarsophalangeal osteotomy for one foot . the results of soft tissue procedures , such as mcelvenny or farmer technique , and those of arthrodesis were satisfactory , but the metatarsal osteotomy produced unsatisfactory results . they reported that metatarsal osteotomy could cause footwear problems due to varus deformity of the second and third toes or shortening of the first ray . even amputations of digits were needed to improve the symptoms in their series.5 ) in the current study , an osteotomy of the first metatarsal or proximal phalanx was more frequently performed than in that of mills and menelaus5 ) and good to excellent results were yielded in five of seven feet . specifically , a soft tissue procedure alone was performed in three feet , an osteotomy alone in three and a combined soft tissue procedure and osteotomy in four feet ( table 1 ) . as result , two of three feet with soft tissue procedure alone had revision surgeries ( patients 1 and 3 ) and two of three feet with osteotomy alone had poor clinical outcomes because of unsatisfactory cosmesis ( bilateral in patient 5 ) . on the other hand , all of four feet with combined procedures had excellent or good results without any complication or subsequent surgery . our study results demonstrated that an osteotomy in conjunction with adequate soft tissue procedure would be a reliable option for the correction of congenital hallux varus . in this series , a soft tissue procedure alone did not provide satisfactory correction and further correction was deemed necessary in case of a combined bony deformity of the metatarsal . our technique , consisting of farmer technique and opening wedge osteotomy , provided satisfactory correction and good clinical results . we also postulated that an osteotomy plays some role in congenital hallux varus surgery , like described as follows : first , an osteotomy can be used to further correct any residual deformity after the soft tissue procedure . it is possible that varus angulation could remain at the first metatarsophalangeal joint or interphalangeal joint due to the severity and complexity of the deformity , and widening of the first web space could still be present after a soft tissue procedure alone . widening of the forefoot should be corrected because it can result in difficulties wearing footwear or painful calluses . a lateral closing osteotomy or medial open wedge osteotomy of the first metatarsal can reduce the first and second intermetatarsal angles and cause relief of symptoms . an osteotomy can not only reduce varus angulation , but can also narrow the gap between the first and second toes . second , a short first metatarsal can be corrected by interposition of a bone graft after osteotomy . uncorrected shortening of the first metatarsal can cause footwear problems and residual deformities.5 ) graft positioning following a medial open - wedge osteotomy may be used for lengthening as well as to reduce varus angulation.8 ) leb involving the first metatarsal is a common cause of hallux varus.2,6,13,15,16 ) in the current study , it was clear that congenital hallux varus in four feet resulted from a leb of the first metatarsal or proximal phalanx . all of these feet had a residual hallux varus even after previous removal of polydactyly approximately 12 months after birth.13,14,15,19 ) therefore a leb could not have been considered at the initial surgery until two years of age as it is the time point where the ossification of the proximal and distal ossification centers occurs and it can not be detected on radiographs before.13,14,15,19 ) a longitudinal growth of the digit is impossible because the longitudinal bony bracket can not elongate sufficiently if a leb is not treatedand the growth occurs in a c - shape along the shortened side of the bone . bracketing along the medial shaft of the bone probably led to varus deviation with growth in these patients . sobel et al.13 ) emphasized that a leb should not be overlooked when treating pediatric deformities , including congenital hallux varus . we agree that a bracket epiphysis should be suspected if a bone appears short or wide on radiographs in conjunction with one of the known associated anomalies or syndrome.13,19 ) diagnostic modalities , including magnetic resonance imaging or ultrasonography , have been shown to be effective in children less than two years of age before the occurrence of ossification of the bracket.14,19 ) numerous surgical procedures to treat a metatarsal leb have been described ; these include bracket chondro - osteotomy accompanied by fat interposition , resection of the leb with silicone polymer or polymethylmethacrylate interposition , simple bracket excision before ossification , distraction osteogenesis and metatarsal osteotomy after complete closure of the leb.6,15,16,17,20 ) the overall goal of any of these procedure is to eliminate the tethering effect of the growth plate by removing the bar and therefore to promote growth in a lengthwise fashion.6,19 ) we performed a medial open - wedge osteotomy at the diaphysis of the first metatarsal with interposition of an allogenous strut graft to resect the abnormal longitudinal section of the epiphysis , to correct varus angulation and to lengthen the short first metatarsal . three of four feet with a leb were sufficiently corrected and showed favorable clinical outcomes using this method . however , in patient 5 ( poor results ) , the varus angulation of the first metatarsal shaft still remained and the longitudinal growth of the first metatarsal was not sufficient at the final follow - up , although the hallux varus did not have a gross physical appearance . the radiographic results of this patient were inferior to those reported by mubarak et al.21 ) ; these authors demonstrated successful longitudinal growth and a normally - shaped metatarsal after central physiolysis for metatarsal leb . although the small size of our series precludes a definitive conclusion on the optimal treatment strategy , based on our findings we only regard an osteotomy in conjunction with soft tissue reconstruction as being of importance . furthermore , patient 's age is another factor that must be assessed when considering surgery . as shown in our series , a leb is one of the causes of the deformity and should be taken into consideration at the initial evaluation . resection of a bracket epiphysis should be performed as with other procedures to prevent recurrent deformity . we also recommend further evaluation with magnetic resonance imaging or ultrasonography before surgery if the presence of a leb is in doubt , especially for patients aged two years or less .

the discovery of undifferentiated , actively proliferating neural stem cells ( nscs ) in the mature brain opened a brand new chapter in the contemporary neuroscience . adult neurogenesis appears to occur in specific brain regions ( including hypothalamus ) throughout vertebrates life , being considered an important player in the processes of memory , learning , and neural plasticity . in the adult mammalian brain , nscs are located mainly in the subgranular zone ( sgz ) of the hippocampal dentate gyrus and in the subventricular zone ( svz ) of the lateral ventricle ependymal wall . besides these classical regions , hypothalamic neurogenesis occurring mainly along and beneath the third ventricle wall seems to be especially well documented . neurogenic zones in sgz , svz , and in the hypothalamus share some particular common features like similar cellular cytoarchitecture , vascularization pattern , and extracellular matrix properties . hypothalamic neurogenic niche is formed mainly by four special types of radial glia - like tanycytes . they are characterized by distinct expression of some neural progenitor and stem cell markers . moreover , there are numerous suggestions that newborn hypothalamic neurons have a significant ability to integrate into the local neural pathways and to play important physiological roles , especially in the energy balance regulation . newly formed neurons in the hypothalamus can synthesize and release food intake regulating neuropeptides and they are sensitive to the leptin . on the other hand , high - fat diet positively influences hypothalamic neurogenesis in rodents . the nature of this intriguing new site of adult neurogenesis is still so far poorly studied and requires further investigations .

over the last hundred years , neuroscience has approved a presumption that the process of neurogenesis is not present in the structures of the adult brain . beginning with the pioneer studies by santiago ramon y cajal and camillo golgi , it was commonly thought that neural cells forming mature cns are definitively postmitotic , do not arise de novo , and that plasticity adaptation mechanisms are based only on the origin of projections and synaptic connections between neurons formed in early brain development stages ( de castro et al . mitotic divisions of ependymal cells located along lateral ventricles of mature rat brain were found for the first time by ezra allen already in 1912 . however , only after recognition of molecular mechanisms of dna synthesis , it was possible to unequivocally identify undifferentiated , proliferatively active neural stem cells ( nscs ) in mature brain ( allen 1912 ; balu and lucki 2009 ) . it was achieved in 1965 by joseph altman group ( altman and das 1965 ) using tritium labeled thymidine to detect replicating dna of precursor cells . this discovery opened the brand new , promising , and intriguing chapter of contemporary neurobiology . nevertheless , the significance of the adult neurogenesis is still far from being explained . a number of recent studies unequivocally reveal the role of this phenomenon in the processes of cns plasticity , learning , and memory ( deng et al . the problem of relations between adult neurogenesis and pathogenesis or / and course of psychiatric diseases like bipolar disorder ( walton et al . 2012 ; braun and jessberger 2014 ) , schizophrenia ( reif et al . 2007 ; eisch et al . 2008 ; christian et al . 2010 ; decarolis and eisch 2010 ; de koning et al . 2012 ) , and particularly depression ( krishnan and nestler 2008 ; lucassen et al . 2010 ; mahar et al . experiments on animal models have proven that elevated levels of stress hormones , observed in depression , often correlated with neurogenesis process reduction . numerous antidepressants , commonly used in the therapy of patients suffering from depression , have strongly stimulating influence on new neurons formation in particular brain structures ( santarelli et al . 2003 ; boldrini et al . 2009 ) . however , there are some doubts and assumptions stating that attenuation of neurogenesis is not a cause of depression , but it is rather one of its functional outcomes ( airan et al . for quite a long - time postulated association between disturbances in formation , proliferation , and differentiation of nscs and pathogenesis of neurological diseases , alzheimer s ( veereraghavalu et al . 2009 ; rodriguez et al . 2008 ) and parkinson s ( bertilsson et al . 2008 it is suggested that decrease of self - renewal rate of certain neural cells populations , e.g. , in substantia nigra , hippocampus , or some neocortex areas ( bonfanti and peretto 2011 ) , could constitute one of the causes of mentioned diseases . based on this assumption , intensive search for the substance selectively stimulating neurogenesis in cns is currently in progress . relatively promising and quite well - studied aminopropyl carbasole and its newly synthesized derivatives ( pieper et al . 2013 ) might become potential , but still hypothetical drugs being able to improve clinical state of patients with neurodegenerative disorders . a decrease in proliferation rate and survival of mature brain neural progenitors is probably related to chronic stress ( mirescu and gould 2006 ) and long - lasting insomnia ( meerlo et al . on the other hand , some data suggest cautiously that among consequences of ischemic stroke and traumatic brain injury , there is also stimulation of adult neurogenesis as one of potential brain repair mechanisms ( kernie and parent 2010 ; bellenchi et al . up till now , two distinctive regions of active and constant neurogenesis process in adult mammalian cns have been identified : subgranular zone ( sgz ) in dentate gyrus structure and subventricular zone ( svz ) located subependymally in the vicinity of brain lateral ventricles ( fig . 1 ) . sgz stem cells are the source of dentate gyrus granular cells , whereas svz forms progenitor cells migrating as rostral migratory stream ( rms ) to olfactory bulb , where they differentiate into interneurons suitable for this brain area . the neural precursor cells ( npcs ) must be located in neurogenic niche , a permissive milieu , which maintain their constant ability to divide and form mature neurons even in the adult brain . the hippocampal sgz niche is created mainly by astrocytes and blood vessel endothelium , numerously represented in this layer of dentate gyrus ( rolando and taylor 2014 ) . in sgz cytoarchitectonics three types of proliferatively active cells have been identified : ( 1 ) radial glia - like stem cells , type i cells ; ( 2 ) non - ciliated cells with nestin expression , type ii cells also described as transiently activated progenitor cells , tap cells ; and ( 3 ) neuroblasts with doublecortin protein ( dcx ) and ki67 expression . on the other hand , in svz structure , four mitotically active and self - renewing cell populations have been distinguished : ( 1 ) ependymocytes with cd133 expression ; ( 2 ) gfap - positive astrocytic stem cells , type b1 cells ; ( 3 ) transiently activated progenitor ( tap ) cells with mash1 expression , type c cells ; and ( 4 ) dcx protein expressing neuroblasts , type a cells ( okano and sawamoto 2008).fig . 1the canonical sites of adult neurogenesis in the rat brain and the scheme of subventricular zone ( svz ) stem cell niche . a subgranular zone of dentate gyrus ( sgz , dark blue ) , subventricular zone ( svz , red ) surrounding the lateral ventricle ( lv , black ) , red arrow indicates direction of neuron migration from svz to olfactory bulb ( rostral migratory stream , rms ) . b cellular composition of svz niche sectioned vertically to ependymal surface ; e1 cells ( yellow ) and e2 cells ( red ) concentrically surround centrally located cilia of b1 cells ( dark blue ) forming the pinwheel structure . neuroblasts a cells ( gray ) and transiently activated stem cells c ( green ) are also presented . c horizontal view of the mentioned svz niche ( color figure online ) the canonical sites of adult neurogenesis in the rat brain and the scheme of subventricular zone ( svz ) stem cell niche . a subgranular zone of dentate gyrus ( sgz , dark blue ) , subventricular zone ( svz , red ) surrounding the lateral ventricle ( lv , black ) , red arrow indicates direction of neuron migration from svz to olfactory bulb ( rostral migratory stream , rms ) . b cellular composition of svz niche sectioned vertically to ependymal surface ; e1 cells ( yellow ) and e2 cells ( red ) concentrically surround centrally located cilia of b1 cells ( dark blue ) forming the pinwheel structure . neuroblasts a cells ( gray ) and transiently activated stem cells c ( green ) are also presented . c horizontal view of the mentioned svz niche ( color figure online ) ependymocytes play a fundamental role in svz niche structure formation . here , three cell subpopulations have been distinguished : previously mentioned cd133 cells functioning as nscs as well as e1 and e2 ependymal cells with cd24 expression . multiciliated e1 cells are dominating , whereas e2 ependymocytes make up less than 5 % of ependymal cell population and possess only two cilia with particularly big basal bodies . studies of rat brain svz niche cytoarchitecture have revealed unusually peculiar spatial pattern of stem cells and ependymocytes . central part of the niche is formed by gfap - positive b1 stem cells surrounded in their apical , ciliated part by several e1 and e2 ependymocytes forming a unique rosette or pinwheel - like structure ( mirzadeh et al . on the other hand , central and basal part of b1 cells remain in contact with concentrically located c and a cells . b1 cells have long cytoplasmatic projections reaching the surface of blood capillaries ( fig . 1 ) . extracellular matrix , in the form of three - dimensional spatial structures called fractones , plays also an important role in the niche functioning . in human and rodent brain , fractones are characterized by collagen iv , 1 and 1 laminin , perlecan and nidogen expression , and lacking laminin 2 ( kerever et al . limited fractone subpopulation surrounding highly mitotically active cells additionally demonstrates immunoreactivity toward n - sulfate heparan sulfate proteoglycan ( n - sulfate hspg ) . fractones and subependymal capillaries are probably capable of binding fibroblast growth factor 2 ( fgf-2 ) , which substantially promotes nscs proliferation ( kerever et al . matrix metaloproteinases mmp-1 and mmp9are also considered to play a significant role in internal niche environment modification and nscs fate determination ( tonti et al . the origin of neurons in the sgz and svz is probably strictly regulated by various growth factors ( duan et al . 2008 ; balu and lucki 2009 ; llorens - martn et al . 2010 ) , classical neurotransmitters ( petrus et al . 2009 ; okeeffe et al . 2009 ; yanpallewar et al . 2010 ) , neuropeptides ( howell et al . 2005 ; garza et al . 2008 ; moon et al . 2009 ; manda and reiter 2010 ) , and cytoskeleton proteins ( jiao et al . 2010 ) . a large spectrum of commonly administered pharmaceuticals such as selective serotonin reuptake inhibitors ( encinas et al . 2009 ) , antipsychotics ( wang et al . 2004 ; keilhoff et al . 2010 2013 ) , hypnotic and normothymic drugs ( yu et al . 2009 ; boku et al . 2010 ) , lithium ions ( wexler et al . 2008 ) and even steroid hormones ( brummelte and galea 2010 ) , and cyclooxygenase-2 inhibitors ( goncalves et al . 2010 ) can also significantly affect the course of adult neurogenesis . there are numerous evidences , that new neural cells can also be formed , usually in limited number , outside sgz and svz , in potential niches located in distinct structures of mature brain . 2002 ; luzzati et al . 2003 ) , striatum ( emsley et al . 2005 ; bdard et al . multipotent neural precursors have been also isolated from certain neocortical areas ( dayer et al . currently , there is not enough evidence proving that these putative , non - classical neurogenic sites are stable in time and space , and that the continuous origin of new neurons is really taking place . therefore , the presence of established and permanently functional nscs niches in mentioned brain areas is still very controversial . however , hypothesis suggesting existence of a stable hypothalamic neurogenic site located in subependymal zone of the third ventricle ( hypothalamic ventricular zone , hvz ) seems to be particularly intriguing and relatively well documented ( kokoeva et al . 2005 ; more importantly , it is also suggested that neurogenesis process is significantly involved in various hypothalamic regulatory mechanisms , especially in the energy balance regulation ( pierce and xu 2010 ) . the key role of ependymocytes lining lateral ventricles has become a source of presumptions that these cells could also take part in hypothalamic nscs niche formation , located in the vicinity of the third ventricle . studies held on rodents have revealed that proliferatively active cells are present not only in ependyma , but also in surrounding neuropilus ( kokoeva et al . 2007 ; migaud et al . 2010 ) . to date , two adult hypothalamic neurogenesis regions have been distinguished : located in lateral walls of the third ventricle at the level of paraventricular and arcuate nuclei ( hypothalamic ventricular zone , hvz ) and hypothalamic proliferating zone ( hpz ) formed by tanycytes located at the bottom of the third ventricle in median eminence region ( fig . 2 ) . tanycytes that form rat and human hvz are characterized by expression of proteins typical for neural precursor cells e.g. , nestin ( wei et al . 2002 ) , vimentin ( bolborea and dale 2013 ) , and doublecortin - like protein ( dcl ) ( saaltink et al . 2012 ) . they are also enriched for neural stem and progenitor genes such as sox9 , notch 1 and 2 , he s 1 and 5 , cd63 , fzd5 , dirc , ntrk-2t1 , and thrsp ( rodrguez et al . 2005 ; shimogori et al . recently , the expression of sox2 , a selective marker of nscs , was also reported ( lee et al . 2012 ; li et al . 2012 ) . four types of radial glia - like tanycytes have been identified , differing from each other in gene profile and location in the third ventricle wall . tanycytes 1 are present at the level of ventromedial nuclei , whereas 2 subpopulation in the vicinity of arcuate nuclei . both cell types send their long processes toward blood vessels , hypothalamic neurons , and glial cells . elongated 1 tanycytes form the lateral part of infundibular recess , while 2 cells line the floor of third ventricle inside the median eminence forming the hpz . the basal processes of 1 tanycytes are in contact with endothelial cells and terminals of gnrh - expressing neurons , whereas processes of 2 cells head toward blood vessels of hypothalamo - pituitary portal system and also pia mater ( lee and blackshaw 2012 ) ( fig . 2 ) . noteworthy , the 2 tanycytes show particularly high level of aforementioned hes1 and hes2 marker expression ( lee et al . some authors report the presence of the following zones of the third ventricle wall : ventrally located tanycytic zone composed of both and 2 tanycytes , transition zone with 1 tanycytes , and the most dorsal ependymal zone which contains only ependymal cells ( mathew 2008 ) . most recently , two kinds of tanycytes have been found , dorsally located fgf10 , blbp , gfap+ , glast+ , s100+ subtype and ventral fgf10 + , blbp+ , gfap , glast subtype ( haan et al . 2013 ) . probably , the fgf10 + tanycytes are the neural stem / progenitor cells and can continuously proliferate and form parenchymal neurons and glial cells . importantly , tanycytes located on the median eminence are sensitive to the hormones and nutritional substances carried by the blood flowing in capillary vessels . in turn , the somata of tanycytes lining the third ventricle are able to receive the molecular signals e.g. , growth factors from the cerebrospinal fluid.fig . 2schematic representation of the subependymal npcs niche in the animal hypothalamus . the neurogenic zones are located in the vicinity of the third ventricle at the level of paraventricular , ventromedial , and arcuate nuclei ( hypothalamic ventricular zone , hvz ) , while tanycytes of the median eminence form the hypothalamic proliferating zone ( hpz ) . ciliated and non - ciliated ependymocytes line the third ventricle as well as four types of tanycytes . various populations of glial cells ( fibrous astrocytes , subependymal astrocytes , and oligodendrocyte precursor cells ) and neural cells ( peptidergic neurons , parenchymal proliferative cells , gnrh - releasing neurons , and doublecortin - expressing immature neurons ) are located beneath the ependymal layer forming the npcs niche . neuropeptides and other regulatory factors are released from the capillary network schematic representation of the subependymal npcs niche in the animal hypothalamus . the neurogenic zones are located in the vicinity of the third ventricle at the level of paraventricular , ventromedial , and arcuate nuclei ( hypothalamic ventricular zone , hvz ) , while tanycytes of the median eminence form the hypothalamic proliferating zone ( hpz ) . ciliated and non - ciliated ependymocytes line the third ventricle as well as four types of tanycytes . various populations of glial cells ( fibrous astrocytes , subependymal astrocytes , and oligodendrocyte precursor cells ) and neural cells ( peptidergic neurons , parenchymal proliferative cells , gnrh - releasing neurons , and doublecortin - expressing immature neurons ) are located beneath the ependymal layer forming the npcs niche . neuropeptides and other regulatory factors are released from the capillary network the identity of hypothalamic neural progenitor cells ( npcs ) remains so far very controversial . some studies suggest that -tanycytes are the main proliferating cells in the hypothalamus of young adult mice ( haan et al . they are the neural progenitors in the median eminence but not in other hypothalamic regions . in rats , the rate of neurogenesis within hvz and median eminence is significantly higher in the first weeks of postnatal life than in adult animals ( lee et al . 2012 ) . on the other hand , a recent report identifies the lateral -tanycytes as local npcs and crucial cells in the mouse hypothalamic niche . the long - term study using a lineage tracing in vivo shows that -tanycytes form a self - renewing population that differentiate to new tanycytes , astrocytes , and neurons . moreover , this finding demonstrates clearly that only gfap - positive dorsal -tanycytes , but not -tanycytes or parenchymal cells may form neurospheres ( robins et al . 2013 ) . probably , the fibroblast growth factor ( fgf ) signaling is required to maintain -tanycyte proliferation , and increased fgf level enhances the rate of mitotic divisions . interestingly enough , the hypothalamic tanycytes are characterized by selective expression of transcription factor called retina and anterior pituitary neural fold homeobox ( rax ) . this molecule shows a similar pattern of expression to retinoic acid receptor responder ( rarres-2 ) , a marker of third ventricle ependymal cells . the rax action is considered as required for tanycyte and ependymocyte differentiation at the level of hypothalamus ( miranda - angulo et al . the regulatory mechanism by which rax determines the fate of tanycytes is still unknown ; however , some recent studies suggest the involvement of six6 and hmgb2 , the factors selectively expressed in hypothalamic progenitor cells ( andreazzoli et al . acetylcholine , histamine , and atp sensitive tanycytes can activate intracellular calcium signaling regulatory pathway ( frayling et al . it should be noted that atp stimulates ca waves in the subventricular progenitor cells acting via p2y1 receptor ( weissman et al . tanycytes as the radial glial cells can release atp to control neural progenitor cell proliferation , and they also have the aforementioned type of purynergic receptors . moreover , tanycytes synthesize ectonucleoside triphosphate diphosphohydrolase 2 ( ntpdase 2 ) that inactivate the extracellular atp ( braun et al . although tanycytes appear to be the key players in the npcs niche formation , they are not only nscs in the hypothalamus . a population of sox2 expressing cells scattered within the parenchymal regions also has a proliferative activity ( kokoeva et al . undoubtedly , advanced genetic tools such as using of inducible cre lines e.g. , hgfapcreer , pdgfr-crert , or glast - creer should be useful to reveal whether these brdu - positive cells can form a quiescent hypothalamic neural progenitor pool ( ganat et al . the proliferating gfap - positive subependymal astrocytes have an apical process with single cilium and their periventricular basement membranes form a three - dimensional network that is typical for svz microachitecture ( prez - martin et al . numerous granular dcx - positive immature neuroblasts were identified close to the wall of the third ventricle ( fig . 3 ) . on the other hand , the bipolar dcx cells found in the parenchyma of ventromedial hypothalamus have fusiform perikarya and sometimes elongated processes displaying the features of migrating cells that may undergo maturation within this region ( batailler et al . , the neuroblasts located inside the subependymal niche can form a kind of migratory stream spreading to the hypothalamic nuclei ( haan et al . 2013).fig . 3hypothalamic neurogenic zones in the rat brain fluorescence ( 20-m - thick frozen sections ) and classical immunostaining ( 7-m paraffin sections ) of the adult ( 3-month - old ) rat hypothalamus . primary antibodies dilutions : dcx 1:100 ( santa cruz biotechnology ) , nestin 1:500 ( millipore ) , and ki67 1:100 ( abcam ) . brains were fixed with 4 % buffered formalin , dehydrated with ethanol or sucrose solutions , and then embedded on paraffin or mounted in oct medium . the photomicrograph shows the doublecortin ( dcx ) expressing cells in the parenchyma of paraventricular nucleus ( a , b , a single cell in higher magnification nestin expressing cells : tanycytes at the level of ventral hypothalamus , arcuate nuclei and within the median eminence ( d , zone of tanycytes inset d1 , 1 tanycytes , h ) , and ependymocytes in the dorsal hvz ( e g ) . scale bars 50 m , excluding c , d , i ( 100 m ) , and a1 , b1 ( 10 m ) . arc arcuate nucleus , me median eminence , pa paraventricular nucleus , vmh ventromedial hypothalamus , 3v third ventricle hypothalamic neurogenic zones in the rat brain fluorescence ( 20-m - thick frozen sections ) and classical immunostaining ( 7-m paraffin sections ) of the adult ( 3-month - old ) rat hypothalamus . primary antibodies dilutions : dcx 1:100 ( santa cruz biotechnology ) , nestin 1:500 ( millipore ) , and ki67 1:100 ( abcam ) . brains were fixed with 4 % buffered formalin , dehydrated with ethanol or sucrose solutions , and then embedded on paraffin or mounted in oct medium . the photomicrograph shows the doublecortin ( dcx ) expressing cells in the parenchyma of paraventricular nucleus ( a , b , a single cell in higher magnification nestin expressing cells : tanycytes at the level of ventral hypothalamus , arcuate nuclei and within the median eminence ( d , zone of tanycytes inset d1 , 1 tanycytes , h ) , and ependymocytes in the dorsal hvz ( e g ) . scale bars 50 m , excluding c , d , i ( 100 m ) , and a1 , b1 ( 10 m ) . arc arcuate nucleus , me median eminence , pa paraventricular nucleus , vmh ventromedial hypothalamus , 3v third ventricle a large spectrum of endogenous regulatory substances , especially growth factors , is able to regulate the course of adult hypothalamic neurogenesis . the brain - derived growth factor ( bdnf ) , insulin - like growth factor 1 ( igf-1 ) , fibroblast growth factor ( fgf ) , epidermal growth factor ( egf ) , and ciliary neurotrophic factor ( cntf ) seem to play a key role as the activators of neuronal progenitor divisions . a recent report suggests that also gonadotropin - releasing hormone ( gnrh ) can increase the proliferating activity of hypothalamic npcs in aged mice ( zhang et al . it should be emphasized that the aforementioned growth factors promote neurogenesis in different hypothalamic centers in a distinctly selective manner ( sousa - ferreira et al . thus , bdnf administration increases cell proliferation in the paraventricular nucleus , both fgf and cntf induce neurogenesis in the parenchymal zone of the arcuate nucleus , while igf-1 in the medial periventricular region ( pencea et al . injection of fgf and egf promotes cell proliferation in the ependymal layer , whereas extended subcutaneous injection increases neurogenesis in the arcuate nucleus ( xu et al . as hypothalamus is responsible for the constant control of many autonomous functions of the organism , it was considered that neurogenesis in this region may support some of these processes or act as a compensatory mechanism in response to different environmental signals . indeed , in the light of latest discoveries , it is possible that neurogenesis in hypothalamus is potentially involved in regulation of energy homeostasis via modulation of eating behavior . neuronal populations located within distinct regions of the hypothalamus express various neuropeptides modulating food intake . in the arcuate nucleus ( arc ) , there is an expression of orexigenic factors like agouti - related peptide ( agrp ) , neuropeptide y ( npy ) , as well as anorexigenic factors like pro - opiomelanocortin ( pomc ) and its derivative-melanocyte - stimulating hormone ( -msh ) . on the other hand , , there is a dynamic balance between regulatory neuropeptides activities , but diet - related factors like obesity and high saturated fatty acid ( sfa ) intake cause hypothalamic neurogenesis inhibition and simultaneous defective energy balance . it suggests a link between eating behavior and intensity of hypothalamic neurogenesis ( fig . the limited neuronal loss , homeostasis , and some growth factors promote the npcs proliferation , while severe cellular loss , obesity , and saturated fatty acids ( sfa ) inhibit the neurogenesis . arc arcuate nucleus , pvn paraventricular nucleus , lha lateral hypothalamus , pomc pro - opiomelanocortin , npy neuropeptide y , agrp agouti - related protein , crh corticotrophin - releasing hormone , trh thyrotropin - releasing hormone , mch melanin - concentrating hormone , bdnf brain - derived neurotrophic factor , cntf ciliary neurotrophic factor , igf-1 insulin - like growth factor 1 , fgf fibroblast growth factor , egf epidermal growth factor hypothalamic neurogenesis plays a role in the regulation of energy balance . the limited neuronal loss , homeostasis , and some growth factors promote the npcs proliferation , while severe cellular loss , obesity , and saturated fatty acids ( sfa ) inhibit the neurogenesis . arc arcuate nucleus , pvn paraventricular nucleus , lha lateral hypothalamus , pomc pro - opiomelanocortin , npy neuropeptide y , agrp agouti - related protein , crh corticotrophin - releasing hormone , trh thyrotropin - releasing hormone , mch melanin - concentrating hormone , bdnf brain - derived neurotrophic factor , cntf ciliary neurotrophic factor , igf-1 insulin - like growth factor 1 , fgf fibroblast growth factor , egf epidermal growth factor it was also shown that newborn neural cells in the hypothalamus are functional they express orexigenic peptides ( agrp , npy)and anorexigenic peptide ( pomc ) ( kokoeva et al . 2005 ; pierce and xu 2010 ) . simultaneously , some of them have ability to respond to leptin administration by inducing strong phospho - signaling transducer and activator of transcription 3 ( pstat3 ) immunoreactivity ( kokoeva et al . 2005 ) . interesting data concerning significance of hypothalamic neurogenesis have been provided with the use of antimitotic agent arabinosylcytosine ( arac ) or focal irradiation procedure both applied in order to silence neurogenesis process . in wild - type mice , however , this effect is not observed in mutant mice with degenerated agrp - positive cells . as such genetic manipulation causes also the increase in cell proliferation rate in arcuate nucleus of the hypothalamus and because inhibition of this proliferation ( by arac ) results in decreased feeding and weight loss , it is suggested that hypothalamic neurogenesis plays a compensatory role in preventing anorectic effects of neurodegeneration ( pierce and xu 2010 ) . in another study , mice were kept on high - fat diet ( hfd ) and infused with ctnf to induce neurogenesis within hypothalamic energy balance centers ( kokoeva et al . ctnf treatment resulted in weight loss , but the long - term effect was present only in mice with undistrupted neurogenesis process . if arac was administered , then mice gained weight 20 days after ctnf treatment , which indicates that neurogenesis in hypothalamus is crucial for maintaining ctnf - mediated metabolism regulation ( kokoeva et al . later , it was found that hfd alone enhances neurogenesis in the median eminence ( me ) of mouse hypothalamus . blocking of this process by focal irradiation leads to attenuation of weight gain and increase in animal activity levels , which proves that neurogenesis in me may promote fat storage and body mass increase ( lee et al . 2012 ; lee and blackshaw 2012 ) . on the other hand , studies on rodent obesity models revealed the significant decrease in hypothalamic neurogenesis process rate in arcuate nucleus of obese mice . it further suggests that regulation of energetic homeostasis can be mediated by hypothalamic neurogenesis ( mcnay et al . are considered as new neural cells precursors , detailed studies have been performed to characterize these cells and trace the fate of their progeny . experiments held on mice confirmed that tanycytes positive for fibroblast growth factor 10 ( fgf10 ) give rise to cells that are located mainly in arcuate nucleus . some of them expressed npy and were sensitive to fasting alone or followed by leptin administration , which was proven by c - fos and pstat immunostaining . moreover , newly formed neurons were able to significantly expand their axonal / dendritic arborization ( haan et al . tanycytes are also known to respond to glucose by releasing atp ca waves , especially if it is applied selectively to cell bodies ( frayling et al . another interesting feature of tanycytes , suggesting their contribution to energy metabolism , is expression of orphan receptor gpr50member of melatonin receptor family ( sidibe et al . gpr50 is functionally linked to energy homeostasis , which was proven by observation of knock - out ( gpr50 ) mice having more stable body weight they were more resistant to obesity induced by high - energy diet and to fasting - induced weight loss ( ivanova et al . 2007 ; bolborea and dale 2013 ) . another intriguing point of view , from which adult hypothalamic neurogenesis can be analyzed , is the relationship between this process , obesity , and macronutrients dietary content . diet rich in saturated fatty acids ( sfa ) not only induced obesity and pre - diabetes in mouse model , but also caused reduction in the number of nscs and their proliferation potential , which was associated with inhibitor kappa/nuclear factor kappab ( ikk/nfb ) activation ( li et al . 2012 ; yon et al . different effects were observed when rats were fed with sfa - rich diet during pregnancy . resulting offspring showed higher proliferation rate of npcs together with the increase in blood circulating lipids and orexigenic peptides expression ( chang et al . 2008 ; yon et al . 2013 ) . additionally , some researchers linked neurogenesis process with endocannabinoid system . treatment with the cannabinoid-1 receptor inverse agonist ( am251 ) reduced food intake and modulated neurogenesis in rats , but the neurogenic change was present only in hfd - fed animals there was a reduction of neurogenesis levels in svz and hypothalamus , but induction in sgz . it suggests that hfd sensitizes the endocannabinoid system to modulate neurogenesis ( rivera et al . apart from well - documented contribution of neurogenesis in hypothalamus to energy balance regulation , some other potential functions of newly formed neural cells have been reported ( yuan and arias - carrion 2011 ) . it is supposed that social environment especially pheromones influences hypothalamic neurogenesis in adult female prairie voles . two - day - long male exposure increased the number of new formed cells in amygdala and hypothalamus in comparison to condition of female exposure and social isolation ( fowler et al . 2002 ) . moreover , immunohistochemical staining of female pig hypothalamus have revealed that new oxytocin - containing neuron formation rate in paraventricular nucleus ( pvn ) is greater in lactating sow and adult gilts in comparison to puberty ones ( raymond et al . formation rate of new neurons expressing vasopressin in vasopressin and oxytocin - containing nucleus ( von ) was positively correlated with the growth of this brain region more newly formed vasopressin positive neurons were shown in adult pigs , than in adolescent individuals ( rankin et al . it may support the hypothesis that adult hypothalamic neurogenesis is involved in sexual maturation process and can be sensitive to environmental conditions . the pubertal increase in anteroventral periventricular nucleus ( avpv ) volume seems to be connected with the hormone - dependent formation of new cells ( juraska et al . ( 2008 ) , brdu was administered to rats that were at different stages of puberty , and the labeled cell was quantified 3 weeks later . a number of avpv cells that had been born during puberty had transformed into mature neurons . interestingly , more brdu cells were found in the female than in the male avpv . unexpectedly enough , brdu - labeled cells were also found in the sexually dimorphic nucleus of the preoptic area ( sdn ) , with greater numbers of pubertally born cells in males than in females ( ahmed et al . however , these brdu - positive cells coexpressed neither neural nor glial markers , so their phenotype remains unclear . possibly , they require more than 34 weeks to become the fully differentiated neurons or astrocytes , as suggested for cortical interneurons ( cameron and dayer 2008 ; juraska et al . recently , accumulating reports seem to confirm the real existence of adult hypothalamic neurogenesis in the animal brain and highlight its potential role in the energy balance regulation . the cellular architecture of hypothalamic subependymal neurogenic niche is quite well described ; however , many crucial questions remain so far unanswered . first of all , the presence of npcs in the human hypothalamus is not yet reliably proven . moreover , we also do not know whether hypothalamic neurogenesis can contribute to energy homeostasis in humans . both eating disorders and obesity pathogenesis are strictly related to the disturbances at the level of hypothalamic neuronal populations . the blockage of hypothalamic neurogenesis may alter the body weight in rodents supporting the hypothesis that local npcs play role in the control of energy expenditure . having in mind that hypothalamic neurogenesis may be regulated by various factors , an intriguing idea of potential pharmacomodulation of this process should not be excluded . it would be interesting to study the influence of some drugs modulating appetite ( as their main or side effect ) on hypothalamic neurogenesis process in order to better understand their mechanism of action . in the future , it could have some practical implication allowing modification and improvement of existing therapies interfering with organism energetic homeostasis for example , treatment of eating disorders and schizophrenia . taking to the account recent results from experiments on pigs and prairie voles , it is also justified to perform research on hormonal regulation of hypothalamic neurogenesis and its impact on various physiological processes connected with hormonal activity . finally , promising future direction of research concerns hypothalamus - derived nscs cultures . it would contribute to detailed characterization of neural precursors and resulting application of hypothalamic regenerative potential .

background : scrub typhus is one of the differential diagnoses for fever with thrombocytopenia . ards associated with scrub typhus has high morbidity and mortality.aims:to evaluate clinical features , lab values , and outcome in patients with scrub typhus and comparison in patients with or without ards.methods:a prospective observational study was conducted on 109 patients with febrile illness and thrombocytopenia during a period of 12 months . all 109 patients were tested with both immune - chromatography test and weil felix test . patients having either immune - chromatography test / weil felix test positive have been included and considered as scrub typhus positive whereas negative for both immune - chromatography and weil felix test were excluded . clinical features , lab parameters , and outcome were evaluated in all patients with scrub typhus . statistical analysis used in this study was t-test.results:among 58 patients who were included ( after exclusion of 51 patients among total of 109 patients ) 34 patients had no ards and 24 patients had ards . the clinical feature like dyspnoea , cough , low blood pressure ( map<65 mmhg ) , ivc collapsibility ( by ultrasound ) and laboratory parameters like decreased hemoglobin , hematocrit , serum albumin , and increased serum creatinine , serum total bilirubin , sgot , sgpt , ldh , cpk , and serum lactate were statistically significant ( p < 0.0001 ) in scrub typhus patients group with ards . the higher titers of weil - felix can be correlated with more severe form of disease according to our observation . all 34 scrub typhus patients without ards recovered completely . among 24 scrub typhus patients with ards , 22 patients recovered , and 2 patients died.conclusion:scrub typhus is an important differential diagnosis in a patients having fever with thrombocytopenia . scrub typhus associated with ards has high morbidity and mortality . early diagnosis and treatment with doxycycline can prevent the occurrence of ards

a billion people are at risk , and nearly a million cases are reported every year . o. tsutsugamushi is an obligate intracellular gram - negative bacterium ; it grows freely in the cytoplasm of infected cells since it lacks vacuolar membrane . it has 5 major serotypes - boryon , gilliam , karp , kato , and kawazaki . scrub typhus is one of the differential diagnosis for fever with thrombocytopenia or hemorrhagic fever . scrub typhus can manifest with either nonspecific febrile illness or constitutional symptoms ( fever , rash , myalgia , and headache ) or with organ dysfunction such as kidney ( acute kidney injury [ aki ] ) , lungs ( acute respiratory distress syndrome [ ards ] ) , heart ( myocarditis ) , liver ( hepatitis ) and central nervous system ( meningitis ) . the mortality of scrub typhus in untreated patients range from 0% to 30% and tends to vary with age and region of infection . the involvement of lungs has been described which range from bronchitis and interstitial pneumonitis to ards . ards is one of the serious complications of scrub typhus , which has high morbidity and mortality . the occurrence of ards is high in scrub typhus patients who were diagnosed late and received antibiotics late . this study may be useful in the early diagnosis of scrub typhus ( using clinical features and initial lab values ) so that the early initiation of specific antibiotic ( doxycycline ) can prevent the occurrence or reduce the severity of ards . after informed consent either from patient or patient attender ( in case where patient is not able to give consent ) and ethical clearance from the institutional ethical committee , a prospective observational study was conducted on 109 patients with febrile illness and thrombocytopenia during a period of 12 months between june 2012 and may 2013 in acute medical care unit of a super specialty hospital . all 109 patients were tested with both immune - chromatography test ( ict ) and weil - felix test ( wft ) . patients having either ict / wft ( of 1:80 titer and above ) positive have been included and considered as scrub typhus positive whereas negative for both immune - chromatography and wft were excluded from this observational study . 51 patients were excluded as they were negative for both ict / wft and remaining 58 patients were positive for either ict / wft . area of residence ( andhra pradesh ) , detailed clinical examination , presence or absence of eschar , rash and lymphadenopathy were noted . tropical and other co - endemic diseases such typhoid , dengue , leptospirosis and malaria tests were done in all patients . additional tests such as ultrasonography of abdomen , arterial blood gas analysis , chest x - ray , electrocardiogram , two - dimensional echocardiogram , cerebrospinal fluid study , electroencephalography , computed tomography / magnetic resonance imaging brain , blood , and sputum / bronchoalveolar lavage cultures , etc . , were sent as indicated . apache - ii and sequential organ failure assessment ( sofa ) were calculated for all these patients . ( 1 ) onset within 1-week of a known clinical insult or new or worsening respiratory symptoms , ( 2 ) bilateral lung opacities - not fully explained by effusions , lobar / lung collapse , or nodules , ( 3 ) pulmonary edema not fully explained by cardiac failure or fluid overload need objective assessment ( echocardiography ) to exclude hydrostatic edema if no risk factor present , ( 4 ) pao2 /fio2 300 mm hg with positive end expiratory pressure or continuous positive airway pressure 5 cm h2 o ) . various complications associated with disease ( aki , hypotension , ards , hepatitis , meningitis ) were managed accordingly . among 58 patients proved to be having positive for scrub typhus , 24 patients had ards and 34 patients had no ards . the incidence of aki , acute hepatitis and delay in starting treatment with doxycycline were significant in ards scrub typhus group compared to non ards scrub typhus group . the clinical characteristics of scrub typhus patients with or without ards table 2 shows pao2 /fio2 ratio , intensive care unit ( icu ) length of stay , and severity of infection . in our observational study among 24 patients with ards ( mild ards : 8 patients , moderate ards : 16 patients , and severe ards : 2 patients ) the lung injury score , intubation rate , ventilator days , icu length of stay , apache and sofa score were statistically significant as compared to non ards scrub typhus group . pao2/fio2 ratio and lis with mortality the clinical features of scrub typhus patients at the time admission have been shown in table 3 . the most common clinical feature in both groups were fever , headache , nausea , vomiting , myalgia , generalized weakness , pain abdomen , chest pain , conjunctival congestion , hepato - splenomegaly , eschar and macula - papular rashes . the clinical feature such as dyspnea , cough , low blood pressure ( mean arterial pressure < 65 mmhg ) , and inferior vena cava collapsibility of > 50% by ultrasound ( hypovolemia ) were statistically significant in scrub typhus patients with ards . the clinical features of scrub typhus patients with or without ards table 4 shows the initial laboratory parameters of scrub typhus patients at the time admission . the laboratory parameters such as decreased hemoglobin , hematocrit , serum albumin , and increased serum creatinine , serum total bilirubin , serum glutamic oxaloacetic transaminase , serum glutamic pyruvic transaminase , lactate dehydrogenase ( ldh ) , creatine phosphokinase ( cpk ) , and serum lactate were statistically significant ( p < 0.0001 ) in scrub typhus patients group with ards . the laboratory parameters ( at admission ) of scrub typhus patients with or without ards the correlation between wf and ict of scrub typhus patients at the time admission have been shown in table 5 . in our observational study all patients were positive for scrub typhus by ict but wf titers were insignificant ( < 1:80 ) in 7 of them . the mortality of scrub typhus in untreated patients range from 0% to 30% and tends to vary with age and region of infection . generally seen in people whose occupational or recreational activities bring them into contact with ecotypes favorable with vector chiggers . scrub typhus manifest as either nonspecific febrile illness or multi - organ dysfunction such as aki , ards , myocarditis , hepatitis , and meningo - encephalitis . in our prospective observational study conducted over 12 months , the involvement of lungs has been described which range from bronchitis and interstitial pneumonitis to ards . the pathogenesis of ards in scrub typhus is not known , thought to be immunological response of the lung to previous o. tsutsugamushi infection without direct invasion of the organism and diffuse alveolar damage without evidence of vasculitis . when the patients presented with atypical symptoms , initially they were not diagnosed ( delay in diagnosis ) as scrub typhus at outside hospital or when the patient came to our hospital it was average 11.333 0.570 days and 5.264 0.431 days from the onset of signs and symptoms in scrub typhus patients with and without ards , respectively . probably delay in treatment might have caused a high incidence of ards . chest x - ray showing acute respiratory distress syndrome in patient with scrub typhus table 2 shows pao2 /fio2 ratio , icu length of stay and mortality . the majority of patients in ards group had moderate ards , the number of ventilator days and icu mortality were high which was statistically significant ( p < 0.05 ) . eschar is a vesicular lesion at the site of mite feeding is the first sign of disease and also pathognomonic of scrub typhus often found in the groin , axilla , genitalia , and neck . this is rarely seen in south east asia and indian subcontinent . in our observational study , 6 out of 34 scrub patients without ards and 4 out of 24 scrub patients with ards had eschar . figure 2 shows the eschar seen near right axilla in one of our patient coming from an endemic area . eschar over anterior chest wall near axilla - on right side table 3 shows the clinical features of scrub typhus patients with or without ards . fever , headache , conjunctival congestion , myalgia , generalized weakness , vomiting , and pain abdomen were the common clinical features . in ards group , the hypovolemia was more common which was statistically significant ( p < 0.05 ) . table 4 shows the laboratory parameters ( at admission ) of scrub typhus patients with or without ards . patients in ards group had more severe disease in the form of deranged liver parameters , increased serum creatinine , elevated ldh , cpk , and serum lactate . serology is the mainstay of diagnosing scrub typhus , immuno - florescence antibody test or indirect immuno - peroxidase assay is the gold standard . due to high - cost factor and nonavailability of above two tests we have done both ict and wft in all 109 patients . based on either ict / wft positivity patients we considered as scrub typhus positive . remaining 58 patients were positive for interstitial cystitis test whereas 7 patients who are positive for ict had wf titer < 1:80 . this shows that the wft , which is cheap and commonly used in resource limited countries , has a sensitivity of 30% and specificity of 100% at titer breakpoint of 1:80 in detection of scrub typhus . wft results among patients of immuno - chromatography proven scrub typhus with or without ards have been shown in table 5 . the higher titers of wf can be correlated with more severe form of the disease according to our observation . treatment of scrub typhus was initiated with doxycycline in 56 patients and combination of doxycycline and azithromycin was used in 2 patients with ards and multiple organ dysfunction syndrome ( mods ) . most of the patients responded to doxycycline therapy within 2 - 3 days except 2 patients who died due to severe mods ( four organ involvement ) and they were referred fairly late to our hospital . azithromycin has shown to have comparable efficacy when compared to doxycycline in a small trial . rifampicin can be used in combination with azithromycin or doxycycline in cases of poor response to doxycycline alone . all 34 scrub typhus patients without ards recovered completely and discharged . among 24 scrub typhus patients with ards , 22 patients recovered , and 2 patients died ( refractory septic sepsis with severe mods ) . early diagnosis using ict in a patient from the rural background with a history of fever , thrombocytopenia and multiorgan dysfunction and treatment with doxycycline in these patients can help to reduce mortality .

objectivechronic exposure to calcineurin inhibitors and corticosteroids poses renal transplant recipients ( rtr ) at high risk for development of new - onset diabetes after transplantation ( nodat ) . pancreatic -cell dysfunction may be crucial to the pathophysiology of nodat and specific markers for -cell dysfunction may have additive value for predicting nodat in this population . therefore , we prospectively investigated whether proinsulin , as a marker of pancreatic -cell dysfunction , is associated with future development of nodat and improves prediction of it.research design and methodsall rtr between 2001 and 2003 with a functioning graft for 1 year were considered eligible for inclusion , except for subjects with diabetes at baseline who were excluded . we recorded incidence of nodat until april 2012.resultsa total of 487 rtr ( age 50 12 years , 55% men ) participated at a median time of 6.0 ( interquartile range [ iqr ] , 2.611.5 ) years after transplantation . median fasting proinsulin levels were 16.6 ( iqr , 11.024.2 ) pmol / l . during median follow - up for 10.1 ( iqr , 9.110.4 ) years , 42 ( 35% ) rtr had development of nodat in the highest quartile of the distribution of proinsulin versus 34 ( 9% ) in the lowest three quartiles ( p < 0.001 ) . in cox regression analyses , proinsulin ( hazard ratio , 2.29 ; 95% ci , 1.852.83 ; p < 0.001 ) was strongly associated with nodat development . this was independent of age , sex , calcineurine inhibitors , prednisolone use , components of the metabolic syndrome , or homeostasis model assessment.conclusionsin conclusion , fasting proinsulin is strongly associated with nodat development in rtr . our results highlight the role of -cell dysfunction in the pathophysiology of nodat and indicate the potential value of proinsulin for identification of rtr at increased risk for nodat .

study design and inclusion / exclusion criteria have been described previously ( 10 ) . in brief , for this prospective cohort study all adult allograft recipients between august 2001 and july 2003 who survived with a functioning allograft beyond the first year after transplantation were eligible to participate at their next visit to our outpatient clinic . a total of 606 from an eligible 847 rtr ( 72% consent rate ) signed written informed consent . we excluded 105 recipients with existing diabetes ( defined as fasting plasma glucose 7.0 or antidiabetic medication ) at baseline from analysis . baseline data were collected between august 2001 and july 2003 , and rtr were followed - up for several years . the groningen renal transplant database contains information on all renal transplantations performed at our center since 1968 . relevant transplant recipient characteristics such as age , sex , and date of transplantation were extracted from this database . we found current medication information in the medical record and obtained information on employment status , living situation , smoking and alcohol consumption , and cardiovascular history by self - report questionnaire . standard immunosuppressive treatment consisted of the following : prednisolone and azathioprine ( 100 mg / day ) from 1968 to 1989 ; cyclosporine standard formulation ( trough levels of 175 to 200 mg / l in the first 3 months , 150 mg / l between 3 and 12 months after transplantation , and 100 mg / l thereafter ; sandimmune ; novartis pharma b.v . , arnhem , the netherlands ) and prednisolone ( starting with 20 mg / day , rapidly tapered to 10 mg / day ) from january 1989 to february 1993 ; cyclosporine microemulsion ( trough levels idem ; neoral ; novartis pharma b.v . ) and prednisolone from march 1993 to may 1997 ; and mycophenolate mofetil ( 2 g / day ; cellcept ; roche b.v . , woerden , the netherlands ) , which was added from may 1997 to present date . in some specific situations , cyclosporine was converted to tacrolimus in the event of acute rejection , hypertrichosis , gingival hypertrophy , or intolerance of cyclosporine . waist circumference was measured on bare skin midway between the iliac crest and the 10th rib . blood pressure was measured after a 6-min rest in the supine position as the average of three automated measurements at 1-min intervals ( omron m4 ; omron europe b.v . ) . plasma glucose , insulin , hdl cholesterol , ldl cholesterol , high - sensitivity c - reactive protein , and serum creatinine were measured as described previously ( 10 ) . homeostasis model assessment ( homa ) was calculated as : [ glucose ( mmol / l ) insulin ( in microunits / ml)]/22.5 ( 12 ) . in this study , metabolic syndrome ( ms ) was defined according to the definition of the national cholesterol education program expert panel ( ncep - atp ) ( 13 ) . mercodia - proinsulin elisa is a solid phase , two - site enzyme immunoassay based on the sandwich technique , in which two monoclonal antibodies are directed against separate antigenic determinants on the proinsulin molecule . proinsulin in the sample reacts with antiproinsulin antibodies bound to microtitration wells and peroxidase - conjugated anti - insulin antibodies in the solution . cross - reactivity for insulin is < 0.03% and cross - reactivity for c - peptide is < 0.006% . the international expert panel meeting ( 14 ) proposed recommendations to define nodat based on the american diabetes association criteria 2003 ( 15 ) . the diagnosis of nodat was based on one of the following criteria : symptoms of diabetes ( classic symptoms of diabetes include polyuria , polydipsia , and unexplained weight loss ) plus casual plasma glucose concentration 200 mg / dl ( 11.1 mmol / l ) ; fasting plasma glucose 126 mg / dl ( 7.0 mmol / l ) ; or use of antidiabetic medication . fasting is defined as no caloric intake for at least 8 h. nodat was recorded until april 2012 . data were analyzed with spss version 16.0 ( spss , chicago , il ) , stata version 11 ( statacorp lp ) , and graphpad prism version 4.03 ( graphpad software , san diego , ca ) . for analyses , we combined quartiles 13 as one group and compared this group with quartile 4 . differences between groups were tested for statistical significance with student t test for normally distributed variables , mann - whitney test for skewed distributed variables , and test for categorical variables . we performed multivariate cox regression analyses to investigate whether proinsulin is independently associated with nodat . in subsequent multivariate analyses , we investigated whether the association of proinsulin is independent of age , sex , use of cyclosporine , tacrolimus , dose of prednisolone , trough levels of cyclosporine and tacrolimus , homa , or components of the metabolic syndrome . patients were censored at date of last follow - up or death . because there are no validated clinical models for the prediction of nodat in rtr this clinical model includes the following : sex , smoking , waist circumference , hypertension , and family history of diabetes ( 16 ) . to assess the added value of proinsulin , we examined improvement of diabetes prediction in terms of discrimination and integrated discrimination improvement ( idi ) . discrimination was evaluated using the harrell c - index for censored data , a statistic similar to the area under a receiver - operating characteristic curve ( 17 ) . in general , discrimination refers to the ability of a model to distinguish well between individuals with and without incident diabetes ; a value of 1 implies a perfect discrimination and a value of 0.5 implies performance no better than chance . we used idi as a continuous measure of reclassification , calculated by subtracting the mean difference of predicted risk between the clinical model ( 16 ) and the model including different biomarkers . study design and inclusion / exclusion criteria have been described previously ( 10 ) . in brief , for this prospective cohort study all adult allograft recipients between august 2001 and july 2003 who survived with a functioning allograft beyond the first year after transplantation were eligible to participate at their next visit to our outpatient clinic . a total of 606 from an eligible 847 rtr ( 72% consent rate ) signed written informed consent . we excluded 105 recipients with existing diabetes ( defined as fasting plasma glucose 7.0 or antidiabetic medication ) at baseline from analysis . baseline data were collected between august 2001 and july 2003 , and rtr were followed - up for several years . the groningen renal transplant database contains information on all renal transplantations performed at our center since 1968 . relevant transplant recipient characteristics such as age , sex , and date of transplantation were extracted from this database . we found current medication information in the medical record and obtained information on employment status , living situation , smoking and alcohol consumption , and cardiovascular history by self - report questionnaire . standard immunosuppressive treatment consisted of the following : prednisolone and azathioprine ( 100 mg / day ) from 1968 to 1989 ; cyclosporine standard formulation ( trough levels of 175 to 200 mg / l in the first 3 months , 150 mg / l between 3 and 12 months after transplantation , and 100 mg / l thereafter ; sandimmune ; novartis pharma b.v . , arnhem , the netherlands ) and prednisolone ( starting with 20 mg / day , rapidly tapered to 10 mg / day ) from january 1989 to february 1993 ; cyclosporine microemulsion ( trough levels idem ; neoral ; novartis pharma b.v . ) and prednisolone from march 1993 to may 1997 ; and mycophenolate mofetil ( 2 g / day ; cellcept ; roche b.v . , woerden , the netherlands ) , which was added from may 1997 to present date . in some specific situations , cyclosporine was converted to tacrolimus in the event of acute rejection , hypertrichosis , gingival hypertrophy , or intolerance of cyclosporine . waist circumference was measured on bare skin midway between the iliac crest and the 10th rib . blood pressure was measured after a 6-min rest in the supine position as the average of three automated measurements at 1-min intervals ( omron m4 ; omron europe b.v . ) . plasma glucose , insulin , hdl cholesterol , ldl cholesterol , high - sensitivity c - reactive protein , and serum creatinine were measured as described previously ( 10 ) . homeostasis model assessment ( homa ) was calculated as : [ glucose ( mmol / l ) insulin ( in microunits / ml)]/22.5 ( 12 ) . in this study , metabolic syndrome ( ms ) was defined according to the definition of the national cholesterol education program expert panel ( ncep - atp ) ( 13 ) . mercodia - proinsulin elisa is a solid phase , two - site enzyme immunoassay based on the sandwich technique , in which two monoclonal antibodies are directed against separate antigenic determinants on the proinsulin molecule . proinsulin in the sample reacts with antiproinsulin antibodies bound to microtitration wells and peroxidase - conjugated anti - insulin antibodies in the solution . cross - reactivity for insulin is < 0.03% and cross - reactivity for c - peptide is < 0.006% . the international expert panel meeting ( 14 ) proposed recommendations to define nodat based on the american diabetes association criteria 2003 ( 15 ) . the diagnosis of nodat was based on one of the following criteria : symptoms of diabetes ( classic symptoms of diabetes include polyuria , polydipsia , and unexplained weight loss ) plus casual plasma glucose concentration 200 mg / dl ( 11.1 fasting is defined as no caloric intake for at least 8 h. nodat was recorded until april 2012 . mercodia - proinsulin elisa is a solid phase , two - site enzyme immunoassay based on the sandwich technique , in which two monoclonal antibodies are directed against separate antigenic determinants on the proinsulin molecule . proinsulin in the sample reacts with antiproinsulin antibodies bound to microtitration wells and peroxidase - conjugated anti - insulin antibodies in the solution . cross - reactivity for insulin is < 0.03% and cross - reactivity for c - peptide is < 0.006% . the international expert panel meeting ( 14 ) proposed recommendations to define nodat based on the american diabetes association criteria 2003 ( 15 ) . the diagnosis of nodat was based on one of the following criteria : symptoms of diabetes ( classic symptoms of diabetes include polyuria , polydipsia , and unexplained weight loss ) plus casual plasma glucose concentration 200 mg / dl ( 11.1 fasting is defined as no caloric intake for at least 8 h. nodat was recorded until april 2012 . data were analyzed with spss version 16.0 ( spss , chicago , il ) , stata version 11 ( statacorp lp ) , and graphpad prism version 4.03 ( graphpad software , san diego , ca ) . recipient - related characteristics were analyzed separately for quartiles of proinsulin . for analyses , we combined quartiles 13 as one group and compared this group with quartile 4 . differences between groups were tested for statistical significance with student t test for normally distributed variables , mann - whitney test for skewed distributed variables , and test for categorical variables . we performed multivariate cox regression analyses to investigate whether proinsulin is independently associated with nodat . in subsequent multivariate analyses , we investigated whether the association of proinsulin is independent of age , sex , use of cyclosporine , tacrolimus , dose of prednisolone , trough levels of cyclosporine and tacrolimus , homa , or components of the metabolic syndrome . patients were censored at date of last follow - up or death . because there are no validated clinical models for the prediction of nodat in rtr this clinical model includes the following : sex , smoking , waist circumference , hypertension , and family history of diabetes ( 16 ) . to assess the added value of proinsulin , we examined improvement of diabetes prediction in terms of discrimination and integrated discrimination improvement ( idi ) . discrimination was evaluated using the harrell c - index for censored data , a statistic similar to the area under a receiver - operating characteristic curve ( 17 ) . in general , discrimination refers to the ability of a model to distinguish well between individuals with and without incident diabetes ; a value of 1 implies a perfect discrimination and a value of 0.5 implies performance no better than chance . we used idi as a continuous measure of reclassification , calculated by subtracting the mean difference of predicted risk between the clinical model ( 16 ) and the model including different biomarkers . the study cohort was composed of 487 rtr ( 55% men ) aged 50 12 years at a median time of 6.0 ( interquartile range , 2.611.5 ) years after transplantation . baseline characteristics of the rtr according to the two groups of proinsulin are shown in table 1 . high proinsulin was positively associated with use of -blocker , use of statin , history of bmi , high - sensitivity c - reactive protein , glucose , insulin , homa , and proinsulin - to - insulin ratio . we found other differences in lipid - profile with lower hdl and ldl cholesterol and higher triglycerides in subjects with high proinsulin . no differences were found in other components of immunosuppressive treatment . out of the 487 rtr , 309 ( 75% ) fulfilled the criteria for ms . prevalence of ms was 101 ( 86% ) in the highest quartile of proinsulin compared with 208 ( 57% ) in the lowest three quartiles ( p < 0.001 ) . recipient characteristics according to groups of proinsulin nodat developed during median follow - up for 10.1 ( interquartile range , 9.710.4 ) years in 76 ( 16% ) rtr . incidence of nodat was 42 ( 35% ) in the highest quartile compared with 34 ( 9% ) in the lowest three quartiles of proinsulin ( p < 0.001 ) ( fig . 1 ) . cumulative percentages of nodat at 1 , 3 , 5 , and 10 years after baseline were 1.2 , 4.6 , 7.4 and 14.8% , respectively . kaplan - meier curve of de novo diabetes in quartiles of proinsulin tested with log - rank test ( p < 0.001 ) . cut - off points for quartiles of proinsulin were as follows : quartiles 13 , 2.424.4 ( pmol / l ) , and quartile 4 , > 24.5 ( pmol / l ) . subsequently , we proceeded with prospective analyses for proinsulin and development of nodat during follow - up . proinsulin ( hazard ratio [ hr ] , 2.29 ; 95% ci , 1.852.83 ; p < adjustment for age and sex did not materially influence the associations ( model 2 ) . we adjusted for use of cyclosporine , tacrolimus , and prednisolone dose in model 3 . this adjustment also did not materially influence the association of proinsulin with nodat development . of note , use of tacrolimus was significantly associated with nodat development ( hr , 2.84 ; 95% ci , 1.375.89 ; p = 0.005 ) in this cox regression model . this was independent of proinsulin , age , sex , use of cyclosporine , and prednisolone dose . we found no significant association of use of cyclosporine with development of nodat . in further analyses in which we additionally adjusted for trough levels of tacrolimus and cyclosporine ( model 4 ) however , trough levels of cyclosporine were associated with increased risk for higher concentrations ( hr , 1.07 ; 95% ci , 1.011.13 ; p = 0.02 ) . in further analyses , it appeared that the association between proinsulin and nodat was independent of homa ( model 5 ) . interestingly , in this model , hr for homa also was associated with nodat independent of proinsulin ( hr , 1.23 ; 95% ci , 1.091.40 ; p = 0.001 ) . adjustments for factors of ms ( model 6 ) slightly weakened the association , but proinsulin remained independently associated with nodat . waist circumference ( hr , 1.02 ; 95% ci , 1.011.04 ; p = 0.01 ) , triglycerides ( hr , 1.21 ; 95% ci , 1.051.40 ; p = 0.01 ) , and glucose ( hr , 2.26 ; 95% ci , 1.633.11 ; p < 0.001 ) were significantly associated with nodat , independent of proinsulin . proinsulin independently predicts nodat in rtr evaluation of prognostic value of proinsulin is summarized in table 3 . harrell c - index of discrimination improved from 0.71 ( interquartile range , 0.650.77 ) to 0.80 ( interquartile range , 0.750.85 [ see table 3 ] ; p < 0.01 ) after adding proinsulin to a clinical prediction model including sex , smoking , waist circumference , hypertension , and family history of diabetes ( 16 ) . idi analysis shows that the clinical risk score with proinsulin predicted nodat more accurately than the clinical risk score alone . we found similar results when homa or glucose was added to the clinical model . when proinsulin was added on top of glucose , idi was positive and remained significant . these results show that proinsulin is a promising biomarker for predicting nodat beyond established clinical risk predictors in rtr . proinsulin levels predicted development of nodat in rtr after adjustment for risk factors for nodat . our findings emphasize the importance of -cell dysfunction in the pathophysiology of nodat in rtr . in our study , adjustment for ms ( waist circumference , triglycerides , hdl cholesterol , blood pressure , and glucose concentration ) attenuated the association of proinsulin with nodat . this supports the notion that ms and particularly waist circumference , triglycerides , and glucose , partly contribute to this association . factors of the ms could be modified by regular physical activity and a healthy diet , which shows the importance of lifestyle interventions after renal transplantation . the association between proinsulin and nodat was independent of homa , which suggests that the relationship is driven by -cell dysfunction . it has become clear that both insulin resistance and -cell dysfunction are present early in the natural history of diabetes . there is a hyperbolic relationship between insulin sensitivity and insulin secretion that depends on a negative feedback loop . pancreatic -cells compensate for changes in insulin sensitivity ( 18,19 ) . in healthy subjects , plasma glucose levels are maintained near to normal , even with low insulin sensitivity , as a consequence of a compensatory increase in insulin secretion . ( 20 ) showed that obesity , waist - to - hip ratio , and prednisolone treatment are the predominant determinants of insulin resistance after transplantation . furthermore , proinsulin can be used as a marker of pancreatic -cell dysfunction . in this light , increased circulating levels of proinsulin are seen as a marker of -cell stress when insulin demands required for maintenance of glycemic control are relatively high for the prevailing -cell capacity . this is accompanied by increased spill - over of proinsulin ( 5 ) . calcineurin inhibitors impair insulin secretion , produce -cell toxicity , cause insulin resistance , and thereby contribute to an increased risk for nodat ( 2123 ) . various studies comparing cyclosporine and tacrolimus showed that use of cyclosporine is associated with a significantly lower incidence of nodat than tacrolimus after renal transplantation ( 3,8,24 ) . interestingly , proinsulin levels tended to be lower in rtr receiving tacrolimus , despite the fact that use of tacrolimus was significantly associated with increased risk for nodat development . use of tacrolimus increased the risk for nodat by almost three - fold ( hr , 2.84 ; 95% ci , 1.375.89 ; p = 0.005 ) . this observation could point to the known interference of tacrolimus with insulin production by pancreatic -cells at the level of synthesis rather than at the level of conversion of proinsulin to insulin ( 2123 ) . the low variation in steroid doses in the population we investigated did not allow for us to find a relationship between steroid dose and nodat . based on the results from the united kingdom prospective diabetes study , it was suggested that -cell dysfunction was reduced up to 50% at time of diagnosis ( 25 ) . therefore , proinsulin can be used to identify patients at risk for nodat development several years later . proinsulin can still bind to the insulin receptor and has a glucose - lowering effect of 1020% compared with insulin ( 26 ) . because of this minor but evident effect , patients with -cell dysfunction do not always have diabetes diagnosed . although no cut - off points for proinsulin are described in the literature , there is one study in which cut - off values are given for defining insulin resistance . data from the iris - ii ( study on insulin resistance and insulin sensitivity ) show that elevated proinsulin levels ( > 10 pmpl / l ) are a good indirect marker for insulin resistance ( 27 ) . in our study , 80% of the rtr had proinsulin levels above this threshold of > 10 pmpl / l . this may reflect the fact that -cell function is impaired in almost all rtr because of increased metabolic demands on the -cells and the chronic exposure to immunosuppressive drugs , which places rtr at high risk for development of nodat ( 2 ) . sharif et al . ( 28 ) validated important insulin resistance indexes in rtr using tacrolimus . rodrigo et al . ( 29 ) analyzed the performance of two general population risk scores for prediction of diabetes in rtr . ( 30 ) recently developed a pretransplant risk score for the prediction of post - transplant diabetes . ( 30 ) was modest , with areas under receiver - operating curves varying between 0.70 and 0.72 . our study is the first to include a marker of -cell dysfunction in addition to insulin resistance indexes in the prediction of nodat in rtr . in our analyses , we found that both proinsulin as a marker of -cell dysfunction and homa as a marker for insulin resistance are independently associated with increased risk for nodat . the prediction model with proinsulin had good discrimination , showing that 80% of the rtr were adequately classified as at risk for nodat . proinsulin is a promising marker for early detection of patients at risk for nodat and , possibly , future studies also may identify it as useful in the clinic to monitor -cell function early after transplantation . monitoring -cell functions could allow for early intervention and treatment strategies to preserve -cell function . ( 31 ) recently showed in a randomized controlled study that basal insulin therapy may be a good strategy to reduce hba1c and may decrease the incidence of nodat , presumably by protection of the -cells . patients were randomized to immediate postoperative isophane insulin ( treatment group ) or short - acting insulin with or without oral antidiabetic agents ( standard care ) . the treatment group had 73% lower odds for nodat and hba1c was 0.38% lower than in the control group . besides pharmacological strategies , lifestyle interventions could play an important role in prevention of nodat beyond the first year after transplantation . exercise training decreases insulin resistance and the risk of diabetes in the general population , and it can be assumed that it has similar effects in rtr . sharif et al . ( 32 ) showed that lifestyle modification is beneficial for high - risk rtr with glucose intolerance . intensive lifestyle modification ( dietitian , exercise program , and weight loss advice ) resulted in 15% improvement in 2-h postprandial glucose ( 32 ) . lifestyle interventions targeting physical activity and diet after transplantation as well as individualized choice of immunosuppressive agents could help in the prevention of nodat . rtr in this study were closely monitored through regular check - ups at our clinic , which provide complete information on patient status . our study population is a cross - cut of all the rtr who visited our outpatient clinic , giving a variation of rtr with different times after transplantation and including stable rtr late after transplantation . however , our study is limited by the heterogeneousness of our study population , with variable time after transplantation and immunosuppressant medication . this modeling can not fully correct for the fact that rtr without nodat late after transplantation may be a healthier group . our results highlight the role of -cell dysfunction in the pathophysiology of nodat in rtr . considering that the development of nodat is associated with a higher risk of complications and worse survival , identifying rtr at increased risk for development of nodat proinsulin as a marker of -cell dysfunction has potential value for identification of rtr at increased risk for nodat . prevention of nodat by lifestyle interventions , early identification of patients at risk , and choice of immunosuppressive medication are all important to control and manage nodat in rtr .

tuberculosis remains a major global health problem worldwide , and hence there is a need for novel vaccines that better induce cellular - mediated immunity ( cmi ) . in search of a better vaccine target , the quantiferon - tb gold in - tube test ( qft - git ) and the interferon- elispot assay ( elispot ) were used to compare the magnitude of cmi in patients . results of the elispot assay led to the discovery of specific epitopes within the early secreted antigenic target 6 kda ( esat-6 ) and culture filtrate protein 10 kda ( cfp-10 ) proteins . both peptides showed a strong association with several hla class ii drb1 molecules in the japanese population . using esat-6-specific hla class ii tetramers , we determined that the expression of esat-6-specific cd4 + lymphocytes was significantly decreased in treated patients compared with active patients . in addition , programmed death-1 ( pd-1)/killer cell lectin - like receptor g1 ( klrg-1 ) double positive cells were found only in treated patients and not in those with active tb . these data could provide clues for the development of novel tuberculosis vaccines .

tuberculosis ( tb ) , caused by mycobacterium tuberculosis ( m. tuberculosis ( mtb ) ) , is still a major health problem around the world . it is second only to aids as the leading cause of death from infectious diseases . the world health organization estimates that there were 9 million new cases of tb and 1.4 million tb - related deaths in 2011 . the effectiveness of the only available tb vaccine , bacillus calmette - gurin ( bcg ) , is limited . protection in children has only been demonstrated for disseminated tb and tuberculosis meningitis , while the efficacy for adult pulmonary tb is very variable . in fact , a controlled trial of bcg failed to demonstrate its effectiveness in protecting against the development of tb . lack of efficacy of bcg for adult tb is a serious problem in controlling the disease and may be partly related to the fact that the efficacy of bcg vaccinations declines over time . the results of several epidemiological studies suggest that the effectiveness of bcg vaccinations lasts for around twenty years [ 4 , 5 ] . this decline of efficacy would be associated with a decrease of cell - mediated immunity ( cmi ) to mtb . an assessment of the degree of cmi over time would require objective , repeatable , and economical assays . the tuberculin skin test ( tst ) , an assay for type iv - delayed hypersensitivity reaction , is the primary screening method used to determine tb exposure . the presence or absence of induration following the tst injection can be used as objective proof of active cmi against mycobacterial infection . however , the tst can not distinguish mtb from bcg or nontuberculous mycobacteria ( ntm ) . therefore , an in vitro ( or ex vivo ) interferon- ( ifn- ) release assay ( igra ) , which utilizes antigens within the region of difference 1 ( rd1 ) of m. tuberculosis , is used in conjunction with the tst . these antigens , such as early secreted antigenic target 6 kda ( esat-6 ) ( rv3875 ) , culture filtrate protein 10 kda ( cfp-10 ) ( rv3874 ) , and tb7.7 ( rv2654c ) , are not present in bcg or most environmental ntm . these antigens are also very potent inducers of cmi , even in an in vitro setting [ 9 , 10 ] , which enables the establishment of an elisa - based assay system to measure ifn- secreted into the culture supernatant after antigenic stimulation . quantiferon - tb gold ( qft - g ) is an igra that utilizes two mtb - specific antigens , esat-6 and cfp-10 . its successor , the quantiferon - tb gold in - tube test ( qft - git ) , incorporated tb7.7 in addition to esat-6 and cfp-10 . a simultaneous and longitudinal comparison of the qft - g and qft - git assays among healthcare workers showed that qft - git is the more sensitive of the two , probably due to the addition of tb7.7 . however , t-spot.tb uses only two antigens , esat-6 and cfp-10 , to stimulate samples . results from previous methods were significantly affected by the lymphocyte count in the peripheral blood of each patient . therefore , we aimed to analyze precise epitopes of mtb - specific antigens in cd4 + cells , which are the predominant cell type that secretes ifn- upon stimulation with mtb antigens . we developed an elispot assay ( elispot ) using multiple , overlapping peptides from three proteins , esat-6 , cfp-10 , and tb7.7 , to stimulate memory t cells . in this study , the sensitivity of two igra assays , qft - git and elispot , was compared using blood samples from the same tb patients and the same tuberculosis antigen sets to determine the activity of cmi in patients . elispot , using the same antigen peptide sets , was then used to analyze t cell response to mtb - specific antigens to identify restricted epitopes . finally , mtb - specific cd4 + lymphocytes were segregated to evaluate their phenotype in tb patients . patients of tokyo national hospital in tokyo , japan , were consecutively enrolled in the study , after giving informed consent , from april 2010 to april 2011 . a total of 177 japanese patients ( age : 58.5 18.2 yr ; male : 68.5% ) were recruited . the following information was obtained from all patients at the time of enrollment : history of prior tb disease , work history in any healthcare settings or recent exposure to a patient with active tb , and other tb risk factors such as taking immunosuppressive drugs . information on previous medical history , any clinical symptoms and signs , and radiological and microbiological data were also collected . the patients were then divided into three categories : ( 1 ) active disease : patients having positive symptom(s ) and positive smear results and/or positive demonstration of mtb in culture ; ( 2 ) past disease : previously diagnosed with tb , treated , and currently free from symptom(s ) ; and ( 3 ) latent tb infection ( ltbi ) : no symptoms with normal chest x - ray but having positive results from an igra . among the 177 patients recruited , 56 ( 32% ) had active disease , 103 ( 58% ) were in the past disease group , and for those patients classified as having the disease in the past , the average time period since disease onset was 2605 605 ( mean se ) days . the research protocol was approved by the institutional review board of tokyo national hospital and by the research ethics committee of the national institute of infectious disease , tokyo , japan . the qft - git assay was performed using fresh whole blood in accordance with the manufacturer 's instructions ( cellestis , chadstone , australia ) . the results were interpreted with software provided by cellestis . results were scored as positive if the ifn- concentration in the tube with tb - specific antigen was > 0.35 iu / ml after subtracting the value of the nil control and at least > 25% of the negative control value . if the net ifn- response was < 0.35 iu / ml for the antigens and the response to the mitogen - positive control was > 0.5 iu / ml , the response was considered peptides 15 amino acids in length , with nine overlapping residues , were synthesized to cover the entire length of esat-6 , cfp-10 , and tb7.7 . imajoh - ohmi in the medical proteomics laboratory , institute of medical science , university of tokyo , tokyo , japan . the purity of the peptides was > 95% after purification with reversed - phase hplc . the cfp-10 peptides were labeled as c1 to c16 , esat-6 peptides as e1 to e15 , and tb7.7 peptides as t1 to t13 . supplemental table 1 ( see supplementary material available online at http://dx.doi.org/10.1155/2014/764028 ) lists the sequence of each peptide . peripheral blood mononuclear cells ( pbmcs ) were separated from heparinized blood samples by density centrifugation using bd vacutainer cell preparation tubes ( becton , dickinson and company , franklin lakes , nj , usa ) . genomic dna was isolated from pbmcs with qiaamp dna blood kits ( qiagen , germantown , md , usa ) for hla typing using the luminex multi - analyte profiling system ( xmap ; luminex , austin , tx , usa ) . the ifn- elispot assay was performed to compare the sensitivity between igras and to find an immunological epitope to tuberculosis - specific antigens . the peptide mixture of each protein was used to obtain sensitivity measurements , while a single peptide was applied to each well for epitope determination . pbmcs were seeded into precoated ifn- elispot plates ( becton , dickinson and company , franklin lakes , nj , usa ) with 2.5 10 cells per well in aim - v medium ( gibco ) and incubated with one of a series of peptides ( 10 m ) or a peptide mixture ( 10 m ) of each antigen at 37c in 5% co2 for 16 h. a negative control ( no mitogen or antigen ) and a positive control ( phytohemagglutinin , pha , 5 g / ml ) were also included . after incubation , the wells were washed and developed with a conjugate against the antibody used and an enzyme substrate . spot - forming units were counted using a ks elispot imaging system ( carl zeiss , hallbergmoos , germany ) as spot - forming cells ( sfc ) . elispot results were interpreted according to the following criteria : the test result was positive when ( 1 ) the negative control had 05 spots and ( 2 ) the ( antigen spot count ) ( negative control spot count ) was greater than six . the test result was negative if the above criteria were not met and the positive control was valid ( 20 ) . hla class ii tetramers conjugated with apc - labeled streptavidin were provided by the tetramer core laboratory at the national institutes of health , bethesda , md , usa . pbmcs were incubated with class ii tetramers for 2 h at room temperature . the following fluorescence - labeled monoclonal antibodies ( mabs ) were used in this study : anti - cd3-apccy7 ( hit3a ) , anti - cd8-percp - cy5.5 ( mab11 ) ( biolegend , san diego , ca , usa ) , and anti - cd4-pacific blue ( okt4 ) ( ebioscience , san diego , ca , usa ) , anti - pd-1-pe ( bd bioscience ) . anti - klrg-1-alexa488 was kindly provided by professor h. pircher ( university of freiberg , germany ) . where necessary , the relevant isotype control mab was used . cell viability was assessed using the live / dead kit ( invitrogen , carlsbad , ca , usa ) . following a 30 min incubation at 4c , the cells were washed and acquired using a facs canto ii flow cytometer ( bd bioscience ) . facs data were reanalyzed using flowjo software , version 8.8.7 ( treestar , san carlos , ca , usa ) . group medians and distributions were analyzed using the wilcoxon matched - pairs signed - rank test and the mann - whitney u test . all analyses were performed using graphpad prism software , version 5 ( san diego , ca , usa ) . all patient samples ( n = 177 ) were analyzed by qft - git and 115 were analyzed by ellispot . as expected , when the results from both assays were compared , there was a positive correlation between qft - git values and the number of spots obtained in the elispot assay ( r = 0.532 ; p < 0.001 ) ( figure 1 ) . the detection rate of each assay was compared across the three groups of patients : active disease , past disease , and ltbi . qft - git failed to detect many patients with active and past disease ; elispot detection was more consistent ( overall detection rate of 93.9% versus 65.5% ) ( table 1 ) . the average duration from disease onset to the date of the assay was 2 , 086 743 days in qft - git - negative past tb patients and 4 , 920 3042 days in elispot - negative past tb patients . when both assays were compared in the same patients , 31 of 37 qft - git - negative cases ( 84% ) were positive by elispot ( table 2 ) , suggesting that the elispot assay developed in our laboratory was better at detecting a broader range of tb+ patient populations . consequently , the elispot was chosen to carry out the detection of antigenic peptides in mtb - specific proteins . each mtb - specific antigen used in the igra comparison was further evaluated to identify the peptide(s ) that most efficiently induced antigenicity to mtb and activation of host cmi . we found that 77% of patients responded to esat-6 peptides and 66% to cfp-10 , while no single case responded to tb7.7 ( data not shown ) . elispot data provided an indication of the prevalence and strength of the esat-6 and cfp-10 mtb antigens . the next step was to map the precise epitope(s ) of the mtb - specific antigens recognized by cd4 + lymphocytes . two sets of peptides were synthesized , 15 overlapping esta-6 peptides ( e1e15 ) and 16 overlapping cfp-10 peptides ( c1c16 ) ( supplemental table 1 ) , to evaluate patient samples using elispot . among the 15 peptides from esta-6 , e1 , e4 , e5 , e10 , and e13 elicited a response in multiple patients ( figure 2(a ) ) . the same peptides induced a higher average number of spot - forming cells ( sfc ) in the elispot assay ( figure 2(b ) ) , suggesting that these are major epitopes that stimulate cd4 + cells in peripheral blood . similarly , peptides c1 , c5 , c9 , c10 , and c13 from cfp-10 were identified as responsible epitopes that stimulated multiple patients ( figure 3(a ) ) to induce a stronger ifn- response ( figure 3(b ) ) . although cmi to tb involves both cd4 + and cd8 + positive lymphocytes [ 21 , 22 ] , the primary source of ifn- as measured by elispot is cd4 + cells [ 14 , 23 ] . cd4 + t lymphocytes are activated by specific antigens presented by antigen presenting cells ( apcs ) through major histocompatibility complex ( mhc ) class ii molecules ( or hla - dr in humans ) . therefore , to ascertain specificities to each esat-6 and cfp-10 peptide , human hla drb1 haplotypes were examined by employing a high - resolution luminex - based method . as shown in table 3 , the proportion of drb1 identified in tb patients was most similar to that found in the japanese population ( n = 916 ) . the predominant haplotype was drb1 * 0405 ( 13.5% ) , followed by drb1 * 0901 ( 12.9% ) , drb1 * 1502 ( 12.6% ) , and drb1 * 0803 ( 8.0% ) . however , some haplotypes were significantly higher or lower than those in the japanese control population . they included drb1 * 0101 ( 1.8% in tb versus 4.8% in control ) , drb1 * 0406 ( 7.1% versus 3.2% ) , and drb1 * 1502 ( 12.6% versus 8.7% ) . the ability of each esat-6 and cfp-10 peptide to induce ifn- in elispot was examined and its relation to each hla drb1 haplotype was analyzed . haplotypes that showed characteristic results are illustrated in figure 4 , in which positive spots as described in section 2.5 are shown in dark grey . among the esat-6 peptides identified in the previous analysis ( figures 2 and 3 ) , e4 showed a strong association with drb1 * 0405 ( 28 out of 31 cases ) and weak associations with drb1 * 1501 ( 5 out of 17 cases ) and drb1 * 1502 ( 4 out of 21 cases ) ( figure 4 , left panel ) . likewise , the c10 peptide from cfp-10 showed strong associations with drb1 * 1501 ( 17 cases ) and drb1 * 1502 ( 21 cases ) . peptide - mhc binding is the most selective of the events that determine t cell epitopes . thus , peptide - mhc binding motif profiles can be used to predict the identity of t cell epitopes [ 2426 ] . we predicted hla drb1 * 0405-restricted t cell epitopes using in silico analysis of peptide - mhc binding profiles derived from peptides known to bind the relevant mhc molecules [ 2426 ] . a given peptide bound to a specific hla molecule was considered a potential cd4 + t cell epitope when its binding score ranked within the top 3% percentile of scores obtained for 1000 random 9-mer peptides ( average amino acid composition of proteins in the swissprot database ) , using the same profile . peptide - mhc class ii binding profiles only predict the 9-mer core that fits in the binding groove . however , we also provided the three most proximal n - terminal and c - terminal residues , as they can also be the target of t cell recognition . as a result , the minimal core region of the e4 peptide although elispot is a highly sensitive method for the identification of tb patients , it is still difficult to differentiate active patients from past patients using igras . based on the epitope information described above , an esat-6-specific tetramer ( qgnvtsihslldegk ) was synthesized with hla drb1 * 0405 . another tetramer ( pvskmrmatpllmqa ) provided by the nih tetramer facility was used as the negative control . the pmbcs of one active and one past tb patient were stained with drb1 * 0405 esat-6 tetramer using a modification of a previous method . pbmcs were gated with ssc and fsc to isolate the lymphocyte fraction after deletion of doublets using fsc - a and fsc - h . lymphocytes were stained with anti - cd3 , cd4 , cd8 , and live / dead dye to eliminate dead cd4 + t cells ( figure 5(a ) ) . viable cd4 + lymphocytes were then gated with esat-6-specific tetramer ( figures 5(b ) and 5(c ) , left panels ) . a lower proportion of esat-6-specific cd4 + lymphocytes was found in the past tb patient than in the active tb patient ( 0.148% versus 0.939% , figures 5(b ) and 5(c ) , left panels ) . ( pd-1 ) and antikiller cell lectin - like receptor g1 ( klrg-1 ) were then used to distinguish the proliferation and cytokine production phenotypes of the cd4 + cells . pd-1 expressing cd4 + lymphocytes possess proliferative capacity , while klrg-1 expressing cd4 + lymphocytes are relatively short lived but have cytokine secretion capacity . although pd-1 positive and klrg-1 positive cd4 + t cells were identified in both cases , pd-1 and klrg-1 double positive cd4 + lymphocytes were detected only in the past tb patient ( figure 5(c ) versus figure 5(b ) , right panels ) . in this study , we first compared the ability of two igras to detect tb infections in order to evaluate cmi to tb . both assays were mutually acceptable for this purpose , but the elispot was more sensitive than qft - git . the elispot was then used to determine antigenic dominant regions in mtb - specific proteins . several epitopes of mtb - specific antigens were found in cd4 + lymphocytes . using these epitopes , we made an esat-6-specific mhc class ii tetramer to detect the kinetics of tuberculosis - specific cd4 + lymphocytes . there are many studies comparing qft - git and elispot , especially for individuals suspected of having tuberculosis or a latent tb infection [ 2830 ] . in most cases , elispot or the commercially available t-spot.tb was more sensitive than qft - git with a similar specificity . moreover , in this study , we were able to detect specific epitopes of the mtb - specific antigens . a time lapse between bacterial growth and appearance of the adaptive immune response has been observed in mice as well as in humans . around 30% of active patients were negative by qft - git , suggesting that early evaluation of tb might lead to misdiagnosis . next , past tb patients were recruited to investigate the continuation of the host immune response to mtb . qft - git and elispot exhibited differences in time course when the igra results were negative ( 5 yr versus 13 yr ) . however , the age factor could not be adjusted because of the small population size . indeed , among 18 ltbi cases , 4 were diagnosed by qft - git only and 6 by elispot ; only 8 cases were diagnosed by both . with the elispot assay established in our laboratory , we used overlapping peptides from antigenic proteins , not an empirical mixture of peptides , to detect distinct antigenic regions in esat-6 and cfp-10 . identified similar regions ; however , as the ethnicity of the two groups of subjects was considerably different , the regions were not expected to be identical . * 0405 is the most prevalent in the japanese population and , as expected , the most frequent population in tb patients . none of the patients responded to the tb7.7 peptides , which was consistent with the results of a us study . this was surprising , given that this antigen is a recent addition to qft - git tests . this finding suggests that the increased sensitivity of qft - git tests is due to stimulation by the mixture of antigens in the same tube rather than addition of the novel tb7.7 antigen . pd-1 and klrg-1 were used as phenotypic markers for esat-6-specific cd4 + lymphocytes . in the lcmv model , pd-1 expression on cd8 + lymphocytes is a marker of cell exhaustion ; however , in the tuberculosis model , cd4 + lymphocytes that express pd-1 have proliferative potential , suggesting that these are effector cd4 cells . examined several other activation markers such as cd44 , cd62l , cd27 , and cd127 ( il-7r ) and found that they do not differ between the klrg-1- and pd-1- expressing cell populations during tuberculosis infection . our results suggest that , in the active phase , esat-6-specific cd4 + lymphocytes , expressing either pd-1 or klrg-1 , are dominant . however , in the chronic phase , pd-1 expression might decline and klrg-1 + or pd-1+/klrg-1 + cd4 + lymphocytes are dominant , although a recent mouse study showed that a significant portion of esat-6-specific pd-1 expressing cd4 + lymphocytes also express klrg-1 . further precise study with human peripheral blood is required with a larger population of participants . the elispot assay was more sensitive than qft - git in evaluating the adaptive immunity to tb . in addition , the use of overlapping peptides revealed the association between epitopes of two mtb peptides in cd4 + lymphocytes and mhc class ii haplotypes . finally , a significant difference in the expression of esat-6-specific cd4 + lymphocytes was discovered based on stage of treatment .

satisfactory restoration in a patient with a partially edentulous situation can be challenging especially when unilateral or bilateral posterior segment of teeth is missing . successful restoration can be done with various conventional and contemporary treatment options . one such treatment modality is attachment - retained cast partial dentures . a key to success for an attachment retained cast partial denture is the strategic selection of teeth for retention . this clinical report discusses rehabilitation of a patient with the help of a combined prosthesis in the upper arch and stud retained overdenture in the lower arch .

the preservation of teeth to support an attachment - retained prosthesis whether fixed or removable is an appropriate and stable alternative to extractions and complete dentures . combinations of fixed and removable partial dentures using precision / semi - precision attachments represent one high - tech solution in the field of prosthodontics . combined fixed / removable partial denture prosthesis usually refers to the use of precision attachments , double crowns and sometimes overdentures with root attachments . tooth - supported overdentures can be retained with the help of precision attachments and can improve both retention and stability while simultaneously reducing alveolar bone resorption . they may also be more cost - effective and maintain more dental proprioception than implant supported overdentures . it incorporates one component into the removable partial denture , and the connecting component is traditionally incorporated into a cast crown or a fixed partial denture , sometimes referred to as patrix and matrix . the classic indication for precision attachments is in patients with natural anterior teeth and unilateral or bilateral distal extension cases for whom high esthetic demands must be met . attachment - retained cast partial dentures facilitate both esthetic and functional replacement of missing teeth . studies have shown a survival rate of 83.35% for 5 years , of 67.3% up to 15 years , and of 50% when extrapolated to 20 years . this case report describes the rehabilitation of a partially edentulous patient by use of preci - vertix attachment in the maxillary arch and stud attachments in the mandibular arch . a 50-year - old female patient in good general health presented with poor esthetics and compromised masticatory function to the department of prosthodontics and implantology . the clinical examination revealed several missing teeth both in the mandibular and maxillary arch with no loss of vertical dimension . maxillary lateral incisors were found to be supra - erupted , buccally inclined with grade ii mobility [ figure 1a ] . the remaining maxillary canines and 1 premolars presented good periodontal support and mandibular canine / premolar presented with reasonable bone support . radiographs were made [ figure 1b ] , diagnostic casts were articulated at the existing occlusal vertical dimension , and the treatment was carefully planned taking into account patient 's esthetic demand and economical condition . inter - arch space was found to be 14.03 mm [ figure 2 ] , adequate for the use of precision attachments in both the arches . treatment plan included extraction of hopeless teeth ( i.e. , maxillary lateral incisors and second premolar in the second quadrant ) , followed by rehabilitation of maxillary arch with combined fixed / removable prosthesis ( using preci - vertix precision attachment ) and overdenture with stud attachment in the mandibular arch . ( a ) pre- treatment patient presentation ( b ) pre treatment orthopantomogram of the patient showing healthy abutments after extraction of hopeless teeth digital vernier caliper showing available inter - arch space diagnostic impressions were made and mounted on semi adjustable articulator using a face bow , following which diagnostic wax - up was done on the mounted casts . a putty matrix ( express std putty ; 3 m espe , st . paul , minn . ) was made over the completed diagnostic wax - up for evaluation of the existing space for the extra - coronal resilient attachments . maxillary canines and 1 premolars were prepared to receive porcelain - fused - to - metal crowns [ figure 3a and b ] . impression was made in polyvinyl siloxane impression material ( affinis , coltene / whaledent , altsttten , switzerland ) and the cast was poured in die stone ( kalrock , kalabhai karson , mumbai ) . crowns were waxed to full contour and milled in wax for maximum guiding plane surface . burnout plastic male ( preci - vertix standard attachment ) with built in paralleling mandrel was attached to the distal surface of the waxed abutment using a dental surveyor , lingual to the center of proximal contour [ figure 4a and b ] . this ensured that the bulk of the matrix does not interfere with esthetics of the buccal cusp of replacing a tooth . the height of the standard plastic male was 5.0 mm which was sufficient to provide lateral stabilization to the prosthesis . eight unit fixed partial denture along with a male part of preci - vertix attachment was cast in ni - cr alloy [ mealloy , dentsply , uk , figure 5a ] . porcelain build - up of the 8 unit fixed partial denture was completed and tried in the patient 's mouth [ figure 5b ] . after the cementation of the bridge , impression was made in polyvinyl siloxane impression material ( affinis , coltene / whaledent , altsttten , switzerland ) and poured in die stone . wax - up of the cast framework was completed on the master cast , and the entire cast partial framework was cast in co - cr alloy [ jinbego - fh , china , figure 6a and b ] . patient was instructed regarding insertion and removal of the prosthesis . ( a ) abutments prepared to recieve 8 unit pfm bridge ( b ) prepared canine and premolar teeth to recieve cast pivots with short copings burn - out plastic male with paralleling mandrel attached to distal surface of waxed 8 unit segment ( a ) casting with opaque layer : 8 unit metal framework with male preci - vertix attachment ( b ) try - in of 8 unit pfm bridge with male attachment ( a ) acrylized cast partial framework ( b ) acrylized combined prosthesis showing orientation of hader polypropylene clips with eight unit fpd similarly , mandibular abutments ( canine and premolar ) were prepared to receive short copings . postspace was prepared and the impression made in polyvinyl siloxane impression material ( aquasil lv , dentsply , caulk , germany ) for indirect technique . plastic post with sphere were placed in prepared root space and checked for parallelism with the help of ney 's surveyor [ figure 7a ] . wax patterns of the pivots were cast in ni - cr alloy ( mealloy , dentsply , uk ) . retentive nylon caps were placed over master cast , wax block out completed [ figure 7b ] and cast partial framework was fabricated in co - cr alloy [ jinbego - fh , china , figure 8a and b ] . maxilla - mandibular relationships were recorded , and occlusion was evaluated [ figure 9a ] . ( a ) wax milling of copings with plastic posts and sphere to achieve parallelism ( b ) wax block out of master cast with retentive nylon caps ( a ) wax pattern cast partial framework over the refractory cast ( b ) try - in of co - cr cast framework with retentive nylon caps ( a ) post insertion picture with combined prosthesis seated ( b ) overdenture prosthesis with nylon retentive caps overdenture prosthesis for the mandibular arch and cast partial denture prosthesis for the maxillary arch was fabricated in heat - cure acrylic resin [ leucitone 199 denture resin ; dentsply , trubyte , york , pa , figure 9b ] . balanced occlusion was achieved and home care instructions regarding insertion and cleaning of the prosthesis were given to the patient . attachments have always been surrounded by an aura of mystery , primarily because of a lack of familiarity and experience . glossary of prosthodontic terms 8 edition defines attachment as a mechanical device for the fixation , retention , and stabilization of a prosthesis or as a retainer consisting of a metal receptacle and a closely fitting part ; the former ( the female [ matrix ] component ) is usually contained within the normal or expanded contours of the crown of the abutment tooth and the latter ( the male [ patrix ] component ) , is attached to a pontic or the denture framework . attachments may be classified as either precision or semi - precision , depending on the method of fabrication and tolerance of fit . precision attachments have prefabricated , machined components with precisely manufactured metal - to - metal parts with close tolerances . these attachments have a long track record of more than 50 years and have been preferred in cases of reduced tooth support . attachments have a number of desirable qualities that indicate their use in place of conventional clasp retained removable partial dentures . conventional clasp assemblies and rests may be visible and unesthetic whereas preci - vertix and stud attachments get enclosed within contours of the part of the prosthesis . a major advantage of the use of attachments is that the point of force application to the tooth is more apical than for occlusal or incisal rests , thus shortening the lever arm and decreasing torqueing forces . attachments may also allow better cross - arch force transmission and stabilization than clasps , but this is determined by the type of attachment used , the number of guiding surfaces and the design and adaptation of the framework and the attachment . attachment alignment is not as critical in highly resilient extracoronal attachments due to the omniplanar motion possible . poor dental motivation and manual dexterity of the patient may result in earlier failure than with the use of conventional clasping . a minimum of 4 mm of vertical space is necessary for most attachments . in this particular case preci - vertix ( ceka ) attachments and stud attachments ( ot cap , rhein 83 inc . , usa ) were used that provided frictional retention to the maxillary cast partial denture as well as for the mandibular overdenture . preci - vertix ( ceka ) attachments are extra - coronal devices in which exchangeable plastic layers of various sizes are used in the female elements to vary the retention force . the female plastic insert ( made up of polypropylene ) used in this case provided standard retention to the prosthesis . moreover , cross arch splinting of the upper canines and premolars provided better stress distribution thus reducing the rate of alveolar bone resorption . preci - vertix resilient attachments permit vertical movement during mastication reducing stress transfer to the abutments ( stress breaking function ) and direct the forces to the residual ridge acting as stress redirectors . these attachments are based on a broken stress philosophy , thus help to distribute forces equally between soft and hard tissues and are advocated in kennedy class i situations . due to reduced tooth support provided by the mandibular arch and to reduce the masticatory load over canine / premolar teeth , it was decided to use resilient stud attachments that will redirect the forces to the residual ridge thus preventing the torqueing of the abutment teeth , justifying the use of resilient attachments in both the arches . according to feinberg classification of precision attachments , preci - vertix is categorized as passive ( free moving , stress - breaking action type of attachment ) . these attachments are passive , and free - moving that dissipates destructive lateral forces , preventing their infliction on the abutment teeth . thomas forde , in the principles and practice of oral dynamics , theorizes that vertically directed forces drive the hydraulic system of dentitional blood supply to the periodontal structures , whereas rocking or rotational forces disrupt the dentitional blood supply , causing force - induced mouth degeneration and loss of teeth . the tissue under a passive , free - moving attachment case is generally pink and healthy as a result of the vertically - directed physiologic stimulation during function . various stud attachments available are selected based on vertical space available , crown / root ratio , type of coping , number of teeth support , amount and quality of bone support , location of abutments , type of opposing dentition , angulation of the root to the occlusal plane , chewing pattern and the musculature of the patient and patient desire . rheins stud attachments to retain mandibular overdenture were used in this case due to their simplicity in design , ease in maintenance and minimum leverage . canines are the most important proprioceptive organs , the shape and strategic position , and the larger periodontal attachment area make them ideal abutments . the metal denture fabricated to serve as overdenture is less subject to breakage and denture supporting tissues respond more favorably to metal base which may be related to greater ease in maintaining cleanliness of metal base and to effective transmission of thermal changes through the metal base . various cases with esthetic and retention challenges can be solved with correct selection of attachment . whether the need for treatment revolves around health , function or esthetics , attachment retained prosthesis have the capacity to impact patients in life - changing ways . attachment - retained dentures provide long - term prosthetic stability compared to conventional clasp retained removable partial dentures along with support to the oral and facial soft tissue , which can bolster patient 's confidence and alleviate insecurity [ figure 10a and b ] .

objectivecranial nerve palsy ( cnp ) and neck haematoma are complications of carotid endarterectomy ( cea ) . the effects of patient factors and surgical technique were analysed on the risk , and impact on disability , of cnp or haematoma in the surgical arm of the international carotid stenting study ( icss ) , a randomized controlled clinical trial of stenting versus cea in patients with symptomatic carotid stenosis.materials and methodsa per - protocol analysis of early outcome in patients receiving cea in icss is reported . haematoma was defined by the surgeon . cnp was confirmed by an independent neurologist . factors associated with the risk of cnp and haematoma were investigated in a binomial regression analysis.resultsof the patients undergoing cea , 45/821 ( 5.5% ) developed cnp , one of which was disabling ( modified rankin score = 3 at 1 month ) . twenty - eight ( 3.4% ) developed severe haematoma . twelve patients with haematoma also had cnp , a significant association ( p < .01 ) . independent risk factors modifying the risk of cnp were cardiac failure ( risk ratio [ rr ] 2.66 , 95% ci 1.11 to 6.40 ) , female sex ( rr 1.80 , 95% ci 1.02 to 3.20 ) , the degree of contralateral carotid stenosis , and time from randomization to treatment > 14 days ( rr 3.33 , 95% ci 1.05 to 10.57 ) . the risk of haematoma was increased in women , by the prescription of anticoagulant drugs pre - procedure and in patients with atrial fibrillation , and was decreased in patients in whom a shunt was used and in those with a higher baseline cholesterol level.conclusionscnp remains relatively common after cea , but is rarely disabling . women should be warned about an increased risk . attention to haemostasis might reduce the incidence of cnp . icss is a registered clinical trial : isrctn 25337470 .

the combined rate of stroke or death at 30 days following cea in nascet , ecst , and the va trials was 7.1% ( 95% ci 6.3 to 8.1% ) . however , the primary endpoints of these trials did not include cranial nerve palsy ( cnp ) or haematoma . although less extensively studied , the surgical complications of cnp and haematoma have long been recognized following cea , and have been associated with an increased risk of stroke or death . nerves affected include the mandibular branch of the facial nerve , vagal , glossopharyngeal , hypoglossal , and accessory nerves , and therefore cnp has the potential to cause significant postoperative morbidity . carotid angioplasty and stenting ( cas ) was developed as an alternative to cea , in part to avoid these hazards of a surgical incision . however , the results of recent large randomized trials , including the international carotid stenting study ( icss ) , have consistently shown that cas carries a higher risk of non - disabling stroke than cea within 30 days of the procedure , with no significant difference in the rates of disabling stroke or death . icss was a randomized controlled multicentre open clinical trial that randomized patients with symptomatic carotid stenosis to cea or cas . in this study , the incidence and severity of cnp and haematoma in icss , and risk factors for their development , were studied , to identify groups of patients at higher risk and determine whether these complications merit consideration in selection of a revascularization procedure . patients over 40 years old were eligible for randomization in icss if they had more than 50% recently symptomatic carotid stenosis suitable for either cas or cea , and were clinically stable . patients were excluded if they had a major stroke with poor recovery of function , if their vascular anatomy rendered cas or cea unsuitable , if the stenosis was caused by non - atheromatous disease , if cardiac bypass was planned within 1 month of the revascularization procedure , or if there had been previous revascularization of the symptomatic artery . carotid endarterectomy in icss was performed according to the surgeon 's usual practice : local , general , or combined anaesthesia was allowed for the procedure . the type of arterial reconstruction to be carried out was not specified in the protocol , nor was the choice of peri - procedural medication . technical details of the surgical procedure and the occurrence of cranial nerve palsy or haematoma were reported by trial investigators . all patients were then re - assessed at 1 month after the procedure by a neurologist or investigator under their supervision . cnps were adjudicated internally at the icss trial office and judged to be disabling if the patient 's score on the modified rankin scale ( mrs ) increased to 3 or more at 30 days after the procedure , where that increase was attributable to the cnp . investigators were additionally asked to complete a questionnaire ( appendix i ) giving details of the clinical consequences of cnp and whether or not the lesion resolved during subsequent follow - up in the trial . haematoma was classified as severe if it required re - operation , transfusion , or prolonged hospital stay . the data were analysed per - protocol ; only patients in whom the randomly allocated procedure was initiated were included in this analysis . a procedure was deemed to have been initiated if the patient underwent either local or general anaesthesia prior to commencement of surgery . risk factors for cnp and haematoma were examined sequentially in a univariable binomial regression analysis using maximum likelihood estimation . wald tests were used for continuous and binary predictors , with an overall likelihood ratio test for categorical predictors of more than two levels . stata statistical software : release 12 . college station , tx : statacorp lp ) . patients over 40 years old were eligible for randomization in icss if they had more than 50% recently symptomatic carotid stenosis suitable for either cas or cea , and were clinically stable . patients were excluded if they had a major stroke with poor recovery of function , if their vascular anatomy rendered cas or cea unsuitable , if the stenosis was caused by non - atheromatous disease , if cardiac bypass was planned within 1 month of the revascularization procedure , or if there had been previous revascularization of the symptomatic artery . carotid endarterectomy in icss was performed according to the surgeon 's usual practice : local , general , or combined anaesthesia was allowed for the procedure . the type of arterial reconstruction to be carried out was not specified in the protocol , nor was the choice of peri - procedural medication . technical details of the surgical procedure and the occurrence of cranial nerve palsy or haematoma were reported by trial investigators . all patients were then re - assessed at 1 month after the procedure by a neurologist or investigator under their supervision . cnps were adjudicated internally at the icss trial office and judged to be disabling if the patient 's score on the modified rankin scale ( mrs ) increased to 3 or more at 30 days after the procedure , where that increase was attributable to the cnp . investigators were additionally asked to complete a questionnaire ( appendix i ) giving details of the clinical consequences of cnp and whether or not the lesion resolved during subsequent follow - up in the trial . haematoma was classified as severe if it required re - operation , transfusion , or prolonged hospital stay . the data were analysed per - protocol ; only patients in whom the randomly allocated procedure was initiated were included in this analysis . a procedure was deemed to have been initiated if the patient underwent either local or general anaesthesia prior to commencement of surgery . risk factors for cnp and haematoma were examined sequentially in a univariable binomial regression analysis using maximum likelihood estimation . wald tests were used for continuous and binary predictors , with an overall likelihood ratio test for categorical predictors of more than two levels . stata statistical software : release 12 . college station , tx : statacorp lp ) . patients over 40 years old were eligible for randomization in icss if they had more than 50% recently symptomatic carotid stenosis suitable for either cas or cea , and were clinically stable . patients were excluded if they had a major stroke with poor recovery of function , if their vascular anatomy rendered cas or cea unsuitable , if the stenosis was caused by non - atheromatous disease , if cardiac bypass was planned within 1 month of the revascularization procedure , or if there had been previous revascularization of the symptomatic artery . carotid endarterectomy in icss was performed according to the surgeon 's usual practice : local , general , or combined anaesthesia was allowed for the procedure . the type of arterial reconstruction to be carried out was not specified in the protocol , nor was the choice of peri - procedural medication . technical details of the surgical procedure and the occurrence of cranial nerve palsy or haematoma were reported by trial investigators . all patients were then re - assessed at 1 month after the procedure by a neurologist or investigator under their supervision . cnps were adjudicated internally at the icss trial office and judged to be disabling if the patient 's score on the modified rankin scale ( mrs ) increased to 3 or more at 30 days after the procedure , where that increase was attributable to the cnp . investigators were additionally asked to complete a questionnaire ( appendix i ) giving details of the clinical consequences of cnp and whether or not the lesion resolved during subsequent follow - up in the trial . haematoma was classified as severe if it required re - operation , transfusion , or prolonged hospital stay . the data were analysed per - protocol ; only patients in whom the randomly allocated procedure was initiated were included in this analysis . a procedure was deemed to have been initiated if the patient underwent either local or general anaesthesia prior to commencement of surgery . risk factors for cnp and haematoma were examined sequentially in a univariable binomial regression analysis using maximum likelihood estimation . wald tests were used for continuous and binary predictors , with an overall likelihood ratio test for categorical predictors of more than two levels . of 858 patients randomized to cea , the allocated procedure was initiated in 821 ( 95.7% ) . four of 821 patients ( 0.5% ) died between initiation of the procedure and 30 days post - procedure . forty - five of 821 patients ( 5.5% of initiated ceas ) were reported to have cnp within 30 days of the procedure . the results of adjudication of which cranial nerves were affected are presented in table 1 . a total of 50 cnps were reported : facial ( n = 23 ) , vagus ( 6 ) , hypoglossal ( 13 ) , glossopharyngeal ( 4 ) , accessory ( 1 ) , and trigeminal ( 1 ) ; in two patients it was not possible to determine which cranial nerve was affected . one cnp was judged to be disabling with an mrs of 3 at 1-month follow - up . this patient had glossopharyngeal nerve palsy with impairment in swallowing requiring placement of a naso - gastric feeding tube . two cranial nerve palsies were reported in each of five trial participants . in those patients with cnp where symptomatic resolution was confirmed ( n = 20 ) , the median duration of symptoms before resolution was 30 days ( minimum 2 days , maximum 520 days ) . the exact duration of symptoms in the remainder was undetermined . in only two of 821 patients ( 0.2% ) were the symptoms reported to have not resolved during follow - up of 6.4 and 3.1 years , respectively . one of these patients experienced voice hoarseness caused by vocal cord paresis following vagal nerve injury . the other experienced uplifting of the mouth on one side as a result of facial nerve injury . 1 . statistically significant predictors of cnp in univariable analysis were female sex ( risk ratio [ rr ] 1.90 , 95% ci 1.08 to 3.36 , p = .03 ) and a high degree of contralateral carotid artery stenosis . other demographic and technical factors , including the type of arterial reconstruction , type of anaesthesia or shunt use did not predict cnp . independent predictors of cnp in multivariable analysis , summarised in table 2 , were cardiac failure ( rr 2.66 , 95% ci 1.11 to 6.40 , p = .03 ) , female sex ( rr 1.80 , 95% ci 1.02 to 3.20 , p = .04 ) , the degree of contralateral carotid stenosis , and time to operation of > 14 days after the day of randomization ( rr 3.33 , 95% ci 1.05 to 10.57 , p = .04 ) . of 821 patients in whom the surgical procedure was initiated , 50 ( 6.1% ) developed neck haematoma . the results of univariable regression analysis for the risk factors for haematoma development are presented in appendix ii . statistically significant predictors of increased risk of haematoma were : anticoagulant prescription preoperatively ( rr 1.83 , 95% ci 1.04 to 3.23 , p = .04 ) , previous cardiac bypass graft surgery ( cabg ) ( rr 2.46 , 95% ci 1.37 to 4.42 , p < .01 ) , atrial fibrillation ( rr 2.29 , 95% ci 1.08 to 4.85 , p = .03 ) , and the duration of arterial clamping in minutes ( rr per each extra 20 minutes 1.13 , 95% ci 1.04 to 1.24 , p < .01 ) . factors associated with a decreased risk of postoperative haematoma were shunt use ( rr 0.54 , 95% ci 0.29 to 0.99 , p = .05 ) , antiplatelet agent prescription prior to the procedure ( rr 0.44 , 95% ci 0.21 to 0.93 , p = .03 ) and each 1 mmol / l increase in cholesterol at baseline ( rr 0.69 , 95% ci 0.55 to 0.88 , p < .01 ) . the results of multivariable analysis of predictors of risk for haematoma are presented in appendix iii . independent predictors of increased risk were being female ( rr 2.03 , 95% ci 1.13 to 3.62 , p = .02 ) , having atrial fibrillation ( rr 2.38 , 95% ci 1.07 to 5.27 , p = .03 ) , and the prescription of anticoagulant pre - procedure ( rr 1.86 , 95% ci 1.01 to 3.42 , p = .05 ) . independent factors reducing the risk of haematoma were shunt use ( rr 0.40 , 95% ci 0.21 to 0.80 , p < .01 ) and each 1 mmol / l increase in the patient 's baseline cholesterol level ( rr 0.68 , 95% ci 0.54 to 0.86 , p < .01 ) . twelve of 45 ( 26.7% ) patients with cnp also suffered haematoma , versus 38/776 ( 4/9% ) of patients without cnp . there was a significant association between these complications as detailed in table 3 ( p < .01 , fisher 's exact test ) . table 4 details the impact of adding cnp to the combined incidence of stroke , myocardial infarction ( mi ) , or death in icss in a post - hoc analysis comparing cea with cas . there was no significant difference in the combined risk of stroke , mi , death , or cnp , nor was there a significant difference in the incidence of disabling stroke , disabling cnp , or death between the two trial arms . of 858 patients randomized to cea , the allocated procedure was initiated in 821 ( 95.7% ) . four of 821 patients ( 0.5% ) died between initiation of the procedure and 30 days post - procedure . forty - five of 821 patients ( 5.5% of initiated ceas ) were reported to have cnp within 30 days of the procedure . the results of adjudication of which cranial nerves were affected are presented in table 1 . a total of 50 cnps were reported : facial ( n = 23 ) , vagus ( 6 ) , hypoglossal ( 13 ) , glossopharyngeal ( 4 ) , accessory ( 1 ) , and trigeminal ( 1 ) ; in two patients it was not possible to determine which cranial nerve was affected . one cnp was judged to be disabling with an mrs of 3 at 1-month follow - up . this patient had glossopharyngeal nerve palsy with impairment in swallowing requiring placement of a naso - gastric feeding tube . two cranial nerve palsies were reported in each of five trial participants . in those patients with cnp where symptomatic resolution was confirmed ( n = 20 ) , the median duration of symptoms before resolution was 30 days ( minimum 2 days , maximum 520 days ) . the exact duration of symptoms in the remainder was undetermined . in only two of 821 patients ( 0.2% ) were the symptoms reported to have not resolved during follow - up of 6.4 and 3.1 years , respectively . one of these patients experienced voice hoarseness caused by vocal cord paresis following vagal nerve injury . the other experienced uplifting of the mouth on one side as a result of facial nerve injury . 1 . statistically significant predictors of cnp in univariable analysis were female sex ( risk ratio [ rr ] 1.90 , 95% ci 1.08 to 3.36 , p = .03 ) and a high degree of contralateral carotid artery stenosis . other demographic and technical factors , including the type of arterial reconstruction , type of anaesthesia or shunt use did not predict cnp . independent predictors of cnp in multivariable analysis , summarised in table 2 , were cardiac failure ( rr 2.66 , 95% ci 1.11 to 6.40 , p = .03 ) , female sex ( rr 1.80 , 95% ci 1.02 to 3.20 , p = .04 ) , the degree of contralateral carotid stenosis , and time to operation of > 14 days after the day of randomization ( rr 3.33 , 95% ci 1.05 to 10.57 , p = .04 ) . of 821 patients in whom the surgical procedure was initiated , 50 ( 6.1% ) developed neck haematoma . the results of univariable regression analysis for the risk factors for haematoma development are presented in appendix ii . statistically significant predictors of increased risk of haematoma were : anticoagulant prescription preoperatively ( rr 1.83 , 95% ci 1.04 to 3.23 , p = .04 ) , previous cardiac bypass graft surgery ( cabg ) ( rr 2.46 , 95% ci 1.37 to 4.42 , p < .01 ) , atrial fibrillation ( rr 2.29 , 95% ci 1.08 to 4.85 , p = .03 ) , and the duration of arterial clamping in minutes ( rr per each extra 20 minutes 1.13 , 95% ci 1.04 to 1.24 , p < .01 ) . factors associated with a decreased risk of postoperative haematoma were shunt use ( rr 0.54 , 95% ci 0.29 to 0.99 , p = .05 ) , antiplatelet agent prescription prior to the procedure ( rr 0.44 , 95% ci 0.21 to 0.93 , p = .03 ) and each 1 mmol / l increase in cholesterol at baseline ( rr 0.69 , 95% ci 0.55 to 0.88 , p < .01 ) . the results of multivariable analysis of predictors of risk for haematoma are presented in appendix iii . independent predictors of increased risk were being female ( rr 2.03 , 95% ci 1.13 to 3.62 , p = .02 ) , having atrial fibrillation ( rr 2.38 , 95% ci 1.07 to 5.27 , p = .03 ) , and the prescription of anticoagulant pre - procedure ( rr 1.86 , 95% ci 1.01 to 3.42 , p = .05 ) . independent factors reducing the risk of haematoma were shunt use ( rr 0.40 , 95% ci 0.21 to 0.80 , p < .01 ) and each 1 mmol / l increase in the patient 's baseline cholesterol level ( rr 0.68 , 95% ci 0.54 to 0.86 , p < .01 ) . twelve of 45 ( 26.7% ) patients with cnp also suffered haematoma , versus 38/776 ( 4/9% ) of patients without cnp . there was a significant association between these complications as detailed in table 3 ( p < .01 , fisher 's exact test ) . table 4 details the impact of adding cnp to the combined incidence of stroke , myocardial infarction ( mi ) , or death in icss in a post - hoc analysis comparing cea with cas . there was no significant difference in the combined risk of stroke , mi , death , or cnp , nor was there a significant difference in the incidence of disabling stroke , disabling cnp , or death between the two trial arms . in icss , cnp developed in 5.5% of icss patients undergoing cea , and haematoma in 6.1% . the largest available series of patients studied pre- and post - operatively , in the european carotid surgery trial ( ecst ) , found a motor cnp rate of 5.1% and a long - term cnp rate of 0.5% at 4 months . likewise , nascet reported an overall risk of postoperative cnp of 8.6% , of which the majority were mild in severity , suggesting that cnp rates remain constant over time . patients should be made aware of these common complications and the likely clinical effects , including sensory and possible motor consequences . they can be reassured from the evidence presented here that postoperative cnp is rarely disabling ( risk around 1 in 1000 operations ) , but should be warned that the symptoms of the cnp may persist for several weeks or longer . there are several other findings of interest from this study : to the authors ' knowledge , the association between female sex and cnp has not previously been described , and this finding is worth confirming in another cohort of patients . one possible explanation for higher risk in female patients may be more challenging surgical anatomy and the smaller average diameter of the carotid artery . combined with other reports of a higher perioperative stroke risk in symptomatic women undergoing cea versus men , and evidence that the net benefit of cea in women is lower than in men , an increased incidence of cnp should be borne in mind when advising female patients of their risk of complications following cea . other risk factors for cnp identified in this analysis , including the degree of contralateral carotid stenosis , are statistically significant predictors but are more difficult to link clinically with the outcome and have not , to the authors ' knowledge , been reported by other groups . the association of neck haematoma with pre - operative anticoagulation is not surprising , but emphasizes the importance of careful surgical technique to mitigate the risk of haematoma in anticoagulated patients . a risk of haematoma in icss of 6.1% is similar to the rate of wound complications seen in large case series and a severe haematoma risk of 3.4% is similar to the reported risk of re - exploration of the surgical wound required in patients undergoing cea while on antiplatelet medication . however , although there is concern about perioperative bleeding in patients on dual antiplatelet therapy , the incidence of haematoma was actually decreased in this study in patients taking these medications , perhaps because surgeons took more care with haemostasis in these patients . some systematic reviews have included cnp in a composite outcome event of death or neurological complications up to 30 days after treatment . icss compared cas with cea on the assumption that if cas could avoid neck incision , cnp , and haematoma with no excess risk of stroke , then it could provide a beneficial alternative to cea for the prevention of recurrent stroke in patients with symptomatic carotid stenosis . in icss , as reported here , the total number of events in the composite cluster of any stroke , mi , death , or cnp was greater after cea compared with cas , but the numbers in the cluster of disabling stroke , disabling cnp or death were greater after cas than after cea . it is concluded , therefore , that it would not be appropriate to base treatment considerations concerning the choice of cas versus cea on the basis of composite short - term endpoints including cnp . information regarding baseline risk factors was unavailable , and information about the duration of cnp symptoms was limited . multiple comparisons without statistical correction raise the possibility of obtaining a type i ( false positive ) error . this is not a randomized comparison of surgical techniques or perioperative processes of care , and it is possible that unmeasured confounders are associated with the risk of cnp or haematoma . cnp remains a relatively common complication of cea , but in many patients is transient . haematoma is similarly common , and there is a statistical association between haematoma and cnp . fortunately , prolonged disability or permanent symptoms as a result of haematoma or cnp are rare , and thus , in the authors ' opinion , do not warrant inclusion in composite endpoints for future trials of carotid revascularization , but nevertheless one in 821 cea patients in icss had permanent impairment of swallowing caused by cranial nerve palsy . information regarding baseline risk factors was unavailable , and information about the duration of cnp symptoms was limited . multiple comparisons without statistical correction raise the possibility of obtaining a type i ( false positive ) error . this is not a randomized comparison of surgical techniques or perioperative processes of care , and it is possible that unmeasured confounders are associated with the risk of cnp or haematoma . cnp remains a relatively common complication of cea , but in many patients is transient . haematoma is similarly common , and there is a statistical association between haematoma and cnp . fortunately , prolonged disability or permanent symptoms as a result of haematoma or cnp are rare , and thus , in the authors ' opinion , do not warrant inclusion in composite endpoints for future trials of carotid revascularization , but nevertheless one in 821 cea patients in icss had permanent impairment of swallowing caused by cranial nerve palsy . icss was funded by the uk medical research council ( mrc ) and managed by the uk national institute for health research ( nihr ) on behalf of the mrc - nihr partnership ( grant numbers g0300411 and eme 09/800/14 respectively ) . the views and opinions expressed herein are those of the authors and do not necessarily reflect those of the nihr health services research programme of the department of health . additional funding was supplied by grants from the stroke association ( grant numbers tsa2005/01 and tsa2007/12 ) , sanofi - synthelabo and the european union . mmb 's chair in stroke medicine is supported by the reta lila weston trust for medical research . this work was undertaken at university college london , which received a proportion of funding from the uk department of health 's national institute for health research biomedical research centres funding scheme . the funders and sponsors of the study had no role in study design , data collection , data analysis , data interpretation or the writing of this paper .

the three - dimensional configuration of mid - urethral sling tapes is difficult to demonstrate in traditional anatomical dissections or imaging studies . the aim of this study was to test the utility of a novel technique using mesh tapes to assess spatial differences between the in - out and out - in transobturator mid - urethral slings . two independent surgeons performed their usual transobturator mid - urethral sling placement on 10 fresh thawed cadavers , alternating sides in the consecutive cadavers . tantalum wires threaded through the polypropylene - tapes rendered them radio - opaque . following placement , ct scans were obtained to generate 3-d and mips images for analysis . results showed that the mean angle formed by the in - out sling measured 122 ( 95%ci : 107-136 ) ; versus 144 ( 95%ci : 131-151 ) for the out - in sling ( p = 0.02 ) . the paired differences between the tapes inner angles were significantly different ; with a mean difference of 20 ( median 19.0 ) , ( p = 0.008 ) . there was no significant correlation between either approach and bmi or angle of the pubic arch . the images revealed that the tapes lie as a band posterior / dorsal to the urethra rather than inferior . in conclusion : marking mesh with tantalum wire , in combination with 3-d and mips ct - scan reconstruction images , provided a unique method to visualize the entire sling trajectory . the clinical implications of the more horizontal positioning after the out - in approach remain to be determined .

the transobturator approach to treat female stress urinary incontinence by means of a midurethral sling ( mus ) placement was first described by delorme to potentially prevent complications associated with the retropubic mus placement originally described by ulmsten ( delorme , 2001 ; ulmsten et al . , 1996 ) . a similar transobturator passage utilizing an inside - out approach , as opposed to delorme s outside - in approach , was later described by de leval , aiming to further reduce the potential injury to the urethra and bladder ( de leval , 2003 ) . a recent meta - analysis found similar cure rates between the 3 most commonly used surgical approaches for treating stress urinary incontinence , namely the retropubic , outside - in transobturator , and inside - out transobturator mus procedures ( latthe et al . , 2010 ) . multiple studies have discussed the anatomical trajectories of both obturator approaches and confirmed their anatomical safety from a neurovascular perspective ( bonnet et al . , 2005 ; achtari et al . , 2006 ; hinoul et al . , 2007 ; zahn et al . , 2007 ; reisenauer et al . , 2006 ) . the large body of clinical evidence supports these findings . to date , anatomical studies have focused on the mus s trajectory in relation to the obturator foramen , the muscles and especially the neuro - vascular structures . traditional anatomical dissections do not allow the study of the mus s configuration in relation to the urethra , as a progressive dissection of the entire tape s trajectory would be required , inherently leading to a distortion of the relevant anatomy . the aim of this cadaveric study was to compare the specific relationship between the tape and the urethra in both the inside - out and outside - in approach . to allow such a comparison , ct - scan image analysis was performed after tantalum wire marked mesh slings were implanted in a series of cadavers . all 10 cadavers were operated upon , by independent surgeons , one experienced with the inside - out ( in - out ) and one with cadavers were positioned in the dorsal supine lithotomy position with the legs in abduction and at a 100 flexion position ( at the level of the hips ) on the in - outside and at 90 flexion on the out - in side . the surgical technique for the transobturator mus procedures were performed according to the instructions for use as provided by the respective manufacturers ( out - in : monarc , american medical systems , minnetonka , mn and in - out : tvt - obturator system , ethicon inc . , all cadavers were catheterized using a foley catheter with its lumen filled with a radio - opaque solution ( hypaquemeglumine 60% , amersham health , princeton , nj ) to allow subsequent identification of the urethral position . both the in - out and the out - in transobturator procedures were performed on each cadaver , alternating the type of procedure and tape between the right and left sides in the consecutively operated cadavers . to be able to do this with a single tape in each cadaver , a device was conceived for this study in which the tape on one side consisted of an in - out mus device , while an out - in mus device was attached to the other side at the midpoint . to allow radiographic visualization of these tapes , a double tantalum wire was threaded through each side of both midurethral tapes as shown in figure 1 . this design allowed for an intra - cadaveric comparison between 10 independent in - out transobturator mus trajectories and 10 out - in transobturator mus trajectories . care was taken to have a flat positioning of both tape ends at the end of the procedures . the vaginal incision sites were closed and the mus tapes were cut 2 cm from the level of the skin . to exclude the possible influence of the surgeon s right handedness , an equal number of both procedures was performed on the right and left sides ; bmi of the cadavers ranged between 15 and 33 kg / m2 ( mean : 25.3 ; median : 26 ) . post capture image analysis was performed using the vitrea core 5.1.1618.1808 image analysis software ( vital , 5850 opus parkway , suite 300 , minnetonka , mn 55343 - 4414 ) . plane that included the urethra at the point where it crossed the urethra and the two arms of the sling extending from the urethra to the point where it crossed the obturator membrane . this was done to prevent parallax effects that might alter the measured angle ( hoyte and ratiu , 2001 ) . on the mips view of the images , the relative angulations between a line through the urethral axis and the slope drawn through each mus tape s mean upward pathway ( forming the hammock part of its trajectory ) were measured ( table i ) . the angle inside the upward trajectory of the tape constituted the angle under consideration for this study and is referred to as the tape s hemi inner angle , as only half a procedure was performed on each cadaver ( fig . for the study s purposes the assumption was made that a symmetric , bilateral placement of a full length tape would yield the full inner angle of the mus hammock corresponding to double the hemi - inner angle measurement . 2a . ventro - dorsal view ( frontal view ; l = left , r = right ) . note : dotted lines schematically demonstrates the hemi - inner angle but do not correspond to the mips view used to measure the actual angles . statistical analyses were performed using the wilcoxon signed - rank test for matched paired data for non - parametric testing of 2 dependent samples . statistical analyses were performed using the wilcoxon signed - rank test for matched paired data for non - parametric testing of 2 dependent samples . three cadavers were noted to have stage 2 prolapse and 1 had a notably high vaginal sulcus . the 3-dimensional ct images revealed that the mus tapes lie as a band posterior / dorsal to the urethra rather than inferior . the out - in muss were more closely wrapped around the inferior ischio - pubic ramus than the in - out muss . qualitative assessment of the images demonstrated a safe distance from the tapes to the obturator canal for both techniques . distances of the tapes trajectories to the individual obturator nerve branches could not be determined . the mean full inner angle in a strict antero - posterior view formed by the in - out mus measured 122 ( 95%ci : 107-136 ) ; versus 144 ( 95%ci : 131-151 ) for the out - in mus ( p = 0.02 ) . the ( paired ) differences between the tapes inner angles were significantly different ; with a mean difference of 19.8 ( median 19.0 ) , ( wilcoxon signed rank test : p = 0.008 ) . correlation analysis only showed a trend between the out - in and the in - out mus angle ( correlation coefficient : 0.6 , p = 0.08 ) . a similar trend was observed between the patient s bmi and the in - out mus angle ( correlation coefficient : 0.6 , p = 0.08 ) , but not for bmi and the out - in mus angle ( correlation coefficient : 0.08 , p = 0.8 ) . there was no correlation between the infra - pubic angle measurement and the out - in mus angle nor the in - out mus angle . the use of tantalum wires threaded through the polypropylene-/ prolene - tape in combination with three - dimensional ct - scanning yielded a unique method to visualize midurethral tapes throughout the entirety of their trajectory . this technique can be applied to different types of non radio - opaque implants , allowing for a better spatial understanding of the anatomical position within the pelvis . this technique circumvents the technical problems encountered by ultrasound where visualization is partially obstructed by the bony pelvis and the fact that traditional anatomical techniques rely on a progressive dissection intrinsically only allowing visualization at a single level each time . this is the first controlled anatomical study to compare the spatial relationships between the in - out and out - in mus tapes and the female urethra . the difference could be objectified by measuring the angle formed between the two legs of the mus tape . the in - out approach angulated around the urethra in a more acute fashion than the out - in approach , which seemed to follow a more horizontal , flatter , trajectory ( 122 versus 142 ) . these findings were counter - intuitive as the exit - points of both devices at the level of the skin would suggest the opposite . the three dimensional radiographic visualization of the tape s trajectory also allowed for an appreciation of the mus tapes spatial configuration in relation to the bony pelvis and the urethra . contrary to the belief that the urethra is suspended in a cranially directed u - shaped band , both obturator mus approaches were demonstrated to be u - shaped bands that head backwards or dorsally . the tape is not positioned caudally but lies as a band behind the urethra , suggestive of its passive ( non obstructive ) role in achieving continence . as observed in the different measurements of the tapes angles , there seemed to be a higher variability in the in - out s than the out - in trajectory ( 58 versus 40 variation respectively ) , as previously described by hinoul et al . in a traditional anatomic study , underscoring the need for strict adherence to the prescribed operative technique ( hinoul et al . , 2007 ) . the importance of this becomes clear when comparing it to two ultrasound studies investigating the angles formed by the two arms of mus tapes . lin et al described an angle at rest for the in - out tape to be 115 ( sd13 ) while chene et al , measured the same angle at rest also using ultrasound to be 138(sd7 ) ( lin et al . the differences in outcomes obtained in these studies demonstrate that ( inter- and intra - individual ) comparisons may prove to be difficult to interpret . the cadaveric study design was also the study s limitation as no clinical findings could be correlated to the static anatomic findings . a direct meta - analysis comparing both routes of transobturator tapes by madhuvrata et al . ( 2012 ) reported no significant differences in the objective and subjective efficacy but showed that the inside - out route was associated with significantly fewer vaginal angle injuries but with a non - significanttrend towards higher risk of postoperative groin pain . in a small retrospective study , out - in mus tapes were more frequently associated with a finding of superficial placement at the level of the lateral vaginal sulcus , described as paraurethral banding ( cholhan et al . , 2010 ) . coincidentally , the only cadaver with a notably high vaginal sulcus was found to have a very palpable out - in mus tape post placement . increased rates of dyspareunia and tape exposures after the out - in compared to the in - out approach ( 2012 ) , but these finding were not confirmed in an rct by abdel - fattah et al . the more horizontal positioning of the out - in tape s trajectory , taking the mesh closer to the vaginal wall , as described in this study could explain these clinical observations . the radiographic images explain the increased risk of button - holing during an out - in procedure because the more acute in - out mus trajectory buries the tape deeper inside the internal adductor muscles . on the other hand , this study also confirmed that the out - in mus trajectory stayed closer to the ischiopubic ramus . the slightly more lateral in - out mus trajectory causes the tape to be seated in more muscular tissue . the corresponding inflammatory reaction might explain the trend to a slightly higher risk of transient thigh pain . the clinical implications from this cadaveric imaging study are that surgeons must pay more attention to the 2nd half of the helical pass , whichever way they chose to go . for the in - out passage , one must rotate hard and carefully around the ischiopubic ramus and not get lazy with the rotation ; while for the out - in pass , care must be taken that dissection of the peri - urethral tunnel provides for a full thickness vaginal epithelial layer . the advent of three - dimensional ultrasonography will allow for similar measurements of the different tapes trajectories in vivo in a dynamic setting , possibly allowing correlation of these findings to nuances in clinical outcomes . ultrasound will not be able to provide the same holistic visualization within the bony pelvis , but mesh - tape beyond the obturator membrane probably does not contribute to the mus functional effect or possible adverse events at the level of the vagina . recently , a new approach , incorporating iron particles into polymer - based implants , allowed in vivo visualization of mesh by magnetic resonance imaging ( mri ) following inguinal hernia surgery ( hansen et al . , 2013 ) . approval of these types of products for human use in the future will bring new insights into the action mechanism of mesh in the pelvic floor as well as a better understanding of the mesh- tissue interaction .

backgroundneurocutaneous melanosis ( ncm ) is a rare neurocutaneous syndrome characterized by the presence of multiple congenital melanocytic nevi ( cmn ) and the proliferation of melanocytes in the central nervous system , usually involving the leptomeninges . chronic partial epilepsy as a sole manifestation is rare in ncm.case reporta 32-year - old man suffering from chronic partial epilepsy presented with multiple cmn on his trunk and scalp . brain mri demonstrated a focal lesion in the right amygdala that was consistent with interictal epileptiform discharges in the right temporal region on electroencephalography ( eeg ) . an anterior temporal lobectomy was performed , and the pathology investigation revealed numerous melanophages in the amygdala . the patient was seizure - free after surgery.conclusionswe report a patient with ncm presenting as chronic partial epilepsy who was successfully treated by anterior temporal lobectomy .

neurocutaneous melanosis ( ncm ) is a rare neurocutaneous syndrome characterized by the presence of multiple congenital melanocytic nevi ( cmn ) and the proliferation of melanocytes in the central nervous system , usually involving the leptomeninges . a 32-year - old man suffering from chronic partial epilepsy presented with multiple cmn on his trunk and scalp . brain mri demonstrated a focal lesion in the right amygdala that was consistent with interictal epileptiform discharges in the right temporal region on electroencephalography ( eeg ) . an anterior temporal lobectomy was performed , and the pathology investigation revealed numerous melanophages in the amygdala . we report a patient with ncm presenting as chronic partial epilepsy who was successfully treated by anterior temporal lobectomy . neurocutaneous melanosis ( ncm ) is a rare congenital , noninheritable neurocutaneous syndrome , characterized by the presence of large and/or multiple congenital melanocytic nevi ( cmn ) associated with melanosis or melanoma that usually appear in the leptomeninges . although the clinical presentations of ncm vary with age , ncm is generally accompanied by significant neurological deficits . however , ncm as a cause of epilepsy without any neurological deficit is extremely rare . we report a case of chronic partial epilepsy caused by pathologically proven parenchymal ncm in the brain without leptomeningeal involvement , that was successfully treated surgically . a 32-year - old right - handed man visited our epilepsy clinic due to drug - resistant partial epilepsy . his seizures started at the age of 22 years , since when he had suffered from frequent episodes of disgusting odor and light - headedness despite antiepileptic drug treatment . also , he had occasionally experienced motionless staring followed by generalized tonic - clonic seizures when he had not complied with treatment . his familial and own medical histories were not remarkable , and the findings of a neurological examination were normal . electroencephalography ( eeg ) showed frequent right temporal sharp waves with maximal negativity in the ipsilateral nasopharyngeal electrode . brain mri showed a small focal lesion in the lateral part of the right amygdala . the lesion was hyperintense on a t2-weighted image and hypointense on a t1-weighted image without gadolinium enhancement ( fig . 1 ) , which suggested a benign tumor.1 there were multiple large black pigmented nevi on the back , scalp , and posterior neck area , which had existed from birth . the largest black nevus was on his back , covering 9% of his body surface area ( 1.82 m ) . the amygdala lesion was a grayish - to - black friable soft tissue without definite mass formation . the skin biopsy of the scalp lesion showed that it was an intradermal nevus , as expected ( fig . 2 ) . he was seizure - free ( including aura ) during the 15-month postsurgery follow - up period . kadonaga and frieden2 proposed the following criteria in 1991 that are currently used to diagnose ncm : 1 ) large and/or multiple cmn in association with meningeal melanosis or melanoma ; 2 ) no evidence of cutaneous melanoma , except in patients with histologically benign meningeal lesions ; and 3 ) no evidence of meningeal melanoma , except in patients with histologically benign cutaneous lesions . the second and third definitions exclude the possibility of metastases from the skin to the central nervous system ( cns ) and vice versa , respectively . cmn are classified into large , medium , and small nevi according to their size:3 larger than 20 cm in an adult , 1.5 to 19.9 cm , and smaller than 1.5 cm , respectively . kadonaga and freiden2 found that 66% of ncm patients had large nevi , and the remaining 34% had numerous pigmented lesions in the absence of a single large congenital melanocytic nevus . in their study , all ncm patients had either posterior midline nevi or head and neck lesions , which suggests that the posterior axial distribution is an important risk factor for developing ncm . embryologically , melanocytes originate from the neural crest and migrate to various sites in the body , such as the basal layer of the epidermis , pia mater , leptomeninges , medullary reticular formation , and substantia nigra of the midbrain.4 it is thought that cmn and ncm are caused by the arrest or aberrancy of this normal developmental pattern . in ncm , pigmented thickening of the leptomeninges is most frequent in areas with melanocytes.4 parenchymal melanosis is less common than leptomeningeal melanosis , and caused by primary involvement of the melanin - containing macrophages and melanocytes , or secondary spread via virchow - robin spaces from the leptomeninges . the pathology investigation of our patient showed melanin - containing macrophages , and revealed parenchymal melanosis without leptomeningeal melanosis . the neurological manifestations of ncm vary with age.2 before the age of 2 years , the most common initial clinical signs and symptoms of ncm are related to increased intracranial pressure , including headache ( 35% ) , vomiting ( 42% ) , generalized seizures ( 48% ) , increased head circumference ( 23% ) , cranial nerve palsies ( 26% ; in particular vi ) , papilledema ( 10% ) , and meningeal signs ( 3%).6 the subset of patients with a discrete intracranial mass becomes symptomatic when older ( mean age , 12.8 years ; range , from birth to 65 years ) and is more likely to develop focal seizures , localized sensorimotor deficits , difficulties with speech , or psychiatric symptoms.6 our patient became symptomatic with partial seizures when he was 22 years old . most of the 33 ncm patients in a study involving giant cmn7 demonstrated the first neurologic symptom when they were younger than 5 years . eighty - two percent of the patients died by the time of reporting , and the median age at death was 3 years . while symptomatic ncm is estimated to occur in less than 3% of patients with giant cmn,7 one study found mri evidence of cns melanosis in 10 ( 23% ) of 42 asymptomatic giant - cmn patients,8 among whom only 1 had subsequently developed symptomatic disease during an average follow - up of 5 years . because the mri findings of these 10 patients showed parenchymal ncm without diffuse leptomeningeal involvement , the prognosis of parenchymal ncm appears to be much better than that of typical ncm with diffuse leptomeningeal involvement or melanoma . melanin pigment is inherently paramagnetic , and hence melanin deposits in ncm show a high signal intensity on t1-weighted mri and a low signal intensity on t2-weighted mri . this pattern of parenchymal melanosis appears to be most evident and frequent at the amygdala and adjacent anterior temporal cortex.8 in our patient , brain mri showed the typical location but an atypical pattern of hyperintensity on t2-weighted images and isointensity on t1-weighted images . some cases with chronic epilepsy related to ncm have been reported , but most of them had other conditions such as cns melanoma , mental retardation , psychosis , and diffuse leptomeningeal involvement with neurologic deficits.9 to our knowledge , the only surgical case with ncm similar to our patient was a 29-year - old woman with normal psychomotor development and intelligence who had right temporal - lobe epilepsy due to amygdaloid melanosis and underwent right temporal resection.10 here we have reported a surgically successful case of chronic partial epilepsy caused by pathologically proven parenchymal ncm without leptomeningeal involvement . therefore , ncm occurring in the brain parenchyma should be considered as a cause of chronic partial epilepsy .

background : maternal thyroid function alters during pregnancy . inadequate adaptation to these changes results in thyroid dysfunction and pregnancy complications.objectives:this prospective study aimed to evaluate the prevalence of thyroid diseases in pregnancy and its outcomes in south of iran.materials and methods : this prospective study was conducted on 600 healthy singleton pregnant women who aged 18 to 35 years old at 15 to 28 weeks of gestation . we investigated the prevalence of thyroid dysfunctions in women . multivariate analysis was performed to determine the effect thyroid dysfunction on obstetric and neonatal outcome.results:thyroid stimulating hormone ( tsh ) levels of 0.51 , 1.18 , 1.68 , 2.4 , and 4.9 miu / l were at 2.5th , 25th , 50th , 75th , and 97.5th percentile in our population . the prevalence of clinical hypothyroidism , subclinical hypothyroidism , overt hyperthyroidism , and subclinical hyperthyroidism in all pregnant women was 2.4% , 11.3% , 1.2% , and 0.3% , respectively . in addition , 1.4% of patients had isolated hypothyroxinemia . clinical hypothyroidism was associated with increased risk of preterm delivery ( p = 0.045 ) . subclinical hypothyroidism had a significant association with intrauterine growth restriction ( iugr ) ( p = 0.028 ) as well as low apgar score at first minute ( p = 0.022 ) . maternal hyperthyroidism was associated with iugr ( p = 0.048).conclusions : we revealed that thyroid dysfunction during pregnancy was associated with iugr and low apgar score even in subclinical forms . further studies are required to determine whether early diagnosis and treatment of thyroid diseases , even in subclinical form , can prevent their adverse effect on fetus .

the prevalence of overt hypothyroidism was 1% to 1.5% , ( 1 - 9 ) and prevalence of subclinical hypothyroidism was 5% to 8% ( 8 , 10 ) . the main pregnancy complications of hypothyroidism were anemia , preeclampsia , prematurity , low - birth weight ( lbw ) , fetal distress in labor , fetal death , and congenital hypothyroidism , and neurocognitive deficits in children . subclinical hypothyroidism might be associated with preterm delivery and low apgar score ( 1 - 3 , 11 , 12 ) . overt hyperthyroidism and subclinical hyperthyroidism affects about 0.2% to 0.8% and 0.4% to 1% of pregnancies , respectively ( 1 - 3 , 13 ) . maternal hyperthyroidism may cause preterm delivery , intrauterine growth restriction ( iugr ) , and neonatal thyrotoxicosis ( 1 - 3 , 14 ) . according to the last published guidelines on 2012 for the management of thyroid dysfunction during pregnancy and postpartum , " universal screening of healthy women for thyroid dysfunction before pregnancy is not recommended " and " the committee could not reach agreement with regard to screening recommendations for all newly pregnant women . " increased knowledge about the interaction between the thyroid and pregnancy has changed our view about the definition and diagnosis of thyroid dysfunction in pregnancy ( 15 - 18 ) ; for example , we have recently found that thyroid stimulating hormone ( tsh ) level of 2.5 miu / l in the first trimester has been accepted as the upper limit of normal range ( 15 ) . this fact has an important implication in interpretation of the previous literature as well as a critical effect on diagnosis of clinical hypothyroidism . considering these changes , the american thyroid association has published a new guideline in 2011 for an accurate diagnosis and management of thyroid disease in pregnancy . this guideline determined the normal limits of tsh in the second trimester as 0.2 to 3 miu / l and free thyroxin ( ft4 ) as 12 to 30 pmol / l ( 15 ) . subsequent to these changes in definition of thyroid diseases in pregnancy , it is necessary to reevaluate the accurate prevalence of thyroid diseases in pregnancy and their mentioned effects in previous studies ( 1 - 3 , 12 - 14 ) . recent changes in definition of thyroid diseases in pregnancy has resulted in changes in interpretation of previous studies about the prevalence and effect of thyroid diseases on pregnancy outcomes after 2011 . in addition , due to lack of sufficient data about the prevalence and pregnancy outcomes of thyroid disease in iran , this prospective study aimed to evaluate the prevalence of thyroid diseases and its outcomes in pregnancy in fars province , south of iran . this prospective study included 600 singleton pregnant women who were in consecutive follow - up at shiraz university of medical sciences obstetric hospitals ( zeinabieh hospital and hafez hospital ) , shiraz , iran . shiraz is the capital city of fars province in the south of iran . in 2000 , iran was considered as an iodine sufficient country by the world health organization and in previous reports , iranian pregnant women were also iodine sufficient ( 19 , 20 ) . informed consent was obtained from each patient included in the study and the study protocol conforms to the ethical guidelines of the 1975 declaration of helsinki as reflected in a priori approval by the shiraz university of medical sciences human research committee . inclusion criteria were healthy singleton pregnancy , residents in fars province , and pregnancy with gestational age of 15 to 28 weeks . in the first visit , individuals at high risk for thyroid illness according to their medical history , physical examination , or prior biochemical information had been considered and the women were excluded if they had known chronic diseases such as thyroid diseases , usage of thyroid drugs , diabetes mellitus , and hypertension . data about maternal age , parity , obstetric history , gestational age ( determined by last menstrual period ) , medical history of thyroid drug usage , and physical examination of the mother were collected twice : in the second trimester of pregnancy and at delivery . physical examination of the neonate was done in delivery room to determine apgar score , resuscitation at birth , weight , head circumference , and length in the hospital . preeclampsia was diagnosed when a pregnant woman develops both of the followings : 1 ) systolic blood pressure > 140 mm hg and/or diastolic blood pressure > 90 mm hg at two separate visits with at least six hour interval in a previously normotensive woman ; and 2 ) proteinuria 0.3 g/24 hours ( 21 ) . iugr was defined as birth weight 10th percentile for its gestational age ( 22 , 23 ) . preterm delivery was defined as the delivery before the end of 37th week of gestational age ( 24 ) . apgar score was determined by evaluating the newborn on five simple criteria on a scale from zero to two and summing up the five values . the five criteria are appearance , pulse , grimace , activity , and respiration . the first minute apgar score < 7 was defined as low apgar score ( 25 ) . for thyroid function tests ( tft ) , 10-ml blood sample of pregnant women was drawn at the first visit in the second trimester ; then it was centrifuged and stored in aliquots at -70 until assays , which were done after delivery . tft were assessed by quantitative analysis of serum tsh and ft4 ( electrochemiluminescence , cobase411 , japan ) . the inter - assay and intra - assay coefficients of variation were respectively < 8.6% and < 8.7% for tsh ; in addition , these values were < 2.9% and < 6.6% for ft4 , respectively . tsh was mentioned as miu / l and ft4 as pmol / l . according the last guideline of american thyroid association ( ata ) for the diagnosis and management of thyroid disease during pregnancy and postpartum ( 15 ) , the reference range for the second trimester tsh and ft4 was 0.2 to 3 miu / l and 11.84 3.86 pmol / l , respectively . considering this recommendation and according to ata guideline ( 15 ) , we categorized pregnant women into five groups : category 1 ( clinical hypothyroidism ) was defined as an elevated tsh ( > 3 miu / l ) in conjunction with a low ft4 . in women with tsh 10 miu / l , irrespective of their ft4 level , were also considered as clinical hypothyroidism . category 2 ( subclinical hypothyroidism ) was defined if serum tsh was between 3 to 10 category 3 ( normal ) , was those with tsh of 0.2 to 3 miu / l and ft4 of 11.84 3.86 to . category 4 ( subclinical hyperthyroidism ) was defined as suppressed tsh ( range , 0.1 - 0.2 miu / l ) with normal ft4 . category 5 ( overt hyperthyroidism ) was defined as any suppressed tsh ( < 0.2 miu / l ) when accompanied by high ft4 and anyone with tsh < 0.1 miu / l irrespective of ft4 level . in addition , we calculated the prevalence of isolated hypothyroxinemia according to ata guideline in which tsh was in normal range but had low ft4 . analysis was done using spss 18 ( spss inc . , normality of the quantitative variables was assessed by kolmogorov - simonov test . to compare difference across groups , anova was used for normally distributed variables and kruskal - wallis test was used in those without normal distribution . data were mentioned as mean sd or median . for serum tsh , free triiodothyronine ( ft3 ) , and ft4 , 2.5th , 25th , 50th , 75th , and 97.5th percentiles as previously mentioned , category 3 was normal thyroid function pregnant women and was used as control group . the influence of thyroid diseases was analyzed by comparing the frequencies of each outcome in the abovementioned categories . results of analysis were mentioned as relative risk ( rr ) and the corresponding 95% confidence interval ( 95% ci ) . this prospective study included 600 singleton pregnant women who were in consecutive follow - up at shiraz university of medical sciences obstetric hospitals ( zeinabieh hospital and hafez hospital ) , shiraz , iran . shiraz is the capital city of fars province in the south of iran . in 2000 , iran was considered as an iodine sufficient country by the world health organization and in previous reports , iranian pregnant women were also iodine sufficient ( 19 , 20 ) . informed consent was obtained from each patient included in the study and the study protocol conforms to the ethical guidelines of the 1975 declaration of helsinki as reflected in a priori approval by the shiraz university of medical sciences human research committee . inclusion criteria were healthy singleton pregnancy , residents in fars province , and pregnancy with gestational age of 15 to 28 weeks . in the first visit , individuals at high risk for thyroid illness according to their medical history , physical examination , or prior biochemical information had been considered and the women were excluded if they had known chronic diseases such as thyroid diseases , usage of thyroid drugs , diabetes mellitus , and hypertension . data about maternal age , parity , obstetric history , gestational age ( determined by last menstrual period ) , medical history of thyroid drug usage , and physical examination of the mother were collected twice : in the second trimester of pregnancy and at delivery . physical examination of the neonate was done in delivery room to determine apgar score , resuscitation at birth , weight , head circumference , and length in the hospital . preeclampsia was diagnosed when a pregnant woman develops both of the followings : 1 ) systolic blood pressure > 140 mm hg and/or diastolic blood pressure > 90 mm hg at two separate visits with at least six hour interval in a previously normotensive woman ; and 2 ) proteinuria 0.3 g/24 hours ( 21 ) . iugr was defined as birth weight 10th percentile for its gestational age ( 22 , 23 ) . preterm delivery was defined as the delivery before the end of 37th week of gestational age ( 24 ) . apgar score was determined by evaluating the newborn on five simple criteria on a scale from zero to two and summing up the five values . the five criteria are appearance , pulse , grimace , activity , and respiration . the first minute apgar score < 7 was defined as low apgar score ( 25 ) . for thyroid function tests ( tft ) , 10-ml blood sample of pregnant women was drawn at the first visit in the second trimester ; then it was centrifuged and stored in aliquots at -70 until assays , which were done after delivery . tft were assessed by quantitative analysis of serum tsh and ft4 ( electrochemiluminescence , cobase411 , japan ) . the inter - assay and intra - assay coefficients of variation were respectively < 8.6% and < 8.7% for tsh ; in addition , these values were < 2.9% and < 6.6% for ft4 , respectively . according the last guideline of american thyroid association ( ata ) for the diagnosis and management of thyroid disease during pregnancy and postpartum ( 15 ) , the reference range for the second trimester tsh and ft4 was 0.2 to 3 miu / l and 11.84 3.86 pmol / l , respectively . considering this recommendation and according to ata guideline ( 15 ) , we categorized pregnant women into five groups : category 1 ( clinical hypothyroidism ) was defined as an elevated tsh ( > 3 miu / l ) in conjunction with a low ft4 . in women with tsh 10 miu / l , irrespective of their ft4 level , were also considered as clinical hypothyroidism . category 2 ( subclinical hypothyroidism ) was defined if serum tsh was between 3 to 10 category 4 ( subclinical hyperthyroidism ) was defined as suppressed tsh ( range , 0.1 - 0.2 miu / l ) with normal ft4 . miu / l ) when accompanied by high ft4 and anyone with tsh < 0.1 miu / l irrespective of ft4 level . in addition , we calculated the prevalence of isolated hypothyroxinemia according to ata guideline in which tsh was in normal range but had low ft4 . normality of the quantitative variables was assessed by kolmogorov - simonov test . to compare difference across groups , anova was used for normally distributed variables and kruskal - wallis test was used in those without normal distribution . data were mentioned as mean sd or median . for serum tsh , free triiodothyronine ( ft3 ) , and ft4 , 2.5th , 25th , 50th , 75th , and 97.5th percentiles as previously mentioned , category 3 was normal thyroid function pregnant women and was used as control group . the influence of thyroid diseases was analyzed by comparing the frequencies of each outcome in the abovementioned categories . results of analysis were mentioned as relative risk ( rr ) and the corresponding 95% confidence interval ( 95% ci ) . out of 600 enrolled women in our study , 14 refused to follow the study . ultimately , 586 women participated in our investigation to the end . the mean of participants age was 25.6 3.6 years , mean of their weight was 63.4 9.1 kg , and the mean of their height was 162.5 6.7 cm . fourteen women ( 2.4% ) had clinical hypothyroidism , 66 ( 11.3% ) had subclinical hypothyroidism , 2 ( 0.3% ) had subclinical hyperthyroidism , and 7 ( 1.2% ) had overt hyperthyroidism . general characteristics of the mothers ( height , weight , and age ) in each category are summarized in table 1 . there was no significant difference in general characteristics of mothers in different thyroid function categories ( p > 0.05 ) . the mean of the newborns head circumference was 35.4 1.7 cm , mean of their weight was 2599 385 g , and mean of their length was 50.9 2.8 cm . the neonates had an apgar score of 8.4 1.1 at first minute of delivery . general characteristics of the neonates are categorized according to maternal thyroid function in table 2 . categories are defined as follows : i , clinical hypothyroidism ; ii , subclinical hypothyroidism ; iii , euthyroid ( control ) ; iv , subclinical hyperthyroidism ; v , over hypothyroidism . p value across groups according to the diagnosis of thyroid functions ( category i - v ) . abbreviations : bmi , body mass index ; sbp , systolic blood pressure ; dbp , diastolic blood pressure ; cs , cesarean section delivery . categories are defined as follows : i , clinical hypothyroidism ; ii , subclinical hypothyroidism ; iii , euthyroid ( control ) ; iv , subclinical hyperthyroidism ; v , over hypothyroidism . p value across groups according to the diagnosis of thyroid functions ( category i - v ) . there was no significant difference between gestational age , head circumference , weight , length , and apgar score of neonates in different thyroid function categories ( p > 0.05 ) . therefore , the rank numbers of 2.5th to 97.5th percentiles were used to estimate the lower and the upper limits of the reference interval , respectively . 2.5th , 25th , 50th , 75th , and 97.5th percentiles for ft3 and ft4 , which had normal distribution , were calculated by frequency analysis . median ( interquartile range ) for subclinical hypothyroidism and overt hypothyroidism was 3.65 ( 3.18 - 4.93 ) and 12.1 ( 4.42 - 15.22 ) , respectively . abbreviations : tsh , thyroid stimulating hormone , ft3 , free triiodothyronine ; and ft4 , free thyroxine . prevalence rates of preeclampsia , iugr , preterm delivery and low apgar score in participants were 6.3% , 7.5% , 14.5% , and 8% , respectively . rout of delivery in 16.2% of mothers was cesarean section . in women with hypothyroidism , prevalence rate of preeclampsia , iugr , preterm delivery , and low apgar score was 7.5% , 13.7% , 21.2% , and 13.7% , respectively . prevalence rates of preeclampsia , iugr , preterm delivery and low apgar score in women with hyperthyroidism were 0% , 22.2% , 11.1% , and 11.1% , respectively . prevalence of hypothyroidism in pregnant woman was 13.7% ( clinical , 2.4% ; and subclinical , 11.3% ) . hypothyroidism was associated with iugr ( p = 0.017 ) and low apgar score at first minute ( p = 0.04 ) ; it increased the risk of iugr by 2.2 times and low apgar score by 1.95 times . clinical hypothyroidism had no significant association with preeclampsia ( p > 0.05 ) , but was associated with preterm delivery ( p = 0.045 ) . subclinical hypothyroidism had a significant association with iugr ( p = 0.028 ) and low apgar score at first minute ( p = 0.022 ) . it increased the risk of low apgar score by 2.15 times and iugr by 2.18 times . this regression analysis for iugr was adjusted for maternal body mass index ( bmi ) , age of mother , and occurrence of preeclampsia . moreover , logistic regression analysis for neonates with low apgar score was adjusted for delivery gestational age , maternal bmi , age of mother , and occurrence of preeclampsia . after this adjustment , no significant change in associations was found , except for the preterm delivery . the association of clinical hypothyroidism and preterm delivery was not significant ( p value = 0.116 ) after adjustment for confounding factors . abbreviations : iugr , intrauterine growth restriction ; rr , relative risk ; ci , confidence interval . p values are the result of chi square analysis of the frequency of each complication between abnormal thyroid function mothers and euthyroid ones ( as controls ) . no significant change in associations was seen after adjusting the analysis for the confounding factors except for the p value of the analysis of clinical hypothyroidism and preterm delivery , which was change to 0.116 after adjusting this analysis for maternal bmi , age , and preeclampsia . prevalence of hyperthyroidism in pregnant women was 1.5% ( overt , 1.2% ; and subclinical , 0.3% ) . nevertheless , it did not show a significant association with preeclampsia , preterm delivery , and low apgar score . these results for each complication were not significantly changed in logistic regression analysis after covariates were adjusted for abovementioned confounding factors . abbreviations : iugr , intrauterine growth restriction ; rr , relative risk ; ci , confidence interval . p values are the result of chi square analysis of the frequency of each complication between abnormal thyroid function mothers and euthyroid ones ( as controls ) . no significant change in associations was seen after adjusting the analysis for the confounding factors . prevalence rates of preeclampsia , iugr , preterm delivery and low apgar score in participants were 6.3% , 7.5% , 14.5% , and 8% , respectively . rout of delivery in 16.2% of mothers was cesarean section . in women with hypothyroidism , prevalence rate of preeclampsia , iugr , preterm delivery , and low apgar score was 7.5% , 13.7% , 21.2% , and 13.7% , respectively . prevalence rates of preeclampsia , iugr , preterm delivery and low apgar score in women with hyperthyroidism were 0% , 22.2% , 11.1% , and 11.1% , respectively . prevalence of hypothyroidism in pregnant woman was 13.7% ( clinical , 2.4% ; and subclinical , 11.3% ) . hypothyroidism was associated with iugr ( p = 0.017 ) and low apgar score at first minute ( p = 0.04 ) ; it increased the risk of iugr by 2.2 times and low apgar score by 1.95 times . clinical hypothyroidism had no significant association with preeclampsia ( p > 0.05 ) , but was associated with preterm delivery ( p = 0.045 ) . subclinical hypothyroidism had a significant association with iugr ( p = 0.028 ) and low apgar score at first minute ( p = 0.022 ) . it increased the risk of low apgar score by 2.15 times and iugr by 2.18 times . this regression analysis for iugr was adjusted for maternal body mass index ( bmi ) , age of mother , and occurrence of preeclampsia . moreover , logistic regression analysis for neonates with low apgar score was adjusted for delivery gestational age , maternal bmi , age of mother , and occurrence of preeclampsia . after this adjustment , no significant change in associations was found , except for the preterm delivery . the association of clinical hypothyroidism and preterm delivery was not significant ( p value = 0.116 ) after adjustment for confounding factors . abbreviations : iugr , intrauterine growth restriction ; rr , relative risk ; ci , confidence interval . p values are the result of chi square analysis of the frequency of each complication between abnormal thyroid function mothers and euthyroid ones ( as controls ) . no significant change in associations was seen after adjusting the analysis for the confounding factors except for the p value of the analysis of clinical hypothyroidism and preterm delivery , which was change to 0.116 after adjusting this analysis for maternal bmi , age , and preeclampsia . prevalence of hyperthyroidism in pregnant women was 1.5% ( overt , 1.2% ; and subclinical , 0.3% ) . nevertheless , it did not show a significant association with preeclampsia , preterm delivery , and low apgar score . these results for each complication were not significantly changed in logistic regression analysis after covariates were adjusted for abovementioned confounding factors . abbreviations : iugr , intrauterine growth restriction ; rr , relative risk ; ci , confidence interval . p values are the result of chi square analysis of the frequency of each complication between abnormal thyroid function mothers and euthyroid ones ( as controls ) . no significant change in associations was seen after adjusting the analysis for the confounding factors . we have investigated the normal distribution of tsh in our 586 pregnant women and showed that our range for the level of tsh in iran was higher than the ata ranges ( 15 ) . our data were similar to those of previous reported from india and iran in which the ranges of tsh in the second trimester were about 0.43 to 5.78 and 0.5 to 4.1 miu / l , respectively ( 26 - 28 ) . differences in laboratory methods , differences in kits , maternal iodine status , and ethnic , genetic , and environmental factors in our and other similar studies could explain these differences . on the other hand , other studies report a lower range for tsh level ( 29 - 33 ) . after publishing the ata guideline , we have found some studies in which the thyroid function status was evaluated according to this guideline ( 3 , 7 , 21 ) . the present study provides , for the first time , the data about the thyroid function status in iranian pregnant women according to this new guideline . we found that prevalence of hypothyroidism in pregnancy was 13.7% ( clinical , 2.4% ; and subclinical , 11.3% ) . in addition , 1.4% of our pregnant women had isolated hypothyroxinemia . according to previous reports , prevalence of clinical hypothyroidism in pregnant women ranged from 1% to 3.5% and subclinical hypothyroidism ranged from 4% to 31% ( 1 - 8 , 22 ) . prevalence of hyperthyroidism of pregnant women was 1.5% in our study ( subclinical , 0.3% ; and over , 1.2% ) . in previous reports , prevalence of hyperthyroidism was reported as 0.2% to 2% ( 1 - 8 , 22 ) . one of the causes of variation in prevalence of thyroid dysfunction might be variation in definition of normal range for tsh in different studies . the ata recommendations could orchestrate the investigations about the prevalence and effects of thyroid dysfunction and prevent the errors in our interpretations and clinical judgment about the detrimental effects of thyroid dysfunction on pregnancy outcomes . hypertension is one of the most important health problems related to both overt hypothyroidism and hyperthyroidism ( 1 , 34 ) and to a lesser extent , to subclinical thyroid dysfunction ( 35 ) . its prevalence was 6% , 7.5% , and 0% in euthyroid mothers , mothers with hypothyroidism , and mothers with hyperthyroidism , respectively . although the prevalence of preeclampsia was higher in pregnant women with hypothyroidism , this difference was not statistically significant . a few investigations have reported that overt hypothyroidism increases the incidence of preeclampsia ( 8 , 9 ) ; however most of the studies showed no significant association ( 1 , 3 , 7 ) . prevalence of preterm delivery was 13% , 21% , and 11.1% in euthyroid mothers , mothers with hypothyroidism , and mothers with hyperthyroidism , respectively . spontaneous abortion and preterm delivery have been reported in 17% to 31% of all pregnancies ( 36 , 37 ) . the majority of them have no known risk factor ( 6 ) . one of the known risk factors is clinical or subclinical hypothyroidism ( 38 - 40 ) . allan et al . showed that tsh level > 6 miu / l was significantly associated with a higher frequency of pregnancy loss ( 39 ) ; however , one recent study showed no significant associations between tsh level and the risk of preterm delivery ( 41 ) . we showed that the association of clinical hypothyroidism and preterm delivery might be due to secondary confounding factors such as maternal bmi , age , and preeclampsia . consistent with our results , previous reports revealed that subclinical hypothyroidism was associated with iugr ( 5 , 8 , 42 ) . it might be due to the essential role of thyroid hormones in growth and maturation of many tissues of the fetus like the brain , bones , and muscles ( 43 ) . we revealed that hypothyroidism is associated with low apgar score and mothers with subclinical hypothyroid have 2.15 times greater risk for having low apgar score neonates . showed a higher risk of fetal distress in mothers with subclinical or clinical hypothyroidism ( 44 ) . it seems that hypothyroidism exerts irreversible influences on the placenta and fetus during pregnancy and decreases the fetal ability to tolerate stress and therefore , neonates present with low apgar scores at birth ( 45 ) . therefore , future studies should evaluate thyroid autoantibodies in addition to tft . in our study , we found that the hypothyroidism during pregnancy , even in subclinical form , could cause iugr and low apgar score . although prevalence of hyperthyroidism in our pregnant women was very low , it could be associated with iugr . interventional studies are required to determine whether early diagnosis and treatment of thyroid diseases , even in the subclinical form , prevent their adverse effect on the fetus in iranian pregnant women .

the noncoding rna designated as microrna ( mirna ) is a large group of small single - stranded regulatory rna and has generated wide - spread interest in human disease studies . to facilitate delineating the role of micrornas in cancer pathology , we sought to explore the feasibility of detecting microrna expression in formalin - fixed paraffin - embedded ( ffpe ) tissues . using ffpe materials , we have compared fluorescent in situ hybridization ( fish ) procedures to detect mir-146a with ( a ) different synthetic probes : regular custom dna oligonucleotides versus locked nucleic acid ( lna ) incorporated dna oligonucleotides ; ( b ) different reporters for the probes : biotin versus digoxigenin ( dig ) ; ( c ) different visualization : traditional versus tyramide signal amplification ( tsa ) system ; ( d ) different blocking reagents for endogenous peroxidase . finally , we performed mir-146a fish on a commercially available oral cancer tissue microarray , which contains 40 cases of oral squamous cell carcinoma ( oscc ) and 10 cases of normal epithelia from the human oral cavity . a sample fish protocol for detecting mir-146a is provided . in summary , we have established reliable in situ hybridization procedures for detecting the expression of microrna in ffpe oral cancer tissues . this method is an important tool for studies on the involvement of microrna in oral cancer pathology and may have potential prognostic or diagnostic value .

microrna refers to the category of single - stranded small noncoding rnas that are approximately 22 nucleotides in length . more than 1500 human microrna have been identified and registered via various approaches including high throughput screenings ( http://www.mirbase.org/ ) . as more than 30% of human of mrnas are regulated by micrornas , the functional impact of microrna in physiology and pathology has yet to be fully elucidated . generally speaking , microrna imposes its regulatory role by sequence - specific but incomplete complementary binding to its target mrna sequences , which are usually located at the 3 untranslated region [ 1 , 2 ] . this binding may mediate the degradation of target mrna or the inhibition of protein translation efficiency of target mrna . however , due to the loose stringency of this kind of targeting , the exact mechanism of specific microrna function remains undefined and is an actively addressed research topic . the function and target mrnas of individual micrornas can not be reliably predicted via current bioinformatic approaches , thereby warranting continued experimental interrogation . the investigation of microrna expression and its functional relationship with various cancer types has instigated tremendous interest in employing such molecules as novel diagnostic or therapeutic modalities in oncology studies [ 3 , 4 ] . recent developments that show correlation between plasma microrna levels and cancer have further increased enthusiasm for this approach due to the easy accessibility of blood serum and plasma specimens . similarly abundant in clinical settings are archived formalin - fixed paraffin - embedded ( ffpe ) human cancer specimens , which can provide a rich resource for investigating the relationship between microrna expression and cancer progression . studies on such material have the added advantage of maintaining the information content from cancer tissue morphology when in situ hybridization ( ish ) is used to detect microrna expression . unlike the frequently used quantitative reverse transcription polymerase chain reaction ( q - rt - pcr ) method , which requires extraction of rna , ish retains the microscopic topological information content with respect to microrna expression and makes it possible to be related to other factors on the same tissue section . however , formalin fixation causes cross - links between biological molecules , potentially limiting access to microrna molecules . moreover , degradation has been a constant concern for preservation of rna molecules in such tissue samples . the in situ hybridization technique for nucleic acid detection on tissue and cytological preparations was initiated decades ago [ 7 , 8 ] , when radioisotope labeling and autoradiography were the only means to visualize positive hybridization signals . improvement and development of newer technologies , such as the advent of nonradioactive digoxigenin as probe labeling / reporter molecule and the tyramide signal amplification ( tsa ) system , have made in situ hybridization much more accessible . however , due to the short length of microrna molecules , in situ hybridization for microrna detection remains challenging . this paper demonstrates the feasibility and highlights key technical factors of developing a protocol for fluorescence in situ hybridization ( fish ) to detect microrna in archived ffpe human oral cancer tissues . formalin - fixed paraffin - embedded tissues are from lab - archived human oral cancer xenografts ( 10 blocks ) and commercially available human oral cancer tissue microarray ( tma ) sections from us biomax , inc . ( rockville , md ) , consisting of 50 tissue cores including 40 oral squamous cell carcinoma and 10 adjacent normal stratified squamous epithelia . xenograft specimen collection was approved by the animal care and use committees ( northwestern university and university of missouri ) . surgically removed xenograft tissues were immediately fixed in 10% neutral buffered formalin for 1224 hours and passed through dehydration , clearing , and paraffin - embedding steps . sections were cut at 5 m thick and mounted on positively charged slides , baked at 65c for 2 hours , and then stored at room temperature for later use . as for samples which were used to make tma , according to the supplier , they were typically put into formalin within 1530 minutes after surgical resection . some of the tissue samples were snap frozen in liquid nitrogen and stored there for later fixation . either way , fixation in neutral buffered formalin was about 24 hours before they were processed in automatic tissue processor and embedded in paraffin . ( st . louis , mo , usa ) unless otherwise specified . normal goat serum and hrp - conjugated anti - mouse igg were from santa cruz biotechnologies , inc . mouse antidigoxigenin and hrp - conjugated anti - digoxigenin antibodies were obtained from roche applied science ( indianapolis , in , usa ) . tsa - cyanine 5 kit ( nel705a ) was from perkin - elmer ( waltham , ma , usa ) , and contains hrp - streptavidin , blocking reagent , amplification diluents , and cyanine 5-conjugated tyramide . lna - based microrna mir-146a antisense oligonucleotides were labeled with digoxigenin ( dig ) at the 5 end . lna and non - lna - modified 5 biotinylated mir-146a specific probe and scrambled probe with the same sequences were custom made from integrated dna technologies , inc . prolong mounting media containing 4,6-diamidino-2-phenylindole ( dapi ) was from invitrogen ( carlsbad , ca , usa ) . an important consideration is to use diethylpyrocarbonate ( depc)-treated water in all solution preparation and to exercise caution to maintain a rnase - free work environment during all procedures . to generate 20x ssc , dissolve the following in 800 ml of milli - q grade water : 175.3 g of nacl and 88.2 g of sodium citrate , adjust the ph to 7.0 with a few drops of 1 m hcl , and adjust the volume to 1 liter with additional distilled h2o . sterilize by autoclaving . to prepare 50x denhardt 's solution , add the following to 900 ml distilled h2o : 10 g ficoll 400 , 10 g polyvinylpyrrolidone , and 10 g bsa , then fill up to 1 liter . filter the solution prior to storage through a 0.2 m filter and store at 4c ( but warm up to appropriate temperature prior to use ) . the prehybridization solution contains the following : 50% deionized formamide , 2x ssc , 1x denhardt 's , 0.02% sds , yeast trna ( 0.5 mg / ml ) , and salmon sperm dna ( 0.5 mg / ml ) . the hybridization solution contains 50% deionized formamide , 2x ssc , 1x denhardt 's , 10% dextran sulfate , yeast trna ( 0.5 mg / ml ) , and salmon sperm dna ( 0.5 mg / ml ) . for testing the effectiveness of peroxidase inhibition , two methods ( hydrogen peroxide versus hydrochloric acid ) after the sections were dewaxed and rehydrated , two kinds of fresh prepared solutions : 3% hydrogen peroxide in phosphate - buffered saline ( pbs ) and 0.024 m hydrochloric acid in ethanol applied , respectively , 200 l per slide , to two groups of slides and incubated for 10 minutes at room temperature . the cyanine 5-tyramide stock solution was diluted 1 : 50 using the included 1x amplification diluent to make the cyanine 5-tyramide working solution . approximately 100 l of cyanine 5-tyramide working solution was added per slide , incubated for 10 minutes at room temperature , and washed ( 3 times , 5 minutes each ) with pbs . slides were then air dried in the dark and mounted using prolong mounting media with dapi and coverslips . solidified fluorescent slides were observed with the wide field function of an olympus dsu spinning disc confocal microscope and slidebook software version 4.0 . fluorescent filter sets used for dapi are d350/50x and et455/50 m , and for cy5 are 645/30x and et705/72 m . the typical work flow of our fluorescence in situ hybridization for mir-146a microrna detection is described below . the total procedure can be completed within 2 days and is adaptable to detecting proteins of interest at the same time with multicolor labeling . a negative control slide should always be included that will be otherwise treated equally but with scrambled probe or without any probe in the hybridization step . deparaffinize in xylene ( 2x 10 minutes ) , rehydrate in serial ethanol solutions ( 100% , 90% , 80% , 70% ) , and depc - treated water ( 2 minutes each ) and pbs wash ( 2x 5 minutes ) . pretreatment of the slide : cross - linking during fixation can block reagent access to rna / dna molecules , so it is critical to unmask these sites using proteinases . it is also necessary to block certain endogenous active molecules that may interfere with signal development in later steps . to quench endogenous peroxidase : 0.024 m hcl in ethanol , incubate for 10 minutes , then pbs wash ( 2x 5 minutes ) . for proteinase treatment , incubate with proteinase k ( 20 g / ml , 37c for 10 minutes ) followed by a pbs wash ( 2x 5 minutes ) , and complete with 4% paraformaldehyde ( pfa ) fixation for 10 minutes followed by a pbs wash for 5 minutes ; 100 mm glycine incubation for 10 minutes ; pbs wash ( 2x 5 minutes ) and end with a 2x ssc wash ( 5 minutes ) . prehybridization : this is meant to block nonspecific binding of probes and minimize background signals . pre - hybridize for 2 hours at 50c in prehybridization solution , cover with plastic coverslip , and place in a moist chamber . hybridize overnight ( 18 hours ) at 50c with probe at the concentration of 25 nm ( 1 l 2.5 m dig labeled probe to 100 l hybridization solution ) , cover with plastic coverslip and place in a moist chamber . stringency wash : this step is critical to remove nonspecific probe binding and overloaded probes . wash with 2x ssc ( 37c for 15 minutes ) followed by a high temperature 2x scc wash ( at 50c with shaking ) . next wash with 1x ssc ( 2x at 37c for 15 minutes ) , with shaking at 50c in 1x scc for another 15 minutes , and wash with 0.02% sds in 1x ssc ( 2x at 37c for 15 minutes ) , with shaking at 50c in the same buffer for another 15 minutes . then followed by pbs - t ( pbs containing 0.1% tween-20 ) serum block uses 10% normal goat serum in pbs for 1 hour at room temperature , followed by mouse anti - dig ( 1 : 250 ) in the above blocking solution for 0.5 hour at room temperature ; rinse with pbs - t wash ( 4x 5 minutes ) . add hrp - conjugated goat anti - mouse igg ( 1 : 500 ) for 0.51 hour at room temperature . note that the use of 2 antibodies ( mouse anti - dig and hrp - conjugated goat anti - mouse igg ) could be replaced with hrp - conjugated anti - dig only . following antibody incubation , wash with pbs - t ( 4x 5 minutes ) and add cy5-tyramide working solution ( 100 l per slide ) ; incubate at room temperature for 10 minutes . pbs - t wash ( 4x 5 minutes ) ; pbs wash ( 5 minutes ) , air - dry for 10 minutes in the dark , and apply prolong gold mounting medium with dapi and a coverslip . the need to further evaluate the expression and function of specific micrornas in cancer pathology prompted us to establish a method of detecting micrornas in archived ffpe materials . abundant concerns regarding the integrity of rna in ffpe samples have been confirmed by studies showing that mrna in ffpe materials has various degrees of degradation as well as chemical modification by the fixative , rendering downstream analysis of extracted rna difficult . interestingly however , recent studies using microarray analyses to compare rna species between paired ffpe and fresh frozen samples demonstrated closely related microrna profiles , while mrna profiles exhibited signs of degradation in ffpe materials [ 11 , 12 ] . time to fixation , nuclease activity before fixation , chemical modification by formalin and variation in sample - processing procedures likely contribute to the decline in mrna quality in ffpe relative to fresh frozen tissue samples . however , as demonstrated in the aforementioned studies , the small size of microrna as well as its close association with large protein complexes enable the relatively better maintenance of their integrity after formalin fixation and paraffin embedding . this high degree of correlation between microrna signatures in ffpe and fresh frozen specimens encourages further exploration of microrna detection methods on archived pathology samples . after optimizing certain critical conditions , we have found that lna - based probes labeled with digoxigenin and combined with tsa amplification provided satisfactory results for mir-146a fish detection in archived oral cancer tissues . specimen processing in our study was relatively well controlled ; we have to acknowledge that results on specimens from real - life clinical archives may exhibit greater variability . horseradish peroxidase ( hrp ) conjugated antibodies combined with chromogenic substrates are frequently employed for this purpose . however , some tissues and cells contain endogenous peroxidase , especially leukocytes and erythrocytes . in cancer tissues , because of abundant angiogenesis and frequent inflammatory infiltration , peroxidase - containing cells are common . this is a major concern when using the highly sensitive tsa system , the mechanism of which depends on peroxidase activity and which provides up to 1000-fold amplification in detection sensitivity , as endogenous peroxidases produce significant background staining when not inhibited properly . because of this concern , we tested different quenching methods for endogenous peroxidase . hydrogen peroxide is mostly commonly used in immunohistochemistry , at a typical concentration from 0.3% to 3% diluted in methanol or pbs buffer and an incubation time of 10 to 60 minutes ( lower concentrations require longer incubation times but may induce less damage to certain antigens of interest ) . a less known method was reported by weir and colleagues decades ago , that is , to incubate slides with 0.024 m hydrochloric acid ( hcl ) in ethanol for 10 minutes to efficiently destroy endogenous peroxidase . we compared the use of 3% h2o2 ( figures 2(a ) and 2(b ) ) and 0.024 m hcl ( figures 2(c ) and 2(d ) ) on consecutive ffpe sections . the difference is striking , wherein incubation with the hcl solution produced a nearly complete suppression of endogenous peroxidase when incubated only for 10 minutes , but in 3% h2o2-treated samples at the same incubation time , inflammatory cells and even some carcinoma cells are quite positive with highly sensitive cy5-tyramide detection ( figure 2 ) . complete suppression of endogenous peroxidase activity may not be necessary for routine immunohistochemistry as trace peroxidase will not detectably affect the chromogenic reaction . however , with the tyramide signal amplification system , trace amounts of active peroxidase can result in tyramide precipitation , and this high sensitivity dictates the needs for thorough inhibition of such enzyme activity . as demonstrated ( figure 2 ) , dilute hcl solution is a convenient , low cost , and highly effective replacement for the traditional h2o2-blocking step . as for the timing of peroxidase blocking the ready availability of commercially labeled oligonucleotide - based probes enables end users to choose the tracer or reporter that best fits their protocols . biotin , digoxigenin , and fluorescein have been frequently used as reporter molecules on probes . early versions of the commercially available tsa system used streptavidin - biotin affinity for initial signal detection . in this reaction schema , a biotinylated probe hybridizes with a single - stranded target sequence , hrp - conjugated streptavidin binds to the biotin , hrp catalyzes the activation of tyramide conjugated with fluorescein , and active tyramide precipitates in the vicinity of hrp molecule . based on this protocol , we initially used biotinylated probes for in situ hybridization . however , exploratory tests confirmed that this is not appropriate for the type of tissues evaluated , as oral cancer cells are rich in endogenous biotin ( figure 3 ) . indeed endogenous biotin ( or a similar streptavidin - binding activity ) has been reported in many types of human tissues . to circumvent this nonspecific staining problem , digoxigenin ( dig ) has been long proven to be a good option as reporter molecule in nucleic acid probe design [ 1517 ] . as a heptan , the only natural source of dig is digitalis plants , significantly reducing the likelihood that the anti - dig antibody will bind any endogenous antigens in animal tissues . locked nucleic acid ( lna ) is a nucleic acid analog that contains at least one nucleotide monomer with a bicyclic furanose ring locked in a conformation mimicking rna . as the length of microrna is only about 22 nucleotides , many microrna molecules are differentiated from each other by only a few bases , such that it is difficult to achieve appropriate high - level probe sensitivity and specificity . lna - modified oligonucleotides demonstrate much higher thermal stability and higher melting temperatures when hybridized with target rna sequences compared to unmodified counterparts . it improves the mismatch discrimination , increasing the base - pairing selectivity and providing the needed high degree of affinity for effective microrna hybridization . using a commercially available lna - modified dig - labeled probe under the conditions outlined above resulted in appropriate differential staining ( figure 4 ) . prickle cells ( stratum spinosum ) in most normal oral squamous epithelia were positive for mir-146a , while basal cells exhibited negative staining . this kind of good performance is consistent with other studies showing the use of lna - based probes for hybridization - based microrna detection . the introduction of tyramide conjugates as substrates for hrp has revolutionized the sensitivity of any hrp - based detection system . mechanistically , hrp reacts with hydrogen peroxide and the phenolic part of tyramide and produces a quinone - like structure with a radical on the c2 group , becoming activated . activated tyramide then rapidly and covalently binds to all nearby tyrosine residues with proximity to the initially immobilized hrp site such that signal resolution is not compromised . previous studies have shown that the tyramide signal amplification system , which was also known as catalyzed reporter deposition method ( card ) , provides the capability to identify single copy dna or rna with conjugated fluorophore [ 21 , 22 ] . to detect microrna in situ with probe - based approaches requires such highly sensitive signal amplification . we have tested the use of the more traditional chromogenic substrate diaminobenzidine ( dab ) with hrp - labeled anti - dig antibody in the context of lna - based dig - labeled probes but did not get well - differentiated staining in in situ hybridization . the caveat is that when using any tsa system , complete suppression of endogenous peroxidase is essential , as discussed above . another well - recognized way of amplifying positive signal from nucleic acid hybridization is branched dna signal amplification [ 23 , 24 ] , which makes use of multiple layers of probes with the last layer being extensively enzyme labeled . however , for microrna detection , the primary probes still require the lna or lna - like nucleotides to enhance the initial specificity and sensitivity , and signal amplification with the branched dna procedure is less customizable than the tsa system allows . the use of branched dna signal amplification for in situ hybridization microrna detection has not been reported . ffpe tissues initially appeared to be a challenging platform for microrna detection but are actually better suited for microrna than mrna studies as recently revealed [ 10 , 11 , 25 ] . thus , clinically archived cancer tissue specimens can represent buried treasure , as micrornas are well preserved in such materials . our study has demonstrated that after efficient inhibition of endogenous peroxidase , lna - based and digoxigenin - labeled probe , applied together with tyramide signal amplification , significantly improves the results of fluorescence in situ hybridization for microrna detection . in summary , we have established a feasible in situ hybridization procedure for detecting the expression of microrna in ffpe oral cancer tissues . this detection is important for studies on the participation of microrna in oral cancer pathology and may have potential prognostic or diagnostic value as large cohort studies using such material will confirm .

summarydendritic cells ( dcs ) are professional antigen - presenting cells that hold great therapeutic potential . multiple dc subsets have been described , and it remains challenging to align them across tissues and species to analyze their function in the absence of macrophage contamination . here , we provide and validate a universal toolbox for the automated identification of dcs through unsupervised analysis of conventional flow cytometry and mass cytometry data obtained from multiple mouse , macaque , and human tissues . the use of a minimal set of lineage - imprinted markers was sufficient to subdivide dcs into conventional type 1 ( cdc1s ) , conventional type 2 ( cdc2s ) , and plasmacytoid dcs ( pdcs ) across tissues and species . this way , a large number of additional markers can still be used to further characterize the heterogeneity of dcs across tissues and during inflammation . this framework represents the way forward to a universal , high - throughput , and standardized analysis of dc populations from mutant mice and human patients .

conventional dendritic cells ( cdcs ) are found in almost all tissues and lymph nodes ( lns ) and act as sentinels capable of integrating multiple environmental signals and conveying them to cd4 and cd8 t lymphocytes . plasmacytoid dcs ( pdcs ) produce type i interferons and can also develop into antigen - presenting cells , particularly when stimulated by virus or self dna . human and mouse cdcs are derived from committed dc precursors ( pre - cdcs ) produced in the bone marrow ( bm ) . these pre - cdcs migrate from the bm into the blood and then seed the various tissues where they develop into two distinct lineages of cdc . the existence of two distinct dc lineages is supported by the identification of lineage - defining transcription factors ( tfs ) required for development and/or function of cdc1 ( irf8 , batf3 , id2 ) and cdc2 ( irf4 , zeb2 ) ( breton et al . , 2015 , grajales - reyes et al . , 2015 , guilliams et al . , 2014 , , 2006 , schlitzer et al . , 2015 , scott et al . , 2016 ) . a separate e2 - 2-dependent progenitor with prominent pdc potential has been recently described ( onai et al . , 2013 ) . with these recent molecular insights , it is now clear that cdcs belonging to the same lineage are present in various tissues and species ; however , these have been historically characterized by different surface markers . this results from the fact that many murine macs can express the prototypical cdc markers cd11c or mhcii and , conversely , that cdc2 can express the mac marker f4/80 ( bain et al . , 2012 , , 2015 , scott et al . , 2015 , tamoutounour et al . , 2012 , tamoutounour et al . , 2013 ) . distinguishing dcs from macs in human tissues has been equally challenging ( collin et al . , 2013 , mcgovern et al . , 2015 ) . finally , the lack of conserved markers to identify dcs hampered communication between mouse and human experts and was detrimental for fostering translational medicine . the advent of multicolor flow cytometry only aggravated the matter by yielding a seemingly ever - growing list of dc subsets based on different marker combinations . therefore , a rational approach simplifying the classification of dc subsets across tissues and species , yet still permitting the use of additional markers to study tissue- and disease - specific activation states , is urgently needed . it was recently proposed to classify dcs based on their ontogeny before subdividing them based on their micro - anatomical location or specific functional specialization ( guilliams et al . , 2014 ) . this would yield only three subsets of dcs : conventional type 1 dcs ( cdc1s ) , conventional type 2 dc ( cdc2s ) , and pdcs . however , due to a lack of consensus regarding how to define dc subsets experimentally , such classification remains of limited practical use ( guilliams and van de laar , 2015 ) . recent progress in the unsupervised analysis of high - dimensional flow cytometry datasets has rendered the identification process of cell subsets more objective and more reproducible ( saeys et al . , 2016 ) . however , a limitation of those approaches is that they give an equal weight to all the surface markers , not necessarily yielding the most biologically meaningful clusters . for instance , both langerhans cells ( lcs ) and cdc1s express cd207 , cd24 , mhcii , and cd11c , but they have completely different localization , ontogeny , lifespan , and functional specialization ( malissen et al . , 2014 ) . thus , the way forward has to be based on better markers to faithfully identify dc subsets alongside computational approaches that simplify the classification of dc subsets without compromising the multidimensional marker combinations necessary to grasp the fascinating functional heterogeneity of dcs . cd64 is highly expressed on macs and can be used in combination with f4/80 to discriminate these cells from cdc2s ( bain et al . , 2012 , gautier et al . , 2012 , , 2013 , scott et al . , 2015 , tamoutounour et al . , 2013 ) ( figure 1a ) . outgating macs on the basis of their cd64f4/80 phenotype is essential to prevent them from contaminating the cdc2 gate in most tissues ( figure s1 ) . as f4/80 is expressed on a part of the cdc2s , it should not be used alone to exclude macs , as has been proposed ( gurka et al . , 2015 ) . cd3 t cells , cd19b220 b cells , and nk1.1 natural killer ( nk ) cells were next excluded from further analysis using a lineage mix and the remaining cells gated for expression of mhcii molecules ( lineagemhcii cells ) . pdcs can be found among the lineage ( lin ) cells and identified as 120g8(cd317)b220cd11cly6ccd11b cells ( figure s1 ) . to obviate the fact that cd11c , a classical cdc marker , can be downregulated on dcs ( osorio et al . , 2014 ) and is lowly expressed by the double - negative , cd26 was added as an additional cdc marker , since it is highly expressed on all mouse cdcs across tissues ( see below ) . this permitted us to identify a well - defined population of cd11ccd26 cdc across tissues ( figure 1a , cyan gate ) that can be further subdivided into xcr1cd172a ( dark blue gate ) and xcr1cd172a ( green gate ) cdcs . to validate that xcr1cd172a and xcr1cd172a cells corresponded to cdc1s and cdc2s , respectively , we relied on their differential expression on irf8 and irf4 ( murphy et al . , 2015 , sichien et al . , analysis of the co - expression of both transcription factors ( tfs ) by intracellular staining revealed that xcr1cd172a cdc1 and xcr1cd172a cdc2 had an irf8irf4 or irf8irf4 profile , respectively ( figure 1b ) , while macs and pdcs had an irf8irf4 or irf8irf4 profile , respectively ( figure 1b and figure s1 ) . the pulmonary cd11cmhciicd64f4/80cd172a monocyte - derived cells ( often referred to as modcs ) also had an irf8irf4 profile . as expected , cdcs were lacking in flt3l mice ( figure 1c ) and competitive bm chimeras showed that cdc1 development was batf3 dependent and irf4 independent in all tissues analyzed ( figure 1d ) . cdc2s developed independently of batf3 , whereas they were irf4 dependent particularly in the lns . this likely reflects a late requirement for irf4 in cdc2 survival ( persson et al . , 2013 , schlitzer et al . , 2013 ) and is consistent with the fact that irf4 deficiency blocks the migration of dermal cdc2s to cutaneous lns , but not their intradermal development ( bajaa et al . , 2012 ) . therefore , the differential dependence on subset - defining tfs and growth factors validates the correct identification of cdc1s and cdc2s using just eight surface markers . intracellular irf4-irf8 double staining can be further used to validate the correct assignment of cdc1s , cdc2s , pdcs , and macs across tissues . identifying cdc1s and cdc2s across tissues in parallel allowed us to analyze their relative abundance in all tissues ( figure 1e ) . the network of myeloid cells found in the mouse skin and cutaneous lns is particularly complex to dissect due to the presence of lcs . lcs share many markers with cdcs ( mhcii , cd207 [ langerin ] , cd24 , and cd11c ) and macs ( f4/80 ) ( henri et al . presently , lc radioresistance constitutes the best way to distinguish them from radiosensitive dermal cdcs ( ginhoux et al . to distinguish lcs from cdcs , cd64f4/80 macs were first outgated and the broadly cd11ccd26 cells found among linmhcii cells ( figure 2a , cyan gate ) were subdivided into xcr1cd172a and xcr1cd172a cells . owing to their cd64linmhciixcr1cd172a phenotype , lcs must be distinguished from bona fide cdc2s present in the same gate , and cd24 expression allowed this . on a cd26-cd24 dot plot , cd26cd24 cdc2s ( green gate ) the distinction of cdcs from lcs on the basis of this minimalistic surface marker combination was validated using bm chimeras ( figure 2b ) . intracellular irf4-irf8 staining validated the correct identification of dcs and macrophages ( macs ) , in that all cdc1s displayed an irf8irf4 profile , all cdc2s displayed an irf8irf4 profile , and all macs and lcs displayed an irf8irf4 profile ( figures 1 and 2 ) . therefore , our gating strategy allows cdc1s and cdc2s to be identified across mouse tissues when cd24 is added to the panel to separate lcs from cdcs in skin . the dermis has been shown to contain cdc1s with a cd103 and cd103 phenotype and double - negative cdc2s that have low expression of cd11c and cd11b ( henri et al . , 2010 , , cd11b expression permitted cd11bcd11c double - negative cdc2s ( olive green gate ) to be distinguished from cd11bcd11c cdc2s ( green gate ) . nonetheless , all cdc1s had a homogeneous irf8irf4 profile and were batf3 dependent and irf4 independent , whereas all cdc2s showed a homogeneous irf8irf4 profile and were batf3 independent and irf4 dependent ( figures 2a and 2c ) . this illustrates that using a restricted set of lineage - imprinted markers facilitates the correct alignment of cdcs into cdc1s and cdc2s according to their ontogeny while still permitting the inclusion of additional markers to further gauge the presence of different subsets of cdc1s and cdc2s within a given tissue . clec9a - based fate - mapping identified a putative cdc2 subpopulation expressing cd64 in the kidney ( schraml et al . consistent with this , our gating strategy revealed that , unlike in other organs , kidney cd64f4/80 cells were not homogeneously irf8irf4 ( figure 1 ) but contained a small population of irf8irf4 cdc2-like cells ( figure 2d ) . these irf8irf4 cells expressed higher cd26 and cd11c and lower f4/80 and cd64 compared with irf8irf4 cells ( figure 2d ) . furthermore , irf8irf4cd64f4/80 cells , but not the irf8irf4cd64f4/80 cells , were flt3l dependent , identifying the irf8irf4 and irf8irf4 cells as cdc2s and macs , respectively . therefore , regardless of the expression of cd64 on a small fraction of kidney cdc2s , our gating strategy unambiguously identified cdc2s in the kidney . the flow cytometry dataset from distinct tissues was subjected to the unsupervised identification method flowsom ( van gassen et al . , 2015 ) . flowsom uses a self - organizing map ( som ) to cluster cells in different nodes based on the expression of the distinct markers used in a given flow cytometry dataset and subsequently structures the nodes in a minimal spanning tree . we first concatenated live cd45 cells from all tissues and used flowsom to generate a single flowsom tree . to identify the node(s ) corresponding to the cdc1s and cdc2s , we defined them ab initio as xcr1cd24cd26cd11cmhciicd11bcd172af4/80cd64linfscssc and cd11bcd172acd26cd11cmhciixcr1f4/80cd64linfscssc cells , respectively ( figure 3 ) . for each node , we calculated a final score indicating its correspondence with the defined cdc1 or cdc2 profile . first , a marker score was calculated for each node as the difference between the median value of the node for that marker and the minimum or maximum median node value present in the tree . then , for each marker , the score was normalized between zero and one , and the final node score was then computed as the mean of its scores for each individual marker . all nodes with a final node score of at least 0.95 times the highest score separate analysis of each tissue confirmed the presence of discrete cdc1 and cdc2 nodes in all tissues ( figure 3d ) . finally , exporting the cells within the cdc1 and cdc2 nodes identified by flowsom on xcr1-cd172a , cd11c - cd26 , and cd64-f4/80 dot plots confirmed that cdc1 and cdc2 were correctly identified across tissues ( figure 3e ) . notably , although cdc2s are defined as f4/80 , flowsom clustered f4/80 cdc2 with the f4/80 cdc2 ( figure 3e ) , since a small difference in one marker is not sufficient to separate cells into two separate clusters . therefore , unsupervised analysis corroborated our manual gating strategy and allowed the robust and fully automated identification of cdc1s and cdc2s across mouse tissues . an additional advantage of unsupervised identification algorithms such as flowsom or tsne is that each marker on a cell is analyzed simultaneously , compared with manual techniques that rely on sequential gating using pairs of surface markers . we have recently reported that splenic cdc1s lacking xbp1 downregulate their surface expression of cd11c ( osorio et al . , 2014 ) . this results in the loss of a fraction of cd11c cdc1s from the analysis when using a classical manual gating strategy ( figure s2a ) . in contrast , since our proposed manual gating strategy includes identification of cdcs by their cd11ccd26 profile , it was easier to avoid missing the cd11c cdc1s , as these cells maintained their high cd26 profile ( figure s2b ) . more importantly , both flowsom ( figures s2d and s2e ) and tsne ( figure s2f ) readily identified cdc1s regardless of their lower cd11c expression because the remaining xcr1cd172acd64f4/80mhciicd26 profile was sufficient to identify these cells as cdc1s in a fully unsupervised way . this demonstrates that unsupervised gating using flowsom or tsne outperformed classical manual gating for the analysis of the dc compartment of xbp1-deficient mice . we next aimed to align dcs across mouse , macaque , and human tissues ( figures 4 and s3 ) . we first had to overcome a few obstacles , such as the species - specific expression pattern of some markers ( cd64 in human and macaque ) and the lack of cross - reactive antibodies ( cd26 for macaque and xcr1 for human and macaque ) . in human and macaque , cd64 can not be used , as cdcs also express some cd64 ( figure s3e ) . therefore , we opted to exclude monocytes and macs on the basis of cd14 and cd16 expression as classically used . in mouse , human , and macaque and in all organs tested ( spleen , liver , lung for mouse and spleen , blood , lung for human and macaque ) , cdcs were thus defined as cd45linmhciicd11c cells that are f4/80cd64 in mouse and cd14cd16 in human and macaque . as in the mouse , cd26 although mouse cdc1s and cdc2s were both cd11ccd26 , human cdc1s were cd26cd11c , whereas cdc2s were cd26cd11c ( figure s3b ) . we investigated whether cadm1 could be used as a cdc1 marker instead of xcr1 since cadm1 is expressed on porcine and macaque cdc1s ( dutertre et al . 2015 ) . to compare the degree of overlap between the expression of xcr1 and cadm1 , we used a chimeric protein consisting of human xcl1the ligand of xcr1and of the mcherry fluorescent protein ( mcherry - xcl1 vaccibodies [ fossum et al . , 2015 ] ) to detect xcr1 on human and macaque spleen cells ( figure s4 ) . all cadm1cd172a cdc in human , macaque , and mouse spleen displayed a high xcr1 expression and also strongly expressed irf8 , but not irf4 ( figure s4 ) . thus , cdc1s can be defined as cadm1cd172a cdc in mouse , human , and macaque . we next evaluated the use of this panel in multiple mouse , human , and macaque tissues in combination with classical human dc markers such as cd1c ( bdca1 ) ( figure 4 ) . while cadm1cd172a cells comprised only irf4irf8 bona fide cdc2s in mouse , two populations of cadm1cd172a cells were detected in human and macaque lung : a population of bona fide cdc2s with a cd1cirf4irf8 phenotype and a population of cd1c cells showing the typical irf4irf8 expression observed for macs ( figures 4c and s4a ) . therefore , in the case of human and macaque , monocytes or macs were not properly outgated using cd14 and cd16 expression , and cd1c expression was further required to define cdc2s hence , across tissues and species , cdc1s and cdc2s could be identified as cadm1cd172acd11ccd26irf8irf4 and cadm1cd172acd1ccd11cirf4irf8 cells , respectively . akin to mouse pdcs , human and macaque pdcs ( defined as cd45cd11chladr cells ) were irf8irf4 across tissues ( figures s4b s4d ) . finally , in the human and macaque skin , the gating strategy required an additional marker , cd1a , to identify and outgate cd1acd11c lc before cdc1s and cdc2s could be faithfully identified ( figure s4 ) . therefore , using a limited number of flow cytometry markers , it is possible to align cdc subsets across several human , macaque , and mouse tissues , including the skin . we next used tsne to perform an unsupervised analysis of the flow cytometry dataset generated from different tissues of mouse , human , and macaque ( figure 5 ) . for each species ( figures 5a , 5e , and 5i ) , cd45linmhcii cells of the distinct tissues were exported , concatenated , and displayed in a single tsne contour plot ( tsne_dim1-tsne_dim2 ) . heatmap representations of the expression of various markers that define macs , cdcs , and pdcs defined clusters of cells corresponding to the different cell subsets ( figures 5b , 5f , and 5j ) . cells falling in the cdc1 ( blue ) , cdc2 ( green ) , and ( for human and macaque ) pdc ( pink ) tsne cell clusters were overlaid on classical contour plots , indicating that the dc subsets automatically delineated on tsne fit the criteria used to define them by manual gating ( figures 5c , 5 g , and 5k ) . comparison of the different tissues showed that equivalent dc subsets always fell in the same tsne regions ( figures 5d , 5h , and 5l ) . additionally , tsne analysis confirmed that , in human lung , cd14cd16hladrcd172acd1cirf4irf8 cells , identified by manual gating ( figures 4 and s4a ) , were indeed related to monocytes or macs since they clustered with cd14cd16 monocytes ( figures s4c and s4d ) . to elucidate the heterogeneity among the dc subsets , mass cytometry ( cytof ) data were acquired from different mouse and human tissues and total dc events were analyzed using one - sense ( one - dimensional soli - expression by nonlinear stochastic embedding ) ( cheng et al . , 2015 ) . here , manually chosen lineage - imprinted markers define the first dimension , and the other markers define the second dimension ( figure 6 ) . this resulted in the generation of multiple clusters of cadm1cd26 cdc1 ( blue ) , cd172acd11b ( mouse ) , or cd172acd1c ( human ) cdc2s ( green ) and siglechb220 ( mouse ) or cd123cd303 ( human ) pdcs ( pink ) ( figures 6a and 6d ) . binned frequency heatmaps were generated for each dimension . in human tissues , the lineage dimension validated the established dc subsets and also revealed contaminating cd172acd1c cells ( region delineated by an orange rectangle ) , separating them from the bona fide cd172acd1c cdc2s ( figure 6d ) . as before , these contaminating cells were confirmed to be cd11ccd26cadm1cd172acd1c cells when visualized in classical two - dimensional ( 2d ) contour plots ( figure 6e ) . the marker dimension gave key information concerning the degree of heterogeneity that exists among dc subsets ( figures 6a and 6d ) . such heterogeneity can be primarily accounted for by tissue imprinting ( figures 6b and 6f ) . heatmaps ( figures 6c and 6 g ) , 2d contour plots , or histograms ( figure s5 ) illustrated the mean expression of the list of differentially expressed markers for each of the three dc subsets . in mouse , this analysis confirmed that esam cdc2s ( cluster 3 ) were mainly found in the spleen , while spleen cdc1s ( cluster 5 ) had the highest cd8 and the lowest cd103 expression compared with the majority of cdc1s in the lung and gut ( clusters 4 and 6 , figure 6c ) . in humans , cdc phenotypic heterogeneity was also mostly explained by the tissue of residence ( figure 6f ) . furthermore , some heterogeneity could be observed for human pdcs with a subset expressing higher cd141 , cd56 , cla , cd62l , cd5 , cxcr3 , cd2 , cx3cr1 , and cd39 ( cluster 6 ) being observed in the spleen and in lower proportions in the blood and lung . therefore , our analysis reveals a previously unappreciated phenotypic heterogeneity in both mouse and human dcs that can now be mined for functional relevance . to demonstrate the usefulness of our approach in inflammatory settings , we utilized one - sense to track the activation of cdc1s , cdc2s , and monocyte - derived cells upon lps - induced inflammation in mice . linmhciicd11c cells from the lung and mediastinal lns harvested 1 , 2 , or 3 days following intranasal treatment with lps were profiled using cytof followed by one - sense analysis ( figures 7 and s6a ) . linmhciicd11c cells from the different time points post - lps were exported and concatenated , yielding a single one - sense analysis for the lung ( figure 7a ) and for the ln ( figure 7f ) . f4/80 and cd64 were added to the lineage markers to analyze monocyte - derived cells , yielding separated clusters of cd26cadm1cd11bcd172a cdc1 ( blue clusters ) , cd11bcd172af4/80cd64 cdc2 ( green clusters ) , and cd11bcd172af4/80cd64 monocyte - derived cells ( orange clusters ) . these were then further subdivided into multiple smaller clusters along the marker dimension , revealing variations linked to the duration of lps treatment ( figures 7b , 7c , 7 g , and 7h ) , including progressive phenotypic changes in expression of costimulatory receptors and inflammatory cytokines ( figures 7d , 7e , 7i , and 7j ) . this analysis also revealed striking differences in the proportion of cdcs and monocyte - derived cells in both organs after lps challenge . day 1 ( d1 ) following lps - treatment , cdc1s and cdc2s were strongly reduced in the lung , which was paralleled by a massive accumulation of monocyte - derived cells that represented 95% of all lung linmhciicd11c cells by d3 after lps . monocyte - derived cells appeared later in the ln , and their frequency gradually increased from below 1% at d0 and d1 to 36% at d3 . at d0 , both cdc subsets found in the lung and ln were not activated ( low cd40 , cd80 , and cd86 expression ) , and cdcs reached maximal activation only after migration to the lns ( see migratory cdc2 # 3 , cdc1 # 6 , and # 7 at d1 in the ln ) . lung monocyte - derived cells upregulated the costimulatory molecules cd80 and cd86 , as well as the inhibitory receptor pdl1 ( see monocyte - derived cells # 3 at d3 ) . we also observed a progressive increase of fcri , bst2 , and sca1 expression by monocyte - derived cells in the lung post - challenge ( expression : # 1 < # 2 < # 3 ; figures 7d and 7e ) . ln monocyte - derived cells likely represent cells directly recruited from the bloodstream , as previously described ( nakano et al . , 2009 ) . both lung and ln monocyte - derived cells expressed inflammatory cytokines il-12p70 , tumor necrosis factor ( tnf ) , and interleukin-6 ( il-6 ) , whereas high costimulatory molecule expression was limited to those in the lung . altogether , this analysis demonstrates the power of our approach in which cdcs and monocyte - derived cell activation can be tracked automatically during an inflammatory response . this open - ended approach could , in the future , include phospho - stat signaling analysis or a wider range of cytokines , chemokines , or costimulatory receptors . recent developments in computational methods permit more robust analysis of flow cytometry and cytof data . relying on objective mathematical principles to define cellular clusters , automated analyses increase the reproducibility of flow analysis by circumventing manual gating ( saeys et al . , 2016 ) . this constitutes a major improvement , since manual gating is one of the largest variables in the analysis of flow cytometry experiments ( mair et al . , 2015 ) . furthermore , automated analyses assess the expression of all markers simultaneously and are not influenced by the order in which cells are gated , as in manual sequential pairwise comparisons . finally , these techniques simplify the visualization of the multidimensional datasets , which is particularly important when analyzing such data with more than 30 markers . however , application of these techniques for the study of dcs has remained limited . here , we have defined lineage - imprinted surface markers that permit the faithful identification of cdc1s and cdc2s across species and tissues by adding cd26 as a cdc marker complementary to cd11c , combining cd64 and f4/80 to ensure a better distinction between dcs and macs in mice , using cadm1 or xcr1 in combination with cd172a to separate cdc1s from cdc2s , and using irf8-irf4 staining to validate the identification of cdc1s and cdc2s . unsupervised computational techniques , such as flowsom and tsne , confirmed the robustness of our strategy for defining cdc1s and cdc2s across mouse , human , and macaque tissues . these techniques could also identify cdc1 and cdc2 populations automatically , paving the way toward the reliable analysis of the dc compartment of many mutant mice in a high - throughput , unsupervised , and standardized manner . importantly , automated identification can outperform classical manual gating in the analysis of mutant strains when markers change their expression profile , as was exemplified here with the correct identification of cd11c xbp1-deficient cdc1s that were missed by the classical manual gating strategy . 2009 ) and is highly expressed on human and macaque cdc1s , as demonstrated with xcl1-mcherry vaccibodies , no commercial anti - human or anti - macaque xcr1 antibody is currently available . similarly , although cd26 is expressed by macaque dcs at the rna level ( data not shown ) , none of the commercial anti - cd26 antibodies tested showed cross - reactivity with macaque . generating cross - species reactive anti - xcr1 and anti - cd26 antibodies will be invaluable , as it will allow further simplification of the minimalistic and universal cdc phenotyping panel proposed here . ideally , we would need one or two surface markers that allow the identification of all macs ( including lcs ) across tissues and species , an essential issue to avoid contamination of the cdc2 population since macs are also xcr1cd172a . as confirmed by the addition of additional markers to our standard minimalistic panel for instance , mouse skin xcr1cd172airf8irf4 cdc1s comprise both cd103 and cd103 cells ( henri et al . , 2010 ) . in mouse , the foundations of dc subset specialization are imprinted in the bm , as pre - cdcs already contain cells committed toward the cdc1 or cdc2 lineage before colonizing peripheral tissues ( grajales - reyes et al . , 2015 , these lineage - imprinted programs include the mutually exclusive expression of xcr1 versus cd172a and of irf8 versus irf4 . to acquire the phenotype of terminally differentiated tissue cdc1s and cdc2s , precursors likely further integrate tissue - associated programs on the top of those lineage - imprinted foundations . in some organs , cdcs occupy distinct micro - anatomical compartments that can provide distinct environmental cues to developing cdc1s and cdc2s . for example , only those cdc2s that are localized in the splenic bridging channels specifically express cd4 . this subset of splenic cdc2s requires notch2 for their terminal differentiation ( caton et al . , 2007 , lewis et al . , 2011 ) and depends on the expression of chemotactic receptor ebi2 for their localization in the bridging channels ( gatto et al . , 2013 ) . analysis by flow cytometry of the splenic dc compartment of ebi2-deficient or notch2-deficient mice using only xcr1 and cd172a yields a less dramatic phenotype than with the inclusion of cd4 , because a significant fraction of cd172acd4 cdc2s are found in these mice . use of additional , tissue - imprinted markers such as cd4 is therefore required to fully appreciate the functional heterogeneity of the splenic cdc compartment . as such the one - sense approach we have described fulfills such a requirement , as it first aligns the dc across tissues based on lineage - imprinted markers yielding a simplified lineage dimension and subsequently analyzes the heterogeneity resulting from the tissue - imprinted programs through an unsupervised marker dimension . it simplifies the classification of dcs into cdc1s and cdc2s across tissues and species based on conserved lineage - imprinted markers without losing the power that multi - dimensional analyses offer . on that basis , we have gauged the differences that exist in cdc1s and cdc2s according to their tissue of residence and following an inflammatory insult . this type of analysis can be readily expanded to track any parameters of interest , such as the production of inflammatory mediators , or to dissect particular signaling pathways using phospho - flow approaches . in conclusion , the herein described methodology should provide a useful framework to analyze the complexity of the dc compartment , paving the way toward the identification of the best dc subset(s ) to target for specific therapeutic applications such as the development of next - generation vaccines . c57bl/6 mice used in france and belgium were obtained from harlan or janvier laboratories . flt3l and c57bl/6 mice for the experiment in singapore were from the biological resource center ( brc ) , agency for science , technology and research ( astar ) . all animals were housed under specific pathogen - free conditions in individually ventilated cages in a controlled day - night cycle and were given food and water ad libitum . all animal experiments performed were approved by the local animal ethics committee ( vib - ugent ; institutional animal care and use committee of the biological resource center , astar ; ciml ) and were performed according to the guidelines of belgian , french , and european animal protection law and of the agri - food and veterinary authority and the national advisory committee for laboratory animal research of singapore . see supplemental experimental procedures for additional information about the generation of bm chimeras , the intranasal lps treatment , and the digestion of mouse tissues . c57bl/6 mice were treated or not intranasally with 5g lps ( invivogen ) and were euthanized 1 , 2 , or 3 days later . 8 hr before euthanasia , mice were injected intraperiotoneally with 0.25 mg of brefeldina ( sigma ) in 200 l pbs . human samples were obtained with approval from singapore singhealth and national health care group research ethics committees . samples were prepared as described previously for skin ( mcgovern et al . , 2014 ) , lung ( schlitzer et al . , 2013 ) , and colon ( watchmaker et al . , 2014 ) . the antibodies and reagents used for facs analyses of mouse tissues are listed in tables s1 ( extracellular panel ) and s2 ( irf4 and irf8 ) . the antibodies used for facs analyses of human tissues were all mouse anti - human monoclonal antibodies , except the chicken anti - human cadm1 igy primary mab . the list of antibodies and reagents used for human and macaque flow cytometry experiments are listed in table s3 , and details of the antibody combinations ( panels ) can be found in table s4 . , purified antibodies were obtained from invitrogen , becton dickinson , biolegend , ebioscience , bioxcell fluidigm , r&d biosystems abd , and abd serotec using clones as listed in table s5 for mouse experiments and in table s6 for human experiments . the data were exported as a traditional flow cytometry file ( .fcs ) format , and cells for each barcode were deconvoluted using boolean gating . one - sense analysis was performed as recently described ( cheng et al . , 2015 ) . the automated analysis was performed by the flowsom algorithm ( van gassen et al . , 2015 ) or the tsne algorithm ( becher et al . , 2014 , wong et al . , ; clinicians for helping to access samples , multiple advice , and discussion , j.k.y.c . , c.n.m . ; formal analysis , m.g . , c .- a.d . , c.l.s . , n.mcg . ,

here , we present a case of chronic myeloid leukemia for which imatinib therapy was initated . triiodothyronine ( t3 ) , thyroxine ( t4 ) , and thyroid - stimulating hormone was normal , and thyroid microsomal autoantibodies ( tma ) were positive and patient was diagnosed as thyroiditis treated with corticosteroids for 1 months which lead to resolution .

in june 2004 , a 58-year - old asian female presented with anxiety , palpitations and breathlessness . differential leukocyte count revealed blasts , promyelocytes , myelocytes , and metamyelocytes with decreased leukocytes . liver function test ( lft ) and kidney function test ( kft ) were normal . the patient was started on imatinib 400 mg and folic acid 5 mg once daily in august 2004 . peripheral smear remission was seen in 2 months , i.e. , tlc returned to normal value and hemoglobin and platelet counts normalized . one year later , the patient presented with bilateral redness of eyes and facial swelling which was treated with antihistamines . following this , 1 month later , the patient presented with bilateral parotid swelling which was not evaluated . parotid swelling resolved of its own within a month without any treatment . in august 2005 , bone marrow biopsy revealed normocellular marrow spaces with adequate representation of all three marrow elements , i.e. , complete hematological remission . triiodothyronine ( t3 ) , thyroxine ( t4 ) , and thyroid - stimulating hormone were normal , but thyroid microsomal autoantibodies ( tma ) antibody was positive . fine needle aspiration cytology was planned , but the patient did not consent for the investigation . most common adverse events reported with imatinib are edema , nausea , muscle cramps , musculoskeletal pain , diarrhea , rash , fatigue , and headache . this case illustrates a possible occurrence of imatinib - induced autoimmune thyroiditis . in a case of thyroiditis , the differential diagnosis of thyroid pain includes acute , subacute thyroiditis , chronic thyroiditis , hemorrhage into a cyst , malignancy including lymphoma , and rarely , amiodarone - induced thyroiditis or amyloidosis . the absence of small tender asymmetric goiter , fever , dysphagia , and erythema over thyroid rules out bacterial or fungal causes of thyroiditis . the patient had bilateral parotid swelling 6 months before the presentation which resolved of its own . the absence of fever and presence of tma ruled out mumps as a cause of thyroiditis . finally , the positive response to treatment with corticosteroids also ruled out infection and malignancy as a cause of pain in thyroid . thus , only possible cause can be autoimmune thyroiditis which responded well to the treatment with corticosteroids . imatinib was started in august 2004 , and the patient presented with the thyroiditis in november 2005 establishing the temporal association of thyroiditis with imatinib . use of naranjo 's probability scale and who causalty scale assessment indicated imatinib as a possible cause of thyroiditis because the idiopathic cause of autoimmune thyroiditis can not be ruled out . sunitinib , a tk inhibitor , a drug of same class has been implicated as a cause of lymphocytic and destructive thyroiditis . a review the literature of studies of thyroid dysfunction induced by tk inhibitors showed that thyroid dysfunction is not a rare entity with the use of tk inhibitors although not so common with the use of imatinib . as there was possibility of progression of cml when drug is withheld , drug withdrawal was not done to check the possible causation of thyroiditis by imatinib . second , there was no increase in the thyroid hormone levels and the severity of thyroiditis did not mandate drug withdrawal . hence , a possibility of activation of autoimmune diseases with the use of imatinib can not be ruled out . hence , clinicians should be aware of the possibility of thyroiditis with imatinib and should have an eye not to miss the event .

purpose . this study aimed to evaluate the clinical efficacy of using expression levels of ccl24 ( eotaxin-2 ) mrna on the ocular surface as a biomarker in patients with vernal keratoconjunctivitis ( vkc ) and atopic keratoconjunctivitis ( akc ) . methods . eighteen patients with vkc or akc ( vkc / akc group ) and 12 control subjects ( control group ) were enrolled in this study . the vkc / akc clinical score was determined by objective findings in patients by using the 5 - 5 - 5 exacerbation grading scale . all subjects underwent modified impression cytology and specimens were obtained from the upper tarsal conjunctiva . expression levels of ccl24 ( eotaxin-2 ) mrna on the ocular surface were determined using real - time reverse transcription polymerase chain reaction . results . the vkc group was divided into two subgroups , depending on the clinical score : the active stage subgroup with 100 points or more of clinical scores and the stable stage subgroup with 100 points or less . ccl24 ( eotaxin-2 ) mrna expression levels in the active vkc / akc stage subgroup were significantly higher than those in the stable vkc / akc subgroup and the control group . clinical scores correlated significantly with ccl24 ( eotaxin-2 ) mrna expression levels in the vkc group . conclusions . ccl24 ( eotaxin-2 ) mrna expression levels on the ocular surface are a useful biomarker for clinical severity of vkc / akc .

allergic conjunctival diseases ( acd ) are conjunctival inflammatory disorders , characterized by an immediate hypersensitivity reaction associated with antigen - specific ige antibodies . patients with acd are usually categorized into the following types based on clinical criteria : seasonal allergic conjunctivitis ( sac ) , perennial allergic conjunctivitis ( pac ) , vernal keratoconjunctivitis ( vkc ) , atopic keratoconjunctivitis ( akc ) , and giant papillary conjunctivitis ( gpc ) . sac and pac are characterized by conjunctival hyperemia , conjunctival edema , and papillary hyperplasia of tarsal conjunctiva and are classified as mild acd . in contrast , vkc is characterized by the development of proliferative lesions of the conjunctiva , including giant papillary proliferation of the tarsal conjunctiva and gelatinous cell infiltration of the limbal conjunctiva . vkc also complicates corneal disorders , such as shield ulcer and punctate corneal keratitis , and is classified as a severe acd , because the visual prognosis may be poor . akc usually develops in older patients with atopic dermatitis and also complicates severe ocular surface diseases , including giant papillary conjunctivitis , shield ulcer , and dry eye . in shield ulcers of patients with vkc and akc , depositions of major basic protein and eosinophil cationic protein ( ecp ) , which comprise specific granules of eosinophils , have reportedly been observed histologically in the corneal ulcerative lesions [ 3 , 4 ] . furthermore , ecp levels in the tears of vkc and akc patients are reportedly increased , in comparison with those in controls [ 57 ] . therefore , the pathophysiological characteristics of these severe acd , including vkc and akc , include eosinophilic inflammation in the conjunctiva . eotaxin is a member of the cc chemokine family and is divided into three subfamilies , namely , ccl11/eotaxin-1 , ccl24/eotaxin-2 , and ccl26/eotaxin-3 . eotaxin-1 , eotaxin-2 , and eotaxin-3 interact with the cc chemokine receptor 3 ( ccr3 ) . representative inflammatory cells expressing ccr3 on their cell surfaces are eosinophils , type-2 helper t cells ( th2 ) , and basophils . il-13 , which is a th2-derived cytokine , induces eotaxin in vitro through activation of the il-4r receptor / stat-6 pathway [ 8 , 9 ] . therefore , eotaxin-1 , eotaxin-2 , and eotaxin-3 are thought to be allergic inflammation - related chemokines . in the eotaxin subfamily , it has been reported that increased eotaxin-2 levels in tears and expression of ccl24 ( eotaxin-2 ) mrna on the ocular surface are more common in vkc patients than those of eotaxin-1 and eotaxin-3 [ 10 , 11 ] . however , the relationship between the expression levels of ccl24 ( eotaxin-2 ) mrna on the ocular surface and the severity of severe acd , including vkc and akc , has not been fully investigated . in this study , we evaluated the clinical efficacy of using ccl24 ( eotaxin-2 ) mrna expression levels on the ocular surface in patients with severe acd , including vkc and akc , as a biomarker for severe acd . this study was approved by the institutional review board of the nihon university school of medicine and adhered to the tenets of the declaration of helsinki . this study included 18 consecutive patients with vkc or akc treated at the department of ophthalmology , nihon university itabashi hospital , tokyo , japan , from august 2012 to january 2015 ( akc / vkc group ) , and 12 healthy volunteers who did not have any personal or family history of atopic disease , were not affected by ocular surface diseases , or have a history of wearing contact lenses as controls ( control group ) . objective diagnoses were made in vkc and akc patients by means of slit - lamp clinical examination and serum examination for antigen - specific ige antibodies , according to the japanese guidelines for acd . patients with ocular surface disease other than acd , including lagophthalmos , blepharospasm , conjunctival chalasis , dry eye , infectious conjunctivitis , infectious keratitis , stevens - johnson syndrome , and ocular pemphigoid , were excluded . nontreated patients or patients treated with antiallergic ophthalmic solutions alone , such as mast cell stabilizers , histamine h1 receptor antagonists , corticosteroids , and immunosuppressive agents , were included in the study . patients who used oral medicines or injections for treating allergic diseases and those who received immunotherapy were excluded from the study . clinical scores of objective findings in the akc / vkc group were determined using the 5 - 5 - 5 exacerbation grading scale for acd . the akc / vkc group was divided into two subgroups depending on the clinical score : the active stage subgroup with clinical scores of 100 points or more ( n = 6 ) and the stable stage subgroup with 100 points or less ( n = 12 ) . modified impression cytology was performed after instillation of topical oxybuprocaine 0.4% ( benoxil , santen , osaka , japan ) . schirmer 's test strips ( schirmer tear production measuring strips , showa yakuhin kako , tokyo , japan ) were applied to the upper tarsal conjunctiva , pressed gently using a glass rod , and then removed . the membrane was preserved in rnalater rna stabilization reagent ( qiagen , hilden , germany ) until analysis . total rna was harvested from each schirmer tested paper using an rneasy mini kit ( qiagen , hilden , germany ) following the manufacturer 's instructions . cdna was then synthesized using a high - capacity cdna reverse transcription kit ( life technologies japan , tokyo , japan ) , according to the manufacturer 's instructions . to detect expression of ccl24 ( eotaxin-2 ) mrna , real - time reverse transcription polymerase chain reaction ( real - time rt - pcr ) was performed using a commercial pcr master mix ( taqman universal pcr master mix ; life technologies , tokyo , japan ) and predesigned primers ( life technologies ) for ccl24 ( eotaxin-2 ; hs00171082_m1 ) . samples were analyzed using the step one plus real - time pcr system ( life technologies ) and comparative threshold ( ct ) values were obtained . target ct values were normalized to those of gapdh ( hs99999905_m1 ) in the same sample . differences between akc / vkc and control groups were identified using welch 's t - test or the chi - square test . the results for ccl24 ( eotaxin-2 ) mrna expression on the ocular surface were evaluated using the nonparametric steel - dwass test . spearman 's rank correlation coefficient was used to evaluate whether ccl24 ( eotaxin-2 ) mrna expression correlated with the clinical score for ccl24 ( eotaxin-2 ) mrna expression on the ocular surface , 6 of 6 patients in the active stage subgroup of akc / vkc group were ccl24- ( eotaxin-2- ) positive , with median ( range ) levels of 133 ( 27.6232 ) . eleven of 12 patients in the stable stage subgroup of akc / vkc group were ccl24- ( eotaxin-2- ) positive , with median ( range ) levels of 5.99 ( 0.14033.2 ) ; the remaining patient had levels below the lower limit of detection . eight of 12 patients in the control group were ccl24- ( eotaxin-2- ) positive , with median ( range ) levels of 0.98 ( 0.0820.2 ) , while 4 patients had levels below the lower limit of detection . the expression levels of ccl24 ( eotaxin-2 ) mrna on the ocular surface were significantly higher in the active stage than in the stable stage akc / vkc subgroup and the control group ( both p < 0.01 ; steel - dwass test ; figure 1 ) . the median value ( range ) of the clinical scores in the active and stable stage subgroups of akc / vkc group were 13 ( 233 ) and 128 ( 343112 ) , respectively . in patients with akc / vkc , clinical scores were significantly correlated with the levels of ( ccl24 ) eotaxin-2 mrna expression on the ocular surface ( = 0.795 , p < 0.01 , spearman 's rank correlation coefficient ; figure 2 ) . a 10-year - old boy was diagnosed as having vkc and had been under treatment for vkc by a local doctor for 6 years . his clinical observation of vkc repeated exacerbation and remission , and his severity of vkc was different in right and left active giant papillae and exfoliative epithelial keratopathy was present in his right eye ; his clinical score was 212 , and his ccl24 ( eotaxin-2 ) mrna expression levels on the ocular surface were 203.2 . in his left eye , papillary lesions and hyperemia at the upper palpebral conjunctiva were observed , and the clinical score was 2 , while the ccl24 ( eotaxin-2 ) mrna expression level on the ocular surface was 17.2 ( figure 3 ) . a 10-year - old girl was diagnosed as having vkc and had been under treatment for vkc by a doctor for 1 year . she experienced pain in her right eye and also had recurrence of corneal plaque and was referred to our hospital . at her first examination , she demonstrated shield ulcer in her right eye , giant papillae , and palpebral conjunctiva with a velvety appearance . topical 0.1% tacrolimus ophthalmic suspension ( talymus ophthalmic suspension 0.1% , senju pharmaceutical , co. , ltd . , osaka , japan ) and 2% sodium cromoglicate ophthalmic solution ( intal ophthalmic solution 2% , sanofi , tokyo , japan ) were administered . at commencement of treatment with tacrolimus ophthalmic suspension , her clinical score was 213 and the level of ccl24 ( eotaxin-2 ) mrna expression on the ocular surface was 349.6 . during the first 10 weeks of treatment , the clinical score was 3 points and the ccl24 ( eotaxin-2 ) mrna expression level on the ocular surface was 9.6 ( figure 4 ) . in this study , we assessed the usefulness of ccl24 ( eotaxin-2 ) mrna expression levels on the ocular surface as a biomarker of the severity of acd . we found that these levels correlated well with the clinical score reflecting objective findings in patients with akc and vkc . as a method for sampling the ocular surface to test the expression levels of ccl24 ( eotaxin-2 ) mrna this method entailed a membrane biopsy technique , as used for impression cytology , but used filter paper instead of nitrocellulose membrane for the biopsy . in the conventional impression cytology method , the specimen obtained using a nitrocellulose membrane is examined histologically . however , the major concern of this method is the ocular sensation of a foreign body and the ocular pain experienced after the examination ; repeated examinations for follow - up of the biomarker may cause discomfort for the patients . changing to the filter paper for impression cytology sampling and using a quantitative method ( real - time rt - pcr ) allowed assessment of the biomarker on the ocular surface . the specimens obtained by modified impression cytology most likely included conjunctival epithelial and invading inflammatory cells , in addition to tears and mucin . it was therefore considered useful for investigating inflammation - associated factors expressed by conjunctival epithelial cells and inflammatory cells as potential biomarkers of allergic inflammation at the ocular surface . in this study , we elucidated a significant correlation between clinical observations and ccl24 expression on the ocular surface of akc / vkc patients with or without treatment with ophthalmic solutions . these results suggest that ccl24 expression on the ocular surface is suitable as a biomarker of severe allergic conjunctival diseases . in previous reports , it has been demonstrated that eotaxin-1 plays a critical role in eosinophilic infiltration in the conjunctiva and cornea of patients with acd [ 10 , 11 , 13 , 14 ] . however , the concentration of eotaxin-2 in tears has been reported to be higher than those of eotaxin-1 in acd patients . therefore , in this study , we investigated the usefulness of ccl24 ( eotaxin-2 ) mrna expressed on the ocular surface as a biomarker for patients with akc / vkc . we found that expression levels of ccl24 ( eotaxin-2 ) mrna on the ocular surface were significantly increased and correlated with the clinical score in the active stage of vkc . eotaxin-1 is reportedly produced by corneal keratinocytes [ 13 , 14 ] , conjunctival fibroblasts , cd68-positive cells in the conjunctiva , and eosinophils in the conjunctiva . previously , we have reported that the tear levels of eotaxin-2 correlated significantly with those of eosinophil cationic protein and that epithelial cells in conjunctival smears of patients with vkc expressed eotaxin-2 , based on immunohistochemistry . leonardi and colleagues reported that tear levels of eotaxin-1 and eotaxin-2 significantly correlated with the percentage of eosinophils in tears . therefore , conjunctival epithelial cells and eosinophils are thought to be candidate eotaxin-2-producing cells on the ocular surface . furthermore , expression levels of ccl24 ( eotaxin-2 ) mrna in modified impression cytology may be a good biomarker for evaluating allergic inflammation in the conjunctivas of patients with akc / vkc . in these case reports , we showed differences between the right and left eye in the severity of vkc based on the quantitative analysis of ccl24 ( eotaxin-2 ) mrna levels . furthermore , in the 10-year - old girl with vkc , we were able to show the therapeutic effect of treatment by tacrolimus instillation by the reduction of both clinical scores and expression levels of ccl24 ( eotaxin-2 ) mrna . therefore , in akc / vkc patients undergoing treatment , monitoring of the expression levels of ccl24 ( eotaxin-2 ) mrna may provide a useful index of exacerbation and therapeutic response . the limitation of this study included the small sample size , and a lack of patients with mild acd , such as sac and pac . further investigation is necessary for verifying the usefulness of ccl24 ( eotaxin-2 ) mrna levels as a biomarker of acd in a large sample that includes patients with sac and pac . another limitation of this study was that akc / vkc patients not receiving treatment and those only receiving treatment with ophthalmic solutions were enrolled . the ccl24 mrna expression on the ocular surface may be affected by treatment with ophthalmic solutions . however , the efficacy of the therapeutic agent is one of the items measured by a biomarker . therefore , further investigation on the therapeutic effect of antiallergic treatment in a large cohort , including untreated patients with allergic conjunctival diseases , using eotaxin-2 expression as a biomarker , will be necessary in the future . expression levels of ccl24 ( eotaxin-2 ) mrna on the ocular surface are a useful biomarker of the clinical severity of akc / vkc .

the pedant genome database provides exhaustive annotation of 468 genomes by a broad set of bioinformatics algorithms . we describe recent developments of the pedant web server . the all - new graphical user interface ( gui ) implemented in java allows for more efficient navigation of the genome data , extended search capabilities , user customization and export facilities . the dna and protein viewers have been made highly dynamic and customizable . we also provide web services to access the entire body of pedant data programmatically . finally , we report on the application of association rule mining for automatic detection of potential annotation errors . pedant is freely accessible to academic users at .

the pedant genome database was first announced in a short note entitled pedantic genome analysis which appeared in trends in genetics in 1997 ( 1 ) and reported computational analysis of seven completely sequenced and two partial genomes available at that time . from the very beginning the main mission of pedant was defined as filling the gap between manually curated high quality protein sequence databases , such as uniprot / swiss - prot ( 2 ) , and the enormous amounts of other protein sequences produced by genome sequencing projects at an ever increasing pace . over the past decade the pedant genome database was produced by systematically applying an automatic annotation pipeline to genome data released in the public domain . these efforts resulted in one of the most comprehensive currently available genome databases which includes 468 organisms from all three domains of life . we report on three new features of the pedant web server : ( i ) an all - new graphical user interface ( gui ) , ( ii ) availability of web services and ( iii ) rule - based detection of annotation errors . the current version of the pedant software familiar to most users was introduced several years ago and is known as version 2 ( 3 ) . in 2006 we were deploying and testing the all - new version 3 which represents a complete re - implementation of the pedant software in the java programing language . server application for enterprise - scale molecular sequence analysis with advanced features such as highly dynamic workflow - based process management , direct interface to computing grids , support for essentially all existing sql database management systems , open architecture for easy integration of additional algorithms , and powerful scripting interfaces ( d. frishman et al . , manuscript in preparation ) . migration of pedant genomes to the new version 3 is currently in progress and will be accomplished by mid-2007 . so far we have completed a pilot study to annotate 40 genomes to demonstrate the advantages of pedant3 . the new version of the pedant gui can be viewed by selecting any genome with a the pedant gui has been made user - configurable and supports four main types of windows : ( i ) sequence analysis : information about the complete analysis with links to orf lists of the individual algorithms , ( ii ) gene report : information about a selected gene , ( iii ) contig report : information about a selected contig and ( iv ) genetic element report ( where available ) : information about a selected nonprotein coding genetic element . on selection of a pedant3 genome , the analysis window is opened displaying a list of genes and their best - blast hits . the left - hand panel contains a context - dependent navigation tree that makes available different choices dependent on the particular view the user is working with . the report page ( figure 1 ) is split into different sections ( overview , protein function , protein structure , protein location , general properties and export ) and lists the analysis results for a particular gene product , which are also available for download in different formats . the protein viewer displays several panels for various types of evidence that can be configured by selecting the dna viewer allows for easy navigation along the chromosomes and is capable of displaying an unlimited number of features at different zoom levels . navigation tree and report page for the hypothetical secreted protein of helicobacter pylori ( gi_15645712 ) . this figure also illustrates the application of association rule mining for finding potential annotation errors ( see text ) . the interpro domain ipr001440 and pfam domain pf00515 were erroneously assigned to this gene product based on a weak similarity hit to a single tpr_1 repeat whereas the domain definition requires at least three repeat copies . note that all other features marked as suspicious are in fact annotated correctly ; the method does not detect annotation errors as such , but rather incompatible feature combinations which might include annotation errors . any pedant3 dataset can be searched using sequence ids , sequences , prosite - like patterns or free text as the query option . web services technology is becoming increasingly popular within the bioinformatics community as a means to exploit the large amounts of data , software programs and computing power available at various institutions ( 4,5 ) . according to the world wide web consortium ( w3c ) a web service is a software system designed to support interoperable machine - to - machine interactions over a network ( ) . this technology is based on the extensible markup language ( xml ) and open standards , and is platform and programing language independent . this enables clients for a particular service to be written in many languages , such as java or perl , irrespective of the language the service was written in . a web service has an interface that is described in a machine processable format using the xml based web services description language ( wsdl ) . wsdl provides a format for the description of a web service interface , including parameters and data types in sufficient detail for a programer to write a client application for that service . tools are available for various programing languages to generate the required client classes , such as apache axis 's wsdl2java ( ) . the client programs interact with the web service using messages based on the simple object access protocol ( soap ) . as with wsdl , soap messages are xml based , permitting the interoperability of web services . for the transport layer itself , web services typically use the hypertext transfer protocol ( http ) , preventing problems sending the soap messages through firewalls . bioinformatics users can avoid keeping local copies of databases and software and use a client program instead to access remote databases and software via web services . the pedant web service allows the user to query the database in an automated way from client programs and workflows . we provide a number of data retrieval methods in our data retrieval service ( table 1 ) . for example , to fetch the functional and structural annotations of a particular protein , the client program can call the getreportbydbcodeandcontig method . methods available in the data retrieval service currently this web service only retrieves data from pedant2 analyses ; the pedant3 web service is in preparation . the raw ( unparsed ) output from various bioinformatics methods a protein can be uniquely identified with just the database name and protein code . for unfinished genomes , where the proteins were predicted using orpheus ( 6 ) , it is necessary to provide the database name , protein code and contig name to uniquely identify a protein . a list of the bioinformatics methods available for each genome can be obtained by calling the getmethodsbydb method . we have generated a java client program using the apache axis software ( wsdl2java ) which has an example class demonstrating how to make calls to the dataretrievalservice . we have also included pedant web service client functionality in our prompt workbench as a use case to demonstrate the various advantages of the web services . prompt is a standalone application which enables a user to compare protein sequence sets , revealing statistically significant differences in their annotation features ( 7 ) ; ( ) . genome annotation produced by automatic pipelines such as pedant is notoriously prone to various kinds of errors ( 8) . while every effort is being made to select the most reliable , carefully benchmarked bioinformatics tools and to avoid spurious similarity hits by setting conservative similarity thresholds , automatically produced function predictions are still not on a par with the results of manual curation of sequence data in high - quality databases such as uniprot ( 2 ) . we estimate that only 5% of all known proteins have been manually annotated and , given the progress in superfast sequencing technologies ( 9 ) , it is becoming increasingly clear that the overwhelming majority of sequence data will not be processed by human experts . we have recently developed a technology to improve the quality of automatically generated annotation using data mining techniques ( 10 ) . the entire body of annotation available in the pedant database can be considered as a collection of records of variable length , one for each gene product , containing functional and structural attributes , such as predicted functional categories , domain assignments and the like . using a large collection of pedant records as input , we apply an unsupervised learning algorithm called association rule mining ( 11 ) to derive protein features that occur frequently together . specifically , implications in the form a&b c are derived which mean that the majority of proteins possessing features a and b also possess c. some rules have the strength 1.0 which means that they are always fulfilled , while other rules may be true only in a certain percentage of cases . an exception from a reasonably strong rule will have features a and b , but not c , which may be caused either by over - annotation ( features a or b ascribed erroneously ) , or by under - annotation ( feature c missed ) . we have shown that 70% of exceptions from strong association rules found in pedant data point to incompatible or missing annotation and thus may be instrumental in identifying annotation errors . however , since strong association rules detect not annotation errors as such but only incompatible feature combinations , it is not possible to automatically correct errors . instead , we highlight suspicious features and add the features which were putatively missed in a separate section at the bottom of the report page ( figure 1 ) . in a recent pilot project we applied this approach to analyze the pedant annotation of 10 model genomes arabidopsis thaliana , aeropyrum pernix , bacillus subtilis , escherichia coli , helicobacter pylori , mycobacterium tuberculosis , parachlamydia , saccharomyces cerevisiae , synechocystis and thermoplasma acidophilum containing a total of 55 123 protein entries . in the course of 2007 we intend on providing rule - based correction for all pedant genomes . the current version of the pedant software familiar to most users was introduced several years ago and is known as version 2 ( 3 ) . in 2006 we were deploying and testing the all - new version 3 which represents a complete re - implementation of the pedant software in the java programing language . server application for enterprise - scale molecular sequence analysis with advanced features such as highly dynamic workflow - based process management , direct interface to computing grids , support for essentially all existing sql database management systems , open architecture for easy integration of additional algorithms , and powerful scripting interfaces ( d. frishman et al . , manuscript in preparation ) . migration of pedant genomes to the new version 3 is currently in progress and will be accomplished by mid-2007 . so far we have completed a pilot study to annotate 40 genomes to demonstrate the advantages of pedant3 . the new version of the pedant gui can be viewed by selecting any genome with a the pedant gui has been made user - configurable and supports four main types of windows : ( i ) sequence analysis : information about the complete analysis with links to orf lists of the individual algorithms , ( ii ) gene report : information about a selected gene , ( iii ) contig report : information about a selected contig and ( iv ) genetic element report ( where available ) : information about a selected nonprotein coding genetic element . on selection of a pedant3 genome , the analysis window is opened displaying a list of genes and their best - blast hits . the left - hand panel contains a context - dependent navigation tree that makes available different choices dependent on the particular view the user is working with . the report page ( figure 1 ) is split into different sections ( overview , protein function , protein structure , protein location , general properties and export ) and lists the analysis results for a particular gene product , which are also available for download in different formats . the protein viewer displays several panels for various types of evidence that can be configured by selecting for example certain panels can be hidden , resized and the color scheme can be changed . the dna viewer allows for easy navigation along the chromosomes and is capable of displaying an unlimited number of features at different zoom levels . navigation tree and report page for the hypothetical secreted protein of helicobacter pylori ( gi_15645712 ) . this figure also illustrates the application of association rule mining for finding potential annotation errors ( see text ) . the interpro domain ipr001440 and pfam domain pf00515 were erroneously assigned to this gene product based on a weak similarity hit to a single tpr_1 repeat whereas the domain definition requires at least three repeat copies . note that all other features marked as suspicious are in fact annotated correctly ; the method does not detect annotation errors as such , but rather incompatible feature combinations which might include annotation errors . any pedant3 dataset can be searched using sequence ids , sequences , prosite - like patterns or free text as the query option . web services technology is becoming increasingly popular within the bioinformatics community as a means to exploit the large amounts of data , software programs and computing power available at various institutions ( 4,5 ) . according to the world wide web consortium ( w3c ) a web service is a software system designed to support interoperable machine - to - machine interactions over a network ( ) . this technology is based on the extensible markup language ( xml ) and open standards , and is platform and programing language independent . this enables clients for a particular service to be written in many languages , such as java or perl , irrespective of the language the service was written in . a web service has an interface that is described in a machine processable format using the xml based web services description language ( wsdl ) . wsdl provides a format for the description of a web service interface , including parameters and data types in sufficient detail for a programer to write a client application for that service . tools are available for various programing languages to generate the required client classes , such as apache axis 's wsdl2java ( ) . the client programs interact with the web service using messages based on the simple object access protocol ( soap ) . as with wsdl , soap messages are xml based , permitting the interoperability of web services . for the transport layer itself , web services typically use the hypertext transfer protocol ( http ) , preventing problems sending the soap messages through firewalls . bioinformatics users can avoid keeping local copies of databases and software and use a client program instead to access remote databases and software via web services . the pedant web service allows the user to query the database in an automated way from client programs and workflows . we provide a number of data retrieval methods in our data retrieval service ( table 1 ) . for example , to fetch the functional and structural annotations of a particular protein , the client program can call the getreportbydbcodeandcontig method . methods available in the data retrieval service currently this web service only retrieves data from pedant2 analyses ; the pedant3 web service is in preparation . the raw ( unparsed ) output from various bioinformatics methods can be obtained using the methods getrawhitsbydbcodeandmethod and getrawhitsbydbcodecontigandmethod . for completely sequenced genomes , a protein can be uniquely identified with just the database name and protein code . for unfinished genomes , where the proteins were predicted using orpheus ( 6 ) , it is necessary to provide the database name , protein code and contig name to uniquely identify a protein . a list of the bioinformatics methods available for each genome can be obtained by calling the getmethodsbydb method . we have generated a java client program using the apache axis software ( wsdl2java ) which has an example class demonstrating how to make calls to the dataretrievalservice . we have also included pedant web service client functionality in our prompt workbench as a use case to demonstrate the various advantages of the web services . prompt is a standalone application which enables a user to compare protein sequence sets , revealing statistically significant differences in their annotation features ( 7 ) ; ( ) . genome annotation produced by automatic pipelines such as pedant is notoriously prone to various kinds of errors ( 8) . while every effort is being made to select the most reliable , carefully benchmarked bioinformatics tools and to avoid spurious similarity hits by setting conservative similarity thresholds , automatically produced function predictions are still not on a par with the results of manual curation of sequence data in high - quality databases such as uniprot ( 2 ) . we estimate that only 5% of all known proteins have been manually annotated and , given the progress in superfast sequencing technologies ( 9 ) , it is becoming increasingly clear that the overwhelming majority of sequence data will not be processed by human experts . we have recently developed a technology to improve the quality of automatically generated annotation using data mining techniques ( 10 ) . the entire body of annotation available in the pedant database can be considered as a collection of records of variable length , one for each gene product , containing functional and structural attributes , such as predicted functional categories , domain assignments and the like . using a large collection of pedant records as input , we apply an unsupervised learning algorithm called association rule mining ( 11 ) to derive protein features that occur frequently together . specifically , implications in the form a&b c are derived which mean that the majority of proteins possessing features a and b also possess c. some rules have the strength 1.0 which means that they are always fulfilled , while other rules may be true only in a certain percentage of cases . an exception from a reasonably strong rule will have features a and b , but not c , which may be caused either by over - annotation ( features a or b ascribed erroneously ) , or by under - annotation ( feature c missed ) . we have shown that 70% of exceptions from strong association rules found in pedant data point to incompatible or missing annotation and thus may be instrumental in identifying annotation errors . however , since strong association rules detect not annotation errors as such but only incompatible feature combinations , it is not possible to automatically correct errors . instead , we highlight suspicious features and add the features which were putatively missed in a separate section at the bottom of the report page ( figure 1 ) . in a recent pilot project we applied this approach to analyze the pedant annotation of 10 model genomes arabidopsis thaliana , aeropyrum pernix , bacillus subtilis , escherichia coli , helicobacter pylori , mycobacterium tuberculosis , parachlamydia , saccharomyces cerevisiae , synechocystis and thermoplasma acidophilum containing a total of 55 123 protein entries . in the course of 2007 we intend on providing rule - based correction for all pedant genomes .

the small fields in radiotherapy are widely used due to the development of techniques such as intensity - modulated radiotherapy and stereotactic radio surgery . the measurement of the dose distributions for small fields is a challenge . a perfect dosimeter should be independent of the radiation energy and the dose rate and should have a negligible volume effect . the radiochromic ( rc ) film characteristics fit well to these requirements . however , the response of rc films and their digitizing processes present a significant spatial inhomogeneity problem . the present work uses a method for two - dimensional ( 2d ) measurement with rc films based on the reduction of the spatial inhomogeneity of both the film and the film digitizing process . by means of registering and averaging several measurements of the same field , the inhomogeneities are mostly canceled . measurements of output factors ( ofs ) , dose profiles ( in - plane and cross - plane ) , and 2d dose distributions are presented . the field sizes investigated are 0.5 0.5 cm2 , 0.7 0.7 cm2 , 1 1 cm2 , 2 2 cm2 , 3 3 cm2 , 6 6 cm2 , and 10 10 cm2 for 6 and 15 mv photon beams . the ofs measured with the rc film are compared with the measurements carried out with a pinpoint ionization chamber ( ic ) and a semiflex ic , while the measured transversal dose profiles were compared with monte carlo simulations . the results obtained for the ofs measurements show a good agreement with the values obtained from rc films and the pinpoint and semiflex chambers when the field size is greater or equal than 2 2 cm2 . these agreements give confidence on the accuracy of the method as well as on the results obtained for smaller fields . also , good agreement was found between the measured profiles and the monte carlo calculated profiles for the field size of 1 1 cm2 . we expect , therefore , that the presented method can be used to perform accurate measurements of small fields .

the increased use of radiotherapy techniques such as intensity - modulated radiotherapy and stereotactic radiosurgery has generalized the use of small fields . also , some treatment units as the gammaknife or the cyberknife are based on the use of small fields . it is therefore important to have methods for the precise measurement of the dose for these kinds of fields . the measurement of the dose distributions for smalls fields have beam geometries that are quite different from the reference conditions stated in the dosimetry protocols . the radiation field does not produce constant dose volumes to encompass the volume of conventional detectors . this finite detector volume has been identified as the major reason for the measurement of low dose values in the non - constant dose regions . moreover , the spectrum exhibits a dependence on the field size and the dose rate becomes smaller as the field size decreases , due to the loss of lateral electronic equilibrium . it is therefore necessary to use small volume dosimeters , with negligible energy and dose rate dependencies . ionization chambers ( ics ) have a small dependency on the energy of the radiation for the range of energies found in radiotherapy . however , in order to obtain a good signal to noise ratio , small sensitive volumes are not recommended . in this way the volume effect is not negligible for small field sizes , and the measurement in these non - constant dose regions is limited in accuracy . given the high spatial resolution of radiochromic ( rc ) films , the volume effect is not a concern in the dosimetry of radiotherapy beams . however , the response of rc films has a poor spatial homogeneity , and this may be their main drawback for dosimetric applications . lynch et al . , shows that important lateral inhomogeneities are introduced by a flatbed scanner , and these inhomogeneities are function of the optical density . this work uses a method for small fields two - dimensional ( 2d ) measurements with rc films based on the reduction of the spatial inhomogeneity of the film and the film reading . by means of averaging several measurements of a field , , the calibration curve is obtained after averaging several measurements of each dose calibration value . the film pieces used for each dose value are selected from different locations of one sheet of film . in this way the positive characteristics of rc film are then exploited to measure output factors ( ofs ) , dose profiles , and 2d dose distributions . the results obtained with the rc on ofs are compared with the measurements carried out with a pinpoint ic and a semiflex ic , whereas the measured profiles are compared with profiles calculated by monte carlo methods . the ics used in this work were a 0.015 cm pinpoint thimble - type 31006 ( ptw , freiburg , germany ) and a 0.125 cm semiflex thimble - type 31010 . the rc film used was the ebt-2 film ( international specialty products , wayne , usa ) . after irradiation the films were digitized in a flatbed scanmaker 9800xl ( microtek , hsinchu , taiwan ) . the red channel of a 48 bits rgb digitization was then extracted and used as the digitizer reading . ics and films were irradiated with 6 and 15 mv photon beams produced from a varian 2100-dhx linac ( varian medical systems ) . films and ics were placed in a pmma phantom of size 30 30 20 cm . the field sizes investigated were 0.5 0.5 cm , 0.7 0.7 cm , 1 1 cm , 2 2 cm , 3 3 cm , 6 6 cm , and 10 10 cm . ofs for fields ranging from 0.5 0.5 cm to 10 10 cm , and beam profiles and 2d dose distributions for fields of sizes 0.5 0.5 cm , 0.7 0.7 cm , and 1 1 cm were measured with the secondary jaws delimiting the field size , at a depth of dmax ( 1.5 cm for the 6 mv beams and 2.5 cm for the 15 mv beams ) and 100 cm source - to - surface distance ( ssd ) . all measurement were performed with a gantry angle of 0 and a dose rate of 300 mu / min that correspond to 3 gy / min for a 10 10 cm field , at dmax and 100 cm ssd . the pinpoint and the semiflex ics were placed with their stems perpendicular to the beam axis . the maximum lengths of the sensitive volumes of both chambers are 5 and 6.5 mm , respectively ; these lengths being equal or slightly larger than the smallest field lateral dimension , and larger than the constant dose region for some of the fields measured . a number of segments of one sheet of film are used to carry out a calibration of the film response . calibration segments of film are irradiated separately to known doses of 50 , 170 , 250 , 320 , 400 , and 520 cgy . then the segments are digitized and the readings obtained are used to fit a power function relating the digitizer reading to the dose . for each dose value 10 segments of film are read , and the readings are averaged before the curve fitting . the reading for each film calibration segment is obtained as the median of the pixel values in a roi of 1.5 1.5 cm in the center of the imaged segment . a sheet of film is cut into segments of 2 2 cm , conforming a 2d array . for each dose value 10 segments of film are selected , so that each pair of segments is in different columns and rows of the array . the film segments are irradiated separately and 6 h after irradiation the films are arranged in the same way before being cut and read . in this way , after averaging all the pieces belonging to a dose value the effect of the spatial inhomogeneity is minimized . six calibration fields ( 50 , 170 , 250 , 320 , 400 , and 520 cgy ) together with four fields of sizes 0.5 0.5 cm , 0.7 0.7 cm , 1 1 cm , and 2 2 cm were used ( these fields can be seen , for instance , in first row and columns 7 , 8 , 9 and 10 respectively ) . each field was used to irradiate 10 film segments , and the readings of these 10 segments are averaged to minimize film inhomogeneities for each small field ( 0.5 0.5 cm , 0.7 0.7 cm , and 1 1 cm ) , the images obtained after digitizing the pieces of films are registered . after averaging the registered images , the pixel values are converted to dose by applying the calibration curve . then contour plots and transversal profiles are obtained . monte carlo calculations of the 1 1 cm fields were carried out with the penelope code and the spectra data for the 6 and 15 mv photon beams were taken from daryoush and rogers . a simplified simulation was carried out , aimed to simulate the effect of the collimator jaws in the penumbra of the small fields . the photon beam was modeled by a photon point source with the energy spectrum given by daryoush and rogers . figure 2 shows the calibration curve relating the readings of the films and the irradiation doses . it is a power function y = ax + c , where y stands for the dose and x stands for the normalized pixel value . pv = pixel value figure 3 shows the reduction of uncertainty when applying the method of averaging of readings . this figure shows 100 curves ( solid lines ) , randomly chosen from 100,000 possibilities . each curve is obtained by fitting the readings of five pieces of film to the doses 520 , 400 , 320 , 170 , and 50 cgy . the curve obtained by fitting the averaged readings is also shown ( bold and dashed line ) . using this curve , the dose estimation for the films irradiated to 250 cgy ( averaged readings ) is 249.6 cgy . on the other hand , the standard deviation for the estimation based on one single piece of film for each dose value is 19 cgy . calibration curves using one piece of film for dose values ( solid lines ) and averaging 10film pieces for each dose value ( dashed line ) figure 4 shows contour plots of film measurements for the fields 0.5 0.5 cm , 0.7 0.7 cm , and 1 1 cm . the isodoses shown are 80 , 50 , and 20% of the maximum dose for each of the 6 and 15 mv fields . the isodose curves represent the 80 , 50 , and 10% of the maximum dose in each field figure 5 shows the transversal profiles measured with films for the fields of sizes 0.5 0.5 cm , 0.7 0.7 cm , and 1 1 cm , and the energies 6 and 15 mv . the dashed lines correspond to the in - plane direction ( gun - target direction ) and the continuous lines correspond to the cross - plane direction ( transversal to the in - plane ) . this figure shows the differences in the shape of the dose profiles for the in - plane and the cross - plane directions . in the clinac 2100 the jaws delimiting the field in the in - plane are closer to the target , and transversal profiles measured with films for the 0.5 0.5 cm , 0.7 0.7 cm , and 1 1 cm fields . the dotted lines correspond to the in - plane direction , while the solid lines correspond to the cross - plane direction figures 6 and 7 show the transversal profiles for the field of size 1 1 cm and the energies 6 and 15 mv , respectively . the dashed lines correspond to the monte carlo calculations and the continuous lines correspond to the film measurements . film measurements ( solid lines ) and monte carlo calculations ( dashed lines ) transversal profiles for the 1 1 cm 15 mv field . film measurements ( solid lines ) and monte carlo calculations ( dashed lines ) the measurements of the field sizes ( determined as the full width half maximum ) and penumbras 2080% for the 6 and 15 mv beams are shown in tables 1 and 2 , respectively . the monte carlo calculations for the field of size 1 1 cm are also presented . field sizes ( full width half maximum ) and penumbras 20 - 80% in the in - plane ( ip ) and cross - plane ( cp ) directions for the fields shown in figure 5a ( 6 mv beams ) field sizes ( full width half maximum ) and penumbras 20 - 80% in the in - plane ( ip ) and cross - plane ( cp ) directions for the fields shown in figure 5b ( 15 mv beams ) figure 8 shows the measured ofs . the right side shows the measurements for the 6 mv energy beams , while the left side shows the measurements for the 15 mv beams , the error bars in the rc film measurements correspond to 1 standard deviation . in both cases a good agreement is found between both types of chambers ( semiflex and pinpoint ) and the rc films for field sizes of 2 2 cm or larger . rc = radiochromic , pp = pinpoint , sf = semiflex the measurement of the of for the 6 mv field of size 1 1 cm with the pinpoint and semiflex chambers are 10 and 14% lower , respectively , than the of measured with the rc films . in the case of 15 mv , the corresponding differences are 10 and 18% , respectively . for the smallest fields the differences increase . for the 0.7 0.7 cm , the differences with the rc measurements are 19 and 35% for the 6 mv beams and 17 and 33% for the 15 mv , respectively . in the case of the 0.5 0.5 cm field , the differences with the rc measurements are 38 and 52% for the 6 mv beams and 32 and 50% for the 15 mv beams , respectively . in this work a method to measure small radiotherapy fields by means of rc films has been presented . ofs , beam profiles , and 2d dose distributions are accurately measured , taking advantage of the dosimetric properties of the rc media . the small energy and dose rate dependencies shown by the rc films and their high spatial resolution are exploited for measuring small fields . on the other hand , the method presented in this work shows how to deal with the main drawback of rc films : the spatial inhomogeneity of their response . this problem is even bigger when reading the film with a flatbed scanner . in this work , the inhomogeneity is mostly canceled by means of averaging the measurements over a number of segments taken from different parts of one sheet of film . one advantage of using rc films is the possibility of accurately measuring the dose gradient regions . in - plane and cross - plane profiles are shown and the differences in penumbras are obtained , together with the full width at half maximum . good agreement is found between the measured profiles and the monte carlo calculated profiles for the field size of 1 1 cm . for the fields of size 1 1 cm or smaller , the measurements of the ofs by means of the pinpoint and semiflex chambers is affected by the volume effect . the sensitive volume has a length of 5 mm for the pinpoint chamber and of 6.5 mm for the semiflex chamber . those lengths are larger than the constant dose regions of the in - plane profiles [ see figure 5 ] . the measurement of the ics averages the dose on these volumes , and therefore underestimates the dose in the central axis central axis ( cax ) . the results obtained for the ofs measurements show a good agreement between rc films and the pinpoint and semiflex chambers when the field size is 2 2 cm . this agreement and the goodness of fit shown by the calibration curve gives confidence on the accuracy of the method . the ics are accurate when measuring fields of size 2 2 cm or larger . for these fields , the ofs measured with rc films match the ofs measured with the ics ( being the differences smaller than 1.0% ) . it can be argued that , for large fields , the rc films measurements are also accurate . moreover , as reducing the field size is not expected to change the accuracy of the rc films measurements , as demonstrated through the extensive use of the rc films in the measurements of the ofs , the accuracy of the rc film measurements can be extrapolated to the small fields . the same can be said about the measurement depth ; the material media , water instead of pmma ; the collimation system , multileaf collimator ( mlc ) instead of jaws ; or even the irradiation unit . we expect , therefore , that the presented method can be used to perform accurate measurements of small fields in a wide range of situations .

trpv1 is well known as a sensor ion channel that transduces a potentially harmful environment into electrical depolarization of the peripheral terminal of the nociceptive primary afferents . although trpv1 is also expressed in central regions of the nervous system , its roles in the area remain unclear . a series of recent reports on the spinal cord synapses have provided evidence that trpv1 plays an important role in synaptic transmission in the pain pathway . particularly , in pathologic pain states , trpv1 in the central terminal of sensory neurons and interneurons is suggested to commonly contribute to pain exacerbation . these observations may lead to insights regarding novel synaptic mechanisms revealing veiled roles of spinal cord trpv1 and may offer another opportunity to modulate pathological pain by controlling trpv1 . in this review , we introduce historical perspectives of this view and details of the recent promising results . we also focus on extended issues and unsolved problems to fully understand the role of trpv1 in pathological pain . together with recent findings , further efforts for fine analysis of trpv1 's plastic roles in pain synapses at different levels in the central nervous system will promote a better understanding of pathologic pain mechanisms and assist in developing novel analgesic strategies .

transient receptor potential vanilloid subtype 1 ( trpv1 ) is a well - known pain - mediating ion channel expressed in sensory neurons including dorsal root ganglionic ( drg ) neurons , trigeminal ganglionic ( tg ) neurons , and vagal neurons . in response to various harmful stimuli , trpv1 pore opens and cationic flux through the pore into the nerve terminal causes electrical depolarization which may lead to action potential generation . when propagated and transmitted into the brain , the signals finally result in the perception of pain . because of its extreme polymodality compared to other known peripheral sensor molecules that allows trpv1 to cover a large spectrum of pain qualities from chemical through thermal ones and of its famous activator capsaicin which has been traditionally utilized for pain research even before trpv1 discovery , trpv1 has garnered a great deal of attention as a peripheral pain - modulating target . topical application of a trpv1 modulator is currently the mainstream for trpv1-targeting analgesic strategies while systemic approaches have been dropped due to a potential for hyperthermic adverse effect because body temperature regulation is perturbed by antagonism of vagal trpv1 . although the initial report about trpv1 finding suggested that it is specifically expressed in sensory neurons , its wider distribution in various regions including certain regions of the central nervous system ( cns ) and nonnervous tissues has been surmised ( for review , ) . using rodent and human brains , mezey et al . verified the existence of trpv1 protein and mrna in the spinal cord , amygdala , medial and lateral habenula , hippocampus , striatum , hypothalamus , centromedian and paraventricular thalamic nuclei , substantia nigra , reticular formation , locus coeruleus , cerebellum , inferior olive , and certain cortical areas . soon after , valtschanoff et al . , focusing on the spinal cord , showed that both presynaptic ( from the central terminal of sensory neurons ) and postsynaptic regions ( from the dendrites of spinal cord dorsal horn neurons ) exhibit trpv1 positivity , especially in the superficial laminae i and ii , which are the first relaying stations in the pain sensory pathway . using dorsal rhizotomy , they were able to histologically show that postsynaptic trpv1 expression levels were highly dependent on the presence of peripheral inputs , indicating that spinal cord trpv1 expression and function may be dynamically controlled by sensory states . since that time , cns and spinal cord expression of trpv1 these results suggested that trpv1 may play a role in the central areas and in this review we more focus on pain transmission in the spinal cord . altered expression of a protein depending on disease states often implies its importance in disease progression . upregulation of trpv1 in drg or tg neurons under a proinflammatory state has been reported [ 69 ] . differential regulation of trpv1 expression occurs in neuropathic pain states . at the scale of whole drg neuronal collection , including both damaged and undamaged neurons , the amount of trpv1 was reduced in many different neuropathy models including those of sciatic nerve axotomy , partial nerve ligation , chronic constriction injury ( cci ) , spinal nerve ligation , and diabetic neuropathy [ 14 , 15 ] . the loss of total trpv1 expression appears to be at least partially due to the degeneration of damaged trpv1-positive drg neurons . interestingly , in the spinal cord dorsal horn , trpv1 is upregulated in a cci neuropathic pain model . when looking at uninjured drg neurons , higher trpv1 expression was detected even in the neurons at different spinal levels from that for damaged ones [ 11 , 17 ] . different from the peripheral neuropathy models mentioned above , in the spinal cord injury models , an increase in trpv1 proteins or mrnas was consistently detected in the drg [ 1820 ] . the molecular and cellular mechanisms for such increased trpv1 expressions in undamaged neurons in diverse injury models remain undetermined and we dealt with those in unsolved issues below . briefly , the uninjured drg neurons may be affected by inflammatory processes , for example , via increased secretion of inflammatory mediators such as nerve growth factor ( ngf ) from recruited immune components around adjacent damaged drg or spinal cord regions . indirect synaptic mechanisms via collaterals or descending circuits also likely participate in trpv1 upregulation in drgs at different spinal levels . the results of elevated trpv1 levels indicate that peripheral trpv1 expression can be controlled upon injury conditions and that increased amplification of pain signals may involve upregulation of trpv1 , which might serve as a potential leverage for therapeutic modulation . besides expression results , outcomes from pharmacological manipulation of spinal cord trpv1 activity have also consistently emphasized its crucial role in pain transmission and therapeutic advantages . in particular , industrial field hypothesized that selective antagonism to spinal trpv1 could be one option for avoiding adverse malignant hyperthermia since cns trpv1 seems to be free from the hyperthermic mechanism [ 21 , 22 ] . not only demonstrated increased trpv1 levels in the spinal cord of cci rats but also produced a promising analgesic result from intrathecal administrations of the trpv1 antagonist bctc . in the hundreds of nanomolar range , mechanical allodynia and calcitonin gene - related peptide - like immunoreactivity and substance p - like immunoreactivity were attenuated in the spinal cord from the cci - injured rats researchers at abbott labs used three different inflammatory pain models with complete freund 's adjuvant , capsaicin , and sodium monoiodoacetate injections . their cns - penetrable version , a-784168 , more effectively blocked pain than a less penetrable a-795614 , despite being similar in terms of in vitro profiles for trpv1 antagonism . watabiki et al . at astellas pharma demonstrated that mechanical allodynia in their mouse spinal nerve ligation ( snl ) model was alleviated by intrathecal injection of either of bctc or their own trpv1 antagonist as1928370 . wu et al . demonstrated that trpv1 antagonism using intrathecal amg9810 reversed mechanical and thermal hypersensitivities in a contusive spinal cord injury model . two research groups independently demonstrated that intrathecal agonist ( capsaicin or 9-hydroxyoctadecadienoic acid ) injections induced mechanical allodynia [ 25 , 26 ] . since trpv1 is not a mechanosensitive ion channel and thus trpv1 in the periphery has no role in mediating mechanical phenotypes , the mechanical hypersensitivity is purely due to central trpv1 activity on transmission . intrathecal injections of resiniferatoxin ( rtx ) , a potent trpv1 agonist , deactivated voltage - dependent components or ablated trpv1-positive neuronal terminals [ 2931 ] . interestingly , in a carrageenan - induced inflammation model , the thermal threshold but not the mechanical threshold was normalized with this strategy [ 27 , 28 ] . although the model involved an agonistic challenge , body temperatures of the treated animals were largely unaffected . collectively , the results from expression dynamics and pain pharmacology commonly raise the importance of the existence of spinal cord trpv1 . in turn , several groups have begun to explore the next question : how , differently from peripheral trpv1 , spinal trpv1 intervenes pain transmission in pathologic states . spinal synaptic plasticity is a central concept that accounts for pathologic transition from a normal acute pain to a chronically morbid one . both long - term potentiation and depression ( ltp and ltd ) paradigms and technical progress for brain slice electrophysiology from learning and memory research mostly using the hippocampal area were imported to the pain field . those concept and technology have contributed to the understanding of the pathological pain transmission in the spinal cord and to finding painkilling targets : ionotropic and metabotropic glutamate receptors , n - type and t - type voltage - gated ca channels , upstream and downstream signaling molecules of the nitric oxide synthesis pathway , calcium / calmodulin - dependent kinases , neuropeptides and their receptors , and so forth . for validating the spinal trpv1 mechanism , , three important aspects of the roles of spinal trpv1 have been reported in recent years [ 26 , 34 , 35 ] . the discussions about the detailed results follow . gone through simple traditional observations where spontaneous excitatory postsynaptic currents ( sepscs ) were facilitated by the presynaptic actions of capsaicin to understand the circuitry under normal conditions [ 36 , 37 ] , researchers became interested in trpv1 's role in pathologic states . xu et al . found that trpv1 in the central terminal of drg neurons plays an important role in exaggerating pain in an inflammatory state during their analyses of the effects of endogenous proresolving lipids . when they recorded the lamina ii neurons of transverse slices of the murine lumbar spinal cord with a patch clamp technique , sepscs were increased upon tumor necrosis factor- ( tnf- ) exposure . because the frequency but not the amplitude of epscs was increased , tnf- seemed to elevate glutamate release by acting at presynaptic terminals . this perfusion with tnf- ex vivo may represent an inflammatory or neuropathic pain state in vivo . simultaneous treatment of capsazepine reversed this tnf- effect : the changes were limited to numbers of sepsc frequencies . consequently , the results of this study provide multiple implications : the primary action site of the acute tnf- effect is presynaptic sensory neurons ; trpv1 had been presumed to be a major effector of tnf- action , at least in the periphery [ 38 , 39 ] , and the results confirmed it and extended to the central terminals ; regarding capsazepine - affected parameters , trpv1-mediated mechanism may be more important in presynapses . despite being out of the scope of this review , resolvin e1 , a potent endogenous proresolving lipid , appears to disturb the presynaptic signaling between tnf- and trpv1 via its g - protein - coupled receptor ( gpcr ) activation , as a part of its painkilling mechanisms [ 4042 ] . more recently , the wei and dong labs revisited the presynaptic role of trpv1 from the viewpoint of descending excitatory modulation when they tried to explain secondary hyperalgesia that occurs from neighboring but uninjured receptive field under neuropathic conditions . they developed a knock - in mouse line in which the expression of gcamp3 encoding a ca indicator protein is driven by the promoter of pirt ( phosphoinositide - interacting regulator of transient receptor potential channels ) , a pan - drg / tg marker . with this mouse model , changes in intracellular ca levels in cell bodies and peripheral and central terminals can be discerned in drg and tg neurons , even under ex vivo conditions surrounded by other tissue types or buried in complex synaptic circuits . they also created a cheek mechanical hyperalgesia model using cci of the infraorbital nerve , which is the major branch of the maxillary ( v2 ) tg nerve . accordingly , the trigeminal subnucleus caudalis ( vc ) , which is analogous to the spinal dorsal horn in terms of the sensory synaptic circuit , was observed for presynaptic trpv1 functions . although only v2 tg nerve had undergone the cci procedure , heightened pain sensitivities occurred in the cheek , jaw , and ear , the latter two of which are mandibular ( v3 ) tg nerve territories . when intracellular ca fluorescence level due to gcamp3 was analyzed as a surrogate measure for excitability , both v2 and v3 central terminals in vc exhibited larger ca increases than under normal conditions . furthermore , these elevated sensitivities were commonly observed in terminals from superficial through deep laminae , suggesting that not only injured but also adjacent undamaged nerve fibers became hyperactive and that this situation consequently led to secondary hyperalgesia and allodynia . based on their previous observations , wei and dong 's group hypothesized that the rostral ventromedial medulla ( rvm ) in the brainstem relays 5-hydroxytryptamine ( 5-ht , serotonin ) dependent excitatory input to uninjured nerves [ 44 , 45 ] . indeed , 5-ht immunoreactivity was elevated near the gcamp3-positive central sensory terminals of vc . moreover , antagonistic manipulations including treatment using the 5-ht3 receptor antagonist in vc or 5-ht depletion in rvm using rna interference against its biosynthesis alleviated both hyperactivity of trpv1-mediated ca signals and hypersensitive behaviors . since 5-ht receptor - mediated trpv1 facilitation was confirmed in the central presynaptic terminals , the descending excitatory axons from rvm seem to constitute axoaxonal contacts . collectively , trpv1 in the presynapse of sensory neurons that covers the undamaged regions participates in secondary pain amplification through a descending facilitation mechanism ( figure 1 ) . previous positive results of the spinal cord expressions of trpv1 strongly implicated a functional role of trpv1 in the spinal postsynaptic neurons [ 24 ] . the oh lab focused on the spinal cord inhibitory synapse regarding the role of trpv1 . in fact , loss of gabaergic or glycinergic inhibitory control in the spinal synaptic network has long been proposed as a cause of central pain sensitization [ 4648 ] . different from sepscs , evoked epscs have been reported to decrease after capsaicin perfusion [ 49 , 50 ] . trpv1 activation acutely induced increases in frequency in spontaneous inhibitory postsynaptic currents ( sipscs ) in the dorsal horn neurons via gabaergic or glycinergic connections [ 51 , 52 ] . notably , in a mouse model with ablation of trpv1-positive drg neurons , a significant proportion of mechanical hypersensitivity remained following intrathecal capsaicin administration , indicating that postsynaptic trpv1 also contributes to the pain state . indeed , trpv1 expression and agonist - dependent activation in postsynaptic dorsal horn neurons were verified , and over 75% of gabaergic interneurons were trpv1-positive whereas ~75% of non - gabaergic postsynaptic neurons were trpv1-negative . surprisingly , in their epsc profiling of the gabaergic neurons in response to electrical stimulation of the dorsal root entry zone , ltd occurred after postsynapse - specific trpv1 activation . trpv1 activation - induced ltd was dependent on an increase in the intracellular ca concentration and the reduction of alpha - amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ( ampa ) receptor activities , similar to other typical ltd processes . accordingly , ltd caused less excitability and reduced gaba release of the gabaergic interneurons , and therefore the secondary projection neurons ( spinothalamic tract neurons in this study ) received less inhibitory input , resulting in enhanced relay of pain signals to higher brain areas ( figure 1 ) . the analgesic effects of spinal trpv1 antagonism in mice cci model were successfully repeated without malignant hyperthermia , but the authors proposed a novel mechanism engaging ltd of trpv1-positive inhibitory neurons . because the studies on the contribution of trpv1 to the transmission of spinal pain circuit are relatively in their early stage , much of biological information is still unavailable . here , we focus on several important issues as follows . detailing how dynamically the spinal trpv1 expression alters depending on the pathologic process may provide more sophisticated explanation of spinal pain mechanisms and help fine - tuning analgesic strategies . as mentioned above , a promising result from trpv1 expression in the spinal cord has been demonstrated by kanai et al . . in their cci rat model , trpv1 expression of the ipsilateral superficial dorsal horns was gradually elevated for two weeks . this expression location indicates an increase in trpv1 in the presynaptic terminals by neuropathic insult , supporting the enhanced capsaicin sensitivity of the central terminals in the novel trigeminal cci neuropathy model . detected elevated trpv1 levels in the dorsal horn presynaptic regions in a carrageenan - inflammation model . in a relatively chronic inflammation model using complete freund 's adjuvant , despite a statistically ambiguous elevation on days from 1 to 2 after injection , over 50% increased trpv1 levels were maintained for 1 to 3 weeks [ 6 , 8 ] . the increase may depend on the mediator effect including ngf , which is similar to well - known mechanisms for increases in trpv1 expression of the peripheral terminals . however , information on trpv1 dynamics of interneurons or projection neurons in the presence or absence of injury is still lacking . although trpv1 is known as a major heat sensor in the body , its thermosensory functions and related thermoregulatory feedback mechanisms are unlikely to be exerted in the spinal cord because this region only experiences a limited range of temperatures , close to the core body temperature . evidence mentioned above suggests that pharmacological antagonism for trpv1 in the spinal cord does not cause thermal effects . however , that situation leads to another question : what conditions else activate trpv1 there ? inflammatory peptides including tnf- , which was tested by xu et al . , and neurotransmitters such as 5-ht , which proved to be a descending modulator , may utilize trpv1 for a downstream effector . for the tnf--trpv1 signaling axis , hypotheses of prostaglandin production for increased trpv1 activity and extracellular signal - regulated kinase ( erk ) activation for elevated trpv1 expression were raised by studies on drg neurons [ 38 , 54 ] . extracellular prostaglandins are known to enhance trpv1 sensitivity via phosphorylation by protein kinase a or c through their g - protein - coupled receptor - mediated signaling , but it remains elusive whether this paracrine signaling cascade also works for tnf- axis . even if this prostaglandin - mediated mechanism is true , the mechanism can only sensitize but not activate trpv1 . by this sensitization , heat threshold for in addition , while only erk itself has been shown to participate , there has not been sufficient clarification of the further downstream signal in this pathway . moreover , it also needs to be explored in the presynapse whether the releases of neurotransmitter vesicles are facilitated simply by increased intracellular ca ions through trpv1 opening - induced depolarization , or other unknown molecular downstream signals are involved . the 5-ht3 receptor is a major receptor that receives descending excitatory input from rvm . interestingly , 5-ht3 receptor is a cation channel that may be functionally redundant regarding the trpv1 outcome to depolarize the presynaptic area . whether these two cation channels are additive or otherwise functionally or physically coupled for a synergistic action , as shown in recently published observations at the peripheral terminals about trpv1-trpa1 and trpv1-anoctamin cooperation [ 56 , 57 ] , requires further examination . transient receptor potential ankyrin subtype 1 ( trpa1 ) is comparable to trpv1 in terms of the importance of covering pain modalities and transductory roles to initiate nociceptor depolarization in the periphery . if both trp channels share central locations of their expressions , their redundancy , cooperativity , or compensation in the roles in synaptic transmission could not be ignorable . indeed , the presynaptic facilitative role of trpa1 in the spinal cord has been addressed . the effects of antagonistic challenges against spinal trpa1 function were examined and the treatment displayed analgesic outcomes in diverse pain models including snl , rapid eye movement sleep deprivation , capsaicin - paw injection , formalin - paw injection , and diabetic neuropathy [ 6062 ] . it was further demonstrated that the pain - facilitating effect of descending excitatory inputs from rvm stimulation or spinal cord 5-ht3 receptor activation was all blunted by trpa1 antagonism . unlike trpv1 , the analgesic mechanism does not seem to employ the postsynaptic gabaergic disinhibition mechanism , which seems to be inconsistent when comparing the earlier and the most recent observations that spinal trpa1 activation facilitates not only the frequency and amplitude of sepscs but also those of sipscs [ 59 , 63 ] . as mentioned , kim et al . although this can form analgesic proof of concept from a therapeutic viewpoint , it is still poorly understood why the gabaergic neurons need to express trpv1 regarding their ordinary transmission and what naturally stimulates trpv1 in neuropathic conditions . regarding the polymodality of trpv1 kim et al . suggested that 12(s)-hydroperoxyeicosatetraenoic acid ( 12(s)-hpete ) may be a natural trpv1 activator candidate . gibson et al . demonstrated that anterograde metabotropic glutamate receptor activation results in postsynaptic 12(s)-hpete production and that this lipoxygenase metabolite retrogradely diffuses and activates presynaptic trpv1 , causing ltd in the hippocampal ca1 synapses . application of this paradigm to the spinal cord may narrow down the candidate mechanisms to a gpcr - lipoxygenase cascade but measurement of which substances in the spinal cord are a major metabolite is still required , for example , hepoxilins and hydroxyoctadecadienoic acids [ 25 , 42 , 65 , 66 ] . trpa1 also has a wide spectrum for sensing endogenous reactive substances the levels of which are frequently elevated under injury conditions in tissues or within active synapses . those are reactive oxygen and nitrogen species and lipid peroxidation products that are known to covalently bind and activate trpa1 [ 58 , 67 ] . normalizing imbalanced local production of atypical excitatory substances for trp channels by developing specific enzyme inhibitors might be another analgesic strategy to utilize the central trp channel - mediated synaptic mechanism . stemming from the recent accomplishments in spinal trpv1 research , unexplored mechanisms connecting newly uncovered trpv1 's roles and related hypotheses are being raised . like pharmacological approaches , a series of studies using local rna interference techniques have given new lines of firm evidence for the roles of trpv1 in pathologic pain progress and also for its action in the spinal cord [ 6870 ] . as tools to improve the efficiency of interfering gene delivery and to lessen safety concerns are being developed , expectancy about future utility of gene editing therapies for chronic pain modulation is currently forming [ 71 , 72 ] . in the last year , hirai et al . showed that intrathecal administration of adenoassociated virus serotype 9 ( aav9 ) vector carrying short - hairpin rna ( shrna ) against trpv1 resulted in long - term suppression of thermal hyperalgesia in a mouse spared nerve injury model . viral delivery may confer long - term stable generation of shrna and limited exposure to immune protection mechanisms outside of the cns region may help time scale of the effect further since shrnas in themselves have tough permeability to blood brain barrier ( bbb ) . the confinedness by intrathecal injection may also reduce potential adverse effects from nontarget tissues . interestingly , despite spinal targeting , only thermal hyperalgesia , not mechanical or cold allodynia , was blunted , which typically occurs in drg - specific trpv1 impairment . conversely , the parameters of shrna abundance and trpv1 mrna reduction were significantly better in the spinal cord than in the drgs . differential translational compensation or the presence of trpv1 isotypes free from the target sequence may be conceivable regarding the broad analgesic spectrum of the above circuit research . since trpv1 expression is absent in glial components , it is not likely that the effects of pharmacological modulations of spinal trpv1 have a direct link to glial contributions . however , in a follow - up study of kim et al . , the wei lab demonstrated indirect participation of trpv1 . although they did not measure trpv1 activity , guo et al . repeated presynaptic stimulation by mimicking activation of the descending 5-ht pathway , which subsequently activated microglia and astrocytes . by showing pharmacological and histological evidence , they suggested that fractalkine released from the presynapses stimulates microglia and then interleukin-18 from activated microglia boosts astrocyte function , from which released interleukin-1 finally enhances excitability of the dorsal horn neurons by inducing phosphorylation of an n - methyl - d - aspartate receptor subunit . nitric oxide ( no ) is of general importance as a retrograde signal for modifying brain synaptic strength . in pain synapses , postsynaptic no plays a central role for presynaptic activation of the guanylyl cyclase - cyclic guanosine monophosphate- ( cgmp- ) protein kinase g pathway [ 75 , 76 ] . interestingly , trpv1 activity seems to be tolerant of pkg action or even downregulated by pkg [ 77 , 78 ] . not only direct pkg action , but also effects of known substrates of pkg , for example , inositol trisphosphate receptor and myosin light - chain kinase , appear to be independent of the amplification of trpv1 activity . calcium / calmodulin - dependent protein kinase ii ( camkii ) plays a crucial role in ampa receptor facilitation in the postsynapse . it has been reported that phosphorylation of trpv1 of drg neurons by camkii is important for maintaining its sensitivity to ligands , which can be explored regarding the postsynaptic paradigm . -opioid receptor - induced ltp in the spinal cord appears to occur in trpv1-positive presynapses and it might be a future issue whether changes in trpv1 activity in the central terminal are practically involved in opioid signaling . two of many reasons why peripheral trpv1 has received much attention from industry in the decade since its gene discovery seem to be its polymodality integrating painful inputs with diverse qualities and its peripheral location . for the latter , a central advantage of targeting peripheral trpv1 is that its ligand avoids the adverse effects of the cns when it is designed to be bbb - impenetrable . however , assuming that the aim is cns administration and considering the novel nociceptive roles of trpv1 in the spinal circuit , one may need to conceive similar adverse situation as already observed or predicted for other cns analgesic candidates because the target is possibly expressed in other central regions and may have differential actions . as mentioned above , the bulbospinal circuit receives descending input from periaqueductal gray ( pag ) and confers descending excitatory inputs as well as inhibitory ones [ 35 , 81 ] . earlier , mcgaraughty et al . demonstrated that trpv1 activation of dorsal pag gave a hyperalgesic phase via the rvm circuit . however , in ventrolateral pag ( vl - pag ) , the neighboring region , it has been suggested that when -opioid receptor is simultaneously activated trpv1 activation contributes to facilitation of glutamatergic interneuronal activity , which offers gabaergic inhibition of descending excitatory on cells in the rvm circuit , leading to a reduction of nociceptive transmission in the spinal cord . decreased evoked ipscs , increased miniature ipscs and epscs , and contribution of the cannabinoid receptor have also been proposed by a different intra - pag recording study . because this circuit was examined only under acute pain conditions , the analgesic aspect of trpv1 action in vl - pag needs to be further addressed for pathologic pain conditions . exploring the mechanism of analgesic effects of acetaminophen , researchers have found out that trpv1 activation in the central nervous system is involved [ 85 , 86 ] . when acetaminophen is systemically administered , its metabolites are formed in the brain by the action of fatty acid amide hydrolases . those appear to directly activate brain trpv1 , leading to analgesia in formalin - induced pain and acute thermal or mechanical pain . antagonism by intracerebroventricular injection of an antagonist and virtual localization of the drug using methylene blue injection argue that supraspinal trpv1 , but not spinal cord trpv1 , may participate in the central analgesic action . interestingly , some of electrophilic and toxic metabolites produced during the acetaminophen metabolism , different from the metabolites that activate trpv1 , were reported to activate spinal presynaptic trpa1 , resulting in acute antinociception via subsequent inactivation of adjacent presynaptic voltage - gated na and ca channels . other adverse situations due to the potential presence of trpv1 in cns regions might be possible . for example , whether inadvertent diffusion of an antagonist in the ventricular regions may enable access to untargeted areas and affect other brain functions including memory or mood needs to be carefully evaluated [ 64 , 88 , 89 ] . whether trpv1 activation can gain specific delivery of local anesthetics to trpv1-positive nociceptor is being considered as a novel analgesic strategy [ 90 , 91 ] . some membrane - impermeable hydrophilic derivatives of lidocaine species are able to permeate into neurons through dilated trpv1 pore when trpv1 is activated and once inside , they can block voltage - gated na channels , with or without their permeant blockade of trpv1 itself . trpv1-negative neurons should be inert to the blocking effects of these drugs since the drugs can be admitted only through trpv1 , which may allow avoidance of common adverse effects of the local anesthetics including numbness and motor defects via interfering nonpain pathways . such approaches of specific application may be taken into consideration in the future for modulation of spinal cord trpv1 . among trpv1-targeting pain therapies , the rationale is based on the functional incapacitation of the peripheral sensory terminals by agonist - induced trpv1 desensitization and mitochondrial permeability transition which leads to terminal ablation [ 79 , 92 ] . the same mechanism might be possible in the spinal region : analgesia via defunctionalization of trpv1-specific pre- and postsynapses by agonist - induced effects . however , as mentioned , intrathecal administration of trpv1 agonists resulted in some pain phenotypes in animal studies [ 25 , 26 ] . this appears to be predictable because it is an unavoidable side effect that topical capsaicin application in its early treatment stage evokes pain via initial trpv1 activation in humans . one possible option to overcome this hurdle is currently thought to be a substitution by a nonpungent capsaicin analogue free from the initial pain induction . although such a class of trpv1 agonists were recently developed , they have not been thoroughly tested regarding their analgesic effects . in the early stage , more attention was given to the sensory involvement of trpv1 in the peripheral terminal of the nociceptor neurons to harmful environments and to its contribution to neurogenic inflammation [ 94 , 95 ] . however , revisiting traditional capsaicin pharmacology , assessments regarding trpv1 expression patterns and their dynamics in diverse neural regions have provided clues of other nociceptive roles for trpv1 . particularly , recent accumulation of knowledge on trpv1 functions in the spinal presynaptic and postsynaptic locations connecting to exacerbation mechanisms for neuropathic pain has begun to address its roles in the central nervous system . this heightened understanding and new hypotheses also appear to raise the possibility of developing new proof of concept targeting spinal trpv1 . such attempts for new approaches could be extended to other cns locations and other polymodal trp channels such as trpa1 . further analyses regarding the role of trpv1 in plastic changes for pain synapses at the individual level including supraspinal circuits will shed light on the collective contribution to exacerbation of pathologic pain and its analgesic utility .

we have solved the crystal structure of a predicted fructose - specific enzyme iibfruc from escherichia coli ( eceiibfruc ) involved in the phosphoenolpyruvate - carbohydrate phosphotransferase system transferring carbohydrates across the cytoplasmic membrane . eceiibfruc belongs to a sequence family with more than 5,000 sequence homologues with 2599% amino - acid sequence identity . it reveals a conventional rossmann - like -- sandwich fold with a unique -sheet topology . its c - terminus is longer than its closest relatives and forms an additional -strand whereas the shorter c - terminus is random coil in the relatives . interestingly , its core structure is similar to that of enzyme iibcellobiose from e. coli ( eciibcel ) transferring a phosphate moiety . in the active site of the closest eceiibfruc homologues , a unique motif cxxgxaht comprising a p - loop like architecture including a histidine residue is found . the conserved cysteine on this loop may be deprotonated to act as a nucleophile similar to that of eciibcel . the conserved histidine residue is presumed to bind the negatively charged phosphate . therefore , we propose that the catalytic mechanism of eceiibfruc is similar to that of eciibcel transferring phosphoryl moiety to a specific carbohydrate .

in bacteria , the phosphoenolpyruvate - carbohydrate phosphor - transferase system ( pts ) is a chief carbohydrate transport system ( deutscher et al . , a large number of carbohydrates are phosphorylated by the pts generally consisting of cytoplasmic proteins , enzyme ei ( ei ) and heat stable phosphoryl carrier protein ( hpr ) , and a membrane - associated carbohydrate - specific permease complex enzyme ii ( eii ) . in general , eii complexes are composed of hydrophilic enzymes , eiia and eiib , and hydrophobic integral membrane proteins , eiic ( and eiid in a mannose- and fructose - specific pts ) . the catalytic function of ei , hpr and eiia depends on a functional histidine residue transiently phosphorylated during enzyme reaction . the active sites of eiib subunits include either a cysteine or a histidine as a catalytic residue . the phosphoryl group which will be transferred to various carbohydrates is obtained from phosphoenolpyruvate by ei . hpr becomes phosphor - hpr by accepting the phosphoryl group from ei . at the next step , the phosphoryl group from phospho - hpr is transferred to a sugar - specific eii complex . in general , ei and hpr are not selective and are shared by different pts systems . however , there are many different types of eiis present for uptake of different carbon sources , and they are not interchangeable . a phylogenetic analysis of the eii proteins show that the pts permease families are classified into seven families as follows ( marchler - bauer et al . , 2015 ) ; the ( i ) glucose ( including glucoside ) ( glc ) , ( ii ) fructose ( including mannitol ) ( fru ) , ( iii ) lactose ( including n , n - diacetylchitobiose ) ( lac ) , ( iv ) galactitol ( gat ) , ( v ) glucitol ( gut ) , ( vi ) mannose ( man ) , and ( vii ) l - ascorbate ( asc ) families . in addition , pts components directly interact with their target proteins which carry out various cellular functions such as transport proteins or transcription regulators . an open reading frame of e. coli str . mg1655 codes for a frwd gene ( ncbi refseq : np_418388.1 ) which is annotated as putative pts eiib protein ( eceiib ) of 12.6 kda . it belongs to the ncbi conserved domain cd05569 ( pts_iib_fructose ) which includes subunit iib of eii of the fructose specific pts ( barabote and saier , 2005 ) . a psi - blast search ( http://www.ncbi.nlm.nih.gov ) of this sequence revealed around six hundred proteins with sequence identity above 35% ( fig . most of the homologous sequences are annotated as fructose - like specific pts system eiib or fused forms , eiibc or eiiabc . the biochemical study revealed that at least 15 different eii complexes are found in escherichia coli ( saier and reizer , 1994 ) . several putative fructose - like iib components are also detected in the e. coli pts system . since the molecular structure and the functional implication of this eceiib family have not been reported , we have determined the x - ray crystal structure of eceiib . based on the crystal structure analysis , molecular functions of eceiib have been inferred and discussed in this paper . details of the preliminary x - ray study ( shin , 2008 ) and the structure determination ( joo et al . , 2015 ) of eceiib had been previously published . in brief , eceiib crystals were grown using 20% ( w / v ) peg mme2000 in the presence of 10 mm nickel ( ii ) chloride hexahydrate . the x - ray dataset of eceiib was collected to 2.28 resolution and the crystal belonged to the primitive trigonal space group p32 with unit - cell parameters a = b = 33.11 , c = 154.38 . molecular replacement ( mr ) was performed using phenix ( adams et al . , 2010 ) with highly accurate protein 3d models generated by the recently proposed method called got ( global - optimization - based template - based modeling of proteins ) ( joo et al . , 2015 ) . a relatively good electron density map was obtained after automated model - building and refinement with the phenix autobuild wizard ( afonine et al . , 2012 ) . the electron density map clearly showed the presence of two protomers of eceiib in the asymmetric unit . interestingly , they form a dimer bridged by a nickel ion contained in the crystallization solution . the final model exhibited good stereochemical geometry and was refined to r- and r - free values of 22.5% and 29.9% , respectively ( joo et al . , 2015 ) . the atomic coordinates and structure factors of the eceiib structure have been deposited in the rcsb protein data bank under the accession code 4tn5 . details of the preliminary x - ray study ( shin , 2008 ) and the structure determination ( joo et al . , 2015 ) of eceiib had been previously published . in brief , eceiib crystals were grown using 20% ( w / v ) peg mme2000 in the presence of 10 mm nickel ( ii ) chloride hexahydrate . the x - ray dataset of eceiib was collected to 2.28 resolution and the crystal belonged to the primitive trigonal space group p32 with unit - cell parameters a = b = 33.11 , c = 154.38 . molecular replacement ( mr ) was performed using phenix ( adams et al . , 2010 ) with highly accurate protein 3d models generated by the recently proposed method called got ( global - optimization - based template - based modeling of proteins ) ( joo et al . , 2015 ) . a relatively good electron density map was obtained after automated model - building and refinement with the phenix autobuild wizard ( afonine et al . , 2012 ) . the electron density map clearly showed the presence of two protomers of eceiib in the asymmetric unit . interestingly , they form a dimer bridged by a nickel ion contained in the crystallization solution . the final model exhibited good stereochemical geometry and was refined to r- and r - free values of 22.5% and 29.9% , respectively ( joo et al . , 2015 ) . the atomic coordinates and structure factors of the eceiib structure have been deposited in the rcsb protein data bank under the accession code 4tn5 . the crystal structure of eceiib has been determined at the resolution of 2.4 ( supplementary fig . the structure of the eceiib protein is composed of a central six - stranded parallel open twisted -sheet , which is flanked by three -helices ( 1 , 3 , 4 ) on the concave side and two ( 2 , g1 ) on the convex side ( fig . , the crystal structure shows an artificial dimer seemingly being triggered by one nickel ion coordinated to two neighboring his109 residues located on the c - terminus of each protomer ( fig . this coordination may induce a loop - to - strand conversion of the c - terminal loop resulting in the formation of the last -strand 6 . as a result , a twelve stranded -sheet composed of two contiguous -sheets is formed in the dimer . however , eceiib is a monomer as indicated in the monodisperse peak with molecular weight of 13 kda from dynamic light scattering ( dynapro 99 , proterion corporation , usa ) ( shin , 2008 ) and this dimeric form is not found in the other crystal structures of its closest homologues as mentioned below . however nickel ions improved the diffraction limits of crystals ( shin , 2008 ) . in the crystal structure , two histidine residues and two water molecules coordinated the nickel ion . during the refolding step , various metals were tried but a dimeric size was not detected in size exclusion chromatography and dynamic light scattering . furthermore , the cd spectrum showed the same secondary structure contents regardless of the presence of metal ions including zinc and nickel ions ( oganesyan et al . , 2005 ) . however , the metal induced dimerization of eceiib can not be thoroughly excluded and its biorelevance should be further studied inside bacterial cells . eceiib belongs to a sequence family with more than 5000 sequence homologues having 2599% amino - acid sequence identity obtained by a psi - blast search ( fig . the 3d structure alignment search with dali ( holm and rosenstrom , 2010 ) reveals that the crystal structure of eceiib determined in this study , along with the closest homologues ( z - scores above 14 ) , 2r48 ( mannose - specific eiib from bacillus subtilis ) , 2r4q ( fructose - specific eiib from b. subtilis ) , 2m1z ( hypothetical protein lmo0427 from listeria monocytogenes ) and 2kyr ( fructose - like enzyme iib from e. coli ) , forms a unique fold family not found in other protein structures . it is composed of 1 , 1 , 2 , 3 , 2 , 4 , g1 , 5 , 3 and 4 . the root - mean - square ( rms ) deviations of matching c atom positions of eceiib compared with its closest homologues are 0.727 ( 70 c atoms of 2r48 ) 0.965 ( 71 of 2r4q ) , 1.090 ( 86 of 2m1z ) and 0.989 ( 80 of 2kyr ) , respectively . it is noteworthy that the c - terminus of eceiib is longer than its closest homologues and forms an additional -strand 6 whereas the shorter c - termini of the other members are unstructured ( fig . the central -sheet of eceiib has the topology of 6 5 4 1 2 3 instead of 5 4 1 2 3 found in the other members . the dali search revealed that the topology of the six - stranded -sheet , 6 5 4 although this additional -stand is stabilized in our crystal form by an inter - molecular antiparallel -bonding and chelation of a nickel ion by the neighboring two his108 ( fig . 2a ) , it is not yet clear whether the last -strand is an intrinsic structure or an artificial result . in fact , the predicted secondary structure of the c - terminus of eceiib calculated with psipred ( http://bioinf.cs.ucl.ac.uk/psipred/ ) ( buchan et al . , 2013 ) is a loop . therefore , the state of the c - terminus in solution and the possibility of a transition from disordered to ordered or vice versa related to its function are still under investigation . the next homologue group of eceiib is dna - binding response regulators with z - scores above 8 . however , this family has a different -sheet topology from eceiib and contains unique active site residues . the structure of the eiib subunit of fructose permease ( eiib ) , another homologue of eceiib , also has a different -sheet topology ( 3 2 4 1 5 6 7 ) with the active site residue , his15 ( schauder et al . , 1998 ) . interestingly , the dali search revealed that the core -sheet topology , 21 4 5 , of eceiib is closer to that of enzyme iib from e. coli ( eciib , pdb : 1iib ) ( van montfort et al . , 1997 ) with the z - score of 7.2 ( fig . it contains a -sheet with the topology of 2 13 4 lacking two -strands at each end of the sheet . eciib homologues comprises a protein family with the enzyme gatb of the galactitol - specific phosphoenolpyruvate - dependent phosphotransferase system from e. coli ( 1tvm ) , the cytoplasmic b domain of the mannitol transporter iiabc from e. coli ( 1vkr ) , and ptxb from streptococcus ( 3czc ) . they have the -sheet topology of 2134 with a conserved cysteine ( cys10 ) and a threonine / serine ( thr17 ) located at the active site constructed by a p - like loop ( fig . the cysteine residue functions via a cysteine - phosphate intermediate and the threonine residue assists the binding of phosphate ( ab et al . , since these residues are critical in transferring a phosphoryl group in eciib homologues , cys10 and thr17 of eceiib may function in a similar way . as for eciib homologues , the phosphorylation site of eiib is located on this conserved cysteine residue at the n - terminal end of the p - like loop . this cysteine receives the phosphoryl group from eiia and transfers it to the specific carbohydrate when bound at the catalytic site of eiic . the catalytic activity of the cysteine is aided by the p - like loop structurally similar to that of the phosphate binding p - loop of the phosphotyrosine protein phosphatase ( ptpase ) superfamily ( consensus sequence cxxxxxr(s / t ) ) ( su et al . , 1994 ) . the p - loop forms an anion - binding motif together with the helix dipole of helix 1 and thus provides a favorable environment to generate deprotonated cysteine as a nucleophile and to accommodate a negatively charged phosphoryl group . however , the p - like loop of eciib homologues lack the conserved arginine residue at the corresponding position of the p - loop of the ptpase superfamily and thus the insufficient positive charge is thought to be compensated by their partner molecules ( lei et al . , 2009 ) . as mentioned above , the architecture of the core structure of eceiib is quite similar to the overall structure of eciib together with the conserved residues , cys10 and thr17 . when the core structural elements of eceiib are superimposed with those of eciib homologues ( fig . 3b ) , the rms deviations of aligned c atoms are 0.822 ( 25 c atoms of 1iib ) 0.929 ( 28 of 1tvm ) , 1.030 ( 32 of 1vkr ) and 1.253 ( 10 of 3czc ) , respectively . however , unlike eciib , eceiib homologues contain a conserved histidine his16 which may assist accommodating a negatively charged phosphoryl group . the spatial position occupied by his16 in the p - like loop is almost equivalent to that of the functional arginine of the p - loop of the ptpase superfamily ( fig . consequently , the eceiib family has a unique consensus sequence of cxxgxaht at the p - like loop . interestingly , the crystal structures of the nucleotide binding subunit b of a1a0 atp synthase ( sankhala et al . , 2014 ) and human pir1 ( tadwal et al . , 2012 ) revealed that they also contain a catalytic histidine residue in their p - loops which interacts with a phosphate moiety . in summary though the overall fold is quite similar to different iib enzymes and other fold families , eceiib homologues still consist of an independent subfamily due to their unique topological connection . based on the sequence alignment and structural comparison of its homologues , a unique motif cxxgxaht comprising a p - loop like architecture therefore , we proposed that the conserved cysteine on this loop may be deprotonated to act as a nucleophile to transfer phosphoryl moiety to a specific carbohydrate . the bigger size of eceiib homologues together with the presence of extra catalytic histidine over eciib suggests that though their core molecular function is similar , their biological partners and substrates may be different . a further study is going on to find a biological partner and substrate of eceiib .

approximately 2040% of diabetic patients develop nephropathy which is the leading cause of esrd in developed countries . the ace i / d polymorphism is thought to be a marker for functional polymorphism which regulates circulating and tissue ace activity . while the initial study found a protective effect of the ii genotype on the development of nephropathy in iddm patients , subsequent studies have addressed the role of ace i / d polymorphism in the development and progression of diabetic nephropathy . raas blockers are the first line drugs for the treatment hypertension associated with diabetes and have been widely used in everyday clinical practice for the purpose of reducing proteinuria in patients with various renal diseases . however , the antiproteinuric effect of raas blockers is variable and the percentage of reducing proteinuria is in the range of 2080% . the antiproteinuric effect of raas blockers may be related to a number of factors : the type or the dose of raas blockers , the duration of therapy , the level of sodium intake , and the type of patient 's ace i / d genotype . besides the nongenetic factors , drug responses , can be influenced by ace gene polymorphism . in this review , we discuss the relationship between ace i / d polymorphism and diabetic nephropathy and therapeutic response of raas blockers .

chronic kidney disease ( ckd ) is a worldwide public health problem that affects millions of people from all over the world . diabetic nephropathy is a common complication in patients with diabetes and the leading cause of end - stage renal disease ( esrd ) . over the last decade , the prevalence of diabetes has increased worldwide , as a result of the continuous rise in type 2 diabetes incidence . the pathogenesis of this drastic complication is not clearly understood , but available data suggests that multiple factors such as hemodynamic alterations , metabolic abnormalities , various growth factors , and genetic factors contribute to the pathogenesis of diabetic nephropathy . in experimental and human diabetic nephropathy , these hemodynamic changes may be explained in part by alterations in the renin - angiotensin system . consequently , genes involved in the renin - angiotensin system have been suggested as potential genetic predispositions for the development of diabetic nephropathy . many previous publications suggest that genetic predisposition plays a role in the development of diabetic nephropathy which clusters within families , both in type 1 ( iddm ) and type 2 ( niddm ) diabetes mellitus [ 47 ] . dna polymorphism , so far , is known to exist for the great majority of human genes . in diabetes mellitus , several models can be figured out to represent different concepts of the pathogenesis of diabetic nephropathy and to incorporate genetic factors . angiotensin - i converting enzyme ( ace ) is one of the key enzymes in the renin - angiotensin - aldosterone system ( raas ) and the insertion ( i)/deletion ( d ) polymorphism of this gene has been studied extensively with renal and cardiovascular complications of diabetic nephropathy . the ace , which was originally discovered in equine plasma , is a membrane - bound dipeptidyl carboxypeptidase ectoenzyme located in the endothelial lining of blood vessels throughout the body where it plays an important role in proliferation of vascular smooth muscle cells through the conversion of angiotensin - i to angiotensin ii and bradykinin inactivation . the ace is found as a membrane - bound enzyme in endothelial cells and different types of epithelial and neuroepithelial cells as well as in circulating form in biological fluids , such as plasma , cerebrospinal fluid , amniotic fluid , and seminal fluids [ 11 , 12 ] . the mechanisms that lead to the biosynthesis of the circulating form of ace are unclear , but available data indicates that its structure is very similar to that of the cellular form . the circulating form is virtually identical to the cellular form except for the lack of the transmembrane and intracellular sequence but whether it arises from specific proteolytic cleavage from the cell surface or nonspecifically from senescent cells has yet to be determined . ace genes have been cloned in the human , mouse , and rabbit and the enzyme was the product of one gene in man [ 12 , 13 ] . most somatic forms of ace appear to be transcribed from all exons except exon 13 which encodes the unique n - terminal region of the testicular form . cloning of the ace gene had made it possible to identify a 287 bp insertion / deletion polymorphism in intron 16 ( ace i / d polymorphism ) that appears to affect the level of serum ace activity . the genotype with deletion of the 287 base pair resulted in higher plasma ace levels . the plasma ace was predominantly derived from tissue vascular endothelial cells suggesting that patients with the dd genotype might have higher tissue angiotensin ii . angiotensin ii might modulate the growth of smooth muscle cell and induce myointimal hyperplasia after endothelial injury , and administration of ace inhibitors prevented myointimal proliferation after vascular injury . plasma ace concentrations are stable when measured repeatedly in a normal subject but large interindividual variations make it difficult to interpret plasma ace levels in a given patient . the study conducted in a large sample of healthy families showed an intrafamilial resemblance between ace levels and also suggested that they were subject to the effect of major gene . the ace i / d polymorphism accounts for over 40% of interindividual variability of serum or tissue ace activity . patients homozygous for the deletion had the greatest serum ace activity , whereas those homozygous for the insertion had the lowest level . an insertion / deletion ( i / d ) polymorphism of intron 16 of the angiotensin - i converting enzyme ( ace ) gene is largely responsible for variations in plasma ace levels and for explaining approximately 50% of the variability in serum ace activity observed . using the linkage - segregation analysis of the plasma ace , cambien has shown that the ace i / d polymorphism was a marker for an unknown functional polymorphism ( ace s / s ) which appeared to be a new independent risk factor for myocardial infarction . the ace i / d polymorphism is thought to be a marker for functional polymorphism which regulates circulating and tissue ace activity [ 14 , 18 , 19 ] . since 1990 , its association with diabetic nephropathy has been extensively investigated and more than 300 studies have explored genetic associations of this polymorphism in more than 100 conditions including diabetic nephropathy . up to now , the i / d polymorphism of ace gene is the most extensively studied marker for association with diabetic nephropathy . while the initial study by marre et al . found a protective effect of the ii genotype on the development of nephropathy in iddm patients , subsequent studies have yielded inconsistent results . over the past decades , many studies have been conducted to examine the association between ace i / d polymorphism and nephropathy in type 1 and type 2 diabetes , but the results still remain inconsistent . however , recently conducted meta - analysis showed consistent association between ace d allele or dd genotype and esrd risk in diabetic nephropathy patients . in 2005 , ng et al . reported a meta - analysis of 14727 diabetic patients including 47 studies ( 8,663 cases , 6,064 controls ) from 1994 to 2004 . they confirm a statistically significant protective role of the ii genotype in the development of diabetic nephropathy ; the effect was most pronounced in asians with type 2 diabetes , followed by caucasians with types 1 and 2 diabetes . they suggested that these findings may have implications for the management of diabetic nephropathy using ace inhibitors especially among type 2 diabetic asians . in 2012 , they conducted a comprehensive meta - analysis on 63 published studies from 1994 to 2010 with 14,108 cases and 12,472 controls relating variants of the ace i / d polymorphism to the risk of developing diabetic nephropathy . they included all of the studies that determined the genotype distribution of ace i / d polymorphism in cases with type 1 or type 2 diabetes and nephropathy and in diseased controls or in healthy controls . the overall analysis showed a significant association between the ace i / d polymorphism and the risk of diabetic nephropathy for all genetic models ( i d versus ii : or = 1.12 , 95% ci 1.021.24 ; dd versus ii : or = 1.27 , 95% ci 1.131.44 ; allele contrast : or = 1.15 , 95% ci 1.081.23 ; dominant model : or = 1.18 , 95% ci 1.071.31 ; and recessive model : or = 1.18 , 95% ci 1.081.30 , resp . ) . in stratified analysis by ethnicity and diabetes mellitus type , they further found that the asian group with type 2 dm showed a significant association for all genetic models ( i d versus ii : or = 1.25 , 95% ci 1.071.47 ; dd versus ii : or = 1.57 , 95% ci 1.241.98 ; allele contrast : or = 1.30 , 95% ci 1.151.46 ; dominant model : or = 1.37 , 95% ci 1.101.69 ; and recessive model : or = 1.34 , 95% ci 1.151.56 , resp . ) . however , they failed to find any significant effects for different genetic models in other subgroups . the authors suggested that the ace i / d polymorphism may contribute to nephropathy development , especially in the asian group with type 2 diabetes mellitus . in 1996 , yoshida et al . including 168 japanese patients with niddm followed over 10 years have shown that ace dd genotype has a high prognostic value for progressive deterioration of renal function . they also showed that patients with stable renal function had no significant difference in the distribution of ace genotype between patients with albuminuria and patients without albuminuria . analysis of the clinical course of the three ace genotypes revealed that the majority ( 95% ) of patients with the dd genotype who had albuminuria progressed to end - stage renal disease within 10 years of diagnosis of diabetes . moreover , the ace dd genotype had the increased mortality in patients on dialysis and the prevalence of the dd genotype in patients on chronic dialysis decreases year by year . next year , schmidt et al . reported 658 caucasian patients with type ii diabetes , 347 without diabetic nephropathy and 311 with various stages of diabetic nephropathy , and determined the i / d polymorphism of the ace gene . they compared patients at the extremes of renal risk , that is , normotensive patients without antihypertensive treatment and without nephropathy ( n = 144 ) versus patients on dialysis ( n = 61 ) , differed with respect to genotype ( dd 36.8% versus 57.4% ; p = 0.007 ) and allele frequencies ( d 0.59 versus 0.76 ; p < 0.001 ) . in this study , patients with the highest renal risk more frequently had the dd genotype . this would be compatible with a greater risk of ( or rate of ) progression to end - stage renal failure . also compared the ace genotype distributions in 79 caucasian iddm patients with esrd and 82 control patients without microalbuminuria after fifteen years of iddm . the ace genotype distribution in patients was not in accordance with the hardy - weinberg equilibrium due to a significant overrepresentation of the dd genotype ( = 8.9 , p = 0.01 ) . the presence of the dd genotype increased the risk of end - stage renal failure two - fold compared to the other genotypes ( odds ratio 2.1 , 95% ci 1.14.0 ) . they concluded that the risk of end - stage renal failure in patients with iddm is two - fold increased in patients with the dd genotype as compared to patients with other genotypes . we also studied the impact of insertion / deletion ( i / d ) genotypes on the progression of diabetic nephropathy in 239 korean patients with type 2 diabetes ( group 1 , 99 patients with stable renal function ; group 2 , 140 patients with declining renal function ) . the frequency of the dd genotype was significantly greater in group 2 compared with group 1 ( 30.7% versus 9.1% ; p < 0.05 ) . patients with the dd genotype reached the end point ( serum creatinine > 2.0 mg / dl [ 176.8 micromol / l ] ) faster than those with the other genotypes ( dd , 11.38 4.08 years ; i d , 13.85 4.04 years ; ii , 14.04 4.06 years , resp . , p < 0.05 ) and took significantly less time to reach dialysis therapy ( dd , 13.10 4.45 years ; i d , 16.21 4.74 years ; ii , 15.13 4.09 years , resp . in multiple logistic regression analysis , systolic blood pressure and dd genotype showed significant correlations with the progression of diabetic nephropathy . in patients with the dd genotype , the odds ratio was 3.881 ( 95% confidence interval , 1.564 approximately 9.628 ; p = 0.003 ) compared with that with the ii genotype . we suggested that the ace dd genotype might be a significant risk factor for the progression of diabetic nephropathy . recently , yu et al . published meta - analysis of 12 previous studies containing 4,015 diabetic nephropathy patients ( 981 cases , 3,034 controls ) . in this study , they showed that in overall populations , there was a notable association between d allele or dd genotype and esrd susceptibility ( d : or 1.32 , 95% ci 1.111.56 , p = 0.002 ; dd : or 1.67 , 95% ci : 1.252.21 , p = 0.004 ) . in the subgroup analysis according to ethnicity , d allele or dd genotype was associated with esrd risk in asians . in caucasians , the association of dd genotype with esrd risk was observed , but the d allele was not . furthermore , ace i / d gene polymorphism was associated with esrd risk in patients with diabetic nephropathy due to type 2 dm , but the association was not found for patients with diabetic nephropathy due to type 1 dm . they concluded that d allele or dd genotype is associated with the esrd susceptibility in diabetic nephropathy patients . earlier studies repeatedly showed that patients with dd genotype or d allele have elevated circulating and tissue ace activity [ 14 , 19 ] compared to patients with i allele . this may contribute to the interindividual variability in the antiproteinuric responses to inhibition of the raas using either ace inhibitors ( aceis ) or angiotensin ii type 1 receptor blockers ( arbs ) . raas blockers are the first - line drugs for the treatment of hypertension associated with diabetes in the fact that these drugs not only lower systemic blood pressure but also reduce intraglomerular pressure . imanishi et al . showed that the mechanism by which an ace inhibitor caused a short - term decrease in albuminuria in early diabetic nephropathy involved a glomerular hemodynamic change , namely , a decrease in intraglomerular capillary pressure . these drugs have greatly improved the renal prognosis and survival of diabetic patients with nephropathy over the last decade . however , despite therapy targeting elevated blood pressure , albuminuria , hyperglycemia , and lipid abnormalities , patients with diabetic nephropathy still on average have a rate of decline in renal function three to six times that seen in individuals without renal disease [ 31 , 32 ] . consequently , diabetic nephropathy is still one of the principal causes of end - stage renal disease , leading to dialysis and death in developed countries . although current therapeutic strategies have alleviated the huge burden of diabetic nephropathy , many patients still progress to esrd . one reason for the inadequacy of current antiproteinuric ( renoprotective ) therapy and the persistent poor renal prognosis is the large interindividual variation in response to first - line therapy including antihypertensive drugs blocking the renin - angiotensin - aldosterone system . furthermore , overt proteinuria seen in diabetic nephropathy is itself a risk factor that may adversely affect renal function , and it is associated with a faster rate of renal disease progression . therefore , the reduction of proteinuria is an important tool for retarding the progression of renal disease in diabetic nephropathy patients . traditionally , ace inhibitors ( aceis ) and angiotensin ii type 1 receptor blockers ( arbs ) have been widely used in everyday clinical practice of nephrology for the purpose of reducing proteinuria in patients with various renal diseases including diabetes mellitus . however , the antiproteinuric effect of ace inhibitors on proteinuria is variable and the percentage of reducing proteinuria is in the range of 2080% in a variety of renal diseases [ 3537 ] . the antiproteinuric effect of ace inhibitors and/or angiotensin ii type 1 receptor blockers may be related to a number of factors : the type or dose of the raas blockers , the duration of therapy , the level of sodium intake , and the type of patient 's ace genotype [ 29 , 3840 ] . the antiproteinuric mechanisms of raas blockers are thought to decrease intraglomerular capillary pressure by reducing both afferent and efferent renal arteriolar resistance , predominantly dilating efferent arteriole and systemic blood pressure [ 41 , 42 ] . as demonstrated in previous studies , raas blockade has been superior to other antihypertensive agents in reducing albuminuria and slowing rate of decline in gfr despite similar blood pressure controls . besides the nongenetic factors , the basic concept of pharmacogenomics is a drug interacting with its target , for instance , an enzyme or a receptor . when genetic polymorphism leads to modified target availability or function , the drug response is modified as well . it has been reported that ace i / d genotypes appeared to be a major determinant of plasma and tissue ace activities [ 14 , 18 , 19 ] . individuals with the dd genotype have the greatest and those with ii genotype have the least ace concentrations . so , it is expected that the differences in plasma and tissue ace activities associated with ace i / d genotype might affect the antiproteinuric ( renoprotective ) responses to raas inhibition . i / d polymorphism in diabetic nephropathy were reported so far [ 34 , 4453 ] . however , the antiproteinuric ( renoprotective ) effect of raas blockers and ace i / d genotype in diabetic nephropathy is inconclusive . these results may be due to incomplete blockade of raas by suboptimal doses and/or compensatory mechanisms occurring during long - term treatment with raas blockers . after initiation of acei treatment , angiotensin ii level in plasma is lowered initially . however , during long - term treatment of acei , angiotensin ii level tends to increase as a result of ace escape and angiotensin ii generation through non - ace dependent pathways such as chymase or other serine proteases . incomplete raas blockade during chronic acei therapy may be overcome by inhibiting the action of angiotensin ii at the site of the angiotensin ii type 1 receptor by an arb . on the contrary , in treatment with arb , there also tends to be a compensatory increase in renin and angiotensin ii levels , thereby increasing the competition at the angiotensin ii type 1 receptor site . so , in this situation , we can reduce compensatory increased angiotensin ii by adding acei . several previous studies suggested that ace genotype may predict the response of patients to antiproteinuric and renoprotective effect with ace inhibitors ( aceis ) . , in their observational followup study of type 1 diabetic patients with diabetic nephropathy receiving ace inhibitor captopril ( n = 35 and n = 169 ) , have reported that the dd genotype reduces the long - term beneficial effect of ace inhibition on the progression of diabetic nephropathy in patients with iddm . jacobsen et al . tested the potential role of ace i / d polymorphism on the early antiproteinuric responsiveness in an observational followup study with sixty young hypertensive type 1 dm nephropathy patients . they showed more albuminuria reduction with captopril therapy in ii genotype than in i d or in dd genotypes . data from a eurodiab randomized controlled trial lisinopril in iddm showed that the i / d polymorphism of the ace gene modulates the therapeutic effect of ace inhibition on the progression of urinary albumin excretion in iddm patients . patients with the ii genotype showed the fastest rate of aer progression on placebo but had an enhanced response to lisinopril . albumin excretion rate ( aer ) at 2 years ( adjusted for baseline aer ) was 51.3% lower on lisinopril than placebo in the ii genotype patients ( 95% ci , 15.7 to 71.8 ; p = 0.01 ) , 14.8% lower in the i d group ( 7.8 to 32.7 ; p = 0.2 ) , and 7.7% lower in the dd group ( 36.6 to 37.6 ; p = 0.7 ) . only in the ii group the difference was statistically significant even after adjustment for gender , baseline and followup bp , and metabolic control . the authors concluded that knowledge of ace genotype may be of value in determining the likely impact of ace inhibitor treatment on the albuminuria reduction . , which included 169 iddm patients with overt nephropathy , showed that the d allele of the ace i / d polymorphism in addition to nongenetic risk factors independently accelerated progression of diabetic nephropathy during ace inhibition . also , in this study of patients with type 1 diabetes , the i allele was associated with a slower progression to doubling of serum creatinine or esrd . taken together , in type 1 diabetes with nephropathy , the i allele increases the responsiveness to the antiproteinuric ( renoprotective ) effect of ace inhibitor therapy . data in type 2 dm nephropathy in relation to ace i / d polymorphism and antiproteinuric ( renoprotective ) effect of ace inhibitors are scarce . our group investigated the antiproteinuric effect of ace inhibition ( benazepril 10 mg / day or perindopril 4 mg / day ) in relation to ace i / d polymorphism in a short - term observational followup study in 83 niddm patients with overt nephropathy ( urinary protein excretion over 500 mg / day ) classified into three groups in accordance with ace genotypes ( 17 dd ; 33 i d ; 33 ii ) and prospectively followed up for 3 months . before entry , previously used ace inhibitors were withdrawn for at least 2 weeks and baseline proteinuria was measured . our study showed that the percentage reduction in proteinuria for dd genotype was significantly higher than in i d and in ii genotypes ( 50.9 19.2% versus 19.2 16.0% , 20.2 20.4% , p < 0.05 ) . there were no statistically significant correlations between the levels of baseline proteinuria and the magnitudes of the reduction of proteinuria under ace inhibition ( p > 0.05 ) . these results indicated that ace dd genotype is more susceptible than ace ii and i d genotypes to the antiproteinuric effect of ace inhibitors . they found that , in 2089 chinese patients with normoalbuminuria , microalbuminuria , or macroalbuminuria over a median period of 44.6 months , ace inhibitor therapy decreased mortality and renal end point which was defined as death due to renal failure , dialysis , egfr of < 15 ml / min/1.73 m or more than 50% loss of egfr , more effectively in i allele carriers than in dd . this study was the only study in type 2 diabetes with adequate power and followup . however , we have to be cautious in interpreting this finding because the favorable effects of the i allele were restricted to those with normoalbuminuria or microalbuminuria . another study regarding raas gene polymorphisms and renal responsiveness to raas inhibition in type 2 diabetic asian indians they enrolled 810 north indian type 2 diabetics treated with ace inhibitor or arb and were followed up for 3 years . they observed that the ace ii genotype and cumulative genetic risk score of < 1 was associated with better renoprotective response to ace inhibitor in type 2 dm with normoalbuminuria . however , there was no significant difference in renoprotective effect in type 2 diabetics with nephropathy based on ace i / d genotypes with 3-year ace inhibitor therapy ( table 1 ) . data on angiotensin ii type 1 receptor blocker ( arb ) in relation to ace i / d polymorphism and antiproteinuric ( renoprotective ) effects in type 1 diabetes with nephropathy are also scarce . they showed that the antiproteinuric effect of 36- and 4-month arb ( losartan ) had similar beneficial renoprotective and antiproteinuric effect in 54 hypertensive white type 1 dm nephropathy patients with ace ii and dd genotypes . same result was reported by haneda et al . in 127 japanese type 2 dm nephropathy patients . they found that arb ( candesartan ) is useful in reducing proteinuria in type 2 dm nephropathy patients compared with placebo and seems to be effective in subjects with both the ii and dd genotypes of the ace gene . more data on the interactions between ace i / d polymorphism and arb therapy have been provided by analyses of the reduction of endpoints in niddm with the aii antagonist losartan ( renaal ) study , a double blind , multicenter , prospective , randomized , and placebo controlled clinical trial designed to evaluate the renal effects of losartan in 1513 type 2 diabetic patients with overt nephropathy . ace i / d data were available in 1435 of the 1513 renaal study patients . available ace i / d data showed that compared with placebo losartan reduced the risk of reaching the composite end point of doubling of serum creatinine , esrd , or death by 5.8% , 17.6% , and 27.9% among those with the ii , i d , and dd genotypes , respectively . this study demonstrated that the deletion allele of the ace gene had a harmful impact on the composite endpoint . the beneficial effects of losartan were greatest in the ace dd genotype group and intermediate in the ace i d genotype group for nearly all endpoints . the novel clinical importance of this study is that those patients who have the greatest need for renoprotective treatment have the best effect of losartan ( dd and i d genotype ) , whereas those patients with a better renal prognosis ( ii genotype ) also derived renal benefit . first , this is the largest trial evaluating the association of the ace / id polymorphism on renal outcome and death during angiotensin ii - receptor blockade in diabetic nephropathy ; the study may well be underpowered in relation to the individual components of composite endpoint . second , the i d alleles were not in hardy - weinberg equilibrium in the black patients . lastly , despite randomization , higher baseline proteinuria was present in the ii genotype in the losartan group . in spite of the several limitations of this study , the deletion allele of the ace i / d polymorphism showed unfavorable renal prognosis in patients with proteinuric type 2 diabetes , which can be improved by losartan treatment . another recent study by cheema et al . also reported that , in type 2 diabetics with nephropathy , ace dd genotype and a genetic risk score of > 6 were associated with better renoprotective response to arb . they suggested that ace i / d genotypes individually and in interaction with other ras single nucleotide polymorphisms ( angiotensinogen and angiotensin ii type 1 receptor gene ) modulate renoprotective efficacy of ace inhibitor and arb in type 2 diabetics depending on the status of proteinuria ( table 2 ) . the association between the raas activation and the development and progression of diabetic nephropathy has been known for a long period of time . ace is the key enzyme of the raas - cascade that plays a central role of blood pressure regulation and volume homeostasis in the body . despite the large amount of studies looking for candidate genes , the ace i / d polymorphism remains the unique and well - characterized locus clearly associated with development and progression of diabetic nephropathy . , numerous studies have addressed the role of ace i / d polymorphism in the development and progression of diabetic nephropathy . data reported so far showed that ( 1 ) the ace i / d polymorphism directly influences circulating levels of ace , ( 2 ) the ii genotype protects against the development of diabetic nephropathy , ( 3 ) the dd genotype predicts poor renal response to raas inhibitors ( the current strategy of raas inhibition in patients with the dd genotype may be insufficient to block an activated raas ) , and ( 4 ) angiotensin ii type 1 receptor blockers ( arbs ) can ameliorate the adverse effect of the d allele ( no difference between genotypes is observed when patients are treated with an arb ) . evaluating the ace i / d polymorphism in diabetic nephropathy is a reliable tool to identify diabetic patients at risk and identify patients who may benefit from antiproteinuric ( renoprotective ) therapy with ace inhibitors and/or arbs . this may guide pharmacologic therapy in individual patients and help to identify the patients with more aggressive use of raas blockers such as supramaximal dose of individual raas blocker and double or triple blockade of the raas . in case of an insufficient response to raas blockers , we also consider other treatment strategies such as glycemic control , low salt intake , more aggressive proteinuria reduction strategy , and hypercholesterolemia control that halt progression of diabetic nephropathy .

objectives : non - melanoma skin cancer is the most common malignancy in us , with an annual incidence of in excess of 1.5 million cases . in the majority of cases , locoregional treatment is curative and systemic therapy is not indicated . platinum - based chemotherapy regimens have been used most commonly in refractory cases . the use of cetuximab , a monoclonal antibody targeting epidermal growth factor receptor [ egfr ] , has been reported for skin cancer treatment . this current study evaluated eight cases of locally advanced and refractory basal cell or squamous cell cancers which were treated with cetuximab.methods : this is a retrospective study on eight patients who had received cetuximab for treatment of cutaneous carcinoma since 2007 at southern illinois university school of medicine ( siu - som ) medical oncology clinic.results : three of the four patients with basal cell carcinoma and two of the four patients with squamous cell carcinoma maintained remission on treatment .. the main side effect was acneiform rash which required termination of treatment for one patient and dose reduction in another.conclusion : the study indicates that cetuximab may have a beneficial role for patients with non - melanoma cutaneous carcinomas that are refractory to standard therapy .

as per the united nations environment programme ( unep ) , global incidence of non - melanoma skin cancer exceeds 2 million each year 2 . with a 10% decrease in stratospheric ozone , seventy - five percent of the non - melanoma skin cancers are basal cell carcinomas ( bcc ) , which are usually effectively treated with excision . treatment options for scc vary depending on the severity of the disease , but surgical excision alone is generally adequate treatment . however , for high risk scc patients , which include those with inadequately excised and recurrent lesions , lymph node metastasis , and immunosuppression , the current treatment recommendation is combined surgery and radiation 3 . patients with high risk cutaneous scc includes those with inadequately excised and recurrent lesions , lymph node metastasis , and immunosuppression 3 . at the molecular level , over expression of egfr is considered a predictive factor for identification of high - risk scc 3 . the combination of radiation treatment and surgery has reduced the rate of locoregional relapse by 20 - 25% 4 . as per porceddu et al , patients with high - risk pathologic features such as multiple nodes , extra nodal spread , positive margins , and perineural and vascular invasion will benefit from concurrent platinum - based chemotherapy and radiation treatment 5 . but , this is frequently a patient population with advanced age and multiple co - morbidities , in which platinum - based regimens are least tolerated . cetuximab is a chimeric human / murine monoclonal antibody that binds competitively to egfr and prevents activation of the receptor 6 . considering the low side effect profile , interest in cetuximab as a treatment for non - melanoma skin cancer is growing area . most adverse skin reactions expected with this treatment are cutaneous and include acneiform eruption , xerosis , paronychia , hair changes , telangectasia and hyperpigmentation . the most common adverse effect is acneiform rash which is seen in 50 - 100% of patients 7 . this is assumed to be due to interference with the physiological role of egfr in the epidermis . there are reported cases of severe acneiform rash , mucositis and dermatitis in people treated with combined radiation and cetuximab 8;9 . there is also one reported case of toxic epidermal necrolysis caused by cetuximab plus minocycline in a patient receiving chemoradiation for recurrent squamous cell carcinoma of head and neck which evolved in the 5th week of treatment with cetuximab 10 . here , we evaluate eight cases of advanced squamous cell carcinoma or basal cell carcinoma patients treated with cetuximab . among the eight patients , four were basal cell carcinoma and four were squamous cell carcinoma . a retrospective analysis was performed using the records of the siu - s between the years 2007 - 2011 . local institutional board approval ( exempt approval on 01/21/2010 ) eight patients were identified who had received cetuximab for treatment of skin carcinoma . collected from the case records were demographic data including age , co - morbidities , and family history . tumor characteristics included histology , stage and treatment factors included previous interventions , dose and frequency of cetuximab , and adverse effects of cetuximab . assessment of disease status was based on physical examination documented in the clinic notes and imaging studies . complete and partial remission ( cr & pr ) has been determined based on recist criteria except the fact that patients may or may not been continuing the treatment during that time 11 . among the eight patients analyzed , four had basal cell carcinoma and four had squamous cell carcinoma . three of the four patients with bcc had gorlin syndrome with a significant family history of basal cell carcinoma . the age group of these patients varied from 45 - 87 , including three above the age of 80 . all of the octogenarians had multiple medical problems and required multiple surgeries and excisions of basal cell carcinomas that had resulted in deformities in the head and neck area . except for one scc patient who had received carboplatin and docetaxel chemotherapy , the fourth bcc patient , without a history of gorlin syndrome , had an aggressive basal cell carcinoma that warranted numerous resections over several years as well as radiation therapy . he ultimately developed regional lymph node metastasis and was referred for further management of his progressive disease that was considered refractory to any further radiation or surgery . four patients , aged 38 to 89 , had recurrent squamous cell carcinoma with a history of multiple recurrences in the past . one was on mycophenolate and presented with a right temple lesion that recurred four times . it was treated initially with surgery and combined cetuximab and radiation for the second recurrence . the third and fourth recurrences were also treated with surgery . the other patient presented with a metastatic squamous cell carcinoma of unknown primary with mediastinal adenopathy , neck mass and pulmonary nodules . he failed carboplatin/ docetaxel chemotherapy and developed progressive disease with a pericardial effusion , necessitating a pericardial window . his transplanted kidney was surgically removed , and all of his immunosuppression was stopped . for five out of the 8 patients , one of the patients with bcc received cetuximab 250 mg / m once a week , and the latter dose was subsequently escalated to 300 mg / m . the starting dose of cetuximab for 2 of the bcc patients was 125 mg /m once a month . the dose was gradually increased up to 250 mg / mfor one of the patients . one of the scc patients started with cetuximab 250 mg / m2 once a week for eight weeks as induction treatment . the same patient continued maintenance treatment for two and half years as cycles of once a week treatment for four weeks with two weeks off . five out of 8 patients ( 63% ) experienced grade ii acneiform skin rash as a side effect , which was usually noted after 2 - 3 treatments . one patient was treated with steroids and minocycline because the rash recurred after each treatment . another patient required premature termination of cetuximab due to persistent skin rash despite treatment with steroids and minocycline . this latter patient was subsequently able to tolerate cetuximab with a 50% dose reduction when it was restarted for disease recurrence . for the remaining three patients , the grade ii skin rash resolved with 2 - 3 weeks of minocycline treatment . this patient later developed grade ii skin induration and grade iii skin pain after two and half years of treatment and stopped therapy . they were treated with magnesium intravenously ( iv ) with each treatment . among the four patients with bcc , fifty percent attained complete remission ( cr ) and three patients relapsed after stopping the treatment ( 2 cr and 1 pr ) and currently have been placed back on cetuximab . one patient remains on maintenance treatment for the last 12 months after attaining an initial partial response . another patient had a local recurrence of the disease after a four - month hiatus from treatment and now remains on maintenance treatment . the third bcc patient had two recurrences after the first treatment with cetuximab , which had to be discontinued after four cycles secondary to skin rash . the first recurrence occurred two months after discontinuing cetuximab but responded to reinstitution of treatment for seven months . the second recurrence occurred after five months off therapy , but remission was obtained again with the reinitiation of cetuximab , and the patient has been on maintenance treatment for the last seven months . the fourth patient , who had a partial response with cetuximab , had a multifocal recurrence soon after being off treatment . he was then started on a multidrug chemotherapy regimen with 5-flurouracil , carboplatin , and cetuximab . he achieved a near complete response to three cycles of chemotherapy but therapy was stopped due to his advanced age and performance status . his disease recurred several months later , and he chose to receive supportive care only at that point . median disease - free survival was 1 month . with a median follow up of 12 months , overall survival was 100% . one relapsed at 6 months following active treatment , and the other two have been in remission for the past 3 years . the latter two patients had a renal transplant and had previously been on mycophenolate or other immunosuppression . the patient who had a partial response died secondary to other co - morbidities before any further follow - up . median follow - up and disease- free survival were 32.5 months and 20.5 months , respectively . this study shows that recurrent non - melanoma skin cancers can be successfully treated with extended cetuximab therapy . 62.5 percent of the study patients obtained a complete remission and a major response ( cr and pr ) was achieved in all of the patients . however , among those whose treatment was held , 63% of the patients subsequently relapsed . for the three patients who had a sustained remission , two have continued the maintenance treatment , and the other one has stopped . 63% and 25% of patients had grade ii skin rash and grade 1 hypomagnesemia , respectively . the skin induration and skin pain experienced by one patient were considered to be unrelated adverse events . no other adverse events were noted , and no one developed grade iii or iv toxicities with treatment . epidermal growth factor receptor [ egfr ] is a member of tyrosine kinase growth factor receptor family . activation of this receptor results in intracellular tyrosine kinase autophosphorylation , which initiates a cascade of intracellular events ultimately leading to cell cycle progression , angiogenesis , metastasis and reduced apoptosis 12 . egfr is normally expressed in human cells , but higher levels of expression have been shown in many malignancies . as per krahn , 80% of scc and 57% of bcc express egfr 13 . as per previous studies , aberration in egfr pathways is associated with poor prognosis in many malignancies . even though bcc also expresses egfr , the key pathology in bcc is an aberration in the hedgehog pathway 14;15 . novel inhibitors of this pathway such as gdc-0449 are on trial but are not yet clinically approved 16 . it has been shown that egfr signaling acts synergistically with the hedgehog pathway in the malignant transformation of cells 17;18 . we were able to obtain either a cr or a pr for all of our patients . however , we had a significant relapse rate for patients when treatment was stopped , which emphasizes the importance of maintenance treatment for long - term disease control . even though median disease free survival was only one month , overall survival over the 30 month follow up period was 75% . all of these patients had refractory disease , and we were able to prolong their life with a relatively low toxicity treatment regimen . compared to currently approved chemotherapies for skin carcinomas , cetuximab is well tolerated . in our study , the most commonly experienced side effect was acneiform skin rash occurring in 63% of patents , which is consistent with a prior study 7 . all of skin rashes were grade ii in severity , which were treated successfully with steroids and minocycline . concurrent radiation treatment increases the incidence of serious adverse events as per the case reports that we discussed earlier . only one of our patients had received radiation treatment along with cetuximab treatment , and he subsequently responded to single agent cetuximab therapy along with immunosuppressant modulation . there have been isolated case reports of using cetuximab in recurrent non - melanoma skin cancers with favorable responses 19 - 22 . however , there have been no studies comparing efficacy of treatment with cetuximab versus currently approved chemotherapeutic agents for cutaneous malignancies . the results of the above reported cases are encouraging for the use of cetuximab in refractory non - melanoma skin cancers and suggest that a randomized phase iii study between chemotherapy and cetuximab in this condition may be useful .

aimsthe aim of this study was to analyze factors related to rejection of care and behaviors directed towards others in nursing home residents with dementia.methodsthe relationship of lack of understanding , depression , psychosis and pain with rejection of care and behaviors directed towards others was explored using four assessments from the minimum data set ( mds ) within a period of 15 months on 1,101 residents with dementia in dutch nursing homes . presence of depressive symptoms was ascertained using a validated mds scale , and presence of lack of understanding , rejection of care , psychosis and pain through the individual mds items . a structural equation modeling approach and latent growth models were used to investigate the longitudinal relationship between changes in rejection of care and physical or verbal behaviors directed towards others , and changes in lack of understanding , pain , depression and psychotic symptoms.resultschanges in lack of understanding predicted changes in rejection of care , and there was also a relationship between changes in depression and rejection of care . changes of behaviors directed towards others were related to changes in lack of understanding and depression . pain and behaviors directed towards others were unrelated , and psychosis was rather stable throughout . a mediation model suggested that the relationship of lack of understanding with behaviors directed towards others was mediated by rejection of care.conclusionthese results indicate that lack of understanding and depression are important factors in development of rejection of care and behaviors directed towards others . the relationship between lack of understanding and behaviors directed towards others is mediated by rejection of care . improvement in communication between residents and caregivers , and perhaps also effective treatment of depression may prevent or ameliorate these behaviors directed towards others .

development of apraxia and other cognitive deficits requires help of others with activities of daily living . unfortunately , the person with dementia sometimes does not cooperate with family or professional caregivers but actually resists care or exhibits behaviors during care that may be labeled abusive . these behaviors may be difficult to handle by family caregivers and may lead to institutionalization . in an institution , rejection of care and behaviors directed towards others may lead to injuries of the resident or staff and to staff burnout . a survey of nursing home physicians showed that the most common problem that they treated in residents with dementia was rejection of care . some of them investigated the effectiveness of pain control , others used personalized psychosocial interventions based on the unmet needs theory for treatment of agitation and found positive results . however , the problem with these studies is that they consider all behavioral symptoms to be agitation and do not differentiate between agitation and rejection of care . this is due to using the cohen - mansfield agitation inventory or neuropsychiatric inventory in which the item agitation / aggression is actually measuring rejection of care . similar problems are also present in drug trials investigating effects of antipsychotics and other medications . therefore , we decided to study specifically rejection of care that is a highly relevant problem in clinical practice . we have previously described strong correlations between rejection of care and lack of understanding , depression and psychotic symptoms . behaviors directed towards others were strongly correlated with rejection of care , depression and delusions . however , these findings were based only on cross - sectional data . in this study , we performed longitudinal analyses to further explore the relationships of these modifiable factors with rejection of care and behaviors directed towards others of nursing home residents . our hypothesis was that longitudinal data would support causal relationships between these modifiable factors and rejection of care . we used minimum data set - resident assessment instrument ( mds - rai ) data collected by 8 dutch nursing homes and 10 residential homes . we included the data of residents within a 12-month time window for each facility separately , resulting in a range from april 4 , 2007 , to december 1 , 2008 . of the records of 2,705 residents available from the 18 facilities , we selected the last records of 1,101 residents aged over 65 with alzheimer or other dementia ( i1q or u ) , who were dependent in decision making ( b4 not equal 0 ) and who were not comatose ( b1 equal 0 ) . four assessments from the mds within a period of 15 months were used for the analyses . the terminology was changed by the introduction of mds 3.0 , which uses rejection of care instead of resist care. other new , but equivalent , items in mds 3.0 are physical behavioral symptoms directed towards others instead of physical abuse and verbal behavioral symptoms directed towards others instead of verbal abuse . presence of rejection of care and behavioral symptoms directed towards others was obtained from mds 2.0 section e with choices from not exhibited to daily. the variables physical behavioral symptoms directed towards others ( e4ca ) and verbal behavioral symptoms directed towards others ( e4ba ) were combined into one variable called behaviors directed towards others. ability to understand ( c6 ) was rated as sometimes and rarely / never. psychotic symptoms [ delusions ( j1e ) and hallucinations ( j1i ) ] were combined into one variable , psychosis , whose values are number of symptoms . clinical diagnosis of depression was obtained from mds item i1ee , and depression symptoms were assessed by the validated depression rating scale using other mds items : negative statements ( e1a ) , anger ( e1d ) , unrealistic fears ( e1f ) , repetitive health complaints ( e1i ) , sad expression ( e1i ) and crying ( e1 m ; score 0 - 14 ) . this scale correlated well with the cornell and hamilton depression scales using at least mild depression as a cutoff point , and with psychiatric diagnosis . it was also more sensitive and specific than the 15-item geriatric depression scale in detecting depression in the nursing home population . data about presence of pain were obtained from the mds item j2a with options of no , less than daily and daily. demographic data were also obtained from the mds . we investigated the longitudinal relationship between changes in rejection of care and behaviors directed towards others , and changes in lack of understanding , pain , depression and psychotic symptoms with the structural equation modeling approach . within this approach , we used latent growth models , bivariate latent growth models and longitudinal latent growth mediation models . since the studied variables were ordinal , we used latent growth models for categorical variables to study change within each variable . this model assumes the existence of latent or nonobserved trajectories , which are observed only indirectly through the repeated measures . the intercept represents the initial level at the beginning of the study , and the slope represents the rate of change . the factor loadings for the slope represent the measurement points , and they were set to the values 0 , 3 , 6 and 9 that represent the months at which the measurements were taken . second , we used bivariate latent growth curve models to study the relationships between rejection of care and understanding , and rejection of care and depression . these models combine two latent growth curve models that describe two separate longitudinal processes , and study the relationship between the initial level of the first process ( y , e.g. rejection of care ) with the initial level and rate of change in the second process ( x , e.g. lack of understanding ) , and the relationship between the rate of change in the first process with the initial level and rate of change in the second process ( fig . 1 , process y and x ) . bivariate latent growth curve models were also used to describe the relationship between behavioral symptoms directed toward others with rejection of care and behavioral symptoms directed towards others with understanding , and depression . frequency distributions of the variables psychotic symptoms and presence of pain remained stable through the whole measurement period , and the latent growth models showed no trend . therefore , we did not use bivariate latent growth models but latent growth models that included these variables as time - dependent covariates . in the model for psychosis symptoms , we included only the measurements of the first and the fourth psychosis symptom because of the high correlation between the first and the second measurement , and the third and the fourth measurement . finally , longitudinal mediation models were used to study whether rejection of care mediated the relationship between lack of understanding and behaviors directed towards others , and whether it mediated the relationship between depression and behaviors directed towards others ( fig . this means that first changes in lack of understanding ( depression ) occurred , and they promoted changes in rejection of care . next , changes in behaviors directed towards others occurred as a consequence of changes in rejection of care . three sets of latent growth factors were specified , one set for the independent variable , one set for the mediator , and , finally , another set for the dependent variable . the mediation model examined whether the growth in the independent variable affected the growth trajectory of the mediating variable which , in turn , affected the growth trajectory of the dependent variable . the value , the root mean square error of approximation ( rmsea ) , the comparative fit index ( cfi ) , and the tucker - lewis ( tli ) index were used to evaluate the model . a model achieves a good fit if the rmsea is lower than 0.05 , and fit is acceptable for values between 0.05 and 0.09 . we used minimum data set - resident assessment instrument ( mds - rai ) data collected by 8 dutch nursing homes and 10 residential homes . we included the data of residents within a 12-month time window for each facility separately , resulting in a range from april 4 , 2007 , to december 1 , 2008 . of the records of 2,705 residents available from the 18 facilities , we selected the last records of 1,101 residents aged over 65 with alzheimer or other dementia ( i1q or u ) , who were dependent in decision making ( b4 not equal 0 ) and who were not comatose ( b1 equal 0 ) . four assessments from the mds within a period of 15 months were used for the analyses . for rejection of care , we used the mds 2.0 item resist care ( e4ea ) . the terminology was changed by the introduction of mds 3.0 , which uses rejection of care instead of resist care. other new , but equivalent , items in mds 3.0 are physical behavioral symptoms directed towards others instead of physical abuse and verbal behavioral symptoms directed towards others instead of verbal abuse . presence of rejection of care and behavioral symptoms directed towards others was obtained from mds 2.0 section e with choices from not exhibited to daily. the variables physical behavioral symptoms directed towards others ( e4ca ) and verbal behavioral symptoms directed towards others ( e4ba ) were combined into one variable called behaviors directed towards others. ability to understand ( c6 ) was rated as sometimes and rarely / never. psychotic symptoms [ delusions ( j1e ) and hallucinations ( j1i ) ] were combined into one variable , . clinical diagnosis of depression was obtained from mds item i1ee , and depression symptoms were assessed by the validated depression rating scale using other mds items : negative statements ( e1a ) , anger ( e1d ) , unrealistic fears ( e1f ) , repetitive health complaints ( e1i ) , sad expression ( e1i ) and crying ( e1 m ; score 0 - 14 ) . this scale correlated well with the cornell and hamilton depression scales using at least mild depression as a cutoff point , and with psychiatric diagnosis . it was also more sensitive and specific than the 15-item geriatric depression scale in detecting depression in the nursing home population . data about presence of pain were obtained from the mds item j2a with options of no , less than daily and daily. demographic data were also obtained from the mds . we investigated the longitudinal relationship between changes in rejection of care and behaviors directed towards others , and changes in lack of understanding , pain , depression and psychotic symptoms with the structural equation modeling approach . within this approach , we used latent growth models , bivariate latent growth models and longitudinal latent growth mediation models . since the studied variables were ordinal , we used latent growth models for categorical variables to study change within each variable . this model assumes the existence of latent or nonobserved trajectories , which are observed only indirectly through the repeated measures . the intercept represents the initial level at the beginning of the study , and the slope represents the rate of change . the factor loadings for the slope represent the measurement points , and they were set to the values 0 , 3 , 6 and 9 that represent the months at which the measurements were taken . second , we used bivariate latent growth curve models to study the relationships between rejection of care and understanding , and rejection of care and depression . these models combine two latent growth curve models that describe two separate longitudinal processes , and study the relationship between the initial level of the first process ( y , e.g. rejection of care ) with the initial level and rate of change in the second process ( x , e.g. lack of understanding ) , and the relationship between the rate of change in the first process with the initial level and rate of change in the second process ( fig . 1 , process y and x ) . bivariate latent growth curve models were also used to describe the relationship between behavioral symptoms directed toward others with rejection of care and behavioral symptoms directed towards others with understanding , and depression . frequency distributions of the variables psychotic symptoms and presence of pain remained stable through the whole measurement period , and the latent growth models showed no trend . therefore , we did not use bivariate latent growth models but latent growth models that included these variables as time - dependent covariates . in the model for psychosis symptoms , we included only the measurements of the first and the fourth psychosis symptom because of the high correlation between the first and the second measurement , and the third and the fourth measurement . finally , longitudinal mediation models were used to study whether rejection of care mediated the relationship between lack of understanding and behaviors directed towards others , and whether it mediated the relationship between depression and behaviors directed towards others ( fig . this means that first changes in lack of understanding ( depression ) occurred , and they promoted changes in rejection of care . next , changes in behaviors directed towards others occurred as a consequence of changes in rejection of care . three sets of latent growth factors were specified , one set for the independent variable , one set for the mediator , and , finally , another set for the dependent variable . the mediation model examined whether the growth in the independent variable affected the growth trajectory of the mediating variable which , in turn , affected the growth trajectory of the dependent variable . the value , the root mean square error of approximation ( rmsea ) , the comparative fit index ( cfi ) , and the tucker - lewis ( tli ) index were used to evaluate the model . a model achieves a good fit if the rmsea is lower than 0.05 , and fit is acceptable for values between 0.05 and 0.09 . the residents were mostly female , and some had diagnoses of both alzheimer 's disease and other dementias ( table 1 ) . over one third of them exhibited rejection of care for at least some days , and almost one third exhibited verbal behavioral symptoms directed towards others , while about 15.0% exhibited physical behavioral symptoms directed towards others . about half of these residents ( 51.0% ) exhibited significant symptoms indicating depression , but only 12.4% had a clinical diagnosis of depression . psychotic symptoms were present in 15.1% of residents , while almost one half of them experienced some pain ( table 1 ) . most common medications used for treatment of residents with rejection of care and behaviors directed towards others were antipsychotics that were administered at the beginning of the study to 32.2% of subjects ( table 1 ) . antidepressants were used in 18.7% of subjects , and antianxiety medications were used in 13.7% of subjects . further , all three classes of psychotropic medications were used more often in residents with depressive symptoms than in residents without depression ( antipsychotics 27.5 vs. 44.0% , antidepressants 13.0 vs. 27.4% , antianxiety 10.2 vs. 19.6% ) . the prevalence of psychotropic drug administration did not change during the study for antipsychotics ( t = 0.22 , p = 0.826 ) and antidepressants ( t = 0.81 , p = 0.416 ) , while the use of antianxiety medications increased ( 13.7 vs. 17.3% , t = 2.51 , p = 0.012 ) . table 2 shows the longitudinal trajectories of all the variables analyzed with latent growth models to investigate the development of the symptoms over time . based on the rmsea criteria , the model fit was good for the lack of understanding , rejection of care and behaviors directed towards others , and acceptable for depression and pain . the cfi and tli coefficients were equal to one for all the models . however , the increase was not significant for pain . the latent growth model for psychosis symptoms could not be estimated because there were almost no cases with change in psychosis scores within the study period . next , bivariate linear latent growth models were used to study the relationships between evolution in rejection of care and evolution in symptoms related to rejection of care . for psychosis and pain , we fitted a model with these variables as time - dependent covariates because previously estimated latent growth models did not show evidence of change for these variables . the model fit columns in table 3 show low rmsea values indicating a good model fit . changes in rejection of care were associated with or can be predicted by changes in lack of understanding and also by changes in depression . finally , lower initial levels of pain were associated with a stronger increase in rejection of care ( c = 0.009 , se = 0.004 , p = 0.029 ) . the relationships between the intercepts or initial level of rejection of care and intercepts of the other variables are not reported in table 3 because intercept parameters depend on the measurement scale of the analyzed variables . the relationships between psychosis symptoms and pain , and initial levels of rejection of care are scale free and can be interpreted . we found a strong relationship between psychosis and initial levels of rejection of care , with higher initial levels of psychosis associated with higher initial levels of rejection of care ( c = 0.207 , se = 0.053 , p < 0.001 ) . we also found that high initial levels of pain were associated with high initial levels of rejection of care ( c = 0.116 , se = 0.045 , p = 0.009 ) . then , bivariate linear latent growth models were used to describe the relationships between evolution in behaviors directed towards others and evolution in symptoms related to those behaviors . the columns reporting relationships between slopes show significant positive relationships between changes in rejection of care and changes in behaviors directed towards others . changes in understanding and changes in depression were also associated with changes in behaviors directed towards others . the relationships between intercepts for behaviors directed towards others are not reported in table 3 because of the same reasons explained above for the rejection of care analyses . regarding the relationship between initial behaviors directed towards others and initial levels in pain or psychotic symptoms , we found a significant relationship between initial behaviors directed towards others and initial psychosis ( c = 0.205 , se = 0.051 , p < 0.001 ) , indicating that initial psychosis was related to more frequent behaviors directed toward others . 1 ) , one to investigate whether rejection of care mediated the relationship between lack of understanding and behaviors directed towards others , and the other to investigate whether rejection of care mediated the relationship between depression and behaviors directed towards others . consistent with the literature on statistical mediation , the parameters illustrating the relationships in figure 1 are called a , b and c. parameter a describes the relationship between the slope for the independent variable ( x ) and the slope for the mediator ( m ) . parameter b describes the relationship between the slope of the mediator ( m ) and the slope in the dependent variable ( y ) . parameter c represents the direct effect , which is the part of the effect of x on y that is independent of the mediator . the fit of the first model was good ( test = 88.6 , d.f . = 61 , p = 0.012 , rmsea = 0.021 , cfi = 1.000 , tli = 1.000 ) . the relationship between lack of understanding and rejection of care was significant ( a = 0.876 , se = 0.124 , p < 0.000 ) . there was a significant relationship between rejection of care and behaviors directed towards others ( b = 1.249 , se = 0.337 , p < 0.001 ) , while the relationship between lack of understanding and behaviors directed towards others after adjusting for rejection of care was not significant ( c = 0.645 , se = 0.465 , p = 0.166 ) . therefore , this model suggests that the relationship between lack of understanding and behaviors directed towards others is mediated by rejection of care . to quantify the indirect effect , the product of the parameters a and b was calculated and tested with the sobel test . we found a significant mediation effect ( ab = 0.979 , se = 0.440 , p = 0.026 , with ab being the change in the outcome per one unit lack of understanding that goes through the mediator rejection of care ; fig . the fit of the second model was also good ( test = 105.1 , d.f . = 58 , p = 0.0002 , rmsea = 0.027 , cfi = 1.000 , tli = 1.000 ) . the relationship between the slope for depression and the slope for rejection of care was significant ( a = 0.828 , se = 0.138 , p < 0.001 ) , but there was not significant evidence that the slope in the mediator rejection of care was associated with the slope in behaviors directed towards others ( b = 0.561 , se = 0.306 , p = 0.067 ) . the adjusted direct effect ( c = 0.427 , se = 0.343 , p = 0.214 ) and the indirect effect were also not significant ( ab = 0.465 , se = 0.277 , p = 0.094 ) . therefore , we can not conclude that the relationship between depression and behaviors directed towards others is mediated by rejection of care . since we did not observe changes in psychosis and pain , we did not consider mediation models for these processes . table 2 shows the longitudinal trajectories of all the variables analyzed with latent growth models to investigate the development of the symptoms over time . based on the rmsea criteria , the model fit was good for the lack of understanding , rejection of care and behaviors directed towards others , and acceptable for depression and pain . the cfi and tli coefficients were equal to one for all the models . in table 2 , positive slopes show that all these variables increased with time . however , the increase was not significant for pain . the latent growth model for psychosis symptoms could not be estimated because there were almost no cases with change in psychosis scores within the study period . next , bivariate linear latent growth models were used to study the relationships between evolution in rejection of care and evolution in symptoms related to rejection of care . for psychosis and pain , we fitted a model with these variables as time - dependent covariates because previously estimated latent growth models did not show evidence of change for these variables . the model fit columns in table 3 show low rmsea values indicating a good model fit . the cfi and tli coefficients were equal to one . changes in rejection of care were associated with or can be predicted by changes in lack of understanding and also by changes in depression . finally , lower initial levels of pain were associated with a stronger increase in rejection of care ( c = 0.009 , se = 0.004 , p = 0.029 ) . the relationships between the intercepts or initial level of rejection of care and intercepts of the other variables are not reported in table 3 because intercept parameters depend on the measurement scale of the analyzed variables . the relationships between psychosis symptoms and pain , and initial levels of rejection of care are scale free and can be interpreted . we found a strong relationship between psychosis and initial levels of rejection of care , with higher initial levels of psychosis associated with higher initial levels of rejection of care ( c = 0.207 , se = 0.053 , p < 0.001 ) . we also found that high initial levels of pain were associated with high initial levels of rejection of care ( c = 0.116 , se = 0.045 , p = 0.009 ) . then , bivariate linear latent growth models were used to describe the relationships between evolution in behaviors directed towards others and evolution in symptoms related to those behaviors . the columns reporting relationships between slopes show significant positive relationships between changes in rejection of care and changes in behaviors directed towards others . changes in understanding and changes in depression were also associated with changes in behaviors directed towards others . the relationships between intercepts for behaviors directed towards others are not reported in table 3 because of the same reasons explained above for the rejection of care analyses . regarding the relationship between initial behaviors directed towards others and initial levels in pain or psychotic symptoms , we found a significant relationship between initial behaviors directed towards others and initial psychosis ( c = 0.205 , se = 0.051 , p < 0.001 ) , indicating that initial psychosis was related to more frequent behaviors directed toward others . 1 ) , one to investigate whether rejection of care mediated the relationship between lack of understanding and behaviors directed towards others , and the other to investigate whether rejection of care mediated the relationship between depression and behaviors directed towards others . consistent with the literature on statistical mediation , the parameters illustrating the relationships in figure 1 are called a , b and c. parameter a describes the relationship between the slope for the independent variable ( x ) and the slope for the mediator ( m ) . parameter b describes the relationship between the slope of the mediator ( m ) and the slope in the dependent variable ( y ) . parameter c represents the direct effect , which is the part of the effect of x on y that is independent of the mediator . the fit of the first model was good ( test = 88.6 , d.f . = 61 , p = 0.012 , rmsea = 0.021 , cfi = 1.000 , tli = 1.000 ) . the relationship between lack of understanding and rejection of care was significant ( a = 0.876 , se = 0.124 , p there was a significant relationship between rejection of care and behaviors directed towards others ( b = 1.249 , se = 0.337 , p < 0.001 ) , while the relationship between lack of understanding and behaviors directed towards others after adjusting for rejection of care was not significant ( c = 0.645 , se = 0.465 , p = 0.166 ) . therefore , this model suggests that the relationship between lack of understanding and behaviors directed towards others is mediated by rejection of care . to quantify the indirect effect , the product of the parameters a and b was calculated and tested with the sobel test . we found a significant mediation effect ( ab = 0.979 , se = 0.440 , p = 0.026 , with ab being the change in the outcome per one unit lack of understanding that goes through the mediator rejection of care ; fig . the fit of the second model was also good ( test = 105.1 , d.f . = 58 , p = 0.0002 , rmsea = 0.027 , cfi = 1.000 , tli = 1.000 ) . the relationship between the slope for depression and the slope for rejection of care was significant ( a = 0.828 , se = 0.138 , p < 0.001 ) , but there was not significant evidence that the slope in the mediator rejection of care was associated with the slope in behaviors directed towards others ( b = 0.561 , se = 0.306 , p = 0.067 ) . the adjusted direct effect ( c = 0.427 , se = 0.343 , p = 0.214 ) and the indirect effect were also not significant ( ab = 0.465 , se = 0.277 , p = 0.094 ) . therefore , we can not conclude that the relationship between depression and behaviors directed towards others is mediated by rejection of care . since we did not observe changes in psychosis and pain , we did not consider mediation models for these processes . the results of this longitudinal study showed that changes in lack of understanding or depression commonly precede changes in rejection of care . changes in behaviors directed towards others are related to changes in lack of understanding , depression and rejection of care . furthermore , a mediation model suggested that the relationship between lack of understanding and behaviors directed towards others was mediated by rejection of care . therefore , our results support conclusions of our previous cross - sectional study , which found that lack of understanding and depression are the two main risk factors for development of rejection of care and behaviors directed towards others . however , our results are more informative because longitudinal models provide more information regarding temporal relationships between the variables , and the mediation model provides information about the order in which the symptoms develop . initial psychotic symptoms and pain were also related to increased rejection of care , but we did not observe changes in the number of psychotic symptoms and pain within our study period . regarding to the absence of observed changes in pain , hendriks et al . found a high proportion of patients with pain persistence . that precluded inclusion of psychotic symptoms and pain in the mediation model . a relationship of depression to resistive behavior ( rejection of care ) and aggression ( abusive symptoms ) was already reported by lyketsos et al . and by leonard et al . . they found , in agreement with our results , that psychotic symptoms play a minor role in the development of these behaviors . the lack of understanding is caused by the progression of dementia , and it increases the prevalence of rejection of care ; this increase in rejection of care contributes to the increasing behaviors directed towards others . it may be improved by communication skills training of professionals and family caregivers that includes verbal skills , nonverbal and emotional skills , behavioral management skills , usage of tools and theoretical knowledge . such a program would be useful also in a hospital where many dementia patients are transferred . the most important intervention is to teach the staff that most people with dementia are not aggressive ; they just defend themselves against unwanted attention from the caregivers and may consider the caregivers to be the aggressors . there is a clear - cut distinction between rejection of care and agitation , and the term agitation should be reserved for behaviors that occur when the resident is solitary , e.g. restlessness , repetitive movements , crying out . we identified depressive symptoms using the mds depression scale , which uses 7 mds items . this scale correlated well with the cornell and hamilton depression scales using at least mild depression as a cutoff point , and with psychiatric diagnosis . this is comparable to 47.4% detected in a large study of a long - term care facility . high prevalence of depression in individuals with alzheimer 's disease can be expected because alzheimer 's disease causes serotoninergic deficit , and some data indicate that this deficit may be related to aggressive behaviors . the relationship between serotoninergic function and aggressive behavior may not be unique to alzheimer 's disease because decreased serotoninergic activity is present also in frontotemporal dementia . our results suggest that depression contributes to both rejection of care and behaviors directed towards others and that the relation between depression and behaviors towards others is not mediated by rejection of care . depression in dementia could be treated with psychological interventions as shown in a recent review . antidepressants could be the first line of medication for individuals exhibiting these behaviors directed towards others if psychotic symptoms are not present . although some studies found improvement of behavioral symptoms after treatment with antidepressants , other studies were negative . this was documented by the depression in alzheimer disease study , which found that treatment with sertraline decreased behavioral disturbance and caregiver distress only in patients whose depression responded to antidepressant treatment . antidepressant treatment was found effective in decreasing behavioral symptoms of dementia in a cochrane data analysis and in a recent citalopram study , but antidepressants may sometimes require augmentation with atypical antipsychotics . the results of this study show that the psychotropic medications administered most frequently to the participants in this study were antipsychotics . it is possible that these drugs somewhat reduced rejection of care by their sedative effects . similar prevalences of antipsychotic use were reported from a survey of nursing home physicians in the us and a dutch survey . in that study , 32% of subjects received antipsychotics , which is comparable with reports from sweden ( 38% ) and from germany ( 32.5% ) . antipsychotics are used frequently for treatment of behavioral symptoms of dementia despite multiple reports that found serious side effects including increased mortality in residents treated with these medications . efforts are made in several countries to decrease the use of antipsychotics in dementia . in the us , the prevalence of antipsychotic use in nursing homes is about 25% , and the use of antipsychotics was recently reduced by 9.1% . in the uk , antipsychotic use in people with dementia decreased from 19.9% in 1995 to 7.4% in 2011 , concomitant with an increase of antidepressants from 10.7 to 25.3% , indicating that antidepressants may be substituted for antipsychotics . our data were derived from mds records that were completed mostly by nurses . however , these were experienced clinicians , and mds data correlated well with other scales . we have studied the relationship of only four factors in abusive behavior of nursing home residents with dementia . however , there are probably also other factors that may be involved , e.g. physical causes such as thirst , hunger or inappropriate environmental temperature . using a discomfort scale in future studies would provide information about the importance of these other factors . our data could not evaluate these causes . a model with all predictors controlling for the influence of each variable would provide more evidence for causality than our models . another limitation is that we did not have data about specific drugs and drug dosages that may have been modified in residents with rejection of care and behaviors directed towards others even when the prevalence of their use did not change . the results of this study together with the results of previous investigations indicate that lack of understanding and depression are important factors in development of rejection of care and behaviors directed towards others , and that rejection of care may escalate into behaviors directed towards others . furthermore , the relationship between lack of understanding and behaviors directed towards others may be mediated by rejection of care . therefore , improved communication between caregivers and persons with dementia and perhaps also effective treatment of depression may prevent or ameliorate rejection of care and behaviors directed towards others .

miriplatin , a cisplatin derivative with a high affinity for iodized ethyl esters of fatty acids from poppy seed oil , is a novel chemotherapeutic agent designed for use in the transarterial treatment of hepatocellular carcinoma ( hcc ) . here , we describe transcatheter arterial infusion ( tai ) using miriplatin to treat a case of advanced hcc with portal vein tumor thrombus ( pvtt ) refractory to tai with epirubicin . a 66-year - old man with advanced hepatitis c virus - related hcc with pvtt in the right lobe of the liver was treated with tai with epirubicin suspended in iodized oil ; however , tumor marker levels ( alpha - fetoprotein and des - gamma - carboxy protein ) did not decrease . next , he was treated twice with tai with miriplatin suspended in iodized oil . the tumor marker levels markedly decreased to a nearly normal range and the size of the main tumor was markedly reduced according to dynamic computed tomography . no serious adverse events occurred during the course of treatment with tai and miriplatin . therefore , we suggest that tai with miriplatin is a safe and effective treatment option for advanced hccs refractory to tai with epirubicin .

miriplatin , a cisplatin derivative with a high affinity for iodized ethyl esters of fatty acids from poppy seed oil , is a novel chemotherapeutic agent designed for use in the transarterial treatment of hepatocellular carcinoma ( hcc ) . miriplatin ( 1 ) inhibits cell proliferation in a similar manner as cisplatin and has superior solubility in ethyl esters of iodized fatty acids from poppy seed oil ; ( 2 ) releases its platinum constituent continuously by remaining at local tumor sites together with ethyl esters of iodized fatty acids from poppy seed oil ( sustained release ) ; and ( 3 ) alleviates adverse effects as it maintains an antitumor effect owing to its sustained release as well as its minimal presence in the general circulation [ 1 , 2 , 3 , 4 ] . despite such characteristics , however , many aspects of its antitumor effects are not clear . we experienced a case of advanced hcc that showed marked decrease in tumor markers following treatment with transcatheter arterial infusion ( tai ) with miriplatin , despite being refractory to tai with epirubicin . the patient , a 66-year - old man , had been diagnosed with chronic hepatitis c and had been followed - up at another hospital for the past 5 years . he had never smoked cigarettes and occasionally drank alcohol . during follow - up for chronic hepatitis c at the other hospital , the levels of tumor markers an advanced hcc with portal vein tumor thrombus ( pvtt ) in the right lobe was observed on the dynamic computed tomography ( ct ) scan , and he was admitted to our hospital for medical treatment in july 2010 . the patient 's height was 157 cm , body weight was 46 kg , blood pressure was 134/74 mm hg , body temperature was 36.6c , heart rate was 84 bpm , and no significant findings were observed upon physical examination . laboratory data are shown in table 1 . a marked increase in the levels of tumor markers dynamic ct showed an irregularly shaped tumor , 5.6 3.8 cm in size , which was enhanced in the early phase ( not enhanced in some areas ) and indicated a region of deficiency in the delayed phase , in liver segment 8/7 . the tumor had invaded the right anterior branch of the portal vein , and was cut off at the portion indicated by the arrow in fig . 1 ( i.e. vp2 ) . in segment 5 , adjacent to the gallbladder , a classical hcc 2 cm in size was observed , which was enhanced in the early phase and indicated a region of deficiency in the delayed phase . as an initial clinical course beginning in late july 2010 , abdominal angiography ( fig . 2 ) was performed . digital subtraction angiography ( dsa ) of the superior mesenteric artery showed disruption of the right anterior branch of the portal vein . the lumen of the posterior branch of the postal vein was narrowed . in the dsa of the common hepatic artery , a large irregular tumor stain in the right lobe of the liver and a nodular tumor stain , 2 cm in size , were found in segment 5 . an emulsion of epirubicin ( 50 mg ) and lipiodol ( 8 ml ) was infused primarily from the right hepatic artery . after tai , a transient fever over 38c was observed ; however , the fever was easily managed using only antipyretics , and there were no other adverse events . conducted in late september revealed a marked increase in tumor marker levels ( alpha - fetoprotein [ afp ] from 2,116 ng / ml to 4,622 ng / ml , and des - gamma - carboxy protein [ dcp ] from 373 mau / ml to 8,394 mau / ml ) . the second clinical course was begun in early october 2010 , during which abdominal angiography was performed . next , an emulsion of miriplatin ( 80 mg ) and lipiodol ( 8 ml ) was infused primarily from the right hepatic artery because the tumors were refractory to tai with epirubicin . one month after tai with miriplatin , the afp level was 1,507 ng / ml and the dcp level was 9,457 mau / ml , indicating that the therapy was not effective . however , 2 months after tai , the afp level was 63.9 ng / ml and the dcp level was 64 mau / ml . a marked decrease in the levels of the tumor markers was observed . because of the good response to tai with miriplatin , an emulsion of miriplatin ( 100 mg ) and lipiodol ( 5 ml ) was again infused primarily from the right hepatic artery in early january 2011 . one month after the 2nd tai with miriplatin , the levels of the tumor markers improved to a nearly normal range ( afp , 16.3 ng / ml and dcp , 34 mau / ml ) . dynamic ct performed 3 months after the 2nd tai with miriplatin showed that the size of the main tumor in segment 8/7 had markedly decreased , resulting in less obstruction of the right branch of the portal vein ( fig . 4 ) . because the tumor in segment 5 exhibited poor response to the treatment , percutaneous therapy to the nodule several intra - arterial chemotherapy regimens using epirubicin , mitomycin , adriamycin , fluorouracil , fluorodeoxyuridine , mitoxantrone , and cisplatin as treatment for hcc have been reported . however , the optimal regimen for intra - arterial chemotherapy for hcc is still unknown . in the case described , the patient obtained a marked decrease in tumor marker levels following treatment with tai and miriplatin , despite being refractory to tai with epirubicin . considering the clinical courses used , it was thought that the main tumor caused an elevation in the tumor marker levels . the tumor enhancement pattern seen on the dynamic ct indicated that the main tumor contained an undifferentiated region ( irregular enhancement with deficit regions in certain areas ) in the early phase . considering the effects of the applied treatment ( i.e. remarkably effective for the former tumor and ineffective for the latter tumor ) , tai with miriplatin may be effective for treating undifferentiated tumors . it is noteworthy that in the ineffective treatment of the tumor in segment 5 as well as in the markedly effective treatment of the main tumor , most of the administered lipiodol was washed out after treatment with tai and miriplatin . tai treatment typically results in tumor necrosis , which is normally evaluated by lipiodol accumulation and ct because lipiodol accumulation in the area of the tumor corresponds to the necrotic area of the tumor [ 12 , 13 ] . the liver cancer study group of japan recommends that the area of lipiodol accumulation should be calculated when evaluating the effects of tai . however , 1 case has been reported in which sufficient treatment effects were obtained despite washing out of most of the infused lipiodol as was observed for the main tumor in this case . the relationship between the degree of lipiodol accumulation and tumor necrosis should be verified . in conclusion , tai with miriplatin is a safe and effective treatment option for advanced hccs refractory to tai with epirubicin .

duodenal adenomas are uncommon , however , when present a proportion have dysplasia associated with the adenoma and therefore require treatment . the options range from less invasive endoscopic treatments to a pancreaticoduodenectomy . this case report describes two patients with adenomas involving the ampulla of vater . one patient had familial adenomatous polyposis , the other was a renal transplant patient with a large adenoma . both patients adenomas contained high - grade dysplasia . both patients underwent a pancreaticoduodenectomy . histology of both specimens demonstrated that the adenoma had migrated up the bile duct for at least 7 mm , and the pancreatic duct for 8 mm in one patient . limited resection of ampullary adenomas may leave residual adenomatous tissue in the bile duct with the risk of recurrent adenomatous disease and malignant transformation .

polyps in the second part of the duodenum are uncommon , however , up to 22% are adenomas . a proportion of these have high - grade dysplasia and need some form of intervention to prevent the progression to carcinoma . there are a range of options to manage the adenomas , from less invasive endoscopic resections [ 2 , 3 , 4 ] to surgical resection and pancreaticoduodenectomy [ 6 , 7 ] . this paper describes the management and outcome of two patients who presented with high - grade villous adenomas of the ampulla . the aim is to highlight the potential pitfalls of a minimally invasive approach to resecting adenomas with high - grade dysplasia involving the ampulla of vater . a 66-year - old woman with familial adenomatous polyposis ( fap ) had a colectomy and ileorectal anastomosis in 1961 , followed by a completion proctectomy in 2004 . as part of the follow - up , a gastroscopy was performed and demonstrated multiple adenomas carpeting the duodenum with high - grade dysplasia on biopsy . a computed tomography scan confirmed that the disease was localised but was associated with mild bile duct dilatation . because of the number and the extent of the lesions , it was elected to perform a pancreaticoduodenectomy . the patient made an uneventful post - operative recovery and was discharged 14 days after surgery . on histology multiple discrete and merging polypoid lesions were seen to stud the duodenal mucosa , ranging in size from 3 mm to 22 15 5 mm , including a 10 mm polyp in the periampullary region . histologically these demonstrated multiple tubular and villous adenomas with focally high - grade glandular dysplasia ( fig . 1 ) . both high- and low - grade dysplasia extended down the common bile duct at the ampulla of vater for a distance of 12 mm and down the separate pancreatic duct for a distance of 8 mm ( fig . low - grade dysplasia involved the jejunal margin but not the pancreatic or bile duct margins . gastroscopy revealed a villous adenoma of the second part of the duodenum and histology confirmed a villous adenoma with low - grade dysplasia . an ercp in october 2004 showed progression of the adenoma , then being 10 cm in length , almost circumferential ( fig . the patient had significant co - morbidities , including ischaemic heart disease and a coronary artery bypass graft ( 2003 ) , chronic renal failure and a renal transplant ( 2000 ) , hypercholesterolaemia , hypertension , gastro - oesophageal reflux disease and recurrent urinary tract infections . despite this the post - operative course was prolonged and complicated by a pancreatic anastomotic leak , resulting in a 4-week hospital stay . on histology , a periampullary villous adenoma was present ( macroscopically 90 60 17 mm ) featuring high- and low - grade glandular dysplasia . both high- and low - grade dysplasia extend down the common bile duct at the ampulla for a distance of 7 mm , and there was colonisation of periampullary glands by dysplastic epithelium without invasion ( fig . the diagnosis of duodenal polyps is likely to increase in frequency as their association with hereditary neoplastic syndromes , such as hereditary non - polyposis colorectal cancer and fap , becomes more recognised . research into the natural history of duodenal polyps is inconclusive , with some authors indicating rapid progression of polyps to carcinoma , and a high incidence of carcinoma at diagnosis , whilst others describe a benign course . pancreaticoduodenectomy is the procedure of choice for malignant lesions of the duodenum , ampulla of vater or head of the pancreas . because of the considerable morbidity and mortality , there has been increased interest in less extensive operations for the management of benign disease . alternate strategies include endoscopic resection , piecemeal , snaring [ 2 , 4 ] , or fulguration , argon plasma coagulation , trans - duodenal resection , and pancreas - preserving duodenectomy . these procedures limit the complications of surgery or post - operative morbidity , whilst attempting to maintain adequate disease control . in all of these situations , except pancreas - preserving duodenectomy , where a biopsy of the bile duct is obtained at the time of surgery the role of endoscopic ultrasound is not well established but may add information regarding invasion and/or ductal involvement . the two cases described here demonstrate one of the pitfalls of treating patients with dysplastic villous adenomas of the second part of the duodenum , involving the ampulla of vater , with a limited resection as the dysplasia extended up the bile duct in both these cases . this would potentially have been missed in a trans - duodenal , endoscopic and pancreas - preserving resection of the duodenum . the first patient presented with multiple duodenal polyps secondary to fap . the migration of dysplastic adenomatous tissue into the ampulla of vater may explain some of the high recurrence rate of these adenomas after local excision . the second patient had just one large villous adenoma , which was not related to a known hereditary polyposis syndrome . in this situation , dysplastic adenomatous tissue had again migrated up the ampulla of vater . in conclusion we present two cases with villous adenomas of the duodenum , both of which displayed migration of the dysplastic adenoma up the bile duct . this would indicate that duodenal adenomas involving the ampulla of vater , with features of dysplasia , should be treated with definitive surgery .

three new homo - adamantanyl type natural products were derived from polyprenylated polycyclic acylphloroglucinol . hypercohones a - c ( 13 ) , along with five other known hypercohones ( 48 ) , were isolated from the aerial parts of hypericum cohaerens . the structures of 13 were elucidated on the basis of comprehensive spectroscopic analysis . the inhibitory activities of these isolates against five human cancer cell lines in vitro were tested . electronic supplementary materialsupplementary material is available for this article at 10.1007/s13659 - 013 - 0032 - 9 and is accessible for authorized users .

supplementary material is available for this article at 10.1007/s13659 - 013 - 0032 - 9 and is accessible for authorized users .

conception is rare in patients on chronic dialysis and diagnosis is often delayed as it is least expected . serum beta hcg levels are elevated in both pregnant and non - pregnant dialysis patients , and pregnant dialysis patients have slightly higher beta hcg levels when compared with normal pregnancy . this can be erroneously interpreted as non - viable pregnancy and so results should be viewed with caution . serial beta hcg levels must be followed when non - viable pregnancy is suspected in a dialysis patient before contemplating termination of pregnancy as described in our case .

conception is unusual in women treated with conventional dialysis , with estimates ranging from 0.3% to 1.5% per year [ 14 ] . the diagnosis of pregnancy has often been made late in gestation because it is unexpected and because amenorrhea and nausea are common in dialysis patients . a review from 1987 found the average gestational age at diagnosis of pregnancy to be 16 weeks . it has long been recognized that beta hcg levels are elevated in both pregnant and non - pregnant dialysis patients . slightly elevated levels occasionally cause problems such as postponement of surgery while waiting to demonstrate that levels are not rising . high levels of beta hcg in pregnant dialysis patients rarely caused a problem in pregnancies diagnosed late because ultrasound was used to determine gestation age , but at least one pregnancy was terminated because of a suspected hydatidiform mole . the elevated levels become more important when the index of suspicion is higher and pregnancy is diagnosed earlier . we present a case in which misinterpretation of beta hcg levels led to consideration of terminating a viable pregnancy . in august 2005 , a 25-year - old woman on hemodialysis was referred to our renal clinic with a positive pregnancy test based on serum beta hcg . she had end - stage renal disease secondary to type 1 diabetes and had started dialysis 9 months prior to her visit . she had had two previous pregnancies , the first resulting in spontaneous abortion in the first trimester and the second resulting in the delivery of a baby at 30 weeks gestation . her past medical history was significant for diabetes since age 9 with retinopathy and nephropathy . she was not aware of any kidney disease until she reached the point of needing dialysis in september 2004 and had been on thrice weekly dialysis since then . on exam , her bp was 112/67 mm hg and she did not have any edema . her beta hcg levels were 13 270 miu / ml at the time of her pregnancy test and increased to 30 232 miu / ml within 3 days . the plan was to increase her dialysis regimen to more than 20 h per week and she was referred to the care of high - risk obstetrics . she had a transvaginal ultrasound scan , which showed an intra - uterine gestational sac of 5.3 weeks , but no fetal heart tone could be detected . given that she had hcg levels consistent with 7 or more weeks gestation without visible cardiac activity , she was thought to have a non - viable pregnancy and surgical termination was contemplated once her blood sugars were well controlled . beta hcg levels were followed with the expectation that they would drop in the setting of a non - viable pregnancy . her beta hcg levels are charted below : weeks gestationpatient s beta hcgnormal beta hcg5 weeks13 2707526005 weeks 3 days30 32385020 8005 weeks 5 days44 68585020 8007 weeks133 7084000100 000 a transvaginal ultrasound was repeated in 10 days and showed an intra - uterine pregnancy consistent with a gestational age of 7 weeks and with good fetal heart tones at a rate of 128 bpm . although , in the second trimester , she had an abnormal triple antigen screening test for fetal abnormalities , she delivered a healthy baby weighing 3.5 kg at 32 weeks without any further complications . pregnancy in dialysis patients is a major challenge to physicians involved in care , given the rarity of occurrence , complications involved and the distressing observation that < 50% of pregnancies in patients receiving conventional dialysis result in surviving infants . outcomes in patients receiving nocturnal hemodialysis have been more encouraging , making accurate interpretation of beta hcg levels more important . serum beta hcg levels should be interpreted with caution in dialysis patients as levels tend to be slightly elevated even in non - pregnant patients and can be erroneously interpreted as intact pregnancy in a non - pregnant patient or a non - viable pregnancy in a pregnant patient as in our case . hcg is secreted in small amounts by all cells , and since the hormone is largely excreted by the kidneys , its level can be elevated in dialysis patients even if not pregnant . in a study conducted by schwarz et al . , beta hcg levels were increased by 10-fold in two of the 19 non - pregnant women who were on dialysis with post - dialysis levels being significantly higher than pre - dialysis levels . this observation has an important implication in clinical practice when monitoring serial beta hcg levels ( pre- or post - dialysis blood sample should be specified when results are reported ) . further studies need to be conducted in pregnant dialysis patients as data are limited on pre- and post - dialysis values of hcg . since serum beta hcg levels are not reliable in dialysis patients , pregnancy is normally confirmed by an ultrasound . fetal cardiac activity can be first detected at 5.56 weeks and can be missed if imaging is done earlier . if a non - viable pregnancy is suspected in a dialysis patient early in the first trimester , the diagnosis should be confirmed by measuring serial beta hcg as it decreases in a non - viable pregnancy , albeit more slowly than in a woman with normal renal function . the triple antigen test which is used for screening fetal abnormalities might not be reliable in dialysis patients . the patient reported in our case would have had a therapeutic abortion in her first trimester if not for the poorly controlled diabetes . pregnancy in a dialysis patient might represent the last opportunity for child bearing and every effort should be made to ensure a successful outcome .

background : the objectives of this study were to investigate the incidence of cervical enamel projection ( cep ) in molars of indian dry human skulls and to evaluate its relationship with furcation involvement ( fi).materials and methods : the material consisted of 944 upper and lower first , second and third permanent molars from 89 indian dry human skulls . ceps were investigated from the buccal aspect of the tooth and classified according to a system describeddescribed by masters and hoskins . fi was measured horizontally from the buccal aspect into the furcation with a graduated probe to the nearest millimeter . any measurement 2 mm was considered to have positive fi.results:the results showed that ceps was found more frequently in the mandibular than in the maxillary molars ( 2:1 ) . the highest incidence of cep was found in the mandibular second molar ( 14.7% ) followed by the maxillary second molar ( 14.6% ) . the mandibular third molar showed the lowest incidence ( 5.5% ) . the association between cep and fi ( 87.5% ) was statistically significant . this favors the view of the possible role played by such anomalies in the progression of periodontal diseases . cep in male skulls ( 77.4% ) was significantly more prevalent than in female skulls ( 20.4% ) . no significant difference was found between the right and left side of maxillary and mandibular teeth with cep and fi.conclusion:the findings suggested the role of ceps as a local contributing factor in localized chronic periodontitis and fi in molars . detailed examination as well as early diagnosis of periodontal disease at the region of furcation is clinically very important .

periodontitis is primarily a bacterial - induced disease that can be modified by tooth related local factors . enamel , which is normally restricted to the anatomical crown of human permanent teeth , may be found ectopically on the root either as enamel pearl or enamel projections . among the anatomical factors cervical enamel projections ( ceps ) are a common tooth anomaly that can act as a contributing factor in the development and progression of periodontitis . ceps are flat , ectopic deposits of enamel apical to the normal cemento - enamel junction ( cej ) level in molar furcation areas . these enamel deposits usually have a triangular shape and a tapering form , extending apically into furcation areas . they are most commonly found at the buccal surfaces of mandibular molars . a possible relationship between cep and periodontal breakdown he suggested that the anatomy and location of cep might act as probable causes for rapid pocket formation . , the gingival tissue adjoining the cep is attached to the tooth by epithelial attachment , which is less resistant to the insult of bacterial plaque . this , together with a reduced access for oral hygiene measures and the proximity to the furcation dome , might enhance periodontal breakdown in the furcation . the clinician must be aware of the presence of such aberrations and their importance in the etiology of local periodontal breakdown . although reports vary , about 15 - 24% of mandibular molars and 9 - 25% of maxillary molars have ceps . considering both arches , they are most likely to be found on buccal surfaces of second molars . swan and hurt , in their study on indian skulls , noticed a direct association between the contour of the marginal bone and the position of cep . they also found widening of the periodontal ligament adjacent to the alveolar crest , which allowed the apical portion of the cep to merge toward the furcation . they concluded that it was the bone 's excellent remodeling properties that enabled it to adjust to the varying tooth surface anatomy . masters and hoskins reported the incidence of ceps in extracted human teeth and suggested their possible implication in isolated furcation involvement ( fi ) . they feel that even a slight gingival inflammation could produce a deep constricted periodontal pocket almost overnight . this could explain why destruction of the periodontium occurs at one given location , while bypassing adjacent areas however , an attempt by leib et al . to statistically associate these enamel extensions with the purpose of this study is to re - evaluate the incidence and distribution of ceps and to correlate their possible relationship with fis present in dry human indian skulls . a total of 944 upper and lower first , second and third permanent molars were examined from 89 dry human indian skulls for the presence of cep dipping into the root furcation area . ceps were investigated from the buccal aspect of the tooth and classified according to a system given by masters and hoskins [ figure 1 ] . classification of cervical enamel projection grade i : the enamel projection extends from the cej of the tooth toward the furcation entrance . fi was measured horizontally from the buccal aspect into the furcation with a graduated probe to the nearest millimeter . male and female skulls were identified on the basis of the following anatomical landmarks : male skulls typically have more prominent supraorbital ridges , a more prominent glabella and more prominent temporal lines . female skulls generally have rounder orbits and narrower jaws.male skulls on average have larger , broader palates , squarer orbits , larger mastoid processes , larger sinuses and larger occipital condyles than those of females.male mandibles typically have squarer chins and thicker , rougher muscle attachments than female mandibles . male skulls on average have larger , broader palates , squarer orbits , larger mastoid processes , larger sinuses and larger occipital condyles than those of females . male mandibles typically have squarer chins and thicker , rougher muscle attachments than female mandibles . the data collected were subjected to statistical analysis using a chi - square test for determining the incidence of each variable and tabulated in tables 15 . distribution of cervical enamel projections in relation to maxillary and mandibular molars distribution of cep with furcation involvement according to their grade number of cervical enamel projections and furcation involvements in study sample distribution of molars involving cep & furcation according to the sex distribution of cep with furcation according to the side of the arch correlation of cervical enamel projection and furcation involvement presence of furcation involvement without presence of cervical enamel projection presence of cervical enamel projection without involving the furcation the incidence of ceps was found to be 11.9% ( 112 out of 944 molars examined ) . cep and furcation were present on 98 ( 10.3% ) of the 944 molars examined . a total of 308 ( 32.6% ) of the 944 molars examined , were with fi . cep without furcation were present on 14 ( 1.4% ) of the 944 molars examined . ceps were found almost twice as frequently in the mandibular molars as in the maxillary molar , which was found to be highly significant ( p = 0.000 ) [ table 1 ] . tooth with grade iii cep shows the highest number ( 82 ) of fi as compared with grade ii and grade i cep , which was found to be highly significant ( p = 0.000 ) [ table 2 ] . number of molars with cep and fi were 98 out of 944 ( 10.3% ) which was statistically significant ( p = 0.000 ) [ table 3 ] cep with fi was significantly more in male skulls as compared with female skulls ( p = 0.001).[table 4 ] there was no significant difference between the right and left maxillary and mandibular teeth with cep and fi ( p = 0.751 ) [ table 5 ] . periodontitis is primarily a dental plaque induced inflammatory disease but the local factors that facilitate the accumulation of bacteria may contribute to the progression of the disease . factors such as tooth anatomy and restorative / endodontic considerations have been linked to gingival inflammation and attachment and tooth loss . of all anatomic factors , the cep is probably the most common and associated with attachment loss in the molar furcation area . this study investigated the incidence and distribution of cep and attempted to correlate their possible relationship to fis present in dry human indian skulls . cep were present on 112 ( 11.9% ) of the 944 molars examined ( 224 maxilla , 720 mandible ) . in previous reports , the incidence of cep ranged from 15% to 30% . a possible explanation for the low incidence in the present investigation may be the difference in racial origin of the specimens . as shown in table 1 , ceps were found almost twice as frequently in the mandibular molars as in the maxillary molars . the predominance in the mandibular molars is in general agreement with previous reports . the incidence of cep varied between the first , second , third molars [ table 1 ] . mandibular second molars showed the highest incidence ( 14.7% ) , followed by maxillary second molar ( 14.6% ) . this pattern of frequency is in accordance as that reported by swan and hurt , and bissada et al . we found that 87.5% of molars with enamel projections ( 98 of 112 ) had an associated fi . such a high percentage of association between the enamel projection and fi supports the view of a possible relationship , which may lead to the progression of periodontal disease . whether or not , there is a cause and effect relationship , requires further investigation . a total of 14 molars out of 112 molars ( 2.5% ) with cep had no associated fi . this was to be expected since there are a number of factors such as the presence of occlusal facets ( trauma from occlusion ) , thickness of alveolar housing , variations in the root - trunk length and severity and extension of gingival inflammation which can contribute to periodontal breakdown in these areas . master and hoskins , suggested a classification system based on the extent of cep into the furcation area . they used the adjacent cej and the furcation area as reference points to classify cep as grade i , ii and iii [ figure 1 ] . hou and tsai and swan and hurt , reported cep grade iii to be the most commonly found cep . in the present specimens , grade iii was the most prevalent ( 83.7% ) and was associated with fi which shows the maximum potential of grade iii cep for the fi . this can be explained by the mechanism that connective tissue can not form an attachment to enamel . instead the junctional epithelium is present in these areas and consists of hemidesmosomes and basal lamina . as a result , when enamel forms on roots ( more in grade iii ) , it may predispose the area to increased probing depth in the presence of gingival inflammation . together with its plaque one more new result found in the present study was the significantly more prevalence of cep in male skulls ( 77.4% ) than in female ( 20.4% ) . no significant difference was found between the right and left maxillary and mandibular teeth with cep and fi . exact reason behind this finding is unclear , but the possible role of genetics should be ruled out in future studies as bilateral occurrence of cep was reported by one case report . these results are not comparable as none of the previous studies have evaluated this aspects of cep . since bissada et al . had experienced great difficulty in detecting selected teeth with cep radiographically , in the present study the authors have not included such an identification in this manner ; in fact , through clinical examination is more reliable for their detection . a total of 944 maxillary and mandibular molar teeth present in indian human dry skulls were examined for the incidence and location of cep . under the limitations of the present study , we have drawn the following conclusions : the prevalence of ceps in molars was 11.9%ceps were found more frequently in the mandibular than in the maxillary molars ( 2:1)the highest prevalence of cep was found in the mandibular second molar ( 14.7% ) followed by the maxillary second molar . the mandibular third molar showed the lowest incidence ( 5.5%)the association between cep and fi ( 87.5% ) was statistically significant . this favors the view of the possible role played by such anomalies in the progression of periodontal diseasescep in male skulls ( 77.4% ) was significantly more prevalent than in female skulls ( 20.4%)no significant difference was found between the right and left maxillary and mandibular teeth with cep and fi . the prevalence of ceps in molars was 11.9% ceps were found more frequently in the mandibular than in the maxillary molars ( 2:1 ) the highest prevalence of cep was found in the mandibular second molar ( 14.7% ) followed by the maxillary second molar . the mandibular third molar showed the lowest incidence ( 5.5% ) the association between cep and fi ( 87.5% ) was statistically significant . this favors the view of the possible role played by such anomalies in the progression of periodontal diseases cep in male skulls ( 77.4% ) was significantly more prevalent than in female skulls ( 20.4% ) no significant difference was found between the right and left maxillary and mandibular teeth with cep and fi .

feed is a risk factor for poisoning the farm environment thus also fresh milk with pathogenic microorganisms of listeria genus species . listeria ivanovii , listeria innocua , and listeria seeligeri were isolated from 9.2% , but listeria monocytogenes from 20.0% of feed samples . most often different fodders ( 9.3% ) and silage ( 4.7% ) were contaminated with listeria monocytogenes . listeria genus species were isolated more often from feed prepared and used in organic dairy farm than from that used in conventional dairy farm , correspondingly 44.4% and 18.3% . no listeria monocytogenes was found in bulk milk samples of organic dairy farm .

we had 7 040 dairy herds with 121 945 cows in latvia at the 2010 . production level of milk products has been increased from 93.9 million lvl at 2002 to 218.2 million lvl at 2008 . enhanced nutrition qualities , taste , and health benefits have all been advocated as reasons for increase interest in raw milk consumption . unfortunately , milk is a good source of nutrients and edible energy not only for humans but also for numerous microorganisms , which thus can grow in milk . these microorganisms are primarily bacteria , but some moulds and yeasts can also grow in milk . the presence of several species of microorganisms in raw milk is undesirable , either because the organisms can be pathogenic , or because their growth results in undesirable transformations in the milk [ 2 , 3 ] . it is known that bacterial toxins may act as very danger food poisoning substances . the often listed pathogens in raw milk are staphylococcus aureus , escherichia coli , salmonella spp . , yersinia enterocolitica , aeromonas hydrophila , brucella abortus , campylobacter jejuni , bacillus cereus , and listeria monocytogenes ( l. monocytogenes ) , among them [ 3 , 5 , 6 ] . monocytogenes , l. ivanovii , l. innocua , l. seeligeri , l. welshimeri , and l. grayi . the first contains l. monocytogenes , l. innocua , and l. welshimeri , the second group l. ivanovii and l. seeligeri , and the third l. grayi . only two , l. monocytogenes and l. ivanovii , are pathogenic for humans and animals [ 8 , 9 ] . the cellular behaviour of l. ivanovii is quite similar to that of l. monocytogenes , and the virulence gene cluster of l. monocytogenes is present in the other pathogenic species l. ivanovii . reveal that the ability to grow in the host cytoplasm cause vasodilator - stimulated phosphoprotein , which is characteristic both for l. monocytogenes and l. ivanovii . l. monocytogenes is a high adaptable environmental bacterium capable of existing both as animal pathogen and plant saprophyte with powerful array of regulated virulence factors . at the same time it is potentially lethal foodborne pathogen commonly found in dairy cows ' environment in cow feces , silage , soil , water , and so forth . it is proved that l. monocytogenes grows into biofilms attached to the surfaces in food - processing plants [ 14 , 15 ] and milking systems in dairy farms . the common treatment of surfaces is not effective to eliminate this dangerous foodborne pathogen , and it easily can pass into raw milk . l. monocytogenes can cause a rare but serious disease called listeriosis , especially among pregnant women , the elderly , or individuals with a weakened immune system . l. monocytogenes is more likely to cause death than other bacteria that cause food poisoning . 20 to 30% of foodborne listeriosis infections in high - risk individuals may be fatal . the presence of pathogens depends on ingestion of contaminated feed followed by amplification in bovine hosts and fecal dissemination in the farm environment . the final outcome of this cycle is a constantly maintained reservoir of foodborne pathogens that can reach humans by direct contact , ingestion of raw contaminated milk or cheese , or contamination during the processing of milk products . therefore it is essential to gather information about microbial risk factors and hazards associated with raw milk production . risk assessment and microbial monitoring will continue to play important role in ensuring food safety . critical control point management programmes created for individual milk production farms based upon risk analysis , total quality management and hazard analysis , and critical control point principles are essential for obtaining safe and healthy raw milk for consumers and for processing . the objective of this study was to clarify incidence of bacteria from the genus listeria int . foodborne pathogen l. monocytogenes in the feed and raw milk from one organic and three conventional dairy farms in latvia . the research was carried out from june 2008 to may 2010 . in total , 130 feed samples and 244 bulk tank milk samples from organic farm grantskalni and three conventional farms palsa , lacplesa piens , and robeznieki were analyzed . l. monocytogenes from feed and milk samples were isolated in accordance with international standard lvs en iso 11290 - 1+a1 microbiology of food and animal feeding stuffs horizontal method for detection and enumeration of listeria monocytogenes presumptive l. monocytogenes isolates were purified and confirmed by the fourier transform infrared spectroscopy ( ft - ir ) technique . identification of listeria species using ft - irsample preparation and measurement of ft - ir spectra were performed according to manufacturer 's instructions using an infrared spectrometer tensor 27 and software opus version 6.5 ( bruker optic gmbh , germany ) . the bacterial strains were subcultured on tryptone soya agar ( tsa , oxoid ) by incubating the plates at 37c for 24 h. bacteria inoculum was transferred from the tsa preculture onto the surface of the tsa plate with a loop and spread with spatula until homogeneity bacterial lawn ( one half of the agar plate is enough for each strain ) . prepared plates were incubated for 24 h at 30c . after incubation suspensions with 2 full loops of the bacteria scraped from the confluent lawn in 100 l distilled water in the eppendorf tubes were prepared . afterwards we homogenized this suspension with a vortex for 10 sec . , transferred 25 l of the suspension on the microtiter plate , and dried about 45 min . at 42c . for the ft - ir absorption measurements we used 32 scans , 6 cm resolution , 24 phase resolution , and repeated background position measurements between 4,000 and 500 identification of bacteria was based on bruker 's spectral library of listeria species which includes reference strains of l. monocytogenes , l. innocua , l. ivanovii , l. welshimeri , and l. seeligeri . sample preparation and measurement of ft - ir spectra were performed according to manufacturer 's instructions using an infrared spectrometer tensor 27 and software opus version 6.5 ( bruker optic gmbh , germany ) . the bacterial strains were subcultured on tryptone soya agar ( tsa , oxoid ) by incubating the plates at 37c for 24 h. bacteria inoculum was transferred from the tsa preculture onto the surface of the tsa plate with a loop and spread with spatula until homogeneity bacterial lawn ( one half of the agar plate is enough for each strain ) . prepared plates were incubated for 24 h at 30c . after incubation suspensions with 2 full loops of the bacteria scraped from the confluent lawn in 100 l distilled water in the eppendorf tubes were prepared . , transferred 25 l of the suspension on the microtiter plate , and dried about 45 min . at 42c . for the ft - ir absorption measurements we used 32 scans , 6 cm resolution , 24 phase resolution , and repeated background position measurements between 4,000 and 500 cm . identification of bacteria was based on bruker 's spectral library of listeria species which includes reference strains of l. monocytogenes , l. innocua , l. ivanovii , l. welshimeri , and l. seeligeri . l. monocytogenes prevalence were analyzed with statistical package for social sciences 17.0 for windows ( spss inc . , the bacteria of listeria species were detected in feed samples ( n = 130 ) in 38 cases or 29.2% . in 12 ( 9.2% ) these were l. innocua , l. ivanovii , and l. seeligeri , but in 26 cases ( 20.0% ) l. monocytogenes . bacteria of listeria genus can be widely found in nature in soil , on plants , in waters , on animal hair and birds ' bodies , and so forth . . l. ivanovii shares certain characteristics with l. monocytogenes ( e.g. , hemolysis ) and is occasionally associated with abortion in ruminants . therefore incidence of these two species in animal feed is a risk factor for presence of listeria in the farm environment , cow infections , their presence in milk and thus also in human body causing infections . animal and human pathogen l. monocytogenes was isolated from 26 feed samples ( 20.0% ) . milk plays important role in l. monocytogenes epidemiology [ 18 , 19 ] ; therefore it must be kept in mind that these dangerous bacteria are brought in the farm environment by contaminated feed and thus also on cow hair , udder , and teat skin and then also in milk . l. monocytogenes is known as cow mastitis , conjunctivitis , and other disease - causing pathogen microorganisms . it has been proved that listeria strains isolated from infections have been found also in farm environment in feces and silage which means that l. monocytogenes strains found in nature are virulent . as it can be seen , the data summarized in table 1 show that none of 14 different grass samples contains bacteria of listeria species . thus , grass has not been in contact with dung , wild animal feces , which can cause the presence of listeria in the soil and on plants . silage is considered as the main source of l. monocytogenes and other listeria genus bacteria in farm environment . however , neither silage ( n = 48 ) nor haylage ( n = 16 ) were highly contaminated in our study ; it ranges from 1.5 to 4.7% ( table 1 ) . dry food products were contaminated with l. monocytogenes : feed mixture prepared in the farm1.5% , different fodder products ( different corns , rape cakes , etc.)9.3% , straw1.5% , and hay1.5% of all tested samples ( the tested straw and hay were stored at the field during winter therefore exposed to long - term impact of environment . hay used as fodder might have been contaminated on the field by bird and animal feces . listerias are gram - positive bacteria with different structure and chemical content of its cell wall and thus more resistant than gram - negative ones . listeria cell wall protects the inner content of the cell against impact of external mechanical and osmotic force , insufficient humidity and ph level , and other damaging growing and reproduction factors . the cell wall of gram - positive bacteria consists of peptidoglycan which is associated with teichoic acids and lipoteichoic acids in complex multilayer structure , while the cell wall of gram - negative bacteria consists of one peptidoglycan layer which is covered with a membrane [ 5 , 23 ] . thus , the cell wall of gram - positive bacteria , including listeria is tenfold thicker than the cell wall of gram - negative bacteria , and they are much viable in external environment , as well as considerably resistant to disinfectants use for cow teat treatment before milking . it is able to grow at temperatures from + 1c , they can be considered as psychrophilic microorganisms capable of surviving , growing , and multiplying in feed also in autumn and winter , when straw , hay roll , and hay are on the field or under open sheds . table 2 shows summarized obtained data on incidence of listeria spp . and l. monocytogenes in organic and conventional farms and feed used in different seasons . the data summarized in table 2 show that feed was free of listeria during summer in both organic and conventional farms . is most likely isolated from feedstuff in winter and autumn than it is in summer [ 25 , 26 ] . l. monocytogenes are found more often in feed prepared in organic farm ( correspondingly 14.8% and 29.6% ) than in feed used in conventional farms ( correspondingly 5.2% and 13.1% ) . results are logical as , according to the legislative acts of the european union , organic farms should use as less chemical substances as possible ; for example , ferments , yeast , and bacteria should be used as silage additives instead of chemical preservatives thus limiting multiplication of pathogenic bacteria . although different feed samples contained l. monocytogenes , no such bacteria were found in samples of bulk milk from organic farm ( n = 33 ) , but in samples of bulk milk from conventional farm l. monocytogenes was found three times or in 1.4% of all cases ( fernandez et al . has isolated l. monocytogenes from 3.0% ( n = 140 ) , jayarao and henning from 3.8% , but vilar et al . from 6.1% of bulk milk samples . the obtained results show that accurate observing of hygiene standards concerning treatment of cows ' udder and teats , as well as proper washing and cleaning process of milking system pipe lines and cooling tanks , protects the milk against bacteria . different type of feed is a risk factor for poisoning the farm environment thus also poisoning fresh milk with pathogenic microorganisms of listeria genus species in both organic and conventional farms . listeria ivanovii , listeria innocua , and listeria seeligeri were isolated from 9.2% , but listeria monocytogenes from 20.0% of feed samples . most often different feed concentrates ( 9.3% ) and silage ( 4.7% ) were contaminated with listeria monocytogenes . listeria genus species were isolated more often from feed prepared and used in organic dairy farm than from the feed used in conventional dairy farms , correspondingly 44.4% and 18.3% . no listeria monocytogenes were found in bulk milk samples from organic dairy farm ( n = 33 ) , but they were found three times in samples of conventional dairy farms ( n = 211 ) .

mucous membrane pemphigoid describes a rare heterogeneous group of chronic , inflammatory , mucous membrane - dominated , subepithelial blistering diseases that manifest a varying constellation of oral , ocular , skin , genital , nasopharyngeal , esophageal , and laryngeal lesions . life - threatening airway obstruction and sight - threatening ocular scarring can occur in this condition , which is rarely reported in indian literature . vitiligo is another acquired autoimmune disorder characterized by loss of melanocytes . vitiligo is associated with a number of disorders also considered to be autoimmune . here we report a very rare coexistence of mmp and vitiligo , the first such report from india .

mucous membrane pemphigoid ( mmp ) is a putative autoimmune , chronic inflammatory , subepithelial blistering disease predominantly affecting mucous membranes . mucous membranes that may be involved include the oral cavity , conjunctiva , nasopharynx , larynx , esophagus , genitourinary tract and anal canal . a 37-year - old male patient presented with complaints of redness and pain in his left eye since two years , painful erosions in the oral cavity since six months , and ulcers over the scrotum and right knee since two months . on ocular examination , there was congestion of the left sclera mainly on the lateral side along with a fibrotic band connecting the upper palpebral conjunctiva with the bulbar conjunctiva near the lateral canthus [ figure 1 ] . on examination of the oral cavity there were widespread erosions involving the inner side of lips , gingival , and labial mucosa [ figure 2 ] . detailed examination revealed a single depigmented macule over the glans penis [ figure 3 ] , which according to the patient appeared insidiously around 8 years ago and remained static . a tzanck smear from the oral erosions , done to rule out pemphigus vulgaris , failed to show any acantholytic cells . in addition there was involvement of nasal mucosa , larynx , scrotal skin , perianal skin , and right knee [ figure 2 ] . a skin biopsy from the right ear showed subepidermal bullae and fibrosis beneath it [ figures 4 and 5 ] . the direct immunofluroscence of perilesional uninvolved skin showed strong linear deposits of igg along the basement membrane zone . detailed hematological , biochemical , and imaging studies did not reveal any evidence of malignancy . the long - standing depigmented macule over the glans penis did not show any evidence of atrophy , ruling out lichen sclerosus et atrophicus . mmp lesions affecting the left eye mmp lesions affecting the oral mucosa , scrotum and knee ( clockwise ) classical vitiligo affecting the glans penis subepidermal bulla with fibrosis beneath it , black arrow demonstrates the site of blistering ( h and e , 10 ) subepidermal bulla ( h and e , 40 ) mmp is described as a heterogeneous group of chronic , inflammatory , mucous membrane - dominated , subepithelial blistering diseases that manifest a varying constellation of oral , ocular , skin , genital , nasopharyngeal , esophageal , and laryngeal lesions . life - threatening airway obstruction and sight - threatening ocular scarring can occur in this condition , also known as cicatricial pemphigoid , benign mmp and incorrectly as ocular pemphigoid . these include bullous pemphigoid antigen 1 ( bpag1 , 230 kda ) , bullous pemphigoid antigen 2 ( bpag2 , 180kda ) , laminin 5 , laminin 6 , 6-integrin subunit , 4-integrin subunit , collagen vii , and other proteins of unknown identity and/or function . scarring is common at non - oral sites of involvement , contributing to disease - related morbidity . the choice of agents for treatment of mmp is based upon the sites of involvement , clinical severity , and disease progression . found there is an association with autoimmune diseases , both organ and non - organ - specific in a group of 34 patients with cicatricial pemphigoid suggesting a possible genetic basis for the association . autoimmune mechanisms with an underlying genetic predisposition are the most likely causes of vitiligo , although neurohumoral and autocytotoxic hypotheses are alternative theories or contributing mechanisms . previously association of vitiligo with bullous pemphigoid either alone or along with other autoimmune diseases have been reported . gaspar et al . reported a case of a 68-year - old woman with vitiligo , primary hypothyroidism and cicatricial pemphigoid with severe laryngeal involvement necessitating tracheostomy . a thorough search failed to reveal any other report of co - existence of these two conditions in the published literature . we believe that this is the first case report of vitiligo coexistent with mucous membrane pemphigoid from india . the co - existence of these two conditions is probably is related to their common autoimmune etiology .

due to lack of evidence from prospective clinical trials , the diagnostic procedures , their frequency , as well as the length of the follow - up period in cutaneous melanoma patients should be based on the individual risk of disease recurrence , which is strongly dependent on the stage of disease at the time of diagnosis . in the paper we propose the current recommendations for follow - up strategy . nowadays , new effective treatment options with biological agents justify the closer monitoring of high risk melanoma patients .

this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and the source are credited .

purposeto compare the effectiveness of intravitreal injections of bevacizumab and ranibizumab in patients with treatment - naive polypoidal choroidal vasculopathy ( pcv).methodsrecords from 106 consecutive patients who received intraviteral bevacizumab ( n = 58 , 1.25 mg ) or ranibizumab ( n = 52 , 0.5 mg ) for treatment of pcv were retrospectively reviewed . after three initial monthly loading injections , injection was performed as needed . the main outcome measures included best - corrected visual acuity ( bcva ) , foveal central thickness ( fct ) as assessed by spectral domain optical coherence tomography , and the changes in polypoidal lesions based on an indocyanine green angiography.resultsthe average number of injections was 3.31 1.25 in the bevacizumab group and 3.44 0.92 in the ranibizumab group . mean logarithm of the minimum angle of resolution of bcva from baseline to 6 months after injection improved by 0.17 in the bevacizumab group ( p = 0.03 ) and by 0.19 in the ranibizumab group ( p = 0.01 ) . average fct decreased from 322 62.48 m to 274 40.77 m in the bevacizumab group ( p = 0.02 ) and from 338 50.79 m to 286 36.93 m in the ranibizumab group ( p = 0.02 ) . polyp regression rate was 20.7% ( 12 of 58 eyes ) in the bevacizumab group and 21.2% ( 11 of 52 eyes ) in the ranibizumab group . there was no statistically significant difference between groups in bcva improvement achieved , fct improvement achieved , and polyp regression rate between groups.conclusionsintravitreal injections of bevacizumab and ranibizumab have similar effects in stabilizing of visual acuity , macular edema , and regression of polypoidal complex in pcv eyes over the short term .

after obtaining approval by the institutional review board , a retrospective medical record review was conducted using records from 106 consecutive patients ( 110 eyes ) who were treated with intravitreal anti - vegf agents for pcv at kim 's eye hospital , konyang university college of medicine from november 2008 to june 2010 . patients were included if they met all of the following criteria : 1 ) confirmation of pcv with fluorescein angiography ( fa ) and indocyanine green angiography ( icga ) performed using a confocal laser scanning system ( hra-2 ; heidelberg engineering , dossenheim , germany ) at the first visit . we only included patients whose icga showed a branching vascular network with polypoidal shaped choroidal vascular lesions , 2 ) patients who were treated with only one type of anti - vegf agent ( either bevacizumab or ranibizumab ) , and 3 ) a minimum follow - up period of 6 months . exclusion criteria were the following : 1 ) combination therapy of more than one anti - vegf agent , 2 ) prior treatment with pdt , 3 ) pathologic myopia , 4 ) idiopathic choroidal neovascularization ( cnv ) , 5 ) other secondary cnv , 6 ) other ocular disease that could affect visual acuity , 7 ) trauma during the study or in the fellow eye , 8) aphakia , or 9 ) previous vitreoretinal surgery . after the initial loading injections at the time of diagnosis , retreatment for each patient was planned as a ' retreat as needed ' protocol . anti - vegf agents were re - injected on a monthly basis if objective visual deterioration of more than two lines , persistent exudates and hemorrhage , or evidence of an active pcv lesion were observed on fa , icga , or optical coherence tomography ( oct ) , were observed . best - corrected visual acuity ( bcva ) was obtained every one or two months using the snellen chart . patients also underwent an ophthalmic examination , including a slit - lamp evaluation and fundus examination , as well as oct ( spectral oct / slo ; oti ophthalmic technologies inc . , miami , fl , usa ) , fa , icga , or a combination thereof . foveal center thickness ( fct ) was assessed by oct using six diagonal fast and slow 6-mm scans . the off - label nature of the treatment and its potential risks and benefits were discussed in detail with all patients , and signed informed consent was obtained from all patients . patients received 1.25 mg of bevacizumab or 0.5 mg of ranibizumab . prior to administration of the injection , topical anesthesia was applied , and 10% povidone - iodine was used for scrubbing of eyelid and lashes . following the application of povidone - iodine eyedrops ( 1.25% ) , a sterile lid speculum was placed into place . intravitreal injection was performed with a 30-gauge needle at 3.5 to 4 mm from the inferotemporal limbus . pressure was applied to the injection site using a sterile cotton swab , for 1 minute . the changes in bcva and fct between baseline and the 6 month follow - up were analyzed with a 1-tailed , paired t - test . for continuous variables , medians for baseline , final values , change , and percent change were compared between bevacizumab and ranibizumab treatment groups using a t - test or a wilcoxon rank sum tests . the off - label nature of the treatment and its potential risks and benefits were discussed in detail with all patients , and signed informed consent was obtained from all patients . patients received 1.25 mg of bevacizumab or 0.5 mg of ranibizumab . prior to administration of the injection , topical anesthesia was applied , and 10% povidone - iodine was used for scrubbing of eyelid and lashes . following the application of povidone - iodine eyedrops ( 1.25% ) , a sterile lid speculum was placed into place . intravitreal injection was performed with a 30-gauge needle at 3.5 to 4 mm from the inferotemporal limbus . pressure was applied to the injection site using a sterile cotton swab , for 1 minute . the changes in bcva and fct between baseline and the 6 month follow - up were analyzed with a 1-tailed , paired t - test . for continuous variables , medians for baseline , final values , change , and percent change were compared between bevacizumab and ranibizumab treatment groups using a t - test or a wilcoxon rank sum tests . bevacizumab - treated and ranibizumab - treated patients had similar baseline characteristics for age , sex , distribution of baseline bcva , location of polyps , or fct ( table 1 ) . all patients were korean , and no systemic adverse events were recorded for any of the patients treated with intravitreal injection . no complications , including issues such as endophthalmitis , traumatic lens injury , or retinal detachment , were associated with intravitreal injections . the average number of injections was 3.31 1.25 in the bevacizumab group and 3.44 0.92 in the ranibizumab group . at baseline , the mean bcva ( standard deviation , sd ) in the bevacizumab and ranibizumab groups was 0.92 ( 0.54 ; snellen equivalent , 20 / 166 ) and 0.96 ( 0.57 ; snellen equivalent , 20 / 182 ) , respectively . six months after treatment , both bevacizumab and ranibizumab groups had significantly increased bcva to 0.74 ( 0.51 ; snellen equivalent , 20 / 110 ; p = 0.03 ) and 0.78 ( 0.43 ; snellen equiva lent , 20 / 120 ; p = 0.01 ) respectively ( table 2 , figs . 1 and 2 ) . there was no statistically significant difference in bcva improvement achieved between these two groups ( p = 0.83 ) . six ( 10.3% ) eyes out of 58 eyes in the bevacizumab group and 5 ( 10.0% ) eyes ( 10.0% ) out of 52 eyes in the ranibizumab group showed a loss of 3 lines of visual acuity . in either group , no significant difference in proportion of more than 3 lines of visual acuity loss was observed ( p = 0.82 ) . there was also no significant difference in proportion of more than 3 lines of visual acuity gain in either group ( p = 0.12 ) ( table 3 ) . mean ( sd ) fct at baseline in the bevacizumab and ranibizumab groups was 322 ( 62.48 ) m and 338 ( 50.79 ) m , respectively . six months after treatment , fct of both the bevacizumab and ranibizumab groups were significantly decreased to 274 ( 40.77 ) m ( p = 0.02 ) and 286 ( 36.93 ) m ( p = 0.02 ) , respectively . there was no statistically significant difference in reduction of fct in either group ( p = 0.74 ) ( table 2 ) . twenty four of 58 eyes ( 41.4% ) in the bevacizumab - treated group showed a decrease of more than 10% from baseline fct . thirty one eyes out of 52 eyes ( 59.6% ) in the ranibizumab - treated group showed a decrease of more than 10% from baseline fct . no significant difference in the proportion of decrease greater than 10% from baseline fct was observed in either group ( p = 0.35 ) . however , a greater number for 10% decreased fct was observed in the ranibizumab group , and the difference was statistically significant ( p = 0.02 ) ( table 3 ) . polyp regression was found in 12 of 58 eyes ( 20.7% ) in the bevacizumab group and in 13 of 52 eyes ( 25.0% ) in the ranibizumab group . no significant difference in polyp regression rate was observed between groups ( p = 0.87 ) ( table 3 ) . pcv is increasingly recognized as a major cause of vision loss throughout the world ; however , the incidence of pcv is especially high in asian countries and in asian people throughout the world . the most important causes of severe visual loss include repeated subretinal hemorrhage and leakage from pcv lesions , repeated injuries resulting in atrophy of rpe , or scar change . although the pathophysiology of pcv is poorly understood , resolving subretinal hemorrhage and decreasing leakage from pcv lesions may be an important and reasonable approach to stabilize visual acuity . additionally , vegf concentrations in the aqueous have been found to be markedly increased in pcv eyes , and a strong expression of vegf was observed in pre cells of pcv specimens . collectively , evidence appears to support the success of anti - vegf treatment for pcv eyes . ranibizumab , which is specifically for intraocular use , has several theoretical advantages over bevacizumab . ranibizumab is a humanized anti - vegf antibody that inhibits all forms of biologically active vegf - a ; treatment with ranibizumab appears to significantly decrease bleeding and exudation in pcv . bevacizumab ( a humanized full length anti - vegf antibody ) also appears to have a treatment effect in pcv eyes . considering the molecular weight of each medication ( ranibizumab is a 48-kda fab fragment , whereas bevacizumab is a complete 149-kda antibody ) , ranibizumab may be more effective in treatment of pcv because of its smaller molecular weight and possible deeper penetration to choroidal vascular abnormality lesions in those with pcv . moreover , ranibizumab is affinity - matured and may provide better vegf inhibition through stronger molecular binding , compared with bevacizumab . in comparison , penetration of the neural retina up to the choriocapillaries by intravitreal bevacizumab has been demonstrated . and therefore , it may be possible that the clinical efficacies of these two drugs might differ . in the current study , both drugs significantly improved visual acuity in the short term , and showed similar increases in their mean visual acuity from baseline . however , despite the lack of power to determine small changes in visual acuity between bevacizumab and ranibizumab , our results revealed a trend toward a greater number for 3-line improvements in the ranibizumab group . although it is uncertain what factors made this difference in visual acuity between the two groups , these results showing a significantly greater number for 10% decreased cft in the ranibizumab group could have an association . macular edema based on fct significantly improved in both groups and showed similar decreases in mean fct from baseline . a similar decrease in macular edema was noted after three monthly bevacizumab injections in a study by lai et al . . additionally , kokame et al . reported improvement in macular edema after 6 monthly ranibizumab injections . findings from these previous reports as well as those of our study collectively suggest that short - term continuous anti - vegf therapy doses do have a significant effect in reducing macular edema but not in all pcv eyes . in this study , our results revealed a significantly greater number of patients who showed 10% decrease in fct in the ranibizumab group compared to that in the bevacizumab group . it may be that the difference in changes of fct between the two groups resulted from differences in penetration ability ; ranibizumab may have been more anatomically effective than bevacizumab in reducing macular edema with pcv lesions . further , these results imply that there is a distinct difference in short - term biologic activity between bevacizumab and ranibizumab . the choroidal vascular branching network and polypoidal complexes have been resistant and poorly responsive to anti - vegf therapy with bevacizumab or ranibizumab . in the gomi et al . and the gomi et al . studies , choroidal vascular abnormalities remained in ten of 11 eyes after one to three intermittent injections of bevacizumab . kokame et al . found that polypoidal complex decreased in four of 12 eyes ( 33% ) after 6 continuous monthly ranibizumab injections . in the current study , polypoidal lesions appeared to be resistant to both anti - vegf agent , and the polypoidal complex showed a decrease in only 12 of 58 eyes ( 20.7% ) in the bevacizumab group and 13 of 52 eyes ( 25.0% ) in the ranibizumab group . even though ranibizumab has a theoretically better ability to penetrate through the retina and rpe to the choroidal vascular abnormalities of pcv the location of the pcv vessels beneath the rpe may prevent sufficient penetration of anti - vegf drugs to induce pcv regression . this result suggests that pcv may be a different inner choroidal vascular abnormality and not just a variant of choroidal neovascularizatoin ( cnv ) . in recent studies , pdt has shown good results in reduction of leakage and regression of polyps in pcv eyes . particularly , in the everest study , the first randomized and prospective study of pcv treatment , a pdt combination of ranibizumab and pdt monotherapy was effective in completely regressing polyps at month 6 than was ranibizumab monotherapy . however , there was no significant difference in improvement of visual acuity from baseline between pdt combination with the ranibizumab group and the ranibizumab monotherapy group . in addition , severe visual loss due to extensive subretinal hemorrhage is not uncommon after pdt , and pdt itself can result in a temporary increase in vegf . in terms of visual outcome , despite weakness in polyp regression , anti - vegf monotherapy could be considered for pcv in cases with minimal polyp lesions or in cases with only a branching vascular network . we await long - term results of the everest trial , which can confirm which modality is superior for treatment of pcv . more clinical and basic science studies are necessary to clarify the pathogenesis of pcv and to provide therapeutic guidelines . because of the retrospective nature of the study the inherent bias that exists in this study . and because the treatment choice was left to the discretion of the patient and treating physician , some potential for bias does exist . however , in our institute , the preferred pcv treatment with anti - vegf ( except for pdt ) shifted from bevacizumab to ranibizumab from 2008 to 2009 . almost all patients were treated with bevacizumab from 2008 to the first half of 2009 and with ranibizumab from the second half of 2009 to date . as a result , this study could be more comparative . moreover , the similarity in baseline characteristics between the two groups suggests that the groups were well balanced . another limitation in this study was the absence of a strict protocol for measuring visual acuity , which led to some of the variances in visual acuity that were noted in the two groups and may limit interpretation of these visual acuity results . however , we were able to significantly identify trends in visual improvement after anti - vegf injection for pcv eyes . a planned randomized , controlled study would be necessary for a more precise determination of the differences between these two treatments . in summary , bevacizumab and ranibizumab have similar effects on stabilization of visual acuity and macular edema with pcv eyes in the short term . however , ranibizumab appears to be superior to bevacizumab with regard to short - term ability to decrease exudation . additionally , there is a trend suggesting that ranibizumab may also provide superior visual acuity . although this study could not provide definite proof that there were significant differences in the visual acuity as result of pcv eyes treated with bevacizumab or ranibizumab , these results appear to demonstrate a possible difference in the biologic activities of the two anti - vegf agents . these results should be considered clinically when performing combination therapy with pdt or when deciding on a course of anti - vegf agent for treatment of pcv eyes .

despite numerous cases of human infection with streptococcus suis worldwide , human disease is rarely diagnosed in north america . we studied 73 swine - exposed and 67 non swine - exposed us adults for antibodies to s. suis serotype 2 . serologic data suggest that human infection with s. suis occurs more frequently than currently documented .

to test the hypothesis that human infections with s. suis occur more commonly than currently recognized , we examined archived serum samples from 73 swine - exposed and 67 non swine - exposed adults living in iowa ( 9 ) . these persons had all previously completed occupational history questionnaires detailing their pig exposure and use of personal protective gear . use of materials was approved by the institutional review board at the university of iowa . antibodies to serotype 2 s. suis were measured by an elisa that used whole s. suis cells ( serotype 2 , strain 3033606 ) as antigen ( 10 ) . the elisa optical density readings for the 73 swine - exposed study participants and 67 non swine - exposed participants were first compared to the positive - control mean option density read per plate per dilution . optical densities greater or equal to mean positive - control optical density were considered positive . to be conservative , again , when a conservative approach was used , the lowest titer among duplicates was considered as the final antibody titer . we used the fisher exact test to test the null hypothesis that the exposed group does not have higher incidence of antibody titer > 10 than the non swine - exposed group does . we tested a similar hypothesis for specific risk groups exposed to swine ( such as nursing or finishing swine , use of gloves ) compared with groups not exposed to swine . seven ( 9.6% ) of 73 swine - exposed study participants were positive , and 1 ( 1.5% ) of the 67 non swine - exposed participants was positive . study participants who work with both finishing and nursery swine had 8.8 the odds of having a titer > 10 when compared to nonexposed study participants ( exact 95% confidence interval 1.1406.3 ) . we identified no positive persons in the group that worked solely with nursery swine , a somewhat unexpected finding because most s. suis disease occurs in young pigs . however , our study had relatively few persons who worked exclusively with nursery swine ( 11/73 ) ; most participants worked with both nursery and finishing swine . additionally , no farm - level data on prevalence of s. suis where these persons were employed were collected ; therefore , whether those persons worked on farms where s. suis had been confirmed is not known . other factors such as age , gender , use of tobacco products , and use of gloves when working with animals were not statistically significant ( table ) . * an antibody titer was considered positive when its optical density was greater or equal to mean positive control optical density . in this cross - sectional pilot study , we found that more swine - exposed persons had higher titers of antibodies to s. suis than did non these data suggest that human infection with s. suis is more common in the united states than currently thought . two possible reasons stand out regarding the lack of human s. suis disease in the united states . one possibility is underdiagnosis or misdiagnosis , rather than a true absence of the disease ( 11 ) . supporting this hypothesis are reports showing that s. suis has been mistaken for enterococci , listeria spp . , viridans streptococcus , or streptococcus pneumoniae ( 7,8,11 ) . because of this potential misclassification , previous publications have asserted that s. suis should be considered in the differential diagnosis of septicemia , especially when complicated by meningitis in adults with a recent history of contact with pigs or unprocessed pork ( 12 ) . a second possibility is that s. suis strains colonizing swine in the united states may be less virulent than asian strains and therefore unlikely to cause overt human disease even when transferred between species . this possibility is supported by molecular analyses showing that many us strains belong to sequence type ( st ) 25 , whereas most virulent serotype 2 isolates have been st1 ( 13 ) . finally , these hypotheses are not mutually exclusive ; both underdiagnosis / misdiagnosis and the circulation of lower - virulence strains may be occurring , resulting in fewer diagnoses of human s. suis infections in north america ( 11 ) . s. suis infection is an important occupational disease in humans in many countries . in research conducted in s. suis endemic countries , the annual incidence of s. suis meningitis was 3 cases/100,000 swine - exposed people : roughly 1,500 higher than the rate in the nonexposed population ( 14 ) . because of this risk , it has been recommended that persons in daily contact with pigs or pig meat should use protective gloves to avoid skin trauma and subsequent risk for exposure to the bacterium . because no human vaccine against s. suis exists , suitable preventive measures coupled with education and supervision of those who come in contact with live swine or unprocessed pork are important to decrease the transmission of this organism to humans . however , few studies have been conducted to detect subclinical cases of s. suis infection ; therefore , the true incidence of infection among the swine - exposed is unknown . because our findings only examined 1 serotype of s. suis , our results may not accurately reflect antibody prevalence . because we used a whole - cell elisa , some antibody reactions may be due to cross - reacting antibodies to other serotypes of s. suis or other species of streptococcus . however , using a slightly different method and population , other investigators found a higher seroprevalence , particularly among farmers and meat inspectors ( 15 ) . this difference may stem partly from the fact that we used a conservative criterion for considering a sample positive , which may further underestimate seroprevalence in our group . for example , 4 study participants ( 3 swine exposed ) were classified as having a titer < 10 because the first dilution ( 1:10 ) was negative in 1 repeat test . acquisition of human positive control serum as a standard to test our assay would enable us to make more definitive comparisons . finally , because the samples analyzed for this pilot study were not collected to specifically assess s. suis infections , more definitive future prospective studies seem indicated . one limitation of this serologic study is that it does not enable us to distinguish antibodies generated as a result of true infection versus exposure to s. suis antigens present in manure or dust in the facility , for example . additionally , because the questionnaire did not include information on pork consumption or handling of raw pork , those factors could not be examined as potential risks . future studies might include targeted questionnaires , attempts of bacterial isolation , and serial sera collections to examine serologic evidence of infection .

background : the number of women who select cesarean section due to fear of childbirth has increased . role play education seems to be a helpful method to remove or reduce the fear of childbirth . therefore , this study aimed to investigate the effect of role play education on primiparous women 's fear of natural delivery and their decision on the mode of delivery.materials and methods : in this blind clinical trial , 67 primiparous women with natural pregnancy at 3436 weeks of gestational age and with no indication of cesarean section were selected from the health care centers in mashhad . they were randomly assigned to two groups who underwent pre - test and post - test with the help of delivery attitude questionnaire to investigate their fear of childbirth and a researcher - made pregnant women 's decision investigation questionnaire . education through role play was conducted in the form of three scenarios during seven stages . the findings were analyzed by fisher 's exact test and independent t - test through spss.results:the two groups were significantly different concerning the fear of childbirth after the intervention ( p = 0.007 ) , and the fear score showed a higher reduction in the role play group compared to the lecture group . there was a significant difference between the two groups concerning the reduction of elective cesarean section and the decision on the mode of delivery at the time of admission in the labor room ( p = 0.000 ) . about 75% in the lecture group and 100% in the role play group selected natural delivery.conclusions:in the present study , the effect of role play was more in making a decision on natural delivery , reducing the fear of childbirth , and reducing the rate of elective cesarean section . it is suggested to use role play method to educate pregnant women to reduce the rate of cesarean sections .

from the time of adam 's creation , god has gifted humans with delivery to have offspring . it is a spontaneous process that needs no intervention , and cesarean section ( cs ) is conducted only when natural delivery is contraindicated to protect mother 's and infant 's health . nowadays , unfortunately , having a cs has become a culture for escaping pain and has influenced public health . this is when cs imposes more risk to the mother , compared to vaginal delivery , and includes complications such as endomyometritis , bleeding , thromboembolism , preterm labor and mortality ( in mother ) , and respiratory distress syndrome , resistant pulmonary hypertension , and damages like injury , bruise , or other traumas in infant . the incidence of cesarean delivery ( cd ) is 1015% worldwide , while in some iranian cities it is reported as 47% . some studies report that 65% of cs conducted in iran are unnecessary and optional ( elective cesarean ) , which may be due to lake of knowledge , negative attitude toward normal delivery , fetal health , prevention of urogenital lacerations , fear of pain , change in sexual relationships , persistence of spouse , insurance for expenses , and others experiences . nowadays , in most of the developed countries , it has been tried to reduce cs and its complications by interventions such as physicians education and change in their attitude . in iran for instance , tavasoli et al . reported a reduction in rate of cs by 15% through mothers education and their psychological and mental preparation to have a natural delivery . but despite these interventions , the rate of cs is growing in developing societies . one of the important factors that play an unnecessary role in increase of cs is fear of childbirth . there are notable evidences on the primary role of fear of childbirth in the demand for cs , as well as the effect of fear on negative experiences of delivery and emergency cs . about 36% of swedish women claimed fear of pain to be the main reason for selecting cs . the main challenge of a pregnant woman that results in their hesitation is taking decision on the mode of delivery . a person whose decision - making is a part of his / her existence experiences fear as a major problem in life . humans naturally fear of what is going to happen the next day or what he / she can achieve . fear , anxiety , and pain are three factors that play a key role at the time of delivery . some reports in sweden and finland showed that half of the women experiencing the fear of childbirth cancelled their request for cs and had a successful vaginal delivery after attaining the ability to express their anxiety and fear and having an interview with a trained person . one of the most effective ways to cope with fear is non - medicational methods including education and giving proper information to the pregnant women . research showed that an increase in knowledge and information influences individuals ability to recognize key points and enhances their understanding and perception . meanwhile , shortage of knowledge causes fear and anxiety which negatively affect decision - making . melender reported an association between absenteeism of primiparous women in delivery preparation classes and pregnancy anxiety , fear of childbirth , and consequently , a request for cs . recently , selection of the most efficient educational method with emphasis on learners skills , abilities , and active participation has been reported as one of the main and basic principles in health care . one of these methods is use of stories and scenarios which are adopted in role play education . role play is one of the new educational methods that tries to help individuals find their meaning in the social world and get help from a social group to make a decision on the solution of a dilemma . in this method , also , the learners discover human - related issues by facing problematic situations and discussing about these situations . nowadays , role play has found numerous applications such as acting as a tool for emotional drain and expression of fears and feelings , and a method for attitude change or formation of insight in referrals and a method for education of new behaviors . therefore , with regard to high prevalence of cs in iran and the effect of fear of childbirth on selection of this method , as well as the restricted research in this field and the mode of research ( descriptive research ) , this study aimed to investigate the effect of role play education on primiparous women 's fear of natural delivery and their decision on the mode of delivery . this blind clinical trial was conducted on 67 primiparous women ( 35 in role play group and 32 in lecture education group ) referring to the health care centers in three cities of mashhad province after obtaining the approval of ethics committee of mashhad university of medical sciences . as no report has been published in relation to the effect of role play on the fear of childbirth , based on a pilot study , sampling was by cluster sampling conducted among the health care centers of region number three . two centers were assigned to role play method , two to lecture , and from the remaining centers , three subcenters were assigned to role play and three to lecture method . to prevent bias in education , it was tried to conduct education in identical conditions concerning the place of education , level of noise , educational equipments , time of the day , etc . as the routine educational method in health care centers is lecture , there was no need for a control group . inclusion criteria were : no experience of acute psychological emotions , delivery and childbirth fear score > 28 , primiparous , single pregnancy , gestational age of 3436 weeks , age of 1835 years , no history of infertility , no indication for cs , and not having passed educational course for delivery methods . exclusion criteria were : some medical condition in pregnant woman , diagnosis of abnormal fetus / no possibility for delivery of fetus on sonography , and abnormal volume of amniotic fluid or placenta . to investigate pregnant women 's decision , a researcher - made checklist containing one question and to investigate the fear of childbirth , harman childbirth attitude questionnaire ( caq ) which was revised by lowe were adopted . firstly , two english language experts separately translated the questionnaire into persian and two others back - translated that into english . after finalization , the version obtained by the bilingual translator was compared with the original version of the questionnaire . the questionnaire has 14 items rated on a four - point likert 's scale ( never , seldom , sometimes , and often ) . as there was no cut - off point for the fear of childbirth , similar international studies were reviewed to determine the cut - off point and a median score of 28 was considered for fear of childbirth . for checking the validity of caq questionnaire , content validity , and reliability , cronbach 's alpha and internal consistency of the questionnaire were used ( a = 0.84 ) . validity of the decision - making checklist before intervention , 2 weeks after intervention , and of decision - making on the mode of delivery at admission in the labor were confirmed through content validity and their reliability was obtained by evaluators consensus ( r = 0.974 ) . the two groups of lecture and role playing were divided into four subgroups after taking pre - test . the role playing group was divided into two subgroups of 10 subjects each and another two subgroups of 9 subjects each ( 38 subjects ) . each group was instructed in a 90-min session about the advantages and disadvantages of normal delivery and cs . in this method , the researcher with two other co - researchers played three scenarios in seven steps ( for each scenario ) including warm up , selecting the participant , preparing the scene , preparing observers , play , discussion and evaluation , and generalization to education about the advantages and disadvantages of normal delivery and cs . in the warm - up stage , the researcher narrated two true stories about the individuals who were wondering about the selection of the mode of delivery due to fear of childbirth and asked the participants to voluntarily accept to play the role of pregnant woman with the researcher and two co - researchers . then the participants helped the researcher to prepare and process the scene ( scene preparation was conducted with the needed equipments for role play in two scenarios ) , and the observers were asked to pay close attention to the scenarios , taking important notes , and discuss them at the end of scenario . in scenarios , the reasons for mothers fear of natural delivery and cs were discussed . in the first scenario , one of the participants ( a pregnant woman ) played the role of a woman who referred to a midwife 's office to select the mode of delivery and witnessed the events occurring in the office . the midwife talked to her about the two types of delivery impractically and asked her to express her decision after the scenario about choosing the type of delivery . after choosing the type of delivery , participants discussed the pregnant woman 's selection ( same researcher gave no help ) and justified each other to come to a conclusion . the second scenario was about a woman with a normal delivery and the benefits and complications experienced by her . the next step was similar to the first scenario . in the third scenario , one of the co - researchers defended cs and another defended normal delivery at judge ( natural delivery and cs appeared like a human and judge is researcher ) . after these three scenarios , participants were asked to talk about their friends/relatives experiences of the two types of delivery . lecture group ( two subgroups of 10 subjects each and two subgroups of 9 subjects each ) was instructed using powerpoint presentation , marker , and whiteboard in a 90-min session . at the end of the session , two weeks after administration of educational session in each group ( lecture and role play ) , the caq was filled by the pregnant women to measure their fear , and further follow - ups concerning women 's decision on delivery mode continued at the time of admission and in the maternity unit . finally , the researcher recorded the favorite mode of delivery of participants through phone calls . data were analyzed by spss , and paired t - test , independent t - test , and chi - square were used for analysis . participants were aged 24 4 years , of whom 58% were housewives and 14.9% were employees . educational level of most of the participants ( 51.4% ) was up to high school . majority of their husbands had an educational level up to junior high school ( 38.8% ) and they were workers ( 44.3% ) . two groups showed no significant differences in terms of age , educational level of pregnant woman and her husband , income , job of pregnant woman and her husband , income , source of information about the type of delivery , insurance , time of referring for pregnancy care , and suggestion of mother , husband , friends , relatives , and gynecologist about the type of delivery ( p < 0.05 ) . table 1 shows a significant reduction in the mean score of fear about the mode of delivery after intervention in both groups of role play and lecture , compared to before intervention ( p = 0.001 in role play group and p = 0.047 in lecture group ) . the obtained results also showed a significant difference in the mean score of fear after intervention in both groups of role play and lecture ( p = 0.007 ) . this difference was not significant before intervention ( p = 0.074 ) [ table 1 ] . comparison of primiparous women 's mean scores of fear before and two weeks after intervention in two groups of lecture and role play fischer 's exact test was used for comparing the frequency of primiparous woman 's decision - making about the type of delivery before , 2 weeks after intervention , and at admission to maternity department . as to the results , no significant difference was found ; however , it was significant at admission ( p = 0.001 ) [ table 2 ] . comparing frequency of primiparous woman 's decision making about type of delivery before , two weeks after intervention and at admission to maternity department chi - square test showed no significant difference in the frequency of mode of delivery ( pregnant women 's function ) in the two groups ( p = 0.117 ) [ table 3 ] . comparison of frequencies of primiparous women 's mode of delivery in two groups of role play and lecture the obtained results showed that education through role play is effective on reduction of fear of childbirth and increase of natural delivery . although there was no significant difference in the mode of delivery in the present study ( p = 0.117 ) , the rate of elective cs in the lecture group was fivefold more compared to that in the role play group . the goal of antepartum education is to reduce mother 's request for elective cs , which was achieved in the present study . as no study was found on the effect of role play on pregnant women 's decision - making and fear , the results will be compared with the effects of other educational methods on fear and selecting the type of delivery . in the present study , the mean score of fear was higher in the lecture group after intervention , compared to the role play group . ryding states that the fear of delivery in third trimester can be associated with the occurrence of emergency cs . johnson and stlid showed no difference in the fear of delivery between women with natural delivery and those with elective or emergency cs , revealing the effect of fear of childbirth in various cultures . in a study on the effect of group therapy in reducing the fear of childbirth , reported that 85% of women cancelled their request for cs after intervention . mehdizadeh et al . , in a study on the effect of delivery preparation classes on reduction of cs , obtained similar results . meanwhile , fahami , in a study on the effect of lazmar exercises on the outcome of primiparous women 's pregnancy , found no significant difference in the rate of cs between the two educational groups of lazmar technique and control . a person whose decision - making is a part of his / her existence experiences fear as a major problem in his / her life . a fear - captivated mind is always wondering and in conflict , and is the main concern of pregnant women which brings about hesitation in decision - making on their mode of delivery . being bombarded with negative information and losing power , they experience high fear , which reduces their ability of correct decision - making . khorsandi et al . , in a study on the effect relaxation on reduction of cs , showed that relaxation is effective on reducing the fear of childbirth and increasing natural delivery . in khorsandi 's study , the mean scores of childbirth fear in relaxation and control ( receiving conventional care ) groups were 40.71 ( 6.23 ) and 39.35 ( 6.96 ) , respectively , before intervention and 38.03 ( 9.27 ) and 29.82 ( 7.18 ) , respectively , after intervention . the difference between the present study and that of khorsandi et al . was that in their study , each group separately received educational program ( including nine 90-min sessions ) from the second trimester and used actual play , role play , lecture , and educational cd for education on relaxation , but the control group received only routine care . meanwhile , in the present study , role play method was conducted just in one group to reduce pregnant women 's fear of childbirth and the education included one 90-min session . then , the obtained results were compared with those of lecture method of education . in the present study , the difference in fear score was significant in the two groups of role play and lecture , and the score of pregnant women 's fear was reduced leading to a reduction in an elective cs request at admission in the maternity unit and , consequently , a lower number of elective css . therefore , the present study managed to obtain acceptable results with less time consumed in educating mothers and lower educational costs , compared to the study of khorsandi . nowadays , role play has applications such as emotional drain , expression of fears and feelings , attitude change , making an insight in referrals , and education of new behaviors . in the present study , role play could drive pregnant women to select the best mode of delivery by giving them a way to empty their emotions and reduce their fear . in the lecture group , 25% of women probably and 40.6% two weeks after intervention , the possibility of a decision on natural delivery was reduced by 6.2% and its absoluteness increased by 31.3% . but at the time of admission in the labor ward , this possibility decreased by 3.2% , the absoluteness of natural delivery decreased by 12.5% , and the decision on cs increased , compared to before intervention . on the contrary , in role play group , 31.4% and 57.1% of women before intervention would possibly and absolutely select natural delivery , respectively . two weeks after intervention , 11.4% decrease of probable decision and 22.9% increase to absolute decision were observed . at the time of admission in the labor ward , therefore , role play could change pregnant women 's hesitation to decisiveness . in fathian 's study , behavioral intent model ( based on this theory , people firstly make decision and behave based on rational and logical review of available information , and secondly , people consider the outcomes and results of their function before making a decision ) was found to be effective in changing pregnant women 's attitude toward normal delivery , in comparison with routine pregnancy care ( p = 0.007 ) . in fathian 's study , 74.3% of pregnant women of the experimental group ( 25.7% absolute and 48.6% probable ) and 71.4% of the control group ( 30.0% absolute and 41.4% probable ) intended to undergo normal delivery before intervention . after intervention , normal delivery intention was 92.9% in the experimental group and 49.8% in the control group . so , intention to normal delivery has increased by 18.6% in the experimental group and has decreased by 21.6% in the control group . this could be indicated as insufficient routine pregnancy care which has decreased by approaching to delivery time . the difference between the present study and fathian 's study is that in our study , pregnant women 's decision - making was studied in three sections ( before intervention , 2 weeks after intervention , and at admission ) while fathian studied it before and after intervention . at admission , many factors could affect the decision on the type of delivery . in this study , although increase in decision - making for normal delivery after intervention in role play in comparison with behavioral intent model was 11.518.6% , this increase led to 100% final decision for normal delivery , which remained 100% up to admission in the maternity department , while fathian did not compare the effect of behavioral intent model after intervention and at admission . in fathian 's study , the type of delivery performed in the two groups was not significant ( p = 0.002 ) ; however , the elective css in the presence and absence of midwifery problems were not studied . in the present study , although no significant difference was found in the type of performed delivery ( p = 0.17 ) , the elective cesarean section in the lecture group was 5 times as much as in the role play group . the aim of education during pregnancy period was to bring about a decrease in elective cesarean by mothers , which was achieved in this study . lashgari et al . conducted a study entitled effect of training programs of pregnant women on their delivery type selection : a single blind , randomized control trial . the test group used different structural methods such as film , booklet / lecture notes , visiting the maternity department and interviewing the women who had delivery . results showed significant different between the test and control ( no education ) groups in term of the type of delivery selected ( p = 0.03 ) . education could increase the decision taken toward normal delivery to 14% ; however , in the present study , role playing could increase the decision for normal delivery to 100% , which remained unchanged up to time of delivery . in fact , the role play method could increase the decision for normal delivery in a short time ( 90 min ) in comparison with the study of lashgari ( 180 min ) and sustain the decision made . in lashgari 's study , pregnant women 's decision at admission to the maternity department was not studied , so there was no data to be compared in this part . in rahimi kian et al . 's study entitled the effect of education based on health belief model on selecting type of delivery , the test group used different structural methods such as lecture , question and answer , film presentation of normal delivery and cs , and pamphlets about normal delivery and cs . the results showed a significant difference between the two groups in terms of the type of delivery selected ( p = 0.001 ) ( control group received routine pregnancy care ) . as rahimi kian did not report the rate of normal delivery and cs before intervention , there was no data to compare with the present study . although the educational intervention of rahimi kian , fathian , and lashgari could increase the rate of normal delivery , none of them studied the sustainability of this decision at the beginning of labor that is the main situation for decision - making , and finally , the type of performed delivery . of the strengths of the present study is 100% decision - making for normal delivery , sustained decision up to admission to the maternity department , and decrease in elective cesarean in the role play group in comparison with the lecture group . in role play education , it is tried to help people find their internal meaning in the social world and get help from it to make a decision while facing a dilemma . the methods , which need active involvement , including role play , increase the individuals association to the message and lead to individuals emotional drain . therefore , this issue can be one of the reasons for an increase in pregnant women 's decision to undergo natural delivery and reduction of elective cs in role play method , compared to lecture method . it is suggested to conduct further studies on the effect of role play education on pregnant women 's fear of childbirth and selection of mode of delivery , and compare it with other educational methods in order to use the most efficient educational method to reduce elective cs . our results emphasize on the effect of role play education in reducing the fear of childbirth and unnecessary cs . application of active educational methods such as role play , accompanied with lecture method , is suggested to reduce primiparous women 's fear of childbirth and , consequently , reduce unnecessary cs .

congenital diaphragmatic hernia ( cdh ) is a known cause of secondary gastric volvulus . it is also known that bronchopulmonary sequestration ( bps ) may be associated with cdh . an extremely rare case of bps associated with gastric volvulus in a girl with left sided cdh is being reported .

gastric volvulus can be acute , acute on chronic or chronic , and may be primary or secondary to anomalies that affect the fixity of the stomach like hiatus hernia , eventration of diaphragm or congenital diaphragmatic hernia ( cdh ) . the association of bronchopulmonary foregut malformations such as congenital cystic adenomatoid malformation of lung and bronchopulmonary sequestration ( bps ) with cdh has also been well described in the literature . herein , we describe a 4-year - old girl with a rare presentation of gastric volvulus and pulmonary sequestration in association with cdh . a 4-year - old girl presented to our casualty with upper abdominal pain , distension , and nonbilious vomiting for the last two days . her mother gave a history of recurrent episodes of similar symptoms every month for the last six months , and each episode lasted for 4 - 5 h. the symptoms used to diminish spontaneously . her perinatal history was unremarkable . a preliminary work up for her past symptoms in another hospital revealed that she had a suspected left sided eventration as seen on the chest x - ray [ figure 1a ] . on examination , the child was lethargic , obtunded , had cold peripheries with tachycardia , hypotension , and feeble pulse . a nasogastric tube insertion was attempted that failed initially but subsequently drained hemorrhagic gastric contents . a skiagram [ figure 1b ] at this stage revealed a gastric bubble , with a paucity of distal bowel gas and a raised left hemi diaphragm . ( a ) radiograph of the abdomen prior to presentation at our institute showing evidence of viscus in left hemithorax . note the paucity of bowel gas excluding the single stomach gas shadow suggesting the possibility of a volvulus after adequate resuscitation , the child was explored , and the intra - operative findings revealed a mesoaxial gastric volvulus . after successful reduction , it was noted that the stomach was grossly healthy except the greater curvature , which was congested . there was a large defect in the posterolateral aspect of the left hemi - diaphragm , which was covered with a sac and the diaphragm had a reasonably well - developed rim of muscle along the anterior segment [ figure 2a ] . ( a ) defect in the diaphragm is seen with well - defined anterior lip ( arrows ) . ( b ) intraoperative image of the mass seen to occupy the cranial aspect of the diaphragmatic hernia sac ( cut edges indicated by arrows ) to repair the defect the sac was excised and it was noted that the cephalic surface of the sac had a lobular 6 cm 3 cm fleshy pedicled mass flimsily attached to it [ figure 2b ] . the pedicle of the blood supply was traced and it was found to be entering the mediastinum . the repair was completed by primary interrupted sutures , and a three - point gastropexy was done to prevent recurrences . the histopathology report of the mass indicated that it was an extra - pulmonary bps . the stomach is prevented from rotating along its axis by the ligamentous supports like the gastrocolic , gastrohepatic , gastrophrenic and the gastrosplenic ligaments . the gastroesophageal junction and the pylorus are two additional fixed points that afford protection against volvulus . when there is a laxity in these supports and the stomach rotates more than 180 , a primary gastric volvulus results . other conditions like hiatus hernia and cdh predisposes to secondary volvulus . based on the two axes along which this rotation takes place it could produce either an organo - axial , mesentero - axial or a mixed volvulus . it has been noted from the literature that the secondary gastric volvulus in children usually is associated with the mesentero - axial type of volvulus as was seen in our case . an astute clinician can diagnose acute gastric volvulus by the typical complaint of sudden onset upper abdominal pain with retching and inability to pass a nasogastric tube - the borchardt 's triad . this symptom complex was also present in our case except for retching , but the radiographs endorsed our clinical suspicion . the exploration revealed a mass on the superior aspect of the sac , which looked like an extrapulmonary bronchopulmonary sequestration ( ebps ) . these are non - functional pulmonary tissues with the systemic blood supply that seldom communicate with the tracheobronchial tree . bps comprise approximately 6% of all congenital malformations , and these are associated with cdh in 30 - 40% of cases . this distinction is made depending on whether or not the visceral pleura invests the sequestered lesion , with the latter referred to as ebps . although the above associations with cdh are common but cdh presenting with acute gastric volvulus and with an underlying ebps is very rare in pediatric age group and is described only once in english literature . had reported a case similar to ours except that the child had an eventration of the diaphragm . another case report in spanish describes an adult with bochdalek 's hernia with stomach volvulus and extrapulmonary sequestration presenting as acute respiratory distress . the aim of this report is to reinforce pediatric surgeons to look for underlying secondary causes of gastric volvulus as most of them can be tackled simultaneously . an awareness of this triad of cdh , gastric volvulus , and bps can result in early detection and safe resection of bps and thus avoid future diagnostic dilemmas .

most ice in nature forms because of impurities which boost the exceedingly low nucleation rate of pure supercooled water . however , the microscopic details of ice nucleation on these substances remain largely unknown . here , we have unraveled the molecular mechanism and the kinetics of ice formation on kaolinite , a clay mineral playing a key role in climate science . we find that the formation of ice at strong supercooling in the presence of this clay is about 20 orders of magnitude faster than homogeneous freezing . the critical nucleus is substantially smaller than that found for homogeneous nucleation and , in contrast to the predictions of classical nucleation theory ( cnt ) , it has a strong two - dimensional character . nonetheless , we show that cnt describes correctly the formation of ice at this complex interface . kaolinite also promotes the exclusive nucleation of hexagonal ice , as opposed to homogeneous freezing where a mixture of cubic and hexagonal polytypes is observed .

mononuclear nonheme fe(ii ) ( mnh ) and -ketoglutarate ( -kg ) dependent halogenases activate o2 to perform oxidative halogenations of activated and nonactivated carbon centers . while the mechanism of halide incorporation into a substrate has been investigated , the mechanism by which halogenases prevent oxidations in the absence of chloride is still obscure . here , we characterize the impact of chloride on the metal center coordination and reactivity of the fatty acyl - halogenase hctb . stopped - flow kinetic studies show that the oxidative transformation of the fe(ii)--kg - enzyme complex is > 200-fold accelerated by saturating concentrations of chloride in both the absence and presence of a covalently bound substrate . by contrast , the presence of substrate , which generally brings about o2 activation at enzymatic mnh centers , only has an 10-fold effect in the absence of chloride . circular dichroism ( cd ) and magnetic cd ( mcd ) studies demonstrate that chloride binding triggers changes in the metal center ligation : chloride binding induces the proper binding of the substrate as shown by variable - temperature , variable - field ( vtvh ) mcd studies of non--kg - containing forms and the conversion from six - coordinate ( 6c ) to 5c/6c mixtures when -kg is bound . in the presence of substrate , a site with square pyramidal five - coordinate ( 5c ) geometry is observed , which is required for o2 activation at enzymatic mnh centers . in the absence of substrate an unusual trigonal bipyramidal site is formed , which accounts for the observed slow , uncoupled reactivity . molecular dynamics simulations suggest that the binding of chloride to the metal center of hctb leads to a conformational change in the enzyme that makes the active site more accessible to the substrate and thus facilitates the formation of the catalytically competent enzyme substrate complex . results are discussed in relation to other mnh dependent halogenases .

in nature , the selective halogenation of nonactivated carbon atoms is performed by mononuclear nonheme fe(ii ) ( mnh ) , o2 , and -ketoglutaric acid ( -kg ) dependent halogenases . the structural rationale of this chemically challenging reaction has been elucidated rather recently , and to date only a handful of mnh halogenases have been biochemically characterized . the molecular mechanism of these halogenases follows , in principle , that of the closely related -kg dependent hydroxylases . the -kg - dependent hydroxylases have a reactive fe(ii ) center coordinated by a 2-his 1-carboxylate motif and three water ligands . upon coordination of the -kg cofactor , two water molecules are replaced but the six - coordinate ( 6c ) geometry of the fe(ii ) center is retained . only after substrate binding in the outer coordination sphere is the remaining water ligand displaced from the fe(ii ) center and the resulting five - coordinate ( 5c ) species is activated for reaction with o2 . the -kg cofactor is decarboxylated by the activated dioxygen species , which leads to the formation of a high - spin fe(iv)=o intermediate that abstracts a hydrogen atom from the substrate . while in mnh dependent hydroxylases the reaction proceeds via transfer of the hydroxyl group from the fe(iii)oh intermediate onto the substrate radical , in halogenases an iron - coordinated chloride replaces the carboxylate ligand and successfully competes with the hydroxyl moiety to yield the halogenated product ( scheme 1 ) . in the presence of -kg and substrate , o2 binds to the five - coordinate fe(ii ) center and decarboxylates the -kg cofactor . this yields a highly reactive fe(iv)=o intermediate that abstracts a proton from the substrate . chloride rather than the hydroxyl moiety is rebound by the substrate radical , resulting in a chlorinated reaction product . several reasons have been invoked for the strong preference of halogenation over hydroxylation that has generally been observed in halogenases : ( i ) the lower redox potential of cl compared to oh ; ( ii ) possible bicarbonate formation between the metal bound hydroxyl group and the -kg derived co2 which prevents the transfer of the hydroxyl moiety ; ( iii ) possible protonation of the hydroxyl group by a nearby glu - arg proton donor that results in the formation of water , which makes hydroxylation unfavorable ; ( iv ) the difference in binding strengths of the chloride and hydroxyl group to the metal ion and the resulting energetic barrier for hydroxylation ; or ( v ) the positioning of the substrate radical relative to the cl fe(iii)oh center . recently , it has been shown that the o2 reactivity of the -kg - bound halogenase syrb2 results in a 5c trigonal bipyramidal fe(iv)=o intermediate with the fe o vector perpendicular to the c h bond of the substrate . h - atom abstraction by an fe(iv)=o * molecular orbital leads to an intermediate where the fe(iii)oh moiety is oriented away from the substrate carbon radical and the halide is primed for rebound halogenation . while substantial efforts have been made to investigate the second half of the halogenases reaction in order to rationalize the preference of substrate chlorination over hydroxylation after proton abstraction , there has not been much focus on the first half of the catalytic cycle . chloride - bound crystal structures of the substrate - free mnh halogenases syrb2 , and cura - hal show that the halide can coordinate to the iron even before the substrate binds . on the other hand , in cytc3 no halide is present in the crystal structure with fe(ii ) and -kg bound , despite the rather high chloride concentrations in the mother liquor ( 80 mm ) . no study has elucidated how the halogenases prevent alternative oxidation reactions in the absence of halide . recently , a fatty acyl - halogenase , hctb from l. majuscula , has been characterized in our laboratory . the enzyme that modifies middle - chain fatty acyl moieties displays an unprecedented three domain organization , which sets it apart from the monodomain amino acyl - halogenases and the multidomain ketide halogenases : an acyl - coenzyme a ( acyl - coa ) binding protein is n - terminally fused to a halogenase domain , while its c - terminus connects to an acyl - carrier protein ( acp ) domain . the acp domain bears an inherent thiolation site , where the substrate covalently binds via a phosphopantetheinyl bridge . this composition makes hctb self - sufficient in regard to a substrate binding entity and may be prototypical for fatty acyl - halogenases . the trifunctional enzyme introduces 5-oxo- , 5,5-dichloro- , and 5-chloro-4-vinyl moieties into the hexanoyl substrate under chloride saturating conditions . in the course of the enzyme s biochemical characterization we observed that o2 reduction was triggered by the presence of not only the substrate but also chloride under single turnover conditions . based on these observations , a suspected role of chloride in primary o2 activation in hctb is investigated in this study : circular dichroism ( cd ) , magnetic cd ( mcd ) , and variable - temperature , variable - field ( vtvh ) mcd spectroscopies directly observe the geometric and electronic structure of the enzymatic fe(ii ) active site . in combination with stopped - flow kinetics the spectroscopic data reveal that the metal center is constituted in the absence of a halide ion but remains inert toward o2 and that the presence of chloride is essential for triggering o2 reduction at the metal center . molecular dynamics ( md ) simulations of the hctb halogenase domain are employed to gain insights into the role of the protein structure in chloride - dependent o2 activation . recombinant nonacylated hctb was expressed , purified , and acylated with the fatty acyl - coa substrate as described previously except the final enzyme solution was exchanged into 20 mm bis - tris buffer ( ph 7.5 ) for stopped - flow analysis . the enzyme was made o2-free by 20 cycles of evacuation and n2-flushing in an airtight v - vial ( wheaton , millville / usa ) capped with a screw - top septum ( supelco , bellefonte / usa ) , and subsequently 0.95 equiv of fe(ii)so47h2o and -kg ( sigma - aldrich , st . louis / usa ) were added from stock solutions ( 10 mm ) in a n2-purged glovebox . the enzyme preparations ( 440550 m active sites ) were mixed at 10 c with equal volumes of 20 mm bis - tris buffer , ph 7.5 that contained 1.4 mm o2 if not stated otherwise and either 0.1 or 1 m nacl if required , using an sx20 stopped - flow uv / vis spectrophotometer ( applied photophysics , leatherhead / uk ) , which was equipped with a polychromatic light source . note that in a previous study the addition of 1 equiv of -kg to acylated hctb ( 100 m ) yielded maximum initial o2 consumption rates and thus , in the enzyme kinetic measurements performed here , which used 95% cofactor - loaded active sites ( > 100 m ) , saturating conditions were ensured . spectroscopic analyses were carried out by using an sx20 photodiode array detector , whereby absorptions from 270 to 730 nm were recorded with 400 collected data points in the first 0.4 s and 1 datum point per 100 ms in the remaining 40 or 60 s. absorbance traces of 4 measurements per condition were averaged and fit using the following methods : when rates of signal changes were so slow that they had linear characteristics over the monitored time , rates were determined based on the extinction coefficient of the -kg - fe(ii)-hctb complex ( 500 nm = 0.15 mm cm ) . rates were then related to the applied metal concentration to give specific rates . in the case of apparently bi- or triphasic curves , the pro - kineticist 1.0.10 software ( applied photophysics ) was used and the trace at the respective constant wavelength was fit to sequential models in the form of a b c and a b c d respectively , in order to obtain the respective apparent 1 order net rate constants . if not stated otherwise , for cd / mcd spectroscopy , protein samples were exchanged into deuterated 50 mm hepes / naod buffer pd 7.5 , containing 1 m nacl if required , using 4 ml amicon ultra centrifugal filters and concentrated to 1.53.5 mm . o2 was removed from the samples by 20 cycles of evacuation and argon flushing . ferrous ammonium sulfate and -kg were added to the enzyme preparations in microliter quantities from deuterated , anaerobic stock solutions in a n2-purged glovebox , where the final sample was filled into a cd or mcd cell . cd measurements were carried out on a jasco j-730w spectropolarimeter at 283 k. spectra were corrected by subtracting the respective cofactor - free spectrum . mcd samples were prepared from cd samples by adding glassing agent to saturation at approximately 50% sucrose . samples were injected into mcd cells , frozen , and stored in liquid n2 until use . mcd spectra were recorded on a jasco j-730w spectropolarimeter equipped with an oxford instruments spectromag 4000 superconducting magnet and a liquid n2-cooled insb detector . to affirm the authenticity of the mcd signals , field dependencies at 7 , + 3.5 , and + 7 t were recorded at 5 k. the c - term origin of the signals was confirmed by tracking temperature dependencies at 5 , 20 , and 40 k at + 7 t ( 3 scans per condition were averaged ) . the data obtained were corrected by subtraction of the zero - field spectrum at the respective temperature . mcd spectra were compared to their respective cd counterparts in order to verify that transitions had the same energies in cd and mcd and with and without glassing agent . a structural model of the hctb halogenase md simulations were performed with the yasara structure suite , version 12.11.20 ( yasara biosciences ) . a periodic simulation cell , which comprised the whole enzyme and an additional 5 in each dimension , was used with explicit solvent . the amber99 force field was employed and long - range electrostatic potentials were calculated with the particle mesh ewald ( pme ) method , with a cutoff of 7.864 . force field parameters for -kg were generated with the autosmiles utility , which assigned a van der waals radius of 2.27 and a charge of + 2 to the iron cofactor . fe(ii ) parameters were defined to reflect the principal octahedral geometry of the fe(ii ) center . an equilibrium spring length that was derived from dft calculations was used for all ligands with a stretching force constant of 125 n m. ligand fe ligand angles of minimum energy were defined as 180 for opposite ligands and as 90 for all others , with angle bending force constants of 1000 kj mol rad . in this way , force field parameters for n- of his112 , n- of his228 , and -kg coordinating carboxylate and keto - oxygen were defined for the chloride - free hctb model . for the chloride - containing complex note that no force field parameters were defined for glu224 . in order to investigate the impact of a putative strongly bound water at the halide position , in a second model the chloride atom was substituted by a water molecule and the respective parameters were adapted accordingly . in a third halide - free model a strongly bound water was positioned in the sixth coordination site of the iron , while the water in the halide binding position remained unrestricted . the oxidation state of the iron cofactor in all complexes was either + 2 , as assigned by autosmiles , or in order to assess the impact of the effective iron charge on the simulation adjusted to values in the 0 to + 2 range ( see table s2 ) . the hydrogen bonding network was optimized by the method of hooft and co - workers , and yasara s pka values at ph 7.5 were assigned . the simulation cell was filled with water at a density of 0.997 g ml using nacl concentrations of 0.001 and 1 m , respectively . after relaxation of the solvent the system was energy minimized , whereby a steepest descent minimization was applied to remove conformational stress , followed by a simulated annealing minimization until convergence was reached ( < 0.05 kj mol per 200 steps ) . integration time steps were set to 1.33 and 4 fs for intra- and intermolecular forces , respectively . md simulations at 298 k were initiated , whereby integration time steps for intramolecular and intermolecular forces were set to 1.25 and 2.5 fs , respectively . the oxidation of the fe(ii)--kg complex was monitored spectrophotometrically via single turnover stopped - flow measurements by recording the decay of its prototypical metal to ligand charge transfer ( mlct ) transition ( 500 nm = 150 m cm ) . therefore , anaerobic enzyme preparations of acylated hctb ( ac - hctb ) , preloaded with 0.95 mol equiv fe(ii ) and -kg , were mixed with o2-enriched ( 6501400 m ) and , optionally , 1 m nacl - containing buffer at a 1:1 ratio . in the absence of nacl the -kg - fe(ii)-ac - hctb complex decayed slowly with a specific rate of 1.1 10 s ( co2 = 700 m ) . by contrast , under analogous conditions but in the presence of chloride , the absorbance band disappeared within 20 s. the trace displayed three distinct phases that could be resolved : a lag phase of 60 ms ( ksc1 = 35.1 s ) was followed by a phase of fast signal decay , which gave an apparent first - order rate constant ksc2 of 6.74 s and accounted for approximately one - third of the total amplitude . the third phase of the reaction , a slower absorbance decrease ( ksc3 = 0.22 s ) , equaled the o2 depletion rate ( 0.22 s ) that had previously been determined using an o2 sensor under similar conditions ( figure 1 ) . ( note that ksc1 , ksc2 , and ksc3 represent apparent first - order net rate constants . ) stopped - flow absorption kinetic traces of the -kg - fe(ii)-ac - hctb complex decay . the conversion of the chromophoric -kg - fe(ii ) pair was monitored in the absence ( gray ) and presence ( black ) of 0.5 m nacl at 500 nm , and average traces were fit with a linear regression ( green ) and via a three - phasic model using the pro - kineticist software ( applied photophysics , see materials and methods section for details ) , respectively . rates were not significantly dependent on the o2 concentration ( table s1 , figure s1 ) . the total amplitude of signal decay corresponded to 97% of the -kg - fe(ii ) concentration , confirming a quantitative conversion of the complex . it is worth noting that in previous studies it was observed that the fe(iv)=o intermediate had a similar extinction coefficient at 520 nm as the fe(ii)--kg complex but additionally showed a significant absorbance increase at 318 nm ( 1500 m cm ) . an analogous signal increase was not detectable during substrate conversion by ac - hctb . instead , a slow signal increase at 320 nm in the absence ( figure s2b ) and presence ( figure s2d ) of chloride was obtained , which may indicate some fe(ii ) oxidation as a side reaction . when nonacylated hctb ( further on termed hctb ) was subjected to stopped - flow analysis , the chloride - free complex displayed an almost stable signal at 500 nm over the measured time range with a specific rate of 0.1 10 s ( figure s2a ) . in the presence of chloride , the velocity of precipitation was chloride dependent and led to an absorbance increase over the whole recorded wavelength range ( figures s2c , s3 , and s4 ) . this phenomenon , which was not observed during o2 sensor measurements , prohibited the determination of these reaction rates . summarizing , according to stopped - flow measurements the decay of the -kg - fe(ii)-ac - hctb complex is accelerated 200-fold by chloride , while the presence of covalently bound substrate in the absence of chloride enhanced the rate only by an order of magnitude . in order to gain insight into the fe(ii ) active site geometric and electronic structure , combined cd / mcd spectroscopic measurements of the enzymatic fe(ii ) centers of hctb ( i.e. , no substrate ) and ac - hctb in the presence and absence of -kg and saturating nacl concentrations were pursued . the methodology developed in ref ( 31 ) allows for the determination of the fe(ii ) center geometric structure , based on the energies of its associated d d transitions : free high spin fe(ii ) possesses a d ground state , which upon the influence of an octahedral ligand field splits into a t2 g ground state and a eg excited state that are separated by 10 dq 10 000 cm . the eg state is further split in the distorted octahedral geometry of a protein environment , leading to two transitions split by 2000 cm . removal of an axial ligand forms a square pyramidal five - coordinated species resulting in the splitting of the eg state by 5000 cm , yielding transitions at 10 000 and 5000 cm . rearrangement to a trigonal bipyramidal 5c geometry leads to transitions at < 10 000 and < 5000 cm ( the latter frequently undetectable ) . thus , near - ir ( nir ) cd and mcd spectra give information on the number of different fe(ii ) coordination environments present in a sample as well as their geometric structures . vtvh mcd complements these excited state data by providing information on the ground state splitting of the t2 g orbitals of a given site . a non - kramers doublet model developed previously for the fitting of vtvh mcd data provides ground state spin hamiltonian parameters and g|| for negative zero - field - split ( zfs ) systems or axial d and rhombic |e| zfs parameters for positive zfs systems , which in turn are used to determine the tetragonal splitting , , of the dxy orbital from the { dxz , dyz } pair , as well as the rhombic splitting , v , of the dxz and dyz orbitals . the relative energies of the five d orbitals of an fe(ii ) active site can therefore be determined , and the site s geometric and electronic structures characterized . 283 k cd and 5 k mcd spectra of fe(ii)-hctb and fe(ii)-ac - hctb in the absence and presence of chloride are shown in figure 2 . in cd , these four enzyme forms all show two transitions at 8100 and 10400 cm ( figure 2a , c , e , and g ) indicative of distorted octahedral fe(ii ) sites , whereas in mcd two transitions are observed at 9200 cm and 11 400 cm for the four forms ( figure 2b , d , f , and h ) . such shifts upon going to low temperature have been observed for clavaminate synthase 2 ( cs2 ) and factor inhibiting hypoxia - inducible factor ( fih ) and are attributed to stronger ligand the similarity of the spectra for all four forms indicates that no significant change in the eg orbitals of hctb - bound fe(ii ) has occurred in the presence of chloride or substrate . 283 k cd and 5 k mcd spectra of fe(ii)-hctb ( a and b , respectively , dark blue ) , fe(ii)-ac - hctb ( c and d , respectively , light blue ) , fe(ii)/cl - hctb ( e and f , respectively , dark green ) , and fe(ii)/cl - ac - hctb ( g and h , respectively , light green ) . component peaks and resultant fits are in black and colored dashed lines , respectively . however , vtvh mcd data as shown in figure 3 indicate that the t2 g orbitals are perturbed when chloride is bound to fe(ii ) . the vtvh mcd data of fe(ii)-hctb and fe(ii)-ac - hctb in the absence of chloride ( figure 3a and b ) can be fit with the same parameters of a positive zfs system with d = + 10.8 0.1 cm and |e| = 2.6 0.1 cm , and corresponding t2 g orbital splittings of 500 100 cm and |v| 300 50 cm . upon addition of chloride , the vtvh mcd data change outside of error as shown in supporting figure s5a and b. fe(ii)/cl - hctb ( figure 3c ) fits to a positive zfs with parameters of d = + 10.2 0.1 cm and |e| = 2.6 0.1 cm , and corresponding t2 g orbital splittings of 700 100 cm and |v| 450 50 cm . the increased t2 g orbital splittings are attributed to chloride being a stronger donor ligand than the replaced water . in the presence of substrate and chloride ( figure 3d ) , the vtvh mcd data fit to give a positive zfs with d = 3.7 0.1 cm and |e| = 2.4 0.1 cm , and corresponding t2 g orbital splittings of 850 100 cm and |v| 540 50 cm-1 . that substrate binding only appears to perturb the fe(ii ) site when chloride is also present is explored in the md simulation section below . vtvh mcd data and fitted curves for fe(ii)-hctb ( a , 2.2 , 3.3 , 5 , 7.5 , 10 , 15 , and 20 k , dark blue ) , fe(ii)-ac - hctb ( b , 2.2 , 3.4 , 5 , 7.5 , 10 , and 15 k , light blue ) , fe(ii)/cl - hctb ( c , 2.3 , 3.3 , 5 , and 7.5 k , dark green ) , and fe(ii)/cl - ac - hctb ( d , 2.2 , 3.4 , 5 , 7.5 , and 10 k , light green ) . error bars for the data are shown or otherwise are the size of the data points . cd and mcd spectra of -kg - fe(ii)-hctb and -kg - fe(ii)-ac - hctb in the absence and presence of chloride are given in figure 4 . in the presence of -kg both hctb and ac - hctb mcd spectra show two nir transitions at 8800 and 11 800 cm as well as the rising edge of the fe(ii ) -kg * metal - to - ligand charge transfer ( mlct ) starting at 16 000 cm ( figure 4b and d ) . 283 k cd and 5 k mcd spectra of -kg - fe(ii)-hctb ( a and b , respectively , red ) , -kg - fe(ii)-ac - hctb ( c and d , respectively , pink ) , -kg - fe(ii)/cl - hctb ( e and f , respectively , orange ) , and -kg - fe(ii)/cl - ac - hctb ( g and h , respectively , purple ) . component peaks and resultant fits are in black and colored dashed lines , respectively . the presence of substrate does not lead to a significant perturbation of the mcd spectra in the absence of chloride for the the relatively large eg splitting of 3000 cm is similar to that found in -kg / chloride - bound cytc3 ( 3660 cm ) and may be attributed to a weaker water ligand than that found in a site containing the 2his/1-carboxylate facial triad , such as cs2 . this has been explained by the lack of the h - bond between the facial triad carboxylate and the coordinated water , which gives this water partial hydroxide character and strengthens the fe the mcd spectrum of -kg - fe(ii)/cl - hctb ( figure 4f , orange ) shows three nir transitions at 7600 , 9100 , and 12 100 cm , in addition to the mlct shoulder at 16 000 cm . the presence of three transitions indicates that more than one fe(ii ) site is present . in order to determine the nature of these species vtvh mcd data were collected on the three peaks at 7350 , 9500 , and 11 800 cm as shown in figure 5 ( arrows in a , isotherms in b vtvh mcd data collected on the 7600 cm transition are fit to a negative zfs with 1.3 0.1 cm and g|| 8.6 0.1 . the very small value of is indicative of a trigonal bipyramidal site , as was found to be present in the reduced , naphthalene - bound form of the rieske - dependent naphthalene dioxygenase , which possessed a feature at 8165 cm with a 1.2 0.1 cm and g|| 8.5 0.1 . data collected at 9500 and 11 800 cm were fit to the same ground state to give a positive zfs with d = 9.3 0.1 cm and |e| = 2.1 0.1 cm , corresponding to t2 g orbital splittings of 900 100 cm and |v| 560 50 cm . note that the data at 11 800 cm required the addition of a b term of approximately 12% of the total intensity , due to overlap with the mlct transition . -kg - fe(ii)/cl - hctb is therefore a mixture of 6c and 5c trigonal bipyramidal sites . ( a ) mcd spectrum with arrows marking the energies at which vtvh mcd data were collected , at ( b ) 7350 cm ( purple ) , ( c ) 9500 cm ( red ) , and ( d ) 11 800 cm ( orange ) . all data were collected at 2.2 , 3.4 , 5 , 7.5 , and 10 k. error bars for the data are shown or otherwise are the size of the data points . the mcd spectrum of -kg - fe(ii)/cl - ac - hctb ( figure 4h , purple ) shows four transitions : 5000 , 7600 , 9900 , and 12 000 cm . the presence of four ligand field transitions indicates that -kg - fe(ii)/cl - ac - hctb is also a mixture in this case containing sites of square pyramidal ( due to the 5000 cm transition ) and octahedral sites . vtvh mcd data were collected on the low - energy band at 5750 cm as shown in figure 6 ( the other bands were too weak for a meaningful signal - to - noise ratio ) . the vtvh mcd data for this band could be fit using negative zfs parameters of 2.6 0.1 cm and g|| 8.8 0.1 , corresponding to t2 g splittings of 1100 100 cm and |v| 660 50 cm , the larger t2 g splitting being consistent with a square pyramidal fe(ii ) center . thus , upon binding of chloride to -kg - fe(ii)-ac - hctb the site goes from 6c to 5c . ( a ) mcd spectrum with arrows marking the energy at which vtvh mcd data were collected at ( b ) 5750 cm ( purple ) . data were collected at 2.3 , 3.3 , 5 , and 7.5 k. error bars for the data are shown . to determine whether the maintenance of the six - coordinate fe(ii ) site in the chloride - free -kg - fe(ii)-ac - hctb complex was due to a missing negative charge at the halide binding position , we recorded the spectrum of the equivalent sample at a pd of 9.1 . however , the pd shift by 1.6 log units , which previously brought about water deprotonation in a noncarboxylate coordinated fe(ii ) center , did not significantly alter the mcd spectrum ( figure s6 ) . consequently , to elucidate the structural basis of chloride mediated o2 activation , a model of the hctb halogenase domain was constructed based on the crystal structure of syrb2 and subjected to md simulations in the absence and presence of 1 m nacl in the simulation cell . in order to ensure a geometrically appropriate metal center structure , an octahedral iron force field was defined and applied to the ligands , namely his112 , his228 , -kg , and where appropriate chloride , as detailed in the experimental section . 100 ns md simulations of the 2-his 1-chloro -kg ligated fe(ii)-hctb model and its chloride - free analogue at 297 k showed overall structures that were stable over the simulation time . however , in the halide - free model , a rearrangement at the metal center was observed : residue glu224 , which pointed away from the metal center in all starting structures , reoriented and coordinated to the fe(ii ) ion , as demonstrated in a 90 ns snapshot of the active site ( figure 7a ) . by contrast , the respective simulation in the presence of chloride roughly preserved the conformation of glu224 from the starting structure ( figure 7b ) . when the simulation of the hctb domain under chloride - free conditions was repeated with a water molecule kept bound to the metal ion either at the axial , 6th position ( figure s7 ) or at the equatorial position that is otherwise occupied by the chloride ion ( figure s8 ) , glu224 also reoriented in order to point toward fe(ii ) in both cases , whereby the residue s carboxylate moiety h - bonded to the water molecule that occupied the cl binding site or coordinated directly ( figures s7 and s8 ) . ( note that both a displacement and preservation of metal bound water are feasible in principle and , therefore , needed to be considered ) . trajectories of the fe(ii)-glu224 carboxylate oxygen distances show that this reorientation takes place instantaneously ( < 100 ps ) , ( figures 8a , left , s9a , s10a ) . by contrast , in the chloride containing structure the carboxylate moiety of glu224 remained pointing away from the hctb active site ( figure 8a , right ) , presumably due to electrostatic interactions . 90 ns snapshots of the hctb halogenase domains derived from md simulations of the modeled structures at 297 k. while in the chloride - free structure ( a ) glu224 points toward fe(ii ) ( orange ball ) , in the chloride - containing structure ( b ) the halide ( green ball ) coordinates to fe(ii ) and glu224 points away from the metal cofactor . this results in a rearrangement of the neighboring residues val114 , tyr115 , asn140 , and arg225 . water molecules are not displayed . in order to assess the impact of a probable overestimation of electrostatics in our model on the orientation of glu224 , 1 ns md simulations at varying reduced effective charges of fe(ii ) were subsequently performed . results showed that the observed two principal distinct orientations of glu224 , toward and away from fe in the absence and presence of chloride , were not sensitive to the fe(ii ) charge within the studied range of 0 to + 2 . however , for cl - free models with no bound water in the halide binding pocket , increased charges roughly correlated with increased frequency of direct coordination of glu224 to fe ( table s2 ) . the reorientation of glu224 in the chloride - free hctb structures led to major structural changes . the residue s flipping resulted in a h - bonding interaction between its backbone oxygen and the backbone nitrogen of val114 ( figure 8c , left , dark red ) in the model containing the water - free fe(ii ) sphere and drew the respective strands closer together ( figure 8b , left , teal ) . however , in the two models with a water tightly bound to the fe(ii ) center either at the chloride - binding position or at the second water - binding position in the octahedral 2-his -kg complex , no interactions between these residues were observed ( figures s9b and s10b ) . in all three chloride - free models ( figures 7a , s7 , and s8 ) , the side chains of tyr115 and arg225 reduced their distance from an average of 10 to 46 ( figures 8c , s9c , and s10c , pink and red traces ) and became less flexible . in the case of the halide - free model without tightly bound water , the backbone nitrogen of glu224 formed a h - bond with the side chain oxo group of asn140 ( figure 8c , left , orange ) . in all halide - free models , the backbone of asn140 and glu224 came closer together ( figures 8b , s9b , and s10b , blue traces ) . thus , according to our models , the effect of chloride binding to fe(ii ) is to induce conformational changes in hctb . trajectories from 100 ns md simulations depicting distances of residues involved in the chloride - triggered structural rearrangement in hctb in the absence ( left panels ) and presence ( right panels ) of 1 m chloride . panel b displays backbone distances from c-2 of asn140 to n-2 of glu224 ( blue ) ; n-2 of val114 to c-2 of glu224 ( teal ) ; and c-2 of tyr115 to c-2 of arg225 ( green ) . panel c gives the distances of the putative h - bonding atoms o-7 of tyr115 to n- of arg225 ( red ) ; o-7 of tyr115 to n- of arg225 ( pink ) ; n-2 of val114 to o-1 of glu224 ( dark red ) ; and o-4 of asn140 to n-2 of glu224 ( orange ) . this study demonstrates that although an enzymatic -kg bound fe(ii ) center is constituted in the absence of a halide , the latter impacts the geometric structure of the metal center and thereby triggers o2 activation in hctb . cd / mcd spectroscopic studies of the metal centers in fe(ii)-hctb and fe(ii)-ac - hctb show a stable six - coordinate geometry in the absence of any cofactor . this sets hctb apart from the related -kg dependent halogenase cytc3 , where -kg was a prerequisite for fe(ii ) binding to the enzyme in solution . hctb samples with and without chloride show similar excited - state spectral features . however , the d ( t2 g ) orbitals are perturbed upon halide binding as shown by vtvh mcd . also , while the ground state splitting is the same for fe(ii)-hctb and fe(ii)-ac - hctb in the absence of halide , the splittings for halide - bound hctb in the absence of substrate differ from those where the substrate is bound . these results provide experimental evidence for the effect of halide binding on protein conformation and thus are consistent with md simultations . it is interesting to note that in the presence of -kg and substrate , conditions in which most other -kg - dependent enzymes trigger water ligand dissociation and activation of o2 , the fe(ii ) site in hctb remains 6c ( figure 4c and d ) . this is consistent with the idea that the substrate does not sterically interact with the active site until chloride is bound to fe(ii ) . evidence for a 5c fe(ii ) site is found only upon binding of chloride to the -kg - fe(ii ) site . for -kg - fe(ii)/cl - ac - hctb the vacated coordination site allows binding of o2 and results in an active site that is competent for catalysis . this is supported by stopped - flow kinetics , which demonstrate that the presence of chloride results in a 200-fold increase in the rate of -kg - fe(ii)-ac - hctb complex decay . interestingly , the presence of -kg and chloride but absence of substrate leads to the formation of a mixture of trigonal bipyramidal and octahedral sites . this directly reflects the slow but measurable uncoupled consumption of o2 , observed only for this form ( 7% of the rate of -kg - fe(ii)-cl - ac - hctb(11 ) ) . these findings are in contrast to the results for cytc3 , which showed the presence of only 6c sites unless -kg , substrate , and chloride were all present , and kinetic findings for syrb2 which showed a 5000-fold loss in rate in the absence of substrate . in the absence of a crystal structure for hctb , stuctural models of its halogenase domain allowed some interesting insights into a putative role of cl in the activation of hctb for catalysis : md simulations of the hctb structural models suggest major structural differences between the chloride - bound and chloride - free forms . in the absence of chloride , the carboxylate moiety of glu224 is oriented toward the iron center ( either directly binding to fe(ii ) or h - bonding to a coordinated h2o ) . this results in h - bonding interactions between the backbones of glu224 and val114 in models containing a water - free fe(ii ) center . additionally , hydrogen bonds form between glu224 and asn140 in all investigated halide - free models that bring the residues carbon atoms closer together . moreover the distances between the tyr115 and arg225 side chains are significantly reduced in all chloride - free models . notably , previous in silico docking studies suggested that the fatty acyl moiety accesses the active site between the strands that contain residues 114115 and 224225 ( figure s11 ) . as these residues converge in the chloride - free model , an analogous active site access by the substrate s acyl moiety could be severely hampered . such a scenario provides a rationale for the substrate s inability to affect mcd spectra ( i.e. , the 6c 5c conversion ) and to markedly increase o2 consumption rates in the absence of chloride . the apparent transient stability of the -kg - fe(ii)-ac - hctb complex during single turnover kinetics , which is mirrored by the 60 ms lag phase in figure 1 , may thus reflect the reorganization of the enzyme upon chloride binding , involving the opening of the substrate channel , the substrate entrance to the active site , and the formation of the five - coordinate iron center required for the o2 dependent catalysis to take place . in the absence of chloride a bound water could take the halide s position at the metal center , or alternatively , direct coordination of glu224 could take place . a corresponding acidic amino acid residue is present in the other so far annotated fatty acyl - halogenase psm3l , but it is not generally conserved in mnh halogenases . according to sequence alignments , the amino acyl - halogenases have a serine in the respective position and in the structure of syrb2 this residue occupies a similar space as glu224 in the hctb model . therefore , the proposed molecular mechanism may be specific to this subtype of fatty acyl - halogenases . however , a principal role of chloride in the activation of halogenases by triggering changes in the protein structure may be a general feature of the mnh halogenase mechanism . interestingly , in syrb2 a major rearrangement of polypeptide strands was obtained upon binding of fe(ii ) and chloride to an -kg containing structure . in cura , concomitant coordination of -kg and chloride to the iron center also resulted in a structural reorganization . in contrast , cytc3 , which could not be crystallized with a metal coordinated halide , possesses the same conformation in its apo and fe(ii)+-kg - bound forms . even though structural data are scarce and the presence of charged buffer molecules may compromise their interpretation , it is noteworthy that in the available studies conformational rearrangements were accompanied by a switch from a halide - free to a halide - coordinating metal center . particularly for halogenases with low chloride affinity and those that perform multiple halogenations per substrate and therefore consume the bound halide , a chloride - dependent activation would prevent undesired hydroxylation after the primary halogenation step . note that a report was recently published that briefly described the chloride induced formation of an fe(iv ) species in syrb2 , supporting our notion that this may be a general mechanism in halogenases . in summary , the present study has shown that halide binding is key for the o2 reactivity of hctb , as neither is an open coordination position for o2 binding present nor is the substrate allowed to properly align above the active site unless halide is ligated to fe(ii ) . these effects help to maintain halogenase activity in hctb and may be factors involved in the mechanisms of halogenases in general .

background : heart disease is one of the most common chronic disease and leading cause of morbidity and mortality worldwide . adropin , a newly identified protein , is important for energy homeostasis and maintaining insulin sensitivity , and has been referred to as a novel regulator of endothelial cells . endothelial dysfunction is a key early event in atherogenesis and onset of hd . therefore , this review gives a systematic overview of studies investigating plasma adropin level in patient with heart disease.methods:data carried out in pubmed , scopus , web of science , embase , google scholar and medline , from the earliest available online indexing year through 2015 . the search restricted to studies conducted in humans . the keyword search was adropin to apply in title , abstract and keywords . references lists of all original published articles were scanned to find additional eligible studies.results:heart failure ( hf ) , coronary atherosclerosis acute myocardial infarction and cardiac syndrome x ( csx ) were type of heart disease acknowledged in this study . majority of evidences introduced low adropin as an independent risk factor of heart disease . in a case - control study , the plasma level of adropin increased with the severity of hf.conclusion:adropinmay be a potential serum biomarker for early diagnosis of hd .

accurate diagnosis of hd improve prognosis through early treatment . although , current strategy for prognosis of hd is good , however , are not enough to explain all coronary events . therefore , researches for potential novel its risk factors and diagnostic criteria are requirement to be fulfilled ( 14 ) . adropin is a recently identified protein encoded by the energy homeostasis - associated gene ( enho ) identified during an investigation of obese insulin resistant mice as a novel factor linking with metabolic homeostasis ( 5 ) . adropin appears to participate in the maintenance of energy homeostasis and insulin response , closely related to the development and progression of atherogenesis ( 5 ) . endothelial dysfunction is a key early event in atherogenesis and onset of hd ( 7 ) . furthermore , low serum adropin introduced as an independent predictor of clinically relevant coronary atherosclerosis ( 8) . the aim of this present study was to investigate the effects of adropin on hd through systematic review . we performed a systematic search of pubmed , scopus , web of science , embase , google scholar and medline for studies examining the association between serum adropin levels and all type of heart disease . the prisma 2009 guidelines for systematic review and meta - analysis were considered throughout the study . the predetermined inclusion criteria were : 1 ) exposure introduced as serum adropin levels ; 2 ) the outcome interest was all type of heart disease ; 3 ) studies that conducted in adult population were not endocrine disorders . studies were excluded if they were pregnant and lactating women or reviews , editorial , letters , meeting abstract and animal study . studies screened independently by two investigators ( sy , ss ) based on the inclusion criteria with a low probability to exclude irrelevant abstract . overall , 220 abstracts were retrieved by searching in the electronic database . of those , 151 abstracts were duplicated , 27 did not study coronary heart disease as interesting outcome ( 5 , 934 ) , 3 were editorial and review ( 3537 ) , 13 were non - human studies ( 6 , 3849 ) , 8 were meeting abstract ( 5057 ) and 12 conducted in children , pregnant and lactating women ( 5869 ) . after final screening of full text , six articles were eligible to address in this systematic review ( 8 , 7074 ) , which four of those were case - control ( 8 , 7274 ) studies and 2 articles were cross - sectional studies ( 70 , 71 ) . all data extracted we rescanned two times . if there were discrepancies , group consensus and consulted a third reviewer were used to reach a ensure accuracy of data . the data extracted included : study characteristics ( authors , year of publication , study location , study design ) the participants age , number of allocated participants , serum adropin level , type of heart disease , adropin assessment method . the newcastle - ottawa scales , validated scale for non - randomized studies were applied to examine the quality of included evidences . we performed a systematic search of pubmed , scopus , web of science , embase , google scholar and medline for studies examining the association between serum adropin levels and all type of heart disease . the prisma 2009 guidelines for systematic review and meta - analysis were considered throughout the study . the predetermined inclusion criteria were : 1 ) exposure introduced as serum adropin levels ; 2 ) the outcome interest was all type of heart disease ; 3 ) studies that conducted in adult population were not endocrine disorders . studies were excluded if they were pregnant and lactating women or reviews , editorial , letters , meeting abstract and animal study . studies screened independently by two investigators ( sy , ss ) based on the inclusion criteria with a low probability to exclude irrelevant abstract . overall , 220 abstracts were retrieved by searching in the electronic database . of those , 151 abstracts were duplicated , 27 did not study coronary heart disease as interesting outcome ( 5 , 934 ) , 3 were editorial and review ( 3537 ) , 13 were non - human studies ( 6 , 3849 ) , 8 were meeting abstract ( 5057 ) and 12 conducted in children , pregnant and lactating women ( 5869 ) . after final screening of full text , six articles were eligible to address in this systematic review ( 8 , 7074 ) , which four of those were case - control ( 8 , 7274 ) studies and 2 articles were cross - sectional studies ( 70 , 71 ) . two authors conducted independently data extraction using a pre - formatted spreadsheet . all data extracted we rescanned two times . if there were discrepancies , group consensus and consulted a third reviewer were used to reach a ensure accuracy of data . the data extracted included : study characteristics ( authors , year of publication , study location , study design ) the participants age , number of allocated participants , serum adropin level , type of heart disease , adropin assessment method . the newcastle - ottawa scales , validated scale for non - randomized studies were applied to examine the quality of included evidences . table 1 exhibited detail of study and participants characterize . heart failure ( hf ) , coronary atherosclerosis acute myocardial infarction and cardiac syndrome x ( csx ) all studies were conducted on both sex , two studies conducted in turkey ( 70 , 71 ) , and four investigation were from china ( 8 , 7274 ) . characteristic of studies that evaluated of adropin status in patient with coronary artery disease there were significant differences in the plasma adropin level between heart disease patients with healthy participants . majority of evidences introduced low adropin as an independent predictor of heart disease ( 8 , 70 , 71 , 73 , 74 ) . in a case - control study , the plasma level of adropin increased with the severity of hf ( control : 6.00.3ng / ml ; ha functional class ii : 7.60.4 ; ha functional class iii : 9.80.5 ; ha functional class iv : 12.40.6 ng / ml ( p<0.01 ) ( 72 ) . in a similar design , reported the stable angina pectoris ( sap ) and acute myocardial infarction compare with control group have lower serum adropin ( 74 ) . a decrease in adropin level found in patients with csx than control participants ( 1.70.8ng / ml and 3.41.8 ng / ml , respectively ; p<0.001 ) . additionally , adropin was one of the predictor risk factors for csx ( =0.104 , p - value=0.005 ) ( 71 ) . patients with stablecoronaryarterydisease ( scad ) had lower serum adropin levels compared to the controls ( 59.219.3 versus 70.018.2 pg / ml , p<0.001 ) . moreover , low serum adropin level was introduced as a predictor of scad ( 73 ) . in a cross - sectional study , serum adropin was inversely associated with the score of severity of coronary atherosclerosis assessed by gensini , friesinger , and syntax scores . a decrease serum adropin indicated a negative independent predictor of coronary atherosclerosis ( as syntax score>11 ) ( 8) . in similar design study introduced lower serum adropin as an independent predictor of saphenous vein graf disease ( svgd ) after coronary artery bypass grafting ( or=0.558 , 95%ci=0.4330.714 , p<0.01 ) ( 70 ) . adropin is a newly identified protein that its protective role on endothelial cells has been shown previously , and has been referred to as a novel regulator of these cells ( 46 ) . the key findings from the present review study were that there were significant differences in the plasma adropin level between patients with heart disease ( ami , sap , atherosclerosis , cardiac syndrome x , occluded svg ) and control subjects . serum adropin level may be referred to as a novel biomarker for evaluation and follow up in patients with hd . the endothelium plays a crucial role in the maintenance of vascular homeostasis , and endothelial dysfunction contributes to the development and progression of cardiovascular diseases ( 71 ) . nitric oxide , a potent endogenous vasodilator formed in the endothelium by the endothelial isoform of enos , plays an important role in maintaining endothelial homeostasis ( 71 ) . nitric oxide furthermore inhibits monocytes and leukocytes adhesion to the endothelium , aggregation of platelets , oxidation of low density lipoprotein , and smooth muscle cell proliferation ( 7679 ) . adropin could enhance the expression of endothelial nitric oxide synthase ( enos ) , responsible for the production of vascular nitric oxide , in the endothelium , so adropin deficiency is associated with reduced nitric oxide bioavailability in the endothelium ( 74 , 80 ) . loss of no bioavailability is a critical stage in formation of endothelial dysfunction that is an independent predictor of the onset of coronary artery disease ( 81 ) . patients with cardiac syndrome x , characterized by endothelial dysfunction , compared to healthy subjects had significantly lower adropin levels ( 71 ) . besides , the reduced circulating adropin concentrations participates in metabolic disorders such as insulin resistance ( 5 , 13 ) related to the onset and progression of coronary atherosclerosis and increased incidence rate of cardiovascular events ( 82 , 83 ) . c - reactive protein has been widely used for cardiovascular risk stratification ( 2729 ) . a negative correlation between adropin and hs - crp levels atherosclerosis has been regarded as a chronic inflammatory disease , so adropin as a potential anti - inflammatory protein play an important role in the prevention of atherosclerosis ( 8) . however , plasma adropin levels were correlated positively with bmi and the severity of heart failure , which is in contrary to our results ( 72 ) . cardiac cachexia which characterized by weight loss , muscle wasting and cytokine activation frequently occurs in patients with advanced hf ( 84 ) . liver enho expression decreases with either diet or genetically induced obesity ( 5 ) . besides , cardiac cachexia or wasting , which could reflect a decrease in visceral fat , may be result in the elevation of plasma adropin levels in patients with hf . adropin may serve as a potential biomarker for early diagnosis of and severity stratification hd . ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors .

arachnoid cysts are prevalent among the general population . the management options of symptomatic arachnoid cysts each have their own merits and disadvantages . we report a case where a large arachnoid cyst was treated by open fenestration and marsupialization that was complicated by remote intraparenchymal and spinal subdural hemorrhage . the potential physiological changes underlying these complications as well as the related literature are reviewed .

arachnoid cysts are prevalent among the general population and with increased access to brain imaging , incidental cysts are being increasingly diagnosed . surgical options for symptomatic arachnoid cysts include cyst fenestration , either open or endoscopic , insertion of a cysto - peritoneal shunt or marsupialization via a craniotomy . recent studies have shown areas of hypoperfusion related to patients with asymptomatic arachnoid cysts suggesting that these lesions may also require surgical intervention . we report a case where a large arachnoid cyst was treated by marsupialization and was complicated by remote intraparenchymal hemorrhage and a spinal subdural hematoma . a 6-year - old , otherwise healthy boy , presented with a chronic history of generalized headache , which had persisted despite strong analgesia . 1.5 t mr imaging ( ge medical systems , milwaukee , usa ) revealed a large left temporal arachnoid cyst causing mass effect with effacement of the left lateral ventricle , remodeling of the inner table of the calvarium , and mid - line shift [ figure 1 ] . two smaller arachnoid cysts in the left parietal and right temporal regions were also noted . axial t2 fse images show the left arachnoid cyst causing midline shift ( a ) and effacement of left lateral ventricle ( b ) after a lengthy discussion with the family detailing the merits and disadvantages of the various treatment options , the patient was admitted for marsupialization of the large cyst . larger than usual cortical veins were noted postero - medially and were not disturbed during surgery . a subgaleal suction drain was inserted after closure of the dura was carried out . in the post - operative suite , the patient initially moved all four limbs equally and responded appropriately . at 1-h post - procedure , he dropped his conscious level and was flexing to pain with no verbal response and was not eye opening to pain . the drainage bag contained 200 ml of blood - stained fluid and the drain was then clamped . immediate axial computed tomography ( ct ) scan revealed significant decompression of the left middle cranial fossa arachnoid cyst and improvement in the degree of midline shift . [ figure 2 ] focal areas of parenchymal hemorrhage were noted distant from the site of surgery in the left frontal lobe and left cerebellum . immediate post - op unenhanced axial ct shows reduction in arachnoid cyst volume ( a ) but also new left cerebellar ( b ) and frontal hemorrhages ( c ) an intracranial pressure ( icp ) monitor , inserted via a standard right frontal approach revealed the pressure to be 6 mmhg . the patient remained well sedated with an aim to keep the icp and cerebral perfusion pressure within normal limits . magnetic resonance imaging ( mri ) head at 18 h revealed a reduction in size of the left arachnoid cyst but also confirmed hemorrhage in the left frontal lobe and left cerebellar hemisphere [ figure 3 ] . 18-h post - op axial t2 fse ( a ) and swan ( b ) show reduction in the arachnoid cyst and areas of parenchymal hemorrhage ( as evidenced by the susceptibility artifact ) the patient was managed post - operatively in the intensive care unit ( icu ) and gradually weaned from the ventilator over the following week . neurological examination confirmed a right - sided upper limb and bilateral lower limb weakness ( mrc grade 4/5 ) with intact sensation and reflexes . ct head on day 8 revealed bi - frontal subdural collections and an extra - axial collection just beneath the bone flap . his power improved over the next 48 h. on day 10 , he was noted to have difficulty in commencing micturition . subsequent mr spine [ figure 4 ] revealed a subdural hematoma from the mid - thoracic region to the sacrum . day 10 post - op t1 images show the extensive subdural high t1 signal fluid collection in the lumbar ( a ) and thoracic ( b ) spine consistent with a hematoma he continued to improve with respect to the power in his lower limbs and the subdural hematoma was thereby managed conservatively . at 4 weeks post - operatively , the patient 's bladder function , along with limb power , had returned to normal . he was seen in outpatients at 10 weeks post - operatively and his headaches had resolved and he was at school performing normal activities . larger than usual cortical veins were noted postero - medially and were not disturbed during surgery . in the post - operative suite , the patient initially moved all four limbs equally and responded appropriately . at 1-h post - procedure , he dropped his conscious level and was flexing to pain with no verbal response and was not eye opening to pain . the drainage bag contained 200 ml of blood - stained fluid and the drain was then clamped . immediate axial computed tomography ( ct ) scan revealed significant decompression of the left middle cranial fossa arachnoid cyst and improvement in the degree of midline shift . [ figure 2 ] focal areas of parenchymal hemorrhage were noted distant from the site of surgery in the left frontal lobe and left cerebellum . immediate post - op unenhanced axial ct shows reduction in arachnoid cyst volume ( a ) but also new left cerebellar ( b ) and frontal hemorrhages ( c ) an intracranial pressure ( icp ) monitor , inserted via a standard right frontal approach revealed the pressure to be 6 mmhg . the patient remained well sedated with an aim to keep the icp and cerebral perfusion pressure within normal limits . magnetic resonance imaging ( mri ) head at 18 h revealed a reduction in size of the left arachnoid cyst but also confirmed hemorrhage in the left frontal lobe and left cerebellar hemisphere [ figure 3 ] . 18-h post - op axial t2 fse ( a ) and swan ( b ) show reduction in the arachnoid cyst and areas of parenchymal hemorrhage ( as evidenced by the susceptibility artifact ) the patient was managed post - operatively in the intensive care unit ( icu ) and gradually weaned from the ventilator over the following week . neurological examination confirmed a right - sided upper limb and bilateral lower limb weakness ( mrc grade 4/5 ) with intact sensation and reflexes . ct head on day 8 revealed bi - frontal subdural collections and an extra - axial collection just beneath the bone flap . his power improved over the next 48 h. on day 10 , he was noted to have difficulty in commencing micturition . subsequent mr spine [ figure 4 ] revealed a subdural hematoma from the mid - thoracic region to the sacrum . day 10 post - op t1 images show the extensive subdural high t1 signal fluid collection in the lumbar ( a ) and thoracic ( b ) spine consistent with a hematoma he continued to improve with respect to the power in his lower limbs and the subdural hematoma was thereby managed conservatively . at 4 weeks post - operatively , the patient 's bladder function , along with limb power , had returned to normal . he was seen in outpatients at 10 weeks post - operatively and his headaches had resolved and he was at school performing normal activities . whilst our patient has made an eventual recovery , it is unclear as to the mechanism of the complications that occurred . sgouros and chapman studied three children with middle fossa arachnoid cysts using mri and single photon emission computerized tomography before and after surgery . in their paper , they have shown that these cysts may cause global impairment of the brain function by interfering with cerebral blood flow and perfusion . our patient had a large arachnoid cyst and may have had chronic impairment of his autoregulation response . after excision of an arteriovenous malformation ( avm ) , hyperperfusion to the adjacent weakened capillary beds resulting in hemorrhage has been described previously . the rapid decompression from the craniotomy could have led to a rapid rise in cerebral perfusion causing damage to the capillary bed and resulting in parenchymal injury . changes in the intracranial dynamics due to brain shift can cause venous hyperemia . in our patient , the potentially rapid loss of cerebrospinal fluid ( csf ) may have led to brain shift and thus potentially cause the sites of hemorrhage noted distant from our operative site . the changes in venous and csf pressures causing the intracranial complications would equally be attributable to the hemorrhage seen in the spine . spinal subdural hematomas in children are usually associated with spinal puncture and non - accidental injury and/or in the presence of coagulation abnormalities , none of which was the case in our patient . brain shift and hyperemic states however , we believe that this is the first case of a spinal subdural hematoma as a complication from decompression of an intracranial arachnoid cyst . the subsequent brain shift may lead to a hyperperfusion injury . whilst we recognize that a craniotomy may provide the best long - term outcome for treating arachnoid cysts , more gradual decompression with programmable shunts or even endoscopic fenestration of the cyst into an adjacent basal cistern may be a safer approach . the use of a suction drain should be avoided in these cases even in the presence of macroscopic watertight closure of the dura .

to determine prevalence of genital human papillomavirus ( hpv ) infection among men in rural china , we analyzed genital swab specimens . among 2,236 male residents of rural henan province , hpv infection prevalence was 17.5% . the most common oncogenic and nononcogenic types were hpv-16 and hpv-3 , respectively . infection was associated with younger age and multiple sex partners .

in 20072009 , a population - based esophageal cancer cohort study was initiated in 9 villages in rural anyang , henan province , china ( 4 ) . this hpv investigation was added to the original cohort study in 6 of the 9 villages . eligibility criteria were as follows : 1 ) male sex ; 2 ) permanent residency in the target villages ; 3 ) age 2565 years ; and 4 ) no history of cancer , cardiovascular disease , or mental disorder . of 3,571 eligible men , 3,172 ( 89% ) were enrolled . the main reason why the other 399 , who were substantially younger , did not participate was loss of contact because they were employed outside anyang . participants were interviewed , and exfoliated cells were collected from the penile shaft , glans penis , coronal sulcus , and scrotum by using saline - soaked swabs ( 5 ) . the sampling procedure was identical for circumcised and uncircumcised men . to assess the adequacy of the specimens positive specimens were subsequently tested for 13 oncogenic and 37 nononcogenic types of hpv by using pcr - based direct sequencing with a pair of spf1/gp6 + primers ( 6 ) . samples with ambiguous hpv typing signals were subjected to further cloning and sequencing . to evaluate the associations between exposure variables and the presence of hpv dna , we used logistic regression analysis with stepwise backward elimination at p>0.1 . to examine the association across the ordered categorical variables , we used a trend test . of 3,172 specimens tested , 2,236 were positive for -globin and were included in the analysis ( median participant age 42 years ; interquartile range 3552 years ) . we excluded from the study men who were older and reported less risky sexual behavior than those who were included ( technical appendix ) . of the 40 hpv types we tested for , we detected 36 , including 13 oncogenic types oncogenic hpv was detected in 140 ( 6.3% ; 95% ci 5.3%7.3% ) specimens , and nononcogenic hpv was detected in 251 ( 11% ; 95% ci 9.9%13% ) specimens . among 15 hpv - positive specimens that had ambiguous direct sequencing signals and were further cloned and resequenced for genotyping , 3 had a second type and minor types were ignored . among these infections ( table 1 ) , the most common oncogenic type detected was hpv-16 ( 17.4% ) , followed by hpv-18 ( 7.2% ) . the most common nononcogenic type was hpv-3 ( 16.4% ) , followed by hpv-57 ( 7.9% ) . of 391 hpv - positive specimens , 15 displayed ambiguous typing signals by direct sequencing of pcr product . infection with > 1 hpv type was detected in only 3 of 15 specimens that were tested by cloning and sequencing ; the predominant type is shown . oncogenic types tested in this study included hpv-16 , -18 , -31 , -33 , -35 , -39 , -45 , -51 , -52 , -56 , -58 , -59 , and -66 . on the basis of latest published literature and our knowledge of the detection method of hpv dna adopted in this study ( spf1/gp6 + mediated pcr and sequencing ) , nononcogenic types that could be tested for were hpv-3 , -6 , -7 , -10 , -11 , -26 , -27 , -29 , -30 , -32 , -37 , -40 , -42 , -43 , -44 , -53 , -54 , -55 , -57 , -61 , -62 , -67 , -68 , -69 , -70 , -72 , -74 , -75 , -77 , -81 , -82 , -84 , -85 , -87 , -90 , -91 , and -94 . prevalence of infection of any or nononcogenic hpv types decreased significantly with participant s increasing age ( table 2 ) . risk for infection with any hpv type was associated with being unmarried , having had multiple sex partners , and having had oral and anal sex . when the outcome was stratified by oncogenicity of hpv type , the association remained statistically significant for having had multiple sex partners . adjusted ors and 95% cis derived by multivariate logistic regression models including all the listed variables ; backward method was used to select significant variables on the 0.10 level . p values for trend derived by logistic regression analyses , treating categorical variables as continuous variables . this study of genital hpv in a large sample of adult men from rural china addresses the paucity of data on male genital hpv infection in asia . prevalence rates for any type of hpv and oncogenic hpv were lower among these men in china than among heterosexual men elsewhere ( asia pacific area and globally ) ( 2,7 ) . this discrepancy might be partly explained by the relatively more conservative sexual behavior and higher median age of participants in this study . among populations of similar age , prevalence of a specific hpv type is usually lower among men than among women ( 1 ) . however , the 17.5% prevalence found in this study exceeds estimates for married women 1559 years of age in china ( 14.8%16.8% ) ( 8) . this finding is consistent with that of another study , which also reported higher hpv prevalence among men than among women ( 9 ) . these inconsistent findings might be explained by differences in hpv type distribution between male genitalia and the female cervix and by variability of type - specific sensitivity in detection methods . although most studies of hpv infection in men worldwide have found no clear trend with regard to age ( 1 ) , we found that infection mildly decreased with increasing age . a potential explanation for this age - related trend might be that in this population , young adults more commonly work for long periods outside the rural home area than do older adults ( data not shown ) , and the increased mobility might be associated with more unprotected sexual behavior and consequent increased exposure to hpv ( 10 ) . our finding that hpv-16 and -18 were the most commonly detected oncogenic hpv types is in keeping with findings of previous studies ( 2,7,11,12 ) . however , our finding that hpv-3 and hpv-57 were the predominant nononcogenic types is in contrast to the findings of other studies that hpv-6 and hpv-11 were the most predominant ( 2,11,13 ) . this discrepancy might partly be explained by the fact that in our in - house evaluation , the primer set spf1/gp6 + was more sensitive for hpv-57 but less sensitive for hpv-11 than was gp5+/gp6 + . completely opposite to our findings , dai et al . reported that among women in neighboring rural shanxi province , china , oncogenic types were more commonly detected than were nononcogenic types ( 14 ) . one possible explanation is that in our study , a significant portion of the exfoliated cells were collected from skin tissue . therefore , a number of cutaneous hpv types , which are nononcogenic for mucosal lesion ( cervical cancer ) , could be detected . we believe this might have led to the higher proportion of nononcogenic hpv than oncogenic hpv detected in our study . another possible explanation is that sequencing methods used in our study can detect more nononcogenic types , which escape identification in studies that use hybridization with preassigned probes . in contrast to previously reported rates of infection ( 7,12 ) , in our study , infection with multiple types was rare . the previous studies used hybridization , an efficient way to identify co - infections , for genotyping . however , we used sequencing instead of hybridization to maximize demonstration of the spectrum of hpv types . because minor types would probably be covered by the predominant type in the sequencing process , sequencing the low proportion of -globin positivity might have partly resulted from the lower efficiency of cell collection by use of a saline - moistened swab as opposed to other methods such as emery paper ( 15 ) . another possible explanation is that a certain proportion of cells collected from male genitalia are keratinized and contain less nucleated human dna than cells collected from mucosal organs ( e.g. , the cervix ) ( 15 ) . however , the biases potentially imposed by the nonparticipation of 400 younger men ( because of mobility ) and 936 older men ( because of specimen inadequacy ) must be noted . this nonparticipation of younger men might have neutralized the age - related association to some extent . although circumcision is extremely rare in rural china , the lack of accurate data for this variable is another study limitation , which rendered subgroup analysis by circumcision status impossible . as reported in other studies ( 1 ) , having had multiple sex partners was associated with hpv infection in this population . this finding indicates that men with higher mobility ( i.e. , higher risk for multiple sex partners and unprotected sexual behavior ) should receive more attention with regard to future hpv control in this region . characteristics of 3,172 men from rural henan province , by human papillomavirus -globin status of genital specimens , 20072009 .

acute myocardial infarction complicated by cardiogenic shock and left main coronary artery disease is called left main shock syndrome . it is reported that the morbility and mortality of the syndrome is approximately 0.46% and 55%80% , respectively . however , the best treatment strategy in these cases is unknown . in this article , we present a patient with lmss who successively underwent emergency percutaneous coronary intervention and coronary artery bypass grafting with hemodynamic support within 5 days . the patient is now on his three month uneventful out - patient follow - up .

acute myocardial infarction ( ami ) complicated by cardiogenic shock and left main coronary artery ( lmca ) disease is called left main shock syndrome ( lmss ) . it is reported that the morbility and mortality of the syndrome is approximately 0.46% and 55%80% , respectively . , we present a patient with lmss who successively underwent emergency percutaneous coronary intervention ( pci ) and coronary artery bypass grafting ( cabg ) with hemodynamic support within 5 days . the patient is now on his three month uneventful out - patient follow - up . a 59 year - old male was admitted to our hospital experiencing recurrent chest pain for 6 years and severe pain for 10 h. he had been smoking for seven years and had prior 5 years history of hypertension ( 160/90 mmhg ) . on physical examination , the patient was clammy and normal blood pressure ( 115/60 mmhg ) , with normal heart sounds and bilateral pulmonary rales on the lower zone of lungs . his electrocardiogram demonstrated st segment elevation in leads i , avl , avr , v1 through v4 , st segment depression in lead ii , iii and avf ( figure 1 ) . acute anterioseptal and lateral myocardial infarction ( killip class ii ) and hypertension were diagnosed and the patient was immediately transferred to the catheterization laboratory from the emergency unit . 300 mg aspirin , 600 mg loading dose of clopidogrel and 5000 u of intravenous heparin were administered . as soon as the coronary angiography was performed , the blood pressure of the patient had dropped to 80/50 mmhg with dyspnea . coronary angiography disclosed a left main occlusion in the middle portion of its body and a severe stenosis at the proximal posterior descending artery of right coronary artery ( figure 2 ) . intra - aortic balloon pump ( iabp ) was inserted and the blood pressure was increased to 95/50 mmhg , except for severe hypoxemia . so an extracorporeal membrane oxygenation machine ( ecmo ) was inserted via left femoral venoarterial access . after one coronary soft guide wire ( 0.014 inch run through ; teromo , japan ) had been passed into the left anterior descending ( lad ) coronary arteries , the distal left main portion and lad was predilatated with 1.5 15 mm balloon inflated at 16 atm . subsequently , lad was dilatated with sprinter 2.0 12 mm balloon inflated at 14 atm and a fire star 2.5 15 mm balloon inflated at 14 atm . distally to this lesion with thrombolysis in myocardial infarction ( timi ) grade 3 flow , both lad and diagonal branch presented a significant diffuse lesion while circumflex ( cx ) ostia presenting with coronary aneurysm ( figure 3 ) . it demonstrated st segment elevation in leads i , avl , avr , v1 through v4 and st segment depression in lead ii , iii and avf . ( a ) : right coronary artery angiograph shows a severe stenosis at the proximal posterior descending artery ; ( b ) : angiograph of left coronary artery viewed from spider position shows a left main occlusion in the middle portion of its body . the patient was then monitored in coronary heart disease care unit ( ccu ) with 3.4 l / min of extracorporeal membrane oxygenation ( ecmo ) and 1 : 1 counterpulsation of iabp supporting on the first day after pci . the total 24 h urine volume increased from 1050 ml of the second day to 3170 ml of the third day after pci . so the iabp was withdrawn and ecmo volume was turned down to 2.4 l / min . however , the number of platelets in the whole blood of the patient dropped from 140 g / l on the first day to 76 g / l on the fifth day with normal liver , kidney and blood clot function , a successively cabg ( ao - svg - lad and ao- svg - d1-pda ) was then performed . the european society of cardiology for 2012 st - elevation myocardial infarction guideline recommends emergency revascularization with either pci or cabg in suitable patients with cardiogenic shock as class i and a level of evidence b. however , since lmss morbility is so low , we do not know exactly which is better for pci or cabg treatment and what we should do if iabp does not produce an positive effect in these patients . distally to the lesion of the left main , proximal left anterior descending , diagonal branch and proximal circumflex presented significant diffuse lesions while circumflex ostia presenting with coronary aneurysm and timi grade 3 flow . in this case , we selected pci and succedent cabg treatment strategy based on the following considerations . first , as common strategies of unprotected left main revascularization in acs , both pci and cabg were significantly associated with improved discharge and 6 month survival in comparison with no revascularization . pci became more feasible strategy of revascularization in this situation than cabg surgery , which will generally be delayed . third , it has been timi 3 grade flow after balloon inflation in lad , in which presented a significant diffuse and small vessel distal to coronary aneurysm lesion in lcx . as a previous study indicated , a small lumen diameter before the procedure is the independent predictor of death in patients with cardiogenic shock , we performed cabg surgery days later . guidelines also suggested iabp with a recommendation class iib and a level of evidence b as an effective measure in combination with balloon angioplasty in these patients . however , there is insufficient evidence endorsing the current guideline recommendation in the setting of lmss . on the contrary , in the meta - analysis included nine cohorts of st - elevation myocardial infarction ( stemi ) patients with cardiogenic shock ( n = 10529 ) treated with pci , support by iabp was associated with a 6% ( 95%ci : 3%10% ; p < 0.0008 ) increase in 30 day mortality . whereas , a recent study indicated that patients who received primary pci and supported with iabp and ecmo had better 30-day and 1-year survival outcomes than those only with iabp . but the difficult decision to withdraw ecmo may need to be made if the patient is not eligible for conventional corrective surgery , or longer term ecmo . as observational data concerning iabp or ecmo therapy in the setting of lmss is importantly hampered by bias and confounding , therefore , randomized controlled trials in the future are needed to determine the use of iabp and ecmo in these patients treated with pci . all together , this case has shown that successful team treatments , including the prompt and suitable revascularization strategy , potent iabp and ecmo support are important to improve the clinical outcome in patients with lmss .

a simple method is described for growing rat embryos in vitro for 48 hr from head - fold to early limb - bud stages at rates of development and protein synthesis indistinguishable from those in vivo . culture of the embryos can be continued for longer periods but at a reduced growth rate . preheating the culture serum to 56 degrees c for 30 min improves embryonic development , but raising the culture temperature 2 - 3 degrees c or exposing the presomite embryos to 20% o2 ( 160 mm hg ) causes malformations , particularly of the brain and spinal cord . the value of such culture methods for teratology is briefly discussed.imagesfigure 3.figure 6.figure 8.figure 9.figure 10.figure 11 .

dna topoisomerases of kinetoplastids represent a family of dna processing enzymes that essentially solve the topological problems not only in nuclear dna but also in kinetoplast dna . we have , for the first time , identified a leishmania donovani homologue of bacterial and eukaryotic ia type of topoisomerase iii protein and termed as ldtopiii. complementation study of wild - type and mutant ldtopiii with slow - growing topoisomerase iii mutant yeast s. cerevisiae revealed the functional conservation of the leishmanial counterpart of topoisomerase iii protein , the 327 tyrosine being the active site amino acid . a c - terminal deletion construct of ldtopiii could not suppress the slow - growth phenotype of mutant yeast , indicating the requirement of c - terminal region for the enzyme function in vivo.ldtopiii localized inside the nucleus and kinetoplast of the parasite . taken together , our study indicates functional conservation and possible role of ldtopiii in parasite dna processing .

dna topoisomerases are ubiquitous enzymes found in all prokaryotic and eukaryotic cells and in some viruses . they are involved in all aspects of dna metabolism such as replication , transcription , recombination , and chromosome segregation [ 1 , 2 ] . these reactions are based on sequential breakage and rejoining of the dna phosphodiester backbone [ 24 ] . type i dna topoisomerases catalyze the cleavage of one strand of dna , whereas type ii dna topoisomerases catalyze the cleavage of a double - stranded dna , requiring atp as a cofactor . type i dna topoisomerases are further classified in two subfamilies , ia and ib , based on differences in amino acid sequence and reaction mechanisms . they are represented by bacterial topoisomerase i and iii and the eukaryotic topoisomerase iii enzymes . type ib topoisomerases , exemplified by eukaryotic topoisomerase i , in contrast , become attached to 3-phosphate end of the cleaved strand of the dna . while the function of topoisomerase ii and i are quite well established , the role of topoisomerase iii in dna metabolism is still being defined . genes encoding topoisomerase iii enzymes are highly conserved in evolution from bacteria to human , and the phenotypic consequences of loss of topoisomerase iii function are generally quite severe . it has been shown to possess a weak , atp - independent relaxation activity towards negatively supercoiled dna only and strict dependence on magnesium . the e. coli chromosome encodes two type ia topoisomerase , dna topoisomerase i and topoisomerase iii [ 8 , 9 ] . loss of topoisomerase iii in e. coli results in an increase in deletions arising from recombination events between direct repeats [ 10 , 11 ] . yeast cells express a single type ia topoisomerase , topoisomerase iii encoded by the top3 gene . in s. pombe , top3 is essential for viability and plays a role in chromosome segregation . it has been shown that top3-ts mutant s. pombe cells are sensitive to the dna damaging agents uv and mms ( methyl methanesulfonate ) at the restrictive temperature revealing that topoisomerase iii is involved in dna damage survival . in s. cerevisiae , top3 mutants are viable , but very slow - growing and have defects in s phase responses to dna damage and in both mitotic and meiotic recombination [ 14 , 15 ] . in vertebrates , there are two isoforms of topoisomerase iii enzymes termed and [ 1619 ] . deletion of mouse topoisomerase iii gene displayed shortened lifespan and infertility [ 21 , 22 ] . dna topoisomerases of kinetoplastids represent a family of dna processing enzymes that essentially solve the topological problems not only in the nuclear dna but also in the kinetoplastid dna . the ib type of bi - subunit topoisomerase i and topoisomerase ii of the parasites which maintain vital cellular processes , are also proven target for clinically useful antitumor drugs . apart from this ib type of topoisomerase i , three type ia topoisomerases are there in the parasite genome , termed as topoisomerase ia , and two topoisomerase iii . topoisomerase ia of t. brucei has been reported and shown to be mitochondrial and essential for late theta structure resolution . very recently , a topoisomerase iii from t. brucei has been shown to play a critical role in antigenic switching . in the present study , for the first time , we have identified functionally active dna topoisomerase iii from kinetoplastid parasite l. donovani , which localized both inside the nucleus and kinetoplast of the parasite and rescued the topoisomerase iii mutant yeast from slow - growth phenotype . l. donovani strain ag 83 promastigotes were grown at 22c in m199 liquid media supplemented 10% heat inactivated fetal calf serum . transfected cells were maintained under the same conditions with 100 g / ml g418 . if required , ampicillin and chloramphenicol were used at 100 and 34 g / ml final concentrations , respectively . the yeast strains used in the studies were w5909 - 3b ( mat alpha trp1 - 1 his3 - 11 , 15 leu2 - 3 , 112 ura3 - 1 rad5 lys2 met15 ade2 ) and w2633 - 4c ( a / alpha top3 : : trp1/+ ) ( kindly gifted by dr . the yeast cells were grown at 25c on yepd medium containing 1% peptone , 2% yeast extract , 2% dextrose and 1.5% agar or synthetic minimal media as required . ldtopiii gene was pcr amplified from the genomic dna of l. donovani parasites using the sense primer 5-ggaaattccatatgggccgcaatgtgttgatg-3 and antisense primer 5-cgggatcctcacctgcgatcctcgcggttgcc-3 and was cloned in bacterial expression vector pet16b in nde1 and bamh1 restriction sites , termed as ldtopiii-pet16b . multiple sequence alignment of ldtopiii sequences from various species was carried out using clustal w ( http://expasy.org/tools ) . three - dimensional models of ldtopiii based on the crystal structure of e. coli topoisomerase iii were generated using swiss prot ( http://expasy.org/sprot ) . the protein sequences were represented in ribbon format and the active site residues were represented in ball and stick format over the ribbon structure . ldtopiii genes was pcr amplified using ldtopiii-pet16b as templates and was subcloned using the sense primer 5-cgggatccatgggccgca atgtgttgatg-3 and antisense primer 5-gatatccctgcgatcctcgcggttgcc-3 in bamh1 and ecorv sites of leishmania transfection vector pxg - b2863 ( a kind gift from dr . s. m. beverley ) , to produce c - terminal - gfp - tagged full - length ldtopiii protein and termed as ldtopiii-gfp . the constructs and empty vector pxg - b2863 were transfected into l. donovani promastigotes separately by electroporation as described earlier . briefly , late log - phase promastigotes were harvested and washed twice in opti - mem ( gibco ) . cells were finally suspended at a density of 1 10/ml and 0.4 ml was taken into a 0.2 mm ice - chilled electroporation cuvette . thirty microgram of plasmid dna was taken in 100 l of electroporation buffer and added to the cells . after 10 min on ice , the cells were electroporated with a single pulse by bio - rad gene pulsar apparatus using 450 v and 550 f capacitance . the cells were incubated on ice for further 5 min and then added to10 ml of drug - free growth medium . after 24 h of survival 10 g / ml g418 was added and kept at 22c . finally the transfected cells were routinely maintained in medium containing 100 g / ml g418 . localization of c - terminal gfp tagged chimeric ldtopiii-gfp protein was visualized by fluorescence microscopy ( olympus ix81 ) . differential visualization of the fluophores was achieved using a 488 nm excitation filters and 523 nm emission filter for gfp and 258 nm excitation and 361 nm emission filter for dapi . the full - length ldtopiii was subcloned in xbai and bamh1 sites into the yeast shuttle vector pvt100u , a kind gift from dr . rolf sternglanz and termed as ldtopiii-pvt using the sense primer 5-gctctagaatgggccgcaatgtgttgatg-3 and antisense primer 5-cgggatcctcacctgcgatcctcgcggtt-3. for construction of c - terminal deletion construct of ldtopiii , regions corresponding to amino acids 1 - 608 was pcr amplified using the primers 5-gctctagaatgggccgcaatgtgttgatg-3 ( sense ) and 5-cgggatccggcggcggagatggcggagaa-3 ( antisense ) and was cloned in xba1 and bamh1 sites of pvt100u vector . mutagenesis was performed by using the quikchangexl site - directed kit ( stratagene , la jolla , ca ) according to the manufacturer 's protocol . to carry out the desired mutations , ldtopiii-pvt was used as templates for all mutagenesis experiments . for each mutation , the wild - type nucleotide was replaced using a specific pair of mutagenic primers . the following sense primer , along with the antisense counterparts ( with codons in boldface and substitutions underlined ) , were used ; for y327 of ldtopiii , sense primer was 5-ccgcggctatatttcgttccctcgtacccgaatcc-3 and antisense primer was 5-ggattcggtacgagggaacgaaatatagccgcgg-3. the top3 mutant yeast strain w2633 - 4c ( a / alpha top3 : : trp1/+ ) ( a kind gift from dr . r rothstein ) was used for transformation with recombinant topo iii proteins from l. donovani by the lithium acetate and polyethylene glycol method . colonies were picked and cultured in tubes with 2 ml of synthetic minimal media at 30c overnight . in vitro transcription and translation of ldtopiii proteins ldtopiii-pet16b plasmids were used as dna templates for synthesis of the protein . after the reaction is over , the crude bacterial lysate containing the newly synthesized protein was tested for activity . the type ia dna topoisomerases were assayed by decreased mobility of the relaxed isomers of supercoiled pbs ( sk+ ) [ pbluescript ( sk+ ) ] dna in an agarose gel . relaxation assay was carried out with the crude lysates containing the in vitro transcribed and translated ldtopiii. supercoiled pbs dna ( 85%95% were negatively supercoiled with the remaining being nicked circles ) was used as substrate in the relaxation buffer ( 25 mm tris - hcl , ph 7.5 , 5% glycerol , 0.5 mm dtt , 2 mm mgcl2 , 50 g / ml bsa ) . the amount of supercoiled monomer dna band fluorescence after etbr ( 0.5 g / ml ) staining was visualized using gel doc 2000 under uv illumination ( bio - rad quality one software ) . one is on chromosome 21 , annotated as topoisomerase ia ( lmjf21.0125 ) with an orf of 2453 bp . two other type ia topoisomerases are present on chromosome 28 and 36 , respectively , both of which are annotated as topoisomerase iii ( lmjf28.1780 and lmjf36.3200 , resp . ) . one of the two topoisomerase iii genes present in l. major genedb is 2601 bp ( lmjf28.1780 ) and encodes a 95 kda predicted protein . the other topoisomerase iii orf ( lmjf36.3200 ) is 2844 bp , and encodes a 104 kda predicted protein . topoisomerase iii gene with 2601 bp was pcr amplified from the genomic dna of l. donovani , cloned and sequenced ( genebank accession number gq499197 ) . blast analysis of the sequence confirmed the topoisomerase iii lineage of the protein and henceforth referred as ldtopiii. the alignment of ldtopiii with s. cerevisiae and s. pombe topoisomerase iii and human topoisomerase iii is shown in figure 1 . the active site tyrosine is located at the 327 position within a highly conserved gyisyprtes sequence . the protein has 46.22% identity and 76.09% similarity with human topoisomerase iii. it contains seven cxxc sequences instead of eight found in other topoisomerase iii proteins . glycine ( g ) and arginine ( r ) rich clusters at the c - terminus end , which is another hallmark of topoisomerase iii , are also present . it has a continuous stretch of 19 g and r residues in the c - terminus . three - dimensional structure generated by swiss prot has been shown in figure 2(a ) . the conserved amino acid residues are represented in ball and stick format and have been labeled . homology comparisons of ldtopiii with other ia type of topoisomerases have been provided in table 1 , which strongly indicates its topoisomerase iii lineage . in silico search a 0.244 probability of mitochondrial transport was predicted by mitoprot ( http://expasy.org/tools ) analysis and 73.9% cytoplasmic and 17.4% nuclear distribution was revealed by psort ii analysis ( http://expasy.org/tools ) . to determine the precise localization of the protein , full - length ldtopiii ( 865 aa ) was cloned in leishmania expression vector as a c - terminal fusion protein with gfp , termed as ldtopiii-gfp , and the construct was transfected in l. donovani parasites . comparison of dapi and gfp fluorescence and merged images ( figure 3(c ) ) revealed that ldtopiii protein localized both inside the nucleus and kinetoplast of the parasites . figures 3(d ) and 3(e ) show cytoplasmic distribution of control gfp protein in l. donovani parasites . mutation of the s. cerevisiae top3 gene is known to result in several phenotypes , including a growth rate which is only 50% that of wild - type . in order to assess whether the ldtopiii possesses functional similarity to the yeast topoisomerase iii , we have used a functional complementation assay of ldtopiii protein to rescue top3 mutant s. cerevisiae strain from slow - growing phenotype . we have cloned the ldtopiii gene in a shuttle vector pvt100u to generate ldtopiii-pvt and transformed in top3 yeast s. cerevisiae and ura+ colonies were selected . the ldtopiii-pvt partially complemented the slow - growth of top3 yeast ( figure 4(a ) ) . the improved growth rate was not observed in case of the vector control ( figure 4(a ) ) . this observation suggests that ldtopiii can be functionally expressed in yeast and shares functional similarity with s. cerevisiae top3 gene , which is consistent with earlier observations made with drosophila and human topisomerase iii proteins [ 19 , 29 ] . to observe this complementation of ldtopiii in liquid medium equal amounts of the wild - type , topoisomerase 3 mutant yeast cells , topoisomerase 3 mutant yeast cells containing empty vector ( pvt100u ) and topoisomerase 3 mutant yeast cells containing ldtopiii ( grown overnight at 30c ) were inoculated in fresh minimal medium and grown at 30c . at every 2 hr interval up to 12 hrs , the growth was monitored and plotted . tyrosine 327 of ldtopiii was predicted to be the active site amino acid residue from sequence alignment analysis . in order to determine that ldtopiii functionally complements the top3 mutant yeast and the growth recovery was not due to any compensatory mechanism induced by ldtopiii we carried out site directed mutagenesis . we have mutated the active site residue of ldtopiii to phenylalanine ( y327f ) by site directed mutagenesis and transformed in top3 mutant yeast . it was observed that the active site mutant construct could not suppress the slow - growth of top3 mutant s. cerevisiae ( figures 5(a ) and 5(b ) ) confirming role of active site tyrosine 327 in functional conservation of ldtopiii inside mutant yeast cells . the leishmania enzyme has a c - terminal segment of amino acids with no counterpart in yeast protein . the leishmanial protein contains zn - binding motif at its c - terminus , which is absent in the topoisomerase iii proteins of e. coli and yeast . the c - terminus residues of e. coli topoisomerase iii have been previously shown to be involved in dna binding . to determine the role of the c - terminal stretch of ldtopiii in functional complementation , we have made a c - terminal deletion construct ( ldtopiiic258 ) removing the 258 amino acids and transformed in topoisomerase iii mutant yeast . the transformants were grown in plates and it was observed that the c - terminal deletion construct failed to rescue the mutant yeast from slow - growth ( figure 5(a ) ) , suggesting essentiality of the c - terminal segment for functional complementation in vivo . to validate this observation in liquid medium we inoculated overnight grown cultures at 30c in fresh minimal medium and monitored their growth at 3 hr intervals . the growth curve ( figure 5(b ) ) clearly indicates that ldtopiiic258 could not functionally complement the slow - growing topoisomerase iii mutant yeast . this indicates that the conserved c - terminal region between amino acid residues 608866 contains important residues that are required for in vivo function of ldtopiiic258 . to get a better insight into the functional characteristics of the enzyme , we next sought to obtain recombinant ldtopiii protein in vitro . ldtopiii was cloned in bacterial expression vector pet-16b and overexpressed in bl21 ( de3)-plyss strain and induced with iptg . but the overexpressed protein went to inclusion body and were found in the pellet as insoluble protein which could not be recovered in the soluble fraction in active state . however , to test the activity of the recombinant protein , we have used in vitro transcription - translation kit , which is specially designed for in vitro transcription and translation of target dna to protein in a single reaction . the crude lysate containing the newly synthesized proteins were used for dna relaxation assay . figure 6(a ) shows dna relaxation by increasing amount of recombinant ldtopiii ( lanes 28 ) . the results clearly show that the recombinant protein containing lysates were able to relax the negatively supercoiled dna . to test that the activity was not coming from the lysate itself , we have carried out dna relaxation activity with the empty vector containing lysate which contained insignificant amount of activity , shown in figure 6(b ) ( lane 3 ) . the type ia topoisomerases are among the most conserved proteins in nature , and their presence in all organisms is supported by extensive biochemical and genomic sequence data [ 2 , 4 ] . this universal presence suggests that the type ia dna topoisomerases play an indispensable role in one or more fundamental processes involving dna , plausibly in the removal of double holliday junctions . topoisomerases iii and iii of kinetoplastid parasites seem to be orthologues of same kind of enzymes in other eukaryotes , notable for branching early within their respective groups . in the present study , for the first time we have identified functionally active dna topoisomerase iii from l. donovani . blast sequence alignments suggested topoisomerase iii from leishmania has high homology with human and drosophila topoisomerase iii. it shares many features , which are typical for other topoisomerase iii proteins including the cxxc type of motifs and a long stretch of g and r residues at its c - terminus . gfp - fused ldtopiii localized both inside the nucleus and the kinetoplast of l. donovani parasites indicating the involvement of ldtopiii in dna processing inside both the parasite organelle . our results show for the first time the presence of an ia type of topoisomerase in the nucleus , as well as in the kinetoplast of leishmania parasites . previously , a ia type of topoisomerase from bacterial origin has been reported to be mitochondrial in t. brucei . ldtopiii could suppress the slow - growth phenotype of the mutant yeast indicating the functional conservation of topoisomerase iii activity . the result is consistent with the earlier observations made with human and drosophila topoisomerase iii enzymes . the c - terminal deletion construct of ldtopiii lacking its zn binding domain was unable to rescue the topoisomerase iii mutant yeast from slow - growing phenotype revealing that the c - terminal 258 amino acids were indispensable for functional complementation of ldtopiii in vivo . previous report reveals the requirement of the c - terminus region of bacterial topoisomerase iii in substrate specificity . it is possible that c - terminal end of the leishmanial topoisomerase iii protein is essential for dna binding which requires further investigations . site directed mutagenesis study revealed that tyrosine at 327 position within the conserved amino acid stretch is the active site tyrosine of ldtopiii and when this tyrosine is mutated to phenylalanine , the protein failed to complement the slow - growing mutant yeasts . the result indicates towards involvement of the functionally active ldtopiii in rescue of the mutant yeast from slow - growth . our attempts to purify recombinant ldtopiii enzymes in active state from bacteria were unsuccessful as the proteins consistently went to inclusion body . but we were able to study , for the first time , the in vitro dna relaxation activity the recombinant topoisomerase iii protein from the kinetoplastid parasite leishmania , when synthesized using cell free in vitro transcription - translation kit . altogether , this is the first report of functionally active topoisomerase iii protein from unicellular kinetoplastid parasite leishmania . important nonoverlapping function of the two isozymes of topoisomerse iii has been revealed by previous studies . the mouse - knockout experiments suggests , the form is essential for embryonic development , whereas the form is critical for life span [ 20 , 21 ] . genetic experiments in yeast have demonstrated that top3 plays a role in suppressing mitotic recombination and in resolving recombined homologous chromosomes during meiosis i [ 14 , 31 ] . preferential cleavage of plasmid - based r- and d - loops , has been reported by drosophila topoisomerase iii . furthermore , the combined action of either yeast or bacterial topoisomerse iii and the dna helicase recq can promote the formation of dna catenanes . the unwinding action of a recq type helicase appears to generate a dna structure that can be recognized by a topoisomerase iii . the only report of functionally significant topoisomerase iii from kinetoplastide parasite came very recently , which describes that topoisomerse iii from trypanosoma brucei influences antigenic variation by monitoring expression - site - associated vsg switching . existence of functionally active topoisomerase iii protein in leishmania indicates towards its role in dna metabolism in the parasites , which requires further studies and might emerge as a new therapeutic target that can be exploited against the deadly parasites . this work was supported by network project ( nwp038 ) of council of scientific and industrial research ( csir ) , government of india to h. k. majumder .

uterine rhabdomyosarcoma ( rms ) typically presents as a mixed epithelial and mesenchymal tumors . pure rmss of the female genital tract are uncommon . spindle cell variant of rms is a rare morphologic subtype of embryonal rms and mostly occurs in the paratesticular region of children . here , we present a case of uterine spindle cell rms in a 76-year - old woman . the tumor , 20157 cm in size , was highly necrotic and adherent to the colon and rectum . tumor cells were mostly spindle - shaped , and isolated rhabdomyoblasts were scattered . immunohistochemical stains for myoglobin and myo - d1 showed diffuse positivity for tumor cells . the patient died only of disease three months after diagnosis .

a 76-year - old woman , gravida 3 , para 2 , presented with intermittent lower abdominal pain of increasing severity . the patient reported a 10 kg weight loss with associated anorexia and constipation for approximately six months . physical examination revealed abdominal tenderness and a fixed lower abdominal mass at roughly six months in gestational size . abdominal and pelvic ultrasonography showed a multilobulated solid and cystic mass in the pelvic cavity . a computerized tomography scan of the abdomen and pelvis demonstrated a 16108 cm lobulating - contoured and heterogeneously - enhanced mass occupying the lower abdomen and pelvic cavity . serum levels of tumor markers , with normal values in parentheses , were as follows : cancer antigen 125 , 101.2 u / ml ( < 35 u / ml ) ; cancer antigen 19 - 9 , 4.8 u / ml ( < 30 u / ml ) . intraoperatively , a yellowish , hard , friable , 20157 cm mass had replaced the right lateral uterine wall and had adhered to the colon , rectum , and appendix . the right ovary was not recognized , but the left ovary and both fallopian tubes appeared normal . the tumor grossly occupied the entire right myometrium and was bulging into the endometrial cavity . the cut surface of the tumor had a variegated appearance , including components of greyish soft , whitish fish - flesh , and yellowish myxoid with small cystic changes ( fig . microscopic findings exhibited highly cellular and necrotic tumor consisting of bundles or fascicles of spindle cells in a myxoid stroma ( fig . mitotic figures were frequently counted with more than 100 mitotic figures per 10 high power fields . right ovarian tissue was identified in the fibrous tumor capsule without direct invasion of tumor cells . 5 ) , myo - d1 , desmin , and vimentin . no stains for smooth muscle actin , pan - cytokeratin , epithelial membrane antigen , or estrogen receptor were postive . rms is a common soft tissue tumor seen in children , but pure uterine sarcomas arising in the female genital tract are rare , particularly in adults.1 in the pediatric population , the most commonly involved site is the vagina , whereas the uterine cervix and corpus are the most frequent locations of rms in adult women.2,3 in postmenopausal women , uterine rmss appear to be entirely the pleomorphic type with extremely poor clinical outcome.4,5 rmss can be morphologically classified into three categories : embryonal , alveolar , and pleomorphic . the spindle cell variant is a recently characterized and rare subtype of embryonal rms first described in 1992.6 spindle cell rms predominately occurs in the paratesticular region in children and young adults , and carries a relatively favorable prognosis . after rubin et al.7 reported the first two cases of spindle cell rms in adults , limited reports of adult spindle cell rms have been subsequently published.8 according to the largest review performed by nascimento and fletcher,9 the head and neck region were the most common sites for adult spindle cell rms followed by the retroperitoneum and lower extremity . these adult tumors seem to have a more aggressive clinical course compared to pediatric cases . only one case of uterine spindle cell rms has been previously reported in a 28-year - old woman who presented at international federation of gynecology and obstetrics ( figo ) stage i and was well after two years of follow - up . the case here , was figo stage iiia at presentation , and the patient died of disease three months after diagnosis . differential diagnosis for spindle cell rms of the uterus includes leiomyosarcoma , undifferentiated endometrial sarcoma , and rhabdomyosarcomatous components of a mullerian adenosarcoma or carcinosarcoma . immunohistochemical staining for skeletal muscle markers can be helpful in differentiating spindle cell rms from other spindle cell tumors . leiomyosarcoma is the most common malignant mesenchymal tumor of the uterine corpus and has similar histologic finding to spindle cell rms . tumor cells of leiomyosarcoma are spindle - shaped with characteristic cigar - shaped nuclei , or large pleomorphic resembling rhabdomyoblasts . markers such as myo - d1 and myoglobin are more specific than muscle - specific actin , smooth muscle actin , and desmin , because smooth muscle markers have limited value in differential diagnosis . undifferentiated endometrial sarcomas , highly aggressive neoplasms , do not histologically resemble entometrial stroma and may consist of spindle or markedly atypical tumor cells . although cd10 expression has been observed in both uterine rms4 and undifferentiated endometrial sarcoma , immunophenotypic evidence of skeletal muscle differentiation has only been seen with rms . mixed epithelial and mesenchymal tumors including adenosarcoma and carcinosarcoma consist of a mixture of benign or malignant epithelial and sarcomatous components . epithelial components are usually glandular or rarely non - glandular and can be constitute a small portion of the tumor , especially in carcinosarcoma . sarcomatous elements may be either homologous or heterologous with heterologous components frequently exhibiting rhabdomyoblastic differentiation . when sarcomatoid overgrowth predominates , epithelial components can be obscured . extensive tumor sampling with careful investigation for epithelial areas , and immunohistochemical confirmation may be required to differentiate rms from mixed epithelial and mesenchymal tumors . in conclusion , pure rms of the uterine corpus is uncommon , and the spindle cell variant is especially rare . here , we present a case of spindle cell rms of the uterine body in 76-year - old woman . uterine rms should be differentiated from leiomyosarcoma , undifferentiated sarcoma , and mixed epithelial and mesenchymal tumors . extensive tumor sampling , careful microscopic examination , and immunohistochemical staining may aid in a diagnosis of spindle cell rms .

focal motor status epilepticus ( fmse ) is often associated non - ketotic hyperglycemia ( nkh ) . there are no previous reports describing fmse with nkh that was accompanied by an acute cerebral infarction and its long term follow - up result . we describe the case of a patient having focal motor status epilepticus ( fmse ) associated with non - ketotic hyperglycemia ( nkh ) and acute cerebral infarction who later developed recurrent unprovoked seizures . a small acute infarct was observed in the left frontal subcortical area on diffusion - weighted images ( dwi ) . fmse was initially controlled with short term antiepileptic drugs and strict glucose control . two years later , recurrent seizures occurred , and long - term antiepileptic drug treatment was administered . dwi should be considered for acute cerebral infarction in patients having fmse associated with nkh , and careful follow - up should be conducted for such patients .

focal motor status epilepticus ( fmse ) is characterized by repetitive and persistent myoclonic , clonic , or tonic contractions of the arm , face , or neck that can affect an entire side of the body , with or without alteration of mental functions . fmse can be associated with several medical conditions including brain tumors , metabolic disturbances such as hyponatremia or a hyperosmolar state , and focal cerebral lesions resulting from stroke.1,2 fmse is often related to non - ketotic hyperglycemia ( nkh ) and accompanied by an underlying localized brain lesion.3,4 however , we are unaware of any report describing fmse with nkh that was accompanied by an acute cerebral infarction . furthermore , little is known as to whether it is epileptogenic or not . here , we report the case of a patient who presented with fmse associated with nkh and a magnetic resonance image ( mri)-documented acute cerebral infarction , and later developed recurrent seizures . a 46-year - old woman presented with sudden and progressive erratic movements of the right hand and arm that had developed 3 days before visiting our hospital . on inspection , initial myoclonus of the right hand turned into a tonic contraction that spread to the wrist and forearm and persisted for approximately 30 seconds . the frequency of seizures gradually increased from once an hour at initial symptom onset , to once every 5 minutes 3 days later . she reported feeling tingling sensations in her right hand and arm prior to seizure onset . she was having diabetes mellitus for more than 15 years , but reported no previous stroke or seizure . no focal neurological abnormality , other than partial seizures , was observed . serum glucose level , osmolality , and sodium level were 690 mg / dl , 312 mosm / l , and 128 meq / l , respectively . mri demonstrated a small focal acute infarction in the left frontal subcortical area on a diffusion - weighted image ( dwi ) . a focal cortical hyperperfusion in the left central area was observed on 99m - tc ecd single photon emission computed tomography ( spect ) , which disappeared 2 weeks later . magnetic resonance angiogram ( mra ) and transfemoral cerebral angiogram ( tfca ) revealed atherosclerotic stenosis of the left internal carotid artery ( ica ) ( fig . we administered antiepileptic drugs ( carbamazepine cr [ 600 mg / d after loading with 20 mg / kg ] and valproate [ 1,000 mg / d ] ) for 2 weeks and maintained her glucose levels below 250 mg / dl with insulin . the clinical seizures were not observed after 2 days , and she was discharged without sequel . two years later , she visited the department of neurology because of recurrent generalized tonic - clonic seizures . during the previous month her fasting and 2-hour postprandial glucose levels were 125 mg / dl and 198 mg / dl , respectively . routine chemistry and assays for electrolytes and serum osmolality did not indicate any abnormality , except for mild elevation of serum creatinine level ( 1.4 mg / dl ) . a follow - up mri failed to indicate recurrent strokes or lesions other than a previous small infarction , and interictal eeg was normal . such seizures can be a presenting symptom of hyperglycemia without ketoacidosis.5 increased metabolism of gamma - aminobutyric acid triggered by hyperglycemia may lower seizure threshold.6 associated metabolic disturbances , such as mild hyperosmolality and mild hyponatremia , may contribute to the seizures.3,7 ketosis is known to have an anticonvulsant effect , and the direct stabilizing effect of ketone bodies and accompanying acidosis may play an important role in preventing seizures.8 therefore , seizures are more frequently developed in patients with nkh than in patients with diabetic ketoacidosis . even though the underlying mechanisms are not fully understood , seizures associated with nkh are not rare in clinical practice . clinical symptoms typically originate from a focal cerebral lesion , whereas hyperglycemia affects the entire brain . these symptoms may result from previous underlying structural lesions that are more susceptible to the proconvulsant effects of nkh . a previous study reported that the majority of patients examined showed evidence of localized structural cerebral lesions.3,4 in contrast , pre - existing or acute structural lesions were not found in other case reports.7,912 our patient had a small focal subcortical infarct , as revealed by dwi , the location of which was relevant to the patient s symptoms . in previous reports , dwi was not used for evaluation , and small acute infarctions could have been missed on ct or routine mri . limb shaking resulting from transient ischemic attacks ( tias ) should be differentiated from fmse in patients who have carotid artery disease . in our case , this differentiation can be made with several points . firstly , limb - shaking tias usually do not present with jacksonian march as the attack experienced by our patient did . secondly , tia symptoms persist less than 5 minutes and are often accompanied by hemiparesis , which was inconsistent with our patient s symptoms.13,14 moreover , brain spect showed hyperperfusion in the area relevant to the patient 's seizures . based on the above findings , the abnormal arm movements observed in our patient appear unrelated to tia . however , conventional ictal scalp eeg often fails to detect seizure activities in fmse because of a limited discharges originated from central sulcus.2,15 in these cases , myoclonus - locked eeg back - averaging technique may be helpful in demonstrating the correlation of the myoclonus with paroxysmal discharges in the motor cortex.16 however , typical clinical manifestations with relevant spect finding of our patient are sufficient for the diagnosis . patients with histories of status epilepticus have a higher risk of recurrent unprovoked seizures , and the risk is much higher in patients with acute structural brain lesions than in patients with metabolic disturnbances.17,18 we usually administer antiepileptic drugs during the acute period of status epilepticus . however , long - term pro - phylactic antiepileptic treatment is not generally recommended because evidence for effectiveness of such long - term treatment in preventing epileptogenesis is still lacking.16,1719 in conclusion , our patient experienced acute symptomatic fmse and subsequent recurrent unprovoked seizures , that is , epilepsy . on the basis of our experience , we suggest that patients with nkh and fmse may have small cerebral infarctions , and dwi is crucial for detecting such lesions . furthermore , clinicians should pay careful attention to the emergence of epilepsy during follow - up .

one of the mysteries in prion research is the structure of the infectious form of mammalian prion protein , prpsc . we used ms analysis of h / d exchange to examine brain - derived prpsc . our data indicate that , contrary to popular models , prion protein conversion involves refolding of the entire region c - terminal to residue ~8090 , and that this region in prpsc consists of - strands and relatively short turns / loops , with no native -helices present .

gynecologic malignancies occur in approximately 1 in 20 women in the united states . until recently , clinical management of these cancers has focused almost exclusively on prolonging the survival of patients . a recent literature search using medline revealed relatively few research studies that reported data on quality of life ( qol ) in a gynecologic cancer population . reports in the literature have been conflicting , with some studies finding deterioration in qol and some finding stability or improvement in qol over time . until recently , the impact of various treatments ( surgery , radiation , chemotherapy ) on qol in this population was unknown . recently , the qol of women with gynecologic cancer has been compared to that of women with other types of cancer . also , risk factors for poor adjustment in gynecologic cancer are beginning to be investigated . this presentation will attempt to 1 ) summarize the relevant literature on qol in a gynecologic cancer population , 2 ) compare qol in this population to other types of cancer , 3 ) examine risk factors for poor adjustment and 4 ) describe the limitations of the literature and future research directions.overall , it appears that qol is most negatively affected from time of diagnosis through completion of treatment . following treatment , qol appears to improve over the course of 612 months , but then appears to remain stable from that time through two years post - treatment . compared to breast cancer patients , it appears that gynecologic cancer patients experience poorer qol on several domains during active treatment , but that after completion of treatment , overall qol is similar between groups . risk factors for maladjustment include treatment with radiotherapy or multi - modality treatment , increased length of treatment , younger age , and coping using a disengaged style . other risk factors include lower education , poor social support and lower levels of religious belief . the significance of these findings and future research directions will be discussed .

until recently , treatment for gynecologic malignancies has focused almost exclusively on prolongation of life , and few research studies have adequately addressed issues related to quality of life . quality of life ( qol ) typically involves the assessment of several dimensions : physical well - being , emotional well - being , social well - being , and functional well - being . as recently as 1993 , andersen published an article acknowledging a grave lack of research on quality of life and challenging " institutions and study groups to support quality of life research for women with gynecologic cancer " . to date , few studies have utilized qol as a primary endpoint . . often , there are few , if any , symptoms until the tumor is in an advanced stage . further , these symptoms are often non - specific and may consist of things like abdominal distention , vaginal bleeding , abdominal or low back pain , often leading treating professionals to misinterpret early signs or defer further work - up . treatment for gynecologic malignancies is often quite morbid and may involve multiple modalities ( surgery , radiation and chemotherapy ) . changes in bowel , bladder , hormonal , sexual and reproductive function are common . in addition , palliation is often difficult in the terminal stage , and death from a slow , obstructive , intra - abdominal process is not unusual . women frequently must adjust to physical changes after treatment including loss of ovarian function , hot flashes , vaginal dryness , hair and skin changes , and mood changes . surgical scarring may be another hurdle to adjustment , as are the need for urostomy or colostomy . sexual functioning may be impaired , and difficulties with desire and sexual response are common . infertility is an issue for many young women diagnosed with gynecologic cancer , and concerns may include adoption , egg donation , and surrogacy . the goals of the present article are to summarize what is known about qol in women treated for gynecologic malignancies , compare qol in women with gynecologic versus other malignancies , attempt to draw tentative conclusions about variables affecting quality of life and risk factors for maladjustment , and suggest directions for future research . bodurka - bevers and colleagues assessed the prevalence of depression and anxiety in 246 women diagnosed with ovarian cancer . these patients were at all stages of disease , and also were in various phases of active treatment or surveillance . results suggested that 21% met criteria for depression and 29% scored above the 75percentile for anxiety . the authors conclude that the prevalence of depression and anxiety may be higher than expected in an ovarian cancer population , and this clearly highlights the need for further assessment of qol in this group of patients . miller , pittman and strong also highlighted the need for assessment of quality of life and emotional functioning . these researchers administered questionnaires to 95 patients with gynecologic cancer at least 6 months after completion of treatment . a majority of patients wanted their physicians to ask questions dealing with spirituality , death and dying , and emotional problems . studied 115 women between the ages of 21 and 83 years who were referred to a university hospital for ovarian , endometrial and cervical cancer . women completed the sf-36 questionnaire , a widely used and well - validated qol instrument . the authors then compared these scores with age - specific expected mean values in published data from a healthy population of women . results of this research suggested that women with primary ( as opposed to recurrent ) gynecologic cancer had qol scores that were similar overall to healthy women . however , patients with recurrent disease scored an average of 10 points lower on each scale of the sf-36 . also notable was the fact that women with primary gynecologic cancer scored lower than healthy women on scales measuring emotional and physical role functioning . patients undergoing palliative chemotherapy treatment had the lowest scores overall , as would be expected . the authors also used linear regression to adjust for age and primary vs. progressive / recurrent disease status . results of this analysis showed that the poorest qol scores were reported by the youngest women with cervical cancer . this was in opposition to women with ovarian and endometrial cancer where age was negatively correlated with qol . studied the qol of long - term ( over 5 years ) survivors of ovarian cancer . 49 women were assessed and the results indicated that this disease - free sample enjoyed a good qol compared to other cancer survivors and non - cancer cohorts . however , approximately 20% of survivors reported significant long - term treatment related side effects , including abdominal , gynecologic and neurologic toxicity . furthermore , greater than half of the women surveyed indicated that they would have attended a support group if one were available to them at the time of diagnosis and treatment . qol data ( eortc-30 , spitzer ql - i ) was collected at six points from pre- to post - treatment . the first assessment of qol was conducted at one day prior to initiation of treatment . of the subjects , 26.2% were diagnosed with breast cancer , 31.9% with cervical cancer , 25.8% with ovarian cancer , and 16.1% with endometrial cancer . at pre - treatment , there were no statistically significant differences between breast cancer and gynecologic cancer patients on any qol domain . during active treatment , breast cancer patients had significantly higher qol scores , particularly in the areas of physical functioning and role functioning . at completion of treatment , breast cancer patients scored significantly lower on emotional functioning compared to patients with ovarian cancer . at six - month and one year post - treatment follow - up visits , there were no significant differences between breast cancer and gynecologic cancer patients on any of the qol domains assessed . overall , the researchers conclude that during active treatment patients with gynecologic cancer are significantly more physically impaired compared to breast cancer patients . however , qol is comparable between groups at one - year follow - up , suggesting that gynecologic cancer survivors experience significant improvement in qol following treatment . predictors of long - term qol included pre - treatment performance status and severity of surgery . not predictive was family support , number of treatments , age , stage or site of disease . miller , pittman , case & mcquellon compared qol in disease - free gynecologic cancer patients ( n = 85 ) to that of 42 unmatched healthy women seen for standard gynecologic screening exams . their data showed no overall difference in fact - g scores between gynecologic cancer patients and normal women . in fact , cancer survivors scored slightly higher on the emotional well - being subscale . within the cancer survivors patients who had treatment over a longer period of time reported decreased functional well being and total fact - g scores . it should be noted that these patients tended to have an ovarian cancer diagnosis and most had been treated with surgery and approximately 6 months of combination chemotherapy . patients treated with surgery only had better overall qol , probably due to short treatment time and less advanced disease . the authors note that prior research has shown that acute treatment effects are resolved after 6 months , and there is only little change expected thereafter . the authors propose that lower levels of education may be predictive of a less supportive social environment , limited knowledge of health issues and poor general health . eisemann & lalos assessed well - being in women with endometrial and cervical cancer at pre - treatment and also at 6 month and 1 year post - treatment . furthermore , well - being before treatment was significantly predictive of post - treatment well - being . chan and colleagues performed a prospective , longitudinal study of 74 newly diagnosed gynecologic cancer patients . qol was measured at 4 points from pre - treatment to 18 months post - treatment . a structured interview was used to measure self - esteem , outlook on life , self - role and femininity . it should be noted that this study only included individuals who had no recurrence of their disease , so this may not be indicative of all patients diagnosed with gynecologic malignancies . also notable is the fact that all psychosocial variables were assessed at pre - treatment primarily by clinical interview ( as opposed to more standardized assessment tools ) . the incidence of depression in this sample was twice that seen in a healthy population . subjects reported no change in relationship with spouse and sexual activity ( though this may have been under - reported due to the fact that this variable was assessed by clinical interview ) . the authors found three high - risk groups for maladjustment ; those with low religious belief , those who had received surgical treatment , and those with low educational level . lutgendorf and colleagues assessed 98 women with early stage or regionally advanced gynecologic cancer . prospective assessments were done measuring qol ( fact - g ) , coping style ( cope ) and mood ( poms ) at pre - treatment and one year post - diagnosis . sleep disturbance was common throughout the study , and occurred in approximately 40% of the sample . surprisingly , medical factors such as disease extent and treatment intensity did not significantly predict physical well being at one year . however , coping strategies contributed significantly to the variance of physical well being , even when medical factors were controlled . over the course of the first year following diagnosis , emotional and functional the authors note that this improvement occurred even in the absence of significant increases in physical well being , suggesting possible adaptation to residual physical limitations . decreases in anxiety , depression and confusion were seen in both groups , but regionally advanced patients had poorer qol and mood compared to early stage patients . interestingly , coping strategies appeared to be very predictive of qol at one year post - treatment . greater disengagement ( avoiding problems , giving up attempts to cope ) was associated with poorer relationship with physician , poorer functional and physical well being , and greater mood distress . another study by chan and colleagues assessed 144 women with newly diagnosed gynecologic cancer . these subjects were assessed at pre - treatment , immediately post - treatment , 6 month , 12 month and 24 month post - treatment . they assessed the impact of age , symptoms , disease parameters , and treatment type on qol using the eortc-30 . in this study as well , women with recurrent disease were dropped from final analysis , so this likely represents a sample of the most medically healthy patients . this is important , as this study may not accurately represent the qol of all newly diagnosed women , but may represent the qol of newly diagnosed women with the most favorable prognosis . in contrast to their earlier study , the results suggested that patients treated with surgery alone reported the best qol ( compared to those treated with multi - modality treatment ) . site and stage of disease had no significant effect on qol after treatment , and qol remained stable between 624 months following treatment . furthermore , overall qol appeared to improve after treatment , and this improvement was seen in global health status , functional scales and symptom report scales . there was a strong correlation between pre - treatment qol and that at 24 months post - treatment . in another compelling study , lutgendorf and colleagues assessed 48 women on qol ( fact - g ) , mood ( poms ) and coping style ( cope ) . 24 women had received one year of extensive chemotherapy , and 24 women had received no chemotherapy . overall , extensively treated women reported substantial , lasting decrements in physical , functional and emotional well being . avoidant coping again seemed to predict poorer qol , specifically in domains of physical and emotional well being . social well being appeared unimpaired in both groups . surprisingly , social support was not associated with any of the outcome variables , and the authors suggest that perhaps social support is not as important in maintaining qol post - treatment as it is during pre- and active treatment . bodurka - bevers and colleagues assessed the prevalence of depression and anxiety in 246 women diagnosed with ovarian cancer . these patients were at all stages of disease , and also were in various phases of active treatment or surveillance . results suggested that 21% met criteria for depression and 29% scored above the 75percentile for anxiety . the authors conclude that the prevalence of depression and anxiety may be higher than expected in an ovarian cancer population , and this clearly highlights the need for further assessment of qol in this group of patients . miller , pittman and strong also highlighted the need for assessment of quality of life and emotional functioning . these researchers administered questionnaires to 95 patients with gynecologic cancer at least 6 months after completion of treatment . a majority of patients wanted their physicians to ask questions dealing with spirituality , death and dying , and emotional problems . studied 115 women between the ages of 21 and 83 years who were referred to a university hospital for ovarian , endometrial and cervical cancer . women completed the sf-36 questionnaire , a widely used and well - validated qol instrument . the authors then compared these scores with age - specific expected mean values in published data from a healthy population of women . results of this research suggested that women with primary ( as opposed to recurrent ) gynecologic cancer had qol scores that were similar overall to healthy women . however , patients with recurrent disease scored an average of 10 points lower on each scale of the sf-36 . also notable was the fact that women with primary gynecologic cancer scored lower than healthy women on scales measuring emotional and physical role functioning . patients undergoing palliative chemotherapy treatment had the lowest scores overall , as would be expected . the authors also used linear regression to adjust for age and primary vs. progressive / recurrent disease status . results of this analysis showed that the poorest qol scores were reported by the youngest women with cervical cancer . this was in opposition to women with ovarian and endometrial cancer where age was negatively correlated with qol . studied the qol of long - term ( over 5 years ) survivors of ovarian cancer . 49 women were assessed and the results indicated that this disease - free sample enjoyed a good qol compared to other cancer survivors and non - cancer cohorts . approximately 20% of survivors reported significant long - term treatment related side effects , including abdominal , gynecologic and neurologic toxicity . furthermore , greater than half of the women surveyed indicated that they would have attended a support group if one were available to them at the time of diagnosis and treatment . qol data ( eortc-30 , spitzer ql - i ) was collected at six points from pre- to post - treatment . the first assessment of qol was conducted at one day prior to initiation of treatment . of the subjects , 26.2% were diagnosed with breast cancer , 31.9% with cervical cancer , 25.8% with ovarian cancer , and 16.1% with endometrial cancer . at pre - treatment , there were no statistically significant differences between breast cancer and gynecologic cancer patients on any qol domain . during active treatment , breast cancer patients had significantly higher qol scores , particularly in the areas of physical functioning and role functioning . at completion of treatment , breast cancer patients scored significantly lower on emotional functioning compared to patients with ovarian cancer . at six - month and one year post - treatment follow - up visits , there were no significant differences between breast cancer and gynecologic cancer patients on any of the qol domains assessed . overall , the researchers conclude that during active treatment patients with gynecologic cancer are significantly more physically impaired compared to breast cancer patients . however , qol is comparable between groups at one - year follow - up , suggesting that gynecologic cancer survivors experience significant improvement in qol following treatment . predictors of long - term qol included pre - treatment performance status and severity of surgery . not predictive was family support , number of treatments , age , stage or site of disease . miller , pittman , case & mcquellon compared qol in disease - free gynecologic cancer patients ( n = 85 ) to that of 42 unmatched healthy women seen for standard gynecologic screening exams . their data showed no overall difference in fact - g scores between gynecologic cancer patients and normal women . in fact , cancer survivors scored slightly higher on the emotional well - being subscale . within the cancer survivors patients who had treatment over a longer period of time reported decreased functional well being and total fact - g scores . it should be noted that these patients tended to have an ovarian cancer diagnosis and most had been treated with surgery and approximately 6 months of combination chemotherapy . patients treated with surgery only had better overall qol , probably due to short treatment time and less advanced disease . the authors note that prior research has shown that acute treatment effects are resolved after 6 months , and there is only little change expected thereafter . the authors propose that lower levels of education may be predictive of a less supportive social environment , limited knowledge of health issues and poor general health . eisemann & lalos assessed well - being in women with endometrial and cervical cancer at pre - treatment and also at 6 month and 1 year post - treatment . furthermore , well - being before treatment was significantly predictive of post - treatment well - being . chan and colleagues performed a prospective , longitudinal study of 74 newly diagnosed gynecologic cancer patients . qol was measured at 4 points from pre - treatment to 18 months post - treatment . a structured interview was used to measure self - esteem , outlook on life , self - role and femininity . it should be noted that this study only included individuals who had no recurrence of their disease , so this may not be indicative of all patients diagnosed with gynecologic malignancies . also notable is the fact that all psychosocial variables were assessed at pre - treatment primarily by clinical interview ( as opposed to more standardized assessment tools ) . the incidence of depression in this sample was twice that seen in a healthy population . subjects reported no change in relationship with spouse and sexual activity ( though this may have been under - reported due to the fact that this variable was assessed by clinical interview ) . the authors found three high - risk groups for maladjustment ; those with low religious belief , those who had received surgical treatment , and those with low educational level . lutgendorf and colleagues assessed 98 women with early stage or regionally advanced gynecologic cancer . prospective assessments were done measuring qol ( fact - g ) , coping style ( cope ) and mood ( poms ) at pre - treatment and one year post - diagnosis . sleep disturbance was common throughout the study , and occurred in approximately 40% of the sample . surprisingly , medical factors such as disease extent and treatment intensity did not significantly predict physical well being at one year . however , coping strategies contributed significantly to the variance of physical well being , even when medical factors were controlled . over the course of the first year following diagnosis , emotional and functional the authors note that this improvement occurred even in the absence of significant increases in physical well being , suggesting possible adaptation to residual physical limitations . decreases in anxiety , depression and confusion were seen in both groups , but regionally advanced patients had poorer qol and mood compared to early stage patients . interestingly , coping strategies appeared to be very predictive of qol at one year post - treatment . greater disengagement ( avoiding problems , giving up attempts to cope ) was associated with poorer relationship with physician , poorer functional and physical well being , and greater mood distress . another study by chan and colleagues assessed 144 women with newly diagnosed gynecologic cancer . these subjects were assessed at pre - treatment , immediately post - treatment , 6 month , 12 month and 24 month post - treatment . they assessed the impact of age , symptoms , disease parameters , and treatment type on qol using the eortc-30 . in this study as well , women with recurrent disease were dropped from final analysis , so this likely represents a sample of the most medically healthy patients . this is important , as this study may not accurately represent the qol of all newly diagnosed women , but may represent the qol of newly diagnosed women with the most favorable prognosis . in contrast to their earlier study , the results suggested that patients treated with surgery alone reported the best qol ( compared to those treated with multi - modality treatment ) . younger patients reported poorer physical health compared to older patients . site and stage of disease had no significant effect on qol after treatment , and qol remained stable between 624 months following treatment . furthermore , overall qol appeared to improve after treatment , and this improvement was seen in global health status , functional scales and symptom report scales . there was a strong correlation between pre - treatment qol and that at 24 months post - treatment . in another compelling study , lutgendorf and colleagues assessed 48 women on qol ( fact - g ) , mood ( poms ) and coping style ( cope ) . 24 women had received one year of extensive chemotherapy , and 24 women had received no chemotherapy . overall , extensively treated women reported substantial , lasting decrements in physical , functional and emotional well being . avoidant coping again seemed to predict poorer qol , specifically in domains of physical and emotional well being . social well being appeared unimpaired in both groups . surprisingly , social support was not associated with any of the outcome variables , and the authors suggest that perhaps social support is not as important in maintaining qol post - treatment as it is during pre- and active treatment . gynecologic malignancies pose special risks for quality of life . despite the fact that gynecologic malignancies occur in approximately 1 in 20 women in the united states , qol has not been widely researched , with the bulk of research devoted to prolongation of life . reports in the literature have been conflicting , with some finding deterioration in qol and some finding stability or improvement over time . little has been known about the impact of various treatments , diagnoses , stages of illness , and other risk factors on qol in these patients . given the challenges and changes that women must face after a diagnosis of gynecologic cancer , qol is an especially pertinent issue on which to focus . before concluding anything regarding qol , however , several caveats are important to note . first , it is important to utilize a well - validated measure of qol in order to compare qol in patients with gynecologic malignancies to any other group of patients or healthy subjects . the studies reviewed in the current paper have generally done so , with the exception of three . second , due to the relatively small number of gynecologic cancer patients seen at any one cancer center , most of the research studies above have grouped gynecologic cancer patients into one group , as opposed to separating out by diagnosis . because of this , any interpretation of the impact of diagnosis on qol must be tentative . related to this is the fact that type of treatment may be reflective of stage of disease ( i.e. patients with advanced disease will be more likely to have multi - modality treatment , while patients with early stage disease may have surgery alone ) . as such , any interpretation of the impact of treatment type on qol must also be done cautiously and in the context of disease stage . there have been no prospective studies in which gynecologic cancer patients have been randomly assigned to any psychological or psychiatric treatment before , during or after treatment . the author of this review is currently planning such a study to investigate how a structured , psychological support group will impact levels of a growth factor ( vegf ) which has been linked to cancer progression in women with ovarian cancer . despite the limitations of the current research , there appears to be a higher than expected incidence of depression and anxiety in patients undergoing treatment for gynecologic malignancies . this is likely related to the high treatment toxicity and often poor prognosis of many of these illnesses . it appears that qol is most negatively affected from the time of diagnosis through the completion of treatment . it also appears that qol is more substantially impaired during treatment of gynecologic malignancies than during treatment of other cancers . 12 years after treatment , disease free patients report qol that is generally equivalent to other cancer survivors and healthy women . further , emotional and functional well - being increase over the first year following treatment , even in the absence of corresponding increases in physical well - being , suggesting adaptation to residual physical limitations . despite these positive results , a significant minority of patients continue to report lasting emotional problems and treatment related toxicities . risk factors for maladjustment include treatment with radiotherapy or multi - modality treatment , increased length of treatment , younger age , and coping using a disengaged style . lower levels of education and spiritual / religious belief , as well as lack of help at home , are also risk factors for poor qol . future research should include large , multicenter studies , which would allow comparative analysis of qol by diagnosis . also , studies measuring the impact of various chemotherapeutic agents on qol should be undertaken , as there appear to be long - lasting toxicities from many of the chemotherapeutic regimens , which are only beginning to be understood . due to the difficulty in palliating terminal illness , any studies focusing on improvement of qol in end - stage disease patients would be welcome and clinically relevant . finally , prospective studies of the impact of psychological treatments on qol and prognostic factors should be undertaken . future studies will hopefully assist women in coping with the challenges and rigors of treatment and post - treatment toxicities . ideally , these studies would determine risk factors not only for psychological morbidity , but also medical mortality and morbidity , and attempt to modify psychosocial variables to improve survival time and quality of life .

aim : the purpose of this study was to clarify detection characteristics of the secondary mesio - buccal canal in maxillary first molars using various methods.materials and methods : the root canal system of 86 extracted human maxillary first molars was inspected using micro - focus - computed tomography to accurately determine the number of canals . radiographs or floors of the pulp chamber for all samples were observed for the secondary mesio - buccal canal with computed tomography for dentistry , digital dental radiography , magnifier , or the naked eye . sensitivity , specificity , positive , and negative predictive values and diagnostic accuracy for these four methods were investigated using the results from the micro - focus - computed tomography inspection as the gold standard . all samples of each method were observed by 10 endodontists . using these results , the 2 test was used to compare and analyze differences between the various conditions ( p<0.05).results : the secondary mesio - buccal canal could be recognized in 60.9% of samples with the micro - focus - computed tomography . no significant difference was seen between efficiencies of the computed tomography for dentistry and the micro - focus - computed tomography . the computed tomography for dentistry was superior to the other three methods.conclusion:detectability of the secondary mesio - buccal canal in the maxillary first molar was superior using dental - computed tomography compared to digital dental radiography , magnification telescope , and the naked eye .

the success ratio of root canal treatment is reportedly around 90% for pulpectomy and approximately 80% for infected root canal.[15 ] however , success ratios display large fluctuations according to periapical lesion , condition of infection in the root canal , periodontal disease , and pre- or postoperative occlusive condition.[68 ] some reports have described no difference in results depending on the tooth kind.[2579 ] however , swartz reported low success rates for the mandibular first molar . likewise , yamaki and yamamoto reported significantly inferior treatment results for the maxillary first molar . the reason for inferior treatment results on the maxillary first molar are the high rates of occurrence of curved canal and advanced curved canal . krithkadatta et al . using the cone - beam - computed tomography , reported unusual root canal morphology in the mandibular first molar . while employing the cone - beam - computed tomography , kottoor et al . furthermore , treatment of the secondary mesio - buccal canal ( mbii ) is often insufficient or overlooked . research into mbii has mainly examined the rate of occurrence and morphological classifications.[1721 ] weine defined four types according to the divergence state of the canal in the mesio - buccal root : type i , single canal from the pulp chamber to the root apex ; type ii , two canals leaving the chamber and merging to form a single canal short of the root apex ; type iii , two separate and distinct canals from the pulp chamber to the root apex ; and type iv , one canal leaving the chamber and dividing into two separate and distinct canals . however , few reports have examined detection and effective treatment methods.[2327 ] radiographic techniques offer effective , nondestructive examination . the observation by dental radiography does not always agree with that by naked eye or some other anatomical assessments , because the two canals of mesio - buccal root are overlapped in the direction of irradiation . computed tomography for dentistry ( dental ct ) has recently been developed , allowing an anatomical view of the microstructural organization of the tooth and periodontal hard tissues with three - dimensional images.[2831 ] dental ct offers high picture resolution compared with conventional high - speed spiral - type ct , but the imaging range is restricted . dental ct is used to examine lesions inside bone , tumors , implants , and tooth transplantation , providing effective imaging information . however the purpose of this study was to identify detection characteristics of the mbii for maxillary first molars according to four test methods : dental ct ; digital dental radiography ; magnifier ; and the naked eye . the root canal systems of 86 extracted human maxillary first molars without root canal treatment were completely inspected using micro - focus computed tomography ( micro ct ) ( mct100-mfz ; hitachi medical , tokyo , japan ) to accurately assess the number of canals . two researchers experienced in diagnostic imaging observed that the number of orifices , root canals , and apical foramina of all experimental teeth on micro - ct , using these results as the gold standard . furthermore , canal systems in mesio - buccal roots were classified as types i - iv using the anatomical classification defined by weine . for all samples , radiographs or floors of the pulp chamber were observed by 10 dentists using the four methods after accessing the cavity preparation . sensitivity , specificity , positive , and negative predictive values and diagnostic accuracy for the four test methods were investigated using the results of micro - ct as the gold standard . specifically , the four methods comprised dental ct ( mean effective does per once : 0.03 msv . ; psr9000 ; asahi roentgen industry , kyoto , japan ) ; conventional digital dental radiography ( mean effective does per once : 0.02 msv . ; dfw-20 ; asahi roentgen industry , or denooptix ; dentsply - sankin , tokyo , japan ) ; magnification telescope with 2-times magnification ( surgitel gl275n ; gsc , michigan , usa ) ; and the naked eye . imaging conditions for dental ct were dental ct mode ; peak voltage , 60 kv ; peak current , 2 ma ; and radiation time , 20.48 seconds . conditions for digital dental radiography were peak voltage , 65 kv ; peak current , 20 ma ; radiation time , 0.1 seconds . the radiation direction was set from the buccal side at right angles to the longitudinal axis of the tooth . with the magnifier or naked eye , orifices of the root canal of all samples were searched using # 08 root canal instruments on the floors of the pulp chambers . imaging conditions for micro - ct when establishing gold standard results were peak voltage , 65 kv ; peak current , 100 a [ figure 1 ] . the time intervals of observation between conditions for a sample were set up long enough to avoid the examiner 's preconception [ figure 2 ] . sensitivity , specificity , positive , and negative predictive values and diagnostic accuracy were calculated from measured values of various observations . using these results , the test was used to compare analyze differences between the various conditions . mbii ( arrows ) in typical images from the micro - focus - computed tomography ( micro ct ) ( a ) mbii ( arrows ) in typical images with the x - ray - computed tomography for dentistry ( dental ct ) ; ( b ) mbii ( arrow ) in typical images of digital dental radiography . mbii was not verified on the image of photography from buccal - palatal direction ; ( c ) mbii ( arrow ) in the image of the floor of the pulp chamber ( 3 ) as the gold standard , micro ct - revealed mbii ( types ii , iii , or iv ) in 52 of 86 teeth ( 60.5% ) in all samples . anatomical classification showed type i in 34 teeth ( 39.5% ) , type ii in 13 teeth ( 15.1% ) , type iii in 24 teeth ( 27.9% ) , and type iv in 15 teeth ( 17.5% ) [ figure 3 ] . dental ct showed 56.4% sensitivity , 76.4% specificity , 78.9% positive predictive value , 47.1% negative predictive value , and 64.2% diagnostic accuracy . digital dental radiography images showed 13.4% sensitivity , 84.6% specificity , 58.9% positive predictive value , 36.0% negative predictive value , and 40.3% diagnostic accuracy . magnification telescope observation showed 26.1% sensitivity , 72.0% specificity , 59.3% positive predictive value , 38.5% negative predictive value , and 44.1% diagnostic accuracy . naked eye observation showed 20.3% sensitivity , 76.0% specificity , 56.8% positive predictive value , 37.9% negative predictive value , and 42.0% diagnostic accuracy [ figures 48 ] . dental ct offered superior results compared with the other three methods for all inspection ratios , and significant differences were seen between the results of dental ct and the other three methods ( p<0.01 ) . digital dental radiography was significantly superior to the naked eye in terms of sensitivity , specificity , and positive predictive value , but the naked eye was superior for negative predictive value and diagnostic accuracy in detecting mbii ( p<0.01 ) . in this study , a classification result of the mesio - buccal root canal by the micro - focus - computed tomography ( micro ct ) inspection n=86 sensitivity rate in each observation method as the standard in micro - ct ( n=86 ) specificity rate in each observation method as the standard in micro - ct ( n=86 ) positive predictive value rate in each observation method as the standard in micro - ct ( n=86 ) negative predictive value rate in each observation method as the standard in micro - ct ( n=86 ) diagnostic accuracy rate in each observation method as the standard in micro - ct ( n=86 ) micro - ct as used for setting the gold standard was developed using industrial equipment for the purpose of nondestructive inspection of various materials . very clear images can be obtained by irradiation with extremely small doses of radiation to the target . however , clinical application of micro - ct is impossible , as an extended period of radiation is required for imaging . one is the dental ct mode that revolves around the circumference of the subject and photographs the region at 42.7 mm height and 30 mm width . the other method is the panorama mode , in which the x - ray tube rotates five times in a spiral orbit . furthermore , these images can be reconstructed to form multiplanar reconstructions and three - dimensional images . the radiation dose involved in dental ct investigation is basically comparable with conventional panoramic ( mean effective does per once ; 0.025 msv . ) however , depending on the anatomical location and the setting of ct device , the radiation dose exposed to the patient could be kept to a minimum . the cone beam ct , which is a variant of conventional ct , is even more useful in dentistry as its radiation can be restricted only to the anatomical area being investigated . in addition , the radiation dose required by the cone beam ct is only one - fifth of the conventional ct and time needed is as short as 1015 seconds . as for cone beam ct application , although there are many advantages than risks , it is important to have a clear objective for using this technique and then to define the anatomical area . surgetel is a magnifier that was developed for dentistry , offering superior resolution with a three - layer lens coating , in addition to enlargement power . determining the anatomical condition of the mesio - buccal root of maxillary first molars before root canal treatment is difficult . weine and kulilid et al . reported that the second mesio - buccal canal is located 1 - 3 mm toward the palatal canal from the larger mesio - buccal canal . however , discovery has been difficult in many cases with searches using thin reamers , since the root canal orifice of mbii is quite small and of variable position . in terms of diagnostic accuracy and negative predictive value , in digital dental radiography , the two root canals of the mesio - buccal root can not be distinguished in many cases , since these canals overlap with the direction of irradiation . the detection rate considerably decreased due to the interviewing of maxilla or periodontium in clinical situations . good diagnostic accuracy was obtained under observation by sergitel or the naked eye because the orifice of the root canal was searched for using reamers on the basis of anatomical knowledge . reported mbii in 54% of maxillary first molars , while seidoberg et al . reported an mbii detection rate of 62% for extracted teeth and 33% in clinical situations the detection rate by the naked eye was 51.4% of a result of micro - ct , which was 30% in all experimental teeth . detection by magnification telescope or the naked eye is impossible for type iv . in other words the root canal treatment has often been finished without discovery of mbii , including types ii and iii . even if mbii is detected however , success rate does seem to improve with detailed knowledge of the anatomical features . radiographic and visual information generally can not be directly compared , because type iv mbii can not be absolutely detected by magnification telescope or the naked eye . the existence of type iv has a same adverse effect with mbii when its not discovered . the present results clearly show that detection rate of mbii is higher for dental ct than for digital dental radiography . few differences in the detection rate of mbii were seen between surgitel and the naked eye , although sergitel was superior in terms of the time required and ease of detection . detectability of mbii in the maxillary first molar was superior using dental - computed tomography compared to digital dental radiography , magnification telescope , and the naked eye .

in an accompanying paper ( nagy , g. ; oostenbrink , c. dihedral - based segment identification and classification of biopolymers i : proteins . j. chem . inf . model . 2013 , doi : 10.1021/ci400541d ) , we introduce a new algorithm for structure classification of biopolymeric structures based on main - chain dihedral angles . the disicl algorithm ( short for dihedral - based segment identification and classification ) classifies segments of structures containing two central residues . here , we introduce the disicl library for polynucleotides , which is based on the dihedral angles , , and for the two central residues of a three - nucleotide segment of a single strand . seventeen distinct structural classes are defined for nucleotide structures , some of which to our knowledge were not described previously in other structure classification algorithms . in particular , disicl also classifies noncanonical single - stranded structural elements . disicl is applied to databases of dna and rna structures containing 80,000 and 180,000 segments , respectively . the classifications according to disicl are compared to those of another popular classification scheme in terms of the amount of classified nucleotides , average occurrence and length of structural elements , and pairwise matches of the classifications . while the detailed classification of disicl adds sensitivity to a structure analysis , it can be readily reduced to eight simplified classes providing a more general overview of the secondary structure in polynucleotides .

since the first elucidation of three - dimensional models of protein structures and polynucleotides , a wealth of structural information has become available . for proteins , different secondary structure elements have been described , and also for dna , two different helices were proposed very early on . while proteins are readily classified in terms of helices , sheets , and various kinds of turns , the full structural diversity of dna and rna is only recently becoming clear . on the basis of our previous work on the classification of protein structures using an algorithm called disicl ( dihedral - based segment identification and classification ) , we here propose a definition of polynucleotide structural elements based on two dihedral angles of the nucleotide backbone complemented by the dihedral angle linking the sugar and the base . while studies of backbone dihedral angles are available for polynucleotides , secondary structure prediction and classification of dna and rna models are more often based on sequence- or knowledge - based approaches such as sequence alignments , context free grammar , and machine learning or empirical energy functions and dynamic programming to determine the most stable secondary structure or an ensemble of structures with a central member . there has been significant effort to combine these methods to predict and construct both the secondary and tertiary structure of rna molecules . structure - based analysis and classification methods on the other hand rely on three - dimensional models to evaluate the shape and intramolecular and intermolecular interactions between dna , rna , and other molecules such as proteins . established structure - based analysis methods for polynucleotides are commonly based on complex helical parameters , such as in the program schnaap ( structure and conformation of helical nucleic acids : analysis program ) . the x3dna analysis tool also relies on helical parameters but performs a local dna classification based on phosphate coordinates of dinucleotide base pair steps . another very useful and effective package , curves , can analyze global helical curvature and local base pair parameters , as well as groove dimensions . while the recently reimplemented curves+ program can effectively analyze molecular dynamics trajectories , its intrabase and interbase pair parameters , which can be used to assign local structure information , are almost identical to those reported by x3dna . almost all of the structure - based approaches for dna or rna classification require double helical structures ( originating from a single or from multiple strands ) and seem relatively limited in terms of the diversity of the structural classes that are being considered . in the current work , we define an extensive library for the classification of nucleotide sequences and apply this classification on databases containing 260,000 trinucleotide segments . after a description of the data sets to be analyzed the suggested classifications are discussed in more detail and demonstrated by selected examples of dna and rna structures obtained from the brookhaven protein databank . the performance of disicl is compared to that of x3dna , and finally , some conclusions are drawn . for the purpose of testing and comparing different classification algorithms , two large scale polynucleotide data sets were obtained from the brookhaven protein databank ( pdb , www.rscb.org ) . both data sets were selected from all pdb entries available on october 23 , 2012 , using the following criteria ( 1 ) entries show at most 30% sequence identity . ( 3 ) entries obtained from x - ray crystallography have a resolution of 0.82.0 . separate dna and rna data sets were defined ( dna_comb and rna_comb , respectively ) , containing structural models determined by both x - ray crystallography and nuclear magnetic resonance ( nmr ) spectroscopy , because the number of entries for polynucleotides was considered too small for further partitioning . the resolution range for x - ray structures was chosen such that the relevant dihedral angles can be reliably determined , but the number of alternative locations for groups of atoms in the data set is kept low . prior to the analysis , multiple chains and multimeric structures were retained , but residues were renumbered to avoid identical residue numbers from different chains . if any of the classification algorithms failed on a particular entry , it was completely discarded from the full analysis to ease the comparison of methods . this approach yielded approximately 80,000 and 180,000 segments for dna and rna models , respectively . further details are provided in the database summary section of table 1 , where the number of downloaded pdb entries ( file number ) , number of extracted individual structures ( model number ) , and total number of classified nucleotides / residues ( total data set ) is provided , along with the average number of structures per pdb entry ( ave . model length ) , and amount of base - paired nucleotides ( base pairing ) . x3dna is a freely available analysis , reconstruction , and visualization tool for dna modeling ( www.x3dna.org ) . it has many smaller modules that can be used to produce idealized dna models based on their sequence and the required helix type , as well as to analyze existing dna and rna structures . the analysis package can determine base pairing and obtain helical parameters ( such as roll , twist , displacement , and groove dimensions ) based on simple geometric calculations , and it also features a dinucleotide segment - based local helix classification algorithm . this classification takes the mean phosphorus atom z coordinates and helix inclinations ( zp and zph , respectively ) of a - dna to distinguish a - dna , b - dna , and transitory ta - dna forms ( often found in tata boxes ) . this particular algorithm does not recognize z - dna forms ( unless the full helix is left handed ) , and the classification should still be verified by other helical parameters also printed by the analysis program . other structure - based programs focus on full helical descriptions of dna sequences and global hydrogen bonding , while the x3dna analysis program performs more localized dinucleotide segment classification , which is better suited to capture the structural diversity of , for example , molecular simulations . the disicl algorithm for protein structure classification first , relevant ( backbone ) dihedral angles are calculated and attributed to regions in the dihedral angle space . the pair of regions occupied by the two central residues of the segment determines the structural class to which the segment is assigned . while the disicl algorithm was originally designed for protein analysis , the purely dihedral - based classification can be applied to other biopolymers as well . using a study of dihedral angles in selected dna backbones published one- and two - dimensional distributions on eight backbone dihedrals of dna oligomers crystallized in different helical structures ( a , b , z ) . this work provided an excellent base for our classifications , while other papers confirm that helical structures and their subconformations are important factors when dna interacts with proteins and drug - like molecules . finally , rna molecules , which often fold into complex structures , also have a tendency to form dna - like helical segments . on the basis of the two - dimensional distributions , we chose three dihedral angles that can characterize helical structures of nucleotides : backbone dihedrals and and base torsion angle . while some helical parameters ( like groove dimensions ) can be more easily measured for full helix turns ( 45 base pair segments ) , a pair of the triplets ( , , ) provided by a base triplet has a sufficiently high resolution to separate the polynucleotide helices . the backbone dihedral angle definitions and the 14 region definitions of the dihedral angle space are shown in figure 1 . similar to the first and last amino acid of the protein segments of disicl , the third nucleotide only provides one atom for the calculations , and as such , the first two nucleotides were used as central residues for the comparison studies . on the basis of the ( , , ) definitions , 17 detailed ( table 3 ) and eight simplified ( table 4 ) classes were defined for dna and rna structures . their region mapping and precise region definitions are shown in table 2 and table s1 of the supporting information , respectively . representation of region definitions used for polynucleotide classification ( on the left ) based on subsequent ( , , ) values within a trinucleotide segment ( on the right ) . atoms and bonds that define 1 , 2 , and 2 are marked in red . segments are assigned to a class if their central residues fall into regions separated by a dot in the segment definitions . the same data is given for the x3dna classes at the bottom of the table . the region definitions of the dna classes are not as straightforward as the protein region definitions , so a summary is provided here . the 14 region definitions can be divided into five groups marked by greek letters . regions ( 1 , 2 , 3 ) have high density in rna structures , with 1 containing the most densely populated area associated with the a - helix . regions ( 1 , 2 , 3 ) are dominant in the dna data set and are associated with the different forms of the b - helix . the experimentally derived subconformations of b - dna , and bi and bii , fall in the 1 and 2 regions , respectively . three regions ( 1 , 2 , 3 ) contain local density peaks normally found in z - dna , although 1 residues are also regularly found in dna quadruplexes , and 2 residues regularly appear in sharp backbone turns . regions ( 1 , 2 , 3 ) have populations comparable to the regions and are separated from the , , and regions by their low value . the 1 region appears in distorted a - helices , and the 2 region regularly appears in backbone turns , while the 3 region is almost exclusive found in dna quadruplexes . the fifth group represents a / b transitions and contains the regions ab1 and ab2 . the ab1 region represents the intersect volume of the 1 and 1 regions ( which contain the maximal peak densities in the rna and dna data sets , respectively ) and is densely populated for both rna and dna structures . region ab2 is a moderate density volume surrounded by the regions 1 , 3 , 1 , 2 , and 3 . 1 , 2 , 1 , 2 , and 3 are based on the angular distributions of schneider et al . but were modified to better fit the density distribution of both dna and rna data sets . this procedure was based on the classification of a data set containing 150,000 nucleotides consisting of approximately equal amounts of dna and rna . the rectangular regions were adjusted to include 75% of the data points including the nearest local density maxima . afterward , selected subsets of structures with common structural elements ( dna quadruplexes , junctions , rna tetraloops , riboswitches , etc . ) were analyzed . if structurally important nucleotides were repeatedly observed near unassigned peaks in the dihedral angle space , additional regions were assigned to those areas ( resulting in regions 2 , 3 , 3 , 1 , 2 , and 3 ) . on the basis of chemical intuition , visual checks concerning the shape of the backbone , directionality of the bases , and the annotation provided in the pdb entries , segment definitions ( pairs of regions ) were associated with structural classes . associated segment definitions were assigned to a class after a careful visual analysis of 20100 randomly picked structures . segment definitions were assigned if at least 50% of the examples were of one particular class . finally , the borders of neighboring regions were fine - tuned in an iterative process , where the effect of shifting the border was determined by performing structural analyses of structures containing the segments that were reassigned to a different class . all structural models were analyzed separately by both classification algorithms . as the different programs produced output in different formats , all results were ordered into identically formatted data series . the data series contained the name of the class along with all the segments in the model that belonged to that class . second , the data series of all models were collected and combined into a single data set for each of the individual algorithms containing elements anj , which was assigned the value 1 if nucleotide n was classified to belong to class j. tables 3 and 4 show the abundance ( occj ) and average length ( lj ) of each structural element ( in nucleotides ) , which were calculated based on the number of residues in the class ( nj ) , number of interruptions ( nj ) , and total number of residues ( nsum ) according to eqs 13 . the number of interruptions was increased by one whenever a gap was found in a continuous chain of dinucleotide segments of class j.123to compare the classification algorithms , the correlation matrices of algorithms were calculated containing the correlation scores cij where i and j mark the i class of the first algorithm and the j class of the second algorithm , respectively . three types of correlation scores were used : pearson correlation ( rij ) , match score ( mij ) , and scaled match score ( mij ) . the pearson correlation ( rij ) is calculated from eq 4 , where ani is the average occurrence of the class i ( ani = ni / nsum).4while the r - score drops quickly with the amount of mismatches ( or different occurrences of classes i and j ) , a large positive r - score is still a good measure to determine agreement between algorithm classes . the unscaled match score ( mij ) is calculated using eq 5 and represents the absolute number of residues assigned to class i in one algorithm and to class j in the other algorithm.5the m - score is additive , which makes it possible to group classes or track distributions of correlations for one class . the scaled match score ( mij ) provides a better comparison between algorithms and is calculated by eq 6.6 in words , the scaled match score is obtained by dividing the observed match ( mij ) between two classes with the maximal theoretical match ( mmax ) . here , mmax is equal to size of the smaller data set.7 to summarize comparisons , the weighted average of the scaled match scores were calculated for a - helical , b - helical , and transitory dna or rna forms for nucleotides ( table 1 , methods agreement ) . additionally , the weighted average of all these superclasses and the scaled match score for unclassified residues were calculated to obtain an overall match between methods . the grouping for superclasses is provided in table s2 of the supporting information . for dna classifications , dna groove dimensions were measured with a simple algorithm using a similar basic idea as used in x3dna ( see figure 2 for a schematic representation of relevant nucleotides for this calculation ) . because the full turn of the b - dna structure consists of approximately five ( base - paired ) nucleotides on each of the two strands , helical fragments of a given classification with five consecutive base pairs identified were used to determine the groove dimensions . sj-1 , paired with central residues i j and ( i + 1)(j - 1 ) , such that base pair i as a rough estimate for major and minor groove widths , the distance between phosphorus atoms p(i-2 ) and p(j-2 ) yields the major groove width , while the distance between atoms p(i+2 ) and p(j+2 ) provides the minor groove width . groove depths were estimated by the distance between the midpoint of the vector defining the width and the midpoint of the vector pi pj . schematic representation of the calculation of groove dimensions in double - stranded dna helices . the classification of polynucleotides was performed on two data sets containing models of dna molecules ( dna_comb ) and rna molecules ( rna_comb ) using two segment - based algorithms , namely , x3dna and disicl . disicl defines 17 detailed classes , which can be grouped into classical helical structures ( a- , bi- , bii- , biii- , and z - helices ) , special loops and turns ( a - loop , tetraloop bulge , b - loop , sharp turns , and quadruplex loops ) , and transitory classes ( ab , ab2 , ad , az , bd , bz , and zd ) ( see table 3 for the average occurrences and lengths of these structural elements ) . in the simplified version of the nucleotide disicl library ( table 4 ) , this is reduced to eight classes ( a- , b- , z - helices , irregular a and irregular b structures , quadruplex loops , ab transitions , and other transitory segments ) . the majority of dna and rna molecules in the databases assume double helical structures . dna under physiological conditions assumes a right - handed double helical form usually referred to as b - dna . the nucleotides in the b - dna form have two identified subconformations ( bi and bii ) mostly differing in their and angle . under certain salt concentrations , rna and dna can form a different helix , normally referred to as the a - form , while some dna structures can assume left - handed helices , normally referred to as the z - form . the bi class ( bi ) contains the dna ( , , ) density maximum associated with continuous repeats of the bi subconformation ( located in the region 1 ) . occurrence of longer stretches of the bi class on both strands forms the classical b - helix , which makes up 35% of all dna nucleotides . the bii class ( bii ) contains definitions for ( 12 ) alternating segments , which are an alternate form of the b - helix ( 3.5% of dna segments ) . while the bii class rarely appears in longer stretches in both strands , bii - rich areas of dna form helices that are more varied in their groove dimensions and on average have wider and more shallow grooves ( table 5 ) , which might be important for dna the bii class in longer stretches also appears in single strands for dna loops and three - way junctions . the biii class ( biii ) was defined for pure 3 segments , which occur in 2.5% of the analyzed nucleotides . biii segments ( often accompanied by b - loop segments ) distort the b - dna helix leading to a wider major groove but narrower minor groove . examples of bi- , bii- , and biii - rich dna models are depicted in panel a of figure 3 . examples of dna structures and structure classification by disicl . for each model , the pdb identification code is given followed by the abbreviation of classes according to table 3 , which are color coded to match the structures they mark . helices are sorted based on the assigned disicl classification for the central segment of the helix turn on both strands . groove dimensions are given as averages ( mean ) and root - mean - square fluctuation ( rmsf ) in . the a - helix class ( ah ) relates to the bent a - form helix of dna ( 2% ) , and it is the predominant form of ribonucleic acids ( 50% ) . the class is defined by segments with pure 1 conformation , which usually appear in fully a - helical models or prior to turns in more complex dna structures . the z - helix class ( zh ) appears predominantly in z - helical dna structures and consists of definitions with an alternating pattern of either 12 or 13 . the z - helix class appears consecutively only in z - helical dna models ( see , for example , panel b of figure 3 ) , but it is observed isolated in segments of dna loops and quadruplexes . in rna structures , the predominant class by far is the a - helix , containing over 50% of rna residues , building up the helices and stem loops that form the majority of the more complex structures . segments which are classified as b - helix appear with less than 2% occurrence and mostly at isolated positions . these segments sometimes have a backbone shape different from the normal helical forms appearing in dna , resembling more the sharp turn class ( this is especially common for segments of the b2 class ) . z - helical segments also appear at isolated positions in rna structures , mostly at the end of stem - loops with receptor functions , suggesting an important functional role ( as shown in figure 4 panel a ) . examples of rna structures and structure classification by disicl . for each model , the pdb identification code is given followed by the abbreviation of classes according to table 3 , which are color coded to match the structures they mark . apart from the classes associated with the classical dna helices , a number of special classes were defined for functionally important segments mostly found in more complex rna and dna structures . while the classes defined here help to monitor possible structurally important parts of polynucleotide structures , these structures do not separate sharply in the ( , , ) space , leading to a lower ( 4070% ) selectivity for individual definitions . the quadruplex loop class contains definitions highly specific for dna quadruplexes , which typically appear at the ends of chromosomes . the quadruplex loops rarely appear in longer stretches than three residues and instead are connected by sharp turns and transitory structures to form repeats . as quadruplexes are mainly formed in dna structures , the occurrence of this class is significantly higher in the dna data set ( 3.5% ) than in the rna set ( 0.5% ) while the quadruplex loop class is highly selective for quadruplex structures ( especially for quadruplexes made from one or two strands ) , quadruplexes formed by multiple strands of dna can exist with one , two , or all four parts built from b - helical segments ( see examples in panel c of figure 3 ) . the tetraloop bulge class ( tl ) was defined for a special bulged loop structure , which appears often in rna loops . the model structure of this class derived from tetraloop receptors , where the loop contains at least one 90 turn in the backbone , with a base facing outward from the loop to interact with bases further away in the rna sequence , possibly playing an important structural role . however , based on visual checks , it is only moderately selective for the required shape , and many segments belong to the more general a - loop class . a bulged loop from a tetraloop receptor is shown in panel b of figure 4 . the sharp turn class ( st ) collects definitions , which are enriched in segments with a more than 90 turn in the backbone and/or the torsion of their bases ( defined by the atoms c1i , c1i , c1i+1 , c1i+1 ) . sharp turn segments typically appear where the bases of the stem loops are connected , at the end of certain riboswitch and aptamer rna loops and in dna and rna knot structures . the occurrence of the sharp turn class is less than 2% in both rna and dna data sets , and sharp turns typically appear as isolated segments . for examples of the sharp turn , see panels d and e of figure 4 . the a - loop class ( al ) contains definitions of the region , which were not found to be highly selective for any of the special classes , and they are typically not forming a perfect a - helix either . a - loop structures appear often in and between rna - stem loops , connecting the classical a - helical segments with each other or with tl and st segments . the a - loop class takes up about 10% of all rna structures , but it is rarely found in dna . the al residues can form longer stretches as well , but these stretches are often single stranded or have significant distortions compared to a - helix structures . examples of a - loop segments in different rna structures are shown in panels b , c , and d of figure 4 . the b - loop class ( bl ) contains the atypical definitions of -regions . similar to the al class , b - loop segments usually connect the b - helical parts of dna models and are often found in different junctions ( holliday junctions , kissing complexes , etc . ) , dna - loop structures , and at sites where small molecules are intercalated into a dna helix . longer helical stretches of b - loop structures also appear in single strands , typically complemented by pure biii segments on the other strand . the average occurrence of b - loop segments in dna is 16% and around 1% in rna models . the ab class collects definitions for segments with a transition from the density maxima of rna and dna structures ( 1 and 1 region , respectively ) . the volume bridging these two peaks is also highly populated in both data sets ( around 10% ) and was suggested to have a functional structure of its own . we found that the ab class is often observed in helical structures in three functional roles : ( 1 ) isolated or short ab segments often serve as junctions for a - helical and b - helical parts of both dna and rna ( as shown on the left side of panel d in figure 3 ) . ( 2 ) short stretches of ab segments temper the bending of a - helices in rna stem - loops allowing for less strained loop structures ( panel a in figure 4 ) . ( 3 ) longer stretches of ab segments ( especially pure ab1 stretches ) are often found in three stranded structures , like dna triplexes ( right side of panel b in figure 3 ) and rna pseudoknots ( panel e in figure 4 ) . the ab2 class collects definitions typically transiting between the 3 and 2 regions . unlike the ab class that mostly looks helical , ab2 segments typically appear more linear as the backbone dihedrals are close to 180 with bases looking well aligned or pointing away from each other this nonideal position for stacking interactions agrees well with the observation of the ab2 class near unpaired or mismatched nucleotides and interaction sites of more bulky drug molecules . the remaining five transitory classes , namely , the ad , az , bd , bz , and zd , were defined based on the major areas that their segments connect . no particular selectivity for any of the previous classes was detected for the definitions of which they are comprised . these classes contain 3% of nucleotides in both data sets and have a similar role as the different turn definitions in the protein classification libraries . the simplified disicl library for nucleotides is designed to provide an easier comparison to cd spectroscopy , where a- , b- , z- , and quadruplex forms of dna can be distinguished from each other . for this reason , the detailed disicl classes bi - helix ( renamed to b - helix or bh ) , a - helix , z - helix , and quadruplex loop remain as separate classes in the simplified library . as the bii - helix , biii - helix , and b - loop classes relate to distorted but mostly b - helical forms of the dna , they were grouped together into the irregular b ( ib ) class . the a - loop and tetraloop bulge classes usually appear in rna - stem loops and are not sharply separated in the ( , , ) dihedral angle space . they are grouped together to form the irregular a ( ia ) class in the simplified classification . although the ab class is a transitory class , we decided to keep it as a separate class in the simplified classification because of its high abundance , enrichment in special helical segments , and definition through its own region ( ab1 ) . the remaining seven classes in the detailed classification ( st , ab2 , az , ad , bd , zb , and zd ) typically stand for nonhelical segments , which connect helical parts in stem loops and other complex polynucleotide structures grouped together in the transitory ( tr ) class in the simplified classification . the average structure element length and occurrence of the simplified disicl classes for nucleotides is shown in table 4 , along with the codes of the detailed classes grouped together in each simplified class . full correlation matrices ( pearson scores and scaled match scores ) for the comparison of disicl and x3dna are given in tables s3s6 of the supporting information . here , we provide an overview of the overall correlation analysis in table 6 . the abundance and average lengths of a- and b - helix structures are similar for the two algorithms ( table 3 ) . the average helix length of disicl is shorter due to the more detailed classification and the fact that disicl can classify one residue less for fully base - paired chains ( as x3dna requires a base - paired dinucleotide step for classification , while disicl uses a segment of three nucleotides in one strand ) . for both algorithms , the occurrence of each class is displayed in the first row or column , respectively . correlation analysis reveals that the assigned helical structures show only a partial overlap between the algorithms . a - helix classes of disicl and x3dna show the best agreement both in the dna and the rna data sets amounting to 65% of disicl residues and set , the b - helical classes as determined by disicl , show a comparable amount of correlation with the b - helix in x3dna . highest correlations are observed for the b - helix class or bi - helix ( m score 48% and r - score 0.4 ) , closely followed by the irregular b class , for which 43% of the residues are also classified as b - form by x3dna ( with similar values for b2 , b3 , and bl classes ) , but its lower abundance decreases the r - score to 0.3 . for the rna data set , the amount of segments assigned by x3dna as b - helix is extremely low ( 0.04% ) and shows little or no correlations with the disicl b - helical classes ( or any other class ) , leading to a combined agreement amounting to 6% the x3dna class . the abundance of b - helical segments in rna according to disicl is 1.5% ( mostly due to the b - loop class ) . visual checks reveal that x3dna b - helix segments do not show the shape normally associated with disicl b - helical segments , while disicl b - helix segments appear in rna mostly as bulges in a - helices or at the end of stem - loops ( an example is shown in panel c of figure 4 ) . we found no models in the rna data set , with hydrogen - bonded base pairs for which disicl classified both strands as b - helix , which partially explains the low correlation with x3dna as this program monitors paired bases only . while the z - helix class in disicl has a low abundance ( 1% and 0.4% in dna and rna , respectively ) , it has no overlap with any of the x3dna classes in the dna data set and a minimal overlap with the a - helix class in rna ( due to the isolated z - helix segments in rna stem loops ) . this shows that x3dna very rarely mistakes z - helical segments for a- and b - form segments , even though not explicitly making the classification ( except for full z - helices in dna ) . considering transitory and special classes , the ta - transitory class of x3dna shows moderate correlations ( m scores ) with the bi - helix ( 32% ) , ab ( 13% ) , and b - loop ( 12% ) classes of disicl in dna and with the ab ( 38% ) and a - helix ( 26% ) classes in rna , showing that the peak of its density distribution falls in the ab1 region . the correlation might be low for dna because the ta class was based on special dna segments meant for interacting with polymerase enzymes , and protein nucleotide complexes were filtered out from our data sets . about one - third of the ab class in disicl was considered as b - helix in dna ( 34% ) and a - helix in rna ( 33% ) by x3dna . additionally the bd class shows a moderate correlation ( 30% ) with x3dna b - helix in dna , while ad ( 15% ) and ab2 ( 14% ) classes correlate weakly . in rna models , moderate agreement with the x3dna a - helix is also observed for the a - loop ( 39% ) and ad ( 38% ) classes ( often found in distorted a - helices ) and the tetraloop bulge class ( 25% ) . the rest of the disicl classes remained mainly unclassified by x3dna with no significant correlations . a summary of the correlation analysis is shown in table 1 ( methods agreement ) , which reveals an overall agreement between x3dna and disicl slightly below 60% for both the rna and dna data sets , which is slightly lower than the agreement between protein classification algorithms . the analysis of the dna and rna data sets provides a solid basis to define and characterize the disicl nucleotide structure classes , and their correlations with the classification of x3dna . the higher level of detail in disicl allows us to monitor the structural effects of interactions of nucleotides with small molecules and proteins as well . two examples for rna protein and dna protein complexes are shown in figure 5 . panels a and b show a model of an aaug tetraloop hairpin in complex with a yeast rnase binding domain ( pdb code 2lbs ) . the bulk of the interactions take place between a short -helix of the protein at the end of the tetraloop hairpin . while x3dna steadily recognizes the a - helical conformation at the base of the hairpin , the interaction site remains unclassified disicl assigns a classification for 70% of the nucleotides over the nmr solution models , mainly to the tetraloop bulge or ab2 class . another interesting example is the ternary complex of double - stranded dna and a protein fragment of the polymerase i from t. aquaticus ( pdb code 2ktq ) . in this case shown in panels c and d of figure 5the longer template strand of the mainly b - form dna is bent by the protein , recognized as an a - helical stretch in disicl . as in the previous example , x3dna readily recognizes the b - helical nature of the double - stranded part but leaves the dna at the interaction site unclassified . the examples suggest that fine structural changes might be revealed by disicl , yielding additional information on interactions between nucleotides , proteins , and small molecules . examples of dna / rna protein complexes classified by disicl and x3dna . for each model , the pdb identification code is given , followed by the method of classification and the abbreviation of structural classes according to table 3 . the disicl algorithm for dihedral - based structure classification was extended to allow for the classification of nucleotide structures . starting from previously published distributions of dihedral angles , three dihedral angles ( , , ) were selected to perform the classifications . fourteen distinct regions were defined in the resulting three - dimensional dihedral angle space . a classification is performed based on the assignment of the two central nucleotides in a trinucleotide segment , first to their regions and as a pair to one of 17 structural classes . apart from helical structures , we define loop regions , turns , and transitory structural elements , and examples of these were given with dna and rna models from the brookhaven pdb . newly suggested structural classes include the quadruplex loop , sharp turn , and tetraloop bulge , as well as a number of transitory elements . the detailed classification was simplified into eight more general classes and were compared to the classification in x3dna . overall , disicl seems a very powerful tool for the detailed structural analysis of both proteins and polynucleotides . studies of practical applications for the disicl algorithm are currently the focus of our attention . additionally ,

the effects of a polyherbal mixture containing allium sativum , cinnamomum zeylanicum , citrullus colocynthis , juglans regia , nigella sativa , olea europaea , punica granatum , salvia officinalis , teucrium polium , trigonella foenum , urtica dioica , and vaccinium arctostaphylos were tested on biochemical parameters in diabetic rats . the animals were randomized into three groups : ( 1 ) normal control , ( 2 ) diabetic control , and ( 3 ) diabetic rats which received diet containing 15% ( w / w ) of this mixture for 4 weeks . diabetes was induced by intraperitoneal injection of streptozotocin ( 55 mg / kg ) . at the end of experiment , the mixture had no significant effect on serum hepatic enzymes , aspartate aminotransferase , and alanine aminotransferase activities . however , the level of fasting blood glucose , water intake , and urine output in treated group was lower than that in diabetic control rats ( p < 0.01 ) . also , the levels of triglyceride and total cholesterol in polyherbal mixture treated rats were significantly lower than those in diabetic control group ( p < 0.05 ) . our results demonstrated that this polyherbal mixture has beneficial effects on blood glucose and lipid profile and it has the potential to be used as a dietary supplement for the management of diabetes .

diabetes mellitus , a metabolic disease with manifestation of hyperglycemia and dyslipidemia , is still one of the most leading causes of death and disability . over time , diabetes leads to serious complications such as nephropathy , retinopathy , neuropathy , stroke , and peripheral vascular diseases . currently , beside insulin , the most widely used medications for diabetes are insulin and the oral hypoglycemic drugs . although early onset manifestations of diabetes can be controlled by current antidiabetic drugs , late onset complications appear in many patients . in addition , the clinical uses of the current drugs are usually accompanied with some adverse effects including abdominal discomfort , severe hypoglycemia , lactic acidosis , and peripheral edema . therefore , the search for new antidiabetic agents with more effectiveness and lesser side effects has continued . today , antidiabetic effects of several plants have been supported by results from animal studies or clinical trials . among them , allium sativum , cinnamomum zeylanicum , citrullus colocynthis , juglans regia , nigella sativa , olea europaea , punica granatum , salvia officinalis , teucrium polium , trigonella foenum , urtica dioica , and vaccinium arctostaphylos are widely used as medicinal plants for management of diabetes [ 4 , 5 ] . several studies have shown that each one of these plants is effective in the decrease of plasma glucose and serum lipids in diabetes [ 618 ] . we hypothesized that a combination of them may have more beneficial effect on improving metabolic indexes . the present study is a part of a research effort to develop the best polyherbal mixture from these antidiabetic plants for management of glucose and lipid in diabetes . in our previous work , we observed that administration of a combination of hydroalcoholic extracts of six of them inhibited the progression of hyperglycemia and decreased serum lipids and hepatic enzyme activity in diabetic rats . since nonprocessed herbs are usually more tolerated than extracted or formulated products and patients prefer to consume plants as salad , spice , and so forth , in this work , we aimed to investigate the effect of a mixture of powders of the above mentioned twelve plants on glycemic and lipidemic status of diabetic rats . the air - dried a. sativum ( cloves ) , c. zeylanicum ( bark ) , c. colocynthis ( fruit ) , j. regia ( leaf ) , n. sativa ( seeds ) , o. europaea ( leaf ) , p. granatum ( fruit ) , s. officinalis ( areal parts ) , t. polium ( areal parts ) , t. foenum ( seeds ) , u. dioica ( areal parts ) , and v. arctostaphylos ( fruit ) were powdered and mixed with ratio of 5% , 5% , 5% , 10% , 5% , 5% , 5% , 5% , 5% , 20% , 15% , and 15% , respectively . this polyherbal mixture ( phm ) was added ( 15% w / w ) to the standard pellets diet of animals in treatment group . male albino wistar rats ( 280330 g ) were obtained from laboratory animals house , mashhad medical university ( iran ) and housed in a room with controlled lighting ( 12 h light/12 h darkness ) and temperature ( 22 2c ) . all animal procedures were done according to the ethical guidelines of the animal care of shiraz university of medical sciences ( iran ) . to generate diabetes , some rats received a single dose ( 55 mg / kg , ip ) of streptozotocin ( stz ) ( enzo life , usa ) . induction of diabetes was confirmed by measuring fasting blood glucose ( fbg ) two days after stz injection . rats with fbg level of 250 mg / kg or higher were considered to be diabetic [ 20 , 21 ] . the animals were randomized into three groups : ( 1 ) normal control rats which were fed standard diet ( n = 6 ) , ( 2 ) diabetic control animals which were fed standard diet ( n = 6 ) , and ( 3 ) diabetic rats which received diet containing 15% ( w / w ) of phm ( n = 8) . the treatment was initiated two days after stz injection and continued for 4 weeks . at the end of the 30th day , the rats fasted 16 h and blood samples were collected from retroorbital sinus for biochemical measurements . at the end of the experiment , animals were placed in individual metabolic cages for urinary collection . after acclimatization ( 1 day ) , the 24 h urinary samples were collected from diabetic animals . serum triglyceride and total cholesterol were evaluated with standard enzymatic colorimetric kits from pars azmun ( iran ) . blood glucose was measured using glucose oxidase reagent ( ziest chem diagnostics , iran ) . serum aspartate aminotransferase ( ast ) and alanine aminotransferase ( alt ) activities were evaluated with colorimetric methods by commercially available kits ( pars azmun , iran ) . as shown in figure 1 , before injection of stz , fbg levels of both groups were statistically not different from each other . at day 2 the diabetic rats in control group showed further increase of fbg at the end of experiment ( 374 17 mg / dl in day 30 versus 272 14 mg / dl in day 2 , p < 0.01 ) . however , administration of phm to diabetic rats blocked the increase of blood glucose . at day 30 , the level of fbg in this group was 263 29 mg / dl which was significantly lower than that in diabetic control rats ( p < 0.01 ) . during experiment , both diabetic control and diabetic phm treated groups exhibited a significant reduction in their body weight . at day 30 , the weight reduction reached to 27% ( p < 0.001 versus day 0 ) and 25% ( p < 0.001 versus day 0 ) for control and phm treated animals , respectively ( table 1 ) . prior to diabetes induction , the level of water intake was not significantly different between three groups . however , after stz administration , there was a significant increase in the levels of water intake in both groups of diabetic rats . although the polydipsia condition was evident during the treatment period , the level of water intake in phm group was significantly ( p < 0.001 ) lower than that in diabetic control group . also , at the end of the experiment , the phm - fed diabetic rats showed an improvement in polyurea state and the excretion of urine was significantly lower than that of diabetic control group ( 30 3 ml/24 h versus 72 9 ml/24 h , p < 0.01 ) . there was a significant elevation in the level of triglyceride ( 129 27 mg / dl versus 59 11 mg / dl , p < 0.05 ) and total cholesterol ( 105 9 mg / dl versus 55 3 mg / dl , p < 0.01 ) in diabetic control rats as compared with normal group . the levels of triglyceride and total cholesterol in phm treated group were 66 27 mg / dl ( p < 0.05 versus diabetic control group ) and 83 4 mg / dl ( p < 0.05 versus diabetic control group ) , respectively . at day 30 , diabetic control rats showed higher ast ( 110 18 versus 71 8 u / l ) and alt ( 77 4 versus 28 5 u / l , p < 0.05 ) activity than that of normal group , suggesting hepatic dysfunction in these animals . the level of ast and alt activity in phm treated groups was 107 15 u / l and 58 10 u / l , respectively , which was not significantly different from that of diabetic control rats ( figure 3 ) . although numerous herbs have been suggested for the treatment of diabetes , but at present no one could completely treat diabetic patients . one approach for the development of an effective phytochemical compound can be mixing a number of hypoglycemic and hypolipidemic herbs to produce more potent antidiabetic agent . the present work was a part of a research effort to find the best mixture from some medicinal plants which their hypoglycemic and hypolipidemic effects were supported by several studies . here , we investigated the effect of a diet containing mixture of twelve medicinal plants on glycemic and lipidemic status of diabetic rats . although this mixture failed to completely restore stz - induced hyperglycemia and had no effect on weight reduction , it significantly prevented further elevation of blood sugar and improved the polydipsia and polyurea . this beneficial effect on glycemic status is expected to happen as antihyperglycemic effect of all twelve plants forming phm has been confirmed with repeated studies [ 616 , 22 , 23 ] . antihyperglycemic effect of medicinal plants is achieved by different mechanisms including decreasing glucose absorption from intestine , enhancing insulin secretion from beta cells , increasing glucose uptake by tissues , inhibiting glucose production in liver , and increasing pancreatic tissue regeneration and/or presence of insulin - like agents in plants [ 2427 ] . regarding phm , inhibitory effect of t. foenum on intestinal glucose absorption and the beneficial effects of j. regia and t. polium on regeneration of pancreatic islets also , it has been demonstrated that n. sativa and s. officinalis decrease hepatic glucose production through inhibition of gluconeogenic enzymes [ 31 , 32 ] . furthermore , the insulin secretory effects of c. colocynthis , o. europaea , t. foenum , v. arctostaphylos , and u. dioica were shown in vitro in the isolated pancreas or islets [ 16 , 17 , 3335 ] . therefore , the levels of serum triglyceride and cholesterol are usually elevated in diabetic patients . in our study the hypolipidemic action of phm is in agreement with earlier studies that reported that a. sativum , c. colocynthis , c. zeylanicum , j. regia , n. sativa , s. officinalis , p. granatum , t. foenum , and t. polium decrease the levels of serum triglyceride and cholesterol in diabetic subjects [ 6 , 7 , 9 , 12 , 14 , 3739 ] . also , in our previous work , we showed that t. foenum led to a significant reduction in lipid droplet accumulation in adipocytes . elevation of alt and ast enzyme activities is considered as an evidence for hepatic damage . an increase of these enzyme activities is also associated with fatty liver disease and decreased hepatic insulin sensitivity in type 2 diabetes [ 41 , 42 ] . according to the reports of previous studies , a. sativum , j. regia , s. officinalis , o. europaea , t. foenum , and t. polium could decrease the alt and ast levels in diabetic rats [ 7 , 8 , 37 , 4345 ] . on the other hand , there are some reports that chronic administration of t. polium results in elevation in plasma levels of liver enzymes [ 15 , 46 ] . in our study therefore , consumption of this polyherbal mixture accompanied with no hepatoprotective or hepatotoxic activity . taken together , our results demonstrated that phm has beneficial effects on blood glucose and lipid profile of diabetic rats . in our previous work , we observed approximately the same level of antihyperglycemic and hypolipidemic effect with administration of a polyherbal mixture made from the combination of macerated and soxhlet ( hydroalcoholic ) extracts of a. sativum , c. zeylanicum , n. sativa , p. granatum , s. officinalis , and t. polium . therefore , we did not find any favorable effects on diabetes when the powders of twelve plants were combined instead of giving a combination of hydroalcoholic extracts of six of these plants . also , some studies reported more antihyperglycemic effect when the extracted or formulated products of herbs especially t. foenum were given individually . however , it should be considered that when they are administrated individually , the antidiabetic effect was achieved usually with high doses of the plant extract which may be accompanied with unpleasant effects in the body . therefore , we still believed that the phm , which was constructed simply from mixing plant powders without the need for extraction procedure , has the potential to be used as a dietary supplement for the management of diabetes , particularly diabetic dyslipidemia . obviously , more study is needed to find a more potent polyherbal mixture from antidiabetic plants . further , combination of active ingredients isolated from these plants may be a subject if interest in future perspective related to diabetes management .

vaccine efficacy in aquaculture has for a long time depended on evaluating relative percent survival and antibody responses after vaccination . however , current advances in vaccine immunology show that the route in which antigens are delivered into cells is deterministic of the type of adaptive immune response evoked by vaccination . antigens delivered by the intracellular route induce mhc - i restricted cd8 + responses while antigens presented through the extracellular route activate mhc - ii restricted cd4 + responses implying that the route of antigen delivery is a conduit to induction of b- or t - cell immune responses . in finfish , different antigen delivery systems have been explored that include live , dna , inactivated whole virus , fusion protein , virus - like particles , and subunit vaccines although mechanisms linking these delivery systems to protective immunity have not been studied in detail . hence , in this review we provide a synopsis of different strategies used to administer viral antigens via the intra- or extracellular compartments . further , we highlight the differences in immune responses induced by antigens processed by the endogenous route compared to exogenously processed antigens . overall , we anticipate that the synopsis put together in this review will shed insights into limitations and successes of the current vaccination strategies used in finfish vaccinology .

the central hallmark of vaccination is to prime the adaptive immune system to develop immune responses that will protect the host organism upon a second encounter with the same pathogen . however , priming the adaptive immune system requires activation of nave b- and t - lymphocytes into effector cells that translate into protective immunity . while studies on the immunological basis of vaccine protection have for a long time focused on humoral and cellular responses as measures of protective immunity , growing evidence shows that the mode by which antigens are presented to b- or t - lymphocytes has a significant influence on the outcome of adaptive immune responses induced by vaccination which is also influenced by the mode in which antigens are administered to host cells [ 1 , 2 ] . put together , these elements drive vaccine development into a cross - talk between vaccinology and immunology in which vaccine design and its delivery ( vaccinology ) on one hand have to be optimized in order to gain an effective immune response ( immunology ) on the other . hence , optimization of antigen design and its delivery into host cells is a prerequisite to inducing an optimal protective immune response . unlike b - lymphocytes , which are precursors of antibody secreting cells that can recognize antigens through primed antigen presenting cells ( apcs)/activated b - cells , t - cell receptors ( tcrs ) endogenous peptides derived from intracellular sources such as replicating virus are synthesized and processed for presentation to nave cd8 t - cells by mhc - i molecules while exogenous peptides derived from extracellular sources are processed and presented to nave cd4 t - cells by mhc - ii molecules . an alternative mechanism that permits some extracellular antigens to activate nave cd8 t - cells called cross presentation exists which occurs via the mhc - i pathway [ 3 , 4 ] . for antigens delivered via the endosomal route , proteosomes degrade soluble antigens after ubiquitination which have been synthesized in the cytosol or escaped to the endoplasmic reticulum ( er ) by cross presentation . thereafter , the processed antigens are released after proteosomal degradation to generate peptides that are transported into the er by the transporter - associated antigen processing ( taps ) [ 5 , 6 ] . once in the er , the antigenic peptides are loaded onto mhc - i molecules for presentation on the cell surface where they initiate the activation of nave cd8 t - cells into effector cytotoxic t - lymphocytes ( ctls ) [ 79 ] . in the case of antigens delivered by the exogenous route , lysosomes degrade endocytosed antigens after endosomal fusion with lysosomes . in general , lysosomes can degrade complex structures such as whole viral particles that are delivered to them via endocytosis by the extracellular route . presentation of processed peptides by endosomal degradation leads to maturation of apcs into professional apcs which is characterized by expressing mhc - ii molecules and antigen specific signaling molecules such cd40l , cd80 , and cd86 . the resulting professional apcs are the prime initiators of adaptive immune responses that activate nave t - cells into effector cells through the mhc - peptide complexes and immune modulation molecules . therefore , it follows that , for a vaccine antigen to turn nave b- or t - lymphocytes into protective cell , there has to be an efficient antigen delivery system that stimulates the activation of cell of the adaptive immune system . although studies on antigen presentation in fish immunology have gained prominence in recent years [ 1214 ] , there is still limited research on activation of cells of the adaptive immune system by apcs , which precludes our understanding of the role of innate immunity in optimizing vaccine performance . despite that , several studies have been carried out trying to deliver viral antigens into different compartments of fish cells . hence , in this review we provide an overview of these delivery systems and based on this approach we highlight the different immune responses induced by antigens delivered by the intracellular route compared to antigens delivered by the extracellular route . in addition , we also highlight the differences in vaccine efficacy from fish immunized using antigens delivered by the exogenous route compared to fish vaccinated using the endogenous route . overall , we anticipate that the synopsis of different antigen delivery systems put together in this review will shed new insights into limitations and successes of the current vaccination strategies used in fish vaccinology . in mammals , antigen presentation is carried out by different cell types that include monocytes , macrophages , and dendritic cells . these cells possess pattern recognition receptors ( prrs ) that recognize and bind to pathogen associated molecular patterns ( pamps ) known as danger signals on pathogens . upon binding to pamps using prrs , monocytes mature into macrophages while immature dendritic cells ( dcs ) also transform into mature dendritic ones to become professional apcs , which induce the expression of proinflammatory cytokines that attract more apcs to the sites of antigen deposition [ 15 , 17 ] . upon encounter with the apcs , nave b- and t - cells undergo maturation to become memory cells capable of recognizing the antigens in subsequent encounters thereby creating the basis acquired immunity . in teleosts fish , apcs known to possess prrs having the capacity to bind to different pamps on pathogens have been described in different species and these include monocytes , macrophages , and dendritic - like cells [ 13 , 1826 ] . in addition fish b - cells have been shown to carry out antigen presentation apart from their role as antibody secreting cells . as a result , in vitro methods for culturing fish monocytes , macrophages , and dendritic - like cells have been developed which makes it easy to study antigen presentation using cell cultures [ 18 , 19 , 24 , 25 ] . it is interesting to note that tap genes comparable to those seen in mammals have been identified and mapped to mhc regions in different cartilaginous and bony fish species suggesting that similar mechanisms of endogenous antigen processing seen in higher vertebrates also exist in teleosts fish [ 2831 ] . unlike in mammals where apcs carrying processed antigens migrate to the lymph nodes , in fish apcs carrying antigens migrate to the head , kidney , and spleen , which are the major lymphoid organs . apart from lymphoid organs , apcs have been detected in other organs such as the gills , skin , and intestines in fish [ 34 , 35 ] . in addition , different phagocytic cell types have been characterized in fish although their antigen presentation capabilities have not been investigated [ 36 , 37 ] . similar to their mammalian counterparts , fish apcs possess a wide range of surface markers that include cd80/cd86 , cd83 , cd209 , mhc - i , and mhc - ii proteins [ 20 , 3841 ] . in fish , cd83 has been shown to be an activation marker for macrophages and dendritic - like cells . apart from cd83 , other surface markers identified for fish dendritic - like cells include cd208/lysosomal associated membrane protein ( lamp3 ) . recently , zhu et al . showed that fish b - cells act as pivotal apcs in priming the adaptive immune system using cd80/cd86 molecules . in another study , abs et al . showed upregulation of mhc - ii genes that coincided with upregulation of cd80/cd86 genes in a nonlethal infection ( no cytopathic effects observed ) of viral hemorrhagic septicemia virus ( vhsv ) in igm+ cells which consolidates the notion that fish b - cells use cd80/cd86 molecules to activate the adaptive immune system using the mhc - ii pathway [ 44 , 46 ] . as shown in table 1 , all four t - cell receptor chains ( , , , and ) required for binding to apcs together with the four chains ( - , - , - , and -chain ) of the cd3 coreceptor complex required for t - cell activation in mammals have been reported in fish . in addition , the t - cell costimulatory marker cd28 and the negative regulatory marker ctla-4 , which bind to cd80 and cd86 receptors on apcs , have also been characterized in fish [ 47 , 48 ] . as for cd8 t - cells , two subsets have been characterized , namely , cd8 and cd8 , from different fish species of which cd8 has been the most widely used marker for t - cell activation in different studies [ 4951 ] . moreover , cell mediated cytotoxicity against allogeneic targets and virus infected cells has been reported by different scientists [ 50 , 5254 ] . put together these observations suggest that fish t - cells possess surface receptors essential for the binding to apcs comparable to those found in mammals and that activation of t - cells into effector cytotoxic t - lymphocytes ( ctls ) could be based on similar mechanisms to those seen in mammals . there are three immunoglobulin isotypes characterized in fish , this far , and these include igm [ 55 , 56 ] and igd [ 55 , 57 ] also present in mammals while the recently identified igt is only found in fish where it exists as membrane bound and secreted form in serum [ 59 , 60 ] . igm is the most abundant isotype in serum where it is estimated to be > 1000-fold higher than igt [ 61 , 62 ] . in addition , igm has been detected in the mucus of the skin , gills , and intestines although its levels in these organs are far much lower than levels detected in serum [ 61 , 62 ] . on the contrary , igt is predominantly found in the mucus of the skin , gut , and intestines [ 6163 ] where it is > 100-fold higher than levels detected in sera . it is interesting to note that the costimulatory marker cd40l mostly expressed in activated cd4 + t - cells , which binds to the cd40 receptors on apcs in order to activate b - cell proliferation , has been characterized in fish . in addition , transcription factors involved in specification of cd4 t - cells into different t - helper ( th ) subtypes have also been characterized , which include t - bet [ 66 , 67 ] , gata-3 [ 6870 ] , and ror [ 71 , 72 ] for the differentiation of nave cd4 t - cells into th1 , th2 , and th17 subtypes , respectively . in addition , several cytokines linked to specification of cd4 t - cell into different subtypes have been characterized and these include il-2 , il-4 , il-6 , il-10 , il-12 , ifn , il-15 , il-21 , il-22 , and tgf [ 40 , 41 , 73 , 74 ] . overall , the characterization of different apcs and adaptive immune cells together with their receptors and regulatory cytokine presented here suggests that teleosts fish antigen presentation mechanisms could be comparable to those used by mammals suggesting that antigen presentation mechanisms have been conserved across the vertebrate taxa . intracellular antigen delivery systems involve immunization strategies that administer the vaccine antigens into the cytoplasm ( figure 1 ) and these include the following . live vaccines use attenuated viruses or recombinant antigens encoded by live virus vectors that have the capacity to replicate in host cells with attenuated pathogenicity lacking the ability to cause disease . as analogues of pathogenic viruses , they engage with the cell membrane by binding to surface receptors using epitopes similar to their native virus , thereby gaining entry into endosomal structures and the cytosol where they use the host cell machinery to replicate . consequently , the processed antigens are presented on the cell surface by mhc - i molecules while soluble antigens expressed by replicating virus are engulfed by apcs to induce humoral immune responses ( figure 1 ) . hence , live vaccines induce both cellular and humoral immune responses . in general , different scientists have reported the induction of ctl responses in fish [ 52 , 53 , 75 ] . . showed activation of the ctls by viral hemorrhagic septicemia virus ( vhsv ) infection in rainbow trout , while we recently showed activation of eomesodermin , a transcription factor involved in activation of cd8 cells in atlantic salmon exposed to infectious pancreatic necrosis virus ( ipnv ) . similarly , chang et al . showed activation of cd8 cells after exposing orange spotted grouper ( epinephelus coioides ) to nervous necrosis virus ( nnv ) . flow cytometry analysis of the spleen cells from fish exposed to nnv showed increased mean fluorescent intensity of the cd8 cells and peripheral blood leukocytes ( pbls ) which were linked to increased cytotoxicity and mhc - i restriction of the sorted lymphocytes by recombinant cd8 antibodies . several fish species have shown upregulation of mhc - i and -ii molecules [ 49 , 7780 ] as well as expression of high antibody levels after exposure to viral infections [ 81 , 82 ] suggesting that attenuated viruses administered as live vaccines could evoke both cellular and humoral immunity . based on these observations , several attempts have been made to develop live viral vaccines for fish ( table 2 ) and some of the strategies explored this far are outlined below . roberti et al . discovered a naturally attenuated mutant of infectious hematopoietic necrosis virus ( ihnv ) that conferred protection against ihnv in rainbow trout although the vaccine resulted in causing low level mortality after challenge . used an oral vaccine against vhsv obtained from a naturally attenuated live virus selected using monoclonal antibodies . in their study , they showed high expression levels of mhc - ii and cd4 mrnas . in addition , they detected antibody responses that were linked to significant protection in rainbow trout after challenge . an attenuated ihnv vaccine was developed at oregon state university by multiple passages using a rainbow trout isolate propagated using the steelhead trout cell culture [ 85 , 86 ] . the vaccine showed high protection ( 95% ) in vaccinated chinook salmon while mortality in control fish reached 90% . although the vaccine was highly protective in chinook salmon , when used in rainbow trout it showed significant mortality and as such it was stopped [ 87 , 88 ] . since the ab strain of infectious pancreatic necrosis virus ( ipnv ) was found to be less virulent than the west buxton , sp , or jasper strain , dorson et al . attempted to develop an attenuated strain of ipnv from the ab strain after several passages on rtg cells . reverse genetics has been used in fish vaccinology to generate avirulent strains for use as live vaccines . for example , recombinant ihnv having a deletion of the nv gene resulted in irreversible attenuation of the wild type virulent strain resulting in the induction of high protection levels in rainbow trout . in another study , recombinant ihnv generated by replacing the nv gene with green fluorescent protein ( gfp ) or substituting the ihnv g - gene with the g - gene of vhsv induced heterologous protection in rainbow trout . for ipnv , reverse genetics was used to generate an avirulent strain for use as a live vaccine against the wild type strain by substituting amino acids on positions 217 and 221 of the vp2 capsid . these studies showed that the strain encoding the t217a221 motif caused high mortality in atlantic salmon while the strain encoding the p217t221 motif was avirulent and linked to subclinical infections . infecting atlantic salmon with high and low virulent strains at a nonpermissive physiological state ( presmoltification stage ) did not result in mortality . when the vaccinated fish were challenged at smolt stage ( permissive ) the avirulent strain was less immunogenic than the virulent vaccine strain . in addition , we observed that the avirulent strain reverted to virulence under stress conditions [ 37 , 94 ] . in general , the fear of reversion to virulence has been the major hindrance for the licensure of live vaccines in aquaculture . the strategy of dna vaccination is based on the principle that the encoded immunogenic protein is injected into the muscle or other tissues where it enters the host cells and directs the synthesis of its polypeptide antigen from the plasmid vector . once transfected into host cells , transcribed antigens replicate in the cytosol using the endogenous pathway while soluble or secreted antigens are phagocytized by apc and gain access into the exogenous pathway ( figure 1 ) . in principle , dna vaccines result in the in vivo synthesis of antigenic proteins using the host cell machinery in a manner identical to natural virus infection in the case of dna vaccines made for viral diseases . this culminates into antigenic proteins expressed by plasmid dna gaining access to both the exogenous and endogenous pathways in the activation of both humoral and cellular mediated immune responses . boudinot et al . demonstrated the intracellular delivery of the plasmid dna encoding the recombinant g protein of vhsv inside the muscle cells of vaccinated rainbow trout . intracellular detection of the g - protein was shown up to 45 days at the injection sites . transcription of the g - protein was demonstrated by detection of mrna in muscle tissue extracts , which was linked to expression of high antibody and mhc - ii mrnas levels . in another study , utke et al . showed activation of the ctls following immunization using the g - protein of vhsv in rainbow trout . in their study , they used pbls collected from fish immunized with a dna vaccine encoding the recombinant g -protein of vhsv and showed that pbls from vaccinated fish killed the vhsv mhc - i matched rtg-2 cells indicating that the g - proteins had the capacity to induce ctl responses in vaccinated fish . they also showed the homing of leukocytes to the injection site suggesting that cells expressing the recombinant g - protein had a chemoattractant effect . this observation was recently supported by castro et al . who showed that b - lymphocytes , both igm and igt cells , represent one of the major cell types infiltrating the injection sites expressing the g - protein of vhsv . in their study , they showed upregulation of cxcr3b , a receptor for cxcl11 , together with ck5b and ck6 chemokines , which could play chemotactic roles in the early recruitment of b - cells at the injection sites . put together , these studies show that the intracellular expression of proteins transcribed from dna vaccines in fish cells leads to homing of leukocytes and b - cells to injection sites with possible involvements of chemoattractant chemokines . further , these studies suggest that antigens delivered by this endogenous route evoke both the humoral and cellular mediated immune responses in vaccinated fish . finally , it is important to point out that immunization using dna vaccines exhibits many advantages over the live and inactivated vaccines . intracellular synthesis of the antigenic proteins poses no danger of reversion to virulence and does not require inactivation of viruses using toxic substances . high expression levels of humoral and cellular responses can be achieved at low doses as shown by corbeil et al . that nanogram quantities of a dna vaccine protected rainbow trout against ihnv infection after challenge . in addition , intracellular synthesized antigens tend to fold in their native conformation and correctly glycosylated displaying the neutralizing epitopes in a similar pattern to the native virus . in terms of genetic engineering for example , the use of molecularly encoded cytokine adjuvants like il-2 in dna engineered vaccines has shown the ability to enhance dna delivery and increase the duration and magnitude of plasmid dna expression in vivo . jimenez et al . coinjected recombinant il-8 with plasmid dna encoding the g - protein of vhsv in rainbow trout and showed massive infiltration of neutrophils at the injection site linked to upregulation of proinflammatory cytokines . table 3 shows the dna vaccines explored for use in fish , this far , of which only the dna vaccine for ihnv has been licensed in canada ( novartis ltd . ) . this mode of antigen delivery which has been referred to as the first class ticket to induction of mhc - i responses relies on receptor mediated internalization of viral antigens to the er followed by retrograde translocation into the cytosol . only a few studies have explored this delivery system in fish vaccinology , this far [ 103 , 104 ] . constructed a fusion protein vaccine made by fusing the vp2-vp3 polyprotein of ipnv with the exotoxin of lactobacillus casei , which resulted in reduced viral loads in vaccinated rainbow trout after challenge while in our group we constructed a fusion protein vaccine made by fusing the vp2 of ipnv with the pseudomonas aeruginosa exotoxin a ( ep ) . the exact mechanisms in which viral antigens are translocated into the cytosol are dependent on three bacterial proteins as illustrated from the pe fusion protein in figure 2 . the pe protein has three functional domains , namely , the receptor binding domain - i , transmembrane targeting domain - ii , and the toxic moiety domain - iii . domain - i binds to the 2-macroglobulin receptor on the cell surface . after binding to domain - i after enzymatic cleavage by the protease furin in the endosome , the protein fragment encoding domains - ii and -iii is delivered into the golgi by the er retrograde transport and further into the cytosol using domain - ii , which is responsible for transmembrane translocation of the toxin proteins into the cytoplasm . as shown in figure 2 , domain - iii , which is toxic to cells , is eliminated and is replaced with the immunogenic protein ( vp2 ) of ipnv bound to the kdel signaling peptide . the purpose of including the kdel signaling peptide in the final construct ( pe - vp2-kdel ) is that it enables the binding of the whole construct to the golgi membrane kdel - receptor . once bound to the golgi membrane receptor , the ligand - receptor complex is packaged into vesicles for retrograde transport back to the er where processed peptides are packaged on mhc - i molecules for presentation on the cell surface . in our studies , we employed the pe - vp2-kdel fusion protein to deliver the vp2 immunogenic protein of ipnv intracellularly as a vaccine . although we did not assess the ctl responses induced by this antigen delivery system , our findings show that these vaccines were able to induce a low level antibody response suggesting that antigens delivered using this method could gain access to induction of humoral responses in vaccinated fish . however , there is a need for detailed investigation to determine the role of ctl responses induced by this mode of antigen delivery in vaccinated fish . polymeric nanoparticles formulated from biodegradable polymers have been widely explored as carriers for controlled delivery of vaccine antigens [ 105 , 106 ] . this system can potentially deliver antigens to the desired location at predetermined rates and durations to generate an optimal immune response . for example , tian et al . [ 108110 ] and zheng et al . showed that lymphocytic disease virus ( lcdv ) encapsulated in particles sustained a much longer release of the dna antigen than naked dna injected in japanese flounder . in addition , carriers protect the antigen from degradation until release as shown by rajeshkumar et al . that encapsulated dna antigens were protected from degradation by dnaase for vaccines used in the asian sea bass ( lates calcarifer ) . to deliver the antigens into host cells , nanoparticle materials are internalized by endocytosis . to deliver the antigens into the cytosol , the release of antigens from the acidic endosomes requires membrane disruptive agents , which release the internalized proteins into the cytosol . therefore , encapsulation carriers should include membrane penetrating peptides and polymers that disrupt the membranes when the ph declines in the endosomes . made acid degradable nanoparticles , which were designed to release encapsulated proteins in a ph - dependent manner . in their study , they made nanoparticles that were stable at ph 7.4 but quickly degradable at ph 5.0 in the acidic endosomal environment enabling the release of antigens into the cytosol , ultimately resulting in upregulation of mhc - i . another method explored is the use of amphiphilic polymers [ 115117 ] , which also have ph - dependent membrane disruptive properties protonated at the endosomal ph range [ 115 , 118 ] . upon reduction of the endosomal ph , these particles increase their hydrophobicity to facilitate the disruption and penetration of the endosomal membranes culminating in the release of antigens in the cytosol . these amphiphilic polymers have been shown to increase cd8 + responses and to improve vaccine potency . in summary , these studies show that nanoparticle antigen delivery systems can be designed to deliver antigens through the intra- or extracellular routes to evoke immune responses linked to the mhc - i or -ii pathways . studies in higher vertebrates have shown that apcs easily carry out phagocytosis of nanoparticles and microparticles between 150 nm and 4.5 m [ 120 , 121 ] with the optimal size for phagocytosis being 500 nm while monocytes have been shown to easily phagocytose nanoparticles > 100 nm . and , as pointed out by gutierro et al . , nanoparticles that encapsulate antigens resemble pathogens in terms of their uptake into host cells by mirroring the route of pathogen uptake and the immune response triggered after nanoparticle uptake . have also pointed out that nanoparticles can also be used to carry antigens on their surface which would serve as a good stimulant for the induction of b - cell responses . although antigen delivery using nanoparticle vaccines has been well studied in higher vertebrates , indications are that fish cells use similar mechanisms of antigen uptake from nanoparticle based vaccines . for example , ruyra et al . showed entry of liposome - based nanoparticles in zebrafish hepatocytes and trout macrophages by endocytosis . upon entry , the nanoparticle laden cells initiated specific proinflammatory responses while fredriksen and grip showed intracellular cytoplasmic localization of polylactic - coglycolic acid ( plga ) nanoparticles in to - cells . these findings support earlier observations , which showed that because plga particles are less hydrophilic than alginates , they are easily incorporated into host cells , which makes them suitable vehicles for delivering antigens into intracellular compartments [ 128131 ] . recently , we used plga nanoparticles to deliver inactivated whole viral particles of ipnv as a vaccine , which expressed low antibody levels comparable to those induced by inactivated whole virus ( iwv ) vaccines suggesting that delivery of antigens using plga nanoparticles has the ability to induce humoral immune responses in vaccinated fish . although there are limited studies that categorically demonstrate the intracellular delivery of nanoparticle based vaccines in fish cells , rajeshkumar et al . were able to induce low level cytotoxicity ( tested in vitro ) using chitosan nanoparticle vaccines in asian sea bass vaccinated against vibriosis ( listonella anguillarum ) . table 4 shows that only a few studies have been carried out using nanoparticle based technologies to administer viral antigens in fish . in general , indications show that nanoparticle based vaccines have the potential to deliver antigens into different host cell compartments and thus that they can induce cellular and humoral immune responses in vaccinated fish . this approach involves antigen delivery systems that administer viral antigens into the extracellular compartments using the exogenous pathway ( figure 1 ) . this mode of antigen delivery ensures that the antigenic protein is preserved in its native structure while the virus is rendered nonreplicative using chemical or physical methods . given that inactivated whole virus ( iwv ) vaccines are nonreplicative , it follows that their antigens enter the host cells by the exogenous route and their processed peptides are presented to cd4 cells via the mhc - ii pathway . and , as such , several studies have shown upregulation of mhc - ii genes in response to vaccination using iwv vaccines [ 77 , 132 ] . in terms of cd4 t - cell differentiation , iwv vaccines have been shown to predominantly activate genes linked to the t - helper 2 ( th2 ) responses . for example , we showed upregulation of gata-3 , a transcription factor linked to activation of nave cd4 cells into th2 responses , when genes linked to activation of th1 and cd8 t - cell responses were downregulated . in this study , we showed a high correlation between gata-3 and antibody levels expressed against ipnv . in terms of antibody responses , iwv vaccines have been linked to high expression levels of igm and igt in vaccinated fish . in our studies , we showed a high correlation between postchallenge reduction of mortality and systemic igm levels , suggesting igm levels could serve as a correlate of protection for iwv vaccines . overall , these studies strongly suggest that iwv vaccines are to be considered as exogenous antigens , mainly inducing humoral immune responses . the basic principle for this vaccination strategy is that the gene encoding the antigenic proteins is isolated from the native virus and transferred into a heterologous vector that is nonpathogenic for propagation . table 6 shows the antigenic proteins identified for the major fish viral diseases and the different expression vectors used for propagation . in the case of vhsv and ihnv , production of subunit vaccines has focused on cloning the g - protein into heterologous vectors [ 136138 ] while , for viruses such as ipnv , the strategy has been to clone the entire outer capsid encoding the protective epitopes , instead of protein segments coding the neutralizing epitopes in heterologous vectors [ 103 , 139141 ] . subunit vaccines are nonreplicative and are delivered exogenously to host cells by the extracellular route ( figure 1 ) . similar to iwv vaccines , subunit vaccines induce humoral immune responses and upregulation of mhc - ii genes [ 139 , 142 ] , which is consistent with observations in higher vertebrates in which it has been shown that immune responses induced by subunit vaccines are mainly dependent on the mhc - ii pathway and that they elicit antibody responses . [ 144 , 145 ] showed a high correlation between reduction of viral rna and activation of cd4 markers in atlantic halibut ( hippoglossus hippoglossus l. ) immunized using a subunit vaccine for nodavirus . although cross presentation of exogenously processed peptides from endosomes into the cytosol has been reported in higher vertebrates [ 3 , 4 ] , there is no study demonstrating cross presentation of peptides processed from endosomes into the cytosol for subunit vaccines in fish . structural proteins of most viruses self - assemble to forms capsids in different expression systems that resemble the native virus structure in size and morphology and , hence , they are referred to as virus - like particles ( vlps ) . made vlps of nodavirus expressed in e. coli or spodoptera frugiperda ( sf21 ) insect cells that formed small , nonenveloped t = 3 quasi - symmetric particles [ 147 , 148 ] . they showed that the capsid of malabaricus grouper nervous necrosis virus ( mgnnv ) spontaneously self - assembled into vlps when expressed in sf21 cells infected with a recombinant baculovirus . these vlps were indistinguishable from the native virus particles by electron microscopy and the 3d structure of the vlps was resolved at 2.3 nm by cryomicroscopy . produced vlps that were devoid of the nucleoprotein but resembled the outer capsid of the native grass carp reovirus ( gcrv ) . however , in some cases vlps are formed from replication of surface particulate components that do not form the entire capsid , but they contain elements of the outer capsid that are immunogenic . these protein structures are called subviral particles ( svps ) . both vlps and svps table 7 shows the vlps , svps , and immature virus particles ( ivps ) made from different fish viruses . as shown in table 7 , different expression systems were used to make vlps , svps , and ivps for ipnv . given the similarity to their native viral capsids , this property was demonstrated by lai et al . who produced vlps for nnv expressed in e. coli and showed that nnv failed to infect the asian sea bass cells that were exposed to the vlps prior to infection , suggesting that the cell surface receptors were occupied by the vlp - epitopes blocking the wild type virus from entering the cells and thereby protected the cells from developing cytopathic effect ( cpe ) while control cells not exposed to vlps developed full cpe . liu et al . showed that vlps generated from nnv induced high antibody responses that lasted for more than five months , similar to those produced by the native wild type virus , which were correlated with long - term protection in vaccinated orange spotted grouper . produced vlps in e. coli for nnv that expressed high antibody levels , which were correlated with igm , mhc - ii , and cd4 levels in vaccinated fish . these observations suggest vlps induce the expression of cd4 responses through the mhc - ii pathways in a similar pattern to those induced by subunit vaccines in mammalia . the most explored strategies for the delivery of antigens using the intracellular route in fish vaccinology involve the use of live and dna vaccines . the use of dna vaccines in fish has undergone intense investigation in the last decades as a substitute of replicative antigens for live vaccines . although factors leading to higher performance of dna vaccines for rhabdoviruses compared to other fish viral families have not been elucidated , similar observations seen in higher vertebrates show that dna vaccines for rhabdoviruses are more protective [ 155 , 156 ] than some of the dna vaccines for other viral families . in general , replicative vaccines delivered via the intracellular route have been linked to activation of cellular and humoral immune responses in vaccinated fish , which makes these vaccines be more protective than nonreplicative vaccines delivered by the extracellular route . for antigens administered by the extracellular route , several antigen delivery strategies have been explored in fish vaccinology , which include the use of iwv , subunit , svp , vlp , and imp vaccines . in general , all exogenous antigens induce humoral immune responses . in terms of cellular immunity , exogenous antigens were linked to expression of mhc - ii and cd4 genes . however , new innovations such as the use of fusion protein and nanoparticles vaccines having the potential to deliver nonreplicative antigens into the cytosol are likely to induce ctl responses in vaccinated fish . the use of nanoparticle vaccines has attracted a lot of interest in the delivery of oral vaccines for fish production systems that require a boost vaccination when fish have been transferred in cages to the sea after prime immunization using parenteral vaccines at the freshwater stage . in general , iwv vaccines are superior to subunit vaccines given that they produce high antibody levels , which correlate with protection in vaccinated fish [ 81 , 157 ] . in addition , these vaccines have been shown to activate the expression of cd4 and th2 genes that correlate with high antibody levels consolidating the common notion that exogenous antigens stimulate humoral immune responses orchestrated by th2 cytokines . although we did not review the role of apc prime / activated b - cells in antigen uptake and presentation to cells of the adaptive immune system in detail given the limited studies carried out on this topic in fish vaccinology , we can conclude that the different antigen delivery systems explored in fish this far deliver their antigens into the intra- and extracellular compartments and that they activate either the cellular or humoral immune response or both depending on the route of antigen delivery . as pointed out by howarth and elliot , the most protective vaccines are those that stimulate both the cd4 + and cd8 + t - cells responses and , as such , replicative vaccines such as the live and dna vaccines that stimulate both the mhc - i and -ii pathways are likely to produce better protection in fish ( table 8) . so far only the ihnv - dna vaccine for use in atlantic salmon in canada is the only one licensed while live viral vaccines are feared to revert to virulence . hence , the use of iwv vaccines which accounts for the largest proportion of licensed vaccines is likely to continue dominating the vaccine industry in aquaculture [ 81 , 157 ] . therefore , the search for better antigen delivery systems that stimulate both cd4 + and cd8 + responses that have the potential to induce long - lasting protective immunity has to continue . overall , we anticipate that the synopsis of different antigen delivery systems presented here will shed new insights into the limitations and successes of the current immunization strategies used in fish vaccinology .

we have previously demonstrated that upa is overexpressed in pancreatic tumors . in an attempt to diagnose these tumors earlier , we sought to determine whether upa could be identified in endoscopic retrograde cholangiopancreatography obtained brushings in patients with malignant pancreatic and biliary strictures . secondarily , upa was measured in the serum of this patient population . upa overexpression was identified in the cytologic tissue in 8 of 11 patients ( 72.7% ) . serum analysis demonstrated a 2-fold higher concentration of upa in the pancreaticobiliary cancer patients ( 1.27 versus 0.56 ng / ml ; p = .0182 ) . also , upa overexpression correlated with serum levels ( p < .0001 ) . this study confirms that upa can be detected in the ercp cytologically obtained tissue and is frequently present in a higher concentration in the serum of pancreaticobiliary cancer patients . a larger sample size will be required to address its value as a sensitive marker for the diagnosis of pancreatic or biliary cancers .

pancreaticobiliary cancer remains a lethal disease where treatment continues to be a major oncologic challenge . most patients present with advanced disease in which survival is dismal . for patients diagnosed early with small tumors , surgery offers a chance of cure . in an attempt to diagnose these tumors at an early stage , many studies have evaluated the use of tumor markers , but so far , due to low sensitivity and specificity , the results have not been promising . the high metastatic potential of pancreatic cancer has led researchers to investigate its ability to invade the basement membrane and extracellular matrix ( ecm ) allowing it access to capillaries and lymphatics . one process that has been shown to be involved in invasion of most types of cancer is the urokinase - type plasminogen activator system . upa is a serine proteinase that is best known for catalyzing the conversion of inactive plasminogen to the active proteinase plasmin . upa and its receptor upar have been demonstrated to be involved in tumor invasion , growth , and metastasis . independently or through the activation of plasmin , upa can degrade ecm , activate matrix metalloproteinases , mediate the release of growth factors ( including transforming growth factor , fibroblast growth factor , vascular endothelial growth factor , insulin growth factor , tumor necrosis factor , and hepatocyte growth factor ) , stimulate cellular migration , induce chemotaxis , and promote angiogenesis [ 3 , 4 ] . since the 1980s when upa was postulated to have a role in tumor invasion , there have been multiple studies evaluating it as a prognostic marker of cancer . it was first studied in breast cancer where it has been shown as an independent prognostic marker for predicting survival second only to lymph node status . following the initial results in breast cancer , upa overexpression was shown to confer a worse prognosis in many other cancers including colorectal , esophageal , gastric , hepatocellular , prostate , sarcoma , and head and neck squamous cells among others [ 612 ] . the importance of the upa activator system has also been demonstrated in pancreatic cancer [ 1318 ] . the first study to show overexpression of upa in pancreatic cancer was by takeuchi et al . in 1993 . he demonstrated by immunohistochemical staining that 78% of pancreatic cancers overexpressed upa and this overexpression correlated with decreased survival . another study by cantero et al . in 1997 showed that concomitant overexpression of upa and its receptor upar correlated with shorter survival times . we detected upa overexpression in pancreatic intraepithelial neoplasia ( panin ) and histologically normal ducts / acini that are in the immediate vicinity of the tumor . upa was also found in the vessels of tumor stroma suggesting that upa is in circulation , and therefore should be detectable by serum analysis . it allows further evaluation through imaging of the ductal system and allows to sample pancreatic juice and brush biopsy cytology for further analysis . ercp has also been studied in small numbers to screen high - risk patients with a family history of pancreatic cancer , and it demonstrated that cytologic brushings showing dysplasia correlate with the pathologic specimen after resection [ 20 , 21 ] . despite combination of sampling with brush cytology , fine needle aspiration , and biopsy , the sensitivity of all three combined is only 62% . most practitioners perform only brush cytology which by itself has a sensitivity of only 30% in the diagnosis of pancreaticobiliary malignancies . we hypothesized that molecular markers such as upa may improve our ability to differentiate malignant from benign pancreaticobiliary strictures and assist us in planning therapeutic strategies . we performed a feasibility study of patients undergoing ercp for confirmed or suspected pancreaticobiliary cancers to determine whether upa overexpression could be identified from cytologic brushings . serum levels of upa were measured in this patient group and compared to healthy subjects . eleven patients with known or suspected pancreaticobiliary malignant strictures secondary to either pancreatic cancer or cholangiocarcinoma as determined clinically by radiologic studies were consented to have cytologic brushings obtained during ercp for evaluation of expression of upa . at ercp , one slide was routinely stained for h&e to identify the presence of epithelial cells and the other was stained for upa in a blinded fashion . presence and type of cancer were determined by biopsy and reviewed by a pathologist . reagents used in the immunohistochemical procedure including blocking solution , secondary antibody , and vectastain elite abc reagent were purchased as the r.t.u vectastain universal elite abc kit ( vector laboratories , burlingame , ca ) . color - development reagents were purchased as part of a dab substrate kit for peroxidase ( vector laboratories , burlingame , ca ) . the dab substrate kit contained the reagents required to make a working solution of 3,3-diaminobenzidine ( dab ) for staining tissue sections . upa expression in cytologic brushings obtained during ercp was evaluated by immunohistochemistry ( ihc ) . the upa1 primary antibody was used at a concentration of 20 g / ml . cells were blocked with normal horse serum ( 2.5% ) for one hour and incubated overnight at 4 celsius with the primary antibody . after gently rinsing the cells with triton - tbs , they were treated with the secondary antibody ( r.t.u . biotinylated universal antibody and anti - rabbit / anti - mouse igg made in horse ) , gently washed and treated with vectastain elite abc reagent . a slide of cytologic brushings was also stained by the same procedure , except that an irrelevant isotype igg was substituted for the primary antibody as a negative control . staining intensity for upa was classified as 03 + ( 0 absent ; 1 + , weak ; 2 + , moderate ; 3 + , strong ) . serum levels of upa were analyzed using an enzyme - linked immunosorbent assay ( elisa ) . correlation between ihc grade and serum levels of upa was estimated with the spearman rank correlation coefficient . all tests were carried forth using sas 9.2 , sas institute inc . , cary , nc . given n = 11 subjects per group and assuming exchangeability under the null hypothesis , the wilcoxon rank - sum test would be able to detect approximate shift alternatives of roughly 0.6 ng / ml , 1.3 ng / ml , and 1.7 ng / ml given 0.80 power and common standard deviations under the null hypothesis of no difference of 0.5 ng / ml , 1.0 ng / ml , and 1.9 ng / ml , respectively . 8 patients had pancreatic adenocarcinoma and 3 had cholangiocarcinoma . when immunohistochemistry for upa was performed on the cytology specimens , six of the eight patients ( 75% ) with pancreatic cancer ( figure 1 ) and two of the 3 patients ( 67% ) with cholangiocarcinoma overexpressed upa ( figure 2 ) . the two patients with pancreatic cancer who did not overexpress upa were being treated with chemotherapy and underwent ercp for biliary stent exchange . the mean serum upa in the 11 patients with cancer was 1.27 ng / ml ( sd = 1.54 ) . the mean serum upa in the 11 normal healthy individuals was 0.56 ng / ml ( sd = 0.16 ) . serum analysis demonstrated a 2-fold higher concentration of upa in the pancreaticobiliary cancer patients compared to the healthy controls ( p = .0182 ) . table 1 compares the level of upa staining in cytologic specimens to serum levels of upa for all 11 patients . the ihc grade for upa staining correlated with serum levels for upa ( r = 0.72 ; p < .0001 ) . a definitive diagnosis of cancer can be difficult in patients with pancreaticobiliary strictures with no obvious mass on imaging studies . we have previously shown that upa is an early event in the malignant transformation of pancreatic cancer . therefore we sought to determine the feasibility of determining upa expression in cytologic brushings of established malignant strictures to evaluate its potential as an adjunct to ercp in diagnosis of pancreaticobiliary malignancies . in our small sample , we found it is possible to identify overexpression of upa in cells obtained from brushings during ercp . upa overexpression ( 72.7% ) appears to be a marker for pancreatic or biliary cancers . although there is no current literature to show that chemotherapy directly inhibits upa synthesis , we have postulated that this treatment impedes growth , and thus decreases upa expression . the relationship between upa overexpression and cholangiocarcinoma has not been studied , but in two of the three ( 66% ) samples , the immunohistochemical analysis of the cytologic brushings showed overexpression . as ercp is an invasive technique with potential morbidity , we also looked at serum upa in our patient population , and it was elevated in patients with pancreaticobiliary cancer . the serum levels correlated with the ihc grade of upa in tissue ( p < .001 ) . serum upa levels have been evaluated in breast cancer by rha et al . , who showed blood upa levels correlated with those of tissue . because our study contains only a small number of patients , the value of serum upa as a tumor marker needs to be further evaluated in large numbers of patients including patients with chronic pancreatitis before it can be compared to the standard tumor markers such as ca19 - 9 . currently there are no established screening tests for pancreatic cancer . the united states preventive services task force ( uspstf ) has recommended against routine screening for pancreatic cancer based upon the lack of data demonstrating its clinical value in the general population . familial pancreatic cancer ( fpc ) accounts for up to 10% of patients with pancreatic cancer . iii , and patients with panin iii have been considered for pancreatectomy by some groups . surveillance by ercp and endoscopic ultrasound is performed in an attempt to identify patients with fpc who have developed panin , but the diagnosis is difficult . evaluating upa overexpression from ercp obtained cytologic brushings may have potential application in screening high - risk patients by identifying either panin or invasive pancreatic cancer . although ercp arguably remains the golden standard for pancreaticobiliary evaluation , there is a risk of complications associated with ercp including pancreatitis ( 5.4% ) . endoscopic ultrasound ( eus ) with eus - guided fna has emerged as an extremely sensitive and specific method of diagnosing pancreatic cancer and can detect small lesions more effectively than conventional imaging . for the diagnosis of pancreatic cancer , as we have shown that upa staining can be performed on brush cytology , it can be done on fna specimens as well and may improve diagnostic accuracy . as targeted therapy has stepped to the forefront of cancer research , molecular markers have moved from their traditional roles of diagnosis and staged to possible aims of treatment . already upa has been evaluated as a potential target for treatment to decrease the invasive and metastatic activity of pancreatic tumor cells . evaluated an anti - upar monoclonal antibody in mice and found that it significantly decreased tumor growth , hepatic metastases , and retroperitoneal invasion . small molecule inhibitors of upa have been evaluated in a fibrosarcoma model in mice resulting in decreased metastases and prolonged survival . these results suggest that detecting overexpression of upa in pancreatic cancers may not only be prognostic and diagnostic but may also direct treatment in the future . a serious limitation of the current study is its small sample size and lack of an adequate control group precluding meaningful analysis . in this study , a control group of healthy subjects for the analysis of upa in the serum was obtained . there are obvious ethical issues in obtaining brushing from healthy volunteers and it would appear more suitable to utilize patients with benign pancreatic and biliary strictures . we elected to exclude potential control groups such as those presenting with obstructive jaundice from benign pancreaticobiliary strictures . the possibility that some of these patients harbored occult malignancy would be difficult to discern without long term follow up . even in patients with choledocholithiasis , there may be a potential for the associated inflammatory process to elevate upa levels . thus , we performed this feasibility study in patients with known or suspected pancreaticobiliary malignancies by brush cytology to corroborate our prior findings in resected pancreatic cancer specimens . we performed a feasibility study to determine whether upa overexpression could be identified from cytologic brushings of patients undergoing ercp for confirmed or suspected pancreaticobiliary cancers . serum levels of upa were measured in this patient group and correlated with upa overexpression . larger studies involving all forms of pancreaticobiliary pathology need to be performed before upa overexpression can be used either as a serum or a cytological tumor marker , especially those with no identifiable masses . also comparison with other tumor markers , that is , ca 19 - 9 and cea will be required to substantiate the role of upa as a diagnostic or prognostic tumor marker .

as embarrassment is a known obstacle to condom acquisition , selling condoms from physically inaccessible places that require personnel assistance constitutes a barrier to access . this study investigates the extent of this barrier in the bronx , a high hiv / sti prevalence county of new york . 75 of 320 listed bronx pharmacies were sampled via computer randomization . investigators coded condom placement and physical accessibility within these pharmacies and 140 surrounding stores . 91% of sites sold condoms . in 82% , condoms could not be accessed without assistance . condoms were physically inaccessible in venues most encountered in the community : grocery stores versus pharmacies ( or=15 ; 95% ci , 548 ) , independent versus chain pharmacies ( or=32 ; 95% ci , 6235 ) . they were physically inaccessible more in the lowest ses / highest hiv prevalence areas versus the highest ses / lowest hiv prevalence areas ( or = 4.3 , 95% ci , 1.117 ) . findings can inform efforts to increase accessibility of condoms , distribute condoms in alternative settings , and prompt similar investigations in other high - risk communities .

new york city has one of the highest reported human immunodeficiency virus ( hiv ) and acquired immunodeficiency syndrome ( aids ) rates in the united states , more than triple the national average . approximately one quarter of these hiv - positive individuals live in bronx county ( known as the bronx ) , with areas reaching an hiv / aids prevalence of greater than 2% . relative to the united states , new york state , and the other counties in new york city , the bronx also has a disproportionately higher prevalence of sexually transmitted infections ( stis ) and teen pregnancy . in 2005 , the teen pregnancy rate in the bronx was 140% higher than that of new york city , and the chlamydia case rate was more than double the national rate [ 3 , 4 ] . therefore , condom access , as a means of hiv / aids , sti , and teen pregnancy prevention , is a high priority in this community . although the determinants of condom use are complex and multifactorial , prior work has established that preparedness by possession of condoms is one strong predictor of use [ 57 ] . however , it has been well documented that embarrassment during the purchase of condoms remains a barrier to acquisition and use among young adults and women [ 810 ] . condoms have been described as a socially sensitive product ; a real or imagined social presence around such products creates embarrassment , which in turn affects purchasing behavior . by the same token , research has shown that adolescents prefer to purchase condoms from places where they are clearly visible and quickly purchased , even when given a cheaper option [ 11 , 12 ] . in 2004 , brackett reported that one of several strategies employed by young adults to reduce the embarrassment of condom purchase is to avoid asking for help or for location of condoms within a store . yet the popular press has reported that condoms are often stored in locked cases in drug store aisles . in 2006 , the washington post reported that one national pharmacy sold condoms in locked cases in 22 of 50 of their washington , dc stores . requiring assistance from store personnel to purchase condoms makes the sale more public , lengthy , complicated , and potentially embarrassing , thereby creating a barrier to access . the theory of planned behavior proposes that intentions and perceived control over behavior predict behavioral performance . part of this model suggests that one 's intentions and behavior in performing a given act are related to the control and difficulty one perceives in performing that act . therefore , among individuals intending to acquire condoms , a setting in which those vulnerable to embarrassment are obliged to ask for assistance creates a structural disincentive with a potentially negative outcome . while condom purchase preferences and behavior have been explored , condom placement within physical reach has received narrow attention . in 2006 , scott - sheldon et al . they examined condom placement relative to the proximity of other embarrassing products , and the positive / negative associations such placement fosters . in this study , two thirds of condoms were positioned behind or next to the checkout counter , though the proportion behind the counter and requiring assistance is not specified . klein et al . investigated several aspects of condom availability , including condom visibility and placement within store aisles . this paper found that the majority of drug stores displayed condoms in the aisles , while the majority of private grocery and convenience stores kept them behind the counter . however , the limited data on this barrier have not been validated in a community of such significant risk as the bronx , nor has condom accessibility through the lens of mandatory interaction been researched . the goal of this paper was to describe the prevalence of structural barriers to condom access in the bronx , specifically physical inaccessibility , defined as the sale from locked cases or behind store counters requiring interaction with store personnel . we hypothesized that physical inaccessibility of condoms would be found in the majority of the sites selling condoms in the bronx . in addition , we sought to determine whether or not the prevalence of these structural barriers was associated with the socioeconomic status ( ses ) of the health districts within the bronx . the bronx is a densely populated urban community of approximately 1.4 million people , with commercial areas in close proximity to or interspersed within residential neighborhoods . one - third of bronx residents live below the federal poverty line , though socioeconomic status varies widely between the seven designated health districts of the county . health data for the bronx are listed in table 1 [ 2 , 3 ] . the bronx yellow pages lists 320 pharmacies ( 80 of them are chain pharmacies ) and approximately 900 grocery stores in the 20 zip codes in the county . as pharmacies are largely represented in commercial areas , they were used as focus points for sampling of the two nearest grocery stores or supermarkets . if the pharmacies were so closely clustered that two different grocery stores per pharmacy were not encountered , we would survey the maximum number of stores within walking distance of those pharmacies , operationalized as within 5 blocks . staff entered each selected location in the manner of an ordinary customer and observed the location of condom placement . if condoms were not visible , the representative asked the store clerk whether condoms were sold and if so , where in the store they were located . on leaving the store , the representative provided the following data on a coding form developed by the investigators : the type of store , zip code , availability of condoms , specific condom location within the store , whether personnel assistance was needed to obtain condoms prior to purchase , and the number of interactions required prior to purchase . information regarding ses , hiv , teen pregnancy , and sti prevalence was obtained from the new york city department of health ( doh ) , which provides data for the seven health districts and the 20 zip codes in the bronx . each site was assigned to one of these districts based on its zip code , to determine whether the manner of condom sales correlated with the health statistics of that region . we determined that 75 pharmacies and 90 grocery stores needed to be sampled based on a presumed point estimate of 50% of sites keeping condoms physically out of reach , in order to achieve a 95% confidence interval of 10% around this point estimate . to even the distribution of grocery stores and achieve the minimum sample size , we surveyed the two closest grocery stores to each pharmacy , aiming for 150 stores and 225 total sites . data organization and analysis were performed using epi info version 2000 ( epiinformatics , doraville , ga ) . we sampled a total of 215 sites , 75 pharmacies and 140 stores . because of the close proximity of some of the pharmacies , condoms were sold at 195 ( 91% ) of these sites ( table 2 ) . condom purchase required assistance from site personnel at 160 of the 195 sites selling condoms ( 82% ; 95% ci : 76%87% ) . condoms were more likely to be kept out of reach in the 115 grocery stores ( 96% ) compared to the 75 pharmacies ( 60% ) selling them ( or = 15 , 95% ci : 548 ) . condoms were also kept out of reach in more independent pharmacies ( 78% ) compared to chain pharmacies ( 10% ) ( or = 32 , 95% ci , 6235 ) . four sites required assistance from two or more personnel prior to condom purchase whereas the remainder required assistance from one person prior to purchase . we stratified for the ses / hiv prevalence of each bronx health district relative to the manner of condom distribution . in health districts 1&2 , with the greatest hiv , sti , and teen pregnancy prevalence and the greatest number living below the federal poverty level , condoms were kept out of reach in 90% out of 55 sites sampled ( table 4 ) . in health districts 6&7 , with the lowest hiv , sti , and teen pregnancy prevalence , and the lowest number living below the federal poverty level , condoms were kept out of reach in 70% out of 30 sites ( table 4 ) . condoms were more likely to be kept out of reach in the lowest ses / highest infection and teen pregnancy prevalence districts compared to the highest ses / lowest infection and teen prevalence districts ( or = 4.3 , 95% ci : 1.117 ) . although 91% of stores surveyed in the bronx sold condoms , the vast majority ( 82% ) sold them in locked cases or behind sales counters . in almost all of the convenience stores and in 78% of independent pharmacies , consumers required assistance from site personnel in order to purchase condoms . thus , condom accessibility was poor in the sites most commonly encountered ; most bronx stores are convenience stores and 3 out of every 4 pharmacies are small and privately owned . since the low - ses districts also have the highest rates of hiv , stis , and teen pregnancy , barriers to condom access are of particular concern . the study conducted by klein et al . in 2001 found that almost two - thirds of adolescents who purchased their condoms did so only from pharmacies . the purchasing preferences reported in klein 's work highlight the potential relevance of our finding that the majority of bronx pharmacies sold condoms from behind the counter . our findings also differ from those of scott - sheldon et al . , who report on 66% of condoms being sold from behind the counter or while we do not know what proportion of these condoms was sold exclusively from behind the counter , we found that significantly more condoms were sold in this manner , and this practice was more common in a higher - risk community . this reinforces the negative implication of selling the vast majority of condoms in a manner that obligates assistance from a stranger . a limitation of our study is that condoms can be acquired from sources other than stores , such as high schools or community health centers . there were no data available on the number or rate of condom distribution in local high schools . however , students who have previously reported on condom availability in schools have found them to be inadequate in supply . moreover , distribution in schools does not benefit those who are older or adolescents who are not in school . a further limitation was inadequate information about where individuals in our region actually acquire their condoms . we were unable to quantify whether or not individuals seek condoms from free sources and the number of such sites in the region . though we do not know whether young adults usually purchase condoms from chain pharmacies , where condoms are usually sold with open access , those pharmacies represent the minority of the total in our community and may be difficult for some to access . finally , although the small sample size led to wide confidence intervals around the odds ratios , the results are nonetheless significant as the lower limit of the confidence interval was greater than one . although not confirmed , it has been suggested that the fear of theft is one reason why condoms are sold from locked cases or behind store counters . therefore , efforts to change the sales practices in smaller stores with less financial stability may be unrealistic . however , this does not change the fact that access to condoms is critical , especially in areas which had the highest sti rates ; that there may be a structural barrier to condom purchase in these communities represents an incongruity between what is most needed and what is available . to this end the vast majority of sites surveyed in the bronx sold condoms in locked cases or behind sales counters . failure to make condoms readily accessible for unmediated purchase may disproportionately deter adolescents and women from acquiring condoms and ultimately from adopting recommendations for condom use . this information can prompt similar investigations in other high - risk communities and inform public health efforts to increase condom accessibility and the distribution of free condoms in public and practice settings .

cosmetic expectations have increased with time and current trends speak volumes about gingival esthetics and smile designing . gingival pigmentation especially on the labial aspect of anterior teeth has become an important component of general esthetics . various physiologic and pathologic factors cause gingival pigmentation . the existing indices do not deal with the etiology , extent and severity of gingival pigmentation . hence , we propose a new classification and index for gingival pigmentation to assess the treatment needs for the patient .

the deposition of coloring matter , coloration , or discoloration by a pigment pertaining to the gingiva is gingival pigmentation . our knowledge about gingival pigmentation ( gp ) and their etiologies has increased enormously over the past decade . the racial - physiological pigmentation is not of medical concern , but may at times be of esthetic concern . light brown to black pigmentation may be physiologic or racial in healthy colored - skinned individuals , whereas the same oral pigmentation in caucasians may be abnormal . the intensity of pigmentation is frequently altered by physical , chemical , and hormonal factors . the normal physiologic color of gingiva is coral pink or salmon pink , with physiological variations of melanin pigmentation . it is a nonhemoglobin - derived brown pigment produced by melanocytes and is also a powerful cationchelator . they work independent of the surrounding epithelial cells and behave as unicellular exocrine gland , convert tyrosine to melanoprotein ( melanin ) , which is transferred to keratinocytes by way of melanosomes . benign and malignant lesions , cultural intentional tattooing , drugs , heavy metal ingestions - poisonings , iatrogenic , smoking , and systemic problems can all cause gp . we propose a new improved classification for gp and pigmented lesions , [ table 1 ] based on our experience gained from previous classification systems . we also propose a new index for gp [ table 2 ] to assess the treatment needs for such patients . classification is a mode to arrange the disease(s ) and or condition(s ) in cohorts , to help communicate among the professionals and to study them . classification systems are necessary in order to provide a rational and scientific framework to study the etiology , pathogenesis , and treatment of diseases and to plan the treatment in an orderly fashion . in addition , such systems help us prioritize and organize the health care needs of the patients . they should have clear categories that make it easy to make a decision as to which category a condition should fit into . an index should be valid possessing , in statistical terms , good sensitivity , and specificity . furthermore , the ideal index should also be reliable and reproducible with no variations as a result of internal flaws within the index and give the same result if the condition being assessed has not changed . it should also be able to detect small changes and should be able to measure changes in either direction , that is , whether the condition being measured improves or deteriorates . finally , it should be acceptable and free of discomfort for patients or subjects , with the length of time to complete any assessment and examination taken into consideration . oral pigmentation index ( dopi):this index of oral pigmentation is the commonly used index due to its simplicity and ease of use . the scores are as follows : no clinical pigmentation ( pink - colored gingiva)mild clinical pigmentation ( mild light brown color)moderate clinical pigmentation ( medium brown or mixed pink and brown color)heavy clinical pigmentation ( deep brown or bluish black color ) melanin index : this index has classified pigmentation as follows : no pigmentationone or two solitary unit(s ) of pigmentation in papillary gingiva without the formation of a continuous ribbon between solitary unitsmore than three units of pigmentation in papillary gingiva without the formation of a continuous ribbonone or more short continuous ribbons of pigmentationone continuous ribbon including the entire area between canines melanin pigmentation index : takashi et al . have proposed another index to measure gingival melanin pigmentation . the index is as follows : score 0 : no pigmentationscore 1 : solitary unit(s ) of pigmentation in papillary gingiva without extension between neighboring solitary unitsscore 2 : formation of continuous ribbon extending from neighboring solitary units this index is not equipped to describe the degree of melanin pigmentation.gingival pigmentation index : score 0 : absence of pigmentationscore 1 : spots of brown to black color or pigments.score 2 : brown to black patches but not diffuse pigmentationscore 3 : diffuse brown to black pigmentation , marginal , and attached oral pigmentation index ( dopi ) : this index of oral pigmentation is the commonly used index due to its simplicity and ease of use . the scores are as follows : no clinical pigmentation ( pink - colored gingiva)mild clinical pigmentation ( mild light brown color)moderate clinical pigmentation ( medium brown or mixed pink and brown color)heavy clinical pigmentation ( deep brown or bluish black color ) no clinical pigmentation ( pink - colored gingiva ) mild clinical pigmentation ( mild light brown color ) moderate clinical pigmentation ( medium brown or mixed pink and brown color ) heavy clinical pigmentation ( deep brown or bluish black color ) this index has classified pigmentation as follows : no pigmentationone or two solitary unit(s ) of pigmentation in papillary gingiva without the formation of a continuous ribbon between solitary unitsmore than three units of pigmentation in papillary gingiva without the formation of a continuous ribbonone or more short continuous ribbons of pigmentationone continuous ribbon including the entire area between canines one or two solitary unit(s ) of pigmentation in papillary gingiva without the formation of a continuous ribbon between solitary units more than three units of pigmentation in papillary gingiva without the formation of a continuous ribbon one or more short continuous ribbons of pigmentation one continuous ribbon including the entire area between canines melanin pigmentation index : takashi et al . the index is as follows : score 0 : no pigmentationscore 1 : solitary unit(s ) of pigmentation in papillary gingiva without extension between neighboring solitary unitsscore 2 : formation of continuous ribbon extending from neighboring solitary units score 0 : no pigmentation score 1 : solitary unit(s ) of pigmentation in papillary gingiva without extension between neighboring solitary units score 2 : formation of continuous ribbon extending from neighboring solitary units this index is not equipped to describe the degree of melanin pigmentation . gingival pigmentation index : score 0 : absence of pigmentationscore 1 : spots of brown to black color or pigments.score 2 : brown to black patches but not diffuse pigmentationscore 3 : diffuse brown to black pigmentation , marginal , and attached score 0 : absence of pigmentation score 1 : spots of brown to black color or pigments . score 2 : brown to black patches but not diffuse pigmentation score 3 : diffuse brown to black pigmentation , marginal , and attached in our view , all the aforementioned indices seem to lack the capacity to relate various aspects of gp . moreover , other gingival - pigmented lesions are beyond their scope , as they were intended only for racial pigmentation . an index quantitatively reflects the clinical state or conditions using set of criteria on a graduated numerical scale whereby one can easily define , describe , distinguish , compare , and analyze the status of an individual or a group with reference to that state and/or conditions . dummett in 1944 noted that some dark complexioned individuals possessed perfectly pink gums devoid of any melanogenous pigmentation . in our view , mild shades of gp may require esthetic treatment in light - skinned individuals but may not have esthetic concern in dark - skinned subjects . in our proposed index , [ table 2 ] 0 - 3 is the range available to record the gingival color and its variation within physiological limits . a clinician may recommend a depigmentation procedure when the patient scores 1 - 2 of our index and has up to class 2 of liebart and deruelle . smile line classification , which is as follows : class 1 : very high smile line - more than 2 mm of the marginal gingiva visibleclass 2 : high smile line - between 0 and 2 mm of the marginal gingiva visibleclass 3 : average smile line - only gingival embrasures visibleclass 4 : low smile line - gingival embrasures and cemento - enamel junction not visible . class 1 : very high smile line - more than 2 mm of the marginal gingiva visible class 2 : high smile line - between 0 and 2 mm of the marginal gingiva visible class 3 : average smile line - only gingival embrasures visible class 4 : low smile line - gingival embrasures and cemento - enamel junction not visible . the classification of gp and the proposed new index should provide a workable framework upon which future studies can be performed to develop effective managements for this complex group of diseases . it is expected that as our knowledge progresses ; future revisions to the classification system will be needed . , the present proposed index represents the most comprehensive of all the indices that evaluate the gp and other pigmented lesions . it takes into account the esthetic aspects , that is , the presence of melanin pigmentation in the esthetic anterior smile region and the severity of gp ; it also considers the etiology of various other pigmented lesions in the gingiva . hence , we believe that it will be a useful tool for both clinicians as well as periodontal epidemiologists . due to clarity and simplicity of the proposed index , this classification can be applied even by nave professionals . the broad implementation of this classification and index may facilitate the comparison of gp across the globe and help esthetic management of such presentations . the baseline data obtained by using the parameters of our index can help in studying the prevalence of gp and pigmented lesions .

erythromelalgia is a rare neurovascular pain syndrome characterized by a triad of redness , increased temperature , and burning pain primarily in the extremities . erythromelalgia can present as a primary or secondary form , and secondary erythromelalgia associated with a myeloproliferative disease such as essential thrombocythemia often responds dramatically to aspirin therapy , as in the present case . herein , we describe a typical case of a 48-year - old woman with secondary erythromelalgia linked to essential thrombocythemia in the unilateral hand . as this case demonstrates , detecting and visualizing the hyperthermal area through infrared thermography of an erythromelalgic patient can assist in diagnosing the patient , assessing the therapeutic results , and understanding the disease course of erythromelalgia .

a 48-year - old woman visited our pain clinic for 2-year episodes of the right hand and wrist pain with a pain score of 9 - 10/10 by vnrs ( verbal numeric rating scale ; 0 with no pain , 10 with maximal pain ) . each time she had been diagnosed with different conditions , such as rheumatic arthritis , raynaud 's phenomenon , and other musculoskeletal diseases . they prescribed painkillers such as acetaminophen , tramadol , pregabalin and even alprazolam along with various physical therapies , but her pain became worse so that she could not even do her house work . the pain started with a sudden onset of " burning " as she described it . her vital signs including blood pressure were normal and she had no co - existing diseases such as hypertension or diabetes . electromyography and nerve conduction studies of both upper extremities were normal and showed no evidence of peripheral neuropathy . infrared thermography studies were performed to assist in understanding the patient 's disorder . in the resulting images , the affected areas of her upper extremities appeared red , indicating a higher temperature , while other areas appeared yellow or green , indicating a lower temperature ; this visualization also revealed vascular markings in the affected hand . the difference in temperature for the region of interest ( roi ) was 2.35 ( 35.37 vs 33.02 ) at the most differentiable area in the third and fourth fingers ( fig . laboratory tests were performed including cbc ( complete blood count ) with differential and platelet counts . initial red blood cell count was 4.84 10/l ; white blood cell ( wbc ) count was 8.3 10/l ; hemoglobin level was 13.8 g / dl ; hematocrit was 41.7% , and platelet count was 1,131,000/mm . prothrombin time ( pt ) and partial thromboplastin time ( aptt ) were 11.3 seconds and 31.2 seconds , respectively . erythrocyte sedimentation rate ( esr ) , rheumatoid factor ( rf ) , and antinuclear antibody ( ana ) screening data were within the normal range . we treated her with 500 mg of aspirin per day , and transferred her to a hematologist for further evaluation . she was diagnosed with essential thrombocytosis after a bone marrow biopsy and aspiration study , and chemotherapy was started with anagrelide while continuing to administer a low dose of aspirin ( 100 mg per day ) . a month later , her platelet count measured 340,000/mm , and her symptoms dramatically disappeared with a pain score of 1/10 ( vnrs ) . the temperature had decreased in the affected areas , where the color was now yellow ; the color boundary between the affected area and the remaining areas was negligible , and the temperature was similar to that of the unaffected arm . primary erythromelalgia , also known as weir mitchell 's disease , is an autosomal dominant disorder , and it has recently been accepted as a channelopathy caused by mutations in the voltage - gated sodium channel -subunit nav 1.7 encoding gene ( scn9a ) , hich is selectively expressed within the nociceptive dorsal root ganglion and sympathetic ganglion neurons . primary erythromelalgia is the first human disorder that may serve as a model of the association between an ion channelopathy and chronic neuropathic pain . secondary erythromelalgia can result from a number of diseases such as myeloproliferative disorders ( i.e. pv , et ) , hypercholesterolemia , autoimmune disorder , small fiber peripheral neuropathy , fabry 's disease , mercury poisoning , mushroom poisoning , sciatica and some medications including bromocriptine , verapamil and ticlopidine . diagnosis is mostly based on the clinical picture because there is no confirmatory diagnostic test . the classic description of erythromelalgia is red , painful , warm hands or feet brought on by warming or hanging the limb downward , and relieved with cooling and elevation of the affected area . secondary erythromelalgia often predate myeloproliferative disorders by a median of 2.5 years , thus a blood test should be serially performed once erythromelalgia is diagnosed . abnormal cbc data such as elevated platelets confirms secondary erythromelalgia [ 6 - 8 ] . most patients are subjected to polypharmacy in an attempt to manage pain if it is so severe that it interferes with daily living . medications such as opioids , gabapentin , lidocaine patches , benzodiazepines and nonsteroidal anti - inflammatory drugs ( nsaids ) are used frequently ; however , in most cases , these treatments either have no proven efficacy or provide only limited relief . various nerve blocks including epidural blocks and sympathetic blockades have been introduced to treat erythromelalgia unresponsive to non - invasive therapies , resulting in considerable efficacy , but no treatment is consistently effective in the management of erythromelalgic patients . for secondary erythromelalgia , treatment of the underlying disorder secondary erythromelelgia is prevalent in 3% to 65% of patients with myeloproliferative disorders , especially polycythemia vera and essential thrombocytosis . . pathological signs presenting with secondary erythromelalgia linked to thrombocythemia include arteriolar intimal proliferation with thrombotic occlusions secondary to platelet aggravation . some researchers suggest that both hypoxia and hyperemia occur in the disease process with high levels of arteriovenous shunting in the extremities . secondary erythromelalgia associated with myeloproliferative disease can be dramatically alleviated with high - dose aspirin therapy . aspirin prevents platelet aggravation through an irreversible inhibition of cyclooxygenase ; the effect of a single dose of 500 mg aspirin usually lasts for about three days . this specific long - lasting effect of a single dose of aspirin is pathognomonic and can be used as a diagnostic test in secondary erythromelalgia linked to myeloproliferative disease . a regular low dose of aspirin can also treat erythromelalgia and be a safe method to prevent erythromelalgia . infrared thermography can be used in the diagnosis and assessment of therapeutic results for erythromelalgia . thermography of an erythromelalgic patient may reveal increased temperature in the affected area and the thermal changes can be linked to the relief of symptoms as in our present case . thompson et al . had reported thermographic findings for a case of primary erythromelalgia with increased skin temperature in the affected area . primary erythromelalgia often presents as bilateral and symmetric erythro - hyperthermic congestion in the extremities , while secondary aspirin - responsive erythromelalgia linked to myeloproliferative disease has unilateral or bilateral effects but in an asymmetric area . in our case , the thermography showed a hyperthermal area specifying vascular markings in the unilateral upper extremity , which may suggest an infective or inflammatory disease like vasculitis . however , we could rule out vasculitis or phlebitis due to her normal range in inflammation markers such as her wbc counts and esr . erythromelalgia can be confused with complex regional pain syndrome ( crps ) because crps may also present severe burning pain and/or erythema with thermal change which can be present in thermography . however , these symptoms in patients with crps are often unilateral and can be proximal while those in erythromelalgic patients are primarily distal and symmetric if not associated with myeloproliferative diseases . in thermography , crps can have increased or decreased thermal areas while the affected area of erythromelalgia is usually increased in temperature . in addition , symptoms triggered by warming of the affected area and relieved by cooling and elevation are less common with crps . in this case , we saw a typical case of secondary erythromelalgia associated with thrombocythemia that was treated successfully with aspirin . in conclusion , pain clinicians should suspect erythromelalgia when seeing a patient with a triad of redness , pain and elevated temperature in the extremities . infrared thermography can help in diagnosing and understanding the disease course of erythromelalgia by presenting the affected area . the symptom of secondary erythromelalgia with myeloproliferative disorders , such as essential thrombocytosis can be alleviated with aspirin as in this case .

leaflet escape of prosthetic valve is rare but potentially life threatening . it is essential to make timely diagnosis in order to avoid mortality . transesophageal echocardiography and cinefluoroscopy is usually diagnostic and the location of the missing leaflet can be identified by computed tomography ( ct ) . emergent surgical correction is mandatory . we report a case of fractured escape of edward - duromedics mitral valve 27 years after the surgery . the patient presented with symptoms of acute decompensated heart failure and cardiogenic shock . she was instantly intubated and mechanically ventilated . after prompt evaluation including transthoracic echocardiography and ct , the escape of the leaflet was confirmed . the patient underwent emergent surgery for replacement of the damaged prosthetic valves immediately . eleven days after the surgery , the dislodged leaflet in iliac artery was removed safely and the patient recovered well .

possible causes of mechanical prosthetic valve dysfunctions include obstruction due to valvular thrombosis or pannus formation or regurgitation of valvular or paravalvular origin . in case of regurgitation , major concern is paravalvular leakage , because mechanical prosthetic valves are proven to be durable and valvular regurgitation due to structural valve deterioration is extremely rare . we report a sudden leaflet dislocation of a edward - duromedics mitral valve ( baxter healthcare corp . , cleveland , ms , usa ) corrected by the prompt diagnosis followed by an emergency operation . a 72-year - old woman visited emergency department as she suddenly experienced chest pain with resting dyspnea [ new york heart association ( nyha ) functional class iv ] while washing dishes at home . she underwent mitral valve replacement 27 years ago , at the age of 45 , due to mitral stenosis . mitral valve replacement was done with edwards - duromedics 29 mm mitral valve ( baxter healthcare corp . , apart from valvular heart disease , the patient was on medication for diabetes mellitus and hypertension as well as paroxysmal atrial fibrillation . she remained to be the status of nyha functional class ii and anticoagulation with coumadin was appropriately done ( international normalized ratio 2 - 2.5 ) . the recent transthoracic echocardiography ( tte ) done 4 month ago confirmed normal left ventricular systolic function [ ejection fraction ( ef ) : 69% ] and well functioning of prosthetic mitral valve with mild pulmonary hypertension [ right ventricle systolic pressure ( rvsp ) = 35 mmhg ] and mild tricuspid regurgitation . on physical examination , her vital signs were as follows ; blood pressure 69/51 mmhg , pulse rate 130/min , body temperature 36.7 with respiratory rate 35 breath / min . 1 ) . complete blood count showed white blood cell count of 9820/l , hemoglobin of 9.8 g / dl , and platelet of 386000/l . other labs showed elevated creatinine ( 1.12 mg / dl , estimated glomerular filtration rate 50 ml / min/1.73 ) and slightly elevated cardiac enzymes ( creatine kinase 555 iu / l , creatine kinase - mb 22.29 iu / l , troponin t 0.016 ng / ml ) with elevated n - terminal prohormone of brain natriuretic peptide level of 832 pg / ml . arterial blood gas analysis showed oxygen pressure of 97 mmhg , carbon dioxide pressure of 27.4 mmhg , with ph of 7.353 with oxygen mask of 10 l. after central line through right jugular vein was inserted , central venous pressure was 1 mmhg . the patient was intubated as she became intolerably tachypneic and progressively hypoxic . to evaluate the cause of the shock and acute decompensated heart failure , bed side tte was performed and revealed normal sized left ventricle ( end diastolic dimension : 44 mm ) and hyperdynamic left ventricular systolic function ( ef : 80% ) without regional wall motion abnormality . moderate tricuspid regurgitation ( grade iii ) and severe pulmonary hypertension ( right ventricular systolic pressure 75 mmhg ) with plethora of inferior vena cava were demonstrated . on two - dimensional ( 2d ) echocardiogram , there was suspicious finding of single prosthetic mitral leaflet , but details of the mitral leaflet morphology and color doppler were not sufficient for evaluating the prosthetic mitral valve function due to tachycardia and poor echo window . however , elevated mean diastolic pressure gradient ( 10 mmhg ) across the prosthetic mitral valve without prolongation of pressure half time ( 54 ms ) and low velocity of mitral regurgitation ( mr , 4 m / s ) , and rapid declined in mr velocity suggested existence of severe mr ( fig . the diagnosis of acute severe mr due to escape of prosthetic valve leaflet with embolization was made and the patient immediately went through emergency operation to surgically correct dysfunctions of previously replaced mitral valve . when mitral valve was exposed , there was one leaflet missing without evidence of paravalvular dehiscence and pannus or thrombus formation ( fig . tte after the surgery demonstrated well functioning bioprosthetic mitral valve with decreased tricuspid regurgitation ( grade i ) and resolution of pulmonary hypertension ( rvsp 39 mmhg ) . in an attempt to localize the missing leaflet , computed tomography ( ct ) was done , and a plate like metallic density in infra - renal abdominal aorta was noted ( fig . the remaining fragment of leaflet in infra renal abdominal artery was removed 11 days after bioprosthetic valve replacement . the abdominal aorta was vertically dissected 7 cm , and the fragment was safely removed ( fig . the patient developed mild fever after the surgery but recovered well and was discharged 37 days after the surgery . leaflet escape of bi - leaflet mechanical prosthesis has been reported ( tekna and duromedics ) to be extremely rare.1 - 5 ) the leaflet escape is reported to happen more frequently in mitral than aortic positions . a few factors have been reported to account for material deterioration.6 ) the cavitation , which is the rapid formation of vaporous microbubbles in a fluid by a local reduction of pressure below the vapor pressure7 ) is recognized as the most contributing factor to the series of valve failure in edward - duromedics prosthesis . other factors identified are asymmetric closure with local stresses , inadequate compliance of calcified sewing ring , clustered microporosity of the pyrolytic carbon and surgical mishandling.6 ) the time of leaflet escape varies from 19 days6 ) to 12 years8 ) after the implantation of the mitral valve . the clinical presentation is usually acute pulmonary edema with cardiogenic shock as the result of acute valvular incompetence.1 - 5 ) other causes of the clinical symptoms , such as myocardiac infarction , para - valvular leak , thrombosis of the prosthetic valves , malignant arrhythmia and pulmonary embolism should be considered . tte is usually not helpful for the diagnosed as it may be mis - interpretated as obstructed closure of the prosthetic valve , paravalvular leak or thrombosis.9 ) transesophageal echocardiography ( tee ) is diagnostic most of times . cineflouroscopy may role as non invasive diagnostic tool to determine leaflet escape from valve thrombosis.9)10 ) it is acknowledged that timely diagnosis and emergent surgical replacement of the prosthetic valve is most important.11 ) there are a few cases of mitral leaflet escape reported in korea.12 - 14 ) as a patient deteriorates with symptoms of acute heart failure with unstable vital signs , in most of the reported cases , an emergent operation is performed with tte finding of acute valvular dysfunction . therefore , an exact diagnosis of leaflet escape is made during the surgery , except there was a case reported by kim et al.13 ) which the diagnosis of a leaflet escape was made before an emergency operation by using fluoroscopy . in our case , although the images of tte were not sufficient for evaluating the exact mitral valve morphology and function , a single mitral leaflet was suspicious on 2d echocardiogram . in addition to the ambigious 2d images of single mitral leaflet , elevated mean diastolic pressure gradient with low velocity of mitral regurgitation , we could make diagnosis of acute severe mr by comprehensive interpretation of tte without performing tee . the location of the missing leaflet can be difficult to identify in case the leaflet embolized to distal aorta or its branches . plain x - rays are not helpful because of lack of radio - opacity of the prosthetic valves . removing dislocated leaflet is recommended as it may cause arterial wall damage leading to erosions , infections , and further migrations . this case is notable that the patient who presented with severe cardiogenic shock after the prosthetic valve implanted 27 years ago suddenly dislodged , recovered from the debilitating condition owing to the prompt diagnosis based on tte and immediate surgical correction . although rare , when a patient with previous history of prosthetic valve replacement presents with symptoms of acute decompensated heart failure , possibility of leaflet and escape of the valve leaflet should be contemplated . in cases of the leaflet escape , the urgent diagnosis and emergent surgical replacement is mandatory to prevent the mortality .

dorzolamide ophthalmic drop is one of the most common glaucoma medications but it has a short residence time in the eye . the aim of this study is to develop ocular dorzolamide hcl nanoliposomes ( drz nanoliposomes ) and to evaluate their potential use for the treatment of ocular hypertension . nanoliposomes were prepared using reverse - phase evaporation vesicle ( rev ) and thin layer hydration ( tlh ) method with 7:3 and 7:4 molar ratios of phosphatidylcholine : cholesterol . the physicochemical properties of the formulations were investigated . formulations with 7:4 lipid ratios were evaluated in terms of drug release , physical stability and ex - vivo permeation through the excised albino rabbit cornea . the rabbits in groups of 6 were treated with selected drz nanoliposomes or dorzolamide solution or marketed dorzolamid preparation ( biosopt ) and intraocular pressure ( iop ) was monitored . formulations with 7:4 molar ratio entrapped greater amount of drug compared to those with 7:3 lipid components ratio . drz nanoliposomes with 7:4 lipid ratio showed more transcorneal permeation than dorzolamide solution ( p<0.05 ) ; and the formulation prepared by tlh method exhibited higher permeability than that prepared by rev method ( p<0.05 ) . the selected drz nanoliposomes showed greater iop lowering activity and a more prolonged effect compared to dorzolamide solution and biosopt. drz nanoliposomes prepared by tlh method with 7:4 ratios showed promising results as a candidate for the treatment of ocular hypertension .

dorzolamide hydrochloride ( drz ) was synthesized in 1980 and it was the first carbonic anhydrase inhibitor approved for the topical treatment of glaucoma in humans in 1995 . the drug reduces the production of bicarbonate ions , thereby reduces secretions and lowers intraocular pressure ( 1 , 2 ) . elevated iop may lead to glaucoma in patients with ocular hypertension . nowadays due to their higher efficiency and lower systemic adverse reactions , ophthalmic preparations are considered to be the first choice in glaucoma treatment ( 3 , 4 ) . one of the most important problems of the ophthalmic drops is their short ocular residence time . the drug is quickly diluted by tears and is removed through nasolachrymal ducts ( 5 ) . therefore , it is beneficial to design a biocompatible ocular drug delivery system that can increase the residence time of the drug in the eye . liposomes are self - assembled colloidal particles ; consist of lipid bilayers surrounding an aqueous compartment . due to their cell - like nature , liposomes are able to attach to cellular tissues of the body . today , nanotechnology provides smaller dimension of liposomes ; under the name of nanoliposomes ( 6 , 7 ) . drug - loaded nanoliposomes can maintain a relatively constant drug concentration , increase the drug residence time in the eye and consequently enhance therapeutic efficiency ; therefore , lower doses of the drug with less frequency can be applied ( 8 , 9 ) . the objective of this study is to develop ocular drz nanoliposomes and evaluate their potential use for the treatment of ocular hypertension . dorzolamide hcl supplied by baselux ( s.a - switzerland ) , soy phosphatidylcholine ( phospholipon 85 g ) purchased from lipoid ( germany ) and cholesterol supplied by sigma ( germany ) were used for the preparation of formulations . nanoliposomes reverse - phase evaporation vesicle ( rev ) method : the lipid components , with 7:3 and 7:4 molar ratio of soy phosphatidylcholine ( spc ) : cholesterol ( ch ) , were dissolved in chloroform - methanol solution ( 2:1 v / v ) . the organic solvent was evaporated at 65 c under reduced pressure at 150 rpm using a rotary evaporator ( ev311 , lab - tech , germany ) and the thin lipid film obtained was maintained at 4 c for 24 hours to ensure complete removal of solvents . the film was dissolved in diethyl ether , followed by addition of 10 ml phosphate buffer ( ph 5.8 ) containing 2% ( w / v ) drz . the organic solvent was removed at 37 c and the dispersion was sonicated for 30 min using a bath sonicator ( t710 , elma , germany ) . to ensure full lipid hydration and maturation , . thin layer hydration ( tlh ) method : in this method the thin lipid film obtained by previously described conditions was directly hydrated with phosphate buffer ( ph 5.8 ) containing drz ( 2% w / v ) , sonicated and kept at 4 c overnight for maturation ( 11 ) . nanoliposomes in order to determine the encapsulation efficiency ( ee% ) of drz nanoliposomes , the formulation was transferred into centrifugal filter device ( centritrep-50 kda ) , and centrifuged at 3000 rpm and 25 c1 for 30 min ( clements 2000 , germany ) . then , the supernatant was analyzed for free drz using uv spectrophotometer ( biochorm , england ) at 254 nm . ee% was calculated as follows : ee%=total drug - free drugtotal drug100 the following parameters were measured : the mean particle size , polydispersity index ( pdi ) and zeta potential of the nanoliposomes by nanosizer ( nano zs , malvern , uk ) , viscosity by brookfield viscometer ( model dv - ii+pro , middleboro , usa ) at 100 rpm , surface tension by the de nouy ring method ( csc scientific company , usa ) , ph value , and refractive index ( ri ) at 251 c . in - vitro drug release study nanoliposomal samples enclosed in dialysis bags ( cellulose membrane mw cut- off 12 kda , sigma ) were immersed in 250 ml phosphate buffer ( ph 7.4 ) at 251 c and were stirred at 100 rpm . at predetermined time intervals , samples were withdrawn and analyzed for drz spectrophotometrically at 254 nm . drz solution ( 2% w / v ) ex - vivo drug permeation study the experiment was carried out using franz diffusion cells designed for ocular permeation studies with receiver medium of 10.5 ml phosphate buffer ( ph 7.4 ) at 351 c . albino rabbits were sacrificed by iv injection of sodium phenobarbital and the whole eyes were enucleated . samples ( 1 ml ) were withdrawn at specific time intervals and assayed spectrophotometrically at 254 nm . the permeation behavior of free drug as a control experiment was determined using drz solution ( 2% w / v ) . the steady - state flux ( jss ) of different formulations was determined by the slope of the linear portion of the plots of the amount of drug in the receiving chamber versus time , divided by exposed corneal surface area and the lag time that was estimated from the x - intercept of the linear portion of the graph . apparent corneal permeability coefficient ( papp ) was calculated according to the following equation : cd represents the drug concentration in the donor compartment ( 13 ) . in - vivo iop measurement the animal experiments were conducted in full compliance with regulatory principles of ethics committee of ahvaz jundishapur university of medical sciences . albino rabbits ( 2.53 kg ) were housed at controlled temperature ( 252 c ) , and humidity ( 605% ) , with a 12/12-h light - dark cycle . nanoliposome , group ii was treated with drz solution and group iii received biosopt ophthalmic drop ( bakhtar biochemie co , iran ) . one drop ( 0.05 ml ) each of drz nanoliposome , drz solution or biosopt drop was instilled into the right eye , while the non - treated eye was considered as control . iop of both eyes was determined using iopen tonometer for 8 h. the mean of three consecutive tonometric measurements was calculated for each animal ( 7 ) . rev and tlh nanoliposomes with lipid molar ratio of 7:4 were stored at 4 c ; their particle size and ee% were monitored after 1 and 3 months ( 14 ) . statistical analysis all studies were carried out in triplicate and data were reported as a mean sd . one - way anova and multiple comparison tukey s test were used to assess the significance of the differences between the various groups , p<0.05 was considered statistically significant . nanoliposomes the data showed that nanoliposomal formulations with lipid ratio of 7:4 ( spc : ch ) produced significantly higher ee% than those with lipid ratio of 7:3 ( spc : ch ) ( p<0.05 ) ( table 1 ) . particle size results showed that higher proportion of cholesterol in lipid ratio led to form larger particles ( p<0.05 ) but they were still below 100 nm and in acceptable range ( table 1 ) . comparing the results of formulations with same molar ratio , revealed that nanoliposomes prepared by tlh method had significantly smaller particle size than those made by rev method ( p<0.05 ) . values close to zero indicate a homogeneous dispersion while those greater than 0.3 indicate high heterogeneity ( 15 ) . pdi of all formulations in this study were approximately 0.3 which indicates acceptable uniformity and homogeneity of the formulations ( p>0.05 ) . nanoliposomal formulations 7:4 ( spc : ch ) rev and 7:4 ( spc : ch ) tlh were positively charged with zeta potential value of + 12.8 and + 10.3 mv , respectively . the tonicity of the formulations was equivalent to that of a 0.875% sodium chloride solution . particle size , polydispersity index and encapsulation efficiency of drz - nanoliposomes ( mean sd , n=3 ) the results of viscosity , surface tension , ph and ri values are presented in table 2 . the results showed that method of preparation and lipid molar ratio did not influence the viscosity value of nanoliposomes ( p>0.05 ) . the surface tension of the formulations in decreasing order is : biosopt > rev prepared drz nanoliposomes > tlh prepared drz nanoliposomes ( p<0.05 ) . the ph value measured for biosopt was 5.2 which is significantly lower than that of drz nanoliposomes ( p<0.05 ) . the ri values of drz nanoliposomes were 1.343 which is close to that of tears . viscosity , surface tension , ph and refractive index of formulations ( mean sd , n=3 ) in - vitro drug release this study was carried out only with 7:4 ( spc : ch ) rev and 7:4 ( spc : ch ) tlh drz nanoliposomes which had good in - vitro properties especially regarding ee% . all of the experiments were performed without separation of entrapped and non - entrapped drz . the release profiles of drz from drz nanoliposomes and drz solution shown in figure 1 are almost the same . a burst effect of drug release observed during the first hour ( about 70% ) that followed by nearly complete release ( over 95% ) after 6 hours . release profile of drz from drz nanoliposomes and drz solution ( means.d , n=3 ) ex - vivo drug permeability after 8 and 24 h , corneal permeability of drz for 7:4 ( spc : ch ) rev was 4.5 and 11% , for 7:4 ( spc : ch ) tlh was 6 and 14.5% , and for drz solution was 1 and 3% , respectively ( figure 2 ) . values of drz nanoliposomes were significantly higher than those of drz solution ( p<0.05 ) . significant ( p<0.05 ) differences were observed between permeability results of 7:4 ( spc : ch ) rev and 7:4 ( spc : ch ) tlh in favor of tlh prepared drz nanoliposomes . transcorneal permeation profile of drz from drz nanoliposomes and drz solution ( means.d , n=3 papp , jss and lag time values are shown in table 3 . however , the permeability parameters ( papp and jss ) of 7:4 ( spc : ch ) rev and 7:4 ( spc : ch ) tlh were not significantly different ( p>0.05 ) . a lag time of about 40 min was observed for drz nanoliposomes , whereas drz solution permeated through cornea without any lag time . apparent permeability coefficient , steady - state flux and lag time of formulations from ex- vivo permeation studies ( mean sd , n=3 the drug leakage after 1 and 3 months storage at 4 c were found to be 1.12% and 2.86% from 7:4 ( spc : ch ) rev drz nanoliposomes and 0.2% and 1.64% from 7:4 ( spc : ch ) tlh , respectively . the particle size of drz nanoliposomes showed no statistically significant changes over 3 months storage at 4 c ( p>0.05 ) . considering the higher permeability and the smaller particle size of 7:4 ( spc : ch ) tlh prepared drz nanoliposomes as compared to rev prepared ones , intraocular pressure ( iop ) figure 3 shows that the iop lowering activity of marketed product and drz solution within 1 h after instillation reached maximum values of 2.25 mmhg and 1.5 mmhg , respectively , but this effect quickly decreased and disappeared for both products . the maximum iop decreased value was 4.42 mmhg at the 4 hour , which was significantly higher than those of drz solution and the marketed product ( p<0.05 ) . macroscopic examination did not reveal any inflammation , redness or irritability in the rabbits eyes during the procedure . iop reduction after administration of drz nanoliposomes , drz solution and marketed product ( biosopt ) . the fine size of nanoliposomal ophthalmic preparations tends to cause less abrasion , irritation and sensitivity ( 6 , 16 ) . in this study , other studies revealed that the liposomes with small size and single layer such as suv , adhere more strongly to tissue and perform a better drug delivery ( 17 ) . calculated tonicity of the formulations was nearly close to isotonic solution which is important for formulations intended for ophthalmic use . marketed drz eye drops are in a solution form , the drug is absolutely freely soluble and becomes diluted with tears and is readily discharged from the eyes . hence , viscosity enhancers such as cellulose derivatives are mostly used in marketed products to increase consistency and improve adherence in the eye ( 2 ) . but , it should be considered that high consistency can cause problems in the movement of the eyeball , the process of optical refraction and creation of the image on the retina . the viscosity of an ophthalmic formulation should be in the range of 1550 cps ( 18 ) . in this study , formulation 7:4 ( spc : ch ) tlh prepared drz nanoliposomes had a viscosity equal to 46 cps , which is in the acceptable range ; while the marketed product ( biosopt ) had a significantly higher viscosity equal to 96 cps as measured in our lab.(p < 0.05 ) . surface tension of drz nanoliposomes was lower than that of biosopt which is attributed to the surface activity of phospholipid amphiphilic molecules ( 19 ) . the lower surface tension , the better spreading of the product on the cornea and the more contact between them . but , eye drops with much lower surface tension than lachrymal fluid ( 0.04 to 0.05 n / m ) may destabilize the tear film and cause visual problems ( 2 ) . drz has maximum stability in the range of ph 4 - 6 ( 20 ) . it should be taken into consideration that a shift from neutral ph to either acidic or basic may cause eye irritation . it is recommended that eye drops should have ri close to tears fluid ( 1.34 to 1.36 ) ( 2 ) . drz nanoliposomes in our study , had ri in the acceptable range ( table 2 ) . as cholesterol settles between phosphatidylcholine molecules , it fills empty spaces , increases rigidity of the bilayer films and prevents leakage of the drug . also , it stabilizes nanoliposomes against external pressure such as extreme shaking ( 21 , 22 ) . in this study , the release of the drug from the drz nanoliposomes did not show a significant difference compared with the drug release from drz solution . the high - rate of nanoliposomes drug release during the first hours may be due to the presence of available free drug in the formulations and the high concentration gradient of drz between the contents in the dialysis bag and the large - volume ( 250 ml ) receiver phase . although drz nanoliposomes and drz solution exhibited similar in - vitro drug release , drz this can be explained in terms of the conditions under which the ex - vivo study was performed . this is more similar to in - vivo conditions in relation to the receiver medium volume ( 10.5 ml ) . it is speculated that the high rate of drug permeability of nanoliposomes is a result of high lipophilicity of the vehicle , which leads to increase in residence time on the surface of the cornea ( 23 ) . the nanoliposomes can carry hydrophilic drugs ( such as drz ) in their cores and then attach to the surface of the corneal epithelium and gradually release the drug ( 6 , 24 ) . other possible mechanisms such as fusion of liposome membrane with the cornea or endocytosis of liposomes may lead to the drug release in the anterior eye space after passing through the cornea ( 22 ) . in addition , the lag time observed in ex - vivo permeation studies of drz nanoliposomes may support this hypothesis . nanoliposomes surface had a positive charge because of the cationic nature of drz at ph below 6.4 ( 20 ) . so , this would lead to their adhesion to polyanionic surface of cornea and conjunctiva and improvement in corneal retention and penetration ( 17 , 25 , 26 ) . the greater permeability of formulation 7:4 ( spc : ch ) tlh as compared to 7:4 ( spc : ch ) rev can be attributed to their smaller particle size ( 23 ) . it was also observed that drz nanoliposomes in contrast with drz solution and marketed product induced a remarkable decrease in iop and with respect to the duration of action , the effect of nanoliposomes was prominent . they investigated liposomal acetazolamide and concluded that acetazolamide liposomes composed of egg phosphatidylcholine : cholesterol : stearylamine ( 7:4:1 ) molar ratio prepared by a method almost similar to tlh method used in our study , revealed more prolonged iop - lowering effect compared to the solution form of the drug ( 7 ) . in their in - vivo study on liposomal diltiazem as ocular drug delivery system for glaucoma , mokhtar ibrahim et al . , proved the system enhanced iop - reducing activity as compared to the solution form at equal concentration ( 27 ) . hironaka et al . also investigated the uptake of carboxyfluorescein ( cf ) into conjuctival cells via liposomal eye drops and concluded that the amount of cf incorporated into cells increased when using liposome as a carrier ; and they proposed that the increase in drug uptake was due to the affinity of phospholipid bilayer to the cell membrane that led to effective delivery of hydrophilic drug to the retina ( 28 ) . the higher permeability coefficient and flux of drz nanoliposomes versus solution form provide a good explanation for this higher effectiveness and prolonged therapeutic effect ( 29 ) . nanoliposomes natural structure , their positive surface charge and small vesicle size contribute to their adhesion to the corneal membrane which leads to improved clinical effect . of the two methods that were applied to prepare nanoliposomes systems , tlh method is simpler , faster , cost effective and has less toxic solvent residue than rev method . in this study , formulation 7:4 ( spc : ch ) tlh was selected because of its small particle size , high ee% and high corneal permeability . the characteristics of this formulation in terms of surface tension , viscosity , ph , ri and safety were all in the acceptable range required for ophthalmic preparations . the formulation was stable throughout 3-months period of storage and revealed longer and higher iop - lowering activity in comparison with drz solution and marketed eye drop . the prolonged effect may be explained in term of gradual drug release from nanoliposomes which are depot in the cornea , suspended in aqueous humor or attached to the surface of the anterior space . we recommend the addition of an antioxidant to prevent lipid oxidation and prepare a stable formulation for longer period of time . however , assay of drz in aqueous humor following drug instillation into rabbit eye should be done before clinical trials as well .

backgroundthe evidence supporting the use of blockers in patients with acute coronary syndrome after successful percutaneous coronary intervention has been inconsistent and scarce.methods and resultsbetween march 1 , 2009 , and december 30 , 2014 , a total of 3180 eligible patients with acute coronary syndrome undergoing percutaneous coronary intervention were consecutively enrolled . the primary end point was allcause death and the secondary end point was a composite of allcause death , nonfatal myocardial infarction , heart failure readmission , and cardiogenic hospitalization . patients were compared according to the use of blockers at discharge . compared with the no blocker group , the risk of allcause death was significantly lower in the blocker group ( hazard ratio [ hr ] , 0.33 ; 95% ci , 0.170.65 [ p=0.001 ] ) . a consistent result was obtained in multiple adjusted model and propensity score matched analysis . the use of blockers was also associated with decreased risk of composite of adverse cardiovascular events ( hr , 0.47 ; 95% ci , 0.280.81 [ p=0.006 ] ) , although statistical significance disappeared after multivariable adjustment and propensity score matching . furthermore , we performed post hoc analysis for the subsets of patients and the results revealed that patients with non stsegment elevation myocardial infarction benefited the most from blocker therapy at discharge ( hr , 0.04 ; 95% ci , 0.000.27 [ p=0.001 ] ) , and the use of < 50% of target dose was significantly associated with better outcome compared with no blocker use , rather than 50% of target dose.conclusionsthe administration of relatively low blocker dose is associated with improved clinical outcomes among patients with acute coronary syndrome after successful percutaneous coronary intervention , especially for patients with nonstsegment elevation myocardial infarction .

blockers , as one of secondary prevention medications , can diminish myocardial oxygen demand by reducing heart rate , blood pressure , and myocardial contractility , thereby being widely used to relieve ischemic symptoms in patients with acute coronary syndrome ( acs).1 the updated american college of cardiology / american heart association ( acc / aha ) guidelines recommend the use of blockers for the management of stsegment elevation myocardial infarction ( stemi)2 and non stsegment elevation myocardial infarction ( nstemi).3 however , evidence supporting the clinical benefit of blockers is largely based on studies in patients with acute mi for stemi , and was extrapolated to patients with unstable angina pectoris ( uap ) and nstemi.4 in the percutaneous coronary intervention ( pci ) era , patients with acs , a spectrum of clinical presentations ranging from uap to nstemi and stemi , mostly constituted those undergoing pci.5 however , few studies are available to systematically describe the contemporary pattern of blocker use and determine its impact on clinical outcomes in acs patients after pci . as shachamet et al6 pointed out , many physicians remain unconvinced of either a short or longterm benefit of blocker use following pci . moreover , much less attention has been paid to specifying which subgroup of patients with acs benefits the most from blocker therapy . thus , we sought to evaluate the impact of blocker therapy on clinical outcomes in patients with acs after pci and specified subgroups in a all patients diagnosed with coronary heart disease at tongji hospital in wuhan , china , were consecutively recruited in the clinical outcomes of coronary heart diseases in tongji hospital registry from march 1 , 2009 . demographics , clinical profiles , and concomitant medications were collected with standardized case report forms by professional investigators in the department of cardiology , and all participants were prospectively contacted at 1 , 6 , and 12 months by cardiology nurses and research coordinators through patient interview , chart review , and serial telephone contacts . written informed consent was obtained from each patient at admission . between march 1 , 2009 , and december 30 , 2014 , all patients in the database were searched . for inclusion , patients were required to meet the following criteria : ( 1 ) age older than 18 years ; ( 2 ) have an ascertained diagnosis of acs at admission , and ( 3 ) undergoing pci . in addition , the following patients were excluded from this analysis : ( 1 ) patients discharging unstable , ( 2 ) patients with the absence of blocker information at discharge , or ( 3 ) contraindication to blocker therapy such as significant bradycardia ( heart rate < 50 beat per min ) or hypotension ( systolic blood pressure < 90 mm hg ) . this strategy was approved by the ethics committee of tongji medical college and conducted in accordance with the declaration of helsinki and the international conference on harmonization guidelines for good clinical practice . the study was supported by grants from the national program on key basic research project ( 973 program ) ( no . 2012cb518004 ) , the national nature science foundation key project ( no . 91439203 ) , and the national health and family planning commission of china ( no . 201202025 , no . 2011bai11b04 ) . for the purpose of calculating the proportion of blocker dose administered ( daily dosage of blockers / target dose ) , the target dose was in line with blocker doses used in large randomized trials , defined as follows : metoprolol 200 mg / d7 carvedilol 50 mg / d,8 timolol 20 mg / d,9 bisoprolol 10 mg / d10 atenolol 100 mg / d,11 and propanolol 180 mg / d.12 in addition , patients who were diagnosed with acs must present with ischemic symptoms within 24 hours and have at least one of the following conditions : ( 1 ) electrocardiographic changes consistent with acs , ( 2 ) an increase in serum cardiac biomarkers ( troponin or creatine kinasemb ) , or ( 3 ) documentation of angina pectoris.13 successful pci was identified as a patent vessel at the treatment site with anterograde thrombolysis in myocardial infarction flow 3 and angiographic residual stenosis < 50% . in the present study , we evaluated two study end points : ( 1 ) the primary outcome was allcause mortality , which was regarded as cardiac origin unless obvious noncardiac cause could be identified ; and ( 2 ) the secondary outcome was a composite end point of allcause death , nonfatal mi , heart failure readmission , and cardiogenic hospitalization . of these , mi referred to symptoms with new electrocardiographic changes ( pathologic q waves , persistent stsegment elevation , or stsegment depression ) as well as cardiac markers at least one value above the 99th percentile of the upper reference limit.14 the identification of heart failure readmission was consistent with the guidelines of the european society of cardiology.15 in addition , cardiogenic hospitalization was considered as a hospitalization for cardiovascular cause , including uap , transient ischemic attack , or revascularization procedure . we divided patients into two groups with regard to whether blocker therapy was received at discharge . categorical variables were compared using chisquare test and continuous variables were analyzed by means of wilcoxon ranksum test or student t test according to its distribution . characteristics of study participants were further compared with respect to the following : no blocker use , < 50% of target dose , and 50% of target dose . differences among groups were examined in the same way for categorical variables and 1way anova analysis or kruskal wallis rank test if deviated from normality for continuous variables . survival curves were depicted by kaplan meier method and compared with the logrank test . multivariable cox proportional hazard regression was applied to identify the independent factors associated with end points . the variables entered into the multivariate model were age , sex , hypertension , diabetes , dyslipidemia , stroke , prior infarction , recent infarction within 3 weeks , heart failure status ( canadian heart class or killip heart class ) , arrhythmia , and medications at discharge ( aspirin , clopidogrel , statins , blocker , angiotensinconverting enzyme inhibitors [ aceis]/angiotensin receptor blockers [ arbs ] , nitrates ) . in addition , clinical factors related to treatment selection may confound the event rates , therefore , we performed propensity score matched analysis to address the issue . to estimate the propensity score , a logistic regression model developed with the variables , including age , sex , hypertension , diabetes , dyslipidemia , stroke , prior infarction , recent infarction within 3 weeks , heart failure status ( canadian heart class or killip heart class ) , arrhythmia , and medications at discharge ( aspirin , clopidogrel , statins , aceis / arb , nitrates ) , was used to predict the use of blockers . patients in the blocker group were 1:1 matched to patients in the no blocker group on the basis of their propensity score and the value of caliper equal to 0.2 . absolute standardized differences < 10% for a given covariate indicate a relatively small imbalance . for the propensity score matched cohort , mcnemar test was used for paired categorical variables and paired t test or paired sample wilcoxon rank test for continuous variables , depending on the normality of the variables . the associations of blocker use with clinical outcomes were evaluated by use of cox regression models . spss version 20.0 ( ibm corp , armonk , ny ) was used for statistical analysis . the power of the study was calculated by pass version 11.0 ( ncss , kaysville , ut ) . all patients diagnosed with coronary heart disease at tongji hospital in wuhan , china , were consecutively recruited in the clinical outcomes of coronary heart diseases in tongji hospital registry from march 1 , 2009 . demographics , clinical profiles , and concomitant medications were collected with standardized case report forms by professional investigators in the department of cardiology , and all participants were prospectively contacted at 1 , 6 , and 12 months by cardiology nurses and research coordinators through patient interview , chart review , and serial telephone contacts . written informed consent was obtained from each patient at admission . between march 1 , 2009 , and december 30 , 2014 , all patients in the database were searched . for inclusion , patients were required to meet the following criteria : ( 1 ) age older than 18 years ; ( 2 ) have an ascertained diagnosis of acs at admission , and ( 3 ) undergoing pci . in addition , the following patients were excluded from this analysis : ( 1 ) patients discharging unstable , ( 2 ) patients with the absence of blocker information at discharge , or ( 3 ) contraindication to blocker therapy such as significant bradycardia ( heart rate < 50 beat per min ) or hypotension ( systolic blood pressure < 90 mm hg ) . this strategy was approved by the ethics committee of tongji medical college and conducted in accordance with the declaration of helsinki and the international conference on harmonization guidelines for good clinical practice . the study was supported by grants from the national program on key basic research project ( 973 program ) ( no . 2012cb518004 ) , the national nature science foundation key project ( no . 91439203 ) , and the national health and family planning commission of china ( no . 201202025 , no . for the purpose of calculating the proportion of blocker dose administered ( daily dosage of blockers / target dose ) , the target dose was in line with blocker doses used in large randomized trials , defined as follows : metoprolol 200 mg / d7 carvedilol 50 mg / d,8 timolol 20 mg / d,9 bisoprolol 10 mg / d10 atenolol 100 mg / d,11 and propanolol 180 mg / d.12 in addition , patients who were diagnosed with acs must present with ischemic symptoms within 24 hours and have at least one of the following conditions : ( 1 ) electrocardiographic changes consistent with acs , ( 2 ) an increase in serum cardiac biomarkers ( troponin or creatine kinasemb ) , or ( 3 ) documentation of angina pectoris.13 successful pci was identified as a patent vessel at the treatment site with anterograde thrombolysis in myocardial infarction flow 3 and angiographic residual stenosis < 50% . in the present study , we evaluated two study end points : ( 1 ) the primary outcome was allcause mortality , which was regarded as cardiac origin unless obvious noncardiac cause could be identified ; and ( 2 ) the secondary outcome was a composite end point of allcause death , nonfatal mi , heart failure readmission , and cardiogenic hospitalization . of these , mi referred to symptoms with new electrocardiographic changes ( pathologic q waves , persistent stsegment elevation , or stsegment depression ) as well as cardiac markers at least one value above the 99th percentile of the upper reference limit.14 the identification of heart failure readmission was consistent with the guidelines of the european society of cardiology.15 in addition , cardiogenic hospitalization was considered as a hospitalization for cardiovascular cause , including uap , transient ischemic attack , or revascularization procedure . we divided patients into two groups with regard to whether blocker therapy was received at discharge . categorical variables were compared using chisquare test and continuous variables were analyzed by means of wilcoxon ranksum test or student t test according to its distribution . characteristics of study participants were further compared with respect to the following : no blocker use , < 50% of target dose , and 50% of target dose . differences among groups were examined in the same way for categorical variables and 1way anova analysis or kruskal wallis rank test if deviated from normality for continuous variables . survival curves were depicted by kaplan meier method and compared with the logrank test . multivariable cox proportional hazard regression was applied to identify the independent factors associated with end points . the variables entered into the multivariate model were age , sex , hypertension , diabetes , dyslipidemia , stroke , prior infarction , recent infarction within 3 weeks , heart failure status ( canadian heart class or killip heart class ) , arrhythmia , and medications at discharge ( aspirin , clopidogrel , statins , blocker , angiotensinconverting enzyme inhibitors [ aceis]/angiotensin receptor blockers [ arbs ] , nitrates ) . in addition , clinical factors related to treatment selection may confound the event rates , therefore , we performed propensity score matched analysis to address the issue . to estimate the propensity score , a logistic regression model developed with the variables , including age , sex , hypertension , diabetes , dyslipidemia , stroke , prior infarction , recent infarction within 3 weeks , heart failure status ( canadian heart class or killip heart class ) , arrhythmia , and medications at discharge ( aspirin , clopidogrel , statins , aceis / arb , nitrates ) , was used to predict the use of blockers . patients in the blocker group were 1:1 matched to patients in the no blocker group on the basis of their propensity score and the value of caliper equal to 0.2 . absolute standardized differences < 10% for a given covariate indicate a relatively small imbalance . for the propensity score matched cohort , mcnemar test was used for paired categorical variables and paired t test or paired sample wilcoxon rank test for continuous variables , depending on the normality of the variables . the associations of blocker use with clinical outcomes were evaluated by use of cox regression models . spss version 20.0 ( ibm corp , armonk , ny ) was used for statistical analysis . the power of the study was calculated by pass version 11.0 ( ncss , kaysville , ut ) . between march 1 , 2009 , and december 30 , 2014 , there were 5063 patients recruited in the database , and only 3453 patients underwent the pci procedure . of these , 23 patients were discharged unstably , 183 were not diagnosed with acs at admission , 43 had a contraindication to blocker use , and 24 could not provide complete information about the administration of blockers at discharge , and were excluded from the analysis . acs indicates acute coronary syndrome ; chd , coronary heart disease ; pci , percutaneous coronary intervention . in the overall evaluation cohort , 2423 patients ( 76.2% ) were discharged on blockers , while 757 patients ( 23.8% ) were not . compared with blocker users , patients who were not administrated blockers were older ( 60.7910.39 versus 58.4410.47 , p<0.001 ) , had lower diastolic blood pressure ( dbp ) ( 79.0113.18 versus 80.5713.18 , p=0.005 ) , lower heart rate ( 74.4015.15 versus 75.1012.42 , p=0.009 ) , and were more likely to have arrhythmia ( 11% versus 7.1% , p=0.001 ) . for concomitant medication use , the prescriptions of aspirin , statins , and aceis / arbs were more common in the blocker group compared with the no blocker group ( 99.4% versus 98.4% [ p=0.007 ] ; 98.6% versus 95.5% [ p<0.001 ] ; 81% versus 58.4% [ p<0.001 ] ) . table 1 summarizes the baseline characteristics and other medication management according to the use of blockers at discharge . the differences in the baseline characteristics in the 3 subgroups are also shown in tables 2 , 3 through 4 . in the propensity score matched model , there was no significant difference in the baseline characteristics between the blocker group and the no blocker group . acei indicates angiotensinconverting enzyme inhibitor ; arb , angiotensin receptor blocker ; dbp , diastolic blood pressure ; mi , myocardial infarction ; sbp , systolic blood pressure . acei indicates angiotensinconverting enzyme inhibitor ; arb , angiotensin receptor blocker ; dbp , diastolic blood pressure ; mi , myocardial infarction ; sbp , systolic blood pressure ; stemi , stsegment elevation myocardial infarction . baseline characteristics in patients with nstemi variables are expressed as meansd or percentage . acei indicates angiotensinconverting enzyme inhibitor ; arb , angiotensin receptor blocker ; dbp , diastolic blood pressure ; mi , myocardial infarction ; nstemi , non acei indicates angiotensinconverting enzyme inhibitor ; arb , angiotensin receptor blocker ; dbp , diastolic blood pressure ; mi , myocardial infarction ; sbp , systolic blood pressure ; uap , unstable angina pectoris . at 1 year after index admission , completed followup information was obtained in 3153 patients ( 99.2% ) . a total of 33 patients died of allcause diseases , 14 patients occurred nonfatal mi , 34 patients had heart failure , and 214 patients were readmitted for cardiogenic reasons during followup . blocker therapy was associated with a lower incidence of allcause death ( unadjusted hazard ratio [ hr ] , 0.33 ; 95% ci , 0.170.65 [ p=0.001 ] ) . after adjusting for confounders , the risk of allcause death remained consistently lower in the blocker group ( adjusted hr , 0.38 ; 95% ci , 0.170.83 [ p=0.015 ] ) ( table 5 ) . in the propensity score matched cohort , the blocker group still had decreased allcause mortality ( hr , 0.27 ; 95% ci , 0.080.97 [ p=0.045 ] ) ( table 6 ) . a lower rate of secondary end point was also observed in the blocker users ( unadjusted hr , 0.76 ; 95% ci , 0.590.98 [ p=0.035 ] , although the statistical difference disappeared after adjustment ( adjusted hr , 0.87 ; 95% ci , 0.661.16 [ p=0.355 ] ) . in addition , the associations of blocker use with the rate of nonfatal mi , heart failure readmission , and cardiogenic hospitalization were computed , respectively . the results are illustrated in table 5 , and figure 2 describes the association between the use of blockers and clinical end points . clinical outcomes and unadjusted / multivariable adjusted hrs during 1year followup the event rate at 1 year was estimated by the kaplan meier method . hf indicates heart failure ; hr , hazard ratio ; mi , myocardial infarction ; nstemi , non stsegment elevation myocardial infarction ; stemi , stsegment elevation myocardial infarction ; uap , unstable angina pectoris . clinical outcomes and hrs after propensity score matching during 1year followup hf indicates heart failure ; mi , myocardial infarction ; nstemi , non stsegment elevation myocardial infarction ; stemi , stsegment elevation myocardial infarction ; uap , unstable angina pectoris . the hazard ratio ( hr ) and 95% ci could not be evaluated that no event occurred in the blocker group . the hazard curves for the primary and secondary end points in the overall population ( a ) , in the patients with stsegment elevation myocardial infarction ( stemi ) ( b ) , in the patients with non stsegment elevation myocardial infarction ( nstemi ) ( c ) , and in the patients with unstable angina pectoris ( uap ) ( d ) . the curves were described by kaplan meier methods and the p values were calculated using the logrank tests . at baseline , 728 patients ( 22.9% ) had stemi , 576 patients ( 18.1% ) had nstemi , and 1876 patients ( 59.0% ) had uap . notably , a greater benefit of blocker use was found in patients with nstemi whose incidence of allcause death was significantly lower in the blocker group ( 0.2% versus 6.4% ; unadjusted hr , 0.04 ; 95% ci , 0.000.27 [ p=0.001 ] ) , and the relationship remained even after performing multivariable cox proportional hazard regression analysis ( adjusted hr , 0.00 ; 95% ci , 0.000.14 [ p=0.005 ] ) . in addition , blocker use was associated with a lower risk of the secondary end point ( 7.8% versus 15.7% ; unadjusted hr , 0.47 ; 95% ci , 0.280.81 [ p=0.006 ] ) , but no statistical difference was observed after adjustment ( adjusted hr , 0.65 ; 95% ci , 0.351.21 [ p=0.171 ] ) . in the patients with stemi and uap , however , there was no statistical difference between the two groups for allcause mortality ( 1.1% versus 1.9% ; adjusted hr , 0.40 ; 95% ci , 0.081.94 [ p=0.257 ] in patients with stemi and 0.7% versus 0.9% ; adjusted hr , 0.96 ; 95% ci , 0.293.10 [ p=0.938 ] in patients with uap ) and the secondary end point ( 8.5% versus 16.1% ; adjusted hr , 1.13 ; 95% ci , 0.592.16 [ p=0.720 ] in patients with stemi and 9.0% versus 9.9% ; adjusted hr , 0.97 ; 95% ci , 0.661.41 [ p=0.852 ] in patients with uap ( table 5 and figure 2 ) . the associations of blocker therapy with the clinical outcomes across the 3 subgroups were consistent in the propensity score matched cohorts ( table 6 ) . among the patients discharged on blockers , receiving < 50% of target dose was reported in 2012 patients ( 83.0% ) , while 411 patients ( 17% ) were prescribed 50% of target dose and the administration of metoprolol accounted for the majority ( 85.4% ) . the baseline characteristics according to the treatment of blocker use are exhibited in table 1 , 2 , 3 through 4 . for overall patients , allcause mortality was 0.7% in < 50% of target dose group , significantly lower than in the no blocker group ( 0.7% versus 2.1% ; adjusted hr , 0.40 ; 95% ci , 0.190.82 [ p=0.012 ] ) , while the rate of allcause death was not different between 50% of target blocker dose group and no blocker group ( 0.7% versus 2.1% ; adjusted hr , 0.46 ; 95% ci , 0.131.59 [ p=0.221 ] ) ( figure 3 ) , and no differences were observed in the incidence of secondary end point between the three different blocker dose groups . entire cohort and subgroup analyses of clinical outcomes according to the prescribed doses of blocker therapy at discharge . the adjusted hazard ratio ( hr ) and 95% ci could not be evaluated that no event occurred in the 50% of target dose group . mi indicates myocardial infarction ; nstemi , non stsegment elevation myocardial infarction ; stemi , stsegment elevation myocardial infarction ; uap , unstable angina pectoris . between march 1 , 2009 , and december 30 , 2014 , there were 5063 patients recruited in the database , and only 3453 patients underwent the pci procedure . of these , 23 patients were discharged unstably , 183 were not diagnosed with acs at admission , 43 had a contraindication to blocker use , and 24 could not provide complete information about the administration of blockers at discharge , and were excluded from the analysis . acs indicates acute coronary syndrome ; chd , coronary heart disease ; pci , percutaneous coronary intervention . in the overall evaluation cohort , 2423 patients ( 76.2% ) were discharged on blockers , while 757 patients ( 23.8% ) were not . compared with blocker users , patients who were not administrated blockers were older ( 60.7910.39 versus 58.4410.47 , p<0.001 ) , had lower diastolic blood pressure ( dbp ) ( 79.0113.18 versus 80.5713.18 , p=0.005 ) , lower heart rate ( 74.4015.15 versus 75.1012.42 , p=0.009 ) , and were more likely to have arrhythmia ( 11% versus 7.1% , p=0.001 ) . for concomitant medication use , the prescriptions of aspirin , statins , and aceis / arbs were more common in the blocker group compared with the no blocker group ( 99.4% versus 98.4% [ p=0.007 ] ; 98.6% versus 95.5% [ p<0.001 ] ; 81% versus 58.4% [ p<0.001 ] ) . table 1 summarizes the baseline characteristics and other medication management according to the use of blockers at discharge . the differences in the baseline characteristics in the 3 subgroups are also shown in tables 2 , 3 through 4 . in the propensity score matched model , there was no significant difference in the baseline characteristics between the blocker group and the no blocker group . acei indicates angiotensinconverting enzyme inhibitor ; arb , angiotensin receptor blocker ; dbp , diastolic blood pressure ; mi , myocardial infarction ; sbp , systolic blood pressure . acei indicates angiotensinconverting enzyme inhibitor ; arb , angiotensin receptor blocker ; dbp , diastolic blood pressure ; mi , myocardial infarction ; sbp , systolic blood pressure ; stemi , stsegment elevation myocardial infarction . acei indicates angiotensinconverting enzyme inhibitor ; arb , angiotensin receptor blocker ; dbp , diastolic blood pressure ; mi , myocardial infarction ; nstemi , non acei indicates angiotensinconverting enzyme inhibitor ; arb , angiotensin receptor blocker ; dbp , diastolic blood pressure ; mi , myocardial infarction ; sbp , systolic blood pressure ; uap , unstable angina pectoris . at 1 year after index admission , completed followup information was obtained in 3153 patients ( 99.2% ) . a total of 33 patients died of allcause diseases , 14 patients occurred nonfatal mi , 34 patients had heart failure , and 214 patients were readmitted for cardiogenic reasons during followup . blocker therapy was associated with a lower incidence of allcause death ( unadjusted hazard ratio [ hr ] , 0.33 ; 95% ci , 0.170.65 [ p=0.001 ] ) . after adjusting for confounders , the risk of allcause death remained consistently lower in the blocker group ( adjusted hr , 0.38 ; 95% ci , 0.170.83 [ p=0.015 ] ) ( table 5 ) . in the propensity score matched cohort , the blocker group still had decreased allcause mortality ( hr , 0.27 ; 95% ci , 0.080.97 [ p=0.045 ] ) ( table 6 ) . a lower rate of secondary end point was also observed in the blocker users ( unadjusted hr , 0.76 ; 95% ci , 0.590.98 [ p=0.035 ] , although the statistical difference disappeared after adjustment ( adjusted hr , 0.87 ; 95% ci , 0.661.16 [ p=0.355 ] ) . in addition , the associations of blocker use with the rate of nonfatal mi , heart failure readmission , and cardiogenic hospitalization were computed , respectively . the results are illustrated in table 5 , and figure 2 describes the association between the use of blockers and clinical end points . clinical outcomes and unadjusted / multivariable adjusted hrs during 1year followup the event rate at 1 year was estimated by the kaplan meier method . hf indicates heart failure ; hr , hazard ratio ; mi , myocardial infarction ; nstemi , non stsegment elevation myocardial infarction ; stemi , stsegment elevation myocardial infarction ; uap , unstable angina pectoris . clinical outcomes and hrs after propensity score matching during 1year followup hf indicates heart failure ; mi , myocardial infarction ; nstemi , non stsegment elevation myocardial infarction ; stemi , stsegment elevation myocardial infarction ; uap , unstable angina pectoris . the hazard ratio ( hr ) and 95% ci could not be evaluated that no event occurred in the blocker group . the hazard curves for the primary and secondary end points in the overall population ( a ) , in the patients with stsegment elevation myocardial infarction ( stemi ) ( b ) , in the patients with non stsegment elevation myocardial infarction ( nstemi ) ( c ) , and in the patients with unstable angina pectoris ( uap ) ( d ) . at baseline , 728 patients ( 22.9% ) had stemi , 576 patients ( 18.1% ) had nstemi , and 1876 patients ( 59.0% ) had uap . notably , a greater benefit of blocker use was found in patients with nstemi whose incidence of allcause death was significantly lower in the blocker group ( 0.2% versus 6.4% ; unadjusted hr , 0.04 ; 95% ci , 0.000.27 [ p=0.001 ] ) , and the relationship remained even after performing multivariable cox proportional hazard regression analysis ( adjusted hr , 0.00 ; 95% ci , 0.000.14 [ p=0.005 ] ) . in addition , blocker use was associated with a lower risk of the secondary end point ( 7.8% versus 15.7% ; unadjusted hr , 0.47 ; 95% ci , 0.280.81 [ p=0.006 ] ) , but no statistical difference was observed after adjustment ( adjusted hr , 0.65 ; 95% ci , 0.351.21 [ p=0.171 ] ) . in the patients with stemi and uap , however , there was no statistical difference between the two groups for allcause mortality ( 1.1% versus 1.9% ; adjusted hr , 0.40 ; 95% ci , 0.081.94 [ p=0.257 ] in patients with stemi and 0.7% versus 0.9% ; adjusted hr , 0.96 ; 95% ci , 0.293.10 [ p=0.938 ] in patients with uap ) and the secondary end point ( 8.5% versus 16.1% ; adjusted hr , 1.13 ; 95% ci , 0.592.16 [ p=0.720 ] in patients with stemi and 9.0% versus 9.9% ; adjusted hr , 0.97 ; 95% ci , 0.661.41 [ p=0.852 ] in patients with uap ( table 5 and figure 2 ) . the associations of blocker therapy with the clinical outcomes across the 3 subgroups were consistent in the propensity score matched cohorts ( table 6 ) . among the patients discharged on blockers , receiving < 50% of target dose was reported in 2012 patients ( 83.0% ) , while 411 patients ( 17% ) were prescribed 50% of target dose and the administration of metoprolol accounted for the majority ( 85.4% ) . the baseline characteristics according to the treatment of blocker use are exhibited in table 1 , 2 , 3 through 4 . for overall patients , allcause mortality was 0.7% in < 50% of target dose group , significantly lower than in the no blocker group ( 0.7% versus 2.1% ; adjusted hr , 0.40 ; 95% ci , 0.190.82 [ p=0.012 ] ) , while the rate of allcause death was not different between 50% of target blocker dose group and no blocker group ( 0.7% versus 2.1% ; adjusted hr , 0.46 ; 95% ci , 0.131.59 [ p=0.221 ] ) ( figure 3 ) , and no differences were observed in the incidence of secondary end point between the three different blocker dose groups . entire cohort and subgroup analyses of clinical outcomes according to the prescribed doses of blocker therapy at discharge . the adjusted hazard ratio ( hr ) and 95% ci could not be evaluated that no event occurred in the 50% of target dose group . stsegment elevation myocardial infarction ; stemi , stsegment elevation myocardial infarction ; uap , unstable angina pectoris . in this observational study , we investigated the association of blocker use with the clinical outcomes in patients with acs undergoing pci . we found that nearly 77% of eligible patients with acs undergoing pci were treated with blockers at discharge , and those not prescribed blockers were more likely to be older and have a history of arrhythmia . importantly , blocker therapy at discharge , especially a relatively low blocker dosage , were independently associated with improved survival , and the efficacy was more significant in patients with nstemi . blocker therapy also showed a trend in improved clinical outcomes in the stemi and uap patients . acs as a major cause of emergency medical care and hospitalization worldwide16 has been well improved by the introduction of pci.17 optimal secondary medication remains important after successful pci . predecessors have highlighted the importance of blocker therapy in patients with acute myocardial infarction.18 , 19 , 20 , 21 , 22 , 23 , 24 however , there are a few studies reporting that blocker use is not associated with improved outcome.25 , 26 , 27 one metaanalysis of randomized trials on the clinical outcomes of blocker use indicated no mortality benefit but reduced recurrent myocardial infarction and angina ( shortterm ) at the expense of increased heart failure , cardiogenic shock , and drug discontinuation.28 in this metaanalysis , data used in the reperfusion era were mainly recruited from the clopidogrel and metoprolol in myocardial infarction trial ( commit)29 and the japanese blockers and calcium antagonists myocardial infarction ( jcbami ) trial.30 in commit , the association between metoprolol allocation and risk of clinical outcomes was only assessed in a mean period of 15 days among ami patients . on the other hand , only postmyocardial infarction patients were enrolled in the jcbami trial , which could not reflect the benefit of early blocker therapy on improvement in prognosis . yet , our study proved the benefit of early use of blockers on longterm survival among patients with acs . nevertheless , chan et al31 reported the mortality benefit of blockers in patients undergoing successful elective pci ; however , they did not discuss which type of patients with acs benefited the most . in the present study , our results showed that blocker use was better associated with decreased incidence of allcause death in patients with nstemi . the published can rapid risk stratification of unstable angina patients suppress adverse outcomes with early implementation of the acc / aha guidelines ( crusade)32 and global registry of acute coronary events ( grace)33 studies also revealed that early blocker therapy had a beneficial impact on hospital and 6month mortality in patients with nstemi . in addition , yang et al24 demonstrated that blocker therapy at discharge was associated with improved survival in stemi patients treated with primary pci and recommended longterm blocker therapy in all patients with stemi regardless of risk profile . in our analysis , the use of blockers was not statistically associated with a lower risk of allcause death in stemi patients , but the trend of improved survival was obvious . additionally , the observational data from the outcome of blocker therapy after myocardial infarction ( obtain ) study suggested that increased survival was not observed in patients treated with blocker doses approximating those used in previous randomized clinical trials compared with lower doses,34 which was consistent with our conclusions that relatively low blocker dose actually decrease the rate of allcause mortality . even though several investigators have studied the benefits of blocker use among patients with myocardial infarction , our study stressed the impact of blocker therapy , especially relatively low blocker dose , on reducing allcause mortality in patients after elective pci , and provided the evidence to support the idea that the benefit of oral blocker therapy might be confined to patients with nstemi.35 evidence has suggested that the benefit of blockers for patients with nstemi may be due to the multivessel disease commonly presenting in them and its sympathetic hyperactivity.36 , 37 , 38 , 39 however , further exploration of the clinical usefulness of blocker therapy in patients with acs warrants largescale clinical trials such as a recently registered project ( nct02648243 ) . finally , we can not claim generalizability to patients with stemi / uap for it was underpowered to detect the difference . first , the nonrandomized nature of this observational study could have resulted in selection bias . although randomized controlled trials are considered the highest standard for evaluating treatment efficacy , observational studies can still provide unique and valuable insights into treatment effectiveness and generalizability in practice . our findings imply that the efficacy demonstrated in randomized clinical trials can be translated into tangible clinical benefits in the real world . second , a 1year followup period may be too short for conclusive determination of the longterm efficacy of blockers in the setting of acs . third , the stemi group and the uap group were underpowered to discriminate the benefit of blocker use . first , the nonrandomized nature of this observational study could have resulted in selection bias . although randomized controlled trials are considered the highest standard for evaluating treatment efficacy , observational studies can still provide unique and valuable insights into treatment effectiveness and generalizability in practice . our findings imply that the efficacy demonstrated in randomized clinical trials can be translated into tangible clinical benefits in the real world . second , a 1year followup period may be too short for conclusive determination of the longterm efficacy of blockers in the setting of acs . third , the stemi group and the uap group were underpowered to discriminate the benefit of blocker use . this large observational study has shown that the higher survival rate in patients following pci is associated with the appropriate use of blockers at discharge and this benefit is consistent in the patients with nstemi . this study was supported by grants from the national program on key basic research project ( 973 program ) ( no . 2012cb518004 ) , the national nature science foundation key project ( no . 91439203 ) , and the national health and family planning commission of china ( no . 201202025 , no .

we deposited os atoms on s- and se - doped boronic graphenic surfaces by electron bombardment of micelles containing 16e complexes [ os(p - cymene)(1,2-dicarba - closo - dodecarborane-1,2-diselenate / dithiolate ) ] encapsulated in a triblock copolymer . the surfaces were characterized by energy - dispersive x - ray ( edx ) analysis and electron energy loss spectroscopy of energy filtered tem ( eftem ) . os atoms moved ca . 26 faster on the b / se surface compared to the b / s surface ( 233 34 pms1versus 8.9 1.9 pms1 ) . os atoms formed dimers with an average os os distance of 0.284 0.077 nm on the b / se surface and 0.243 0.059 nm on b / s , close to that in metallic os . the os2 molecules moved 0.83 and 0.65 more slowly than single os atoms on b / s and b / se surfaces , respectively , and again markedly faster ( ca . 20 ) on the b / se surface ( 151 45 pms1 versus 7.4 2.8 pms1 ) . os atom motion did not follow brownian motion and appears to involve anchoring sites , probably s and se atoms . the ability to control the atomic motion of metal atoms and molecules on surfaces has potential for exploitation in nanodevices of the future .

there is a long history of using electron microscopes to image the motion of adatoms on a thin film , but only recent advances have made it feasible to study the chemistry of metals at the level of atomic resolution . in particular the thickness and regularity of the honeycomb network of graphene provides an exceptional support for the deposition of individual atoms and observation of their motion . however , imaging the dynamics of single atoms on graphenic materials , compared to biomacromolecules and small clusters , is technically difficult because of their subnanometric size and their fast motion . studies of the movement of mo and w atoms on graphene and their trapping by defective sites have been reported , as well as pd and te atoms which bind to the free edge states along graphene hole edges , although much of our current knowledge of such migratory behavior of single atoms relies on theoretical elucidation . recently we showed that electron beam irradiation of self - spreading polymer - encapsulated precious metal complexes can generate in situ - doped graphenic surfaces on which the dynamics of single metal atoms can be studied in real time using an aberration - corrected high resolution electron microscope . here we synthesize novel boron sulfur - doped and boron selenium - doped graphenic surfaces and study the atomic trajectories and rate of migration of single osmium atoms and individual os2 molecules . these experiments reveal the dramatic effect of two chalcogenide ( group 16 ) dopants s and se on osmium atom migration . we synthesized two graphenic surfaces ( supporting information figure s1 for a low magnification image of the b / s surface ) . the first was doped with heteroatoms boron and sulfur ( b / s ) following the procedure we described recently , while the second was doped with heteroatoms boron and selenium ( b / se ) . this involved irradiating osms - se micelles made of [ os(p - cymene)(1,2-dicarba - closo - dodecarborane-1,2-diselenate ) ] encapsulated in the triblock copolymer p123 with the electron beam of an aberration - corrected high resolution hr - tem - stem with a schottky thermal field - emission source ( 80 kev ; 1.9 pa cm or 7.6 10 electrons nm s ) . in general there were regions of the surface which were single - layer ( fast fourier transform in supporting information figure s1 ) and on which an idealized hexagonal lattice could be readily overlaid , although the surface was clearly inhomogeneous in some regions as expected from the presence of the b / s and b / se dopants . the presence of boron and chalcogen atoms as well as osmium was confirmed by a combination of energy - dispersive x - ray ( edx ) analysis and electron energy loss spectroscopy of energy filtered tem ( eftem ) ( supporting information figure s2 ) . we investigated single os atom migration by following the positions of individual os atoms on b / s ( over 390 s ; 40 frames with 10 s between each frame ) and b / se surfaces ( over 15 s ; 31 frames with 0.5 s between each frame ) . the positions of each atom were extracted from a series of images from three different areas of the tem grid . successive images were aligned , and the drift was compensated by using the plugin stackreg in fiji - imagej and by using a digital micrograph(tm ) script ( methods section ) . some sections of the surface are clearly hexagonal and of graphitic nature , whereas other areas are highly distorted by the dopants . moreover , the atoms in the surface also undergo motion by absorption of energy from the beam . density functional theory ( dft ) calculations have predicted that the presence of an os atom adsorbed on a graphene matrix disturbs the structure of graphene when located at bridge- and edge - sites . supporting information videos ( figures s3_video 1 and s4_video 2 ) show two examples of time - lapse tem images used to extract the coordinates of the individual atoms on both b / s and b / se surfaces . perhaps surprisingly , since the surface is doped and not homogeneous graphene , it was possible to correlate the atom trajectories with the individual positions of the atom on idealized hexagonal grids as shown in supporting information figures s5_video 3 for the b / s matrix and s6_video 4 for the b / se matrix . the experimental images highlight the existence of anchoring points on both surfaces , positions of apparent long residence times ( figures 1b , c ) . the analysis of the various trajectories suggests that the chalcogen atoms themselves induce the differences in atomic migration and have a direct impact on the trajectory of osmium atoms on the surface . this might involve direct os chalcogen binding , but the effects could also be propagated over a longer distance on the conjugated graphenic lattice . supporting information figure s7 shows the trajectory of an individual os atom on the b / s graphenic surface ( supporting information figure s7a ) , the superimposition of the atomic position on an ideal hexagonal monolayer graphene grid ( supporting information figure s7b ) , the apparent trapping sites ( in blue in supporting information figure s7c ) , and sites from which longer jumps than the average jump size are observed ( acceleration sites in red in supporting information figure s7c ) . from these data , three hypothetical chemical structures of the doped graphenic surfaces used in this study can be proposed ( supporting information figures s7d f ) : either the trapping sites are occupied by sulfur atoms and the accelerating sites are boron atoms ( supporting information figure s7d ) or the opposite ( supporting information figure s7e ) ; alternatively carbon atoms could act as acceleration sites , sulfur atoms act as trapping sites , and all the other sites where the osmium atom is seen moving to and from are occupied by boron atoms ( supporting information figure s7f ) . these three models are hypothetical since it is technically not possible to investigate the actual atomic composition of the surface site - by - site . positions , trajectory , and experimental hopping of an os atom on the b / s- and b / se - doped graphenic surfaces . ( a ) example of experimental tem images and the trajectory of an individual os atom on the b / s surface recorded at different irradiation times ( five frames extracted from a total of 40 pictures over a total irradiation time of 390 s ) . ( b and c ) illustrative trajectories of three individual osmium atoms on the b / s- and b / se - doped graphenic surfaces , respectively . owing to the high contrast of osmium atoms as compared to the graphenic surfaces , the extraction of the coordinates of the atom in each frame is readily achieved from a 3d surface projection for each tem picture ( a ) . ( d ) cumulative apparent distances covered by an individual os atom on b / s ( yellow ) and b / se ( blue ) surfaces . ( e ) apparent speed of motion of an individual os atom on b / s ( yellow ) and b / se ( blue ) surfaces between each frame ( t = 0 for first frame of each stack ) . ( f ) enlargement of the b / s ( yellow ) plot shown in ( e ) . have reported that w atoms are mobile on a graphitic surface but are trapped by defective sites . they showed that below 250 c , when the graphene lattice was heavily damaged by the beam , almost no jumps occurred and the w atoms were pinned by larger irradiation - induced lattice defects . in the range 250500 c , jumping of w atoms was observable , and escape from a trapping center could be induced thermally or by electron irradiation , a combination of thermal and beam effects which explains the observed small oscillations . in related work the same group observed the trapping of mo atoms at defect sites in carbon nanotubes and in graphene , and recently pb and te atoms have been trapped on amorphous graphene and at edge sites of holes in graphene . our experimental data are consistent with these previous observations and also show that such an escape by os atoms from anchoring sites , at a temperature close to ambient , is dependent on the nature of the graphenic dopants . we determined the apparent rate of migration of individual os atoms on the two multidoped graphenic surfaces ( average from observation of 10 single atoms ) . it should be noted that the real speed of motion of the atoms is at least the apparent monitored rate of migration , since we are limited by the speed of the camera and there could be more steps at intermediate times between frame captures . figure 1d shows the dependence of the cumulative path length covered by two individual os atoms ( blue : b / se surface ; yellow : b / s surface ) on irradiation time on the two surfaces . remarkably , the chalcogen dopant ( s vs. se ) dramatically influences the apparent rate of migration . the os atom travels an apparent distance of 3.5 nm in 15 s on the b / se surface , while the same distance is covered by the os atom in 390 s on the b / s surface . 26 higher speed of os atoms on the b / se surface compared to the b / s surface ( 233 34 pm s versus 8.9 1.9 pm s , supporting information table s1 ) is illustrated in figure 1e and in supporting information figure s8_video 5 . on both surfaces , the apparent rate of migration is not constant but varies over time , with significant autocorrelation ( figures 1e , f ) . this confirms the existence of anchors and suggests that the atom must receive a minimum energy from the high - energy electron beam to move from one anchoring site to another adjacent position . we found that an individual os atom required irradiation for 70 9 s on the b / s surface before hopping to another site , whereas on the b / se surface , only ca . we also investigated the dynamics of migration of two close os atoms on the b / s and b / se surfaces . we elucidated the trajectory of each atom ( figure 2a ) and thus the trajectory of the pair ( figure 2b , c ) . the atomic positions were extracted from a series of time - lapse tem images , as for individual atoms . supporting information figures s9_video 6 and s10_video 7 show examples for b / se and b / s surfaces , respectively . notably , the average os os distances of 0.284 0.077 nm on the b / se surface and 0.243 0.059 nm on b / s are both close to the os os distance in metallic os ( 0.27048 nm as the nearest neighbor distance at 20 c ) suggesting that these two os atoms form a true os2 diatomic molecule . furthermore , the two close os atoms undergo concerted migration on the multidoped graphenic surfaces . the dependence of the jumping pattern for each os atom shown in figure 2c on irradiation time is shown in figure 2e and confirms that the two atoms are indeed bonded . positions , trajectory , and experimental hopping of os2 molecules on the b / s- and b / se - doped graphenic surfaces . ( a ) hrtem images showing the position of the two os atoms on the b / s surface at different irradiation times ( time = 0 s corresponding to the first image of the stack ; scale bar = 0.5 nm ) . the black circles and the black dotted line show the alignment of the surface . ( b ) trajectory of the hopping of the two os atoms on the b / s surface over 390 s. ( c ) trajectory of the hopping of the two os atoms on the b / se surface over 15 s. ( d ) distances covered by each os atom of a diatomic molecule on b / s ( black and red ) and b / se ( light and deep blue ) surfaces . ( e ) jump size against time for each atom of the molecule on the b / se surface . similar to the movement of isolated individual os atoms , the distance traveled by each atom of the os2 molecule increases linearly with irradiation time on both surfaces ( figure 2d , supporting information figure s9_video 6 , figure s10_video 7 ) . the average apparent rate of migration of an os2 diatomic molecule on the b / s and b / se surfaces ( 7.4 2.8 pm s , and 151 45 pm s , respectively , supporting information table s1 ) was slightly slower ( 0.83 and 0.65 ) than for individual os atoms . again , as for single os atoms , the apparent rate of migration of os2 on the b / se - doped surface was dramatically faster ( ca . 20 ) than on the b / s - doped surface ( supporting information table s1 ) . we note that defects within graphenic layers can themselves migrate under the influence of an electron beam . although such effects can be quantified on pure graphene , they are more difficult to map on heavily doped lattices such as those studied here . it should be borne in mind however that such movements of carbons or dopants within the support layer could also influence the movement of os atoms , as is apparent in figure 2a . it is interesting to consider the possible magnetic states of os atoms on a graphenic surface . our dft calculations on an os atom on an ideal ( without defects or doping ) graphene subunit , an aromatic 19-ring c54h18 system ( supporting information figure s11 , table s2 ) , suggested that this model system is paramagnetic . lumo gap will go to zero , making it unlikely that paramagnetic states will be supported . experimental magnetic measurements present a challenge for future work . to further confirm that the doped graphitic surface plays a role in the observed os migration , we investigated whether the atoms follow brownian motion ( bm ) . in bm , each jump is independent and identically distributed according to a normal distribution with variance proportional to the time between observations . we looked for evidence of this in the data , consisting of the migration of five os atoms on each kind of each surface . first we checked that the size of the jumps in the x and y directions were the same ( supporting information figure s12 and table s3 ) , pooling together all of the jumps for the five separate atoms . there appeared to be no significant difference in the dynamics of migration on the sulfur - doped surface and only a very small difference on the selenium - doped surface . the histograms shown in figures 3a , b were observed to be similar , and as such x and y jumps were pooled together for further analysis . we also assumed that the os atoms were not drifting in any particular direction and so the mean jump would be zero , which also appeared to be confirmed by the data . next , we fitted a gaussian density to the pooled jump data and plotted a normal quantile quantile plot to assess deviations from normality ( figures 3c , d ) . analysis of the mechanism of hopping of individual os atoms on the two graphenic surfaces . ( a and b ) histograms of pooled jumps of individual os atoms on b / s and b / se surfaces , respectively . quantile plot of os atoms hopping on b / s and b / se surfaces , respectively . it is clear from these plots that there are more long - range jumps than would be expected for a brownian motion , but for completeness we also performed a shapiro wilk test of normality ; both the selenium- and sulfur - pooled data gave p - values below 0.001 , and so we can reject the hypothesis that brownian motion describes the movement of the os atoms . kinetic energy of the e - beam is the main driving force of the dynamic behavior of os atoms observed in tem . returning to the raw data , we see that no large jumps are captured for these two atoms . it is clear from supporting information table s3 that the jump sizes are larger on selenium - doped than on sulfur - doped graphene , but it is nonetheless interesting to investigate whether they come from similar distributions . supporting information figure s13 shows a comparative quantile although there is a small amount of deviation from the line in the lower tail , the plot does suggest that the distributions are indeed very similar . in conclusion , the new generations of electron microscopes with their atomic resolution capability and ultrafast cameras offer the possibility of imaging dynamic processes in real time . however , there is currently little reported data on such dynamic interactions at the level of the individual atom , owing , in particular , to the fast migration of single atoms on graphene . here , we have used a new synthetic methodology for the in situ formation of graphenic surfaces doped with boron and sulfur ( b / s ) or selenium ( b / se ) heteroatoms . the doping creates anchoring points for individual os atoms , slowing down their migration so that it is commensurate with the time scale of image capture on an aberration - corrected transmission electron microscope . we have imaged in real time the migration of individual os atoms and os2 dimers on these surfaces . the rate of movement is > 20 faster on the b / se - doped boronic graphenic surface compared to b / s . indirectly , through choice of the dopants , our methodology can provide control of atomic dynamics and might lead to a wide range of potential applications ( e.g. , patterning on surfaces , security labeling at the atomic level , sealing confidential documents ) . there is much scope for extending the fabrication to a wider range of doped - graphenic matrices , so as to further modify the surface dopants and the metal atom migration rates , and to the deposition of other metal atoms ( e.g. , other precious metals such as au , pt , and pd ) .

aim : the present study is aimed at delineation of different types of rugae in two different populations and developing a discriminant function for the same.materials and methods : a total of 940 subjects were included in the present study . the sample consisted of 466 subjects from south indian population and 474 from north indian population in the age group of 18 - 23 years . neo colloid easy flow( ) alginate impressions of maxillary arch were made and casts were immediately poured with type iv dental stone . a sharp graphite pencil was used to delineate the rugae and patterns were recorded according to the classification given by kapali et al . the association between different population and different sexes was analyzed with chi - square test and a stepwise discriminant function analysis was also performed to develop a discriminant formula.results:wavy , curved and straight rugae were the most common forms in both groups . chi - square analysis for association between rugae shape and population groups showed significant differences among all the rugae patterns at the p < 5% . chi - square analysis for assessing sex differences in the rugae shapes showed significant difference in straight , unification and circular type . five rugae shapes curved , wavy , nonspecific , unification and circular were selected for discriminant function.conclusion:the discriminant function equation obtained from the different rugae shapes in the present study was highly accurate enough to distinguish the southern and northern indian population with the classification accuracy of 87.8% . thus to identify a specific population , separate discriminant function formulae have to be developed . hence , the study of palatal rugae is one of the simple and reliable tools for population identification in forensic science .

palatal rugae or transverse palatine folds are irregular mucosal elevations present in the anterior third of the palate . it is asymmetrical and made from the lateral membrane of the incisive papilla , arranged in transverse direction from palatine raphae located in midsaggital plane . they are stable throughout life following completion of growth , though there is a considerable debate in this regard.[68 ] identification of a severely burnt edentulous body by rugae comparison to those on the victim 's old denture is a standing proof that rugae are stable in adult life . though odontometrics serves much in post mortem dental identification , palatal rugae proves to be great supplements in post mortem identification of edentulous and severely burnt decomposed individuals . odontometrical analysis , fingerprints and dna comparisons are probably the most used techniques , allowing secure identification . however , these can not be applied in certain cases and in such situations , different , simple and less known technique can be used for personal identifications such as palatal rugoscopy study of palatal rugae . in addition , rugae pattern may be specific to racial groups , facilitating population identification which is essential in mass disasters . racial profiling using intraoral features other than the teeth may have relevance in odonto- stomatological identification in india where , credible dental anthropological data is negligible . they provide a preliminary data on rugae shape between two populations in india and its effectiveness in identifying the populations using discriminant function analysis . the present study is performed using a larger sample size to validate the findings of previous studies and to provide a stronger evidence for forensic identification using palatal rugae . thus the palatal rugae are unique in its morphology of every individual having good stability with low utilization cost providing a concrete postmortem resistant evidence for forensic purpose . therefore the present study is aimed at delineation of different types of rugae in two different populations and developing a discriminant function for the same . a total of 940 subjects were included in the present study who belong to two geographically different populations . the sample consisted of 466 south indians from tamil nadu state and 474 north indians from uttar pradesh . the subjects were enrolled by simple random sampling from ksr group of educational institutions ( south india ) and up college of arts and science ( north india ) . the age group of the study subjects were 18 - 23 years , as there is a lack of consensus on rugae stability with respect to aging.[58 ] informed consent was obtained from all the enrolled subjects . exclusion criteria were palatal asymmetries , cleft palate ( or ) a history of palatal surgery . neo colloid easy flow alginate impression of maxillary arch were made and casts were immediately poured with type iv dental stone . a sharp graphite pencil was used to delineate the rugae and patterns were recorded according to the classification given by kapali et al . the categories were straight , wavy , curved , circular and if rugae had two arms it is unification . further , differentiation of unified rugae length based categorization was not considered in our study . the delineation of all the rugae patterns was performed by the same investigator . to estimate intra - observer variability , a stepwise discriminant function analysis was also preformed between the two different populations for different types of rugae to develop a discriminant formula , di = k+di1 z1 + di2 z2 .. dip zp di is discriminant function score , di is discriminant function co efficient , z is the score of the predictor variable and k is the discriminant function constant . the significance of rugae on population identification was observed for test of function using wilks ' lambda statistics . neo colloid easy flow alginate impression of maxillary arch were made and casts were immediately poured with type iv dental stone . a sharp graphite pencil was used to delineate the rugae and patterns were recorded according to the classification given by kapali et al . the categories were straight , wavy , curved , circular and if rugae had two arms it is unification . further , differentiation of unified rugae length based categorization was not considered in our study . the delineation of all the rugae patterns was performed by the same investigator . to estimate intra - observer variability , a stepwise discriminant function analysis was also preformed between the two different populations for different types of rugae to develop a discriminant formula , di = k+di1 z1 + di2 z2 .. dip zp di is discriminant function score , di is discriminant function co efficient , z is the score of the predictor variable and k is the discriminant function constant . the significance of rugae on population identification was observed for test of function using wilks ' lambda statistics . the occurrence of different rugae shapes in the two populations in the present study is presented in table 1 . circular rugae constituted less than 5% of rugae in the entire sample of 940 casts . a few non - specific rugae forms were observed . the intra - observer reliability calculated during the second examination after two months , revealed the kappa value to be 0.93 . hence , the observations made at two different moments showed a negligible difference and therefore were found reliable . frequency of different rugae shapes in southern and northern indians the incidence of all the rugae patterns were more in number in north indian population compared to that of south indian population except non - specific rugae [ table 1 ] . chi- square analysis for association between rugae shape and population groups showed significant differences among all the rugae patterns at the p < 5% [ table 2 ] . chi - square analysis for assessing sex differences in the rugae shapes showed significant difference in straight , unification and circular type [ table 3 ] . chi - square analysis for assessing differences in rugae shapes between southern and northern indians chi - square analysis for assessing sex differences in the rugae shape tables 4 - 6 show the rugae shape that entered , removed and contributed to the discriminant function analysis respectively . table 7 shows that the rugae shape that contributed to the discriminant function analysis were subjected to test of function with wilks ' lambda statistics and it showed overall significance among all rugae shapes . five rugae shapes curved , wavy , nonspecific , unification and circular were selected in five steps . curved rugae entered the analysis first , indicating they had the greatest ability to differentiate the population groups , followed by wavy , unification circular and non - specific rugae . straight did not contribute to the function , implying their inability to differentiate the groups . the best discriminant function was f = -4.372 + 0.901(curved ) + 0.375 ( wavy ) + 0.354(unification ) + 0.334(circular ) table showing the progress of variables entered the discriminant function analysis table showing the progress of variables removed from the discriminant function analysis step - wise discriminant function analysis of the different rugae shapes wilks ' lambda statistics table table 8 depicts the unstandardized and standardized coefficients , structure matrix , group centroids and sectioning point for the discriminant function . to determine the population group to which an unidentified individual belongs to , the number of each type of rugae shape is multiplied with the respective unstandardized coefficient and added to the constant . if the value obtained is greater than the sectioning point , the individual is considered as north indian ; if the value obtained is less than the sectioning point , the individual is considered as south indian . discriminant function coefficients for rugae shapes that entered the analysis let us consider an unidentified individual where the number of curved rugae = 2 , straight rugae = 3 , wavy rugae = 3 , unification = 1 , circular = 0 and nonspecific = 0 . multiplying the number of each rugae shape with the respective unstandardized coefficients and adding the constant , we obtain -4.372 + 0.901(2 ) + 0.375 ( 3 ) + 0.354(1 ) + 0.334(0 ) 0.289(0 ) = -1.09 . since this value is less than the sectioning point -0.04 , the individual is considered as south indian . the discriminant function 's accuracy in population identification the expected accuracy of identifying an individual from a different population is derived from the entire sample in the original result . this may be biased since the plaster casts from which the function was derived are themselves tested for population origin . to overcome this bias , jack - knifing ) where , a function is derived from all but one cast in the sample and the excluded cast tested for population origin . this procedure was performed for each of the 940 casts , i.e. a function was derived from 939 of the 940 dental casts and the 940 cast tested for population origin . classification results consequently , the cross - validated accuracy of the function ( 87.8% ) is lower than that of the original results ( 87.9% ) . while cross - validation may be a theoretical construct and not a true accuracy , it gives a more realistic indication of the precision of the discriminant function . human identification is one of the most challenging subjects that man has been confronted with . sometimes it becomes necessary to apply lesser known and unusual techniques like cheiloscopy and palatoscopy . thomas and kotze studied the rugae patterns of 6 south african populations to analyse the interracial difference . their results indicate that rugae were unique to each ethnic group and can be used successfully as a medium for genetic research . hence , this shows the applicability of rugae in population differentiation and it has been revealed in the present study also . hauser et al . compared the rugae patterns of swazi and greek populations and observed definite differences in the rugae pattern between the two populations . it was evident that the degree of development of rugae was dependent on the growth of the palate . according to english et al . the observation of palatal rugae is a subjective phenomenon and it poses a problem when interpreted at two different moments . hence , the intra observer variability was calculated and it was found to be k = 0.93 ; which shows that the difference attributed to the observation is practically inexistent . the present study showed more than 42% of wavy , curved and straight rugae forms in each population . this is consistent with finding of australian aborigines , caucasians , indians ( southern and western ) for wavy and curved rugae patterns . but the straight form of rugae was also found to be higher in the present study . unification , non - specific and circular was less common . however , circular forms of rugae were very few in number . thomas and kotze and nayak et al . in their study to identify two genetically similar groups , obtained a jack - knife accuracy of 61.8% and 70% respectively . the classification of jack - knife accuracy obtained from the present investigation , observing two relatively similar populations , showed a relatively higher accuracy of 87% when compared to the previous studies and hence , the type of rugae patterns used has got discriminating ability . the rugae shapes which are considered for classification in the present work are discrete variables when compared to that of rugae dimensions ( continuous variables ) used in previous classifications . when genetically similar populations were considered for differentiation continuous variables such as rugae measurements may have its limitations . the present analysis has revealed that incidence of all the rugae shapes were significantly higher in northern indian population when compared to southern indian population except for non - specific rugae . but the rugae shapes used as a predictor variable for southern indian population in the present study showed a variable rate of incidence when compared to the previous study . hence , we believe that even variations within the same country ( uttar pradesh and tamilnadu ) can show significant rugae pattern variations . the predictor variable entered the discriminant function in the present study showed a variance when compared to the study conducted by nayak et al . more number of predictor variables entered the discriminant function in our study which is attributed to the usage of larger sample size for population differentiation . the discriminant function equation obtained from the different rugae shapes in the present study was highly accurate enough to distinguish the southern and northern indian population with the classification accuracy of 87.8% . thus to identify a specific population , separate discriminant function formulae have to be developed . hence , the study of palatal rugae is one of the simple and reliable tools for population identification in forensic science .

selective serotonin reuptake inhibitor medications are one of the most common treatments for mood disorders . in humans , these medications are taken orally , usually once per day . unfortunately , administration of antidepressant medications in rodent models is often through injection , oral gavage , or minipump implant , all relatively stressful procedures . the aim of the present study was to investigate how administration of the commonly used ssri , fluoxetine , via a wafer cookie , compares to fluoxetine administration using an osmotic minipump , with regards to serum drug levels and hippocampal plasticity . for this experiment , adult female sprague - dawley rats were divided over the two administration methods : ( 1 ) cookie and ( 2 ) osmotic minipump and three fluoxetine treatment doses : 0 , 5 , or 10 mg / kg / day . results show that a fluoxetine dose of 5 mg / kg / day , but not 10 mg / kg / day , results in comparable serum levels of fluoxetine and its active metabolite norfluoxetine between the two administration methods . furthermore , minipump administration of fluoxetine resulted in higher levels of cell proliferation in the granule cell layer ( gcl ) at a 5 mg dose compared to a 10 mg dose . synaptophysin expression in the gcl , but not ca3 , was significantly lower after fluoxetine treatment , regardless of administration method . these data suggest that the administration method and dose of fluoxetine can differentially affect hippocampal plasticity in the adult female rat .

plasticity in the hippocampus has long been implicated in the etiology of mood disorders and regulation of stress [ 13 ] . recent work has also suggested that the actions of antidepressant medications , such as selective serotonin reuptake inhibitors ( ssris ) , on hippocampal plasticity may play an important role in alleviating symptoms of depression [ 46 ] . serotonin , itself , is an important neurotransmitter involved in regulating the rate of hippocampal neurogenesis during adulthood , with lower levels of serotonin reducing the number of newly formed neurons in the hippocampus and elevated levels of serotonin increasing the rate of cell proliferation [ 711 ] . more importantly , increased serotonin levels , following ssri treatment , significantly upregulate adult hippocampal neurogenesis [ 7 , 1214 ] and it has been suggested that the actions of ssri medications on hippocampal plasticity ( morphology and neurogenesis ) may be important for alleviating the effects of stress on affect - related behaviours [ 4 , 12 , 15 , 16 ] . although there has been a substantial amount of work investigating the effects of ssri medications on plasticity in the hippocampus and their role in treating depressive - like behavior , very little work has taken into account the effects of these medications on hippocampal plasticity in the adult female . with women being 2 - 3 times more likely to suffer from depression , it is important to include females in research related to depression [ 18 , 19 ] . recent work focusing on females has shown that chronic administration of imipramine , a tricyclic antidepressant , to intact female rats exhibiting depressive - like behavior increases cell proliferation in the dentate gyrus . however , others have shown that administration of fluoxetine , a popular ssri , has no effect on cell proliferation or neurogenesis in the hippocampus of adult female rats [ 21 , 22 ] , but it does trigger spine formation in the hippocampus of the adult female rats . more work is also needed to better mimic the clinical administration method of ssris . in humans , unfortunately , the typical administration of antidepressant medications in rodent models of depression is invasive and stressful , with administration being done most often via injection , oral gavage , or minipump implant [ 24 , 25 ] . administration method alone affects the metabolism and effects of the medication [ 26 , 27 ] . interestingly , recent research in animal models investigating effects of environmental teratogens on development has demonstrated that administration of a solution injected in a wafer cookie may be as effective as injecting a solution into the animal and thus would reduce the stress of the animal , particularly for long - term daily treatment ( greater than 4 weeks ) . however , it remains to be determined whether this method of drug administration is effective for ssris and is comparable to other methods of antidepressant administration that are commonly used . the aim of this project was to understand the role of fluoxetine treatment in hippocampal plasticity in the adult female rat , using two methods of ssri administration . more specifically the proposed study aims to ( 1 ) determine the blood concentration levels of fluoxetine and its primary metabolite , norfluoxetine , in response to two administration types , cookie and minipump , at three fluoxetine doses ( 0 , 5 , and 10 mg / kg / day ) and ( 2 ) determine the effect of these forms of fluoxetine administration on neural and synaptophysin expression in hippocampus . this study will provide important information toward the goal of generating a new and reliable method of antidepressant administration that eliminates the need for surgery , lowers the stress to the animal , can be readily applied to animal models , provides accurate drug levels , and most closely models antidepressant administration in humans . this work will result in a more accurate understanding of the neurobiological and physiological impact of ssris in preclinical models , ultimately improving our understanding of how to treat mood disorders in humans . thirty - six intact adult female sprague - dawley rats ( 250300 g ; charles river laboratories , france ) were used in the present study . rats were kept under standard laboratory conditions in a 12 h:12 h light / dark schedule ( lights on at 07:00 h ) , initially housed in pairs in clear polyurethane bins ( 48 cm 27 cm 20 cm ) with ad libitum access to rat chow ( sniff ) and tap water . females were randomly divided into two conditions ( cookie or minipump ) with three dose options : ( 1 ) fluoxetine ( 10 mg / kg / day ) , ( 2 ) fluoxetine ( 5 mg / kg / day ) , and ( 3 ) vehicle ( 0 mg / kg / day ) , for a total of 6 groups . all experiments were approved by the animal ethics board of maastricht university in accordance with dutch governmental regulations ( dec 2010 - 146 ) . all efforts were made to minimize the pain and stress levels experienced by the animals . on days 1 - 2 , females in the cookie treatment groups were trained for oral ingestion of the medication treatment . for training , females were fed 1/9th of a vanilla wafer cookie ( crousti fondante , delacre , belgium ) , filled with saline . after cookie training , females in the cookie groups were fed a cookie filled with fluoxetine ( fagron , belgium : 5 mg / kg or 10 mg / kg ) dissolved in vehicle ( 25% propylenediol in saline ) or vehicle solution once per day between 8:00 and 9:00 am after cookie feeding females were monitored to ensure that cookies were eaten . females in the minipump treatment group were administered fluoxetine or vehicle via osmotic minipumps ( alzet osmotic pumps , 2ml2 , charles river , the netherlands ) for 2 weeks . minipump implants were filled with either fluoxetine ( fagron , belgium : 5 mg / kg / day or 10 mg / kg / day ) dissolved in vehicle ( 25% propylenediol in saline ) or with vehicle as previously described [ 22 , 3033 ] . minipumps were implanted subcutaneously in the dorsal region , while females were under mild isoflurane anesthesia . the weight of a full 2ml2 minipump was approximately 7.5 g. thirty minutes prior to the minipump implant , the nsaid carprofen ( dose : 2.55 the first drop of blood was removed ; the second drop was placed on a test strip ( glucocard x - sensor test strips ) for immediate glucose measurement with a hand - held glucose meter ( glucocard tm x - meter , a. menarini diagnostics , benelux , n.v . , valkenswaard , the netherlands ) . to determine serum levels of fluoxetine and its active metabolite , norfluoxetine , blood collection , via the tail vein , from females treated with fluoxetine was taken twice on days 6 and 10 between 8 - 9 am , after cookie feeding , and 24 pm . blood from vehicle - treated females was taken on day 6 between 8 - 9 am only . at decapitation , blood samples were stored at 4c overnight and centrifuged at 10,000 g for 10 minutes . serum was collected and stored at 80c until analysis . to investigate whether estradiol levels affected measures of cell proliferation , 17-estradiol ( e2 ) was measured in a subset of animals that were randomly selected ( 18 in total ) . all samples were run in duplicate using commercially available 17-oestradiol ( e2 ) i radioimmunoassay ( ria ) kits from mp biomedicals ( mp biomedicals , belgium ) . the lowest detection limit for e2 was 1.4 pg / ml . drug concentrations were determined from serum using liquid chromatography coupled with mass spectrometry ( lc - chip - ms / ms ) that was used as previously described [ 22 , 33 , 34 ] . briefly , the chromatographic separation was achieved on a 1200 series lc - chip system ( agilent technologies , germany ) using an ultrahigh capacity chip including a 500 nl trapping column and a 150 mm 75 m analytical column , both packed with a zorbax 80sb 5 m c18 phase ( agilent technologies ) . the mobile phase was composed of h2o / fa ( 100 : 0.1 , v / v ) ( a ) and acn / h2o / fa ( 90 : 10 : 0.1 , v / v / v ) ( b ) and used in gradient elution mode . mass spectrometric detection was performed using a 6340 ion trap equipped with a nanoelectrospray ionization source operating in positive mode ( agilent technologies , waldbronn ) . finally , an oasis elution mcx 96-well plate ( waters , uk ) was used to prepare the samples for the analysis . fluoxetine and norfluoxetine levels were averaged across days to provide one morning and one afternoon value . fourteen days after treatment , females were deeply anesthetized with sodium pentobarbital , weighed , and rapidly decapitated . following extraction , the right hemisphere of the brains was stored at 4c in 4% paraformaldehyde for 24 h , then cryoprotected in 30% sucrose / phosphate - buffered saline solution for up to one week , frozen on dry ice , and kept at 80c . brain tissue was sliced in 40 m sections on a cryostat ( leica ) . the level of cell proliferation in the granule cell layer and subgranular zone ( gcl / sgz ) of the hippocampus was assessed using an endogenous marker for cell proliferation , ki67 . every 6th section throughout the right hippocampi was stained as previously described [ 35 , 37 ] . sections were blocked with h2o2 and incubated overnight in rabbit anti - ki67 ( 1:500 ; vector laboratories ) or blocked with h2o2 and ngs and incubated overnight in mouse antisynaptophysin ( 1:500 ; sigma aldrich ) . sections were then incubated for 2 h in biotinylated donkey anti - rabbit ( 1:500 ; jackson immunoresearch laboratories , west grove , pa ) or biotinylated goat anti - mouse ( 1:200 vector ba-9200 ) secondary antibody . brain sections were further processed by using the avidin - biotin complex ( abc elite kit ; 1:1000 ; vector laboratories , usa ) . dab ( 3,3-diaminobenzidine ) peroxidase substrate kit ( sk-4100 , vector laboratories ) was used as a substrate to obtain a color reaction . sections were mounted on gelatin - coated slides and dried overnight , dehydrated , stained with cresyl violet ( ki67-ir only ) , and coverslipped with permounttm ( fisher scientific , usa ) . the number of ki67 immunoreactive ( -ir ) cells in granule cell layer / subgranular zone ( gcl / sgz ) was counted under 40x objective using a nikon microphot sa microscope . cells were considered ki67-ir if they were intensely stained and exhibited medium round or oval nuclear bodies . ki67-ir cells were counted on half of every 6th section throughout the entire hippocampus . for representative ki67-ir cells in the gcl / sgz of the hippocampus , see figure 1 . three dorsal sections of the hippocampus , located between stereotaxic coordinates bregma 2.64 mm to 4.92 mm , were analysed per animal for synaptophysin - immunoreactivity by an observer blind to conditions . photomicrographs were taken for two areas within the ca3 and gcl / sgz of the hippocampus from each of the three sections ( e.g. , see figure 1 ) for a total of 6 photomicrographs per area . immunoreactivity was examined under 40x objective using a nikon microphot sa and nikon ds - qi1mc camera with nikon nis elements f4.00 software . the software imagej64 ( wayne rasband , nih , bethesda , md , usa ) was used for quantification of optical densities of synaptophysin . the relative optical density was defined as the difference between optical density ( grey level ) measures after calibration within the area of interest and in an equivalent adjacent area ( background ) . for representative photomicrographs of synaptophysin density , the fluoxetine and norfluoxetine levels were analyzed using repeated - measures analysis of variance tests ( anova ) with administration type ( cookie versus minipump ) and dose ( 0 , 5 , or 10 mg / kg ) as the between - subjects factors . repeated measure anovas were also used to assess synaptophysin density in the ca3 and gcl / sgz . anovas were conducted on the total number of ki67 cells in the gcl , the percent change in ki67-ir cells from controls , body weight , and glucose levels with administration type ( cookie versus minipump ) and dose ( 0 , 5 , or 10 mg / kg ) as the between - subjects factors . any effects of estradiol on the number of ki67-ir and synaptophysin - ir cells were controlled for . for serum fluoxetine and norfluoxetine levels there was a significant interaction between administration type ( cookie , minipump ) and dose ( 5 mg / kg / day , 10 mg / kg / day ) ( fluox : f(1,20 ) = 7.95 , p = 0.012 , norfluox : f(1,20 ) = 21.16 , p = 0.0002 ) with significantly higher serum levels of fluoxetine / norfluoxetine in the animals receiving 10 mg / kg / day of fluoxetine via minipump ( 0.000001 < p < 0.0005 ; figure 2 ) . there was also a main effect of time ( fluox : f(1,20 ) = 57.70 , p = 0.000001 , norfluox : f(1,20 ) = 7.14 , p = 0.015 ) with morning levels of fluoxetine / norfluoxetine being significantly lower than afternoon levels . there was a main effect of administration method with minipump administration of fluoxetine resulting in significantly higher serum levels of fluoxetine / norfluoxetine compared to cookie administration ( fluox : f(1,20 ) = 14.68 , p = 0.001 , norfluox : f(1,20 ) = 43.46 , p = 0.000001 ) . there was also a significant main effect of dose on norfluoxetine levels ( f(1,20 ) = 22.98 , p = 0.00011 ) . at sacrifice , serum from trunk blood revealed significantly higher levels of fluoxetine / norfluoxetine in the animals receiving 10 mg / kg / day of fluoxetine via minipump ( 0.00001 < p < 0.03 ; interaction effect fluox : f(1,20 ) = 4.52 , p = 0.046 , norfluox : f(1,20 ) = 14.06 , p = 0.0013 , figure 2 ) . there were also main effects of dose ( fluox : f(1,20 ) = 8.29 , p = 0.0093 , norfluox : f(1,20 ) = 19.84 , p = 0.00024 ) and a main effect of administration type ( norfluox : f(1,20 ) = 10.44 , p = 0.0042 ) on drug levels in trunk blood at sacrifice . there were no other main or interaction effects ( 0.24 < p < 0.91 ) . as expected , vehicle - treated animals did not have detectable serum levels of fluoxetine or norfluoxetine . minipump animals receiving the 5 mg / kg / day dose of fluoxetine had significantly more ki67-ir cells in the gcl compared to minipump animals receiving the 10 mg / kg / day dose , when looking at overall change from baseline ( controls ) ( f(1,9 ) = 9.64 , p = 0.013 ; n = 5/group ) . there were no other significant effects of fluoxetine dose or administration type on total number of ki67-ir cells in the gcl / sgz ( 0.3 < p < 0.7 ; figure 3 ) . there was also no effect of estradiol levels on number of ki67-ir cells in the gcl / sgz ( p = 0.98 ) . synaptophysin density in the gcl / sgz was significantly greater in vehicle - treated animals , regardless of administration method ( 0.000001 < p < 0.03 , figure 4 ; region ( ca3 , gcl ) by dose interaction for synaptophysin density : f(2,30 ) = 8.48 , p = 0.0012 ) . synaptophysin density was also significantly greater in the gcl / sgz than in the ca3 ( main effect of region : f(1,30 ) = 40.35 , p = 0.000001 ) . there were no other significant main or interaction effects between groups in synaptophysin density and no effect of estradiol levels on synaptophysin density ( 0.15 < p < 0.99 ; table 1 ) . as expected , animals fed with the cookie had significantly elevated blood glucose levels compared to animals with minipump implants ( f(1,30 ) = 8.0435 , p = 0.008 ; table 2 ) ; however , these levels were still within the physiological range . for body weight measurements there was no significant main effect of administration type or dose on overall change in weight ( 0.29 < p < 0.58 ) . findings of the present study show that two weeks of fluoxetine treatment results in a significant decrease in synaptophysin expression in the dentate gyrus , but not the ca3 region , of adult female rats . in turn , we found that administration method ( cookie versus minipump ) and fluoxetine dose differentially affected hippocampal cell proliferation , with only females receiving the 5 mg / kg dose via a minipump having increased cell proliferation in the gcl compared to females receiving the 10 mg / kg dose via a minipump . furthermore , administration method differentially affected circulating levels of fluoxetine and its active metabolite , norfluoxetine , with the greatest levels of fluoxetine being evident at a 10 mg / kg dose administered via minipump . in the present study , fluoxetine treatment significantly decreased synaptophysin expressionin the dentate gyrus and had no effect on synaptophysin expressionin the ca3 region of the hippocampus . to our knowledge there is no previous research on the effects of fluoxetine treatment on synaptophysin expression in adult female rats . however , previous research in adult male rats has shown that 7 days of fluoxetine treatment significantly increases synaptophysin mrna levels in the granule cell layer of the hippocampus ; however , they did not measure protein levels . work in hippocampal cell culture , under toxic conditions , and in male ts65dn mice ( model of down syndrome ) also shows that fluoxetine can rescue or improve synaptophysin expression [ 40 , 41 ] . in addition , the decrease in synaptophysin expression in the dentate gyrus of female rats with fluoxetine administration is perhaps counterintuitive given the general idea that ssri medication enhances hippocampal neurogenesis [ 6 , 42 ] . however , it is well documented in males only that ssri medications increase hippocampal plasticity and alleviate depressive - like behaviors , suggesting a significant role of estradiol and progesterone on the effects of ssri medications in adult females . further neurochemical and behavioural data are needed to fully understand the functional significance of ssris on hippocampal plasticity in the adult female . in the present study we did not find a significant effect of fluoxetine treatment of synaptophysin expression in the ca3 region of the hippocampus . previous work has shown that fluoxetine administration ( 5 mg / kg ) significantly increases pyramidal spine formation in both the ca1 and ca3 regions of the hippocampus of adult female rats , with effects in the ca1 region being evident after 5 days of fluoxetine administration and effects in the ca3 region being evident after 2 weeks of fluoxetine administration . however , previous work in adult male rats shows no effect of fluoxetine treatment ( 10 mg / kg ) on synaptophysin mrna density . discrepancies between the present study and the previous work in females may be due to methodological techniques as hajszan et al . administered fluoxetine via intraperitoneal injections and used electron microscopy to quantify spine densities , whereas , in the present study , fluoxetine was administered via a cookie or minipump and synaptophysin immunohistochemistry was measured . furthermore , hajszan et al . used ovariectomized female rats whereas female rats in the present study were cycling . estradiol alone is known to have marked effects on spine density , particularly in the ca1 region of the hippocampus [ 4447 ] , and has been shown to increase serotonin metabolite levels in the ca3 region of the hippocampus . in addition , estradiol has been shown to upregulate serotonin synthesis in the dorsal raphe in a similar manner to fluoxetine . recent work suggests that the effects of fluoxetine on spine density in adult female rats are only evident in ovariectomized females and when the endogenous actions of estradiol on hippocampal spine density are disturbed . thus , there are likely marked interactions between circulating estradiol levels and the effects of fluoxetine on spine density in the hippocampal formation . in the present study , we found effects of fluoxetine on hippocampal cell proliferation only after administration of fluoxetine via minipump . here we show that after 2 weeks of minipump administration females receiving the 5 mg / kg dose of fluoxetine had significantly more proliferating cells in the gcl compared to females receiving the 10 mg / kg via minipump . this work shows that fluoxetine levels can differentially affect cell proliferation in the adult female rat . these findings also replicate previous work showing that fluoxetine ( 5 mg / kg ) has no effect on cell proliferation in the hippocampus of adult female rats when compared to controls [ 21 , 22 ] . interestingly , previous work in adult male rats shows that 2 weeks of fluoxetine administration ( 7 mg / kg ) via minipump , as in the present study , increases hippocampal cell proliferation . this work and a previous one point to marked sex differences in the effect of fluoxetine on hippocampal plasticity . sex / gender must be taken into consideration when investigating neurobiological effects of ssri medications , particularly as these medications are more often used to treat depression in women . apart from sex / gender , exposure to stress and changes in circulating corticosterone levels also play an important role in the effects of fluoxetine on hippocampal neurogenesis [ 22 , 26 ] . for example , previous work shows the effect of fluoxetine treatment on hippocampal neurogenesis in the adult female is markedly increased in females exposed to stress . in the present study fluoxetine administration method differentially affected measures of hippocampal plasticity and also serum fluoxetine and norfluoxetine levels . here we show that serum fluoxetine and norfluoxetine levels , in a dose of 5 mg / kg , do not markedly differ between cookie and minipump administration . however , serum fluoxetine and norfluoxetine levels , after a fluoxetine dose of 10 mg / kg , were significantly elevated in animals treated with a minipump . this difference between administration methods in serum levels of fluoxetine at a higher dose is likely due to metabolism of fluoxetine by cytochrome p450 enzymes in the liver after oral ( cookie ) administration , thus leading to lower circulating levels of fluoxetine and norfluoxetine . in higher doses it may be advantageous to administer fluoxetine twice a day in a cookie in order to increase the circulating levels of fluoxetine and norfluoxetine . we have recently used a twice - a - day cookie administration method ( total fluoxetine dose 10 mg / kg ) during the postpartum period and shown significant effects on offspring development . others have also shown that voluntary fluoxetine administration in a cookie dough ball is an effective and noninvasive technique to chronically administer this , and potentially other , medication . thus , voluntary ssri medication administration is possible and may be a valuable way to further understand how these medications affect neurobiological processes . the present study investigated the effect of fluoxetine , a common ssri antidepressant medication , on hippocampal plasticity in the adult female rat . main findings show that two weeks of fluoxetine treatment results in a significant decrease in synaptophysin expression in the dentate gyrus , but not the ca3 region , of the hippocampus . in turn , administration method ( cookie versus minipump ) and fluoxetine dose ( 5 mg versus 10 mg ) differentially affected hippocampal cell proliferation in the gcl , with the females receiving the 5 mg / kg dose of fluoxetine via minipump having significantly more proliferating cells compared to females receiving the 10 mg / kg via minipump ( when investigating overall change from baseline ) . furthermore , administration method differentially affected circulating levels of fluoxetine and its active metabolite , norfluoxetine , with the greatest levels of fluoxetine being evident with a 10 mg / kg dose given via minipump .

xenotransplantation offers a potentially unlimited source for tissues and organs for transplantation , but the strong xenoimmune responses pose a major obstacle to its application in the clinic . in this study , we investigate the rejection of mouse peripheral nerve xenografts in rats . severe intragraft mononuclear cell infiltration , graft distension , and necrosis were detected in the recipients as early as 2 weeks after mouse nerve xenotransplantation . the number of axons in xenografts reduced progressively and became almost undetectable at week 8 . however , mouse nerve xenotransplantation only led to a transient and moderate increase in the production of th1 cytokines , including il-2 , ifn- , and tnf-. the data implicate that cellular immune responses play a critical role in nerve xenograft rejection but that further identification of the major effector cells mediating the rejection is required for developing effective means to prevent peripheral nerve xenograft rejection .

because peripheral nerve tissue has very limited spontaneous regenerative capacity , the development of strategies to facilitate axonal regeneration is highly important for treatment of peripheral nerve injuries [ 13 ] . although autotransplantation is considered the gold standard approach for bridging nerve gaps , autogenous grafts provide only a limited source of graft material and graft harvesting may result in secondary site morbidity . allogeneic and xenogeneic nerve allografts provide an attractive alternative for patients , for whom the use of autograft is infeasible or undesirable . however , immunological rejection poses a major obstacle to clinical allogeneic and xenogeneic transplantation . unlike organ transplantation , in most cases peripheral nerve injury is not life - threatening , and therefore , peripheral nerve xenotransplantation can only be considered when its benefit is weighted against the risk associated with the therapy , such as the side effects of immunosuppressive drugs . schwann cells and fibroblasts both express mhc antigens and play an important role in eliciting immunological rejection after allogeneic nerve transplantation . however , immunologic responses following nerve xenotransplantation are relatively poorly understood . in this study , we investigate the immunological response and graft survival in rats after nerve xenotransplantation from mice . we show that mouse nerve xenografts were vigorously rejected in rat recipients , and that the rejection was associated with severe intragraft infiltration by mononuclear cells despite only a transient and moderate increase in th1 cytokines detected in the recipients . female sd rats ( weighed between 150 and 200 g ) and adult female balb / c mice were used as the recipients and donors , respectively . all animals were purchased from the second affiliated hospital of harbin medical university ( haerbin , china ) . all surgical procedures and postoperative care of the animals all surgical procedures were performed under anesthesia with ketamine ( 50 mg / kg ; i.m . ) . to prepare donor nerve grafts , the mouse sciatic nerve was exposed through a dorsal gluteal muscle splitting incision , and a segment ( 1 cm ) of sciatic nerve was harvested and used immediately . the skin over the recipient right hindlimb was incised , and the muscle was bluntly dissected to expose the superficial peroneal nerve , and a 1 cm gap was created . the mouse sciatic nerve graft was interposed to the transected nerve and immediately repaired with 10 - 0 nylon epineurial sutures . for autograft recipients , the right superficial peroneal nerve was exposed in an identical fashion , transected with microscissors , and tension - free repair of the nerve gap ( 1 cm ) was then performed with 10 - 0 nylon epineurial sutures . sera were collected from recipient mice at various times , and the levels of il-2 , il-4 , ifn- , and tnf- were determined using elisa kits ( westang biotechnology , shanghai , china ) according to the manufacturer 's instructions . animals were sacrificed at various time points after transplantation , and the nerve grafts were harvested and cut in half horizontally . one segment was immersed in a 10% formaldehyde solution , dehydrated in ethanol , and embedded in paraffin . the paraffin nerve sections were stained with hematoxylin and eosin ( h&e ) and by immunohistochemistry for s-100 . briefly , sections for s-100 staining were first incubated with polyclonal rabbit anti - s-100 antibodies ( westang biotechnology ) , washed , and followed by staining with biotinylated goat antirabbit antibodies ( westang biotechnology ) . another segment of the nerve graft was immersed in a 3% cold gluteraldehyde solution , postfixed with osmium tetroxide , dehydrated in ethanol , and embedded in araldite 502 . ultrathin sections were obtained with an ultramicrotome ( ultracut e , reichert - jung , vienna , austria ) , stained with lead citrate and uranyl acetate , and observed under a jeol transmission electron microscope ( jem-1220 ; jeol ltd . , tokyo , japan ) . differences between group means were tested using student 's t - test by microsoft excel software . recipient rats were sacrificed 2 , 4 , and 8 weeks after transplantation and the nerve grafts were examined macroscopically and histologically ( n = 5 per group at each time point ) . all autografts showed no distension or adhesion to the ambient tissues throughout the 8-week observation period . however , xenografts removed at week 2 markedly dilated and adhered to the surrounding tissues . while some xenografts showed moderately reduced distension at week 4 , graft adhesion to the ambient tissues was progressively overwhelming in all animals during the 8-week observation period . histological examination at weeks 2 and 4 revealed very mild mononuclear cell infiltration with otherwise normal peripheral nerve structure in autografts ( figures 1(a)-1(b ) and 2(a)-2(b ) ) . however , most autografts showed signs of wallerian degeneration at week 8 , with moderate axonal degeneration and nerve fiber fractions ( figures 1(c ) and 2(c ) ) . consistent with the light microscopic analysis , electron microscopy confirmed the preservation of normal fascicular structure in autografts at weeks 2 and 4 ( figures 3(a)-3(b ) ) , but schwann cell cytoplasmic degeneration , chromatin condensation , and nuclear collapse were identified in the autografts at week 8 ( figure 3(c ) ) . severe mononuclear cell infiltration , distension and necrosis were detected in all xenografts as early as 2 weeks after transplantation ( figures 1(d)1(f ) ) . the number of axons in the grafts markedly reduced by week 2 and became almost undetectable at week 8 ( figures 2(d)-2(e ) ) . electron microscopy revealed inflammatory infiltrates in nerve fibers , schwann cell nuclear pycnosis and mitochondrial damage , and progressive destruction and loss of myelinated fibers in all xenografts ( figures 3(d)2(f ) ) . sera were collected from xenograft recipients prior to 2 , 4 , and 8 weeks after transplantation , and the levels of il-2 , il-4 , ifn- , and tnf- were determined by elisa ( table 1 ) . while the serum levels of il-2 , ifn- , and tnf- at week 2 were statistically higher in the xenograft recipients than in the control rats , these increases were relatively less marked and transient . the serum levels of all these cytokines in the xenograft recipients returned to normal by week 4 after transplantation . xenotransplantation provides a possible solution to the severe shortage of allogeneic donors that limits clinical transplantation [ 57 ] , but the virulent xenoimmune responses pose a strong barrier to clinical xenotransplantation . transplantation of discordant xenografts results in hyperacute rejection due to the presence of natural antibodies ( nabs ) in the recipient sera . the major nabs that mediate hyperacute rejection are those which recognize the 1 , 3gal antigens [ 8 , 9 ] . however , anti-1 , 3gal abs are unlikely to mediate the rejection of nerve xenografts , as neural cells do not express 1 , 3gal . thus , cellular immune responses , which play a critical role in nerve allograft rejection , are likely to be more important than humoral immune responses in nerve xenograft rejection . indeed , nerve xenograft rejection has been relatively poorly studied compared to other cellular or organ xenografts . here , we observed that mouse nerve xenografts can be vigorously rejected in rats , and the rejection is associated with a severe mononuclear cell infiltration in the grafts . schwann cells are critical for nerve regeneration , but also the major cells trigger the rejection of nerve allografts because of their mhc expression . it has been reported that cold preservation can decrease the number of schwann cells and reduce the immunogenicity of nerve allografts [ 13 , 14 ] . although recipient schwann cells may migrate into the graft and support axon regeneration , donor schwann cells in the graft are required for the regeneration of a long nerve graft . given the possibility that the recipient schwann cells may be less efficient in migrating into nerve xenografts than into allografts , cold preservation could have an even greater limitation for preventing nerve xenograft rejection . although histological analysis confirmed severe mononuclear cell infiltration and complete nerve xenograft rejection , the recipient rats showed only a transient and moderate increase in th1 cytokine production . further studies are needed to determine whether peripheral nerve xenografts are less efficient than other types of xenografts in eliciting the systemic t cell immune responses . it is possible that other effector cells , such as th17 cells , nk cells , and macrophages , may play an important role in the rejection of mouse nerve xenografts in rats . previous studies have shown that nk cells and macrophages contribute significantly to xenograft rejection due to the genetic incompatibility in mhc class i and cd47 [ 17 , 18 ] , which are needed for inhibiting nk cell and macrophage activation , respectively . further studies are clearly needed to identify the major effector cells in the rejection of peripheral nerve xenografts .

positional cloning in bread wheat is a tedious task due to its huge genome size and hexaploid character . bac libraries represent an essential tool for positional cloning . however , wheat bac libraries comprise more than million clones , which makes their screening very laborious . here , we present a targeted approach based on chromosome - specific bac libraries . such libraries were constructed from flow - sorted arms of wheat chromosome 7d . a library from the short arm ( 7ds ) consisting of 49,152 clones with 113 kb insert size represented 12.1 arm equivalents whereas a library from the long arm ( 7dl ) comprised 50,304 clones of 116 kb providing 14.9x arm coverage . the 7ds library was pcr screened with markers linked to russian wheat aphid resistance gene dnci2401 , the 7dl library was screened by hybridization with a probe linked to greenbug resistance gene gb3 . the small number of clones combined with high coverage made the screening highly efficient and cost effective .

bread wheat ( triticum aestivum l. ) is one of the most important crop species providing the staple food for 40% of the world 's population . as with other crops , the yields of wheat are annually significantly reduced due to attack of a large variety of pests and diseases . supplementation of conventional wheat breeding for pathogen resistance with marker - assisted breeding and direct gene transfer by molecular methods promises to enhance the efficiency of plant breeding . prerequisites of this approach are the saturation of genetic maps in the region of interest and isolation of the resistance gene . positional or map - based cloning is an experimental approach to isolate unknown genes based on their position in the genome . this approach involves construction of a high - density genetic map covering the target locus and a physical map spanning the region of interest . physical maps are produced by ordering dna clones from large - insert ( usually bac ) libraries into contigs on the basis of clone fingerprint pattern . whereas some wheat genes can be mined from smaller genomes of related or model species , many agronomically important genes , including those for pathogen resistance , yield , or grain quality factors , however , only a few bread wheat genes , including those conferring pathogen resistances , grain protein content , vernalization requirement , and domestication traits [ 111 ] , have been successfully cloned . t. aestivum is an allohexaploid species ( 2n = 6x = 42 , aabbdd genome ) , which originated from a spontaneous hybridization of three diploid wheat ancestors , donors of the a , b , and d genomes , respectively . they contributed to the enormous size of the bread wheat genome ( 1c17 gbp ) , which is composed of ~1% of genes [ 12 , 13 ] interspersed by huge amounts of repetitive elements . a variety of genomic resources has been developed to enable positional cloning in bread wheat including a number of bac libraries [ 1418 ] . while fingerprinting so many clones is technically feasible using the snapshot - based hicf procedure and recent results indicate that existing computational techniques may allow for reliable assignment of fingerprinted bac clones to particular homoeologous chromosomes , handling and screening libraries composed of more than million clones remain expensive and tedious tasks . these obstacles can be overcome by working with a library derived from a smaller part of the wheat genome . one option is to use diploid genome - donor species or their relatives as surrogates , taking advantage of smaller genome size and absence of polyploidy . several bac libraries of diploid wheat progenitors have been constructed including those of t. monococcum and ae . tauschii physical map ( see , http://www.wheat.ucdavis.edu ) were employed in cloning agronomically important genes [ 1 , 2 , 47 , 11 ] . however , clone numbers in these libraries are still too large to be screened easily . moreover , wheat genomes have undergone revolutionary changes following the polyploidization events including losses of dna . this partial diploidization and other genomic changes [ 2629 ] suggest that physical maps and genomic sequences of wheat diploid ancestors , although useful resources for wheat genomics , can not fully substitute for the genomic sequence of hexaploid wheat itself . recently , we proposed an alternative approach utilizing flow cytometry to dissect the hexaploid wheat genome into small fractions chromosomes or chromosome arms , which represent only a few percent of the hexaploid wheat genome . the chromosome - based strategy was made possible by developing procedures for purification of particular wheat chromosomes by flow sorting and a protocol for preparation of intact dna from sorted chromosomes suitable for cloning . this allowed us to create the first ever bac library derived from a single chromosome of a higher eukaryote . our ability to purify single chromosome arms from hexaploid wheat enabled us to construct bac libraries from 13 of the 21 wheat chromosomes thus far ( see [ 3537 ] , http://olomouc.ueb.cas.cz/dna-libraries/cereals ) , as well as from the short arm of rye 1r chromosome , which is a component of a number of wheat varieties . here , we report on the construction of bac libraries from both arms of wheat chromosome 7d , which harbors numerous genes underlying agronomically important traits , mainly resistance genes . the long arm of 7d is known to carry several greenbug ( schizaphis graminum ) resistance genes [ 39 , 40 ] as well as qtls influencing russian wheat aphid ( rwa , diuraphis noxia ) resistance . the short arm of 7d comprises several major genes and qtls for rwa resistance [ 4144 ] , the recently dissected lr34 locus underlying resistance to leaf rust , yellow rust , stem rust and powdery mildew , genes stb4 and stb5 underlying resistance to septoria tritici blotch [ 45 , 46 ] as well as several yield - related qtls [ 47 , 48 ] . in order to demonstrate how the chromosome - arm - specific bac libraries can facilitate positional gene cloning in a supersized plant genome , we report on a hybridization - based screening of the 7dl library with markers for the greenbug resistance gene gb3 , and a pcr - based screening of the 7ds library with markers for russian wheat aphid resistance gene dnci2401 . chinese spring carrying both arms of chromosome 7d as telosomes ( 2n = 40 + 2t7ds + 2t7dl ) was used to sort the arms . four thousand two hundred seeds subdivided into 184 batches of 2025 were germinated in the dark at 25c on moistened filter paper for 5560 h to reach root length of 2 - 3 cm . cell - cycle synchronization , accumulation of metaphases in root tips and preparation of chromosome suspensions were performed as described . briefly , chromosome suspensions were prepared by mechanical homogenization of 2025 root - tip meristems enriched for metaphase cells in 1 ml ice - cold isolation buffer ( ib , ) . chromosomes in suspension were stained with 2 g / ml dapi ( 4,6-diamidino-2-phenylindole ) and analyzed using a facsvantage se flow cytometer ( becton dickinson , san jose , usa ) . both arms were sorted separately from the same sample in batches of 200,000 into 320 l of 1.5xib . the purity in sorted fractions was checked regularly by fish with probes for telomeric and gaa repeats as described . preparation of high molecular weight dna ( hmw dna ) and library construction were performed as described [ 32 , 33 ] with some modifications . briefly , each batch of flow - sorted arms was spun down and the pelleted chromosomes were mixed with low - melting point agarose to form 20-l miniplugs , which were incubated in lysis buffer containing proteinase k to purify the chromosomal dna . the isolated hmw dna was partially digested with hindiii ( new england biolabs , beverly , mass . , usa ) and subjected to two rounds of size selection . at the first round , the partially digested dna was size - separated in 1% seakem gold agarose gel ( lonza , rockland , ill . , usa ) in 0.25x tbe under the following conditions of pulsed - field gel electrophoresis ( pfge ) : voltage 6 v / cm , switch time 150 s , run time 17 hours . size fraction of 100210 kb was excised from the gel and split into two parts . fraction b comprised fragments of 100150 kb whereas fraction m represented a fraction of 150210 kb . both fractions were subjected to a second round of size selection in 0.9% seakem gold agarose gel in 0.25x tbe under the following conditions : voltage 6 v / cm , switch time 3 s , run time 17 h. a gel zone corresponding to 100150 kb was excised from the lane containing the b fraction and was subdivided into two slices comprising a fraction of 100120 kb ( b1 ) and 120150 kb ( b2 ) , respectively , whereas only one gel slice ( ~150200 kb ) was excised from the m lane . the dna of particular fractions was electroeluted from the gel and ligated into hindiii - digested dephosphorylated pindigobac-5 vector ( epicentre , madison , wisc . , usa ) in 1 : 4 molar ratio ( dna : vector ) . the recombinant vector was used to transform escherichia coli electromax dh10b competent cells ( invitrogen , carlsbad , calif . the library was ordered by qbot ( genetix , new milton , uk ) into 384-well plates filled with 75 l freezing medium consisting of 2yt , 6.6% glycerol and 12.5 g / ml chloramphenicol . the clones were stored at 80c . a total of 121 bac clones from the 7dl ( 63 from the b1 , 32 from the b2 , and 27 from the m fraction ) and 184 clones from the 7ds library ( 56 from the b1 , 76 from the b2 and 52 from the m fraction ) were analyzed to estimate average insert size and percentage of empty bac clones . the bac dna was isolated after overnight incubation of particular bac clones in 1.5 ml 2yt supplemented with 12.5 g / ml chloramphenicol using a standard alkaline lysis method . resulting dna fragments were separated in a 1% agarose gel in 0.25x tbe buffer by pfge . the size of the fragments was estimated by comparing with two size markers : lambda ladder pfg marker and midrange marker i ( new england biolabs ) . high - density colony filters were prepared from the 7dl - specific bac library by spotting bac clones in duplicate in a 4 4 gridding pattern onto hybond n+ nylon membranes ( ge healthcare , piscataway , nj , usa ) using the genetac g3 robot ( genomic solutions , ann arbor , mich . an est - derived sts marker ( sts - aug-08 - 28 ) was mapped 0.08 cm proximal to the greenbug resistance gene gb3 ( azhaguvel et al . the pcr product was separated by agarose gel electrophoresis and a fragment of desired size was eluted from the gel using qiaquick gel extraction kit ( qiagen , germantown , md . the fragment was then p - datp radio - labeled by neblot kit ( new england biolabs ) using manufacturer 's protocol . the labeled probe was purified in a sephadex g50 column ( ge healthcare ) and denatured at 100c for 10 min . for prehybridization , overnight incubation of colony filters in hybridization solution ( 2x sspe , 0.5%sds , 5x denhardt 's reagent , 40 g / ml herring sperm dna ) was done in rotary glass tubes at 65c . the labeled probe was mixed with 5 ml of hybridization solution and colony filters were incubated at 65c overnight . to remove the unbound probe , we washed the filters twice in washing solution containing 2x sspe and 0.5% sds and rinsed with 1x ssc . the washed filters were exposed to x - ray film for one to three days based on the signal intensity to identify positive clones . to complete the assembly of the contig spanning the gb3 region , we conducted three rounds of 7dl bac library screening . the sts - aug-08 - 28 marker was used for the first round of the screening whereas probes derived from protruding ends of bac no . 25 , respectively , were used for the second and third round of screening ( figure 3 ) . the positive bac clones resulting from each bac library screening were grown overnight in lb media supplemented with 12.5 g / ml chloramphenicol . bac dna was extracted by standard alkaline lysis procedure and subjected to hindiii digestion to create a fingerprint for each positive clone . five micrograms dna of all positive clones in each screen were extracted with qiagen plasmid kit ( qiagen ) and used for direct cycle sequencing of bac ends using t7 ( 5taa tac gac tca cta tag gg 3 ) and m13r ( 5 cag gaa aca gct atg acc 3 ) primers . moreover , three bac clones ( bac 22 , bac 25 and bac 72 ) were sequenced completely by sanger technology at the genome center , washington university , st . the alignment of bac end sequences with the fully sequenced bac clones was done with seqman module in the software dnastar ( lasergene corp . , the insert size of the positive bac clones was determined by aligning bac end sequences with the reference sequences of the completely sequenced bac clones . bac end sequences were used to develop sequence tagged site ( sts ) or cleaved amplified polymorphic sequence ( caps ) markers , which were used for further screening of the library to identify and/or confirm overlapping bacs to span the region of interest . to enable pcr screening of the 7ds library , we generated three types of bac pools : plate pools , superpools and three - dimensional ( 3d ) pools . first , 128 plate pools were prepared by pooling clones from each of 384-well plates comprising the library . rectangular dishes ( nunc , rochester , ny , usa ) containing agarose ( 1.6% ) 2yt medium with 12.5 g / ml chloramphenicol were inoculated with all 384 clones of single plates using a genetac g3 robot . after 16-h incubation at 37c , the clones were suspended in 510 ml te buffer ( 10 mm tris , 1 mm edta , ph 8.0 ) , bacteria were pelleted by centrifugation and subsequently resuspended in 600 l get buffer ( 50 mm glucose , 25 mm tris , 10 mm edta , ph 8.0 ) . dna was isolated using standard alkaline lysis protocol supplemented with rnase treatment and precipitation of contaminating compounds by ammonium acetate . the dna of each aliquot was dissolved in 20 l sterile deionized water ; the aliquots were combined and stored at 20c . for pcr screening these stocks were diluted to 10 ng/l dna . to prepare the 3d pools we created 48 pools ( 8 plate , 16 row and 24 column pools ) for each stack , thus totally 768 pools represented the entire library . to prepare the pools , we spotted bacterial clones in duplicate on plates with agarose medium as described above . bacteria grown for 48 h were suspended in 5 ml te buffer and boiled for 30 min to release dna . remnants of bacteria were pelleted at 3000 g for 60 min and 1.4 ml of the supernatants were deposited at 20c as the particular pools . for pcr superpools were created for each of the 8-plate stacks by combining all clones from the respective 8 plates . spotting , growth of bacteria and dna isolation were done as for the 3d pools . the 7ds - specific library was screened with microsatellite markers xcfd68 , xgwm473 and xbarc214 closely linked to the dn gene . primer sequences were retrieved from the graingenes database ( http://wheat.pw.usda.gov/gg2/index.shtml ) . the pcr reaction mix ( 10 l volume ) consisted of 2 l template dna , 1 buffer for dynazyme dna polymerase ( finnzymes , espoo , finland ) , 0.01% cresol red , 1.5% saccharose , 0.2 mm dntps , 1 m primers and 0.4 u dynazyme ii dna polymerase ( finnzymes ) . the pcr reaction was performed under the following conditions : denaturation for 5 min at 95c followed by 35 cycles of denaturation at 95c for 30 s , annealing at the appropriate temperature for 30 s , extension at 72c for 30 s , and a final extension at 72c for 10 min . the presence of pcr products was detected by electrophoresis in 1.2% agarose gel run in 0.5x tbe buffer . the precise size of individual pcr products was determined using abi 3730xl sequencer ( applied biosystems , foster city , calif . the fragment sizes were estimated relative to the genescan-500 liz size standard ( applied biosystems ) by genemarker v1.75 ( softgenetics , llc , usa ) . all clones of the 7ds - specific library were fingerprinted using the snapshot - based high - information - content fingerprinting technology . the fingerprints were automatically edited with the computer program package fpminer ( www.bioinforsoft.com ) and genoprofiler and assembled using fpc v9.3 at an initial cutoff of 1 10 , followed by various steps of dqing and end merging . the short and the long arms of the 7d chromosome were sorted simultaneously from the 7d double - ditelosomic ( ddt ) 7d line . the flow karyotype obtained from this line consisted of two peaks representing 7dl and 7ds telocentric chromosomes , respectively , three composite peaks comprising groups of various wheat chromosomes and the rightmost peak corresponding to the largest wheat chromosome 3b ( figure 1 ) . the leftmost peak , representing a chromosome with the smallest relative dna content , actually comprised telosome 7dl and not 7ds , as the arm designated 7ds based on homology with the wheat chromosome arms 7as and 7bs is physically longer and hence has greater relative dna content . the purity of sorted telosomes as estimated by fish varied between 8492% ( average 88.8% ) for the 7dl and between 7986% ( average 84.1% ) for the 7ds telosome . the 7d chromosome is metacentric and the 7dl and 7ds arms are of similar size ( 2.04% of the wheat genome alias 346 mbp for 7dl and 2.25% of the wheat genome alias 381 mbp for 7ds , [ 37 , 53 ] ) . the 7ds constituted 1.3% of the sorted 7dl whereas 7dl represented 1.1% of the sorted 7ds fraction . the remaining contamination was composed of a mixture of chromosomes without a prevalence of a particular type . in total , 31 agarose miniplugs comprising 5,900,000 7dl telosomes corresponding to ~4.18 g dna were prepared . in parallel , 6,000,000 7ds telosomes ( ~4.67 g ) were embedded in 30 agarose plugs . bac libraries were created from both telosomes using the hindiii cloning site . the library derived from the long arm of the chromosome 7d called taacsp7dlha comprised 50,304 clones ordered in 131 384-well plates . the average insert size of the whole library reached 116 kb and , excluding 0.5% empty clones and considering the 11% contamination by other chromosomes and the size of 346 mbp , the library provided 14.9x coverage of 7dl . the library derived from the short arm of the chromosome 7d named taacsp7dsha consisted of 49,152 clones ordered in 128 plates . having 113 kb mean insert size and 1.4% empty clones , the library constituted 12.1 arm equivalents if the 16% contamination with other chromosomes and 7ds molecular size of 381 mbp were considered . the representation and mean insert size of the particular fractions are shown in table 1 . the overall distribution of insert sizes , which were estimated for 121 clones from the 7dl library and 184 clones from the 7ds library , ranged from 10 to 200 kb and differed slightly between the libraries as shown in figure 2 . whereas most clones ( 39% ) were found in the fraction of 125149 kb in the 7dl library , the most numerous fraction ( 42% of clones ) of the 7ds library is in the size range of 100124 kb . this was due to greater representation of b2 compared with b1 fraction in the 7dl library . on the other hand , the 7ds library contained a greater percentage of large clones more than 150 kb ( 9% versus 7% for 7dl ) although the average insert size of the m fraction was significantly greater in the 7dl library ( 147 kb for 7dl - m versus 130 kb for 7ds - m ) . this seeming discrepancy was due to a greater proportion of the m fraction in the 7ds library ( 11% for 7dl - m versus 18% for 7ds - m ) . the portion of bac clones smaller than 50 kb , which are usually noninformative in hicf analysis applied in construction of physical maps , was negligible in both libraries ( below 2% ) . on the other hand , presence of short clones less than 75 kb in all fractions of both libraries suggested that the ligation ratio between the wheat dna and the vector was set up correctly preventing ligation of more than a single dna fragment into one vector due to excess wheat dna . an improper ratio would have led to the creation of chimeric clones , which significantly compromise the quality of libraries . absence of chimeric clones in the 7d libraries was further supported by the fact that no inserts exceeding sizes excised from the gel at the size selections were found for the b1 , b2 and m fractions , respectively . the mean insert size was estimated to be 116 kb for the 7dl and 113 kb for the 7ds library ; both were significantly larger than for early wheat chromosome - specific bac libraries , which reached 82 kb in the 1bs - specific library , 85 kb in a composite library constructed from chromosomes 1d , 4d and 6d and 103 kb in the 3b - specific library . the increase in insert size was achieved by including a second size - selection step in the bac library construction procedure . available genomic bac libraries created from hexaploid wheat , which can be used as a standard for evaluating the quality of the 7d - specific libraries , varied significantly in their average insert size , which ranged from 75 kb for a library constructed from wheat cv . however , in a comparison of the coverage of the libraries , even the most representative of the wheat genomic libraries constructed by allouis et al . providing 9.3x wheat genome coverage does not reach the coverage of our chromosome - specific libraries ( 14.9 and 12.1x for the 7dl and 7ds libraries , resp . ) . est - derived marker sts - aug-08 - 28 ( azhaguvel et al . in preparation ) tightly linked with the gb3 gene was used as a hybridization probe to screen the 7dl - specific bac library ( figure 3 , screen i ) providing 14 positive clones . marker sts - bac 22-r developed from the protruding end of bac 22 clone was used as a probe for a second round of library screening and detected 22 positive bac clones including two selected in the previous screen ( figure 3 , screen ii ) . 25-t7 derived from the protruding end of bac no . 25 ( figure 3 , screen iii ) . this marker identified 23 positive bac clones ( including five bac clones detected in the screen ii ) . three bac clones ( bac 22 , bac 25 and bac 72 ) were fully sequenced and assembled . the sizes of bac 22 , bac 25 and bac 72 were 200.56 kb , 110.95 kb and 117.09 kb , respectively . markers that were continuously derived during the contig assembly from available bac sequences cosegregated with the resistance gene . only a marker derived from the end of the bac 72 showed recombination with the gene and flanked the gb3 region from the opposite side . thus three rounds of screening the 7dl library were sufficient to build a contig spanning the region between markers flanking the gb3 gene . positional cloning of gb3 and a detailed annotation of this contiguous sequence will be described elsewhere ( azhaguvel et al . the number of positive clones selected by hybridization with single - copy probes and confirmed by contig assembly and sequencing outputs indicates library coverage greater than estimated based on insert size analysis . in fact , the number of positive clones confirmed by fingerprinting was 20 or more in all screens ( 21.7 at average ) but in the screen i , bac - end sequences were available for 14 of 20 clones , so only these clones were included in the final contig assembly . as all these screens were conducted in one region of the 7dl arm , we do not conclude that this number ( 21.7 ) shows evidence of overall underestimation of coverage based on insert size analysis but rather indicated overrepresentation of this region in the library . differences in local coverage were observed also in the 7ds library for particular markers ( table 2 and figure 4 ) . the above mentioned numbers of positive bac clones were obtained after screening only three high - density filters that comprised the whole 7dl library . for comparison , bac libraries of diploid wheat relatives t. urartu , ae . speltoides and ae . tauschii representing 3.7 , 5.4 and 4.1 equivalents of the respective genomes occupied 9 , 13 and 10 high - density filters , respectively . consequently , one filter comprising 18,432 clones of a diploid library represents only 0.41x genome coverage . an even greater workload would be required in hybridization screening of the polyploid wheat libraries . the bac library of tetraploid t. turgidum with 5.8x coverage occupies 28 high - density filters ; thus one filter carries just 0.2x genome equivalents . glenlea was spotted on 24 filters with a greater density of 27,360 clones per filter . despite of that , one filter represented only 0.13x genome equivalents . on the other hand , the 7dl library with coverage of 14.9x was placed on only three filters and thus the 18,432 clones spotted on one filter cover the 7dl arm 5.46x . such coverage would be sufficient for positional cloning providing 99.5% probability of recovering any sequence present on the arm and poses a strong argument for the chromosome - based genomics in wheat . microsatellite markers xcfd68 , xbarc214 and xgwm473 were found to be tightly linked to the dn gene ( fazelnajafabadi and lapitan , unpublished ) . these markers were applied to screen the 7ds library using the set of bac pools . as the first step , pcr was run on the superpools representing stacks of 8 plates , followed by screening on the plate pools and finally 3d pools . although some of the superpools did not provide a pcr product , screening was performed on all plate pools to verify the reliability of the information obtained from superpools . the results from superpools and plate pools , respectively , were in full accordance for all markers tested . this implies the superpools can be used to preselect stacks of plates to be subjected to further screening and thus reduce the number of pcr reactions needed for library screening . however , considering the small number of 3d pools and the high coverage of the library due to which 37.575% of the superpools ( depending on the locus ) were found to be positive , this step is not essential . similarly , pcr was run on all plate pools and 3d pools , respectively , to compare the results . in 6 of 47 cases ( 12.8% ) the information from the plate pools helped in identifying the positive plates in the 3d pool set . these were cases when the pcr product obtained from 3d pools , which were prepared by a simplified procedure , was too weak for an unambiguous identification of the positive plate . thus the set of plate pools prepared by a more advanced dna - isolation procedure proved to be useful in resolving the position of the positive clones . however , as demonstrated below , its role can be substituted by availability of data from the bac contig assembly . among the positive bac clones selected by xbarc214 and xgwm473 , several bac clones were found that provided products of an unexpected size . by comparing these products with products obtained by pcr on dna of flow - sorted 7ds arms we verified that these products did not relate to the 7ds arm . these bac clones remained as singletons in the contig assembly , which implies that they come from contamination of the sorted fraction by other chromosomes . such clones were not considered positives . however , screening the 7ds library with the xgwm473 marker provided an even more complex spectrum of pcr products . besides clones bearing a double - band of 220 and 226 bp , respectively , which has been characteristic of the dnci2401-linked marker , and a few clones of unexpected size , we also found numerous clones that generated a pcr product of 200 bp . this product was also visible as a weaker band when amplifying dna of flow - sorted 7ds . this presumption was supported by finding a bac contig ctg135 comprising clones with the 200 bp pcr product ( figure 4(d ) ) . sequencing the locus both from positive clones of ctg285 and ctg135 confirmed that the two loci differed . comparing sequences of the 226 and the 200 bp band revealed a 26 bp deletion and 92% homology in the remaining sequence . this locus is probably not polymorphic in available mapping populations as there is no evidence about it in databases as graingenes or gramene . the results of the screening the 7ds library with xcfd68 , xbarc214 and xgwm473 are shown in figure 4 and table 2 . most of the positive clones were identified unambiguously based on pcr screening of plate and 3d pools . the xcfd68 marker selected nine positive clones in the screening and two additional bac clones were revealed after integrating data from the bac contig assembly as row or column information for these clones was missing ( figure 4(a ) ) . similarly , for xbarc214 15 positive bac clones were identified by library screening whereas bac addresses of another two clones could be completed only after considering data from the bac contig assembly ( figure 4(b ) ) . in case of the xgwm473 marker , nine and 16 positive clones , respectively , were identified in ctg285 and 135 , respectively ( figures 4(c ) and 4(d ) ) . all bac addresses were complete ; however , three of ten bac clones providing the 220 and 226 bp products were absent from the contig assembly , thus their position in ctg285 could not be verified . they might have been missing in the library replica used for fingerprinting or excluded by the genoprofiler software due to a low quality of fingerprint or cross - contamination . our results imply that the proposed system of bac pools is sufficiently powerful to reveal positions of positive bac clones . having contig assembly data in hand makes the deconvolution of bac addresses easier and enables further reducing the number of clones to be screened . the local coverage differed among the tested loci and ranged between 10 and 17x , averaging 13.5x . this is in agreement with the coverage estimate based on insert size ( 12.1x ) . various pooling strategies have been proposed for pcr - based screening of wheat libraries for cloning of genes . employed the analogous pooling strategy as applied in this study involving superpools ( combining clones from ten consecutive plates ) and 3d ( plate , row , and column ) pools created for stacks of ten plates . the glenlea wheat library comprising 3.1 genome equivalents was represented by 171 superpools and 8,550 3d pools . in their case , assaying superpools played a substantial role in reducing the overall number of pcr reactions . in this two - step screening , a single positive bac clone could be reached in 221 ( 171 superpools + 50 3d pools for the selected superpool ) pcr reactions . however , for obtaining several clones , which is preferable in positional cloning , significantly more pools would have to be assayed . a different pooling strategy was proposed by febrer et al . who screened a part of the wheat genomic library constructed by allouis et al . for a wheat dwarfing gene rht . the screen of 715,776 clones from the library ( ~5 genome equivalents ) was based on pooling of dna from bac clones into 675 superpools arrayed in a three - dimensional configuration . a second round of pcr was used to detect a specific bac clone within the candidate plate that corresponded to the gene of interest . so assaying at least three 384-well plates was needed to identify a single copy of the target gene . for identifying all three homoeologues of the rht gene , 17 candidate plates were screened providing 13 rht - containing clones . for comparison , assaying one 384-well plate with 3d pools of the 7ds library ( representing half of the library ) supplemented with deconvolution of the positive clones was sufficient to identify four to 12 copies of a particular microsatellite locus . once a bac contig assembly has been completed , screening can be further simplified by preparing bac pools from mtp clones only . in case of the wheat 3b - chromosome - specific bac library consisting of 67,968 clones , thus the library was represented by 60 three - dimensional pools , which were sufficient for screening the whole library . in the case of a library from the short arm of chromosome 3d containing 36,864 clones , the mtp comprised 3,823 bac clones occuring in 50 three - dimensional pools . screening this reduced number of pools can identify a bac contig containing the marker ; however , as the mtp may not comprise the complete chromosomal sequence and its coverage is ~1x , the risk of losing some information is relatively high and thus screening the 3d mtp pools of the 3b library was combined with screening plate pools prepared from the whole library . microsatellites represent a frequently used marker system as they can be easily extracted from genomic ( e.g. , bac - end or shotgun ) sequences , are abundant and highly polymorphic . they are a typical marker of choice in efforts to place genes of interest on a genetic map . however , microsatellites proved to be of limited use in large - scale screening of a soybean genomic bac library to anchor the physical contig map because of their multilocus character . screening by microsatellites in a hexaploid wheat library would be even more complicated due to the presence of homoeologous genomes . this view is supported by findings of nilmalgoda et al . who screened a hexaploid wheat library of 3.1x coverage both with gene - derived and microsatellite markers . whereas gene sequences identified 2.7 positive bac clones on average , the average number of clones hit by microsatellites was twofold ( 5.5 clones ) . our small - scale screening detected two loci relating to one of the microsatellite markers ( xgwm473 ) even on one chromosome arm . similarly , multiple loci were found for another microsatellite marker ( xgwm44 ) when screening the 7ds library and analyzing the selected clones using the available contig assembly ( imkov , unpublished ) . this indicates that results of screening bac libraries with microsatellite markers must be interpreted with caution even in the case of chromosome - arm specific libraries . the screening of the library with markers for the dnci2401 gene is a first step towards positional cloning of this gene . the work is in progress to derive new markers from colinear regions of related genomes ( rice , brachypodium ) that were identified based on sequencing bac clones from contigs containing the markers . two bac libraries specific for both the long and the short arms of the bread wheat chromosome 7d were constructed with parameters challenging available wheat genomic libraries . the libraries represent the first subgenomic bac resources available for wheat homoeologous chromosome group 7 and will facilitate advancement in many areas of wheat genomics , including physical map construction and map - based cloning of genes . due to the small number of clones , high genome coverage and absence of clones from homoeologous genomes , these libraries make screening with markers for genes of interest highly efficient and cost - effective . for example , one high - density filter prepared from the 7dl library provided coverage of 5.4x , which ensures 99.5% probability of finding any sequence located on the 7dl chromosome arm . a simple pooling strategy was elaborated based on which 6x equivalents of the 7ds arm could be screened by 384 pcr reactions . after completing physical map assembly of the 7ds arm , the number of pools to be screened can be further reduced . all these features and the results of screening these libraries with markers for aphid resistance genes indicate that chromosome - arm - specific bac libraries are a powerful resource for cost - effective positional gene cloning in bread wheat .

in general practice , short films of the knee are used to assess component position and define the entry point for intramedullary femoral alignment in tkas ; however , whether it is justified to use the short film commonly used in research settings and everyday practice as a substitute for the whole leg view is controversial and needs clarification . in 138 long leg ct scanograms we measured the angle formed by the anatomic axis of the proximal fourth of the tibia and the mechanical axis of the tibia , the angle formed by the anatomic axis of the distal fourth of the femur and the mechanical axis of the femur , the bow of the tibia ( as reflected by the offset of the anatomic axis from the center of the talus ) , and the bow of the femur ( as reflected by the offset of the anatomic axis from the center of the femoral head ) . because the angle formed by these axes and the bow of the tibia and femur have wide variability in females and males , a short film of the knee should not be used in place of the whole leg view when accurate assessment of component position and limb alignment is essential . a previous study of normal limbs found that only 2% of subjects have a neutral hip - knee - ankle axis , which can be explained by the wide variability of the bow in the tibia and femur and the lack of correlation between the bow of the tibia and femur in a given limb as shown in the current study .

it generally is accepted that wear and loosening depend on proper alignment of the components and the limb , however , review of the methods of previous research of limb alignment in tka has revealed two limitations that cause us to question the relationship between wear and loosening . these limitations include the use of a short film of the knee instead of a whole leg view to assess limb alignment , and the study of a knee component with size , shape , and fixation features that no longer are deemed desirable . the use of the standard short knee film as a substitute for the whole leg view for assessing component and limb alignment is commonplace in everyday practice but controversial . limb alignment in the coronal plane is quantified by the hip - knee - ankle axis . neutral alignment is said to exist when the line connecting the center of the femoral head and center of the ankle passes through the center of the knee , and varus or valgus alignment is said to exist when this line passes medially or laterally , respectively , to the center of the knee [ 4 , 25 ] . one study concluded short knee images can not substitute for whole leg views when accurate assessment of the hip - knee - ankle axis is essential , whereas other studies suggested the anatomic axis of the knee on short knee film appears to be a valid alternative to the hip - knee - ankle axis of the limb on full leg radiographs [ 8 , 16 ] . for the short knee film to accurately substitute for the whole leg view , the variability of the angle formed by the anatomic and mechanical axes in the tibia and femur and the variability of the bow of the tibia and femur should be small . studies have qualitatively reported variability in the bow of the tibia and femur [ 15 , 20 , 24 , 27 , 28 ] ; however , these studies did not describe a method for quantifying the bow . a previous study of normal limbs found that only 2% of subjects have a neutral hip - knee - ankle axis , which might be explained by variability in the angle formed by the anatomic and mechanical axes and the bow in the tibia and femur and a lack of correlation between the bow of the tibia and femur in a given limb . accordingly , we hypothesized ( 1 ) the angle formed by the anatomic axis of the proximal fourth of the tibia and the mechanical axis of the tibia and the angle formed by the anatomic axis of the distal fourth of the femur have wide variability ; ( 2 ) the bow of the tibia and femur has wide variability ; ( 3 ) there are no differences in the angle and bow between females and males ; and ( 4 ) there is no correlation between the bow of the tibia and femur in a given limb . we considered all patients who underwent tkas from june 19 to december 1 , 2007 , for inclusion . the inclusion criterion was the presence of primary arthritis of the knee with or without previous open or arthroscopic meniscectomy or ligament reconstruction . we excluded patients with a treated leg with evidence of a hip disorder ( ie , developmental dysplasia , perthes , or slipped epiphysis ) , an osteotomy , a healed fracture , internal fixation , arthroplasty of the hip , knee , or ankle , or a malaligned computer tomogram of the leg . four patients were excluded because of developmental dysplasia of the hip ( n = 1 ) , tha ( n = 1 ) , internal fixation of a femoral neck fracture ( n = 1 ) , and a malaligned computer tomogram of the leg ( n = 1 ) . therefore , the study consisted of 138 patients ( 90 women , 48 men ) with an average age of 68 10 years . all patients received an unconstrained tka ( vanguard ; biomet , inc , warsaw , in ) . , we acquired a postoperative , anteroposterior scanogram with a field of view from the hip to the ankle with use of ct ( ge lightspeed 16 ; ge healthcare , www.gehealthcare.com ) . because simultaneous flexion of the knee and rotation of the leg causes large changes in projected angles [ 2 , 15 ] , we limited the projection error of the mechanical axis to approximately 1 by iteratively rotating the limb and repeating the scanogram until the two augment holes in the posterior condyles were at least partially visible on either side of the flange of the femoral component . one of us ( kk ) made measurements under a magnified view using screen measurement software ( screen caliper , compass , and protractor ; iconico inc , www.iconico.com ; adobe photoshop , adobe inc , www.adobe.com ) with a reported accuracy of 0.5 . because the short knee film often is used intraoperatively and postoperatively to assess component position , especially when access to full limb radiographs is limited [ 14 , 16 , 24 , 30 ] , and because the short knee film typically shows only the proximal fourth of the tibia and the distal fourth of the femur [ 8 , 16 ] , we defined the anatomic axes of the tibia and femur based on axes centered in the proximal fourth of the tibia and the distal fourth of the femur . the anatomic axis of the tibia was a line joining the midpoints of the tibia at the joint line and at the junction of the proximal one - fourth and distal three - fourths of the tibia [ 15 , 16 , 21 , 26 ] ( fig . 1 ) . the mechanical axis of the tibia was a line joining the midpoint of the tibia at the joint line and the center of the talus [ 12 , 15 , 16 , 18 , 21 , 25 , 27 , 32 ] . the angle formed by the mechanical axis and the anatomic axis of the tibia was measured . we defined the bow of the tibia in the coronal plane as the offset in centimeters of the anatomic axis of the tibia and the center of the talus ( fig . 1 ) . a positive value ( + ) indicated a valgus tibia with the apex of the bow pointing medial and with the anatomic axis passing medial to the center of the talus . a negative value ( ) indicated a varus tibia with the apex of the bow pointing lateral and with the anatomic axis passing lateral to the center of the talus . the anatomic axis of the femur was a line joining the midpoints of the femur at the joint line and at the junction of the distal one - fourth and proximal three - fourths of the femur ( fig the mechanical axis of the femur was a line joining the midpoint of the femur at the joint line and the center of the femoral head . the angle formed by the anatomic axis and the mechanical axis of the femur was measured . we defined the bow of the femur in the coronal plane as the offset in centimeters of the anatomic axis of the femur from the center of the femoral head . the larger the offset , the larger the bow with a positive value ( + ) indicating the anatomic axis passed lateral to the center of the femoral head and a negative value ( ) indicating the anatomic axis passed medial to the center of the femoral head.fig . 1a bthe anatomic axis of the tibia ( longitudinal white line ) was a line joining the midpoints of the tibia at the joint line and at the junction of the proximal one - fourth and distal three - fourths of the tibia ( transverse black line ) . the mechanical axis of the tibia was a line joining the midpoint of the tibia at the joint line and the center of the talus ( longitudinal black line ) . the bow of the tibia was quantified by the offset ( d ) measured from a line ( transverse white line ) drawn perpendicular from the anatomic axis of the tibia to the center of the talus . a positive value ( + ) indicated a valgus tibia with the apex of the bow pointing medial and with the anatomic axis passing medial to the center of the talus . ( a ) the offset of the tibia with the greatest valgus bow was 3.5 cm . a negative value ( ) indicated a varus tibia with the apex of the bow pointing lateral and with the anatomic axis passing lateral to the center of the talus . ( b ) the offset of the tibia with the greatest varus bow was 2.2 cm . the use of an extramedullary tibia guide that references the ankle would place the tibial cut in six additional degrees of varus in the valgus tibia ( left ) and four additional degrees of valgus in the varus tibia ( right ) requiring lateral and medial soft tissue release to balance the knee , respectively.fig . 2a bthe anatomic axis of the femur ( longitudinal white line ) was a line joining the midpoints of the tibia at the joint line and at the junction of the distal one - fourth and proximal three - fourths of the femur ( transverse black line ) . the mechanical axis of the femur was a line joining the midpoint of the femur at the joint line and the center of the femoral head ( longitudinal black line ) . the bow of the femur was quantified by the offset ( d ) measured from a line ( transverse white line ) drawn perpendicular from the anatomic axis of the femur to the center of the femoral head . the larger the offset , the larger the bow with a positive value ( + ) indicating the anatomic axis passed lateral to the center of the femoral head and a negative value ( ) indicating the anatomic axis passed medial to the center of the femoral head . ( a ) the offset of the femur with the greatest lateral bow was 6.4 cm . ( b ) the offset of the femur with the least lateral bow was 0.4 cm . because of the variability in the lateral bow of the femur , the mechanical axis of the femur does not form a 5 to 6 angle with the anatomic axis of the distal fourth of the femur in either leg . the anatomic axis of the tibia ( longitudinal white line ) was a line joining the midpoints of the tibia at the joint line and at the junction of the proximal one - fourth and distal three - fourths of the tibia ( transverse black line ) . the mechanical axis of the tibia was a line joining the midpoint of the tibia at the joint line and the center of the talus ( longitudinal black line ) . the bow of the tibia was quantified by the offset ( d ) measured from a line ( transverse white line ) drawn perpendicular from the anatomic axis of the tibia to the center of the talus . a positive value ( + ) indicated a valgus tibia with the apex of the bow pointing medial and with the anatomic axis passing medial to the center of the talus . ( a ) the offset of the tibia with the greatest valgus bow was 3.5 cm . a negative value ( ) indicated a varus tibia with the apex of the bow pointing lateral and with the anatomic axis passing lateral to the center of the talus . ( b ) the offset of the tibia with the greatest varus bow was 2.2 cm . the use of an extramedullary tibia guide that references the ankle would place the tibial cut in six additional degrees of varus in the valgus tibia ( left ) and four additional degrees of valgus in the varus tibia ( right ) requiring lateral and medial soft tissue release to balance the knee , respectively . the anatomic axis of the femur ( longitudinal white line ) was a line joining the midpoints of the tibia at the joint line and at the junction of the distal one - fourth and proximal three - fourths of the femur ( transverse black line ) . the mechanical axis of the femur was a line joining the midpoint of the femur at the joint line and the center of the femoral head ( longitudinal black line ) . the bow of the femur was quantified by the offset ( d ) measured from a line ( transverse white line ) drawn perpendicular from the anatomic axis of the femur to the center of the femoral head . the larger the offset , the larger the bow with a positive value ( + ) indicating the anatomic axis passed lateral to the center of the femoral head and a negative value ( ) indicating the anatomic axis passed medial to the center of the femoral head . ( a ) the offset of the femur with the greatest lateral bow was 6.4 cm . ( b ) the offset of the femur with the least lateral bow was 0.4 cm . because of the variability in the lateral bow of the femur , the mechanical axis of the femur does not form a 5 to 6 angle with the anatomic axis of the distal fourth of the femur in either leg . we used the arithmetic mean , standard deviation , 95% confidence interval , frequency distribution , and quantile box plot to describe the variability of the angle formed by the anatomic and mechanical axes of the tibia and femur and the bow of the tibia and femur . the small ticks indicate the 10% and 90% quantiles , the intermediate ticks indicate the 2.5% and 97.5% quantiles , and the large ticks indicate the 0% and 100% quantiles . an unpaired t - test was used to determine whether the angle formed by the anatomic and mechanical axes of the tibia and femur and the bow of the tibia and femur were different between females and males . a correlation coefficient was computed to assess the correlation between the bow of the tibia and the bow of the femur . each analysis was performed with software ( version 7.0.2 , jmp for macintosh ; spss , chicago , il ; www.jmp.com ) . the angle formed by the anatomic axis and the mechanical axis of the tibia and the bow of the tibia varied widely ( fig . 3 ) . the angle formed by the anatomic axis and the mechanical axis of the femur and the bow of the femur also varied widely ( fig . the angle formed by the anatomic and mechanical axes of the tibia varied 11 from 4 to 6 and averaged 1.1 1.4 ( valgus tibia ) . the angle was less than 1 or greater than 1 in 61% , less than 2 or greater than 2 in 34% , and less than 3 or greater than 3 in 13% of the subjects . the angle formed by the anatomic and mechanical axes of the femur varied 10 from 1 to 8 , averaged 3 1.6 , and the 95% confidence interval ( 2.83.4 ) did not include 5 or 6.fig . 3a bfrequency distributions of the ( a ) angle formed by the anatomic and mechanical axes of the tibia and ( b ) bow of the tibia as quantified by the offset of the anatomic axis from the center of the talus are shown . descriptive statistics include the quantile plot and the number of patients in each column.fig . 4a bfrequency distributions of the ( a ) angle formed by the anatomic and mechanical axes of the femur and ( b ) bow of the femur as quantified by the offset of the anatomic axis of the femur from the center of the femoral head are shown . frequency distributions of the ( a ) angle formed by the anatomic and mechanical axes of the tibia and ( b ) bow of the tibia as quantified by the offset of the anatomic axis from the center of the talus are shown . frequency distributions of the ( a ) angle formed by the anatomic and mechanical axes of the femur and ( b ) bow of the femur as quantified by the offset of the anatomic axis of the femur from the center of the femoral head are shown . the bow of the tibia , defined by the offset of the anatomic axis from the center of the talus , varied 5.7 cm from 2.2 cm medial to 3.5 cm lateral to the center of the talus and averaged 0.3 1.1 cm . the bow of the femur , defined by the offset of the anatomic axis from the center of the femoral head , varied 7.2 cm from 6.8 cm lateral to 0.4 cm medial to the center of the femoral head and averaged 2.5 1.3 cm . we observed no difference in the angle formed by the anatomic and mechanical axes of the tibia ( p = 0.8784 ) and femur ( p = 0.7706 ) and the bow of the tibia ( p = 0.8578 ) and femur ( p = 0.8101 ) between females and males ( table 1).table 1comparison of the angle formed by the anatomic and mechanical axes and the bowdependent variablefemale ( n = 90)male ( n = 48)p valueangle of the anatomic and mechanical axes in the tibia1.2 1.50.9 1.40.8784 , nsangle of the anatomic and mechanical axes in the femur3.0 1.53.2 1.70.7706 , nsbow of the tibia ( ie , offset of the anatomic axis from the center of the talus)0.4 1.1 cm0.2 1.1 cm0.8578 , nsbow of the femur ( ie , offset of the anatomic axis from the center of the femoral head)2.5 1.2 cm2.7 1.4 cm0.8101 , nsns = nonsignificant . comparison of the angle formed by the anatomic and mechanical axes and the bow we found no correlation between the bow of the tibia and femur in a given limb ( r = 0.0185 , p = 0.8286 ) , which means the degree and direction of the bow in the tibia is not related to the degree of bow of the femur in a given limb . for the short film of the knee to accurately substitute for the whole leg view , the variability of the angle formed by the anatomic and mechanical axes in the tibia and femur and the variability of the bow of the tibia and femur should be small . we hypothesized ( 1 ) the angle formed by the anatomic axis of the proximal fourth of the tibia and the mechanical axis of the tibia and the angle formed by the anatomic axis of the distal fourth of the femur has wide variability ; ( 2 ) the bow of the tibia and femur has wide variability ; ( 3 ) there are no differences in the angles and bows between females and males ; and ( 4 ) there is no correlation between the bow of the tibia and femur in a given limb . we examine several limitations that might have affected the observed variability of the angle formed by the anatomic and mechanical axes and the bow of the tibia and femur . first , while we used nonweightbearing , rotationally controlled ct scanograms obtained in knees with osteoarthritis after tka instead of weightbearing scanograms of normal limbs , we believe this unlikely affected the variability because the weightbearing status , presence or absence of components , and removal of osteophytes back to the normal edge of the joint at the time of surgery do not affect the shape of the tibia and femur . second , the variability from our study of a western population is likely to be different from the variability of an occidental population , which has a high prevalence of tibias with a varus bow and a high prevalence of femurs with a large lateral bow . the wide variability of the angle formed by the anatomic and mechanical axes of the tibia and femur in our study agrees with some previous studies [ 18 , 21 , 27 , 28 ] , but does not agree with the historic , pioneering principles in tka [ 13 , 15 , 19 , 29 , 32 ] . in terms of the tibia , the principle of aligning the varus / valgus osteotomy of the proximal tibia in tka was based on an idealized depiction of the tibia in which the shaft of the tibia was straight and the mechanical and anatomic axes of the tibia were assumed to be the same [ 13 , 15 , 19 , 29 , 32 ] . we found only 11% ( 16 of 138 ) of the tibias were straight with 34% of the subjects having an anatomic axis that diverged greater than 2 varus or valgus from the mechanical axis of the tibia . in terms of the femur , the principle of aligning the varus / valgus osteotomy of the distal femur in tka was based on the assumption that the angle formed by the anatomic and mechanical axes of the femur is consistently between 5 and 6 [ 15 , 19 , 31 ] . in contrast to these previous studies , we found the angle averaged 3 with only 6% of the femurs having an angle of 5 to 6. the difference might be explained by our use of the distal one - fourth of the femoral shaft to define the anatomic axis , which is a shorter linear length for defining the anatomic axis of the femur than used in other studies [ 3 , 4 , 12 , 19 ] . the clinical consequences of this variability are the use of a short knee film to check the varus / valgus osteotomy intraoperatively and component alignment and to predict the hip - knee - ankle axis postoperatively is unreliable because of the inconsistent relationship between the anatomic and mechanical axes of the tibia and femur ( fig b(a ) the short view of the knee suggests the tibial component is malaligned in varus . ( b ) however , the long leg view shows the limb has a neutral hip - knee - ankle axis . the use a short knee radiograph to assess component and limb alignment is not recommended . ( a ) the short view of the knee suggests the tibial component is malaligned in varus . ( b ) however , the long leg view shows the limb has a neutral hip - knee - ankle axis . the use a short knee radiograph to assess component and limb alignment is not recommended . the offset of the anatomic axis of the tibia and femur from the center of the talus and femoral head , respectively , is a new method for quantifying the degree of bow of these two bones . in the tibia , the bow typically starts at the junction of the proximal one - fourth and distal three - fourths of the tibia ( fig . 1 ) . the extramedullary tibial guide strives to reproduce the mechanical axis of the tibia by referencing the ankle and making a classic tibial osteotomy that centers the ankle on the knee . making a classic tibial osteotomy with an extramedullary guide only maintains the normal plane between the knee and ankle when there is no bow in the tibia ( ie , anatomic and mechanical axes of the tibia are the same ) , which occurred in only 11% of the patients in our study . the use of an intramedullary tibial guide results in a less than ideal cut in a bowed tibia because aligning the intramedullary tibial guide parallel to the mechanical axis of the tibia is impossible . in the femur , the factors determining the level of the bow are more complex than in the tibia . the bow in the femur is affected by variations in the bow of the shaft of the femur , the neck - shaft angle , and the length of the femoral neck ( fig . much has been written about how the degree of bow affects the starting point for insertion of the intramedullary rod and the accuracy of alignment of the varus / valgus osteotomy of the distal femur [ 17 , 23 , 24 , 26 , 28 ] . the wide variability of the bow of the femur in our patients further underscores the need to determine , for each patient , the best angle for making the distal femur varus / valgus osteotomy . determining the best angle for each patient is a challenge because guidelines for placing the starting point for insertion of the intramedullary rod in the distal femur and selecting the angle , although available , lack agreement . we identified no difference in the variability of the angle formed by the anatomic and mechanical axes and the bow in the tibia and femur between females and males . the lack of a gender difference between the angle formed by the anatomic and mechanical axes in the femur has been confirmed in the chinese population . a previous study of normal limbs found that only 2% of subjects have a neutral hip - knee - ankle axis , which can be explained by the wide variability of the bow in the tibia and femur and the lack of correlation between the bow of the tibia and femur in a given leg observed in the current study . a leg with a valgus hip - knee - ankle axis typically has a tibia with a medial bow combined with a femur with a small lateral bow ( ie , offset ) . a leg with a varus hip - knee - ankle axis typically has a tibia with a lateral bow combined with a femur with a large lateral bow ( ie , offset ) ( fig . the independent pairing of any shaped tibia with any shaped femur does not agree with the classic assumption that the bow of the tibia and femur compensate for each other to form a neutral hip - knee - ankle axis of the limb [ 13 , 22 , 29].fig . 6a f(a , d ) the anatomic axis ( longitudinal white lines ) of the knee measured on the radiographic view of these two knees are similar ; however , ( b , e ) the hip - knee - ankle axis of these two limbs are dissimilar . ( c , f ) the reason the anatomic axis of the knee does not predict the hip - knee - ankle axis of the limb is because of the wide variability in the bow of the tibia of the femur and because of the lack of correlation between the bow of a tibia and femur in a given limb . ( a , d ) the anatomic axis ( longitudinal white lines ) of the knee measured on the radiographic view of these two knees are similar ; however , ( b , e ) the hip - knee - ankle axis of these two limbs are dissimilar . ( c , f ) the reason the anatomic axis of the knee does not predict the hip - knee - ankle axis of the limb is because of the wide variability in the bow of the tibia of the femur and because of the lack of correlation between the bow of a tibia and femur in a given limb . the variability in the angle formed by the anatomic and mechanical axes and bow in the tibia and femur has changed our method for choosing the correct angle for making the proximal tibia and distal femoral cut . because few normal limbs have a neutral hip - knee - ankle axis ( ie , 2% ) , and because changing the hip - knee - axis from normal to neutral changes the three kinematic axes of the knee from normal and requires collateral ligament and retinacular releases that can lead to instability , we align the components kinematically [ 6 , 7 , 10 , 11 ] . three interrelated axes , none of which share any relationship to the center of the femoral head or the center of the ankle , describe the kinematics of the knee [ 5 , 6 , 9 ] . the primary axis is a tibial - femoral axis in the femur about which the tibia flexes and extends and is nonorthogonal to the three anatomic planes ( ie , sagittal , coronal , and axial ) [ 57 , 9 ] . one secondary axis is the patellofemoral axis in the femur about which the patella flexes and extends that is aligned parallel to the primary axis . the other secondary axis is a tibial - femoral axis in the tibia about which the tibia internally and externally rotates on the femur that is perpendicular to the tibial - femoral axis and the patellofemoral flexion axis in the femur [ 5 , 9 ] . therefore , the foundation for restoring normal kinematics in tka is aligning the axis of the femoral component coincident with the axis in the femur about which the tibia flexes and extends [ 57 , 9 ] . virtual , preoperative planning is used to align the axis of the femoral component coincident with the tibial - femoral axis in the femur by shape - matching the articular surface of the femoral component to the articular surface of the normal femur . a three - dimensional model of the normal femur is reconstructed from mr images of the patient s knee , which are each restored by filling in areas of focal wear . the surgical technique uses custom - made tibial and femoral cutting guides machined to the topography of the patient s knee to position the femoral and tibial components in all six degrees of freedom . this technique , which surface - matches the components to a knee that virtually has been restored to normal , adheres to the measured resection principle of tka and restores the hip - knee - ankle axis to the natural prearthritic alignment of the limb the subject had before arthritis and deformity developed [ 10 , 11 ] . conventional instrumentation and the current iterations of surgical navigation systems do not account for the wide intrasubject and intersubject variations in the angle formed by the anatomic and mechanical axes and the bow of the tibia and femur that are important in the selection of the ideal surgical planes in tka . use of the short knee film to judge placement of intramedullary and extramedullary rods , component alignment , and predict the hip - knee - ankle axis of the limb can not be justified .

introduction : in an aging population , new strategies are required to identify individuals at risk of adverse health outcomes . frailty syndrome is related to negative health events . this increased risk may be used to identify individuals in which interventions can delay the onset of physical and functional complications . the aim of the study was to determine the relationship between the onset of frailty and the beginning of functional disability.materials and methods : this was a cross - sectional observational study with consecutive sampling to analyze 146 patients aged seventy and older who come to the primary care center . the level of frailty was registered according to fatigue , resistance , ambulation , illnesses , and loss of weight scale . disability for instrumental activities of daily live dependency , comorbidity , and social risk factors was registered too.results:the prevalence of frailty and prefrailty was 17.8% and 39% , respectively , and were associated with age , level of disability , and the presence of gastrointestinal disease . prefrail patients had initial levels of dependency , while those who were not frail were mostly independent.conclusion:frailty syndrome is easily detectable . the intermediate stage known as prefrailty is related to the start of the functional disability . the syndrome screening identifies individuals at risk in whom we can potentially intervene to delay the onset of the syndrome and delay functional disability . control of comorbidity in frail patients must be studied . screening age could be set in patients between 75 and 81 years old .

the aging of a population leads to a higher incidence and prevalence of various health and social problems related to age , as well as the increased rates of dependency , comorbidity , and economic and social costs associated to those entities . it is necessary to identify individuals at risk in order to delay the onset of health problems and the economic problems related to aging . frailty appears as a clinical syndrome associated with greater vulnerability of the individual to adverse health outcomes , a higher level of disability , and increased mortality . sarcopenia is considered the factor that initiates the pathophysiological cycle of frailty , generating structural , and metabolic changes through insulin resistance , together with the tendency to inflammatory conditions and an increased cardiovascular risk . the lack of a definition of frailty common to different authors makes it difficult to calculate the real prevalence of the syndrome , which is said to be around 10.7% of the elderly , with 41.6% in the prefrailty situation . fatigue , resistance , ambulation , illnesses , and loss of weight ( frail ) scale may be used to detect frailty . it establishes three frailty levels according to the responses obtained to five questions on comorbidity , fatigue , resistance , aerobic capacity , and unintended weight loss . patients with 0 points are considered nonfrail , with 1 or 2 points for prefrail , and frail are those with 3 or more points . frail people have less capacity for daily living activities than nonfrail patients , but this relation is not well defined since it is not known if these entities are time - related . the possibility of using frailty as a screening method to detect people at risk of being unable to cope with their daily life activities might be useful to establish interventions to prevent or delay this loss . the objective of this study was to describe the functional capacities of the elderly in each frailty state according to frail scale . a time - related relationship between these entities may indicate when to start rehabilitation measures by detecting the frailty level . a descriptive observational study was accomplished , taking as target population 70-year - olds or older patients assigned to a single primary care center . the chosen primary care center was located in an urban area of the eastern part of the city of madrid , of a low - medium socioeconomic status . patients who did not sign the informed consent , were institutionalized and/or had language barriers or cognitive impairment and could not understand the information provided and required were excluded . based on an estimated prevalence of frailty of 10.7% in previous studies with similar tools , in a similar population , the calculated sample size was 145 subjects from a reference population of 3806 patients , ( 5% precision , confidence interval ( ci ) - 95% ) . these individuals were selected using consecutive not randomized sampling during the months of october and november 2014 until the sample was complete . subjects selected were verbally informed about the study features and were asked to complete the informed consent in writing by a letter . this enabled the investigators to consult their medical records and proceed with a personal interview . the following variables were recorded : sex , age ( grouped into 5-year groups ) , frailty level according to frail scale , comorbidities , and pluripathology following the instructions for development of clinical guidelines of the spanish primary care societies ( illnesses are classified in eight groups , depending on the organ or system affected ) , instrumental daily life activities capacity through lawton - brody scale , and the social risk following the gijn social risk scale adapted to the spanish population . age was also measured as a continuous quantitative variable , calculating the median and standard deviation . bivariate analysis was performed with chi - square test for measuring the association between variables , taking frailty as the dependent variable , and using the pearson 's regression ratio to quantify the association power if any . a logistic regression was performed in order to reject those variables that worked as confounding factors . spss 22.0 ( spss 22.0 , copyright by ibm , usa ) program was used for the statistical analysis . ethical issues were considered , respecting personal privacy , obtaining patients consent for the recruitment and the use of the data only for the purpose of this study . this investigation was approved by the investigation committee of the east health assistance area of madrid . classified as frail was 17.81% ( ci 1224% ) of the population and 39.72% ( ci 3248% ) as prefrail . the median age of frail individuals was 84 years with a standard deviation of 1-year . the median age of prefrail individuals was 81 , with a standard deviation of 1-year . prevalence of frailty by age group of the population , 45.20% were male and 54.8% were female . the distribution of age groups and variable frequencies by sex is shown in table 1 . percentage distribution and 95% ci for sociodemographic characteristics of the study population by gender total disability for instrumental daily living activities comprised 3.42% ( ci 0.5 - 6.4% ) of the population , severe 8.22% ( ci 3.9 - 12.7% ) , moderate 15.07% ( ci 9.8 - 20.9% ) , and mild 27.4% ( ci 20.3 - 34.6% ) . there was no limitation in those activities for 45.20% ( 37.1 - 53.3% ) of the population . a marked increase in the level of dependence occurred when the nonfrail and prefrail groups were compared as shown in figure 2 . distribution of dissability for instrumental activities of daily live by different levels of frailty of the total sample , 0.68% ( ci 0.2 - 2.02% ) presented a high social risk , 23.29% ( ci 16.4 - 30.1% ) moderate , and for 76.03% ( ci 69.1 - 82.9% ) there was no risk at all . the bivariate analysis between frailty and the different variables showed p values [ table 2 ] which allowed the establishment of a possible association between the frailty and the level of dependence in instrumental activities of daily living , the presence of comorbidity such as cardiovascular , musculoskeletal and digestive diseases , and age . the only association found was between frailty and disability , age dependency and diseases of the digestive system [ table 3 ] . p - values for association between frailty ( dependent variable ) and the rest of the study variables in a bivariate analysis p - values for association between frailty ( dependent variable ) and variables previously associated in the bivariate analysis within a multivariate model the prevalence of frailty similar to the previous studies with similar scales and population ( 5.827.3% ) was found although the criteria for the diagnosis of this entity are not the same in all of the literature . further research in this field must be done to establish common criteria so that the real impact of this syndrome can be described . although we have used a different methodology in sampling , the use of the same scales makes the comparison with other studies plausible . the results are applied to the population studied , but further research will help us to compare the differences with the general population . it is important to attend primary care to detect frailty properly as certain studies have demonstrated differences in the way family physicians and geriatricians detect this entity . in a descriptive analysis , frailty has been more frequent in relative terms in men than in women although they have presented greater relative frequency of prefrailty . our findings are in agreement with those studies that indicate that frailty is more common in women and that they persist in this condition longer than men . this may be due to the attendance of frail women at a different health - care center from the rest of population , delegating some activities to other family members . the ideal time for screening frailty can be set between 75 and 81 although the sample size limits the interpretation of results , and other studies have shown that there may be younger frail individuals . the transition from a normal situation to prefrailty status entails deterioration in instrumental activities of daily living . this relationship has been described in other articles , but the temporal association between the onset of prefrailty and the start of the loss of functional capacities has not been recounted in the consulted literature . indicate that frailty can be used as a method that allows targeted interventions to retard disability . mijnarends et al . reflected on the relationship between frailty and sarcopenia , recognizing the similarities between the frail scale and fried 's frailty phenotype , and encouraged the study of the relationship of these entities with a disability . cardiovascular diseases showed no significant relationship with frailty , but this result may change with further research , in accordance with previous literature and remove the limitations of this investigation . detection of frailty syndrome , especially its intermediate stage or prefrailty status , identifies individuals with greater risk of disease and negative prognosis . this condition can assist in making clinical decisions based on the timing of benefits of the different diagnostic and therapeutic procedures . establishment of the relationship between frailty and functional dependency as well as the identification of those individuals more likely to require the use of social resources later are vital so that social and health policies can be determined . it is necessary to establish the temporal relationship of the events associated with the onset of frailty , such as the time of evolution from the prefrail status to frailty , or the prognosis of frail and prefrail patients . studying the relationship between frailty and different pathology groups can strengthen the understanding of the pathophysiology of the syndrome in order to find the possible ways of prevention and treatment . it is possible that intervention in the gastrointestinal or cardiovascular field can delay the development of the syndrome , relying in each case on their pathophysiology . patients included in the study did not cover the entire population spectrum because of the sampling . using charts and data collection in a medical environment can produce biases that have to be borne in mind when the results are interpreted . detecting frailty in the early stage called prefrailty may be useful to identify patients at risk of losing their functional capacities . this relationship is important not only to maintain the best health level in the population , but also for the organizational implications that can be developed from it . the identification of prefrail individuals may be useful in the implementation of health and social programs that prevent the development of frailty and disability , and its economic and social costs . further research might be done to describe the effects of different interventions on prefrail patients .

a talking and singing robot which adaptively learns the vocalization skill by means of an auditory feedback learning algorithm is being developed . the robot consists of motor - controlled vocal organs such as vocal cords , a vocal tract and a nasal cavity to generate a natural voice imitating a human vocalization . in this study , the robot is applied to the training system of speech articulation for the hearing - impaired , because the robot is able to reproduce their vocalization and to teach them how it is to be improved to generate clear speech . the paper briefly introduces the mechanical construction of the robot and how it autonomously acquires the vocalization skill in the auditory feedback learning by listening to human speech . then the training system is described , together with the evaluation of the speech training by auditory impaired people .

a voice is the most important and effective medium employed not only in daily communication but also in logical discussions . only humans are able to use words as means of verbal communication , although almost all animals have voices . vocal sounds are generated by the relevant operations of the vocal organs such as a lung , trachea , vocal cords , vocal tract , tongue , and muscles . the airflow from the lung causes a vocal cord vibration to generate a source sound , and then the glottal wave is led to the vocal tract , which works as a sound filter as to form the spectrum envelope of a particular voice . the voice is at the same time transmitted to the auditory system so that the vocal system is controlled for the stable vocalization . different vocal sounds are generated by the complex movements of vocal organs under the feedback control mechanisms using an auditory system . as infants grow they acquire these control methods pertaining to the vocal organs for appropriate vocalization . these get developed in infancy by repetition of trials and errors concerning the hearing and vocalizing of vocal sounds . any disability or injury to any part of the vocal organs or to the auditory system may result in an impediment in vocalization . people who have congenitally hearing impairments have difficulties in learning vocalization , since they are not able to listen to their own voice . a speech therapist helps themtotrain their speech by teaching the vocal organs to learn vocalization and clear speech [ 14 ] . we are developing a talking robot by reproducing a human vocal system mechanically and based on the physical model of the vocal organs in the human . the fundamental frequency and the spectrum envelope determine the principal characteristics of a voice . fundamental frequency is a characteristic of the voice source that is generated by the vibration of vocal cords . the resonance effects that get articulated by the motion of vocal tract and nasal cavity cause the spectrum envelope . for the autonomous acquisition of vocalization skills by the robot , an adaptive learning using an auditory feedback control the robot consists of motor - controlled vocal organs such as vocal cords , a vocal tract , and a nasal cavity to generate a natural voice imitating a human vocalization [ 58 ] . by introducing auditory feedback learning with an adaptive control algorithm of pitch and phoneme , the robot is able to autonomously acquire the control skill of the mechanical system to vocalize stable vocal sounds imitating human speech . in the first part of the paper , the construction of vocal cords and vocal tract for the realization of the robot is briefly presented , and then the analysis of the autonomous learning of how the robot acquires the vocalization skill by using the neural network will be described . then , a robotic training system for the hearing - impaired people is introduced , together with the evaluation of the interactive speech training conducted in an experiment . the talking robot mainly consists of an air pump , artificial vocal cords , a resonance tube , a nasal cavity , and a microphone connected to a sound analyzer , which , respectively , correspond to a lung , vocal cords , a vocal tract , a nasal cavity , and an audition of a human , as shown in figure 1 . an air from the pump is led to the vocal cords via an airflow control valve , which works for the control of the voice volume . the resonance tube as a vocal tract is attached to the vocal cords for the modification of resonance characteristics . the nasal cavity is connected to the resonance tube with a sliding valve between them . it realizes the pitch extraction and the analysis of resonance characteristics of generated sounds in real time , which are necessary for the auditory feedback control . the system controller manages the whole system by listening to the vocalized sounds and calculating motor control commands , based on the auditory feedback control mechanism employing a neural network learning . the relation between the voice characteristics and motor control parameters is stored in the system controller , which is referred to in the generation of speech and singing performance . vocal cords with two vibrating cords molded with silicone rubber with the softness of human mucous membrane were constructed in this study . two - layered construction ( a hard silicone is inside with the soft coating outside ) gave the better resonance characteristics , and is employed in the robot . the vibratory actions of the two cords are excited by the airflow led by the tube , and generate a source sound to be resonated in the vocal tract . the tension of cords can be manipulated by applying tensile force to them . by pulling the cords , the relationship between the tensile force and the fundamental frequency of a vocal sound generated by the robot is acquired by the auditory feedback learning before the singing and talking performance , and pitches during the utterance are kept in stable by the adaptive feedback control . the human vocal tract is a non - uniform tube about 170 mm long in man . its cross - sectional area varies from 0 to 20 cm under the control for vocalization . a nasal cavity with a total volume of 60 cm is coupled to the vocal tract . in the mechanical system , a resonance tube as a vocal tract it works as a resonator of a source sound generated by the vocal cords . it is made of a silicone rubber with the length of 180 mm and the diameter of 36 mm , which is equal to 10.2 cm by the cross - sectional area as shown in figure 1 . the silicone rubber is molded with the softness of human skin , which contributes to the quality of the resonance characteristics . in addition , a nasal cavity made of a plaster is attached to the resonance tube to vocalize nasal sounds like /m/ and /n/. a sliding valve as a role of the soft palate is settled at the connection of the resonance tube and the nasal cavity for the selection of nasal and normal sounds . for the generation of nasal sounds /n/ and /m/ , the motor - controlled sliding valve is open to lead the air into the nasal cavity . by actuating displacement forces with stainless bars from the outside of the vocal tract , the cross - sectional area of the tube is manipulated so that the resonance characteristics are changed according to the transformations of the inner areas of the resonator . compact servo motors are placed at 8 positions xj ( j = 18 ) from the lip side of the tube to the intake side , and the displacement forces pj(xj ) are applied according to the control commands from the motor - phoneme controller . an adaptive learning algorithm for the achievement of a talking and singing performance is introduced in this section . first in the learning phase , the system acquires two maps in which the relations between the motor positions and the features of generated voices are established and stored . one is a motor - pitch map , which associates motor positions with fundamental frequencies . it is acquired by comparing the pitches of vocalized sounds with the desired pitches , which cover the frequency range of speech . the other is a motor - phoneme map , which associates motor positions with phonetic features of vowel and consonant sounds . second in the performance phase , the robot speaks and sings by referring to the obtained maps , while pitches and phonemes of generated voices are adaptively maintained by hearing its own output voices . the neural network ( nn ) works to associate the sound characteristics with the control parameters of the nine motors settled in the vocal tract and the nasal cavity . in the learning process , the network learns the motor control commands by inputting 10th - order linear predictive coding ( lpc ) cepstrum coefficients derived from vocal sound waves as teaching signals . the network acquires the relations between the sound parameters and the motor control commands of the vocal tract . after the learning , the neural network is connected in series into the vocal tract model . by inputting the sound parameters of desired sounds to the nn , the corresponding form of the vocal tract is obtained . in this study , the self - organizing neural network ( sonn ) was employed for the adaptive learning of vocalization . figure 2 shows the structure of the sonn consisting of two processes , which are an information memory process and an information recall process . after the sonn learning , the motor control parameters are adaptively recalled by the stimuli of sounds to be generated . the information memory process is achieved by the self - organizing map ( som ) learning , in which sound parameters are arranged onto a two - dimensional feature map to be related to one another . weight vector vj at node jin the feature map is fully connected to the input nodes xi[i = 1 , , 10 ] , where 10th - order lpc cepstrum coefficients are given . a competitive learning is used , in which the winner c as the output unit with a weight vector closest to the current input vector x(t ) is chosen at time t in learning . by using the winner c , the weight vectors vj - s are updated according to the rule shown below ; ( 1)vj(t+1)=vj(t)+hcj(t)[x(t)vj(t)],hcj(t)={(t)exp(rcrj222(t))(inc),0(i nc ) . here , rc rj is the distance between units c and j in the output array , and nc is the neighborhood of the node c. (t ) is a learning coefficient which gradually reduces as the learning proceeds . then , in the information recall process , each node in the feature map is associated with motor control parameters for the control commands of nine motors employed for the vocal tract deformation , by using the three - layered perceptron . in this study , a conventional back - propagation algorithm was employed for the learning . with the integration of the information memory and recall processes , the sonn works to adaptively associate sound parameters with motor control parameters . in the current system , 25 25 arrayed map v = [ v1 , v2 , , v25 25 ] is used as the som . for testing the mapping ability , after the self - organizing learning , five japanese vowels vocalized by six different people were mapped onto the feature map . same vowel sounds given by different people were mapped close with each other , and five vowels were roughly categorized according to the differences of phonetic characteristics . we found that , in some vowel area , two sounds given by two different speakers fell in a same unit in the feature map . it means that the two different sounds could not be separated , although they have close tonal features with each other . redundant sound parameters which were not used for the japanese speech were buried in the map , since the 150 inputted sounds were generated randomly by the robot . furthermore , two different sounds given by two different speakers were occasionally fallen in the same unit . after the sonn learning , five japanese vowel sounds given by 6 different speakers with normal audition were applied to the supervised learning as the reinforcement signal to be associated with the suitable motor control parameters for the japanese vocalization . figure 3 shows the result of the reinforcement learning with five japanese vowels given by five speakers no . 1 to 5 . the distribution of same vowel sounds concentrated with one another , and the patterns of different vowels were placed apart . after the learning of the relationship between the sound parameters and the motor control parameters , we inputted human voices from microphone to confirm whether the robot could speak autonomously by mimicking human vocalization . with the comparison of spectra between human vowel vocalization and robot speech , we confirmed that the first and second formants f1 and f2 , which present the principal characteristics of the vowels , were formed properly as to approximate the human vowels , and the sounds were well distinguishable by listeners . the transition between two different vowels in the continuous speech was well acquired by the sonn learning , which means that all the cellsoninthe som are associated with motor control parameters properly to vocalize particular sounds . voices of hearing - impaired people then were given to the robot so as to confirm that the articulatory motion would be reproduced by the robot . figure 4 shows the mapping results of six different voices given by hearing - impaired speakers no . a , no . f. the same colors indicate the vocal sounds generated by the same vowels . in figure 5 , vocal tract shapes estimated by the robot from voices of hearing - impaired person no . a are presented , together with the comparison of the vocal tract shapes estimated by the able - bodied speaker no . 1 voices . from the observation of the robot 's reproduced motions of the vocal tract , the articulations of auditory - impaired people were apparently small , and complex shapes of vocal tract were not sufficiently articulated . furthermore , in the map shown in figure 4 , /u/ sound given by the hearing - impaired speaker no . a is located inside the /e/ area of able - bodied speakers , and his /o/ vowel is located close to the /u/ area of able - bodied speakers . these articulatory characteristics also appear in the vocal tract shapes shown in figure 5 . in the figures , the vowel /u/ shape of speaker no . a shown in ( b-2 ) is almost the same with the /o/ shape of speaker no . 1 presented in ( c-1 ) . likewise , the /o/ shape shown in ( c-2 ) appears close to the shape of ( b-1 ) . thus , these results proved that the topological relations of resonance characteristics of voices were well preserved in the map , and the articulatory motion by the robot was successfully obtained to reproduce the speech articulation by listening arbitrary vocal sounds . in the speech training , the robot interactively shows the articulatory motion of vocal organs as a target to a trainee so thats / he repeats his / her vocalization and the observation of the robot motion . the trainee is also able to refer to the som to find the distance to the target voice . the training of speech articulation by an auditory - impaired subject is shown in figure 7 . an experiment of speech training was conducted by six hearing - impaired subjects : no . a f ( four males and two females ) , who study in a high school and a junior high school . in figure 8 , the training results of three subjects no . f are shown by presenting the trajectories of voices appeared in the som during the training experiments . figure 8(a ) shows a result of successful training with less trials conducted by the subject no . a. by observing the articulatory motion instructed by the robot , this subject recognized the difference in his articulation and effectively learned the correct motion . e , however , he had achieved the vocalization by repeating several trials and errors , especially for the vowels /i/ and /e/ as presented by the arrows from i1 to i5 and e1 to e5 , respectively . in the case of the training conducted by the subject no . the clarity of his voices was quite low , and the original voices were mapped far from the area of clear voices . he could not understand the shape of the robot 's vocal tract , nor realize the correspondence between the robot 's motion and the motion of his inner mouth . this subject tried to articulate his vocal tract following the articulatory motion indicated by the robot , however , his voice moved to the different direction in the som as shown by arrows in figure 8(c ) . he failed the acquisition of vocalization skill and could not achieve the training . in the questionnaire after the training , he pointed out the difficulties of moving a particular part of the inner mouth so as to mimic the articulatory motion of the robot . by the experimental training , five subjects could mimic the vocalization following the directions given by the robotic voice simulator , and acquired the better vocal sounds . in the questionnaire after the experiment , two subjects commented that the correspondence between robot 's vocal tract and human actual vocal tract should be instructed , so that they could easily understand which part inside the mouth should be intensively articulated for the clear vocalization . a robotic voice simulator and its articulatory reproduction of voice of hearing - impaired people were introduced in this paper . by introducing the adaptive learning and controlling of the mechanical model with the auditory feedback , the voice robot was able to acquire the vocalization skill as a human baby does in speech training . the robot was applied to introduce a training system for auditory - impaired people to interactively train the speech articulation for learning proper vocalization . the robotic voice simulator reproduces the articulatory motion just by listening to actual voices given by auditory - impaired people , and they could learn and know how to move their vocal organs for the clear vocalization , by observing the motions instructed by the talking robot . the use of som for visually presenting the distance between target voice and trainee 's voice is also introduced . we confirmed that the training using the talking robot and the som would help hearing - impaired people learn the articulatory motion in the mouth and the skill of clear vocalization properly . in the next system , the correspondence between robot 's vocal tract and human actual vocal tract should be established so that a subject could understand which part inside the mouth should be intensively articulated in the training . by analyzing the vocal articulation of auditory - impaired people during the training with the robot , we will investigate the factor of unclarity of their voices originated by the articulatory motions .

diabetes mellitus is associated with cognitive decline and impaired performance in cognitive function tests among type 1 and type 2 diabetics . even though the use of tight glucose control has been limited by a reported higher mortality , few reports have assessed the impact of treatment intensity on cognitive function . we conducted a meta - analysis to evaluate if an intensive glucose control in diabetes improves cognitive function , in comparison to standard therapy . we included 7 studies that included type 1 or type 2 diabetics and used standardized tests to evaluate various cognitive function domains . standardized mean differences ( smds ) were calculated for each domain . we found that type 1 diabetics get no cognitive benefit from a tight glucose control , whereas type 2 diabetics get some benefit on processing speed and executive domains but had worse performances in the memory and attention domains , along with a higher incidence of mortality when using intensive glucose control regimes .

diabetes mellitus ( dm ) is a chronic metabolic condition that affects 8.3% of the world population and causes significant morbidity and mortality . the number of people suffering from diabetes is expected to increase beyond 592 million people over the next 25 years [ 1 , 2 ] . endothelial damage in diabetes leads to damage of multiple organs and an increased risk of myocardial infarction , stroke , and peripheral vascular disease , along with other chronic complications such as kidney disease or retinopathy . diabetes also increases the risk of cognitive dysfunction and both vascular dementia and alzheimer 's disease [ 35 ] . this association is more prominent in elderly diabetics , although mild cognitive impairment may be present also in relatively younger diabetics [ 68 ] . the impact of diabetes in cognitive function may become more apparent as the life expectancy has significantly increased over the past years . a recent meta - analysis determined that type 2 diabetics had worse performance in neuropsychological tests when compared to normal subjects . as for type 1 diabetes , which is less common and there is , however , evidence of an overall decrease in pediatric cognitive performance for diabetic children except in the memory and language domains . a more recent study showed a nonstatistically significant reduction of intellectual function for type 1 diabetics when compared to normal children . although recent data has found that intensive glucose control could be associated with increased mortality among diabetics , the impact on cognitive function is less understood . we conducted a meta - analysis to determine if intensive glucose control can actually prevent or delay the onset of cognitive decline both in type 1 and in type 2 diabetics . as we move to achieve patient centered care , having information for patients regarding the balance between quantity and quality of life will be useful . pubmed ( medline ) database was searched for randomized controlled trials published from january 1 , 1980 , to june 1 , 2014 , using mesh terms and keywords . search terms used included type 1 diabetes mellitus , type 2 diabetes mellitus , drug therapy , and cognitive function . the full search including mesh terms was ( ( ( diabetes mellitus , type 1/drug therapy [ mesh terms ] or diabetes mellitus , type 2/drug therapy [ mesh terms ] ) or diabetes mellitus , type 1/therapy [ mesh terms ] ) or diabetes mellitus , type 2/therapy [ mesh terms ] ) and ( cognitive [ all fields ] and ( physiology [ subheading ] or physiology [ all fields ] or physiology [ mesh terms ] or function [ all fields ] ) ) and ( ( clinical trial [ ptyp ] or randomized controlled trial [ ptyp ] ) and ( 1980/01/01 [ pdat ] : 2014/12/31 [ pdat ] ) ) . we also reviewed the reference list of the identified articles looking for additional studies that might be included in this meta - analysis . we included randomized controlled trials ( rct ) , which analyzed patients with either type 1 or type 2 diabetes , had at least one group of patients receiving intensive glucose control and another receiving conventional antidiabetic treatment , and provided information regarding assessment of cognitive function after a follow - up period using a standardized method . the exclusion criteria we used were as follows : studies which included patients already diagnosed with cognitive dysfunction or established dementia , studies that used only the minimental score examination ( mmse ) as an assessment of cognitive function , and studies that utilized a cognitive testing method which was not comparable to those used in any of the other articles included . we defined interventions as intensive if they tailored care to reach a glycated hemoglobin ( hba1c ) goal of less than 7% or a fasting glucose level of less than 130 mg / dl . conventional treatment was defined simply as the continuation of the regular treatment the patient was already receiving . four of them intended to achieve levels of hba1c below 6% , while another one targeted hba1c levels below 7% [ 1416 , 18 , 19 ] . two more studies did not report a goal level of glycated hemoglobin , one of them targeted preprandial glucose levels below 130 mg / dl instead , and the last one adjusted goals of glycaemia and hba1c individually with each patient [ 13 , 17 ] . the methods used to achieve these goals ranged from multifactorial behavioral interventions to adjusted doses of oral antidiabetics to 3 or more insulin injections per day or continuous insulin infusion with an external pump . the main outcome was cognitive dysfunction classified into the following domains based on standard domain definitions : information processing speed , executive function , attention / concentration , verbal memory , and motor function . information processing speed was assessed through the digit - symbol substitution subtest ( dsst ) of the wechsler assessment of intelligence scale ( wais ) , in which the participant is initially shown a key containing symbol - digit pairs and must later copy the corresponding symbol under a series of numbers with empty boxes below . as measures of executive functioning , participants were assessed using the trail making test part b ( tmt - b ) and the similarities subtest of the wais . the tmt - b measures the time a subject needs to draw lines connecting 25 encircled letters and numbers distributed over a sheet of paper in alternating order . for the similarities subtest , subjects are asked in what way two words are alike ( i.e. , poem and statue ) . memory function was evaluated using the rey auditory verbal learning test ( ravlt ) , a verbal learning task where the participant is given a list of 15 unrelated words repeated over 5 trials . a delayed - recall trial is administered 30 minutes after the initial learning phase and the number of freely recalled words is recorded . reaction time to auditory and visual stimuli was measured through computerized tasks where participants had to press a key immediately after presentation of visual ( light ) or auditory ( tone ) stimuli . the finger tapping test was administered as a measure of motor function . in this test , participants place their hand on a board with a lever and tap their index finger on the lever as quickly as possible , using their dominant and nondominant hands , within a 10-second time interval . scores are calculated by averaging the number of taps over five consecutive trials within a 5-point range with each hand . the stroop test is a measure of selective attention , cognitive flexibility , and cognitive inhibition . read a list of color names printed in black ink . in the second part they must name the color of a list of x 's or color patches , depending on the version used . in the third part of the task the subject must name the color of a color word written in nonmatching ink color ( e.g. , the word green printed in red ) . a stroop interference effect occurs when color - naming speed is significantly reduced as the subject must inhibit an automatic reading response to produce a more effortful color - naming response . we reported relevant baseline characteristics for each study as mean and percentage as reported . to aggregate unweighted results for all studies we report the median and interquartile range for continuous variables and for hba1c we report the mean values before and after the intervention per randomized group . to assess for heterogeneity across studies we used the cochran q chi - square ( significance level < 0.10 ) and the i - squared statistic ( > 50% ) . for the mathematical pooling we stratified the analysis by type of diabetes and calculated the standardized mean difference ( smd ) with the corresponding 95% confidence interval and p value . the smd represents the difference between the mean and standard deviation of the cognitive test in the intensive control group minus that of the conventional group for each study . the smd was weighted by the sample size of each individual study per randomized group . our search strategy yielded 82 articles , from which we excluded 73 abstracts because they were not rct or did not meet inclusion criteria . from the remaining 9 studies from the original search , we removed 3 more articles after exclusion criteria were applied . one additional study was retrieved from the references of the articles reviewed and was included for analysis as it did not meet exclusion criteria . a total of 7 articles were finally included in the meta - analysis , of which 4 analyzed type 1 diabetics and 3 studied type 2 diabetics ( figure 1 ) . the combined sample size was 6056 patients ( 3011 under intensive glucose control and 3045 under conventional treatment ) . the median age was 27 years for type 1 diabetics and 62.4 years for type 2 diabetics . median baseline levels of hba1c were 9.24% for the intensive treatment group and 9.07% for the conventional treatment patients , while hba1c levels after treatment follow - up were 7.43% for the intensive group and 8.17% for the conventional treatment patients . the most commonly reported tests where the dsst , trail making , finger tap , and ravlt . the univariate results show that on each test there is a difference between the intensive treatment group and the control group . diabetes the dsst smd had a positive direction ( 0.71 ) , while the trail making test , stroop test , and ravlt had a negative direction . however , a negative direction on the smd for trail making test also favors intensive glucose control due to the nature of the test . these results are summarized in figure 2 , where results for tmt have been mirrored to a positive sense for a better presentation . our meta - analysis demonstrates that tight glucose control is not superior to conventional care at preventing cognitive decline among type 1 diabetics and has a positive impact only on the information processing speed and executive functions among type 2 diabetics . the lack of effect seen for type 1 diabetics could be related to the nonsignificant differences described to date in cognitive function between diabetics and healthy control groups . among young diabetic patients who are free of multiple comorbidities , the effect of hyperglycemia may not be severe enough to cause a significant cognitive impairment , and thus the glucose lowering regime used to treat diabetes becomes unimportant regarding prevention of cognitive function loss . an alternative explanation is that the small cognitive decline reported among type 1 diabetics is related to the effect of repeated hypoglycemic events which may cause white matter damage but would not be reduced by tight glucose control [ 10 , 26 ] . in contrast , the effect of tight glucose control varied across cognitive domains among type 2 diabetics . the intensive control group performed significantly better on the dsst and tmt but did worse than the conventional treatment group on the stroop test and the ravlt . from these results we can conclude that tight glucose control favors the domains of information processing speed and executive function , but at the cost of negatively affecting attention and memory functions . the presence of comorbidities at the age of onset of diabetes , which is much later than that for type 1 diabetics , may help explain these results . also , it has been described that insulin is one of the molecules that regulate tau protein phosphorylation in neurons , and thus insulin resistance may disrupt this process , causing tau to bind to microtubules , giving rise to the pathogenesis of alzheimer 's disease and dementia . educational level is also an important confounding factor in this population , as it has been observed that cognitive performance correlates directly with the amount of years of completed study . however , there are no enough data to test the impact of these confounders in the current meta - analysis . in regard to the higher risk of mortality previously reported for tight glucose control regimes , only two of the studies included reported a mortality outcome [ 15 , 18 ] . thus , evaluating the relationship between cognitive decline , mortality , and tight glucose control was not possible . to date , age , the increased risk of hypoglycemia , and the presence of important comorbidities are factors that favor the increased incidence of deaths in type 2 population , while in type 1 diabetics , though there was an increased risk of hypoglycemic events , the great majority were nonfatal [ 12 , 15 ] . first , while there is significant evidence on the relationship of diabetes and cognitive decline , very few trials have addressed the impact of different glucose control regimes on cognitive function . more so , many studies evaluating this question could not be included because they either used noncomparable tests or reported cognitive decline using only the mmse [ 26 , 2830 ] . also , the large variation in sample size among type 2 diabetes studies caused one of the studies to carry more significant weight than the others . in conclusion , we observed that there is no benefit from intensive glucose lowering regarding cognitive function for the young type 1 diabetics , while the older type 2 diabetics benefit from this therapy in the domains of information processing speed and executive function but find their attention and memory hindered . these findings provide insight into the pathophysiology of different types of cognitive impairment and possible therapeutic avenues in the future . some studies have shown an increased risk of cardiovascular mortality and hypoglycemia when using intensive glucose control regimes . thus , each case should be evaluated individually to assess the benefits of a tight glycemic control against the observed risks . since these complications are more common in older diabetic patients , intensive control of the glucose levels might be safer and more recommendable in type 1 diabetics , most of which are children or young adolescents , regarding noncognitive benefits .

the isotopic composition of mercury ( hg ) was determined in cinnabar ore , mine - waste calcine ( retorted ore ) , and leachates obtained from water leaching experiments of calcine from two large hg mining districts in the u.s . this study is the first to report significant mass - dependent hg isotopic fractionation between cinnabar ore and resultant calcine . data indicate that 202hg values relative to nist 3133 of calcine ( up to 1.52 ) in the terlingua district , texas , are as much as 3.24 heavier than cinnabar ( 1.72 ) prior to retorting . in addition , 202hg values obtained from leachates of terlingua district calcines are isotopically similar to , or as much as 1.17 heavier than associated calcines , most likely due to leaching of soluble , byproduct hg compounds formed during ore retorting that are a minor component in the calcines . as a result of the large fractionation found between cinnabar and calcine , and because calcine is the dominant source of hg contamination from the mines studied , 202hg values of calcine may be more environmentally important in these mined areas than the primary cinnabar ore . measurement of the hg isotopic composition of calcine is necessary when using hg isotopes for tracing hg sources from areas mined for hg , especially mine water runoff .

mercury is a common contaminant in the environment due to both natural and anthropogenic sources . it is of environmental concern because elevated concentrations can be toxic to living organisms ( 1 ) . mercury has no known metabolic function in animals and is not easily eliminated by organisms , including humans ( 2 ) . areas around mined and unmined deposits of hg contain some of the highest concentrations of hg worldwide ( 35 ) . abandoned and inactive hg mines are of environmental concern because of the high concentration of hg present in discarded wastes at these sites . there are presently no hg mines operating in the united states , primarily because of low demand for hg and environmental concerns ( 6 ) . cinnabar ( hexagonal , hgs ) is the dominant hg - bearing ore mineral in hg mines worldwide , although minor ore minerals such as elemental hg ( hg ) , metacinnabar , ( isometric hgs , metastable relative to cinnabar ) , calomel ( hg2cl2 ) , kleinite ( hg2n(cl , so4)n(h2o ) ) , montroydite ( hgo ) , and terlinguaite ( hghgclo ) ) are found in some deposits ( 710 ) . extraction of hg during mining is generally carried out in a retort or a rotary furnace where hg ore is heated to 600700 c , which converts cinnabar to elemental hg , the final hg product that is sold commercially ( 6,9,1113 ) . retorting of hg - bearing ore is known to be an incomplete process , and as a result , calcine found at most hg mines contains unconverted cinnabar , elemental hg , metacinnabar , and other ionic hg byproduct compounds formed during ore retorting ( 5,7 ) . one consequence of hg mining is that considerable calcine is generated during ore processing and this calcine is typically discarded on the surface at mined sites . even after many years of inactivity , hg mining areas continue to be characterized by highly elevated hg concentrations as a result of the incomplete hg extraction process ( 3,14 ) . microscopic fine - grained cinnabar and several byproduct hg compounds found in calcine can adversely affect surrounding environments due to sediment and water runoff containing high concentrations of hg . minor byproduct hg compounds such as chlorides , oxychlorides , and sulfates , which are water - soluble are generally released downstream in mine runoff , potentially contaminating nearby ecosystems ( 1517 ) . unprocessed ore and tailings containing cinnabar also remain at some mines , and can be a significant source of hg to surrounding environments . new analytical methods for isotopic analysis have been developed to help characterize environmental cycling of hg . measurements of the isotopic composition of hg hold promise as a basis for tracing sources of hg contamination in the environment and evaluation of isotopic fractionation during geochemical processes ( 1822 ) . before hg isotopes can be evaluated as a tracer of hg in mined areas , the variability in hg isotopic composition of the hg sources involved must be measured . therefore , the objective of this study was to measure the isotopic compositions of hg in ( 1 ) cinnabar from two areas of past hg mining to evaluate the variability of hg isotopic composition within hg deposits ; ( 2 ) the minor hg ore minerals metacinnabar , calomel , kleinite , montroydite , and terlinguaite to evaluate potential isotopic fractionation during formation of these hg minerals ; ( 3 ) calcine to evaluate potential fractionation of hg resulting from retorting of ore during mining ; and , ( 4 ) water leachates of calcine to evaluate the variability in isotopic composition of the hg in simulated runoff from hg mines . two mining districts of significant past hg production , mcdermitt , nevada , and terlingua , texas were selected for study . mines of the terlingua district are found in southwest texas , in and around the town of terlingua ( 29 19.170 , 103 37.000 ) . total production from this region was > 5 000 000 kg of hg ( 150 000 flasks of 34.5 kg each ) ( 23 ) . consequently , of the hg mines in the u.s . , the terlingua district produced the third largest amount of hg and only mines in the california coast ranges ( 120 000 000 kg of hg ) and mcdermitt , nevada ( 10 000 000 kg of hg ) produced more ( 4,25 ) . the terlingua district includes over 30 separate mines ( 10 ) , but our work focused on the mariscal , study butte , mariposa , and terlingua ( also known as the chisos ) mines . as is true for most hg mines worldwide , the hg ore in the terlingua district is dominantly cinnabar , but minor hg minerals , including metacinnabar , elemental hg , calomel , montroydite , terlinguaite and kleinite were identified at some mines ( 10,23 ) . the district has been estimated to contain over 2 000 000 m of calcine ( 15 ) . this calcine contains hg concentrations as high as 19 000 g / g ( 15 ) , some of which are water - soluble ( 5,7 ) . leaching , runoff , and downstream transport of hg from calcine potentially deliver significant hg to surroundings streams , which eventually flow into the rio grande ( 15 ) . mercury was mined at mcdermitt ( also known as cordero , 41 54.970 , 117 48.600 ) from 1935 to 1992 , when the mine closed ( 6 ) . mcdermitt was the largest hg mine in nevada , and production of hg was about 10 000 000 kg ( 25 ) . ore was dominantly cinnabar , but minor elemental hg and oxychlorides ( e.g. , hg2clo and hg4cl2o ) were found locally ( 9 ) . numerous piles of calcine are scattered throughout the mcdermitt mine site , but have been estimated as > 1 000 000 m(8 ) . similar to the terlingua mines , high hg concentrations ( up to 1400 g / g ) in calcine at mcdermitt potentially lead to hg leaching and transport of hg downstream from the mined areas ( 8) . all of the calcine and most of the cinnabar samples from the mcdermitt mine and terlingua district analyzed in this study were collected during previous studies ( 8,15,26 ) . all calcine was collected as grab samples about 2550 cm below the surface to avoid the highly oxidized near - surface environment , and generally at this depth , the calcines were dark gray in color as opposed to more oxidized red - brown at the surface . all mcdermitt mine cinnabar samples were physically separated from bulk ore by crushing and hand picking . samples of metacinnabar , calomel , cinnabar , terlinguaite , kleinite , and montroydite from the terlingua district were obtained from the colorado school of mines ( csm , golden , colorado ) geology museum . these minerals were physically separated from the host rock and other minerals . previously separated and powdered calomel , montroydite , and a mixed terlinguaite - kleinite sample from the terlingua district were obtained from a u.s . geological survey ( usgs , denver , colorado ) the terlinguaitekleinite sample from the usgs was fine - grained and these minerals could not be separated , thus , this sample was analyzed as a mixed mineral . the concentration of total hg ( thg ) was determined in pulverized ore and calcine samples using a 3:1 hcl : hno3 ( aqua - regia ) leach ( 1.0 g sample leached in 10 ml aqua - regia ) and cold - vapor atomic fluorescence spectrometry following epa method 1631 ( 27 ) . quality control for thg was established using method blanks , blank spikes , matrix spikes , standard reference materials ( srms ) , and sample duplicates . for the srm s , nist 2704 and pacs-2 analyzed in this study , recoveries ranged from 96 to 107% of certified values for thg . method blanks were below the thg limit of determination , which was 0.005 g / g . pulverized calcine and hg mineral samples were leached in aqua - regia ( 3:1 hcl : hno3 ) overnight in borosilicate glass test tubes ( 1.0 g sample leached in 10 ml aqua - regia ) at room temperature and diluted immediately prior to analysis with 3% ( v / v ) hcl to a final hg concentration of 1 g / l ( all minerals and calcines ) or 2 g / l ( calcine leachates ) . a blank digestion was performed , diluted to 50 ml with 3% ( v / v ) hcl and confirmed to contain no hg . hg was introduced into a multiple collector inductively coupled plasma mass spectrometer ( mc - icp - ms ) ( nu plasma hr , nu instruments , wrexham , north wales , uk ) as a cold vapor , which was generated from hg in solution using stannous chloride in a commercially available cold vapor ( cv ) generation system ( hgx-200 , cetac , omaha , ne ) . argon carrier gas ( sample gas ) was introduced into the cv generator at a flow rate of 5060 ml / min and stannous chloride reductant and sample solution , each at a rate of 1.5 ml / min were mixed with the ar gas . a makeup ar gas flow of 0.91.0 l / min was added prior to introduction into the plasma . background signal ( 3% ( v / v ) hcl blank ) was measured on each isotope peak for 60 s and subtracted from the peak integrated data . signal data were measured via time - resolved analysis software for approximately eight minutes and data were integrated for approximately five minutes . the sensitivity of the mc - icp - ms for hg was 7001000 mv ( g / l ) and data were collected using solution thg concentrations of 12 g / l . each sample was bracketed by nist 3133 hg standard at the same thg as the sample and sample data were corrected for mass bias using standard sample bracketing correction . the standard sample bracket method was optimized to ensure that the precision was comparable to previously published studies that used either standard - sample bracketing or tl correction for mass bias ( 18,28,29 ) . sensitivity for hg was comparable to or greater than that of other published methods ( 2830 ) . studies were performed to determine how closely standard and sample concentrations and matrix should to be matched to obtain optimal precision . common matrix constituents did not cause mass bias in the measurements when no matrix matching between standards and samples was performed . no mass bias was introduced when sample and standard concentrations were varied over a 25-fold range , but method precision decreases as sample concentration is decreased . also , when concentrations greater than 2 g / l were used washout times increased . isotopic ratioshg / hg , hg / hg , hg / hg , and hg / hg were measured and hg isotopic compositions are reported in standard delta notation as hg in per mil ( ) , referenced against nist 3133 ( 18 ) where x = 199 , 200 , 201 , or 202 . values of hg are calculated as follows : all hg values and 2 standard deviation ( sd ) reproducibility of replicate isotopic measurements are reported in supporting information ( si ) table s-1 , but only hg data are discussed below . all data were assessed for mass independent fractionation ( mif ) by calculating hg , hg , and hg using a method previously described ( 18 ) . the long - term reproducibility of the method was determined to be 0.09 ( 2sd ) for hg and 0.05 ( 2sd ) for hg by measurement of the isotopic composition of 2 g / l thg in secondary standard um - almadn ( 18 ) , but was 0.24 ( 2sd ) for hg and 0.10 ( 2sd ) for hg at a thg concentration of 1 g / l ( 31 ) ( si table s-1 contains all and values for this standard ) . laboratory water leaching studies were conducted on samples of calcine using an operationally defined procedure modified from epa method 1312 ( 32 ) . eight mine - waste calcine samples ( seven from terlingua and one from mcdermitt ) of 3050 g were leached with 150 ml of deionized water acidified with ultrapure h2so4/hno3 to a ph of 5.0 . leachate fluid of ph 5 is recommended to simulate rainwater ph in the western u.s . the samples were leached on a shaker table using 250 rpm for 18 h. the leachate was isolated and filtered using 0.45 m nitro - cellulose disposable filters . concentrated ultrapure hcl was added to the filtrate to yield 3% ( v / v ) hcl content . immediately prior to analysis , leachates were diluted with 3% ( v / v ) hcl to yield a hg concentration in the range of 12 g / l . to establish the variability of hg isotopic composition of cinnabar within a single hg deposit , hg isotopic compositions were determined in ( 1 ) several cinnabar crystals separated from a single sample of ore from the mcdermitt mine and ( 2 ) several additional cinnabar samples collected throughout the mcdermitt mine site . the total range in the isotopic composition of hg measured for a single sample of mcdermitt cinnabar was 0.42 to 0.69 , which is indistinguishable within analytical precision ( 0.24 , si tables s-1 and s-2 ) . a smaller range in isotopic hg compositions was found for the multiple samples of cinnabar , 0.57 to 0.70 , also indistinguishable within analytical precision . previous studies report hg compositions for various hg - bearing mineralized rocks and ore deposits in california and nevada as 3.88 to 2.10 , a variation of 5.98 ( 21,33 ) . other studies reported a smaller variation in hg values for cinnabar samples collected from numerous hg mines and deposits worldwide , 1.73 to 1.33 , a variation of 3.06 ( 20,31 ) . it is unclear if the hg compositions measured in hg - bearing mineralized rocks ( 21,33 ) show a wide variation due to isotopic hg sources from varying rock types or isotopic fractionation during ore formation . although the above studies reported a wide variation in hg compositions for various hg deposits worldwide , our hg isotopic analysis of numerous samples of cinnabar collected throughout the mcdermitt mine indicates that cinnabar at the mcdermitt mine has an isotopically narrow range of hg composition of 0.60 0.20. the range in hg was 1.60 to 1.72 , indicating a statistically insignificant variation of 0.12 , for various cinnabar samples from the terlingua district ( figure 1 ) . the hg of other hg - bearing ore minerals from the terlingua district ( montroydrite , kleinite , terlinguaite , metacinnabar , and calomel ) ranged from 2.70 to 1.39 , a variation of 4.09 ( figure 1 ) , a much wider variation than found for the terlingua district cinnabar samples . in addition , mass - independent fractionations in hg ranging from 0.09 to 0.36 were observed in these hg minerals ( si table s-1 , figure 2 ) . the differences in hg isotopic composition among the various hg minerals from the terlingua district are statistically significant ( figure 1 ) . isotopic compositions of hg for various sources of hg from the mcdermitt mine and the terlingua district . data shown for cinnabar , metacinnabar , terlinguaite , and kleinite are isotopic measurements made in duplicate or triplicate on one sample of each mineral and symbols represent the average . for calomel and montroydite , duplicate or triplicate isotopic measurements were made on two separate samples of each mineral from different sources ( colorado school of mines museum and u.s . hg vs hg of minerals , mine waste calcine , and leachates of mine waste calcine . the slope of the line is 1.66 for the minerals and 1.11 for the calcines . the slope of the line for the leachates is 1.35 although the mif is not statistically significant for any of the leachates based on the method reproducibility and 2 of the mean . the hg - bearing ore minerals of the terlingua district are primary , hypogene in origin , formed by hydrothermal mineralizing fluids ( 34 ) . deposition of ore minerals from a hydrothermal fluid is complex , potentially resulting from several geochemical processes including changes in ph , temperature , fluid boiling and mixing , redox reactions , and reactions with surrounding wallrocks ( 35 ) . formation conditions are unknown for the individual hg ore minerals in the terlingua deposit , and thus , it is not possible to explain their wide varying isotopic compositions . however , possible factors controlling mass - dependent isotopic variability in these minerals include varying formation temperatures , paragenesis , or variable redox states or bond strengths among the different minerals ( 36,37 ) . mass independent isotopic variability is generally caused by the nuclear field shift effect or the magnetic isotope effect . previous studies have shown mif due to photochemical reduction of hg and methyl - hg to hg . when hg vs hg is plotted for each of these photochemical reduction processes a slope of 1.36 is obtained for methyl - hg and 1.00 for hg reduction ( 39 ) . the same plot of the terlingua hg minerals has a slope of 1.66 indicating that the source of mif for these minerals is not photochemical reduction ( figure 2 ) . the hg values of two samples of calomel vary significantly between the two samples analyzed ( 0.75 and 2.05 ) . it is formed under a variety of temperatures , and is also known to contain microscopic amounts of elemental hg(40 ) . the hg values of calomel suggest isotopic fractionation as a result of variable formational processes , formation temperatures , or redox conditions . the difference in hg values also could indicate the presence of elemental hg in different proportions to calomel in the samples . this hg would be expected to have a different isotopic composition than calomel , causing variations in the measured bulk hg isotopic composition of the calomel samples . the hg data obtained from the analysis of calcine in the terlingua district show isotopic fractionation of the cinnabar due to the retorting process . the hg values for terlingua calcine vary from 1.34 ( study butte mine ) to 1.52 ( mariscal mine ) , a variation of 2.86 ( figure 1 ) . thus , the hg values for samples of terlingua calcine are isotopically heavier than the original cinnabar , recording hg isotopic fractionation . mif in hg range from 0.17 to 0.23 in the terlingua calcines ( figure 2 ) , which is small or statistically insignificant . during the retorting process , hg in cinnabar the retorting process is often incomplete , and not all of the cinnabar is converted to hg . thus , some of the original cinnabar survives the retorting process and is present as microscopic grains in calcine , often as fine - grain cinnabar encapsulated in quartz and calcite gangue , but there is also metacinnabar present in calcine ( 5,7 ) . concentrations of thg up to 19 000 g / g in samples of calcine from the terlingua district probably are due to the presence of residual cinnabar ( 15 ) ( see si table s-3 for hg concentrations in calcines and calcine leachates ) . therefore , calcines with hg values ( i.e. , study butte mine calcine sample 03sb3 , 1.34 ) that are isotopically similar to terlingua cinnabar ( 1.66 0.12 ) , may contain significant residual cinnabar that has not been altered by the retort process . conversely , terlingua calcines that are enriched in the heavy isotopes of hg ( relative to cinnabar ) require that the volatilized hg was enriched in the light isotopes . however , no correlation between thg in calcines and isotopic composition was found ( si figure s-1 ) . the lack of correlation between thg in calcines and hg values is partly due to a cinnabar nugget effect . for example , a calcine containing cinnabar may have the isotopic composition of cinnabar , but samples with more residual cinnabar will have a higher thg , but also have the isotopic composition of cinnabar , thus , hg will not generally correlate with higher thg in many calcine samples . calcine contains byproduct hg compounds formed when cinnabar is converted to elemental hg during the retorting process ( 12,15 ) and these newly formed hg compounds are enriched in the heavier isotopes of hg . consequently , previous research has shown that there are several natural and anthropogenic processes that will fractionate hg isotopes and result in the formation of an isotopically light hg(gas ) including ( 1 ) biotic , photochemical , and chemical reduction of hg to hg , ( 2 ) volatilization of hg(gas ) from aqueous solution and hg(liquid ) , and ( 3 ) retorting of hg ore ( 31 ) . the results for calcines in this study are consistent with previous studies that have shown that the hg remaining after incomplete reduction and volatilization will be enriched in the heavier isotopes of hg . in addition , theoretical calculations indicate that heavier isotopes of hg will be preferentially partitioned into the oxidized species ( hg ) relative to the reduced species ( hg ) ( 46 ) . if a kinetic process during retorting causes rayleigh - type fractionation , then a range in hg of 0.289.25 represents fractionation between the ore and calcine for retort efficiencies between 50 and 99% ( 47 ) , using published fractionation factors for reduction and volatilization of hg over a temperature range of 2237 c ( 38,39,4143 ) . a much smaller range of fractionation is observed in this study for retorting , which is expected since the retorting process is carried out at much higher temperatures . while the observed fractionation of hg in the calcine relative to cinnabar is consistent with reduction and volatilization as the relevant processes , there could be other processes that fractionate hg in the calcine . for instance , formation of other hg minerals such as metacinnabar during retorting also could cause fractionation . similar to the results for the terlingua calcine samples , the hg data obtained for calcine samples from the mcdermitt mine also reflect fractionation processes due to ore retorting . the hg values vary from 0.64 to 0.22 for three of the five mcdermitt calcine samples ( figure 1 ) ; these samples are isotopically similar to the mcdermitt cinnabar ( 0.60 0.20 ) . mif in hg range from 0.05 to 0.17 and are not statistically significant ( figure 2 ) . these three hg compositions indicate the presence of significant residual cinnabar that has not been altered isotopically by the retort process in these mcdermitt calcine samples . another mcdermitt calcine sample was isotopically heavier ( hg = 0.28 ) than mcdermitt cinnabar , and similar to the results for the terlingua calcine samples , this is likely a result of hg isotopic fractionation during retorting ( as discussed above ) . conversely , the fifth mcdermitt calcine sample has a significantly lighter hg value of 1.49. previous studies have reported the presence of elemental hg in calcine from the mcdermitt mine due to inefficient removal of elemental hg during retorting ( 48 ) . we interpret the lighter hg in this calcine sample to be due to the presence of elemental hg . leaching experiments were designed to examine the isotopic composition of hg minerals that are soluble in weakly acidic water ( ph 5 , which is similar to rainwater ph in this region of the us ) and likely to be mobilized during weathering of the calcine piles . the hg values for leachates of terlingua calcine vary from 0.17 to 2.09 , and in general , the leachates have hg values that are isotopically heavier than the associated bulk calcine ( figure 3 ) . in only one sample ( 03mar3 , mariposa mine ) was the leachate hg ( 0.20 ) isotopically lighter than the bulk calcine sample ( 0.46 ) . the results of the calcine leachate studies suggest the possibility that minor , soluble , byproduct hg compounds , formed during retorting and present in the calcine , were leached during these experiments . this study was designed to determine the isotopic composition of rainwater leachate of the calcine , not that of any particular soluble compounds present in the calcine , thus , our data only indicate that these leachates are isotopically heavy in comparison to the hg of the associated calcine and cinnabar from an individual mine . for example , for sample 03sb1 ( study butte mine ) the leachate hg was 0.71 , whereas the calcine hg was 0.46. thus , the hg measured for the leachate was 1.17 heavier than that of the calcine sample that was leached . variability in isotopic composition of total hg in mine - waste calcine ( open box ) and ph 5 water - leachable hg ( filled box ) of those calcines from mines of the terlingua district and mcdermitt mine . mar = mariposa mine , msm = mariscal mine , sb = study butte mine , and mcd = mcdermitt mine . cinnabar from the terlingua district and mcdermitt mine ( gray box ) are shown for reference . because byproduct hg compounds are a minor component of the calcines , these compounds are often difficult to identify . however , previous studies using edge extended x - ray absorption fine structure analysis have identified several hg oxides , chlorides , oxychlorides , and sulfates ( 12 ) in various calcine samples , and many of these hg compounds are water - soluble . in this study , calcine samples were leached with weak acidic water and compounds such as sulfates , chlorides , and oxychlorides were dissolved , whereas cinnabar has limited solubility at this ph . in summary , the results of our leachate studies indicate that runoff from the hg mines studied is most likely to carry a hg isotopic composition that is similar to or heavier than that of calcine present at the site due to the presence of the minor , byproduct hg compounds formed during or after retorting of the calcine . because of the high solubility of these compounds at the ph of the leachate used , they dominate the hg composition in the water runoff . recent studies reported that the hg isotopic compositions of river sediment collected downstream from the idrija hg mine , slovenia , were similar to that of cinnabar ore at the mine upstream ( 49 ) . in the idrija study , downstream river sediments were found to contain large amounts of cinnabar , and thus , isotopic hg tracing was used successfully to link downstream hg contamination to cinnabar from the idrija mine ( 49 ) . we have not measured the isotopic hg compositions of stream sediments proximal to the mines studied , however , we focused on examining anthropogenic hg isotopic fractionation in calcine at these sites . calcines are voluminous and primary cinnabar ore minor at the mines studied ( e.g. , > 2 000 000 m in the terlingua district ) , and sediment transport away from such mines , especially in dry , desert environments also is generally minor ( 15 ) . therefore , due to the large volume of calcine at these hg mines , the hg isotopic composition of water runoff is most likely to be dominated by the hg isotopic composition of calcine , not the primary cinnabar ore . studies using hg isotopic tracing in hg mined areas should include the measurement of the hg isotopic composition of calcine in areas where calcine predominates volumetrically over primary cinnabar ore . the measurement of the hg isotopic compositions of cinnabar and leachates of the calcine are also necessary to adequately trace hg sources from areas mined for hg . in addition , tracing of mining - related hg may be complicated by microbial , chemical , and photochemical transformations in water and sediment ( 16,17,50 ) that potentially fractionate hg isotopes . such fractionation processes have not been well characterized in ecosystems downstream from areas mined for hg .

objectivecerebral microvascular disease associated with type 2 diabetes may exacerbate the effects of aging on cognitive function . a considerable homology exists between the retinal and cerebral microcirculations ; a hypothesized association between diabetic retinopathy ( dr ) and cognitive decline was examined in older people with type 2 diabetes.research design and methodsin the population - based edinburgh type 2 diabetes study , 1,046 men and women aged 6075 years with type 2 diabetes underwent standard seven - field binocular digital retinal photography and a battery of seven cognitive function tests . a general cognitive ability score ( g ) was generated by principal components analysis . the mill - hill vocabulary scale was used to estimate premorbid cognitive ability . dr was graded using a modification of the early treatment of diabetic retinopathy scale.resultsafter age and sex adjustment , a significant relationship was observed with increasing severity of dr ( none , mild , and moderate to severe ) for most cognitive measures . participants with moderate - to - severe retinopathy had the worst g and the worst performances on the individual tests . there was a significant interaction between sex and retinopathy for g. in male subjects , the associations of retinopathy with g ( and with tests of verbal fluency , mental flexibility , and processing speed but not memory and nonverbal reasoning ) persisted ( p < 0.05 ) when further adjusted for vocabulary ( to estimate lifetime cognitive decline ) , depression , sociodemographic characteristics , cardiovascular risk factors , and macrovascular disease.conclusionsdr was independently associated with estimated lifetime cognitive decline in older men with type 2 diabetes , supporting the hypothesis that cerebral microvascular disease may contribute to their observed accelerated age - related cognitive decline . a sex interaction with stronger findings in men requires further confirmation .

the et2ds is a population - based cohort study designed to investigate potentially modifiable risk factors for cognitive decrements in type 2 diabetes . the study commenced in 2006/2007 as a cross - sectional survey of 547 men and 519 women aged 6075 with type 2 diabetes . participants were recruited at random , in 5-year age bands , from the lothian diabetes register , which is a computerized database containing clinical details on over 20,000 patients with known type 2 diabetes living in lothian , scotland . the lothian research ethics committee approved the study , and fully informed written consent was obtained from each participant . details of the study recruitment and examination procedures have previously been described in detail ( 16 ) . study participants have been shown to be representative of the target population of older men and women with type 2 diabetes living in the general population ( 17 ) . specially trained research assistants administered a detailed battery of cognitive tests in a standard order during a single session in a quiet and well - lit room following tests for adequate visual acuity and capillary blood glucose levels ( > 4.0 mmol / l ) . cognitive ability was assessed using tests of immediate and delayed nonverbal memory and verbal declarative memory ( faces and family pictures subtest [ faces ] and logical memory i ( lm ) from the wechsler memory scale - iii ) ; nonverbal reasoning , working memory , information processing speed ( matrix reasoning [ mr ] , letter - number sequencing [ lns ] , and digit symbol test [ dst ] from the wechsler adult intelligence scale 3rd edition ) ; executive function ( borkowski verbal fluency test [ vft ] ) and mental flexibility ( trail making test - part b [ tmtb ] ) . vocabulary ( crystallized intelligence ) was measured using the combined version of the junior and senior form a synonyms of the mill hill vocabulary scale [ mhvs ] ( 22 ) . as results on vocabulary - based tests vary little with aging , they can be used to estimate peak prior cognitive ability ( 23,24 ) . late - life cognition adjusted for vocabulary correlates highly with actual cognitive change ( 25 ) . the mini - mental state examination ( mmse ) is often used as a screening for dementia and was included as a general mental assessment for possible cognitive pathology ( 26 ) . a self - administered questionnaire was used to collect data on age , sex , educational attainment , diabetes history and treatment modality , smoking history , alcohol consumption , medication use , and history of cardiovascular disease , including the world health organization ( who ) chest pain ( 28 ) and edinburgh claudication ( 29 ) questionnaires . clinical measurements included plasma a1c and fasting total and hdl serum cholesterol , height , weight , waist and hip circumferences , a resting 12-lead electrocardiogram , and brachial blood pressures as previously described ( 16 ) . additional information on macrovascular disease was obtained from the information and services division of nhs scotland on all medical and surgical discharges from scottish hospitals since 1981 ( smr01 scheme ) , and any k09 or k010 codes indicating cardiovascular or cerebrovascular disease were extracted . retinal photography was performed approximately 23 weeks after each subject 's initial visit to the research clinic for cognitive and physiological testing . of the 1,046 participants who underwent both cognitive and retinal examinations , two were excluded who did not have gradable photographs for dr severity in either eye , leaving 1,044 who provided data for this analysis . after pupillary dilatation , standard seven - field nonstereoscopic color photographs were taken of both eyes at 35 using a high - resolution digital retinal camera . all photographs were graded by two trained optometrists , working independently and according to the scale described by the early treatment diabetic retinopathy study ( etdrs ) research group ( 30 ) . inter- and intraobserver variations and the validity of this grading system have previously been evaluated ( 31 ) . for each eye , the maximum grade in any of the seven photographic fields was determined for each of the characteristic lesions of dr and was used in defining the final retinopathy levels , varying from level 10 ( no retinopathy ) to level 81 ( advanced proliferative retinopathy ) . for the purpose of this study , a score of 81 was added for panretinal photocoagulation scars only if the laser treatment was for dr . the retinopathy level for a participant was assigned on the basis of the severity scores of the worse eye . if the photographs from an eye could not be graded , the scores for the other eye were used . discrepancies in the final level score at subject level between the graders were resolved in the first instance by discussion between them . the average weekly alcohol intake was defined as standard drinks per week ( 32 ) . diabetes treatment was classified into three groups : 1 ) diet control only , 2 ) oral antidiabetes agents without insulin , and 3 ) insulin injection with or without oral agents . waist - to - hip ratio ( whr ) was calculated as the waist circumference divided by the hip circumference in centimeters . bmi was defined as weight in kilograms divided by the square of height in meters . mmol / l or use of medication prescribed by a doctor to lower blood lipids level . hypertension was defined as systolic blood pressure 140 mmhg or diastolic blood pressure 85 mmhg or use of medication prescribed by a doctor to lower blood pressure . coronary heart disease ( myocardial infarction and/or angina ) was defined if two of the first three of the following criteria were met or if both the first and last criteria were met : 1 ) subject recall of a doctor 's diagnosis of myocardial infarction or angina , 2 ) positive who chest pain questionnaire , 3 ) electrocardiogram evidence of ischemia , and 4 ) prior hospital discharge ( icd ) code for ischemic heart disease . cerebrovascular disease ( stroke and/or transient ischemic attack [ tia ] ) was defined if two of three of the following criteria were met : 1 ) subject recall of a doctor 's diagnosis of stroke or tia , 2 ) prior hospital discharge code consistent with stroke or tia , and 3 ) confirmation by clinical notes review . intermittent claudication was based on a positive response to the edinburgh claudication questionnaire . any macrovascular disease was defined as a history of myocardial infarction , angina , stroke , tia , or peripheral arterial disease . dr was defined as an etdrs level of 20 , i.e. , the presence of microaneurysms alone or with any of the following lesions : hemorrhages , cotton wool spots , intraretinal microvascular abnormalities , hard exudates , venous beading , venous loops and/or reduplication , fibrous proliferations , preretinal hemorrhage , vitreous hemorrhage , and new vessels . this was further divided into mild nonproliferative dr ( npdr ) ( levels 2035 ) , moderate - to - severe npdr ( levels 4353 ) , and proliferative dr ( levels 6181 ) . due to small numbers in the proliferative retinopathy group , severity of dr was collapsed into three categories ( none , mild , and moderate severe ) ; the two most severe categories were combined into moderate a measure of general cognitive ability , representing the variance common to all the cognitive tests except mhvs , was generated . this was done by subjecting the seven cognitive tests ( faces , lm , mr , lns , dst , vft , and tmtb ) to a principal - components analysis . scree slope analysis and the eigenvalues - greater - than-1 rule both indicated a single component ( which accounted for 44% of the total variance , on which all tests had high loadings ) , thus validating the use of a general factor . scores on this first unrotated principal component were saved as standardized scores ( mean sd 0 1 ) . all continuous variables were normally distributed except for the tmtb for which a natural logarithmic transformation of scores was used , as well as depression scores ( normalized through a n + 1 logarithmic transformation ) , duration of diabetes , and alcohol intake ( square root transformations ) . severe ) were compared on all demographic and medical variables using tests of linearity included in anova for continuous data and analyses for categorical data . ancova was used for comparing cognitive test scores in subjects according to severity of dr and in assessing the effects of retinopathy severity on the imputed cognitive change from estimated peak prior cognitive function ( mhvs scores ) at the same time as adjusting for possible confounders . adjustment variables were introduced into the models in three cumulative steps . in the first step finally , education level , vascular risk factors ( alcohol intake , smoking status , whr , systolic blood pressure , and total cholesterol ) , the presence of macrovascular disease , and depression symptoms were additionally adjusted . these variables were selected as possible confounders if they showed a significant association with dr on univariate analysis or if , in previous literature , a variable was reported to be associated with both dr and cognitive test performance . we did not include duration of diabetes or a1c in the main multivariate model because it is likely that prolonged exposure to hyperglycemia underlies the development of dr and their inclusion in the model could result in overadjustment . all tests were two tailed , and a two - sided p value 0.05 was taken to indicate statistical significance . all analyses were performed using spss , version 14.0 , for windows ( 33 ) . specially trained research assistants administered a detailed battery of cognitive tests in a standard order during a single session in a quiet and well - lit room following tests for adequate visual acuity and capillary blood glucose levels ( > 4.0 mmol / l ) . cognitive ability was assessed using tests of immediate and delayed nonverbal memory and verbal declarative memory ( faces and family pictures subtest [ faces ] and logical memory i ( lm ) from the wechsler memory scale - iii ) ; nonverbal reasoning , working memory , information processing speed ( matrix reasoning [ mr ] , letter - number sequencing [ lns ] , and digit symbol test [ dst ] from the wechsler adult intelligence scale 3rd edition ) ; executive function ( borkowski verbal fluency test [ vft ] ) and mental flexibility ( trail making test - part b [ tmtb ] ) . vocabulary ( crystallized intelligence ) was measured using the combined version of the junior and senior form a synonyms of the mill hill vocabulary scale [ mhvs ] ( 22 ) . as results on vocabulary - based tests vary little with aging , they can be used to estimate peak prior cognitive ability ( 23,24 ) . late - life cognition adjusted for vocabulary correlates highly with actual cognitive change ( 25 ) . the mini - mental state examination ( mmse ) is often used as a screening for dementia and was included as a general mental assessment for possible cognitive pathology ( 26 ) . a self - administered questionnaire was used to collect data on age , sex , educational attainment , diabetes history and treatment modality , smoking history , alcohol consumption , medication use , and history of cardiovascular disease , including the world health organization ( who ) chest pain ( 28 ) and edinburgh claudication ( 29 ) questionnaires . clinical measurements included plasma a1c and fasting total and hdl serum cholesterol , height , weight , waist and hip circumferences , a resting 12-lead electrocardiogram , and brachial blood pressures as previously described ( 16 ) . additional information on macrovascular disease was obtained from the information and services division of nhs scotland on all medical and surgical discharges from scottish hospitals since 1981 ( smr01 scheme ) , and any k09 or k010 codes indicating cardiovascular or cerebrovascular disease were extracted . retinal photography was performed approximately 23 weeks after each subject 's initial visit to the research clinic for cognitive and physiological testing . of the 1,046 participants who underwent both cognitive and retinal examinations , two were excluded who did not have gradable photographs for dr severity in either eye , leaving 1,044 who provided data for this analysis . after pupillary dilatation , standard seven - field nonstereoscopic color photographs were taken of both eyes at 35 using a high - resolution digital retinal camera . all photographs were graded by two trained optometrists , working independently and according to the scale described by the early treatment diabetic retinopathy study ( etdrs ) research group ( 30 ) . inter- and intraobserver variations and the validity of this grading system have previously been evaluated ( 31 ) . for each eye , the maximum grade in any of the seven photographic fields was determined for each of the characteristic lesions of dr and was used in defining the final retinopathy levels , varying from level 10 ( no retinopathy ) to level 81 ( advanced proliferative retinopathy ) . for the purpose of this study , a score of 81 was added for panretinal photocoagulation scars only if the laser treatment was for dr . the retinopathy level for a participant was assigned on the basis of the severity scores of the worse eye . if the photographs from an eye could not be graded , the scores for the other eye were used . discrepancies in the final level score at subject level between the graders were resolved in the first instance by discussion between them . the average weekly alcohol intake was defined as standard drinks per week ( 32 ) . diabetes treatment was classified into three groups : 1 ) diet control only , 2 ) oral antidiabetes agents without insulin , and 3 ) insulin injection with or without oral agents . waist - to - hip ratio ( whr ) was calculated as the waist circumference divided by the hip circumference in centimeters . bmi was defined as weight in kilograms divided by the square of height in meters . mmol / l or use of medication prescribed by a doctor to lower blood lipids level . hypertension was defined as systolic blood pressure 140 mmhg or diastolic blood pressure 85 mmhg or use of medication prescribed by a doctor to lower blood pressure . coronary heart disease ( myocardial infarction and/or angina ) was defined if two of the first three of the following criteria were met or if both the first and last criteria were met : 1 ) subject recall of a doctor 's diagnosis of myocardial infarction or angina , 2 ) positive who chest pain questionnaire , 3 ) electrocardiogram evidence of ischemia , and 4 ) prior hospital discharge ( icd ) code for ischemic heart disease . cerebrovascular disease ( stroke and/or transient ischemic attack [ tia ] ) was defined if two of three of the following criteria were met : 1 ) subject recall of a doctor 's diagnosis of stroke or tia , 2 ) prior hospital discharge code consistent with stroke or tia , and 3 ) confirmation by clinical notes review . intermittent claudication was based on a positive response to the edinburgh claudication questionnaire . any macrovascular disease was defined as a history of myocardial infarction , angina , stroke , tia , or peripheral arterial disease . dr was defined as an etdrs level of 20 , i.e. , the presence of microaneurysms alone or with any of the following lesions : hemorrhages , cotton wool spots , intraretinal microvascular abnormalities , hard exudates , venous beading , venous loops and/or reduplication , fibrous proliferations , preretinal hemorrhage , vitreous hemorrhage , and new vessels . this was further divided into mild nonproliferative dr ( npdr ) ( levels 2035 ) , moderate - to - severe npdr ( levels 4353 ) , and proliferative dr ( levels 6181 ) . due to small numbers in the proliferative retinopathy group , severity of dr was collapsed into three categories ( none , mild , and moderate severe ) ; the two most severe categories were combined into moderate severe dr and used in all subsequent analyses . a measure of general cognitive ability , representing the variance common to all the cognitive tests except mhvs , was generated . this was done by subjecting the seven cognitive tests ( faces , lm , mr , lns , dst , vft , and tmtb ) to a principal - components analysis . scree slope analysis and the eigenvalues - greater - than-1 rule both indicated a single component ( which accounted for 44% of the total variance , on which all tests had high loadings ) , thus validating the use of a general factor . scores on this first unrotated principal component were saved as standardized scores ( mean sd 0 1 ) . all continuous variables were normally distributed except for the tmtb for which a natural logarithmic transformation of scores was used , as well as depression scores ( normalized through a n + 1 logarithmic transformation ) , duration of diabetes , and alcohol intake ( square root transformations ) . severe ) were compared on all demographic and medical variables using tests of linearity included in anova for continuous data and analyses for categorical data . ancova was used for comparing cognitive test scores in subjects according to severity of dr and in assessing the effects of retinopathy severity on the imputed cognitive change from estimated peak prior cognitive function ( mhvs scores ) at the same time as adjusting for possible confounders . adjustment variables were introduced into the models in three cumulative steps . in the first step finally , education level , vascular risk factors ( alcohol intake , smoking status , whr , systolic blood pressure , and total cholesterol ) , the presence of macrovascular disease , and depression symptoms were additionally adjusted . these variables were selected as possible confounders if they showed a significant association with dr on univariate analysis or if , in previous literature , a variable was reported to be associated with both dr and cognitive test performance . we did not include duration of diabetes or a1c in the main multivariate model because it is likely that prolonged exposure to hyperglycemia underlies the development of dr and their inclusion in the model could result in overadjustment . all tests were two tailed , and a two - sided p value 0.05 was taken to indicate statistical significance . all analyses were performed using spss , version 14.0 , for windows ( 33 ) . characteristics of the total et2ds population ( n = 1,066 ) and comparisons with nonparticipants from the target population have previously been presented ( 17 ) . these subjects did not differ significantly from subjects without photographs either available or suitable for grading ( n = 22 ) with respect to diabetes duration or treatment , mean a1c , cardiovascular risk factors , or prevalence of macrovascular disease ( data not shown ) . however , they did perform significantly better on tests of nonverbal memory ( mean sd faces scores 66.0 7.8 vs. 58.3 7.0 ; p < 0.001 ) , verbal declarative memory ( mean lm scores 25.4 8.2 vs. 20.0 5.9 ; p = 0.003 ) , and general cognitive ability ( mean g scores 0.00 1.00 vs. 0.53 0.77 ; p = 0.014 ) . of the 1,044 type 2 diabetic participants , 339 ( 32.5% ) subjects of these , 292 ( 86.1% ) had mild npdr , 32 ( 9.4% ) had moderate - to - severe npdr , and 15 ( 4.4% ) had proliferative retinopathy ( n = 19 and n = 8 for men in the latter two categories , respectively ) . subjects with dr were more likely to be receiving insulin treatment and had higher mean a1c , diabetes duration , and whr . characteristics of study participants according to severity of dr data are means sd , % ( n ) , or median ( interquartile range ) unless otherwise indicated . age- and sex - adjusted general cognitive ability ( g ) was significantly lower in the group with moderate - to - severe dr ( mean 0.44 [ 95% ci 0.73 to 0.16 ] ) compared with the group without dr ( 0.05 [ 0.03 to 0.12 ] ; p = 0.003 ) , with an intermediate score for the mild dr group ( 0.04 [ 0.17 to 0.07 ] ) ( p for trend = 0.003 ) . no significant associations were found between dr and estimated premobid cognitive ability ( mhvs score ) . the association between dr and current general cognitive ability ( g ) remained statistically significant after adjustment for mhvs , but in this model there was a significant interaction between sex and retinopathy for g ( p = 0.030 ) . the total study population was stratified by sex , and the effects of retinopathy on g and individual cognitive tests were examined separately for men and women ( table 2 ) . following age adjustment , significant associations were only found in men between dr and g , vft and tmtb , and in both sexes with dst . when adjusted for mhvs to estimate lifetime change in cognitive ability , the retinopathy - dst association lost statistical significance in women but remained significant in men . when further adjusted for a wide range of potentially confounding variables , including macrovascular disease , associations in men persisted between dr and g ( p for trend < 0.001 ; = 0.020 ) , vft ( p for trend = 0.001 ; = 0.020 ) , tmtb ( p for trend = 0.009 ; = 0.012 ) , and dst ( p for trend = 0.001 ; = 0.032 ) tests ( assessing executive function , mental flexibility , and processing speed , respectively ) . no statistically significant differences across any of the retinopathy categories were found for the lm , mr , or lns tests for either sex . there was not any significant interaction of retinopathy and any of the variables other than sex . estimated mean ( 2 se ) of general factor ( g ) scores of men and women according to severity of dr . multivariable - adjusted mean ( se ) of cognitive scores by severity of dr in men and women further adjusted for education , alcohol intake , smoking status , whr , systolic blood pressure , total cholesterol , major macrovascular disease , and depression symptoms . * effect size of dr as indexed by 2 ( the proportion of variance ) in the fully adjusted model . analyses were repeated after excluding individuals with prevalent stroke or history of tia and those with mmse scores < 24 . none of the associations reported were essentially altered by these exclusions ( data not shown ) . in a final analysis adjustment for both attenuated the association between dr and tmtb to borderline significance ( p = 0.059 ) in men but did not change other results . in this representative population of older people with type 2 diabetes , increasing severity of dr was associated with poorer general cognitive ability . when analyzed separately by sex , a significant dose - response relationship was found only in men and for individual tests of verbal fluency , information processing speed , and mental flexibility ( but not memory and nonverbal reasoning ) . these associations persisted after adjustment for estimated premorbid cognitive ability ( vocabulary scores ) , suggesting that in men , dr was not only associated with cognitive ability in later life but also with increased estimated lifetime cognitive decline . to estimate cognitive decline , it was not possible to adjust for the same cognitive test applied at an earlier age , but all the nonvocabulary tests were significantly correlated and vocabulary scores were strongly loaded on general cognitive ability ( coefficient 0.57 ; p < 0.001 ) . after additional control for education , vascular risk factors , macrovascular disease , and mood , associations between dr and estimated cognitive decline remained significant , suggesting that they were not simply due to confounding by other vascular mechanisms . to our knowledge , no previous studies have specifically examined the relationship between dr and cognitive functioning in a large group of older people with type 2 diabetes from the general population using comprehensive and detailed assessment of retinal signs . two previous studies did not show an association between dr and cognitive function in older diabetic patients ( 34,35 ) , but these were based on either a direct ophthalmoscopic examination of the retina or a single - field retinal photograph . these methods image only a small portion of the retina and with considerably less rigor compared with multiple - field photographs and as a result , the prevalence of dr signs may have been underestimated . our findings are consistent with those from studies of predominantly nondiabetic older subjects from the general population . in the blue mountains eye study ( 14 ) , subjects with hypertension aged 49 years and older who had evidence of retinopathy signs were significantly more likely to score poorly ( < 23 ) on the mmse , and in the cardiovascular health study there was a significant association between retinopathy and worse performance on information - processing speed in subjects aged > 69 years ( 36 ) . the atherosclerosis risk in communities study ( aric ) also reported that retinopathy was independently associated with poorer cognitive function in middle - aged people free of stroke ( 37 ) , both in those with and in those without diabetes and hypertension . longitudinal findings from the 14-year follow - up of the same cohort showed that people with retinopathy had a greater 10-year decline in verbal fluency and information - processing speed ( but not in delayed verbal memory ) compared with those without retinal microvascular abnormalities ( 15 ) . due to the considerable homology between retinal and cerebral microvasculature , retinal vascular changes are likely to provide an indirect marker of concomitant changes in the brain microvasculature . it is possible that accelerated cognitive aging associated with type 2 diabetes may arise , at least in part , from the cumulative impact of a disruption of blood - brain barrier and/or the ischemic injuries leading to diverse changes in brain parenchyma . as our findings persisted after subjects with recognized clinical stroke were excluded , perivascular brain damage and/or ischemia from asymptomatic cerebral small vessel disease ( svd ) could explain the cognitive associations . for example , microinfarcts and mri signs of cerebral svd ( e.g. , lacunar infarcts and white matter lesions ) have been shown to contribute importantly to cognitive impairment and vascular dementia ( 3841 ) . furthermore , increased blood - brain barrier permeability has been demonstrated in people with type 2 diabetes ( 4 ) , and alteration in the blood - brain barrier secondary to microvascular endothelial dysfunction may be an important pathophysiological mechanism in the initiation or worsening of cerebral svd ( 42 ) . cerebral svd predominantly affects the subcortical associative areas of deep gray matter ( the basal ganglia and thalamus ) and white matter structures , with disruption of integrity of frontal subcortical circuits . our results in type 2 diabetes are consistent with a cognitive profile that is associated with cerebral svd . a striking finding of the present study was a significant interaction of dr with sex such that the negative associations of dr with several cognitive measures were statistically significant only in men . , no previous study has examined whether sex modifies the relation of retinopathy with cognition . the sex - specific effect of dr on cognitive functioning could be influenced by lower prevalence of dr ( 29.7 vs. 35.1% ; p = 0.058 ) , coronary heart disease ( 24.4 vs. 37.8% ; p < 0.001 ) ; and stroke and/or tia ( 5.5 vs. 11.6% ; p < 0.001 ) in women compared with men , but adjusting for macrovascular disease did not alter the sex - specific effects . it is also possible that other unknown risk factors that were not examined in this analysis ( e.g. , subclinical atherosclerosis , estrogen use , or physical activity ) could play a mediating role . although several hypotheses have been proposed in an attempt to explain sex difference observed in some studies , including male - female differences in brain reserve and differential susceptibility to normal or pathological age - associated changes , there is no clear empirical evidence on the effect of sex on cognition in the general population or in people with diabetes . despite the representativeness of our study participants to the target population in terms of demographic and clinical characteristics , indeed , it has been hypothesized that diabetes may be a more potent risk factor for cognitive dysfunction in women than in men due to a higher risk of diabetes - related macrovascular disease and early loss of possible protective effects of estrogen ( due to earlier menopause ) on cognitive functioning ( 43 ) . it is possible that women with more severe diabetes ( those more likely to have retinopathy and cognitive decline ) may have died of macrovascular disease before they could be recruited into the study , thereby leading to conservative estimates of associations between retinopathy and cognitive decline . however , the degree of variation of g scores in men and women in the study population was similar and , as shown in table 1 , people with any retinopathy were only slightly more likely to be male . it therefore seems unlikely that survival bias could be the full explanation for the sex difference . given that we had no specific a priori reason for assuming that our association of dr with cognition would be different between males and females , and given that other studies have not suggested the existence of such a sex difference , interpretation of the interaction between sex and dr must be treated with caution until it has been replicated in other large studies . one of the main strengths of this study was the use of high - quality retinal photographs and detailed grading of dr using full seven - field photography and the low amount of missing retinal grading data . other strengths included the application of a detailed cognitive test battery covering the major cognitive domains , although use of a single test to assess each particular cognitive domain could mean that only limited aspects of what may be considered complex mental functions were actually determined ( 20 ) . the study population had a verified clinical diagnosis of type 2 diabetes and has been shown to be representative of the target population of elderly men and women with the full range of severity of type 2 diabetes living in the general population . the use of a general cognitive factor , g , helped avoid potential problems caused by multiple testing and residual confounding by peak prior mental ability was minimized by controlling for performance on a test of vocabulary rather than relying solely on level of education as in many previous studies . however , the presence of microvascular complications other than retinopathy in the no dr group ( e.g. , neuropathy and nephropathy ) , leading to shared microvascular risk burden between the retinopathy and retinopathy - free subjects , may have tended to reduce differences toward the null . the effect size for retinopathy in these older men with type 2 diabetes was small after multivariate adjustment . such a small effect size for risk factors associated with cognitive decline is not uncommon , including the effects of other potentially important factors ( e.g. , apolipoprotein e genotype and smoking ) ( 44,45 ) . it is possible that even very modest effects of a risk factor may lead to larger cognitive deficits and possibly dementia with further aging . furthermore , reduction of such a risk factor could have a significant impact on cognition in the diabetic population as a whole by producing a positive shift in the overall population distribution of cognitive ability . however , while our findings support the hypothesis that reduction of microvascular disease might reduce the rate of cognitive decline , the clinical significance of our findings remains to be established . the major limitation of the study is that analyses were cross - sectional , with measures of retinal and cognitive function made almost simultaneously , limiting our ability to determine whether dr preceded or occurred in parallel with cognitive decline . such a follow - up project involving the present study population for the actual cognitive change is underway . in conclusion , dr was independently associated with estimated lifetime cognitive decline in older men with type 2 diabetes , supporting the hypothesis that cerebral microvascular disease may contribute to the accelerated age - related cognitive decline observed in diabetic populations . if the above findings are substantiated , diabetes - associated cognitive dysfunction may be amenable to therapeutic and preventive strategies that are targeted specifically at protecting the cerebral microvasculature and reducing the risk of developing even mild microvascular disease in an aging diabetic population .

background . optical coherence tomography ( oct ) allows quantitative analysis of the anterior segment of the eye with a noncontact examination . the aim of this study is to analyze associations of central corneal thickness ( cct ) as measured by oct with ocular and systemic cardiovascular parameters . methods . a cross - sectional study of 734 persons was performed in a working age population . only healthy eyes were included . a comprehensive ophthalmological examination including refraction , noncontact tonometry , and imaging of the anterior segment by sd - oct was performed . in parallel , a broad range of systemic cardiovascular parameters were measured . associations were analyzed using a generalized estimating equations ' model . results . cct measurements showed a significant association with corneal curvature and intraocular pressure : a thinner cct was associated with a flatter cornea and with lower intraocular pressure ( p < 0.001 ) . age was positively associated with cct ( p < 0.001 ) ; all other cardiovascular parameters were not associated . conclusion . a thinner cornea is associated with a flatter surface and with lower intraocular pressure readings , while there are no independent associations with refraction and systemic cardiovascular parameters . our findings highlight the value of sd - oct cct measurements as a standard tool in anterior segment analysis .

optical coherence tomography ( oct ) enables high - resolution imaging of the anterior segment of the human eye . in a noncontact examination it is possible to analyze layers and shape of cornea in cross - sectional images central corneal thickness itself affects the accuracy of intraocular pressure measurements . in the last decades , correlations between central corneal thickness and intraocular pressure readings have been evaluated in many studies establishing the correlation between these two ocular parameters [ 212 ] . many of these studies have used optical or ultrasonic pachymetry for corneal thickness measurements and applanation tonometry for intraocular pressure measurement . nowadays , noncontact tonometry is widely used in clinical daily routine for screening purposes . therefore , the relationship of this measurement technique with central corneal thickness readings as determined by spectral - domain oct ( sd - oct ) imaging should be further investigated . for clinical evaluation of central corneal thickness as determined by sd - oct , it is essential to know physiological factors that are associated with the dimensions of the tissue . based on this background , this study aimed to investigate the associations of distinct central corneal thickness measurements determined by sd - oct with ocular and systemic cardiovascular parameters in a caucasian cohort . our hypothesis is that higher central corneal thickness may be associated with a flatter corneal curvature and with higher intraocular pressure , while systemic cardiovascular parameters are not associated . the study was designed as a cross - sectional , observational study conducted within the scope of a health promotion project conducted within a major industry company . the study population consists of working age subjects ( age range from 17 to 64 years ) of the miph eye & health study . exclusion criteria were any manifest eye disease , either self - reported or detected on fundus photographs , and insufficient oct scan quality of the anterior segment oct . 1460 healthy eyes of 734 subjects ( 730 right eyes , 730 left eyes ; 514 men , 220 women ) were included in the analysis . the study was conducted according to the tenets of the declaration of helsinki and fully approved by both the ethics approval committee of the university of heidelberg ( institutional review board ) , the company 's advisory board , and the board of employees . written informed consent as to the scientific objectives of the study blood pressure was recorded from the dominant arm in the seated position after a standardized 20 min rest period by sphygmomanometry . blood samples were drawn to determine triglycerides ( tri ) , low density lipoproteins ( ldl ) , high density lipoproteins ( hdl ) , and glycosylated hemoglobin ( hba1c ) using routine laboratory analyzers . the cornea was imaged with the anterior segment mode of the 3d oct-2000 ( topcon corp . , automated calculation of corneal curvature and central corneal thickness ( cct ) with the integrated software was performed and all oct images were checked for correct identification of the corneal surface . quality of oct scans were graded in a four categories : high , , tokyo , japan ) and noncontact tonometry was performed ( ct-80 tonometer , topcon corp . , fundus photographs of the macula and optic nerve head were obtained from all participants and images were evaluated by two independent ophthalmologists . eyes with previous intraocular or refractive surgery were excluded from the study , as were eyes with known and confirmed ocular disease . raw data results were processed by statistical analysis software ( spss 21.0 , chicago , il ) and plots were performed with stata software ( version 13.1 se , college station , tx : statacorp lp ) . associations with central corneal thickness were analyzed by multivariable linear regression algorithms with parameter estimation performed with generalized estimation equations . this statistical model takes the relationship between the right and left eye into account by linking the individual pair of eyes . as dependent variable central corneal thickness was included in the statistical model , mean corneal radius , intraocular pressure , refraction , age , gender , body mass index , mean arterial blood pressure , hba1c , hdl , ldl , and triglyceride values were evaluated as associated factors . association analysis between central corneal thickness and intraocular pressure , respectively , means corneal curvature was performed with univariate linear regression as well to determine comparable parameter estimates with literature reports . in addition , a reverse analysis with intraocular pressure as dependent and central corneal thickness as explanatory variable was computed . spearman correlation coefficient was computed to determine the relationship between central corneal thickness readings and oct image quality . , we corrected this parameter to 0.004 using the bonferroni method . for further interpretation of the results , all p values above 0.001 considering all eyes together , mean cct was 561.1 32.3 m ( mean standard deviation ; right eyes : 560.1 31.8 m ; and left eyes : 562.2 32.8 m ) . association analysis of cct measurements found a statistically significant relationship between corneal curvature and intraocular pressure : cct was found to be negatively associated with intraocular pressure readings ( p < 0.001 ; figure 1 ) and positively associated with a flatter corneal curvature ( p < 0.001 ; figure 2 ) . reversely , univariable analysis between intraocular pressure as dependent variable and central corneal thickness as explanatory variable revealed that an increase of 22 m in central corneal thickness is linked to an increased measurement of intraocular pressure of 1 mmhg . in the multivariable model , an increase of 10 m in central corneal thickness was associated with an increase of 3 mmhg in intraocular pressure reading . refraction of the eye did not show a significant association with cct ( table 2 ) . regarding anthropometric characteristics of the study sample , age was independently associated with cct : older subjects had a higher central corneal thickness ( table 2 ) . central corneal thickness values from oct measurements increase by 0.34 m per year of age . regarding cardiovascular parameters , none of the investigated parameters ( gender and bmi and mean arterial blood pressure and biochemical parameters : hba1c and hdl and ldl and triglycerides ) was significantly associated with cct after bonferroni correction for multiple testing ( p < 0.004 ) ( table 2 ) . a weak association between lower scan quality and thinner central corneal thickness measurement was determined ( spearman correlation coefficient = 0.09 , p < 0.001 ) . nevertheless , upon incorporating this parameter as sensitivity analysis in the multivariable model , our findings remained unaltered . our study in a caucasian cohort demonstrates that central corneal thickness readings as measured with the topcon 3d oct-2000 are independently associated with corneal curvature and with intraocular pressure readings . previously published studies have yielded congruent findings using optical or ultrasound pachymetry and association with intraocular pressure [ 24 , 7 , 8 , 10 ] . however , we used sd - oct for determining central corneal thickness which makes a technical difference to other studies where analyses were based on ultrasound pachymetry or scheimpflug imaging . sd - oct accurately measures central corneal thickness as reported by prior publications within the physical limitations of the method itself as determined by wavelength , optical system , and sensor characteristics . furthermore , our finding of a relationship of corneal curvature with central corneal thickness as measured by oct is new . nevertheless , there are several studies reporting such a relationship using other methods [ 1518 ] . also , our study may contribute to deepening the understanding of associations between cct readings obtained from oct and intraocular pressure readings obtained from noncontact tonometry and add to knowledge being generated with different methods . in the range between 11 and 21 mmhg intraocular pressure and 500 to 625 m central corneal thickness , we found an approximately linear relationship between these two parameters , but this relationship can not be extrapolated to higher or lower values as measurements were not sufficiently available there . with an increase of 22 m in cct , intraocular pressure ( as dependent variable ) increased by 1 mmhg , and reported associations of central corneal thickness with male gender , higher body height , and body mass index in a german population . the differences with our study might be explained by the underlying study population : we included far more male than female participants , a different age span ( 17 to 64 years versus 35 to 74 years by elflein et al . ) , and a different mean body mass index ( 22.9 versus 27.2 ) , while both studies included mainly caucasians . in a similar age span to elflein et al . , nangia and coworkers found associations of central corneal thickness with male gender , younger age , and higher body mass index in an indian population . in chinese people , central corneal thickness is associated with male gender and urban region , while it is not associated with age , body mass index , and body height . a study in a japanese population found similar findings for asian people reporting an association of central corneal thickness with male gender but none for age , body weight , and body height . the ambiguities to our study results may arise from the use of different cct assessment methods and variations in age , body measurements , and genetic background of the study populations . in our study population consisting of a cohort of caucasians with an age range from 17 to 64 years , central corneal thickness was associated with age : each decade of age went along with an increase of 3.4 m in central corneal thickness over the entire age span . upon comparing our findings to those published by other groups , it must be considered that our age range corresponds to the working age and that we have only included healthy eyes with the purpose of reporting physiological conditions . this means that our study group starts at a juvenile age and terminates at retirement , whereas many other studies have appeared with a much older starting age reaching into senility . there are some studies in european cohorts with different age ranges that did not find an association between central corneal thickness and age [ 9 , 10 , 20 ] . in contrast , we found an association with age : central corneal thickness increased slightly up to the age of 64 years . in other ethnicities and especially in persons aged 70 years and older , chua et al . reported a decrease of central corneal thickness with age ; similar results were reported by other research groups [ 2224 ] . this indicates that central corneal thickness may gradually increase in the decades covered by our cohort , while over 70 years a decrease may be observed . in contrast to earlier studies , gender and body mass index were not related to central corneal thickness , nor was any other cardiovascular parameter . interestingly , mean arterial blood pressure showed some association with cct . however , this association was only small and failed to reach the level of significance after bonferroni correction . sensitivity analysis revealed that systolic blood pressure is rather associated with central corneal thickness compared to diastolic blood pressure . based on these negative findings for associations between central corneal thickness and cardiovascular parameters , the underlying hypothesis seems to be true that central corneal thickness itself is independent of cardiovascular changes . our study has several limitations : first , we did not examine axial length and consequently could not control for it as influencing factor , as previously reported by nangia et al . . as refractive power of the cornea is known to be associated with central corneal thickness , we included both corneal curvature and overall manifest refraction into our analysis model : we found an association of central corneal thickness with corneal curvature , but not with refraction after having corrected for corneal curvature . though not expected , this finding is similar to the study report by zhang et al . , who also did not find an association of central corneal thickness with refraction . in addition , due to the fact that our study cohort is a cross section of a working population , this may not strictly reflect the entire caucasian population . nevertheless , we examined a cohort of over 700 subjects with a broad range of age ( from 17 to 64 years ) and refraction ( from 11.75 d to + 7.625 d ) which may come close to this criterion . oct scan quality is a critical issue for further data processing [ 25 , 26 ] . we found that there is a correlation between lower oct scan quality and thinner central corneal thickness measurements ; nevertheless , the findings for associated factors did not alter upon including the oct scan quality parameter . in conclusion , our study evaluated associations of central corneal thickness readings determined by sd - oct in healthy eyes . as our study was explicitly focused on healthy eyes , this approach may be worthwhile for defining norm values for this specific technology . analysis confirmed intraocular pressure and corneal curvature as ocular factors associated with central corneal thickness . regarding cardiovascular factors , central corneal thickness was not associated with any examined parameters except age . these data suggest that clinical evaluation of central corneal thickness as determined by sd - oct should also be performed with respect to individual corneal curvature and intraocular pressure interpretation merits the knowledge of central corneal thickness .

introduction . sarcoidosis is a chronic granulomatous disease , which can involve different organs and systems . coexistence of sarcoidosis and spondyloarthritis has been reported in numerous case reports . purpose . to determine the prevalence of sacroiliitis and spondyloarthritis in patients previously diagnosed with sarcoidosis and to investigate any possible relation with clinical findings . materials and methods . forty - two patients with sarcoidosis were enrolled in the study . any signs and symptoms in regard to spondyloarthritis ( i.e. , existence of inflammatory back pain , gluteal pain , uveitis , enthesitis , dactylitis , inflammatory bowel disease , and psoriasis ) were questioned in detail and biochemical tests were evaluated . sacroiliac joint imaging and lateral heel imaging were performed in all patients . results . sacroiliitis was found in 6 of the 42 ( 14.3% ) sarcoidosis patients and all of these patients were female . common features of the disease in these six patients were inflammatory back pain as the major clinical complaint , stage 2 sacroiliitis as revealed by radiological staging , and the negativity of hla b-27 test . these six patients with sacroiliitis were diagnosed with spondyloarthritis according to the criteria of asas and of essg . conclusion . we found spondyloarthritis in patients with sarcoidosis at a higher percentage rate than in the general population ( 11.9% ) . controlled trials involving large series of patients are required for the confirmation of the data .

sarcoidosis is a systemic disease characterized by the involvement of multiple tissues and organs with a noncalcified granuloma reaction , which is not yet well understood . although the exact pathogenesis of sarcoidosis is not known , it is currently accepted that , in genetically susceptible individuals , it is caused through alteration of the cellular immune response after exposure to an environmental , occupational , or infectious agent . accumulations of th1 and macrophages with increased production of proinflammatory cytokines induce the inflammatory cascade and consecutive impairment in tissue permeability ; increase in cellular influx and local cellular proliferation cause the formation of granulomas . the disease most frequently presents with bilateral hilar lymphadenopathy and infiltrations in the lungs and skin , as well as with eye lesions . acute disease is the most common form and the patient presents with arthralgia and signs of arthritis and/or periarthritis as the first sign of sarcoidosis . chronic sarcoid arthritis usually coexists with pulmonary parenchymal disease or other organ involvement and is rare . spondyloarthropathies are a group of chronic inflammatory diseases primarily characterized by the involvement of axial and peripheral joints . common characteristics of these diseases are inflammatory back pain , enthesitis , uveitis , psoriasis , inflammatory bowel disease , and rf negativity . coexistence of sarcoidosis and sacroiliitis has been published in numerous case reports ; however the prevalence of sacroiliitis and spondyloarthritis has not been reported in sarcoidosis . evidence is collected for the association of sarcoid - like granulomatous disease developing after the initiation of anti - tnf- therapy , with disease reversal after discontinuation . therefore , this study is designed to determine the prevalence of sacroiliitis and spondyloarthritis in patients diagnosed with sarcoidosis and to establish any possible correlation with the findings of the disease . forty - two consecutive sarcoidosis patients followed by our rheumatology outpatients clinic between december 2011 and june 2013 were enrolled in this study . the initial diagnosis of sarcoidosis had been made by the rheumatologists with reference to clinical signs and symptoms and histopathological confirmation of noncalcified granulomas in the biopsies of various organs and tissues . conditions that might cause granulomatous disease ( such as bacterial and fungal infections ) had been ruled out . after given consents were taken , laboratory tests were performed in all patients , including routine biochemistry , acute phase reactants ( esr , crp ) , serum angiotensin converting enzyme ( ace ) , and calcium and hydroxyvitamin d3 levels . detailed questioning in regard to spondyloarthritis ( the existence of inflammatory back pain , gluteal area pain , uveitis , enthesitis , peripheral arthritis , dactylitis , psoriasis , inflammatory bowel disease , the presence of a preceding infection , and family history for spondyloarthritis ) was performed . the diagnoses of spondyloarthritis were made based on the european spondyloarthritis study group ( essg ) and the assessment of spondyloarthritis international society ( asas ) classification criteria . standard pelvic radiographs were obtained in all patients to assess the sacroiliac joints ( sijs ) . each sij was scored on radiographs according to the mny criteria as follows : grade 0 : normal ; grade 1 : suspicious ; grade 2 : minimal abnormality with small localized erosions , sclerosis without joint spondyloarthritis alteration ; grade 3 : definite abnormality with erosion , sclerosis , and joint spondyloarthritis widening or narrowing or partial ankylosis ; grade 4 : total ankylosis of joint . when / if sacroiliitis was determined in radiography , magnetic resonance imaging of sij was performed using the short time inversion recovery ( stir ) method for the confirmation of the existence of active sacroiliitis . crosstabs were produced to analyse the data , and chi - square analysis or mann - whitney u testing was performed as appropriate . forty - two sarcoidosis patients ( ten males , thirty - two females ) were included in the study . the mean age was 45.2 years ( from 20 to 70 years ) , and mean duration of the illness was 3.5 years . when the manifestations of sarcoidosis were reviewed , it has been observed that twenty patients ( 47.6% ) had erythema nodosum , three patients ( 7.1% ) had acute , unilateral , anterior , and nongranulomatous uveitis , one patient ( 2.3% ) had myositis , one patient ( 2.3% ) had neurosarcoidosis , and thirty - two patients ( 76.2% ) had arthritis . twenty - eight of the patients with arthritis ( 87.5% ) had ankle involvement , three patients ( 9.4% ) had knee joint involvement , and one patient ( 3.1% ) had wrist involvement . the thoracic computerized tomography scans revealed that twelve patients ( 28.5% ) had stage 1 sarcoidosis , twenty - two patients ( 52.4% ) had stage 2 sarcoidosis , four patients ( 9.5% ) had stage 3 sarcoidosis , and four patients ( 9.5% ) had stage 4 sarcoidosis . laboratory assessments revealed that fifteen patients ( 35.7% ) had elevated serum ace , six patients ( 14.3% ) had elevated serum calcium , two patients ( 4.7% ) had elevated serum d3 , twenty - nine patients ( 69% ) had elevated esr , and thirty patients ( 71.4% ) had elevated crp . sacroiliitis was diagnosed in six of the forty - two ( 14.2% ) sarcoidosis patients . all the patients with sacroiliitis were females . while the average age of the cases with sacroiliitis was 55 year , the average duration of disease was 17.8 months . when the first application symptoms of the patients with sacroiliitis were evaluated , it was observed that two patients applied with erythema nodosum , two patients applied with respiration symptoms , and two patients applied with locomotor system complaints . different stages of sarcoidosis were diagnosed in each of the six patients : a stage 1 diagnosis was given in two patients , a stage 2 diagnosis in two patients , and a stage 4 diagnosis in two patients . in radiological staging of sacroiliitis , stage 2 sacroiliitis was found in 6 patients ( figure 1 ) . when the patients were evaluated for hla b-27 , it was found negative in six patients . comparison between patients with and without sacroiliitis revealed statistically significant differences in terms of some of the parameters ( age , inflammatory back pain , enthesitis , and crp levels ) ( table 1 ) . all the six patients with sacroiliitis were diagnosed with spondyloarthritis according to the criteria of asas ( assessment of spondyloarthritis international society ) and of essg ( european spondyloarthropathy study group ) . involvement of the joints can be in 2 different ways ; while acute , migratory , symmetric arthritis is more common , chronic recurrent erosive form can be especially rare . sacroiliitis is an important finding of the diseases of spondyloarthritis group , but many different causes ( e.g. , pyogenic , mycobacterial , and fungal infections and malign infiltrations ) should also be eliminated . while the prevalence of spondyloarthritis is from 1% to 1.9% in the general population , erb et al . reported prevalence of spondyloarthritis in patients with sarcoidosis as 6% . in our study , the prevalence of sacroiliitis in cases diagnosed with sarcoidosis has been 14.2% . comparisons revealed significant statistical differences in the patient group with sacroiliitis and the group without sacroiliitis , in terms of some of the parameters ( age , inflammatory back pain , enthesitis , and crp level ) . furthermore , all the patients with sacroiliitis were also diagnosed with spondyloarthritis according to the asas and essg criteria . these findings suggest that both diseases may have a common etiopathogenesis and/or sacroiliitis may be an important clinical finding in patients with sarcoidosis . however , the etiopathogenesis of spondyloarthritis is not clear ; it starts with an immunological reaction which develops against an undetermined bacterial antigen . among the findings that support this theory are the dna sequences of the many bacteria ( i.e. , mycobacteria , yersinia , and salmonella ) found in patients with spondyloarthritis . the etiology of sarcoidosis is unknown , but the possibility of chronic bacterial infection similar to spondyloarthritis is under investigation since the dna of mycobacteria strains has been shown . in the light of these discoveries , it is considered that infection - related reactive arthritis and sacroiliitis may develop in patients with sarcoidosis with immune susceptibility . the role of the molecule hla - b27 is obvious in the pathogenesis of spondyloarthritis and the elevated molecule hla - dr5 has been found in patients with sarcoidosis . furthermore , it has been suggested that the hla - b8 and hla - dr3 molecules are related to acute sarcoid arthritis and spontaneous remission . the fact that the two diseases develop on different genetic bases ( msc class 1 in spondyloarthritis and mhc class 2 in sarcoidosis ) suggests a coincidence rather than a common etiopathogenesis . some agents ( e.g. , propionibacterium acnes ) have been detected in tissue samples in both diseases , but their role in pathogenesis has not yet been determined . another study has discovered that spondyloarthritis finding has been determined in 13.6% of hla - b27-positive patients ; the study has reported that there is elevated risk of spondyloarthritis with this allele . furthermore , while bilateral sacroiliitis is seen in hla - b27-positive patients , unilateral sacroiliitis was found in hla - b27-negative patients . a further study has recorded three hla - b27-positive patients among fifteen cases determined to have sarcoidosis spondyloarthritis . in our study , all the cases determined to have sacroiliitis were also hla - b27-negative . this suggests that different mechanisms may be involved in the pathogenesis of sacroiliitis developing in patients with sarcoidosis . firstly , the diagnosis of sacroiliitis was performed using x - radiography in the first instance and further confirmed by mri of sij . however , if mri was performed in all patients , higher prevalence of sacroiliitis could have been detected at earlier stages of the disease . but we are conscious that the mri method is too expensive , and the use of x - radiography for the small number of patients was considered cost - effective . secondly , the number of patients was small in our study and it will be wrong to make a generalization . in conclusion , we detected a higher prevalence of sacroiliitis in patients with sarcoidosis when compared to the general population . patients with sarcoidosis should be questioned in terms of inflammatory back pain and should be assessed with sijs imaging and/or mri . trials that involve large series of patients are needed for this .

the signal transducer and activator of transcription stat3 is a transcription factor which plays a key role in normal cell growth and is constitutively activated in about 70% of solid and hematological cancers . activated stat3 is phosphorylated on tyrosine and forms a dimer through phosphotyrosine / src homology 2 ( sh2 ) domain interaction . the dimer enters the nucleus via interaction with importins and binds target genes . inhibition of stat3 results in the death of tumor cells , this indicates that it is a valuable target for anticancer strategies ; a view that is corroborated by recent findings of activating mutations within the gene . yet , there is still only a small number of stat3 direct inhibitors ; in addition , the high similarity of stat3 with stat1 , another stat family member mostly oriented toward apoptosis , cell death and defense against pathogens , requires that stat3-inhibitors have no effect on stat1 . specific stat3 direct inhibitors consist of sh2 ligands , including g quartet oligodeoxynucleotides ( odn ) and small molecules , they induce cell death in tumor cells in which stat3 is activated . stat3 can also be inhibited by decoy odns ( dodn ) , which bind stat3 and induce cell death . a specific stat3 dodn which does not interfere with stat1-mediated interferon - induced cell death has been designed pointing to the stat3 dbd as a target for specific inhibition . comprehensive analysis of this region is in progress in the laboratory to design dbd - targeting stat3 inhibitors with stat3/stat1 discriminating ability .

stat3 belongs to a family of transcription factors ( tfs ) comprising stat1 , stat2 , stat3 , stat4 , stat5a , stat5b and stat6 . like stat5 , stat3 was found to play an important role in cell growth , and its activation has been described in nearly 70% of solid and hematological tumors , giving good reason for a search for specific direct inhibitors , of which there are unfortunately only a few , and none in the clinic to this day . stat3 comprises several distinct functional domains including : an n - terminal domain containing an oligomerization and a coiled - coil domain , a dna binding domain ( dbd ) , a linker domain , a src homology 2 ( sh2 ) domain involved in the interaction of two monomers via phosphotyrosine 705 resulting in dimerization and a c - terminal transactivation domain ( see fig . 1 ) . stat3 activation occurs following cytokine- or growth factor - receptor activation ; it involves phosphorylation within the cytoplasm , dimerization and nuclear transfer ( fig . 2 ) . nuclear transfer of stat3 requires nuclear localization signals ( nls ) which are in the coiled - coil domain ( comprising arginines 214 and 215 ) and in the dimer - dependent dbd ( comprising arginines 414 and 417 ) . the nlss interact with importin s , yet which of the five importin s ( 1 , 3 , 4 , 5 or 7 ) actually carries stat3 is still debated , the complex interacts with importin and is carried through the nuclear pore complex ( npc ) ( fig . 3 ) . while arginines 214 and 215 appear to be the major importin - binding site , arginines 414 and 417 are thought to be required for stat3 to adopt the proper conformation for importin binding . unphosphorylated forms of stat3 can enter the nucleus and stimulate transcription of a subset of gene targets , apparently via interaction with the tf nfb . however , whether unphosphorylated stat3 interacts on its own with importins for nuclear entry is not entirely clear : tyrosine 705-mutated stat3 can shuttle to the nucleus and phosphotyrosine 705/sh2-independent stat3 dimers were shown to enter the nucleus ( but more slowly than phosphorylated stat3 dimers ) ( fig . 2 ) . interestingly , in the case of stat1 , unphosphorylated monomers enter the nucleus through direct interaction with the npc proteins nucleoporins , not with importins and unphosphorylated stat1 dimers bind dna with a 200-fold lower affinity than phosphorylated stat1 dimers ; in fact , single - molecule imaging showed that interferon ( ifn)--activated stat1 has a reduced mobility and resides longer in the nucleus . in any case , the nucleo - cytoplasmic shuttling of stat3 is a major step of the activation process leading to increased transcriptional activity , suggesting that nuclear transfer of stat3 per se can be a target for inhibition . stat3 monomer showing the n - terminal coiled - coil domain , the dbd ( half site ) , the sh2 domain and the c - terminal domain . the stat3 crystal coordinates were downloaded from the protein data bank ( pdb , file : 1bg1 ) and analyzed using the chimera program . note that the model shown comprises residues 136 to 716 ; hence , the protein s n - terminal and c - terminal domains ( comprising the transactivation domain ) are missing ; the coordinates corresponding to the cdna strand were missing in the model and had to be reconstructed . figure 2 . the transcription factor stat3 is present in a latent inactive non - phosphorylated form in the cytoplasm . activated cytokine receptors activate the kinases jak , which phosphorylate tyrosines located in the cytoplasmic portion of the cytokine receptors creating stat - binding motifs . once bound to these motifs , stat3 becomes in turn phosphorylated by the jaks . the phospho stat3 dimers ( colored in pink ) enter the nuclei and bind stat3 target genes ; note that the dna - bound dimer is drawn differently to indicate the stat3 conformational change suggested by molecular dynamics simulations . stat3 can also be activated by tyrosine kinases of the src family ( sfk ) , the sfks can themselves be activated by g protein - coupled receptors ( gpcr ) or growth factor receptors , the growth factor receptors ( including egf - receptors and vegf - receptors ) can also phosphorylate stat3 . there are also monomers , and dimers with non - phosphorylated stat3 , which can shuttle between the cytoplasm and the nucleus . the tyrosine phosphorylated stat3 dimer interacts with the importins through its two nlss [ one within the coiled - coil ( ml ) domain : arginines 214 and 215 ; one within the dbd : arginines 414 and 417 ] . this complex is carried through the nuclear pore complex ( npc ) by the ran gdp which is formed in the cytoplasm by hydrolysis of ran - bound gtp by gtpase activating protein ( gap ) . the high level of ran - gtp in the nucleus is the result of a high gtp - exchange factor ( gef ) activity in this compartment . the stat3 dimer interacts with the stat3 dna consensus motif and then is released ; once released from dna the dimer is dephosphorylated by a nuclear phosphatase ( ptpase ) . the dephosphorylated stat3 is exported to the cytoplasm in combination with exportins and ran - gtp . constitutive activation of stat3 in tumors can result from upstream activated signaling components , including increased cytokines ( il-6 and il-10 ) production , activated receptor ( cytokine receptors , vegfr and egfr ) and non - receptor tyrosine kinases ( including jaks , src and abl ) . recently , mutated hyperactive forms of stat3 have been detected in tumors , interestingly most of the described mutations are located within stat3 s sh2 ( see ref . due to the involvement of tyrosine protein kinases in the stat3 activating pathways , tyrosine kinase inhibitors indirectly inhibit stat3 resulting in anti - tumor activity . but stat3 can also be activated when suppressors of the stat signaling pathway are inactivated such as the socs ( suppressor of cytokine signaling ) , the pias ( protein inhibitor of activated stats ) and protein tyrosine phosphatases ; furthermore , in cells in which proliferation results from the inactivation of negative regulators of signaling , such as the inhibitor of the pi3kinase pathway pten , or the inhibitor of the proapoptotic tf p53 , mdm2 , the inhibition of upstream signaling pathways may have little effect . thus , when upstream stat3 activators are not identified or do not have any known inhibitor , or when stat3 is itself activated it becomes a relevant target for anti - tumor treatments . this reasoning , reinforced by stat3 s previously noted signaling bottleneck situation , has been used by many authors searching for stat3-specific direct inhibitors . the high similarity between stat3 and stat1 is intriguing . both tfs share a 50% amino - acid sequence homology and share similar activating stimuli ( types i and ii ifns , cytokines and growth factors ) ; yet , their functions differ : stat1 is mostly involved in immunity , host defense against pathogens and cell death ( see refs . 23 and 24 ) , with the notable exception that in certain contexts stat1 exerts proliferative potential , while stat3 is mostly involved in cell growth and proliferation ( see ref . their gene targets are mostly distinct : stat3 stimulates the transcription of cell growth - associated genes , including cyclin - d1 , survivin , vegf , c - myc , bcl - xl , mcl-1 , vascular endothelial growth factor , il-10 , transforming growth factor and bcl2 and stat1 stimulates the transcription of pro - inflammatory and anti - proliferative genes , including caspases , inos , mdm2 , p21waf / cip1 and p27kip1 ; but there is also an overlap of repertoires . in reality , stat3 and stat1 recognize very similar dna consensus sequences , based on a ttcnnn(t , g)aa motif ( see table 1 ) . in this motif , a 3n spacing ( n representing any base ) is most frequently present for stat1 and stat3 , as shown by electrophoretic migration shift assay ( emsa ) . natural binding sites share this consensus with minor variations such as a preference for t at position 5 for stat1 , but there is greater diversity outside this consensus ( see table 1 ) , suggesting that target recognition in vivo requires additional co - factors . thus , when attempting to inhibit stat3 with the aim to kill tumor cell or block their growth , one must use substances that have no effect on stat1 , as has been pointed out and shown in tumor cell lines with sirna - suppressed stat1 . indeed , stat1 is required for the antiproliferative effects of interferons and , apoptosis is defective in stat1-null cells and stat1 is the major effector of ifn- , a cytokine with antitumor and cancer immunosurveillance functions . the recognition that activated stat3 is widely present in tumors and that its inhibition is a valuable anti - cancer strategy led to a search for stat3-targeting compounds . among those the dna - alkylating platinum complexes were found to induce the death of tumor cells with activated stat3 and were thought to act directly on stat3 ; other compounds target the sh2 of stat3 , including g quartet oligodeoxynucleotides and small molecules , some of which are highly specific for stat3 . the dimerization of stat3 through reciprocal phosphotyrosine 705/sh2 interaction can be impaired by a phosphopeptide with the sequence ppylktk . this phosphopeptide inhibits stat3 activity in tumor cell lines , induces cell death and has a high affinity and specificity for stat3 : in particular it had no effect on the sh2-containing tyrosine kinase p56lck , and little effect on stat1 as determined by emsa . despite their efficacy and specificity , the inefficient cell penetration of phosphopeptides led to a search for smaller equivalents using computational docking studies exploring the phosphotyrosine 705/sh2 interaction area ( see fig . these studies yielded several small molecules with high affinity and high specificity for stat3 , including sta-21 , stattic , s31 - 201 and recently bp-1 - 102 , a compound with improved bioavailability and anti - cancer properties . the mechanism of action of these compounds is thought to be based on their interaction with stat3 sh2 ( fig . 4a and b ) , an area where the other monomer s phosphotyrosine 705 docks , thereby impairing the formation of the active dimer , as shown in ( see fig . 44 ; the phosphotyrosine peptide is represented together with the inhibitor s31 - 201 , both form h - bonds with the same residues , including lysine 591 , serine 611 , serine 613 and arginine 609 ) . stat3 sh2-targeting compounds are efficacious stat3 inhibitors , they inhibit stat3 dna binding , reduce stat3-dependent cell proliferation and expression of biological targets . yet , experimental demonstration of their interaction with stat3 is missing , the actual data are based on computational studies , not on actual interaction measurements , as noted earlier . this implies that a compound claimed to interact with sh2 might actually interfere with the binding of stat3 to the receptor s phosphotyrosine site leading to biological effects similar to those of jak - inhibitors . the compound might also interact with the dbd with just the same effect in the cell ( reduced dna binding , cell death ) . besides , compounds usually undergo modifications in a biological environment ( cells or body fluids ) , these modifications can occur before the compounds reach their target . for instance , stattic s inhibitory activity of stat3 increases with time and is temperature- and dithiothreitol - sensitive , this suggests that it interacts with a cysteine , such as cysteine 687 which is next to the phosphopeptide motif of stat3 and faces the phosphopeptide - binding area of sh2 . while this point does not invalidate stattic s specificity for stat3 , it suggests possible limitations for its in vivo utilization . despite considerable progress and clear anti - cancer efficacy the areas in squares are the sh2 region where small molecules interact ( b and c ) and the sh2/dbd region where g quartets interact ( d and e ) . ( b ) close up view of the region of the sh2 of stat3 that interacts with the small molecule inhibitors : the key aminoacids involved in interaction are labeled ; the inhibitors interact particularly in the groove located between arginine 595 and lysine 591 . ( c ) the same area as in ( b ) is shown but stat3 and stat1 are superimposed ; this shows the overall great similarity between these two regions , yet some differences are present , accounting for the capacity of the inhibitors to discriminate between stat3 and stat1 ( arrows ) ; stat1 crystal coordinates used in ( c ) were from pdb file 1bf5 . ( d ) modeled interaction of inhibitor s31 - 201 with stat3 sh2 domain , note the important role of lysine 591 , serine 611 and arginine 609 in the interaction . ( e ) same as ( d ) , with added phosphotyrosine - peptide , showing its overlap with the stat3 sh2/small molecule inhibitors binding area . ( f ) detailed view of the region of stat3 interacting with g quartets , this region includes the phosphotyrosine 705-interacting region of sh2 and a neighboring region including part of the dbd , including glutamic acid 638 , glutamine 644 , aspartic acid 647 , glutamine 643 and asparagine 646 . ( g ) same region as in f is shown with the superimposition of stat1 , the major differences are indicated by arrows . [ panels ( d and e ) are reprinted from ref . 44 with permission ; 2007 national academy of sciences usa . ] g quartets are g - rich oligodeoxynucleotides that form potassium - dependent four - stranded intramolecular g - quartet structures . they inhibit stat3 at micromolar concentrations and induce the death of several tumor cell lines , including head and neck cancer lines , they also arrest the development of breast tumor , prostate tumor or non - small cell lung cancer xenografts in nude mice . these reagents are interesting in that their specificity for stat3 , demonstrated by emsa showing high affinity binding to stat3 and much lower affinity for stat1 , is somewhat unexpected . a computational study of the interaction of the g quartet with stat3 and stat1 showed the following sh2 domain amino - acids to be involved in the binding : glutamic acid 638 , glutamine 644 , asparagine 647 , glutamine 643 and asparagine 646 ( fig . we compared stat3 and stat1 surfaces in the area where the g quartet interacts and found that the surfaces differ significantly ( fig . however , the use of g quartets is problematic due to their large size and potassium dependence , which limit cellular delivery . decoy oligodeoxynucleotides ( dodns ) are short stretches of double stranded dna containing a tf s consensus binding sequence . once in the cells , dodns inhibit the corresponding tf as shown for nfb in animal models ( cardiovascular disease and ischemia - reperfusion injury ) and in cancer cells . this property has been used for stat3 : in cells in which stat3 is constitutively activated , stat3 dodns ( table 1 ) induce cell death . while some studies suggested that dodns must enter nuclei to exert their inhibitory action , our studies of the subcellular location of stat3 in dodn - treated cells have shown that the dodns prevent nuclear translocation of stat3 . the dodns are thought to interact with stat3s dbd within the cytoplasm and to prevent interaction with importins which carry the stat3 dimer within the nucleus . inhibition of importin binding by the dodn is thought to result from the masking of the dbd - located nls ( arginines 414 and 417 ) ( see figs . 1 and 5a ) indirectly confirming its involvement in nuclear entry . in fact , competition between dna binding and importin binding was observed in vitro with stat1 and within cells with stat3 . thus , despite the uncertainty regarding the two stat3 nlss relative requirement for nuclear transfer , it seems that the dbd - located nls plays an important role for nuclear translocation since its masking is sufficient to impair it , confirming that it may be a good target for inhibition , as suggested by others . ( a ) detail of the area of stat3 interacting with the dna consensus sequence . arginines 414 and 417 play a key role in the interaction of stat3 with importins and nuclear translocation , arginine 423 is involved in the interaction of dna with stat3 . ( b ) the same area as in ( a ) is shown , the corresponding area of stat1 has been superimposed , showing the high level of homology between stat3 and stat1 , except for glutamic acid 421 ( in italic ) of stat1 whose interaction with dna is very different from that of arginine 423 of stat3 ( see ref . 65 ) , and other differences indicated by arrows . as discussed above , the opposed cellular functions of stat1 and stat3 , in spite of their similarity , is puzzling . stat1 and stat3 can form heterodimers , whose function is not elucidated to this day , they recognize very similar dna motifs ( table ) , and they have targets in common and regulate one another . indeed , in stat3-deficient cells , stat3-activators such as il-6 trigger an ifn--like response through stat1 activation ; and in stat1-deficient cells , ifn- and ifn- trigger stat3-dependent proliferative responses . thus the stat1/stat3 cross - regulation suggests that stat3 inhibitors may work best in stat1-expressing cells . it is therefore essential to inhibit stat3 without inhibiting stat1 to keep cell death processes operational . in this regard , it should be noted that stat3-dodns inhibit stat3 but also inhibit activated stat1 , thereby abolishing ifn--induced cell death and reducing the dodns anti - stat3 efficacy . computer analysis of the dodn s interaction with stat1 and stat3 dbds showed that within the highly similar dna sequences , there were subtle differences including a t at positions 7 and 5 , a dc at position 0 , a da at position + 5 ( see table 1 , dodn # 22 ) , which had been previously noted by computer analysis and dna - binding studies . this allowed designing a dodn that could inhibit stat3 without inhibiting stat1 , demonstrating that at this level specific interaction can be achieved . therapeutic use of the dodns not only requires specific target recognition , but also stability in biological fluids . modifications , including phosphothioate end modification and hairpin structures considerably increased intracellular stability and efficacy . a recent improvement consisting of a cyclic stat3 dodns comprising a hairpin at both ends was designed and found to reduce xenograft tumors following intravenous administration . furthermore , a stat3 dodn has been found to have few side effects when administered to primates , suggesting interesting therapeutic perspectives . another possibility is to design smaller molecules mimicking the dodn s interaction with stat3 , similarly to what was achieved with the sh2 domain . the stat3 area that interacts with the dna target and the dodn ( fig . this area comprises the dbd - located nls including arginines 414 and 417 that are located closely to arginine 423 , involved in interaction with the dodn ; these three arginines surround an opening in the surface in which small molecules could interact ( fig . furthermore , superimposition of stat3 and stat1 showed that in spite of their striking overall similarity , these areas comprise in the same location glutamic acid 421 in stat1 and arginine 423 in stat3 ( fig . 5b ) , a difference that might be exploited for the design of a stat3 small molecule inhibitor with stat3/stat1 discriminating capacity ; studies are underway in the laboratory to try and design such a reagent . the dimerization of stat3 through reciprocal phosphotyrosine 705/sh2 interaction can be impaired by a phosphopeptide with the sequence ppylktk . this phosphopeptide inhibits stat3 activity in tumor cell lines , induces cell death and has a high affinity and specificity for stat3 : in particular it had no effect on the sh2-containing tyrosine kinase p56lck , and little effect on stat1 as determined by emsa . despite their efficacy and specificity , the inefficient cell penetration of phosphopeptides led to a search for smaller equivalents using computational docking studies exploring the phosphotyrosine 705/sh2 interaction area ( see fig . these studies yielded several small molecules with high affinity and high specificity for stat3 , including sta-21 , stattic , s31 - 201 and recently bp-1 - 102 , a compound with improved bioavailability and anti - cancer properties . the mechanism of action of these compounds is thought to be based on their interaction with stat3 sh2 ( fig . 4a and b ) , an area where the other monomer s phosphotyrosine 705 docks , thereby impairing the formation of the active dimer , as shown in ( see fig . 44 ; the phosphotyrosine peptide is represented together with the inhibitor s31 - 201 , both form h - bonds with the same residues , including lysine 591 , serine 611 , serine 613 and arginine 609 ) . stat3 sh2-targeting compounds are efficacious stat3 inhibitors , they inhibit stat3 dna binding , reduce stat3-dependent cell proliferation and expression of biological targets . yet , experimental demonstration of their interaction with stat3 is missing , the actual data are based on computational studies , not on actual interaction measurements , as noted earlier . this implies that a compound claimed to interact with sh2 might actually interfere with the binding of stat3 to the receptor s phosphotyrosine site leading to biological effects similar to those of jak - inhibitors . the compound might also interact with the dbd with just the same effect in the cell ( reduced dna binding , cell death ) . besides , compounds usually undergo modifications in a biological environment ( cells or body fluids ) , these modifications can occur before the compounds reach their target . for instance , stattic s inhibitory activity of stat3 increases with time and is temperature- and dithiothreitol - sensitive , this suggests that it interacts with a cysteine , such as cysteine 687 which is next to the phosphopeptide motif of stat3 and faces the phosphopeptide - binding area of sh2 . while this point does not invalidate stattic s specificity for stat3 , it suggests possible limitations for its in vivo utilization . despite considerable progress and clear anti - cancer efficacy the areas in squares are the sh2 region where small molecules interact ( b and c ) and the sh2/dbd region where g quartets interact ( d and e ) . ( b ) close up view of the region of the sh2 of stat3 that interacts with the small molecule inhibitors : the key aminoacids involved in interaction are labeled ; the inhibitors interact particularly in the groove located between arginine 595 and lysine 591 . ( c ) the same area as in ( b ) is shown but stat3 and stat1 are superimposed ; this shows the overall great similarity between these two regions , yet some differences are present , accounting for the capacity of the inhibitors to discriminate between stat3 and stat1 ( arrows ) ; stat1 crystal coordinates used in ( c ) were from pdb file 1bf5 . ( d ) modeled interaction of inhibitor s31 - 201 with stat3 sh2 domain , note the important role of lysine 591 , serine 611 and arginine 609 in the interaction . ( e ) same as ( d ) , with added phosphotyrosine - peptide , showing its overlap with the stat3 sh2/small molecule inhibitors binding area . ( f ) detailed view of the region of stat3 interacting with g quartets , this region includes the phosphotyrosine 705-interacting region of sh2 and a neighboring region including part of the dbd , including glutamic acid 638 , glutamine 644 , aspartic acid 647 , glutamine 643 and asparagine 646 . ( g ) same region as in f is shown with the superimposition of stat1 , the major differences are indicated by arrows . [ panels ( d and e ) are reprinted from ref . g quartets are g - rich oligodeoxynucleotides that form potassium - dependent four - stranded intramolecular g - quartet structures . they inhibit stat3 at micromolar concentrations and induce the death of several tumor cell lines , including head and neck cancer lines , they also arrest the development of breast tumor , prostate tumor or non - small cell lung cancer xenografts in nude mice . these reagents are interesting in that their specificity for stat3 , demonstrated by emsa showing high affinity binding to stat3 and much lower affinity for stat1 , is somewhat unexpected . a computational study of the interaction of the g quartet with stat3 and stat1 showed the following sh2 domain amino - acids to be involved in the binding : glutamic acid 638 , glutamine 644 , asparagine 647 , glutamine 643 and asparagine 646 ( fig . we compared stat3 and stat1 surfaces in the area where the g quartet interacts and found that the surfaces differ significantly ( fig . . however , the use of g quartets is problematic due to their large size and potassium dependence , which limit cellular delivery . decoy oligodeoxynucleotides ( dodns ) are short stretches of double stranded dna containing a tf s consensus binding sequence . once in the cells , dodns inhibit the corresponding tf as shown for nfb in animal models ( cardiovascular disease and ischemia - reperfusion injury ) and in cancer cells . this property has been used for stat3 : in cells in which stat3 is constitutively activated , stat3 dodns ( table 1 ) induce cell death . while some studies suggested that dodns must enter nuclei to exert their inhibitory action , our studies of the subcellular location of stat3 in dodn - treated cells have shown that the dodns prevent nuclear translocation of stat3 . the dodns are thought to interact with stat3s dbd within the cytoplasm and to prevent interaction with importins which carry the stat3 dimer within the nucleus . inhibition of importin binding by the dodn is thought to result from the masking of the dbd - located nls ( arginines 414 and 417 ) ( see figs . 1 and 5a ) indirectly confirming its involvement in nuclear entry . in fact , competition between dna binding and importin binding was observed in vitro with stat1 and within cells with stat3 . thus , despite the uncertainty regarding the two stat3 nlss relative requirement for nuclear transfer , it seems that the dbd - located nls plays an important role for nuclear translocation since its masking is sufficient to impair it , confirming that it may be a good target for inhibition , as suggested by others . figure 5 . detailed view of the area of stat3 interacting with dna and importins . ( a ) detail of the area of stat3 interacting with the dna consensus sequence . arginines 414 and 417 play a key role in the interaction of stat3 with importins and nuclear translocation , arginine 423 is involved in the interaction of dna with stat3 . ( b ) the same area as in ( a ) is shown , the corresponding area of stat1 has been superimposed , showing the high level of homology between stat3 and stat1 , except for glutamic acid 421 ( in italic ) of stat1 whose interaction with dna is very different from that of arginine 423 of stat3 ( see ref . 65 ) , and other differences indicated by arrows . as discussed above , the opposed cellular functions of stat1 and stat3 , in spite of their similarity , is puzzling . stat1 and stat3 can form heterodimers , whose function is not elucidated to this day , they recognize very similar dna motifs ( table ) , and they have targets in common and regulate one another . indeed , in stat3-deficient cells , stat3-activators such as il-6 trigger an ifn--like response through stat1 activation ; and in stat1-deficient cells , ifn- and ifn- trigger stat3-dependent proliferative responses . thus the stat1/stat3 cross - regulation suggests that stat3 inhibitors may work best in stat1-expressing cells . it is therefore essential to inhibit stat3 without inhibiting stat1 to keep cell death processes operational . in this regard , it should be noted that stat3-dodns inhibit stat3 but also inhibit activated stat1 , thereby abolishing ifn--induced cell death and reducing the dodns anti - stat3 efficacy . computer analysis of the dodn s interaction with stat1 and stat3 dbds showed that within the highly similar dna sequences , there were subtle differences including a t at positions 7 and 5 , a dc at position 0 , a da at position + 5 ( see table 1 , dodn # 22 ) , which had been previously noted by computer analysis and dna - binding studies . this allowed designing a dodn that could inhibit stat3 without inhibiting stat1 , demonstrating that at this level specific interaction can be achieved . therapeutic use of the dodns not only requires specific target recognition , but also stability in biological fluids . modifications , including phosphothioate end modification and hairpin structures considerably increased intracellular stability and efficacy . a recent improvement consisting of a cyclic stat3 dodns comprising a hairpin at both ends was designed and found to reduce xenograft tumors following intravenous administration . furthermore , a stat3 dodn has been found to have few side effects when administered to primates , suggesting interesting therapeutic perspectives . another possibility is to design smaller molecules mimicking the dodn s interaction with stat3 , similarly to what was achieved with the sh2 domain . the stat3 area that interacts with the dna target and the dodn ( fig . this area comprises the dbd - located nls including arginines 414 and 417 that are located closely to arginine 423 , involved in interaction with the dodn ; these three arginines surround an opening in the surface in which small molecules could interact ( fig . furthermore , superimposition of stat3 and stat1 showed that in spite of their striking overall similarity , these areas comprise in the same location glutamic acid 421 in stat1 and arginine 423 in stat3 ( fig . 5b ) , a difference that might be exploited for the design of a stat3 small molecule inhibitor with stat3/stat1 discriminating capacity ; studies are underway in the laboratory to try and design such a reagent . because stat3 is constitutively activated in nearly 70% of tumors , it provides a valuable target for anti - cancer therapy . the recent findings that tumors harbor activating mutations of stat3 underlines the need for additional stat3 inhibitors , and inhibitors that target other regions of stat3 than its sh2 , especially as most identified mutations are within this region . in addition , studies on cancer cell lines indicate that even when stat3 direct targeting is insufficient to induce cell death ; it can diminish the resistance to other anti - cancer compounds such as doxorubicin or egfr inhibitors . finally , stat3 direct inhibitors are also probably less toxic than many others because inhibition of stat3 in mature cells has minimal effects .

purposethe minimally important difference ( mid ) represents the smallest change in score on patient - reported outcome measures that is relevant to patients . the aim of this study was to introduce the mid for the vascular quality of life questionnaire ( vascuqol ) and the walking impairment questionnaire ( wiq ) for patients with intermittent claudication ( ic).methodsin this multicenter study , we recruited 294 patients with ic between july and october 2012 . patients completed the vascuqol , with scores ranging from 1 to 7 ( worst to best ) , and the wiq , with scores ranging from 0 to 1 ( worst to best ) at first visit and after 4 months follow - up . in addition , patients answered an anchor - question rating their health status compared to baseline , as being improved , unchanged , or deteriorated . the mid for improvement and deterioration was calculated by an anchor - based approach , and determined with the upper and lower limits of the 95 % confidence interval of the mean change of the group who had not changed according to the anchor-question.resultsfor the mid analyses of the vascuqol and wiq , 163 and 134 patients were included , respectively . the mid values for the vascuqol ( mean baseline score 4.25 ) were 0.87 for improvement and 0.23 for deterioration . for the wiq ( mean baseline score 0.39 ) , we found mid values of 0.11 and 0.03 for improvement and deterioration , respectively.conclusionin this study , we calculated the mid for the vascuqol and the wiq . applying these mid facilitates better interpretation of treatment outcomes and can help to set treatment goals for individual care.electronic supplementary materialthe online version of this article ( doi:10.1007/s00270 - 015 - 1060 - 0 ) contains supplementary material , which is available to authorized users .

the online version of this article ( doi:10.1007/s00270 - 015 - 1060 - 0 ) contains supplementary material , which is available to authorized users . since the treatment of patients with intermittent claudication ( ic ) is primarily aimed at improving their walking ability and health - related quality of life ( hrql ) , it is essential that these endpoints are measured when evaluating treatment . walking ability is a part of a patient s functional status ( fs ) , and is frequently assessed using a treadmill test . however , treadmill tests do not correlate well with real - life walking distances , and are not an adequate reflection of the patient s perceived walking impairment.[1 , 2 ] therefore , fs can better be assessed using patient - reported outcome measures ( proms ) , such as the walking impairment questionnaire ( wiq ) . hrql can be defined as the aspects of quality of life that relate specifically to a person s health . the vascular quality of life questionnaire ( vascuqol ) is an example of a disease - specific hrql prom for patients with peripheral artery disease ( pad ) . the importance of proms to evaluate treatment outcomes has been recognized by the vascular community , and they are used as endpoints in many clinical trials [ 69 ] . however , the interpretation of changes in prom scores may be difficult when it is unknown how much change is actually considered relevant by patients . a statistically significant mean change in score after treatment in a sample does nt necessarily imply that an individual patient experiences a clinically meaningful change in his or her hrql or fs . the minimally important difference ( mid ) represents the smallest change in score in the construct to be measured which patients perceive as important . the mid can aid to better appreciate trial results and individual treatment results , can be calculated for all available proms and is relevant in all patient populations . a statistically significant change in mean score for the patient sample from 25 to 33 was found , but it is unknown if this change is relevant to an individual patient . if , however , the mid for that prom was known to be + 10 points on the scale , it would be immediately clear that an individual patient would have to improve from a baseline score of 25 to at least 35 for the improvement to be clinically relevant . the current study aims to introduce the concept of the mid for the vascuqol and the wiq in patients with ic . the institutional review board ( irb ) of the academic medical center decided that this study met the criteria for exemption from irb approval . we used the patient sample of a prospective pilot study to determine the feasibility of proms as indicators of quality of care for patients with pad . patients were enrolled from july 2012 until october 2012 in nine hospitals in the netherlands . patients were eligible if they presented at the vascular surgery outpatient clinic with complaints of ic due to pad and if they had not visited the outpatient clinic for symptomatic pad in the previous year . other inclusion criteria were sufficient knowledge of the dutch language , an independent living situation , absence of psychiatric disorders , and the ability to communicate with the researchers . as recommended by national guidelines , first line treatment was supervised exercise therapy ( set ) in most patients . depending on physician and patient preferences percutaneous transluminal angioplasty ( pta ) was sometimes used as primary treatment , and a few patients were treated with surgical revascularization . therefore in some patients treatment consisted of optimal medical therapy ( omt ) ( antiplatelet drug and a statin , advice to walk , and change lifestyle ) . in each centre , a local investigator was responsible for the execution of the study and data collection at baseline and at 34 months follow - up . patient characteristics and questionnaires were sent to an independent trusted party ( itp ) for further data linking and processing . data on smoking history , diabetes , pulmonary and cardiac diseases , renal function , previous vascular interventions ( pta or surgery ) , ankle brachial index ( abi ) , and number of affected legs were recorded at first visit in a pre - specified database . at follow - up , it was also recorded if a patient had received omt or set . no data on age and gender were recorded in this database , since these were retrieved when the itp linked treatment codes ( conservative , pta or surgery ) in the dutch insurance billing system to patients in each hospital s patient administration . however , because unblinding was impossible due to privacy reasons , it was impossible for the itp to retrieve data on age and gender if no treatment code was listed . if no treatment code was listed , we used the data on treatment modality recorded by the local investigator at follow - up . when necessary , patients were contacted by telephone to remind them and help fill in the prom . only patients with available data on age and gender and a resting abi < 0.9 were analysed in the present study to ensure that the patient sample in the mid analysis had a proven diagnosis of ic due to pad . baseline characteristics and prom scores of patients included and excluded from the mid analysis were compared . the vascuqol is a disease - specific hrql prom , developed for patients with ic and critical limb ischemia . it consists of five subscales ( pain , symptoms , activities , emotional , and social ) with 25 items in total . each item is rated on a 7-point rating scale , with 1 representing the worst and 7 the best score . a total score , also ranging from 1 to 7 , is calculated by dividing the sum of all items by 25 . the wiq is a prom to rate walking impairment and consists of a speed , distance , and stairclimbing subscale with 14 items in total . for example , patients are asked to assign a degree of difficulty with which they can walk 100 meters , with answers ranging from no problems to subscale scores are calculated by adding the weighted scores , and dividing this by the maximum score so that each score ranges from 0 to 1 , with lower scores indicating a higher level of impairment . in addition to the proms , at follow - up patients filled in the following anchor - question : has your condition changed in the past three months? with the following response options : ( a ) improved , ( b ) unchanged , and ( c ) deteriorated . imputation of the vascuqol subscales took place if at least 50 % of the subscale was filled in . missing values were imputed with the mean value of all the filled - in questions if this condition was satisfied , and under the assumption of completely missing at random . when items were missing for the wiq , we calculated a best- and worst - case scenario . we hereby took into account the questions the patients did fill in , and assumed that patients could never score higher on a harder task and never lower on an easier task . if the best- and worst - case scenario scores were no more than 0.25 points apart , we used the mean of these two values as the total wiq score . for the mid analysis anchor - based approaches determine the mid by comparing proms to other measures or phenomena that have clinical relevance . suggested that the mid should be based on an anchor that has a correlation 0.3 with the prom . therefore , pearson correlation coefficients were calculated between the change in prom scores and the anchor - question . the upper and lower limit of the 95 % confidence interval ( ci ) of the mean change of the group who indicated on the anchor - question that their situation had not changed after treatment represent the mid for improvement and deterioration , respectively . differences in baseline characteristics and prom scores were determined with a student s t - test for continuous variables , and with a chi - square or fisher s exact test where appropriate for categorical variables . all analyses were performed using sas enterprise guide version 5.1 ; sas institute , cary , nc , usa . the institutional review board ( irb ) of the academic medical center decided that this study met the criteria for exemption from irb approval . we used the patient sample of a prospective pilot study to determine the feasibility of proms as indicators of quality of care for patients with pad . patients were enrolled from july 2012 until october 2012 in nine hospitals in the netherlands . patients were eligible if they presented at the vascular surgery outpatient clinic with complaints of ic due to pad and if they had not visited the outpatient clinic for symptomatic pad in the previous year . other inclusion criteria were sufficient knowledge of the dutch language , an independent living situation , absence of psychiatric disorders , and the ability to communicate with the researchers . as recommended by national guidelines , first line treatment was supervised exercise therapy ( set ) in most patients . depending on physician and patient preferences percutaneous transluminal angioplasty ( pta ) was sometimes used as primary treatment , and a few patients were treated with surgical revascularization . therefore in some patients treatment consisted of optimal medical therapy ( omt ) ( antiplatelet drug and a statin , advice to walk , and change lifestyle ) . in each centre , a local investigator was responsible for the execution of the study and data collection at baseline and at 34 months follow - up . patient characteristics and questionnaires were sent to an independent trusted party ( itp ) for further data linking and processing . data on smoking history , diabetes , pulmonary and cardiac diseases , renal function , previous vascular interventions ( pta or surgery ) , ankle brachial index ( abi ) , and number of affected legs were recorded at first visit in a pre - specified database . at follow - up , it was also recorded if a patient had received omt or set . no data on age and gender were recorded in this database , since these were retrieved when the itp linked treatment codes ( conservative , pta or surgery ) in the dutch insurance billing system to patients in each hospital s patient administration . however , because unblinding was impossible due to privacy reasons , it was impossible for the itp to retrieve data on age and gender if no treatment code was listed . if no treatment code was listed , we used the data on treatment modality recorded by the local investigator at follow - up . when necessary , patients were contacted by telephone to remind them and help fill in the prom . only patients with available data on age and gender and a resting abi < 0.9 were analysed in the present study to ensure that the patient sample in the mid analysis had a proven diagnosis of ic due to pad . baseline characteristics and prom scores of patients included and excluded from the mid analysis were compared . the vascuqol is a disease - specific hrql prom , developed for patients with ic and critical limb ischemia . it consists of five subscales ( pain , symptoms , activities , emotional , and social ) with 25 items in total . each item is rated on a 7-point rating scale , with 1 representing the worst and 7 the best score . a total score , also ranging from 1 to 7 , is calculated by dividing the sum of all items by 25 . the wiq is a prom to rate walking impairment and consists of a speed , distance , and stairclimbing subscale with 14 items in total . for example , patients are asked to assign a degree of difficulty with which they can walk 100 meters , with answers ranging from no problems to subscale scores are calculated by adding the weighted scores , and dividing this by the maximum score so that each score ranges from 0 to 1 , with lower scores indicating a higher level of impairment . in addition to the proms , at follow - up patients filled in the following anchor - question : has your condition changed in the past three months? with the following response options : ( a ) improved , ( b ) unchanged , and ( c ) deteriorated . imputation of the vascuqol subscales took place if at least 50 % of the subscale was filled in . missing values were imputed with the mean value of all the filled - in questions if this condition was satisfied , and under the assumption of completely missing at random . when items were missing for the wiq , we calculated a best- and worst - case scenario . we hereby took into account the questions the patients did fill in , and assumed that patients could never score higher on a harder task and never lower on an easier task . if the best- and worst - case scenario scores were no more than 0.25 points apart , we used the mean of these two values as the total wiq score . anchor - based approaches determine the mid by comparing proms to other measures or phenomena that have clinical relevance . suggested that the mid should be based on an anchor that has a correlation 0.3 with the prom . therefore , pearson correlation coefficients were calculated between the change in prom scores and the anchor - question . the upper and lower limit of the 95 % confidence interval ( ci ) of the mean change of the group who indicated on the anchor - question that their situation had not changed after treatment represent the mid for improvement and deterioration , respectively . differences in baseline characteristics and prom scores were determined with a student s t - test for continuous variables , and with a chi - square or fisher s exact test where appropriate for categorical variables . all analyses were performed using sas enterprise guide version 5.1 ; sas institute , cary , nc , usa . the vascuqol was sufficiently completed twice by 223 patients , the wiq by 184 patients . after exclusion of patients with unknown age , gender , and resting abi > 0.9 there were 163 patients who were suitable for the mid analysis of the vascuqol , and 134 for the wiq . baseline characteristics of both the patients included and excluded from the analysis are shown in table 1 . all baseline characteristics and scores on proms were comparable for included and excluded patients , except for the abi . missing items for both proms are presented in table s2 ( online only).table 1baseline characteristicsbaseline characteristicstotal population ( n = 294)vascuqol ( n = 163)wiq ( n = 134)excluded patients vascuqol ( n = 131)excluded patients wiq(n = 160)age ( years , sd)66.5 ( 10.4 ) ( n = 271)66.6 ( 10.3)65.9 ( 10.1)66.5 ( 10.6 ) ( n = 108)67.1 ( 10.7)(n = 137)nsmale / female gender163/108 ( n = 271)95/6876/5868/40 ( n = 108)87/50 ( n = 137)nscurrent smoker134 ( 45.9 % ) ( n = 292)72 ( 44.2 % ) ( n = 162)54 ( 40.3 % ) ( n = 129)60 ( 48.4 % ) ( n = 124)78 ( 48.8 % ) ( n = 152)nshistory of smoking271 ( 93.1 % ) ( n = 291)141 ( 93.3 % ) ( n = 150)123 ( 91.7 % ) ( n = 129)97 ( 69.4 % ) ( n = 107)115 ( 71.8 % ) ( n = 128)nsdiabetes74 ( 25.2 % ) ( n = 284)43 ( 26.3 % ) ( n = 161)33 ( 24.6 % ) 31 ( 23.6 % ) ( n = 123)41 ( 25.6 % ) ( n = 150)nscardiac disease74 ( 26 % ) ( n = 285)43 ( 26.3 % ) ( n = 162)35 ( 26.1 % ) 31 ( 25.2 % ) ( n = 123)39 ( 25.8 % ) ( n = 151)nslung disease30 ( 10.2 % ) ( n = 84)17 ( 10.4 % ) ( n = 162)13 ( 9.7 % ) ( n = 134)13 ( 9.9 % ) ( n = 122)17 ( 10.6 % ) ( n = 150)nsegfrns<6055 ( 19.3 % ) 30 ( 18.4 % ) 25 ( 18.6 % ) 25 ( 19.1 % ) 30 ( 18.6 % ) > 60162 ( 55.1 % ) 114 ( 69.9 % ) 98 ( 73.1 % ) 48 ( 36.6 % ) 64 ( 40 % ) unknown7719115866previous vascular intervention97 ( 34.2 % ) ( n = 284)51 ( 31.3 % ) ( n = 160)40 ( 29.9 % ) ( n = 133)46 ( 35.1 % ) ( n = 124)57 ( 35.6 % ) ( n = 151)abi at rest p < 0.0001<0.540 ( 13.6 % ) 30 ( 18.4 % ) 28 ( 20.9 % ) 10 ( 7.6 % ) 12 ( 7.5 % ) 0.50.75112 ( 38.1 % ) 83 ( 50.9 % ) 71 ( 53 % ) 29 ( 22.1 % ) 41 ( 25.6 % ) 0.750.963 ( 21.4 % ) 50 ( 30.7 % ) 35 ( 26.1 % ) 13 ( 9.9 % ) 28 ( 17.5 % ) 0.91.118 ( 6.1 % ) 0018 ( 13.7 % ) 18 ( 11.3 % ) 1.11.34 ( 1.4 % ) 004 ( 3.1 % ) 4 ( 2.5 % ) unknown57005757affected legsnsunilateral125 ( 42.5 % ) 78 ( 47.9 % ) 68 ( 50.7 % ) 47 ( 35.9 % ) 57 ( 35.6 % ) bilateral130 ( 44.2 % ) 84 ( 51.5 % ) 65 ( 48.5 % ) 46 ( 35.1 % ) 65 ( 40.6 % ) unknown39113838received treatmentconservative / optimal medical treatment11347406673set14193734868endovascular1710978surgical treatment2313121011questionnairesfollow - up time ( days , sd)123 ( 27)126 ( 26)baseline vascuqol score4.25 ( 1.2)4.26 ( n = 119 ) nsbaseline wiq score0.39 ( 0.1)0.42 ( n = 120 ) nsdata displayed as number ( percentage ) or mean ( standard deviation ) score was calculated in the number of patients that did sufficiently fill in the baseline questionnaire , but were excluded for not sufficiently filling follow - up questionnaire ns not significant baseline characteristics data displayed as number ( percentage ) or mean ( standard deviation ) score was calculated in the number of patients that did sufficiently fill in the baseline questionnaire , but were excluded for not sufficiently filling follow - up questionnaire table 2 shows that the mean improvement in vascuqol summary score was 0.83 . the correlation between the anchor - question and the vascuqol was 0.47 , thus meeting the criteria of revicki .table 2distribution of scores and mid vascuqolvascuqol ( n = 163)baselinefollow upmean change scorecorrelation with anchor - question4.25 ( 1.20)5.08 ( 1.28)0.830.47anchor - question n mean change on vascuqol95 % ciimproved971.23(1.011.46)unchanged430.55(0.230.87)deteriorated230.36(0.74 to 0.01)mid vascuqolimprovement0.87deterioration0.23 distribution of scores and mid vascuqol the mids calculated by the anchor - based approach were 0.23 and 0.87 , for deterioration and improvement , respectively ( table 2 ) . this means that patients with an increase of 0.87 compared to their baseline score have improved in a clinically relevant way . for deterioration while one might expect a negative mid value for deterioration , the mid value found here indicates that an increase in vascuqol summary score of less than 0.23 points is actually experienced as deterioration by patients . figure 1 shows the proportion of patients with a clinically relevant improvement or deterioration of their hrql on the vascuqol . this figure shows that 44 % of the patients achieved a clinically meaningful improvement at follow - up . 1proportion of patients that show a clinically relevant improvement and deterioration per prom proportion of patients that show a clinically relevant improvement and deterioration per prom distribution of scores and details on mid calculation for the wiq are presented in table 3 . the correlation between the anchor - question and the wiq was 0.41 , also meeting the criteria of revicki .table 3distribution of scores and mid wiqwiq ( n = 134)baselinefollow upmean change scorecorrelation with anchor - question0.39 ( 0.24)0.55 ( 0.28)0.160.41anchor - questionnmean change on wiq95 % ciimproved790.25(0.190.3)unchanged370.04(0.03 to 0.11)deteriorated180.01(0.05 to 0.06)mid wiqimprovement0.11deterioration0.03 distribution of scores and mid wiq the mid values found were 0.03 and 0.11 for deterioration and improvement , respectively . figure 1 shows the proportion of patients that reached a clinically relevant improvement or deterioration on the wiq . this figure shows that 57 % of the patients achieved a clinically meaningful improvement at follow - up . the correlation between the anchor - question and the vascuqol was 0.47 , thus meeting the criteria of revicki .table 2distribution of scores and mid vascuqolvascuqol ( n = 163)baselinefollow upmean change scorecorrelation with anchor - question4.25 ( 1.20)5.08 ( 1.28)0.830.47anchor - question n mean change on vascuqol95 % ciimproved971.23(1.011.46)unchanged430.55(0.230.87)deteriorated230.36(0.74 to 0.01)mid vascuqolimprovement0.87deterioration0.23 distribution of scores and mid vascuqol the mids calculated by the anchor - based approach were 0.23 and 0.87 , for deterioration and improvement , respectively ( table 2 ) . this means that patients with an increase of 0.87 compared to their baseline score have improved in a clinically relevant way . for deterioration while one might expect a negative mid value for deterioration , the mid value found here indicates that an increase in vascuqol summary score of less than 0.23 points is actually experienced as deterioration by patients . figure 1 shows the proportion of patients with a clinically relevant improvement or deterioration of their hrql on the vascuqol . this figure shows that 44 % of the patients achieved a clinically meaningful improvement at follow - up . 1proportion of patients that show a clinically relevant improvement and deterioration per prom proportion of patients that show a clinically relevant improvement and deterioration per prom distribution of scores and details on mid calculation for the wiq are presented in table 3 . the correlation between the anchor - question and the wiq was 0.41 , also meeting the criteria of revicki .table 3distribution of scores and mid wiqwiq ( n = 134)baselinefollow upmean change scorecorrelation with anchor - question0.39 ( 0.24)0.55 ( 0.28)0.160.41anchor - questionnmean change on wiq95 % ciimproved790.25(0.190.3)unchanged370.04(0.03 to 0.11)deteriorated180.01(0.05 to 0.06)mid wiqimprovement0.11deterioration0.03 distribution of scores and mid wiq the mid values found were 0.03 and 0.11 for deterioration and improvement , respectively . figure 1 shows the proportion of patients that reached a clinically relevant improvement or deterioration on the wiq . this figure shows that 57 % of the patients achieved a clinically meaningful improvement at follow - up . these can be assessed using proms , which are common endpoints in trials , have the potential to support clinical management of patients and can help assess provider performance . when interpreting changes in prom scores there are some important points to consider . while physicians have a distinct idea which amount of change in clinical measures such as blood pressure is relevant , interpretation of prom scores is less apparent . this is hampered even more by the fact that many proms have different rating scales ( e.g. , 01 , 17 , 1100 ) , making score changes incomparable . furthermore , it is important to realize that in larger sample sizes the standard deviations of scores become smaller , resulting in earlier significant findings than in a small sample sizes . they can be applied independent of sample size , and are thus useful in both individual care and research . in individual care , caregivers may decide to alter treatment strategy when after a certain period a patient does nt meet a relevant improvement . in research , a big advantage of applying mid values is that it helps display the proportion of patients in a sample that reaches a clinically relevant improvement . concurrently , it can display how many patients show a clinically relevant deterioration despite treatment , as shown in fig . 1 . this would have been missed when only comparing the mean baseline score of the sample with the mean score after treatment , since this would have probably resulted in a positive mean change score , falsely indicating improvement for all patients in the sample . while it was beyond the scope of this paper , in future studies that compare treatment modalities it may be insightful to compare the proportion of patients that reach a clinically relevant improvement and deterioration per treatment group . it may be attributed to a learning effect , i.e. , patients who do not improve ( unchanged group ) may still learn to fill in a prom more accurately by repetition , resulting in a higher follow - up score , and thus a positive mid for deterioration . furthermore , the vascuqol is a disease - specific prom , in contrast to the anchor - question . therefore , the mean prom score may increase , while the anchor - question is rated as unchanged . generally , calculation methods are divided into anchor - based approaches and distribution - based approaches . anchor - based approaches determine the mid by comparing proms to other measures or phenomena that have clinical relevance . this can for example be an anchor - question , as we have shown in this study . distribution - based approaches are based on statistical characteristics of the prom scores in a patient sample . while studies have shown that values found in anchor - based and distribution - based approaches are often comparable , in calculations based on distribution - based approaches it is still not taken into account which amount of change is considered relevant by patients . first , the proportion of patients that did not sufficiently complete the proms twice was substantial . this is a well - known problem and not exclusive to our study , but it should be considered when applying proms , since it limits their overall use . second , to ensure that the study population was representative for all ic patients , we intentionally excluded patients of unknown age , gender , and/or abi , which may have induced bias . yet , the included and excluded patients did not differ in terms of baseline characteristics and prom scores , and despite excluding many patients an acceptable sample was left for the mid analysis . finally , we do not know how many patients refused to participate in the pilot study , and how this may have influenced mid values . we have calculated the mid values for two frequently used proms for patients with ic . as demonstrated in this study , the mid is a helpful tool to interpret the clinical relevance of changes in prom scores , which may be used in research and individual care . below is the link to the electronic supplementary material . supplementary material 1 ( doc 46 kb ) supplementary material 1 ( doc 46 kb ) wilma jonkers , ellen rouwet , anco vahl , jim reekers , mark koelemay have no conflict of interest .

objectiveto measure ghrelin and energy intake in the laboratory after pioglitazone treatment.research design and methodsthis was a parallel , three - arm study with 51 obese diabetic subjects randomized to either 1 ) pioglitazone plus a portion - controlled diet ( pio+pc ) , 2 ) pioglitazone plus american diabetes association ( ada ) dietary advice ( pio+ada ) , or 3 ) metformin plus ada advice ( met+ada ) . energy intake and the suppressive response of a meal on ghrelin were measured at weeks 0 and 16 . mixed models tested if changes from week 0 to 16 differed by group.resultsthe pio+ada group had a significantly larger increase ( p < 0.05 ) in energy intake ( [ adjusted means se ] 207 53 kcal ) compared with the pio+pc ( 50 46 kcal ) and met+ada ( 52 49 kcal ) groups . change in restraint and disinhibition ( variables associated with eating behavior ) mediated weight change . ghrelin suppression increased in the pio+ada group , which gained weight.conclusionsa portion - controlled diet attenuated the increase in energy intake after pioglitazone . ghrelin responded to weight change not pioglitazone exposure .

participants had not been previously treated with tzds and were not using drugs that affect metabolism or body weight ( e.g. , sibutramine ) . participants were randomly assigned to one of three treatment groups for the 16-week study : 1 ) pioglitazone plus standard dietary advice from the american diabetes association ( ada ) ( pio+ada ) ; 2 ) pioglitazone plus a portion - controlled diet ( pio+pc ) ; and 3 ) an active control group , metformin plus ada advice ( met+ada ) . briefly , all participants were prescribed a diet that was 500 kcal / day less than their energy requirements . the pio+pc group drank one glucerna ( 290 kcal ) for breakfast and one for lunch , with a planned evening meal . change from baseline to week 16 ( week 16 minus week 0 ) on the following variables was quantified . four hours after a 371-kcal breakfast , energy intake was measured objectively at lunch in the laboratory using methods that produce repeatable / reliable energy intake measurements ( 6 ) . serum ghrelin levels were measured before and 2 h after the start of lunch to quantify ghrelin response to a meal ( postmeal minus premeal ) . ratings of hunger , desire to eat , fullness , and prospective food consumption were measured with visual analog scales ( vass ) ( 7 ) before and after lunch . the eating inventory quantified dietary restraint ( the intent to restrict energy intake ) and disinhibition ( the tendency to overeat ) ( 8) . the food - craving inventory ( fci ) measured general cravings ( total score ) and cravings for the following specific types of foods : sweets , high fats , carbohydrates / starches , fruits / vegetables , and fast - food fats ( 9 ) . analyses were conducted with = 0.05 using sas version 9.0 ( cary , nc ) . mixed models tested if change on the outcome variables differed by group ( baseline values were covariates ) . regression methods ( 10 ) were used to test for mediators of differential body weight change between the pio+pc and pio+ada groups . the following possible mediators were tested : ghrelin , energy intake , dietary restraint , and disinhibition . pearson correlation coefficients were calculated to determine the amount of variance in body weight ( and energy intake ) change that was accounted for by change in restraint and disinhibition . participants were randomly assigned to one of three treatment groups for the 16-week study : 1 ) pioglitazone plus standard dietary advice from the american diabetes association ( ada ) ( pio+ada ) ; 2 ) pioglitazone plus a portion - controlled diet ( pio+pc ) ; and 3 ) an active control group , metformin plus ada advice ( met+ada ) . briefly , all participants were prescribed a diet that was 500 kcal / day less than their energy requirements . the pio+pc group drank one glucerna ( 290 kcal ) for breakfast and one for lunch , with a planned evening meal . change from baseline to week 16 ( week 16 minus week 0 ) on the following variables was quantified . four hours after a 371-kcal breakfast , energy intake was measured objectively at lunch in the laboratory using methods that produce repeatable / reliable energy intake measurements ( 6 ) . serum ghrelin levels were measured before and 2 h after the start of lunch to quantify ghrelin response to a meal ( postmeal minus premeal ) . ratings of hunger , desire to eat , fullness , and prospective food consumption were measured with visual analog scales ( vass ) ( 7 ) before and after lunch . the eating inventory quantified dietary restraint ( the intent to restrict energy intake ) and disinhibition ( the tendency to overeat ) ( 8) . the food - craving inventory ( fci ) measured general cravings ( total score ) and cravings for the following specific types of foods : sweets , high fats , carbohydrates / starches , fruits / vegetables , and fast - food fats ( 9 ) . analyses were conducted with = 0.05 using sas version 9.0 ( cary , nc ) . mixed models tested if change on the outcome variables differed by group ( baseline values were covariates ) . regression methods ( 10 ) were used to test for mediators of differential body weight change between the pio+pc and pio+ada groups . the following possible mediators were tested : ghrelin , energy intake , dietary restraint , and disinhibition . pearson correlation coefficients were calculated to determine the amount of variance in body weight ( and energy intake ) change that was accounted for by change in restraint and disinhibition . forty - eight of 51 participants completed the trial ( 2 subjects dropped out from the pio+ada and 1 from the met+ada group ) . as previously reported ( 5 ) , pio+ada gained ( means sd ) 2.15 1.09 kg , met+ada lost 3.21 0.7 kg , and pio+pc lost 2.59 1.25 kg . the pio+ada group had a smaller decrease in visceral fat compared with pio+pc group but a larger increase in deep subcutaneous fat compared with the met+ada group ( 5 ) . energy intake increased significantly in the pio+ada ( p < 0.001 ) but not the met+ada ( p = 0.30 ) or pio+pc ( p = 0.28 ) groups . increased energy intake in the pio+ada group was significantly larger than the met+ada and pio+pc groups ( p < 0.05 ) . the difference in least squares ( ls ) means se between the pio+ada and met+ada and pio+ada and pio+pc groups was 155 73 and 157 70 kcal , respectively . baseline ( week 0 ) participant characteristics and change on outcome variables from week 0 to 16 data are means se . ls means se , which are adjusted for baseline values , depict change on the outcome variables from week 0 to 16 and are included below the week 0 values . * lettered superscripts that differ indicate that those groups ' change scores differed significantly from each other ( p < 0.05 ) . the suppression of ghrelin after a meal at week 0 and week 16 is included in the table . the pio+ada group had a significantly larger meal - induced suppression of ghrelin at week 16 compared with week 0 , which was significantly larger than the nonsignificant changes in the met+ada and pio+pc groups . change in appetite ratings did not differ significantly among the groups ( data not shown ) ( p > 0.25 ) . the pio+pc group had a significant increase in dietary restraint and a decrease in disinhibition and hunger . change in these end points differed significantly between the pio+pc and pio+ada groups ( p < 0.01 ) . the difference in ls means se between the pio+pc and pio+ada groups on restraint and disinhibition was 4.8 1.4 and 2.5 0.9 , respectively . the met+ada group experienced a significant decrease in general cravings and cravings for high - fat foods . change in restraint and disinhibition mediated differential weight change between the pio+pc and pio+ada groups . change in restraint and disinhibition were negatively ( r = 0.53 , p < 0.001 ) and positively ( r = 0.39 , p < 0.01 ) associated with change in body weight , accounting for 28.4 and 15.2% of body weight change variance , respectively . change in restraint and disinhibition were negatively ( r = 0.44 , p < 0.01 ) and positively ( r = 0.31 , p < 0.05 ) associated with change in energy intake , accounting for 19 and 9.3% of energy intake change variance , respectively . this is the first study to demonstrate that pairing pioglitazone treatment with a portion - controlled diet ( pio+pc ) attenuates pioglitazone - associated increases in energy intake . suppression of ghrelin in response to a meal increased only in the group who gained weight ( pio+ada ) , indicating that ghrelin suppression is dependent on body weight change and not pioglitazone treatment . the results indicate that pioglitazone - associated weight gain is secondary to increased energy intake . larger increases in restraint and decreases in disinhibition were observed in the pio+pc group , with restraint accounting for 28.4% of the variance in body weight change . strengths of the study include the objective measurement of energy intake in a controlled study design . limitations include measuring energy intake during only one meal before and after short - term treatment . further research is warranted to examine the long - term effect of pioglitazone treatment on energy and macronutrient intake .

the buccal fat pad is relatively large and prominent in neonates , infants and young children . the main function of this fat pad is considered as a cushioning tissue and sucking pad . a minor tear of buccal mucosa and buccinator muscle can result in herniation of large volume of fat into oral cavity that is termed as pseudolipoma . the young children tend to be very playful while brushing their teeth . improper brushing technique resulted in severe trauma to the buccal fat , including soft - tissue between buccinator and retromolar area . this article presents a case - report of a female child who developed traumatic pseudolipoma after faulty tooth brushing for long duration and its management along with its detail review of literature .

the buccal fat pad also called as buccal fat pad of bichat or suctorial pad is relatively large and prominent in neonates , infants and young children . it has great relevance to this area giving fullness to face , aids in cushioning and sucking in neonates and children . it serves to line the masticatory space , separating the muscles of mastication from the zygomatic arch , ramus of the mandible and other muscles . the buccal fat pad consists of a central body and four extensions : buccal , pterygoid , superficial and deep temporal . the buccal extension is encapsulated by a parotidomasseteric fascia and enters the cheek below the parotid duct . the term traumatic pseudolipoma was proposed by brooke and macgregor in their case report involving adipose tissue in a swelling of the buccal mucosa distinguishing it from other benign fatty tumors of the oral cavity . two etiologic factors and pathogenetic links between soft - tissue trauma and adipose tissues growth had been discussed in the literature . the first possibility is the herniation of the buccal fat pad with subsequent epithelialization , termed this usually results from direct impact , has a relatively short natural history of presentation and commonly occurs in young children . the second possibility suggests lipoma formation is due to deposition and differentiation of adipocytes , mediated by cytokine release , secondary to trauma and hematoma formation . the average time between soft - tissue trauma and lipoma formation is almost 3 years and occurs more commonly in the fourth to sixth decade . all the reported cases of traumatic pseudolipoma except one 12-year - old boy , were in infants or young children , with an age range from 5 months to 5 years . in every case , two contributory factors were proposed : the buccal fat pad is relatively prominent in infants and young children , who frequently investigate foreign objects by placing them in their mouths . further sucking action results in fat pad being pulled out . a 3 year old female patient was referred from a pediatrician to the department of pedodontic and preventive dentistry with a chief complaint of extra- and intra - oral swelling and pain on the right side of face . child 's father gave history of appearance of extra - oral swelling 5 days back with pain , discomfort , drooling of saliva from mouth and inability to eat . the intra - oral swelling was first noticed approximately 1 month ago as a small reddish pink growth which increased gradually [ figure 1 ] the patient reported to the department after 3 days with complain of intense pain and increased extra - oral swelling and compressed intra - oral swelling [ figure 2 ] . gross clinical intra - oral examination of the lesion revealed the presence of a reddish pink , ovoid , smooth , pedunculated , non - ulcerated , freely mobile , tender swelling of approximately 3.0 2.0 1.5 cm size , located between the maxillary and mandibular primary second molar regions . the lesion was found to be soft , fluctuant in consistency and freely mobile on palpation . the child was having the habit of placing toothbrush for more than 1h daily while brushing teeth . she used to bite on it while keeping it in the right buccal vestibule from last 6 months . intra - oral swelling noticed 1 month ago compressed intra - oral swelling seen after 3 days differential diagnosis of the mass was given as a pyogenic granuloma , traumatic fibroma , haemangioma , infected lipoma , inflammatory pseudotumour , infected benign minor salivary gland tumor , foreign body granuloma and traumatic neuroma . the child was prescribed antibiotics , analgesic and antiseptic mouthwash for 3 days to reduce infection , inflammation , pain as well as post - operative complications while healing . the intra - oral growth was seen pale yellow in color and shrunken after 3 days . as the patient was highly uncooperative ( frankl rating i ) , after complete hematological investigations , surgical excision of the mass was done under local anesthesia with soft - tissue diode laser ( doctor smile wiser laser ) at 2w continuous pulse . tissue mass was excised in - toto from the base of buccal mucosa and sent for histopathological examination to the department of oral pathology [ figure 3 ] . an attempt was made to express saliva from the parotid duct to identify the location of the parotid papilla and confirm that it is not severed or damaged [ figure 4 ] . patient was recalled after 1 week and 3 months for follow - up of wound healing . manoeuvre to express saliva from stensen 's duct was repeated to make sure that it was not damaged . excised tissue mass from buccal mucosa buccal mucosa after excision of lesion and identification of parotid papilla macroscopic examination revealed one soft - tissue of a reddish pink , ovoid , firm in consistency , smooth surfaced , pedunculated , non - ulcerated mass of approximately 3.0 2.0 1.5 cm size [ figure 3 ] . histological examination of the tissue revealed the presence of adipose tissues with non - ulcerated overlying epithelium . presence of marked edema and diffuse proliferation of capillaries along with formation of granulation tissue , composed of moderately dense polymorphonuclear neutrophillic and lymphoplasmocytic infiltrate [ figure 5 ] . the connective tissue stroma consists of dense collagen bundles and lobules of mature adipocytes with no cellular atypia . compressed blood vessels engorged with red blood cells were also evident in the connective tissue stroma . the overall histo - pathological features were confirmatory of traumatic pseudolipoma ( traumatic herniation of buccal fat pad ) [ figures 5 ] . it is closely associated with the muscles of mastication , the parotid duct and the facial nerve . its importance in masticatory function is best illustrated in infants where it acts as sucking pad . it contributes to the bulging of the infant 's cheeks and usually persists in adults , the body and the buccal extensions of which are largely responsible for cheek contour in adults . literature suggests that traumatic intra - oral herniation of the buccal fat pad is a rare phenomenon . most of the reported cases were observed in infants and young children ranging from age of 5 months to 5 years . relatively increased incidence of traumatic herniation of buccal fat pad in infants and young children is attributed to habit of investigating or frequently placing foreign objects like pencil , toothbrush , chopstick in their mouths . the most characteristic aspect of such a lesion is that the mucosal injury or perforation is very small compared to the size of the extruded mass . as to the anatomic location of this lesion , matarasso suggested that a defect or weakness in the parotidomasseteric fascia of the region contribute to the occurrence . sucking action of an infant might encourage the herniation of fat pad through the wound into the mouth , and may also pose the risk of respiratory embarrassment . majority of cases documented involved a foreign object in the mouth , which subsequently caused the penetrating injury through buccal mucosa and buccinator muscle . in this case , traumatic herniation of buccal fat pad can be thought to be caused by primarily because of improper brushing technique . gradual increase in the size of the mass can be because of prolonged placement and sucking of brush for more than 1 h daily . cases in which there is early evaluation and the protruded mass is small , the tissues are able to be repositioned immediately back to its place , followed by suturing of the mucosal laceration . the approximate period between the injury and the first visit should be less than 4 h as tissues start necrotizing after this duration . if the mass is too large to be replaced in the limited laceration or infected because of prolonged exposure to oral cavity , it is recommended to excise the mass at the base with no recurrence . in this case , it was decided to excise the mass from its base under local anesthesia using diode laser in a continuous wave in a contact mode . diode laser cuts soft - tissues and reduces bacterial counts in at the wound surface . there is evidence that this wavelength may cause a reduction in tissue inflammation and a reduced need for local anesthetic during surgical procedures . thermal necrosis zones of less than 1 mm can be achieved , which provides adequate surgical precision and hemostasis for many soft - tissue procedures . the laser when used in a non - contact mode ; coagulates soft tissues or provides hemostasis over the surgical area . it can be concluded from above discussion that the penetrating injuries of soft tissues of oral cavity or lesions occurring from it should be considered potentially serious . proper evaluation of such lesions or injuries is important before coming to final diagnosis and treatment modality . while excising such a traumatic pseudolipoma and closing the wound care

introductionin most patients with type 2 diabetes mellitus ( t2 dm ) and progressive beta - cell insufficiency , insulin therapy is required to achieve sufficient glycemic control . however , insulin therapy may lead to weight gain and increasing risk of hypoglycemia . glucagon - like peptide-1 receptor agonists are being used as add - on therapy to insulin with favorable metabolic effects . nonetheless , to date only few studies exist reporting on the combination of liraglutide and insulin with a short follow - up period . the aim of this study was to evaluate the efficacy and safety of liraglutide as add - on to insulin in patients with t2 dm over a time period of up to 2428 months.methodsdata of patients with t2 dm , treated with insulin and liraglutide at an outpatient clinic in a tertiary referral hospital from october 2009 until december 2011 were retrospectively examined ( n = 36 ) . glycated hemoglobin ( hba1c ) , weight , total daily insulin dose and side effects were assessed 58 months prior to liraglutide , at baseline and at follow - up visits after 3 , 6 , 1216 and 2428 months.resultsmedian hba1c decreased significantly from 7.7% [ interquartile range ( iqr ) 7.08.6 ] at baseline to 6.8% ( iqr 6.57.7 , p = 0.001 ) at 3 months and 6.9% ( iqr 6.37.6 , p = 0.0001 ) at 6 months , but re - increased thereafter ( at 2428 months , median 7.5% , iqr 7.18.2 , p = 1.0 ) . median weight decreased significantly from 99.8 kg ( iqr 81110 ) at baseline to 97.7 kg ( iqr 81.2108.2 , p = 0.027 ) at 3 months , but rose again thereafter . insulin dosage did not change significantly over time . no severe hypoglycemia or major side effects occurred.conclusionsin this observational study , adding liraglutide to insulin in daily clinical practice reduced hba1c significantly within 6 months , but there may be a non - sustainable effect during long - term treatment.electronic supplementary materialthe online version of this article ( doi:10.1007/s13300 - 014 - 0093 - 8 ) contains supplementary material , which is available to authorized users .

the online version of this article ( doi:10.1007/s13300 - 014 - 0093 - 8 ) contains supplementary material , which is available to authorized users . lifestyle modification and oral antidiabetic drugs are first line treatment measures in patients with type 2 diabetes mellitus ( t2 dm ) . over time , progressive beta cell dysfunction results in deteriorating glycemic control requiring insulin therapy . however , insulin is often associated with weight gain and increased risk of hypoglycemia . together with the need for ( multiple ) injections , insulin therapy frequently meets reluctance in both patients and physicians , and initiation of therapy is often delayed . several trials showed that glucagon - like peptide-1 ( glp-1 ) receptor agonists as monotherapy or in combination with one or more oral antidiabetic agent have beneficial metabolic effects leading to improved glycemic control with a low risk of hypoglycemia , and weight loss . they are now recommended as one of several two - drug combination therapies , if metformin monotherapy does not achieve / maintain the defined glycated hemoglobin ( hba1c ) target over 3 months . several reports demonstrated favorable effects of a combination therapy of a glp-1 receptor agonist with insulin . however , only few studies included the use of liraglutide [ 610 ] , and follow - up time was short . despite these limited data , liraglutide is widely used ( off - label ) as add - on therapy in patients on various insulin regimens and oral antidiabetic drugs . in 2013 , liraglutide was officially approved for use in combination with basal insulin in europe ( and the us ) . the aim of this observational study was to retrospectively assess the efficacy and safety of liraglutide as add - on therapy to insulin in a daily clinical practice setting for a duration up to 2428 months . eligible participants were adults ( 1880 years ) with t2 dm , bmi 28 kg / m and hba1c values of 6.510% treated with metformin another oral antidiabetic drug and insulin . exclusion criteria were pregnancy and intolerance for liraglutide . the authors reviewed the charts of all patients with t2 dm with liraglutide being added - on to insulin from october 2009 until december 2011 at their outpatient clinic in a tertiary referral hospital . weight , body mass index ( bmi ) , hba1c and insulin dose ( units / day , units / kg / day ) were assessed 58 months prior to liraglutide , at baseline , after 3 , 6 , 1216 and 2428 months . starting dose of liraglutide was 0.6 mg once daily , with an increase to 1.2 mg after 2 weeks . during the treatment period , oral drugs and insulin dosage were individually reduced by the attending physician according to current glucose control . as the outpatient clinic is part of a teaching hospital , all physicians followed the same treatment strategies according to the guidelines of the swiss society of endocrinology and diabetes . hypoglycemic events were assessed by patient interview and patients blood glucose meter readings were imported into the diabass pro software , mediaspects gmbh , konstanz , germany . severe hypoglycemia was defined as an event requiring assistance of another person to actively administer carbohydrate , glucagon or other resuscitative actions . all procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation ( institutional and national ) and with the helsinki declaration of 1975 , as revised in 2000 and 2008 . informed consent was obtained from all patients for being included in the study . for statistical analysis linear mixed models with random intercept were used to examine changes in endpoints over time from baseline , after 3 , 6 , 1216 and 2428 months . such regression models do not require complete data ( unlike in analysis of variance ) , so all available data was analyzed . all analysis was performed in the r programming language ( version 3.0.1 , r core team 2013 center for statistics , copenhagen , denmark ) . the package lme4 was used to estimate the mixed models , while the multcomp was used to compute the p values and confidence intervals . a total of 36 patients was on this combined therapy regimen . after the baseline visit , four patients preferred to be followed up by their family physician and were therefore not included in this follow - up . three patients stopped liraglutide due to exanthema at the injection site , nausea and abdominal pain , respectively . among the remaining 29 patients , median age was 59 years [ interquartile range ( iqr ) 5367 ] , disease duration 11 years ( iqr 513 ) , hba1c 7.7% ( iqr 7.08.6 ) , weight 99.8 kg ( iqr 81110 ) and bmi 33.5 kg / m ( iqr 29.437.6 ) . at baseline , 17 were on basal insulin , 2 on prandial insulin and 10 on multiple insulin injections . in addition , 24 patients were on metformin , 12 on sulfonylureas , and 6 on other oral antidiabetic agents . median hba1c decreased significantly from 7.7% ( iqr 7.08.6 ) at baseline to 6.8% ( iqr 6.57.7 , p = 0.001 ) at 3 months and 6.9% , ( iqr 6.37.6 , p = 0.0001 ) at 6 months , but re - increased at 1216 months ( median 7.2% , iqr 6.77.9 , p = 0.32 ) and 2428 months ( median 7.5% , iqr 7.18.2 , prior to intervention was lower ( median 7.3% , iqr 7.17.9 ) than at baseline . weight and bmi decreased significantly from 99.8 kg and 33.5 kg / m ( iqr 81110 and 29.437.6 ) respectively at baseline to 97.7 kg and 31.9 kg / m ( iqr 81.2108.2 and 29.436 ) , ( p = 0.023 and 0.027 ) at 3 months . however weight and bmi increased again after 6 months ( median 97.5 kg and 31.5 kg / m , iqr 84.5.5111.5 , p = 0.16 and 29.435.1 , p = 0.15 ) , at 1216 months ( 100.5 kg and 34.1 kg / m , iqr 100.5109.5 , p = 0.17 and 30.736.7 , p = 0.16 ) and at 2428 months ( 106.4 kg and 36.4 kg / m , iqr 96.9118 , p = 1.0 and 30.938.4 , however , during follow - up , three patients ( 10.3% ) were able to completely stop insulin therapy , and two patients ( 6.9% ) could simplify their multiple daily insulin injection regimens to basal insulin at bedtime only . furthermore , sulfonylureas were stopped in 5 of 12 patients ( 41.7% ) and all other antidiabetic drugs beside metformin were stopped ( e.g. glitazones , glinides ) . overall , 5 patients ( 15.6% ) out of 32 followed - up patients discontinued liraglutide due to side effects . three patients stopped liraglutide due to exanthema at the injection site , nausea or abdominal pain at the beginning and two patients discontinued liraglutide due to nausea / vomiting and pain at the injection site after 3.5 and 12 months.fig . 1course of glycated hemoglobin ( hba1c ) , insulin ( total units and units per kg body weight ) , weight and body mass index ( bmi ) prior ( t-1 ) , at baseline ( t0 ) and during follow - up after 3 ( t1 ) , 6 ( t2 ) , 1216 ( t3 ) and 2428 months ( t4 ) course of glycated hemoglobin ( hba1c ) , insulin ( total units and units per kg body weight ) , weight and body mass index ( bmi ) prior ( t-1 ) , at baseline ( t0 ) and during follow - up after 3 ( t1 ) , 6 ( t2 ) , 1216 ( t3 ) and 2428 months ( t4 ) this retrospective study shows that liraglutide as add - on therapy to insulin therapy decreases hba1c and weight during the first 6 months with a fading effect thereafter . the most frequently reported side effects were nausea / vomiting and pain / exanthema at the injection site , leading to discontinuation of liraglutide in 15.6% of the patients . the short - term findings on hba1c and weight reduction are in accordance with earlier randomized and observational studies over 12 weeks on liraglutide added to insulin , which found a reduction of hba1c by 1.41.9% and body weight by 5.15.6 kg , respectively [ 7 , 8 ] . a recent report showed a decrease in hba1c levels of 0.77% and 4.5 kg weight reduction over 26 weeks . a most recent meta - analysis of 15 studies on glp-1 agonists in combination with basal insulin with a maximum observation time of 36 weeks ( mean 24.8 weeks ) yielded an hba1c reduction of 0.44% and weight loss of 3.22 kg . the longest follow - up was in a retrospective study until 38 weeks , where body weight also re - increased in 20 patients on liraglutide or exenatide in combination with insulin , similar to the patient population in the current study . results with longer follow - up time are only available in the reverse situation of basal insulin added on to metformin and liraglutide in a prospective study , showing sustained hba1c and weight reduction even after 52 weeks . several reasons may account for the unsustained effect of liraglutide on body weight and hba1c beyond 6 months in the patients presented here . firstly , reduction of insulin dose or termination of oral antidiabetic drugs may have led to the re - increase in hba1c . secondly , no antidiabetic treatment has been shown to prevent deterioration of -cell function , which drives the natural history of the disease . thirdly , the authors often observe that patient adherence , especially regarding dietary restrictions , fades after a few months of starting liraglutide , which may be explained by the disappearing gastrointestinal side effects . subjects willing to participate in a prospective study are supposedly more motivated and adherent to improve their glycemic control in general . finally , subjects early achieving adequate glycemic control within a few weeks or months usually return to their referring physicians , whereas the more challenging patients remained in the tertiary clinic and were eligible for this study . the clinical setting reflects a real world situation and shows the difficulties which diabetologists face in their daily work . in contrast to other studies with glp-1 receptor agonists and insulin , there was no statistically significant reduction in daily insulin dose in the present study . however , a remarkable proportion of the patients either completely ceased insulin or at least were able to omit prandial insulin injections . moreover , in many of the patients , oral antidiabetic drugs ( beside metformin ) could be stopped , which is especially beneficial for patients with sulfonylureas bearing an increased risk of hypoglycemia . in accordance with earlier reports the dropout rate of 15.6% due to side effects was comparable to other reports [ 18 , 19 ] . limitations of this study are the small patient number and potentially heterogeneous patient characteristics , including patients with long - standing diabetes as well as the retrospective study design . as this was an observational study , there was no control group . the fact that hba1c was increasing before baseline may point to a regression towards the mean . the setting reflects the real life situation in an outpatient clinic of a tertiary referral hospital covering around 500,000 inhabitants . during the observational period , combination therapy of liraglutide and insulin was not yet approved by swissmedic ( swiss agency for the authorisation and supervision of therapeutic products ) , which explains the small number of patients . however , a major strength of this study is the long observational period of up to 2428 months , as compared to most earlier studies with a follow - up time of usually 24 weeks with a few lasting up to 38 weeks . in the meantime , add - on therapy of liraglutide to basal insulin was approved due to its unequivocal positive effect . the combination of liraglutide with multiple insulin injections , however , is still awaiting approval . adding liraglutide to pre - existing insulin therapy in t2 dm reduces hba1c and body weight significantly during the initial 6 months of treatment , but there may be a non - sustainable effect during long - term treatment . lisa sze , ina krull , michael brndle declare they have no conflicts of interest . all procedures followed were in accordance with the ethical standards of the committee on human experimentation ( institutional and national ) and with the helsinki declaration of 1975 , as revised in 2000 and 2008 . the study was approved by the local ethics committee and informed consent was obtained from all patients for being included in the study . this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and the source are credited .

objective : to directly compare the diagnostic utility of growth differentiation factor15 ( gdf-15 ) with our previous fibroblast growth factor21 ( fgf-21 ) findings in the same adult mitochondrial disease cohort.methods:serum gdf-15 levels were measured using a quantitative elisa . statistical analyses of gdf-15 data were compared with our published fgf-21 findings.results:median serum gdf-15 concentrations were elevated in patients with mitochondrial disease and differed between all experimental groups , mirroring group results for fgf-21 . there was a difference between patients diagnosed by muscle biopsy and genetic diagnosis , suggesting that serum gdf-15 measurement may be more broadly specific for mitochondrial disease than for muscle manifesting mitochondrial disease , in contrast to fgf-21 . gdf-15 showed a markedly higher diagnostic odds ratio when compared with fgf-21 ( 75.3 vs 45.7 ) , was a better predictor of disease based on diagnostic sensitivity ( 77.8% vs 68.5% ) , and outperformed fgf-21 on receiver operating characteristic curve analysis ( area under the curve 94.1% vs 91.1% ) . combining both biomarkers did not improve the area under the curve remarkably over gdf-15 alone . gdf-15 was the best predictor of mitochondrial disease ( p < 0.002 ) following multivariate logistic regression analysis.conclusions:gdf-15 outperforms fgf-21 as an indicator of mitochondrial diseases . our data suggest that gdf-15 is generally indicative of inherited mitochondrial disease regardless of clinical phenotype , whereas fgf-21 seems to be more indicative of mitochondrial disease when muscle manifestations are present.classification of evidence : this study provides class iii evidence that serum gdf-15 accurately distinguishes patients with mitochondrial diseases from those without them .

this case - control study provides class iii evidence that gdf-15 is a better diagnostic indicator of mitochondrial diseases when compared with fgf-21 ( diagnostic sensitivity , 77.8% vs 68.5% ; diagnostic odds ratio [ or ] , 73.5 vs 45.7 ; area under the receiver operating characteristic curve , 0.941 vs 0.911 ) . briefly , the adult cohort consisted of 66 controls , 20 nonmitochondrial neuromuscular disease controls , and 54 consecutive patients with mitochondrial disease . mitochondrial disease patients were recruited between november 2011 and october 2012 from the adult mitochondrial disease clinic at royal north shore hospital , sydney , australia , if they met the walker criteria for mitochondrial disease and had a confirmed genetic diagnosis or changes on muscle biopsy indicative of mitochondrial myopathy . participants ranged in age from 20 to 84 years and 57.9% of participants were female . of the 54 patients with mitochondrial disease , 27 had a genetic diagnosis and 27 had muscle biopsy changes indicative of a mitochondrial myopathy ( 9 patients with a genetic diagnosis also had a muscle biopsy showing changes consistent with mitochondrial myopathy ) . we refer to the standards for reporting of diagnostic accuracy diagram published previously for this cohort for inclusion and exclusion details . when possible , the newcastle mitochondrial disease scale for adults in addition , patients were routinely assessed for proximal muscle weakness according to medical research council ( mrc ) criteria . written informed consent was obtained from all participants at the time of sample collection and ethical approval was granted by the northern sydney local health district human research ethics committee . serum creatine kinase , lactate , and pyruvate levels ( along with other biochemical parameters ) measured previously were used here for analysis with serum gdf-15 levels . gdf-15 levels were measured in archived serum samples by quantitative sandwich elisa ( biovendor , laboratorni medicina a.s . , brno , czech republic ) , according to the manufacturer 's instructions . absorbance readings at 570 nm were subtracted from absorbance readings at 450 nm before normalizing to the mean absorbance of blank wells . triplicate measurements for each sample were averaged and the concentration determined from a 4-parameter logistic standard curve ( www.myassays.com ) . elisa measurements were performed in triplicate for each assay and repeated in at least duplicate assays . the scientist who performed the assays ( r.l.d . ) statistical analyses were performed , as previously described , using spss version 20 ( ibm corporation , armonk , ny ) and clinical research calculators on the vassar university statistics web interface ( http://vassarstats.net/ ) . differences between groups were considered statistically significant when 2-tailed p values were less than 0.05 . all p values below 0.0001 the reference cutoff concentration for gdf-15 was set at the 95th percentile of control group serum concentrations , as for fgf-21 . we used mann - whitney u and kruskal - wallis nonparametric testing to determine statistical differences between groups . we calculated the diagnostic sensitivity and the corresponding 95% confidence intervals . an unadjusted diagnostic or was determined for comparison with fgf-21 . receiver operating characteristic ( roc ) curves were plotted using sensitivity vs 100 specificity on a continuous scale and the area under the curve ( auc ) was used to determine the relative diagnostic capacity . biomarker levels were correlated with other biochemical measurements and clinical assessment tools using nonparametric spearman correlation testing . linear regression analysis was used to determine if statistically correlated parameters could predict gdf-15 concentration . multivariate linear regression analysis was performed to determine factors capable of predicting disease while controlling for confounders that may influence serum gdf-15 concentration . this case - control study provides class iii evidence that gdf-15 is a better diagnostic indicator of mitochondrial diseases when compared with fgf-21 ( diagnostic sensitivity , 77.8% vs 68.5% ; diagnostic odds ratio [ or ] , 73.5 vs 45.7 ; area under the receiver operating characteristic curve , 0.941 vs 0.911 ) . briefly , the adult cohort consisted of 66 controls , 20 nonmitochondrial neuromuscular disease controls , and 54 consecutive patients with mitochondrial disease . mitochondrial disease patients were recruited between november 2011 and october 2012 from the adult mitochondrial disease clinic at royal north shore hospital , sydney , australia , if they met the walker criteria for mitochondrial disease and had a confirmed genetic diagnosis or changes on muscle biopsy indicative of mitochondrial myopathy . participants ranged in age from 20 to 84 years and 57.9% of participants were female . of the 54 patients with mitochondrial disease , 27 had a genetic diagnosis and 27 had muscle biopsy changes indicative of a mitochondrial myopathy ( 9 patients with a genetic diagnosis also had a muscle biopsy showing changes consistent with mitochondrial myopathy ) . we refer to the standards for reporting of diagnostic accuracy diagram published previously for this cohort for inclusion and exclusion details . when possible , the newcastle mitochondrial disease scale for adults ( nmdas ) clinical rating scale was performed on patients with mitochondrial disease . in addition , patients were routinely assessed for proximal muscle weakness according to medical research council ( mrc ) criteria . written informed consent was obtained from all participants at the time of sample collection and ethical approval was granted by the northern sydney local health district human research ethics committee . serum creatine kinase , lactate , and pyruvate levels ( along with other biochemical parameters ) measured previously were used here for analysis with serum gdf-15 levels . gdf-15 levels were measured in archived serum samples by quantitative sandwich elisa ( biovendor , laboratorni medicina a.s . , brno , czech republic ) , according to the manufacturer 's instructions . absorbance readings at 570 nm were subtracted from absorbance readings at 450 nm before normalizing to the mean absorbance of blank wells . triplicate measurements for each sample were averaged and the concentration determined from a 4-parameter logistic standard curve ( www.myassays.com ) . elisa measurements were performed in triplicate for each assay and repeated in at least duplicate assays . the scientist who performed the assays ( r.l.d . ) statistical analyses were performed , as previously described , using spss version 20 ( ibm corporation , armonk , ny ) and clinical research calculators on the vassar university statistics web interface ( http://vassarstats.net/ ) . differences between groups were considered statistically significant when 2-tailed p values were less than 0.05 . all p values below 0.0001 are represented as p < 0.0001 . the reference cutoff concentration for gdf-15 was set at the 95th percentile of control group serum concentrations , as for fgf-21 . we used mann - whitney u and kruskal - wallis nonparametric testing to determine statistical differences between groups . receiver operating characteristic ( roc ) curves were plotted using sensitivity vs 100 specificity on a continuous scale and the area under the curve ( auc ) was used to determine the relative diagnostic capacity . biomarker levels were correlated with other biochemical measurements and clinical assessment tools using nonparametric spearman correlation testing . linear regression analysis was used to determine if statistically correlated parameters could predict gdf-15 concentration . multivariate linear regression analysis was performed to determine factors capable of predicting disease while controlling for confounders that may influence serum gdf-15 concentration . median serum gdf-15 concentrations varied considerably among the 3 cohort groups : the control group had a median serum gdf-15 concentration of 1,183.5 pg / ml ( interquartile range [ iqr ] 1,037.51,475.3 ) , disease controls had a median concentration of 1,791.0 pg / ml ( iqr 1,148.03,406.8 ) , and patients with mitochondrial disease had a median concentration of 3,956.0 pg / ml ( iqr 2,516.58,676.8 ) . the interassay coefficient of variation was 9.6% for repeated samples ( 40 samples repeated in duplicate and 42 samples repeated in triplicate from across the 3 experimental groups ) and 10.7% for assay quality controls . on average , serum gdf-15 concentrations were 2 and 4.6 times higher than control levels for the disease control and mitochondrial disease groups , respectively . when compared to mean fgf-21 levels , disease control and mitochondrial disease groups were 2.2 and 7.3 times higher than control levels , respectively . as with fgf-21 ( threshold cutoff 350 pg / ml ) , the 95th percentile of control subjects was set as the threshold cutoff for gdf-15 ( threshold cutoff 2,330 pg / ml ) ( figure 1 ) . the same relationship in median biomarker concentration between groups was observed for both biomarkers and nonparametric mann - whitney u testing showed serum biomarker concentrations to differ between the groups ( figure 1 ) . serum fgf-21 ( red ) and gdf-15 ( green ) concentrations are displayed for the 3 experimental groups on a logarithmic scale . the same relationship between median group concentrations can be seen for both biomarkers , with higher levels in the mitochondrial disease group compared to disease controls and controls . threshold cutoffs ( determined from the 95th percentile of control group biomarker concentrations ) are indicated by blue horizontal lines at the level of 350 pg / ml for fgf-21 and 2,330 pg / ml for gdf-15 . in addition , mann - whitney u ( muscle biopsy vs genetic diagnosis , mtdna vs ndna mutation , muscle manifesting vs non muscle manifesting , mitochondrial myopathy , encephalopathy , lactic acidosis , and strokelike episodes [ melas ] vs non - melas , male vs female , diabetic vs normal , cardiovascular involvement vs no cardiovascular involvement ) and kruskal - wallis ( walker criteria [ possible , probable , definite ] , diabetic status [ normal , type i diabetes , type ii diabetes , impaired fasting glucose , impaired glucose tolerance , insulin resistance ] , body mass index [ anorexic , normal , obese ] , cardiovascular status [ normal , cardiomyopathy , ischemic heart disease , rhythm disturbances , and periprocedural myocardial infarction , as well as cardiomyopathy , rhythm disturbances , and periprocedural myocardial infarction ] ) nonparametric analyses revealed only one statistical difference among all the groups and categories compared . the criterion of muscle biopsy diagnosis vs genetic diagnosis showed a marginal difference in serum gdf-15 concentration ( 3,206 pg / ml [ iqr 2,1526,028 ] vs 6,415 pg / ml [ iqr 2,70412,345 ] , respectively ; p = 0.046 ) . of note , in our previous fgf-21 study , there was a difference between muscle manifesting and non muscle manifesting disease patients , indicative of fgf-21 elevation in myopathic disease states . this difference was not evident when analyzing gdf-15 ( p = 0.324 ) , suggesting gdf-15 production may not be related to tissue - specific pathways , but rather pathways common to all tissues . this highlights a potentially broader indication of mitochondrial diseases by gdf-15 that is not evident for fgf-21 . the vast majority of literature discussing gdf-15 as a disease biomarker pertains to cardiovascular abnormalities , in particular ischemic heart disease . although cardiac involvement is a relatively common comorbidity of mitochondrial disease , the majority of patients in this study did not have cardiac pathology . when analyzing gdf-15 against all cardiac involvement and also against subcategories of cardiac involvement , there was no apparent statistical difference in serum gdf-15 concentration between those with ( n = 11 ) or without ( n = 43 ) cardiac involvement ( p = 0.788 ) , or when subcategorized ( kruskal - wallis , p = 0.451 ) . serum biomarker concentration exceeding the threshold cutoff was considered indicative of disease for statistical analyses . as the threshold cutoffs were set at the 95th percentile of control concentrations , the specificity was artificially determined to be 95.5% ( 95% confidence interval [ ci ] 86.4%98.8% ) for fgf-21 and gdf-15 in both disease control and mitochondrial disease groups . the sensitivity for detecting mitochondrial disease if serum gdf-15 concentrations were raised was 77.8% ( 95% ci 64.1%87.5% ) , as compared to fgf-21 at 68.5% ( 95% ci 54.3%80.0% ) . for the disease control group , the sensitivity for predicting mitochondrial disease when there was none was 30.0% ( 95% ci 12.8%54.3% ) using gdf-15 , compared to 35.0% ( 95% ci 16.3%59.1% ) using fgf-21 . when subcategorizing the mitochondrial disease group by diagnosis , the sensitivity for patients diagnosed by muscle biopsy was 59.3% ( 95% ci 39.0%77.0% ) using serum fgf-21 concentration and 70.4% ( 95% ci 49.7%85.5% ) using serum gdf-15 concentration . for patients diagnosed with a genetic mutation , the sensitivity was 77.8% ( 95% ci 57.3%90.6% ) using serum fgf-21 concentration and 85.2% ( 95% ci 65.4%95.1% ) using serum gdf-15 concentration . nine of the patients with genetic diagnoses also had a muscle biopsy showing changes consistent with mitochondrial disease . the sensitivity when considering these patients was 66.7% ( 95% ci 30.9290.96 ) using serum fgf-21 concentrations and 88.9% ( 95% ci 50.6799.42 ) using serum gdf-15 concentration . the likelihood of having disease if fgf-21 levels were raised above the threshold cutoff of 350 pg / ml , known as the diagnostic or , was calculated in our previous study to be 45.7 ( 95% ci 12.5166.5 , p < 0.0001 ) . for gdf-15 in this study , the diagnostic or was calculated to be 73.5 ( 95% ci 19.6276.3 , p < 0.0001 ) . further to this , roc curve analysis of fgf-21 and gdf-15 showed gdf-15 to be slightly better ( auc 0.941 [ 95% ci 0.8930.989 ] ) than fgf-21 ( auc 0.911 [ 95% ci 0.8550.968 ] ) at predicting disease across combinations of sensitivity and specificity , although the aucs were not statistically different . combining both biomarkers did not increase the auc ( 0.944 [ 95% ci 0.8950.994 ] ) remarkably over that for gdf-15 alone , indicating that there was little synergistic diagnostic benefit when considering the biomarkers together . statistical dependence between gdf-15 and all other measured parameters from the initial study was assessed for the disease group using spearman rank correlation coefficient test . gdf-15 correlated with lactate ( spearman correlation coefficient [ rs ] = 0.583 , p < 0.0001 ) , lactate : pyruvate ( rs = 0.544 , p < 0.0001 ) , fgf-21 ( rs = 0.554 , p < 0.0001 ) , body mass index ( rs = 0.322 , p < 0.02 ) , mrc proximal muscle weakness ( rs = 0.302 , p < 0.05 ) , and the walker criteria ( rs = 0.322 , p < 0.02 ) . interestingly , unlike in other gdf-15 studies , those patients in the mitochondrial disease group for whom an nmdas had been completed at the time of recruitment ( n = 30 ) showed no correlation between the total nmdas score or scores for subsections ( i iii ) and serum fgf-21 or gdf-15 concentration . following multivariate linear regression analysis for each of the groups to determine any variables that could predict serum gdf-15 levels , only lactate : pyruvate ( p < 0.002 ) and fgf-21 ( p < 0.01 ) remained associated in the mitochondrial disease group . despite a moderate correlation between gdf-15 and fgf-21 ( rs = 0.554 ) , an association that persisted after linear regression , there was no benefit to disease prediction when the 2 biomarkers were combined for roc curve analysis ( figure 2 ) , as mentioned above . finally , when considering gdf-15 levels from control and mitochondrial disease groups in a multivariate logistic regression model that included possible confounders ( age , lactate : pyruvate , insulin , glucose , cholesterol , triglycerides , and fgf-21 ) , gdf-15 was the best predictor of mitochondrial disease ( p < 0.002 ) . gdf-15 ( green ) outperformed fgf-21 ( red ) across varying sensitivities at high specificities ( area under the curve [ auc ] = 0.941 vs 0.911 , respectively ; no difference ) . combining gdf-15 and fgf-21 ( blue ) had little benefit over gdf-15 alone ( auc = 0.944 vs 0.941 , respectively ; no difference ) . in our cohort of adult patients with mitochondrial disease , gdf-15 presents as a sensitive indicator of mitochondrial diseases and outperforms fgf-21 and classical markers . median serum concentrations of gdf-15 showed the same relationship between experimental groups as was observed for fgf-21 ( figure 1 ) , but gdf-15 was a better predictor of disease based on diagnostic sensitivity , diagnostic or , roc curves ( figure 2 ) , and multivariate linear regression . despite a moderate correlation between serum gdf-15 and fgf-21 concentration , a relationship that persisted after linear regression analysis , there was no synergistic benefit for considering gdf-15 and fgf-21 together ( figure 2 ) . furthermore , gdf-15 was more broadly indicative of mitochondrial diseases , rather than preferentially indicating muscle manifesting mitochondrial disease like fgf-21 . first , we surveyed an adult - only cohort of patients with mitochondrial disease , whereas other fgf-21 and gdf-15 diagnostic studies have looked at combined adult and pediatric cohorts . as pediatric patients are known to exhibit higher circulating biomarker levels , analyses combining these populations may artificially skew serum biomarker concentrations and therefore the analysis of results . second , our cohort included a combination of patients with genetic and muscle histology diagnoses , representing current clinical diagnostic methods . as such , our study represents a more stringent estimation of biomarker diagnostic validity , compared to other studies that have considered only genetically diagnosed cohorts . given our results , we propose that biomarker triaging of patients suspected of a mitochondrial disorder may prove to be a more efficient diagnostic paradigm when performed in combination with exhaustive genetic sequencing analysis using next - generation sequencing . third , our cohort represents a wider range of mitochondrial diseases in comparison to other studies that have used limited genetic mitochondrial disease groups , such as m.3243a > g , kearns - sayre syndrome , mitochondrial dna deletions , or tk2 mutations . finally , we used assay kits from the same manufacturer ( fgf-21 and gdf-15 ; biovendor , brno , czech republic ) , as compared to other studies that used assays from different manufacturers ( fgf-21 ; biovendor and gdf-15 ; r&d systems , minneapolis , mn ) , to reduce potential variation in methodology and experimental output . the median gdf-15 concentration for controls in this study ( 1,183.5 pg / ml [ iqr 1,037.51,475.3 ] ) was in good agreement with median gdf-15 concentrations for controls ( 1,020 pg / ml [ iqr 8031,362 pg / ml ] in men and 1,017 pg / ml [ iqr 8091,297 pg / ml ] in women ) from the framingham offspring study , despite differing methods of quantification . however , when age- and sex - matched 97.5th percentile values from the framingham study were used as the threshold cutoff for a genetically diagnosed m.3243a > g european patient cohort study , a diagnostic sensitivity of only 53% was calculated . by comparing the 97.5th percentile values from the framingham study ( range 1,0855,006 pg / ml , age- and sex - dependent , using precommercial automated electrochemiluminescence immunoassay ) with the threshold cutoff of a recent japanese mitochondrial disease patient gdf-15 diagnostic study ( 710 pg / ml , using r&d systems quantikine elisa ) , obtained using the same elisa assay as the european study , it becomes apparent that the use of different assays can give markedly different results and the use of data from one assay type in association with a different assay can potentially be misleading . despite this , the correlation between serum gdf-15 and fgf-21 concentrations was in agreement between this study and the european study ( rs = 0.554 vs rs = 0.54 , respectively ) and close to that reported in the japanese study ( rs = 0.64 ) . our study demonstrates that elevated gdf-15 levels were indicative of mitochondrial disease regardless of whether there was muscle involvement . this is in contrast to the japanese study , which claimed that gdf-15 may be a better indicator of muscle manifesting disease than fgf-21 , even though a measure of muscle involvement was not used in that study and therefore comparisons relating muscle involvement with biomarker concentration were not possible . furthermore , that study used different threshold values for gdf-15 : they agreed with our previously determined fgf-21 threshold cutoff value of 350 pg / ml , when using the same elisa from biovendor , but the threshold cutoff values for gdf-15 were set at a lower level ( r&d systems = 710 pg / ml vs biovendor = 2,330 pg / ml in this study ) , possibly owing to the difference in elisa used . in this study and our previous study on fgf-21 , we found no correlation with the total or subcategory nmdas scores for patients who completed this rating scale at the time of recruitment . this is in contrast to other studies that have found moderate to strong correlations between biomarker concentrations and disease severity measured using the nmdas and the japanese mitochondrial disease rating scale . in the european study , a correlation was identified between gdf-15 and nmdas - rated severity for asymptomatic patients , moderately affected patients , or moderately affected patients ( rs = 0.54 , p < 0.001 ) , but not for severely affected patients . this disparity may be due to the method of analysis , as it was reported that a parametric correlation test ( pearson ) was used for the severe group and then compared to a nonparametric correlation test ( spearman ) for asymptomatic , mild and moderate severity groups . a lack of correlation between serum biomarker concentration and disease progression may indicate that the current means of assessing disease progression are inadequate or too narrow for the phenotypic diversity of these disease groups . alternatively , the lack of association may indicate the inability of these biomarkers to infer disease progression on the basis of being surrogate markers and not mediators of disease , a detail that warrants further investigation . finally , the single follow - up sampling of both fgf-21 and gdf-15 in the european m.3243a > g cohort , 2 years after the initial sample , may not provide adequate frequency or time scales for these markers to reliably track disease progression in combination with the nmdas . estimation of progression may therefore be improved by more frequent sampling in addition to more sensitive means of assessing severity . thus , larger studies with more sensitive clinical tools to measure disease progression , performed over longer follow - up periods and with more frequent sampling points , may be required to clarify whether biomarkers such as gdf-15 and fgf-21 are able to indicate disease progression . the clinical diagnostic outlook for mitochondrial disease has been dramatically improved over the past 5 years with the identification of fgf-21 as a useful indicator of muscle manifesting mitochondrial disease and now more so with gdf-15 as a more sensitive and broadly indicative marker of mitochondrial disease . continued investigation into the pathophysiologic relationship of these markers with mitochondrial disease front - line diagnostic indicator tests , such as serum gdf-15 concentration , coupled with exhaustive genetic sequencing methods , afforded by advances in next - generation sequencing technologies , herald a potentially new age for mitochondrial disease diagnosis . it is hoped that the clinical , fiscal , and personal burden of mitochondrial diseases may be improved through better management and treatment resulting from improved diagnostic capabilities . statistical analysis was carried out by r.l.d . with assistance from j. patterson ( both from the kolling institute of medical research , st . conceptualized and designed the study , acquired data , analyzed data , carried out statistical analyses , interpreted data , drafted the manuscript , and revised the manuscript . professor c.m.s . designed and funded the study , interpreted data , and revised the manuscript .

clusters of gm1 gangliosides act as platforms for conformational transition of monomeric , unstructured amyloid ( a ) to its toxic -structured aggregates . we have previously shown that a(140 ) accommodated on the hydrophobic / hydrophilic interface of lyso - gm1 or gm1 micelles assumes -helical structures under ganglioside - excess conditions . for better understanding of the mechanisms underlying the -to- conformational transition of a on gm1 clusters , we performed spectroscopic characterization of a(140 ) titrated with gm1 . it was revealed that the thioflavin t- ( tht- ) reactive -structure is more populated in a(140 ) under conditions where the a(140 ) density on gm1 micelles is high . under this circumstance , the c - terminal hydrophobic anchor val39-val40 shows two distinct conformational states that are reactive with tht , while such a species were not generated by smaller lyso - gm1 micelles . these findings suggest that gm1 clusters promote specific a-a interactions through their c - termini coupled with formation of the tht - reactive -structure depending on sizes and curvatures of the clusters .

conformational transitions of unstructured proteins into -structure - based oligomeric or amyloid states are crucial processes in the onset and development of a variety of neurodegenerative disorders such as alzheimer 's disease ( ad ) and parkinson 's disease [ 1 , 2 ] . amyloid (a ) , a major player in ad , is a 40- or 42-amino acid peptide cleaved from its precursor membrane protein by sequential actions of - and -secretases and has a high propensity for toxic aggregation to form cross--fibrils [ 3 , 4 ] . accumulated evidence indicates that the gm1 ganglioside , a glycosphingolipid abundant in neuronal cell membranes , interacts with a and promotes its assembly , resulting in pathogenic amyloid formation [ 57 ] . for example , high - density gm1 clustering , which is exclusively observed in synaptosomes , is suggested to accelerate a deposition . in vitro experiments have indicated that the a-gm1 interaction depends on the clustering of gm1 , and its carbohydrate moiety alone can not induce conformational changes of a [ 15 , 30 , 31 ] . furthermore , it has been suggested that each of the heredity variants of a reported thus far has its own specificities for gangliosides , which have been supposed to be associated with their ectopic deposition [ 9 , 10 ] . promotion of amyloid formation in membrane - bound states has also been reported for prion and -synuclein [ 11 , 12 ] . for example , prion protein has been reported to be localized in the membrane microdomains and caveolae enriched with ganglioside , which interacts with prion protein and thereby promotes its -to- structural conversion [ 13 , 14 ] . therefore , detailed conformational characterization of a interacting with the ganglioside clusters not only provides structural information as cues for drug development in preventing and treating ad but also offers general insights into the mechanisms underlying the disease - associated amyloid formation facilitated in membrane environments . in previous papers , we have reported nuclear magnetic resonance ( nmr ) studies of the interactions of a ( 140 ) with ganglioside clusters using lyso - gm1 micelles ( approximate molecular mass 60 kda ) as model systems [ 15 , 16 ] . our nmr data showed that a(140 ) is accommodated on the hydrophobic / hydrophilic interface of the ganglioside cluster exhibiting an -helical conformation under ganglioside - excess conditions . in this state , a(140 ) shows an up - and - down topological mode in which the two -helices at segments his - val and ile - val and the c - terminal val - val dipeptide segment are in contact with the hydrophobic interior of the micelles , whereas the remaining regions are exposed to the aqueous environment . a similar tendency of a(140 ) has been observed using excess amounts of gm1 , which forms micelles with an approximate molecular mass of 140 kda [ 15 , 17 ] . these findings indicate that ganglioside clusters offer unique platforms at their hydrophobic / hydrophilic interfaces for binding coupled with -helix formation of a molecules . to gain further insights into the underlying mechanisms of the amyloid formation of a , it is necessary to characterize the conformational transition from -helices to -structures on the ganglioside clusters . on the basis of the circular dichroism ( cd ) data , kakio et al . demonstrated that a/gm1 ratios influence the secondary structure of a(140 ) on the raft - like lipid bilayers composed of gm1 , cholesterol , and sphingomyelin [ 18 , 19 ] . namely , a adopts an -helical structure at lower a/gm1 ratios ( 0.025 ) , while it assumes a -sheet - rich structure at higher ratios ( 0.05 ) . although more detailed structural information on a bound to the gm1 cluster is highly desirable , the small unilamellar vesicles used for the cd measurements are still too large to investigate with solution nmr techniques . in the present study , we attempt to characterize conformational states of a(140 ) in the presence of varying amounts of gm1 aqueous micelles using stable - isotope - assisted nmr spectroscopy in conjunction with synchrotron - radiation vacuum - ultraviolet cd ( vuvcd ) spectroscopy . we found that gm1 micelles also induce distinct secondary structures of a(140 ) depending on the a/gm1 ratios . on the basis of the spectroscopic data , we will discuss a behaviours on the ganglioside clusters from a structural point of view . the plasmid vector encoding a(140 ) was constructed and cloned as a fusion protein with hexahistidine - tagged ubiquitin ( his6-ub ) using the pet28a(+ ) vector ( novagene ) , subsequently transformed into escherichia coli strain bl21-codonplus ( stratagene ) . transformed bacteria were grown at 37c in lb media containing 15 g / ml of kanamycin . for the production of isotopically labelled a(140 ) protein , cells were grown in m9 minimal media containing [ n ] nh4cl ( 1 protein expression was induced by adding 0.5 mm isopropyl--d - thiogalactopyranoside ( iptg ) when the absorbance reached 0.8 at 600 nm . after 4 hours , cells were harvested and then suspended into buffer a ( 50 mm tris - hcl , 150 mm nacl , ph 8.0 ) containing 4-(2-aminoethyl ) benzenesulfonyl fluoride hydrochloride , subsequently disrupted by sonication . after centrifugation , the pellet was dissolved in buffer a containing 8 m urea . his6-ub - a(140 ) was purified by a ni - nitrilotriacetic acid affinity column ( ge healthcare ) . recombinant glutathione s - transferase- ( gst- ) tagged yeast ubiquitin hydrolase-1 ( yuh-1 ) was grown until the absorbance reached 0.8 at 600 nm and then induced to express by iptg . cell pellets were dissolved in buffer b ( 50 mm tris - hcl , 1 mm edta , 1 mm dtt , ph 8.5 ) and disrupted by sonication . gst - yuh-1 was purified by a glutathione affinity column ( ge healthcare ) . a(140 ) protein was enzymatically cleaved from his6-ub by incubation with gst - yuh-1 for 1 h at 37c at a molar ratio of his6-ub - a(140 ) : gst - yuh1 = 10 : 1 . the cleaved a(140 ) was purified by reverse - phase chromatography using an octadecylsilane column ( tskgel ods-80tm , tosoh ) with a linear gradient of acetonitrile . synthetic a(140 ) labelled with n selectively at val or val was purchased from anygen co. both of recombinant and synthetic a(140 ) proteins were dissolved at an approximate concentration of 2 mm in 0.1% ( v / v ) ammonia solution then collected and stored in aliquots at 80c until use . the residual ganglioside was suspended at a concentration of 12 mm in 10 mm potassium phosphate buffer ( ph 7.2 ) and then mixed by vortexing . micelle sizes were determined by dynamic light scattering using a dynapro titan ( wyatt technology ) . a(140 ) was dissolved at a concentration of 0.2 mm in 10 mm potassium phosphate buffer ( ph 7.2 ) in the absence or presence of 0.49 mm gm1 or lyso - gm1 . 980 l of 5 m tht ( sigma ) solution in 50 mm glycine - naoh buffer ( ph 8.5 ) was added to an aliquot of 20 l of each sample . fluorescence was measured immediately after mixing at the excitation and emission wavelengths of 446 and 490 nm , respectively , using spectrofluorophotometer ( hitachi f-4500 ) at 37c . a(140 ) was dissolved at a concentration of 0.2 mm in 10 mm potassium phosphate buffer ( ph 7.2 ) . the cd spectra of a(140 ) in the presence or absence of gm1 were measured from 265 to 175 nm under a high vacuum ( 10 pa ) at 37c using the vuvcd spectrophotometer constructed at beamline 15 ( 0.7 gev ) of the hiroshima synchrotron radiation center ( hisor ) . details of the spectrophotometer and optical cell were described previously [ 21 , 22 ] . the path length of the caf2 cell was adjusted with a teflon spacer to 50 m or 100 m for measurements . the vuvcd spectra were recorded with a 1.0-mm slit , a 16-s time constant , a 4-nm min scan speed , and nine accumulations . the molar ellipticities of a(140 ) were calculated with the average residue weight of 107.5 . the secondary structure contents of a(140 ) were analysed using the modified selcon3 program and the vuvcd spectra down to 160 nm for 31 reference proteins with known x - ray structures [ 24 , 25 ] . the secondary structures of these proteins in crystal form were assigned into four classes ( -helices , -strandes , turns , and unordered structures ) using the dssp program based on the hydrogen bonds between adjacent amide groups . in this analysis , the root - mean - square deviation ( ) and the pearson correlation coefficient ( r ) between the x - ray and vuvcd estimates of the secondary structure contents of the reference proteins were 0.058 and 0.85 , respectively , confirming the high accuracy of the vuvcd estimation . nmr spectral measurements were made on a bruker dmx-500 spectrometer equipped with a cryogenic probe as well as a bruker avance iii-400 spectrometer . isotopically labelled a(140 ) was dissolved at a concentration of 0.2 mm in 10 mm potassium phosphate buffer ( ph 7.2 ) containing 10% ( v / v ) h2o in the presence or absence of gm1 . for h - n heteronuclear single - quantum correlation ( hsqc ) measurements , the spectra were recorded using a(140 ) labelled with n uniformly or selectively at the amide group of val or val at a h observation frequency of 500 mhz with 128 ( t1 ) 1024 ( t2 ) complex points and 256 scans per t1 increment . the spectral width was 1720 hz for the n dimension and 6000 hz for the h dimension . one - dimensional carbonyl c spectra were recorded using uniformly c- and n - labelled a(140 ) at a h observation frequency of 400 mhz with a spectral width of 22,000 hz . in these experiments , we examined whether tht fluorescence is enhanced by a(140 ) in the presence of varying concentrations of gm1 or lyso - gm1 . as shown in figure 1 , gm1 exhibited a bell - shaped dependence on a/gm1 ratios regarding tht fluorescence enhancement , while lyso - gm1 showed virtually no enhancement . maximum enhancement was observed at a 1:15 molar ratio of a(140 ) to gm1 . the dynamic light scattering data confirmed that the gm1 and lyso - gm1 micelles exhibited an approximate hydrodynamic radius of 6 nm and 4 nm , respectively , irrespective of the a/ganglioside ratios . the observed fluorescence intensity remained almost constant up to 12 h. these data indicated that gm1 micelles at appropriate a/gm1 ratios promote some aa interaction with formation of their -sheet - like conformation , which , however , does not result in irreversible fibril formation . we characterized the conformational transition of a depending on a/gm1 ratios by cd measurements . the short - wavelength limit of cd spectroscopy can be successfully extended using synchrotron radiation as a high - flux source of photons , which yields much more accurate data than those obtained with a conventional cd spectrophotometer [ 28 , 29 ] . the spectral data indicated that a(140 ) undergoes conformational transitions depending on gm1 to a(140 ) ratios ( figure 2 ) . the secondary structure contents of a(140 ) at a/gm1 molar ratios of 1 : 0 , 1 : 15 , and 1 : 30 were estimated on the basis of the spectral data ( table 1 ) . the -helix content of a(140 ) in the presence of gm1 at an a/gm1 molar ratio of 1:30 was calculated to be 40.0% , which is consistent with our previous estimation based on the backbone chemical shift data of lyso - gm1 , thus confirming close similarity of the binding modes of a(140 ) between gm1 and lyso - gm1micelles . at an a/gm1 molar ratio of 1:15 , where the maximum tht fluorescence enhancement was observed , the cd data consistently indicated a significantly increased content of -strands . the conformation of a(140 ) in the presence of varying amounts of gm1 micelles was further characterized by c nmr spectroscopy . the carbonyl c nmr spectral data of uniformly c - labelled a(140 ) indicated that the peaks shifted upfield , roughly corresponding to -structures , are more populated at an a/gm1 molar ration of 1 : 15 in comparison with the gm1-excess conditions ( figure 3 ) . intriguingly , intensities of these peaks were selectively reduced upon the addition of tht . these nmr data are again consistent with the vuvcd data as well as the results of the tht assay . to provide more detailed information on the conformational transition of a(140 ) on gm1 micelles , we observed h - n hsqc spectral changes of a(140 ) upon titration with gm1 . interestingly , at an a/gm1 molar ratio of 1 : 15 , a(140 ) exhibited hsqc peaks that were not observed in the spectra of free or fully micelle - bound forms ( supplementary figure 1 ) . by using site - specifically n - labelled a , these extra peaks were assigned to val and val ( figure 4 and supplementary figure 1 available online at doi:10.4061/2011/925073 ) . namely , the amide groups of these c - terminal residues of the micelle - bound a species show double hsqc peaks under the condition where a/gm1 ratio is relatively high . more interestingly , these double peaks were perturbed upon the addition of tht , while the corresponding peaks originating from the free and fully micelle - bound forms showed little or no change ( figure 4 ) . on the other hand , many of the h - n hsqc peaks from a(140 ) , including val and val , were not observed at an a/lyso - gm1 molar ratio of 1 : 15 due to intermediate chemical exchange between free and micelle - bound states of a(140 ) ( data not shown ) . accumulating evidence , including our previous reports , indicates that the interaction of a with gm1 involves multiple steps including the initial encounter complex formation and the accommodating process on the hydrophilic / hydrophobic interface of the ganglioside clusters [ 1517 , 30 ] . nmr spectral data of a(140 ) titrated with gm1 micelles under a-excess conditions indicated that they form a weak complex presumably through an interaction between the n - terminal segment of a(140 ) and the outer carbohydrate branch of gm1 [ 15 , 30 ] . thus , it is conceivable that the outer - branch structures of the carbohydrate moieties of gangliosides influence the association phase of the interaction and thereby determine the ganglioside specificities of a. nongangliosidic micelles and vesicles are barely or not capable of trapping a(140 ) effectively [ 15 , 18 , 31 , 32 ] . on the other hand , the -helical conformation of a(140 ) accommodated on sugar - lipid interface of the gm1 and lyso - gm1 micelles have been characterized by nmr under ganglioside - excess conditions ( a/ganglioside molar ratio of 1 : 30 ) . because the structure of the inner part is common among the gangliosides , non - gm1 ganglioside , for example , gm2 , thus , the spectroscopic characterization of the interactions of a with gangliosidic micelles has so far been performed only under the extreme conditions of the a/ganglioside ratios . the present study attempts to bridge the gap in our understanding of a behavior on gm1 micelles by carrying out spectroscopic analyses of a in the presence of varying amounts of gm1 micelles . the present data all indicated that -structure is more populated in micelle - bound a(140 ) under the condition where the a/gm1 ratio is higher . although the binding mode of tht to amyloid fibrils has yet to be fully elucidated , it has been suggested that tht is more likely to bind perpendicularly to parallel -strands in a -sheet [ 3335 ] . in addition , recently reported solid - state nmr data indicate that a tht - reactive , neurotoxic amyloid intermediate of a(140 ) is composed of parallel -structures . these data suggest that formation of parallel -strands is the minimum prerequisite for tht fluorescence enhancement . with this in mind , the bell - shape dependence of tht fluorescence enhancement ( figure 1 ) can be interpreted as follows . at an extremely low concentration of gm1 , most of a(140 ) exists as a free form , which is an unstructured monomer and therefore is not reactive with tht . fraction of the micelle - bound form of a(140 ) increases with increase of the gm1 amounts . to some extent , the micelles promote intermolecular interaction of a(140 ) , giving rise to the tht - reactive a(140 ) species . under gm1-excess conditions , however , a(140 ) molecules are presumably relatively isolated from one another and therefore are not capable of forming an intermolecular -structure . the a/gm1 molar ratio , where the maximum enhancement was observed , was 1 : 15 , which corresponds to average number of a/micelle of 11.2 with the assumption of the micellar gm1 aggregation number of 168 4 . thus , the a density on gm1 micelles is a crucial factor determining the occurrence of the tht - reactive a species . under the circumstance where the a(1 - 40 ) density on gm1 micelles is high , the c - terminal dipeptide of a(140 ) shows , at least , two distinct conformational states that are reactive with tht . in a previous paper , we demonstrated that the c - terminal val - val dipeptide is inserted into the hydrophobic interior of the gangliosidic micelles . this c - terminal segment is involved in the parallel -structure in the amyloid fibril and intermediate [ 36 , 38 ] . on the basis of these data , we suggest that gm1 clusters promote intermolecular a-a interactions coupled with the conformational transition of their c - terminal hydrophobic anchors into the tht - reactive parallel -structure , in which the local chemical environments of the c - terminal segments are different in different -strands . this may account for the multiple hsqc peaks originating from the c - terminal segments ( figure 4 ) . it has been reported that a exhibits tht - reactive -sheet - rich aggregates in the presence of sodium dodecyl sulfate ( sds ) at submicellar concentrations [ 39 , 40 ] . under these conditions , all the amide peaks of a(140 ) disappeared from the h - n hsqc spectrum because of the formation of large aggregates , except for those from the c - terminal residues that should still be mobile in this assembly state . on the basis of the nmr data obtained using paramagnetic probes , the c - terminal segment of a(140 ) bound to sds micelles taking into account these data in conjunction with our present data , we suggest that different -like structures of a(140 ) are induced by gm1 aqueous micelles and submicellar concentrations of sds . lyso - gm1 micelles could not induce the formation of the tht - reactive -structure of a(140 ) although the micelle - interacting modes of a(140 ) are almost identical between gm1 and lyso - gm1 micelles under ganglioside - excess conditions . by inspection of the dynamic light scattering data on an assumption of their globular shapes , the diameters of gm1 and lyso - gm1 micelles it is plausible that the sizes and curvatures of the gangliosidic micelles are determining factors for the number of a molecules that can be accommodated on their hydrophilic / hydrophobic interface and the occurrence of a-a interactions coupled with tht - reactive -structure formation . indeed , gm1 clusters with flatter curvature such as gm1-containing unilamellar vesicles induce enhanced a fibrillogenesis in comparison with gm1 micelles . lipid composition can also be a determining factor for assembly states of gm1 molecules and their interaction with a. most importantly , there is growing evidence that cholesterol and sphingomyelin contribute to gm1 assembly and thereby influence a deposition promoted by its cluster [ 8 , 18 , 42 , 43 ] . elucidation of the structural basis of these molecular events is an important subject for the forthcoming stage of the research . in conclusion , in the present study , we firstly identified and characterized the tht - reactive -structure of a(140 ) promoted on gm1 micelles . our findings offer structural insights into the mechanisms underlying the -to- conformational transition of a on gm1 clusters , which is associated with the nucleation process in the a aggregation .

by using ultrasound biomicroscopy ( ubm ) , the cross - sectional structures of the entire iridocorneal angle ( ica ) which are unable to assess with gonioscopic examination were evaluated objectively and quantitatively in live healthy and glaucomatous dogs . the icas of normotensive eyes in healthy dogs with normal open angle ( nor ) , a predisposition to primary closed angle glaucoma ( pcag ) ( predis ) and suffering from unilateral pcag ( uni ) , as well as the icas of hypertensive eyes with acute and chronic pcag ( acg and crg ) , were assessed . the opening of the ciliary cleft in predis was smaller than that in nor . in uni , the opening and area of the ciliary cleft were significantly decreased compared with those of nor and predis . acg had widespread structural abnormalities including marked decrease in the ciliary cleft and scleral venous plexus , and a thinner sclera than those in normotensive eyes , whereas the ica collapsed in crg with the thinnest sclera . medical therapy - responsive glaucomatous cases had wider ciliary cleft and scleral venous plexus than unresponsive ones . these findings suggest that the ciliary cleft and scleral venous plexus of the ica are key structures contributing to not only the pathophysiology of canine glaucoma but also the responsiveness to medical therapy in glaucomatous eyes , and cross - sectional entire structures of the ica should be evaluated quantitatively with ubm when diagnosing and managing canine glaucoma .

animals : two hundred and eighty - seven dogs that were presented at osaka prefecture university veterinary medical center ( opuvmc ) , animal eye center and izumi animal hospital for examination and treatment of ophthalmic , medical and surgical diseases were used in this study . all the dogs underwent a general ophthalmologic examination including gonioscopy with a goldmann two - mirror lens and slitlamp biomicroscopy to confirm ocular conditions . laboratory examinations including blood works , urinalysis and diagnostic imagings were also carried out to exclude systemic disorders , if necessary . animals suspected of having secondary glaucoma , which was indicated by their history and ophthalmic conditions , such as the presence of uveitis , cataract , lens luxation , ocular neoplasia and infections including systemic diseases , were excluded from the study . the examined eyes were divided into 5 groups as follows : ( i ) normal ocular group ( nor ) including 336 normotensive eyes of 168 dogs ( 57 beagle , 18 miniature dachshund , 15 shih tzu , 12 pembroke welsh corgi , 11 chihuahua , 9 mongrel and labrador retriever , 8 yorkshire terrier , 6 papillon , 5 pomeranian , 4 shetland sheepdog , 3 cavalier king charles spaniel and miniature schnauzer , 2 bernese mountain dog , doberman pinscher and pug , and 1 afghan hound and french bulldog ) with normal open angle ( fig . 1afig . 1.typical canine iridocorneal angles in the five ocular groups examined in this study : ( a ) wide open angle in a normal normotensive eye ; ( b1 ) open angle with goniodysgenesis in a primary closed angle glaucoma ( pcag)-predisposed normotensive eye ; ( b2 ) narrow angle with severe goniodysgenesis in a pcag - predisposed normotensive eye ; ( c ) very narrow angle in a normotensive fellow eye of unilateral pcag ; ( d ) closed angle in an acute glaucomatous eye ; and ( e ) closed angle in a chronic glaucomatous eye . ) , normal intraocular pressure ( iop ) of 1025 mmhg and no ocular disorders up to the date of ubm examination ; typical canine iridocorneal angles in the five ocular groups examined in this study : ( a ) wide open angle in a normal normotensive eye ; ( b1 ) open angle with goniodysgenesis in a primary closed angle glaucoma ( pcag)-predisposed normotensive eye ; ( b2 ) narrow angle with severe goniodysgenesis in a pcag - predisposed normotensive eye ; ( c ) very narrow angle in a normotensive fellow eye of unilateral pcag ; ( d ) closed angle in an acute glaucomatous eye ; and ( e ) closed angle in a chronic glaucomatous eye . ( ii ) primary closed angle glaucoma ( pcag)-predisposed ocular group ( predis ) including 116 normotensive eyes of 58 dogs ( 18 shiba , 14 american cocker spaniel , 13 toy poodle , 7 golden retriever , 2 maltese terrier , and 1 akita , english springer spaniel , flat - coated retriever and newfoundland , which were recognized as breeds with pcag [ 9 , 18 , 19 ] ) with some kind of ica abnormalities containing goniodysgenesis and open to narrow angles ( figs . 1-b1 and b2 ) , normal iop of 1025 mmhg and no ocular disorders up to the date of ubm examination ; ( iii ) normotensive fellow ocular group in unilateral pcag ( uni ) including 22 normotensive eyes of 22 dogs suffering from unilateral pcag ( 6 shiba , 5 shih tzu , 3 american cocker spaniel , 2 flat - coated retriever , miniature dachshund and mongrel , and 1 chihuahua and welsh terrier ) , which had narrow angle with moderate to severe ica abnormalities ( fig . 1c ) , normal iop of 1025 mmhg and no clinical signs of glaucoma up to the date of ubm examination ; ( iv ) acute glaucomatous eye group ( acg ) including 19 hypertensive non - buphthalmic eyes of 19 dogs ( 6 shiba , 3 miniature dachshund and shih tzu , 2 american cocker spaniel , and 1 chihuahua , english setter , flat - coated retriever , mongrel and welsh terrier ) with closed angle ( fig . 1d ) , iop of over 25 mmhg ( 3158 mmhg ) and signs of acute glaucoma , such as tenderness about the head and eyes , ocular pain ( blepharospasm ) , serous discharge , episcleral congestion ( red eye ) , corneal edema , dilated pupil and mild depression of optic nerve head , in which their clinical signs were observed less than 24 hr after the onset of symptoms on the date of ubm examination ; ( v ) chronic glaucomatous eye group ( crg ) including 20 hypertensive buphthalmic eyes of 20 dogs ( 6 miniature dachshund , 5 american cocker spaniel , 3 shiba , 2 mongrel and shih tzu , and 1 flat - coated retriever and welsh terrier ) with closed angle ( fig . 1e ) , iop of over 25 mmhg ( 4278 mmhg ) and signs of chronic glaucoma , such as globe enlargement with or without descemet streaks , mydriasis , tapetal hyperreflectivity with vascular attenuation , optic nerve cupping and blindness on the date of ubm examination . examination procedure : ultrasonographic images of the ica were obtained with ubm by using a ud-1000 with 40-mhz probes ( tomey corp . , nagoya , japan ) with background room illuminance of 1,2001,300 lux in a general examination room . non - anesthetized or non - sedated dogs were examined using manual restraints in sternal recumbency . when sedation was required , it was achieved following a protocol described previously ; these animals were assessed in a lateral recumbent position . ubm examination was also performed by a procedure described previously , and the following 7 parameters were evaluated with the measurement software of the ubm : ( i ) sld [ the distance between schwalbe s line ( the borderline between the cornea and sclera ) and the anterior lens capsule ] ( fig . 2.typical canine images of ultrasound biomicroscopy ( ubm ) on the iridocorneal angle ( ica , a ) and the same images of the ica with indication of ubm parameters evaluated in this study ( b to d ) . the following 7 parameters were evaluated with the measurement software included in the ud-1000 : ( i ) sld [ the distance between schwalbe s line ( sl , the borderline between the cornea and sclera ) and the anterior lens capsule ( alc ) ] ; ( ii ) the width of the ciliary cleft ( ccw ) , measured from the superior surface of the root of the iris ( ssri ) to the inner surface of the sclera ( iss ) on a perpendicular line ; ( iii ) the area of the ciliary cleft ( cca ) , measured as the area surrounded by the width of the ciliary cleft , the line of the inner scleral side of the ciliary cleft from the inner surface of the sclera to the angle recess ( ar ) and the line of the superior side of the root of the iris from the angle recess to the superior surface of the root of the iris ; ( iv ) the angle of the ciliary cleft ( acc ) , the angle of the inner surface of the sclera to the angle recess and the superior surface of the root of the iris to the angle recess ; ( v ) the scleral thickness at the position of the width of the ciliary cleft ( st ) , as measured from the inner surface of the sclera on a perpendicular line to the outer surface of the sclera ; ( vi ) the minimum distance between the angle recess and the scleral venous plexus ( svp ) [ asd ] ; and ( vii ) the total area of the scleral venous plexus ( svpa ) on the ubm image of the ica . ) ; ( ii ) the width of the ciliary cleft ( ccw ) , measured from the superior surface of the root of the iris to the inner surface of the sclera on a perpendicular line ( fig . 2b ) ; ( iii ) the area of the ciliary cleft ( cca ) , measured as the area surrounded by the width of the ciliary cleft , the line of the inner scleral side of the ciliary cleft from the inner surface of the sclera to the angle recess and the line of the superior side of the root of the iris from the angle recess to the superior surface of the root of the iris ( fig . 2b ) ; ( iv ) the angle of the ciliary cleft ( acc ) , the angle of the inner surface of the sclera to the angle recess and the superior surface of the root of the iris to the angle recess ( fig . 2c ) ; ( v ) the scleral thickness at the position of the width of the ciliary cleft ( st ) , as measured from the inner surface of the sclera on a perpendicular line to the outer surface of the sclera ( fig . 2c ) ; ( vi ) the minimum distance between the angle recess and the scleral venous plexus ( asd , fig . 2c ) ; and ( vii ) the total area of the scleral venous plexus ( svpa ) on the ubm image of the ica ( fig . raw ubm measurements were detected from all obtained images in the examined dogs . in order to exclude differences of raw ubm values caused by taking different breeds with varied ocular size , the ubm measurements except for the angle of the ciliary cleft were corrected by using the ratio with sld on the distance / length , that is , ccw / sld , st / sld and asd / sld , or sld on the area , that is , cca / sld and svpa / sld . the mean of ubm values was calculated to obtain a ubm measurement of each parameter of the ica in an individual eye . typical canine images of ultrasound biomicroscopy ( ubm ) on the iridocorneal angle ( ica , a ) and the same images of the ica with indication of ubm parameters evaluated in this study ( b to d ) . the following 7 parameters were evaluated with the measurement software included in the ud-1000 : ( i ) sld [ the distance between schwalbe s line ( sl , the borderline between the cornea and sclera ) and the anterior lens capsule ( alc ) ] ; ( ii ) the width of the ciliary cleft ( ccw ) , measured from the superior surface of the root of the iris ( ssri ) to the inner surface of the sclera ( iss ) on a perpendicular line ; ( iii ) the area of the ciliary cleft ( cca ) , measured as the area surrounded by the width of the ciliary cleft , the line of the inner scleral side of the ciliary cleft from the inner surface of the sclera to the angle recess ( ar ) and the line of the superior side of the root of the iris from the angle recess to the superior surface of the root of the iris ; ( iv ) the angle of the ciliary cleft ( acc ) , the angle of the inner surface of the sclera to the angle recess and the superior surface of the root of the iris to the angle recess ; ( v ) the scleral thickness at the position of the width of the ciliary cleft ( st ) , as measured from the inner surface of the sclera on a perpendicular line to the outer surface of the sclera ; ( vi ) the minimum distance between the angle recess and the scleral venous plexus ( svp ) [ asd ] ; and ( vii ) the total area of the scleral venous plexus ( svpa ) on the ubm image of the ica . nineteen acg cases received first medical therapy including an intravenous injection of 20% mannitol ( yoshindo , toyama , japan ) at a dose of 1.5 g / kg on the first admission and administrations of 0.005% latanoprost ( xalatan , pfizer , tokyo , japan ) , 1% dorzolamide hydrochloride ( trusopt , santen pharmaceutical co. , ltd . , osaka , japan ) and 0.5% timolol maleate ( timoptol , santen pharmaceutical co. , ltd . then , the responsiveness to the therapy , which means keeping reduction in normal iop of under 25 mmhg , and the seven parameters of the ica structures described above were evaluated simultaneously . statistical analysis : to exclude unexpected bias effects of pooling ubm measurements of right and left eyes , the average ubm values calculated from both eyes were used as ubm measurements in nor and predis . comparison between ocular groups was performed using non - repeated measurement one - way analysis of variance ( anova ) or kruskal - wallis h test followed by scheffe s test ( statcel 2nd ed . ; oms publishing co. , tokyo , japan ) . a p - value reference ranges of ubm measurements in nor and predis were calculated by interative truncation method with correction ( usui s method ) using stss / excel ver . 3.typical images of the iridocorneal angle ( ica ) detected by ultrasound biomicroscopy ( ubm ) in ( a ) normotensive eyes with no ocular disorders , intraocular pressure ( iop ) of 1025 mmhg and normal open angle ( nor ) , ( b ) normotensive eyes of primary closed angle glaucoma ( pcag)-predisposed dogs with no ocular disorders , iop of 1025 mmhg , open to narrow angle and some kind of abnormalities of the ica ( predis ) , ( c ) normotensive fellow eyes of dogs suffering from unilateral pcag , which had iop of 1025 mmhg , narrow angle with moderate to severe abnormalities of the ica and no clinical signs of glaucoma ( uni ) , ( d ) hypertensive non - buphthalmic eyes with acute pcag having iop of over 25 mmhg , closed angle and clinical signs of acute glaucoma ( acg ) and ( e ) hypertensive buphthalmic eyes with chronic pcag having iop of over 25 mmhg , closed angle and clinical signs of chronic glaucoma ( crg ) . the width of the ciliary cleft ( ccw ) of predis was narrower than that of nor ( an arrow in b ) . the area of the ciliary cleft ( cca ) of uni was smaller than those of nor and predis , and significantly narrow ccw was observed in the case of uni ( an arrow in c ) . the ciliary cleft was almost or completely collapsed , and it was difficult or impossible to detect ccw and cca in acg and crg ( arrows in d and e ) . although the scleral venous plexus could be observed in ubm images of acg ( asterisks in d ) , it was not found in a dog with crg . the basal iris of the anterior part of the ciliary cleft would be slightly inserted into the sclera of acg ( an arrowhead in d ) . the sclera became thin , and the basal iris was inserted into the sclera in crg ( arrowheads in e ) . , cross - sectional microstructures of the entire ica could be observed clearly by ubm examination in all ocular groups . the width of the ciliary cleft of predis was narrower than that of nor . it was difficult to identify the width of the ciliary cleft of uni , and the area of the ciliary cleft of uni would be smaller than those of nor and predis . the ciliary cleft was almost collapsed , and it was very difficult to detect the width and area of the ciliary cleft in acg . the scleral venous plexus could be observed in ubm images of most acg , although it was not found in crg . the basal iris of the anterior part of the ciliary cleft was slightly inserted into the sclera of acg , whereas it was inserted into the sclera in crg . typical images of the iridocorneal angle ( ica ) detected by ultrasound biomicroscopy ( ubm ) in ( a ) normotensive eyes with no ocular disorders , intraocular pressure ( iop ) of 1025 mmhg and normal open angle ( nor ) , ( b ) normotensive eyes of primary closed angle glaucoma ( pcag)-predisposed dogs with no ocular disorders , iop of 1025 mmhg , open to narrow angle and some kind of abnormalities of the ica ( predis ) , ( c ) normotensive fellow eyes of dogs suffering from unilateral pcag , which had iop of 1025 mmhg , narrow angle with moderate to severe abnormalities of the ica and no clinical signs of glaucoma ( uni ) , ( d ) hypertensive non - buphthalmic eyes with acute pcag having iop of over 25 mmhg , closed angle and clinical signs of acute glaucoma ( acg ) and ( e ) hypertensive buphthalmic eyes with chronic pcag having iop of over 25 mmhg , closed angle and clinical signs of chronic glaucoma ( crg ) . the width of the ciliary cleft ( ccw ) of predis was narrower than that of nor ( an arrow in b ) . the area of the ciliary cleft ( cca ) of uni was smaller than those of nor and predis , and significantly narrow ccw was observed in the case of uni ( an arrow in c ) . the ciliary cleft was almost or completely collapsed , and it was difficult or impossible to detect ccw and cca in acg and crg ( arrows in d and e ) . although the scleral venous plexus could be observed in ubm images of acg ( asterisks in d ) , it was not found in a dog with crg . the basal iris of the anterior part of the ciliary cleft would be slightly inserted into the sclera of acg ( an arrowhead in d ) . the sclera became thin , and the basal iris was inserted into the sclera in crg ( arrowheads in e ) . 4.box-plots of each ultrasound biomicrosopic measurement in the five ocular groups : ( a ) corrected width of the ciliary cleft ( ccw ) [ ccw / sld ( the distance / length of schwalbe s line to the anterior lenticular capsule ) ] ; ( b ) the angle of the ciliary cleft ( acc ) ; ( c ) corrected area of the ciliary cleft ( cca ) [ cca / sld ] ; ( d ) corrected minimum distance / length from the angle recess to the scleral venous plexus ( asd ) [ asd / sld ] ; ( e ) corrected total area of the scleral venous plexus on the ultrasound biomicrosopic image of the iridocorneal angle ( svpa ) [ svpa / sld ] ; ( f ) corrected scleral thickness ( st ) [ st / sld ] . the boxes define the 25th and 75th percentiles , with the median value indicated by a horizontal bar . the whiskers delineate the 5th and 95th percentiles , with outliers indicated as open circles . not available means no detectable data due to complete collapse of the ciliary cleft and/or the scleral venous plexus . different letters / alphabets indicate statistically significant differences between groups ( p<0.05 ) . , and table 1table 1.reference ranges of corrected ultrasound biomicroscopic values in healthy dogscorrected ccw ( ccw / sld)acc ( )corrected cca ( cca / sld)corrected asd ( asd / sld)corrected svpa ( svpa / sld)corrected st ( st / sld)nor ( n=168)0.0640.1498.322.20.0190.0420.1100.3170.0140.0580.2530.384predis ( n=58)0.0280.1314.819.00.0060.0430.1180.3020.0120.0520.2240.339nor ; normotensive eyes in normal open angle , predis ; normotensive eyes in predisposition to primary closed angle glaucoma . ccw ; the width of the ciliary cleft , sld ; the distance between schwalbe s line ( the borderline of the cornea and sclera ) and the anterior lens capsule , acc ; the angle of the ciliary cleft , cca ; the area of the ciliary cleft , asd ; the minimum distance between the angle recess and the scleral venous plexus , svpa ; the total area of the scleral venous plexus on the ultrasound biomicroscopic image of the iridocorneal angle , st ; the scleral thickness at the ccw . shows reference ranges of each ubm measurement in nor and predis . in predis , ubm measurements of the corrected width of the ciliary cleft and the angle of the ciliary cleft were significantly lower than those of nor . in uni , significantly low values were found for the corrected width and area of the ciliary cleft , and the angle of the ciliary cleft , when compared with those in nor and predis . in acg , the corrected width and area of the ciliary cleft , the total area of the scleral venous plexus and the scleral thickness , and the angle of the ciliary cleft were significantly smaller than those of nor and predis . the corrected width and area of the ciliary cleft , the total area of the scleral venous plexus and the scleral thickness in acg were also significantly decreased compared with those of uni . in crg , the anatomical structures of the sclerociliary cleft including the scleral venous plexus completely collapsed , and all ubm values except the corrected scleral thickness were zero or undetectable . there were significant differences for the corrected width and area of the ciliary cleft , and the total area of the scleral venous plexus between crg and nor , predis and uni . the angle of the ciliary cleft in crg was significantly lower than those of nor and predis . the corrected scleral thickness of crg was significantly smaller than those of nor , predis , uni and acg , as a result of the buphthalmos . box - plots of each ultrasound biomicrosopic measurement in the five ocular groups : ( a ) corrected width of the ciliary cleft ( ccw ) [ ccw / sld ( the distance / length of schwalbe s line to the anterior lenticular capsule ) ] ; ( b ) the angle of the ciliary cleft ( acc ) ; ( c ) corrected area of the ciliary cleft ( cca ) [ cca / sld ] ; ( d ) corrected minimum distance / length from the angle recess to the scleral venous plexus ( asd ) [ asd / sld ] ; ( e ) corrected total area of the scleral venous plexus on the ultrasound biomicrosopic image of the iridocorneal angle ( svpa ) [ svpa / sld ] ; ( f ) corrected scleral thickness ( st ) [ st / sld ] . the boxes define the 25th and 75th percentiles , with the median value indicated by a horizontal bar . the whiskers delineate the 5th and 95th percentiles , with outliers indicated as open circles . not available means no detectable data due to complete collapse of the ciliary cleft and/or the scleral venous plexus . different letters / alphabets indicate statistically significant differences between groups ( p<0.05 ) . nor ; normotensive eyes in normal open angle , predis ; normotensive eyes in predisposition to primary closed angle glaucoma . ccw ; the width of the ciliary cleft , sld ; the distance between schwalbe s line ( the borderline of the cornea and sclera ) and the anterior lens capsule , acc ; the angle of the ciliary cleft , cca ; the area of the ciliary cleft , asd ; the minimum distance between the angle recess and the scleral venous plexus , svpa ; the total area of the scleral venous plexus on the ultrasound biomicroscopic image of the iridocorneal angle , st ; the scleral thickness at the ccw . nineteen cases received first medical therapy on opuvmc , and the responsiveness to the therapy and the ica structures were evaluated simultaneously . twelve dogs showing no response to the first medical therapy had significant decrease of the corrected width and area of the ciliary cleft , and the total area of the scleral venous plexus , compared with 7 cases responding to the therapy ( fig . 5fig . 5.box-plots of each ultrasound biomicrosopic measurement in 7 dogs responding to first medical therapy ( effect ) and 12 dogs showing no response to the therapy ( no effect ) : ( a ) corrected width of the ciliary cleft ( ccw ) [ ccw / sld ( the distance / length of schwalbe s line to the anterior lenticular capsule ) ] ; ( b ) the angle of the ciliary cleft ( acc ) ; ( c ) corrected area of the ciliary cleft ( cca ) [ cca / sld ] ; ( d ) corrected minimum distance / length from the angle recess to the scleral venous plexus ( asd ) [ asd / sld ] ; ( e ) corrected total area of the scleral venous plexus on the ultrasound biomicrosopic image of the iridocorneal angle ( svpa ) [ svpa / sld ] ; ( f ) corrected scleral thickness ( st ) [ st / sld ] . the boxes define the 25th and 75th percentiles , with the median value indicated by a horizontal bar . data of asd in the group of no effect were calculated from 3 cases , because it could not be detected in the other 9 cases due to complete collapse of the ciliary cleft and/or the scleral venous plexus . ) . box - plots of each ultrasound biomicrosopic measurement in 7 dogs responding to first medical therapy ( effect ) and 12 dogs showing no response to the therapy ( no effect ) : ( a ) corrected width of the ciliary cleft ( ccw ) [ ccw / sld ( the distance / length of schwalbe s line to the anterior lenticular capsule ) ] ; ( b ) the angle of the ciliary cleft ( acc ) ; ( c ) corrected area of the ciliary cleft ( cca ) [ cca / sld ] ; ( d ) corrected minimum distance / length from the angle recess to the scleral venous plexus ( asd ) [ asd / sld ] ; ( e ) corrected total area of the scleral venous plexus on the ultrasound biomicrosopic image of the iridocorneal angle ( svpa ) [ svpa / sld ] ; ( f ) corrected scleral thickness ( st ) [ st / sld ] . the boxes define the 25th and 75th percentiles , with the median value indicated by a horizontal bar . data of asd in the group of no effect were calculated from 3 cases , because it could not be detected in the other 9 cases due to complete collapse of the ciliary cleft and/or the scleral venous plexus . glaucoma is a leading cause of blindness in dogs , and there are no perfect therapies for prevention of vision impairment in glaucomatous dogs [ 16 , 26 ] . this may be attributable to inadequate assessment of entire structural changes within the ica and lack of a predictive system of canine glaucoma . hence , anatomical structures of the ica were evaluated by ubm for comparison of its cross - sectional microstructures in healthy and glaucomatous live dogs . ubm clearly revealed abnormalities deep within the ica , especially on the ciliary cleft and the scleral venous plexus , in not only glaucomatous dogs but also in healthy canines with a predisposition to pcag , which were anatomical changes similar to those described in previous reports [ 4 , 6 , 8 , 10 , 16 , 18 ] . in predis , ubm values of the width and angle of the ciliary cleft were significantly lower than those of nor , although there were no significant differences in terms of the area of the ciliary cleft , the minimum distance between the angle recess and the scleral venous plexus , the total area of the scleral venous plexus and the scleral thickness between predis and nor , indicating that the pathway of the aqueous humor in the pcag - predisposed dogs would function as well as that in dogs with a normal open angle , despite the presence of structural abnormalities of the angular aperture ( the opening of the ciliary cleft ) . significant differences were found for ubm measurements related to the ciliary cleft between non - glaucomatous healthy dogs and animals with unilateral glaucoma ( fig . 4 ) . the width , angle and area of the ciliary cleft of uni were decreased significantly compared with those of nor and predis , and the 75th percentile values of the corrected width ( 0.041 ) and angle ( 6.0 ) of the ciliary cleft in uni were smaller than the lower limits of their reference ranges in nor . these quantitative results suggest unilateral pcag has decreased capacity of aqueous outflow from the anterior chamber in the eyes . however , the aqueous outflow might be able to retain a normal level , because there were no differences in the minimum distance between the angle recess and the scleral venous plexus , the total area of the scleral venous plexus and the scleral thickness between uni and healthy normotensive eye groups ( nor and predis ) . therefore , aqueous humor production should be restricted to decrease the aqueous outflow from the anterior chamber , thereby maintaining the balance of the aqueous inflow and outflow in the ciliary cleft to prevent onset of glaucoma . this proposal has already been accepted as prophylactic therapy in the management of canine glaucoma [ 17 , 18 , 26 ] . prophylactic medical treatment from the time of confirmed or presumed glaucoma in the first eye is helpful for delaying or preventing the onset of the disease in the second normotensive eye , although the median delay was about 22 months , less than 2 years . greater delay of the onset of glaucoma may be provided by early start of prophylactic treatment in dogs with a predisposition to pcag or unilateral type . ubm evaluation of the ica is helpful for providing objective criteria regarding the start of prophylactic treatment in dogs with a risk of glaucoma onset . judging from our results presented here , glaucomatous prophylaxis is suggested for cases in which the ubm value of the corrected width , angle or area of the ciliary cleft decreases and is less than the lower limit of the reference range in dogs with normotensive eyes with open angle ( nor ) . in glaucomatous dogs , ubm also revealed ica abnormalities that were available for management of canine glaucoma . 5 ) , namely , the opening of the ciliary cleft and the total area of the scleral venous plexus . both the opening of the ciliary cleft ( width and area of the ciliary cleft ) and the total area of the scleral venous plexus would represent valuable information for prediction of the responsiveness to medical therapies , because the opening of the ciliary cleft and the scleral venous plexus in cases that were responsive to the medical therapies were significantly larger than in dogs that were unresponsive ( fig . these findings suggest that the presence of observable ciliary cleft and scleral venous plexus is important for maintaining aqueous outflow from the anterior chamber in the eye . these ubm parameters are useful for prediction of the effects of medical therapies with ocular hypotensive agents . the opening of the ciliary cleft and the scleral venous plexus are key structures contributing to not only the pathophysiology of canine glaucoma [ 4 , 8 , 16 , 18 , 21 ] but also the responsiveness to medical therapy in glaucomatous eyes . the minimum distance between the angle recess and the scleral venous plexus was only measured in 10 of 19 hypertensive eyes in acg , because it could not be detected in the nine eyes with loss of the angle recess due to complete collapse of the ciliary cleft . no significant differences of the corrected minimum distance between the angle recess and the scleral venous plexus , and the scleral thickness were detected between acg and the other ocular groups , such as nor , predis and uni ( fig . 4 ) , suggesting that the minimum distance between the angle recess and the scleral venous plexus may only contribute to the aqueous outflow resistance in canine eyes , but may not be involved in the causes of increased iop . meanwhile , there were statistically significant differences in the corrected scleral thickness between acg and crg ( fig . these abnormalities might be associated with structural changes , such as scleral stretching or buphthalmos , induced by increased iop in the long term [ 12 , 16 , 18 ] . although gonioscopic examination is an essential diagnostic tool as a conventional procedure for assessing abnormalities of the ica in the field of veterinary ophthalmology , objective and quantitative information of the ica is needed to establish new diagnostic criteria for improving strategies of glaucomatous managements in animals . since ubm can provide objective and quantitative data of entire ica abnormalities which are unable to assess with classical gonioscopic examination , ubm examination should be more widely applied for detecting quantitative abnormalities of the ica in dogs , especially in glaucoma - predisposed dogs or cases suffering from unilateral glaucoma . the potential for ubm examinations to contribute to analysis of glaucomatous mechanisms and provide advanced diagnosis and management of glaucoma warrants further investigation in canine patients .

in a series of experiments , rhesus monkeys have been given in their diets 0.3 , 1.5 , and 25 ppm of a commercial polybrominated biphenyl ( pbb ) ( as firemaster ff-1 ) . the seven adult female monkeys receiving 0.3 ppm pbb have been on the treatment regime for 15 months and have consumed over 22 mg of pbb . during the initial 6 months of exposure , they lost weight and 2 of the animals develop sterile abscesses . at 6 months , 4 of the 7 animals had flattened and lengthened serum progesterone peaks . this change was correlated with an increase in length of their menstrual cycles . after 6 months of pbb exposure , the animals were bred . two of the 7 animals showed excessive and prolonged implantation bleeding . two abortions and 5 live births were recorded . all of the experimental infants were smaller than the controls at birth . the animals receiving a diet containing 1.5 ppm pbb for 36 weeks ( total intake 70 mg ) have shown a moderate weight loss and decrease in serum cholesterol . similar changes have also been recorded in the group given the 25 ppm pbb diet for 14 weeks ( approximately 500 mg total intake ) . in addition , these animals have also developed a hyperplastic gastritis.imagesfigure 1.figure 2.figure 3.figure 4.figure 5 .