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Biosystems Engineering and Control Group 20

15545700

Plasma concentrations of ir-inhibin, inhibin A, inhibin pro-alphaC, FSH, and estradiol-17beta during estrous cycle in mares and their relationship with follicular growth.

The relationship among plasma levels of immunoreactive (ir)-inhibin, inhibin A, inhibin pro-alphaC, FSH, estradiol-17beta and follicular growth were investigated during the normal estrous cycle in mares. Seven mares were used for two successive normal estrous cycles. Follicular diameters and ovulation were obtained by transrectal ultrasonography, and blood samples were collected daily by jugular venipuncture for hormonal assay. The results showed that inhibin A was inversely correlated (r = -0.59, p < 0.0001) with FSH indicating its contribution to negative feedback control of FSH secretions from the pituitary gland. Estradiol-17beta increased during the follicular phase reaching a peak (37.9 +/- 3.8 pg/mL) 2 d before ovulation. Estradiol-17beta was positively correlated (r = 0.78, p < 0.0001) with inhibin A. The high levels of inhibin A and estradiol-17beta were associated with the growth of the preovulatory dominant follicle and inversely correlated with FSH suggesting that both hormones are products of the large dominant follicles and were responsible for the decline in FSH secretion during the follicular phase of estrous cycle. In conclusion, an inverse relationship between inhibin A and FSH was clearly demonstrated indicating that inhibin A has a key role in the negative feedback control of FSH from the pituitary gland. In addition, inhibin A and estra-diol-17beta secretions were associated with the growth of the preovulatory dominant follicle and were positively correlated.

15545699

Erythrocyte osmotic fragility and oxidative stress in experimental hypothyroidism.

The present study was planned to explain the relation between erythrocyte osmotic fragility and oxidative stress and antioxidant statue in primary hypothyroid-induced experimental rats. Twenty-four Spraque Dawley type female rats were divided into two, as control (n = 12) and experimental (n = 12), groups weighing between 160 and 200 g. The experimental group animals have received tap water methimazole added standard fodder to block the iodine pumps for 30 d (75 mg/100 g). Control group animals were fed tap water and only standard fodder for the same period. At the end of 30 d blood samples were drawn from the abdominal aorta of the rats under ether anesthesia. T3, T4, and TSH levels were measured and the animals that had relatively lower T3, T4, and higher TSH levels were accepted as hypothyroid group. Hormone levels of the control group were at euthyroid conditions. Osmotic fragility, as a lipid peroxidation indicator malondialdehyde (MDA), antioxidant defense system indicators superoxide dismutase (SOD) and glutathione (GSH) levels were measured in the blood samples. Osmotic fragility test results: There was no statistically significant difference found between maximum osmotic hemolysis limit values of both group. Minimum osmotic hemolysis limit value of hypothyroid group was found to be higher than that of control group values (p < 0.02). The standard hemolysis and hemolytic increment curve of the hypothyroid group drawn according to osmotic fragility test results was found to be shifted to the right when compared to control group's curve. This situation and hemolytic increment value, which shows maximum hemolysis ratio, is the proof of increased osmotic fragility of the erythrocytes in hypothyroidism. There is no statistically significant difference found between hypothyroid and control groups in the lipid peroxidation indicator MDA and antioxidant indicators SOD and GSH levels. As a result of our study it may be concluded that hypothyroidism may lead to an increase in osmotic fragility of erythrocytes. But the increase in erythrocyte osmotic fragility does not originate from lipid peroxidation.

15545698

Wound instillation--the next step in negative pressure wound therapy. Lessons learned from initial experiences.

Negative pressure wound therapy uses a reticulated sponge and subatmospheric pressure to facilitate healing of a variety of wounds. The therapy appears to assist wound healing by decreasing wound bacterial burden and edema while facilitating granulation tissue formation. The latest development in negative pressure wound therapy allows clinicians to instill a solution into the wound and is indicated for use when patients receiving negative pressure wound therapy would benefit from the controlled delivery of topical wound treatment solutions and suspensions. A retrospective analysis of five cases was conducted to describe initial experiences with instillation and negative pressure wound therapy (average treatment time on negative pressure wound therapy with instillation 15 days, range 5 to 24 days). In two of the five cases where traditional negative pressure wound therapy was tried, infected wounds improved and culture results became negative following instillation of antibiotics in a saline solution. In all cases where used, instillation of a topical anesthetic appeared to effectively minimize wound pain that is occasionally associated with negative pressure wound therapy. Negative pressure wound therapy with instillation and possibly hyperbaric oxygen therapy may be initiated from the onset of treatment to manage high-risk wounds that appear severely infected and may be limb-threatening if located on an extremity. Controlled clinical studies to ascertain the efficacy and cost-effectiveness of this treatment modality are needed.

15545697

Comparing a foam composite to a hydrocellular foam dressing in the management of venous leg ulcers: a controlled clinical study.

Venous leg ulcers are the most prevalent form of chronic wounds in the Western world. The principles of moist wound healing coupled with the use of graduated compression bandaging have become the cornerstone of treatment for venous leg ulcers but not all moist dressings are alike. To compare the attributes of a foam composite dressing with those of a hydrocellular foam dressing in the management of venous leg ulcers, a prospective, randomized, comparative 12-week study was conducted in 15 centers in the US, Canada, France, Germany, and the UK. Dressings were changed and compression bandages applied per manufacturer recommendations and dressing performance was assessed at every dressing change and at the final evaluation. Patients with venous ulcers were randomized to treatment for 12 weeks with either hydrocellular foam (n = 52) or foam composite (n = 55) dressing. Healing differences between the groups were not statistically significant, with 36% of patients using foam composite dressing healed in a mean of 66 days and 39% of patients using hydrocellular foam dressing healed in a mean of 73 days. However, the foam composite dressing performed significantly better than the hydrocellular foam dressing with regard to condition of the periwound skin - 55% of patients in the foam composite group having healed or markedly improved surrounding skin compared to 37% of patients using hydrocellular foam (P = 0.03). The foam composite dressing was rated significantly better than the hydrocellular foam dressing ("very good" to "excellent") in level of satisfaction with conformability (87% and 75%, respectively, P = 0.05); being non-sensitizing (73% and 52%, respectively, P = 0.02); and ease of application (93% and 81%, respectively, P = 0.01). The findings reported in this study suggest that the foam composite dressing offers significant improvements in the quality of life of patients with venous leg ulcers as well as for their caregivers.

15545696

A three year multiphase pressure ulcer prevalence/incidence study in a regional referral hospital.

Pressure ulcers can have a devastating impact on health and care provision, ranging from patient discomfort and increased healthcare costs to a potential reflection on the quality of care. To evaluate the outcomes of prevention education and skin integrity interventions on the incidence of pressure ulcers, a multiphase project was initiated in an urban 154-bed regional referral community hospital in Ontario, Canada that provides care to an urban and rural population. The prevalence study included 84 adult subjects at baseline, 77 after one year (Phase 1), and 100 after 3 years (Phase 2). The Braden Scale for Predicting Pressure Sore Risk and a data collection form were used to record prevalence, incidence, stage and location of pressure ulcers, and related documented interventions. Incidence data were obtained from patient charts and defined as ulcers that developed over 24 hours following admission. Phase 1 interventions involved staff education and replacement of existing skin care products. Phase 2 interventions included adoption of pressure ulcer prevention protocols, advanced wound care products, improved support surface usage, modification of documentation methods, and staff education. Of the 84 patients assessed at baseline , 15 (17.9 %) developed 22 pressure ulcers compared to 4 of 77 (5.2%) during Phase 1 and 2 out of 100 (2.0%) during Phase 2. The difference between baseline and both subsequent time points was statistically significant (P greater than 0.05). These results suggest that education and the implementation of appropriate skin care products and procedures and pressure ulcer prevention protocols may reduce the incidence of hospital-acquired pressure ulcers.

15545678

Better reporting of harms in randomized trials: an extension of the CONSORT statement.

In response to overwhelming evidence and the consequences of poor-quality reporting of randomized, controlled trials (RCTs), many medical journals and editorial groups have now endorsed the CONSORT (Consolidated Standards of Reporting Trials) statement, a 22-item checklist and flow diagram. Because CONSORT primarily aimed at improving the quality of reporting of efficacy, only 1 checklist item specifically addressed the reporting of safety. Considerable evidence suggests that reporting of harms-related data from RCTs also needs improvement. Members of the CONSORT Group, including journal editors and scientists, met in Montebello, Quebec, Canada, in May 2003 to address this problem. The result is the following document: the standard CONSORT checklist with 10 new recommendations about reporting harms-related issues, accompanying explanation, and examples to highlight specific aspects of proper reporting. We hope that this document, in conjunction with other CONSORT-related materials (http://www.consort-statement.org), will help authors improve their reporting of harms-related data from RCTs. Better reporting will help readers critically appraise and interpret trial results. Journals can support this goal by revising Instructions to Authors so that they refer authors to this document.

15545677

Measuring the quality of physician practice by using clinical vignettes: a prospective validation study.

Worldwide efforts are under way to improve the quality of clinical practice. Most quality measurements, however, are poorly validated, expensive, and difficult to compare among sites. To validate whether vignettes accurately measure the quality of clinical practice by using a comparison with standardized patients (the gold standard method), and to determine whether vignettes are a more or less accurate method than medical record abstraction. Prospective, multisite study. Outpatient primary care clinics in 2 Veterans Affairs medical centers and 2 large, private medical centers. 144 of 163 eligible physicians agreed to participate, and, of these, 116 were randomly selected to see standardized patients, to complete vignettes, or both. Scores, expressed as the percentage of explicit quality criteria correctly completed, were obtained by using 3 methods. Among all physicians, the quality of clinical practice as measured by the standardized patients was 73% correct (95% CI, 72.1% to 73.4%). By using exactly the same criteria, physicians scored 68% (CI, 67.9% to 68.9%) when measured by the vignettes but only 63% (CI, 62.7% to 64.0%) when assessed by medical record abstraction. These findings were consistent across all diseases and were independent of case complexity or physician training level. Vignettes also accurately measured unnecessary care. Finally, vignettes seem to capture the range in the quality of clinical practice among physicians within a site. Despite finding variation in the quality of clinical practice, we did not determine whether poorer quality translated into worse health status for patients. In addition, the quality scores are based on measurements from 1 patient-provider interaction. As with all other scoring criteria, vignette criteria must be regularly updated. Vignettes are a valid tool for measuring the quality of clinical practice. They can be used for diverse clinical settings, diseases, physician types, and situations in which case-mix variation is a concern. They are inexpensive and easy to use. Vignettes are particularly useful for comparing quality among and within sites and may be useful for longitudinal evaluations of interventions intended to change clinical practice.

15545676

Taking glucocorticoids by prescription is associated with subsequent cardiovascular disease.

Glucocorticoids have adverse systemic effects, including obesity, hypertension, and hyperglycemia, that may predispose to cardiovascular disease. The effect of glucocorticoid use on cardiovascular disease has not been quantified. To test the hypothesis that users of exogenous glucocorticoids have an increased risk for cardiovascular disease. A cohort study using a record linkage database. Tayside, Scotland, United Kingdom. 68,781 glucocorticoid users and 82,202 nonusers without previous hospitalization for cardiovascular disease who were studied between 1993 and 1996. The average daily dose of glucocorticoid exposure during follow-up was categorized as low (inhaled, nasal, and topical only), medium (oral, rectal, or parenteral <7.5 mg of prednisolone equivalent), or high (> or =7.5 mg of prednisolone equivalent). Poisson regression model, sensitivity analysis, and propensity score methods were used to investigate the association between glucocorticoid exposure and cardiovascular outcome. 4383 cardiovascular events occurred in 257,487 person-years of follow-up for a rate of 17.0 (95% CI, 16.5 to 17.5) per 1000 person-years in the comparator group, and 5068 events occurred in 212,287 person-years for a rate of 23.9 (CI, 23.2 to 24.5) per 1000 person-years in the group exposed to glucocorticoids (22.1, 27.2, and 76.5 in low, medium, and high groups, respectively). The absolute risk difference was 6.9 (CI, 6.0 to 7.7) per 1000 person-years (5.1, 10.1, and 59.4, respectively). After adjustment for known covariates, the relative risk for a cardiovascular event in patients receiving high-dose glucocorticoids was 2.56 (CI, 2.18 to 2.99). Because the data were observational, residual confounding cannot be excluded. Treatment with high-dose glucocorticoids seemed to be associated with increased risk for cardiovascular disease.

15545675

Comparison of endoscopic ultrasonography and multidetector computed tomography for detecting and staging pancreatic cancer.

Accurate preoperative detection and staging of pancreatic cancer may identify patients with locoregional disease that is amenable to surgical resection. To compare endoscopic ultrasonography and multidetector computed tomography (CT) for the detection, staging, and resectability of known or suspected locoregional pancreatic cancer. Prospective, observational, cohort study. Single, tertiary referral hospital in Indianapolis, Indiana. 120 participants with known or suspected locoregional pancreatic cancer. Endoscopic ultrasonography followed by multidetector CT was performed in all patients. Patients with known or suspected pancreatic cancer deemed potentially resectable by 1 or both tests were considered for surgery. Detection, staging, and resectability of pancreatic cancer. Surgically resected pancreatic cancer with negative microscopic histologic margins was considered resectable. Of 120 patients enrolled, 104 (87%) underwent endoscopic ultrasonography and CT. Of the 80 patients with pancreatic cancer, 27 (34%) were managed nonoperatively, and 53 (66%) treated surgically had resectable (n = 25) or unresectable (n = 28) cancer. For the 80 patients with cancer, the sensitivity of endoscopic ultrasonography (98% [95% CI, 91% to 100%]) for detecting a pancreatic mass was greater than that of CT (86% [CI, 77% to 93%]; P = 0.012). For the 53 surgical patients, endoscopic ultrasonography was superior to CT for tumor staging accuracy (67% vs. 41%; P < 0.001) but equivalent for nodal staging accuracy (44% vs. 47%; P > 0.2). Of the 25 resectable pancreatic tumors in patients recommended for surgery, endoscopic ultrasonography and CT correctly identified 88% and 92%, respectively, as resectable. Of the 28 unresectable pancreatic tumors in patients recommended for surgery, endoscopic ultrasonography and CT correctly identified 68% and 64%, respectively, as unresectable. Radiologists who read the scans and endosonographers were not blinded to previous radiographic information. Because of the modest sample size, CIs of the sensitivity estimates were sometimes wide. Compared with multidetector CT, endoscopic ultrasonography is superior for tumor detection and staging but similar for nodal staging and resectability of preoperatively suspected nonmetastatic pancreatic cancer.

15545674

Advanced age, anticoagulation intensity, and risk for intracranial hemorrhage among patients taking warfarin for atrial fibrillation.

The risk for atrial fibrillation-associated stroke increases at low anticoagulation intensities. However, higher intensities increase hemorrhage risk. Optimal use of warfarin for atrial fibrillation requires precise information on the risk for intracranial hemorrhage as a function of patient age and anticoagulation intensity. To examine the relationship of age, anticoagulation intensity, and risk for intracranial hemorrhage. Case-control study. Academic medical center. 170 case-patients who developed intracranial hemorrhage during warfarin therapy and 1020 matched controls who did not; both case-patients and controls were taking warfarin for atrial fibrillation. The authors performed multivariable conditional logistic regression to determine the odds of intracranial hemorrhage with regard to age and international normalized ratio (INR), controlling for comorbid conditions and aspirin use. Case-patients were older than controls (median age, 78 years vs. 75 years; P < 0.001) and had higher median INRs (2.7 vs. 2.3; P < 0.001). The risk for intracranial hemorrhage increased at 85 years of age or older (adjusted odds ratio, 2.5 [95% CI, 1.3 to 4.7]; referent age, 70 to 74 years) and at an INR range of 3.5 to 3.9 (adjusted odds ratio, 4.6 [CI, 2.3 to 9.4]; referent INR, 2.0 to 3.0). The risk for intracranial hemorrhage at INRs less than 2.0 did not differ statistically from the risk at INRs of 2.0 to 3.0 (adjusted odds ratio, 1.3 [CI, 0.8 to 2.2]). Although duration of anticoagulation has been associated with hemorrhage in other studies, the current study could not control for this potential confounder. The risk for intracranial hemorrhage increases at age 85 years. International normalized ratios less than 2.0 were not associated with lower risk for intracranial hemorrhage compared with INRs between 2.0 and 3.0. Therefore, anticoagulation management should focus on maintaining INRs in the 2.0 to 3.0 range, even in elderly patients with atrial fibrillation, rather than targeting INRs less than 2.0. Similarly, INRs of 3.5 or greater should be avoided.

15545669

Sequencing of tamoxifen and radiotherapy after breast-conserving surgery in early-stage breast cancer.

Tamoxifen (TAM) is thought to exert a cytostatic effect on hormone-sensitive breast cancer cells. Some preclinical studies show reduced radiosensitivity in irradiated malignant mammary epithelial cells when pretreated with TAM; other studies refute these results. Recent randomized clinical trials suggest an antagonistic effect of TAM on cytotoxic therapy, with improved disease-free survival (DFS) with sequential versus concurrent TAM. An exploratory analysis was undertaken to evaluate the optimal sequencing of TAM and radiotherapy (RT) after breast-conserving surgery. Southwest Oncology Group trial 8897 (Intergroup 0102) randomly assigned node-negative women with T1-3 breast cancers to cyclophosphamide, doxorubicin, fluorouracil (CAF); CAF --> TAM; cyclophosphamide, methotrexate, fluorouracil (CMF); and CMF --> TAM. For this analysis, data are reported only in the TAM groups. RT was allowed either before adjuvant therapy (sequential [SEQ] RT; 107 patients) or after chemotherapy but concurrent with TAM (concurrent [CONC] RT; 202 patients). Survival data were adjusted for receptor status, age, and tumor size. With a median follow-up of 10.3 years, 10-year DFS values were 83% and 83% for CONC versus SEQ RT groups (log-rank P = .73; P = .76 adjusted for patient characteristics), and 10-year overall survivals were 88% and 90%, respectively (log-rank P = .59; adjusted P = .65). Patterns of failure showed no increase in in-breast recurrence rates between CONC RT and SEQ RT groups, with 10-year local recurrence rates of 7% for CONC RT and 5% for SEQ RT (hazard ratio, 0.73; 95% CI, 0.26 to 2.04; P = .54). The current analysis does not suggest an adverse effect on local or systemic control with CONC versus SEQ TAM and RT in node-negative breast cancer. A randomized trial is encouraged to validate these results.

15545668

KIT and platelet-derived growth factor receptor alpha tyrosine kinase gene mutations and KIT amplifications in human solid tumors.

Mutated KIT and platelet-derived growth factor receptor alpha (PDGFRalpha) tyrosine kinases are the principal targets for imatinib mesylate in the treatment of gastrointestinal stromal tumors (GISTs). The frequency of activating KIT and PDGFRA gene mutations in most other histologic types of human cancer is not known. KIT exons 9, 11, 13, and 17 and PDGFRA exons 11 and 17 of 334 human cancers were screened for mutations using sensitive denaturing high-performance liquid chromatography (DHPLC). In addition, all KIT exons from 9 to 21 of 115 tumors were screened. Thirty-two histologic tumor types were examined. Samples with abnormal findings in DHLPC were sequenced. Immunostaining for the KIT protein (CD117) was performed in 322 (96.4%) of the 334 cases. Of the 3,039 exons screened, only 17 had mutation. All 17 cases with either mutated KIT (n = 15) or PDGFRA (n = 2) were histologically GIST tumors, whereas none of the other histologic types of cancer (n = 316) harbored KIT or PDGFRA mutation. KIT immunostaining was rarely positive except in GISTs (18 of 18), small-cell lung cancer (10 of 30; 33%), and testicular teratocarcinoma (four of 17; 24%). Wild-type KIT gene amplification or chromosome 4 aneuploidy was common (seven of 12) in non-GIST tumors with strong KIT protein expression when studied with fluorescence in situ hybridization. Despite frequent KIT protein expression in some tumor types, KIT and PDGFRA gene mutations are uncommon in most human cancers. Cancer KIT expression is frequently associated with multiple copies of the wild-type KIT gene.

15545666

Sequence of radiotherapy with tamoxifen in conservatively managed breast cancer does not affect local relapse rates.

To evaluate whether the sequencing of tamoxifen (TAM) relative to radiation (RT) affects outcome in breast cancer patients treated with conservative surgery (CS) plus RT (lumpectomy with RT). Between 1976 and 1999, 1,649 patients with stage I or II breast cancer were treated with CS plus RT at Yale-New Haven Hospital (New Haven, CT). TAM was administered to 500 patients. The timing of TAM relative to RT was documented for each patient. Of the 500 patients, the timing of TAM was unclear in five patients, was administered concurrently with RT in 254 patients (CON-TAM), and was administered sequentially after completion of RT in 241 patients (SEQ-TAM). There were no differences between the CON-TAM and SEQ-TAM group in T stage, estrogen and progesterone status, nodal status, histology, or margin status. The CON-TAM group was slightly older than the SEQ-TAM group (62 v 58 years) and received chemotherapy in addition to TAM less frequently (14% v 38%). As of September 2002, with a median follow-up of 10.0 years, there were no significant differences between the CON-TAM and SEQ-TAM groups in overall survival (84% v 82%; hazard ratio [HR], 1.234; 95% CI, 0.42 to 2.05; P = .45), distant-metastasis-free rate (82% v 78%; HR, 1.55; 95% CI, 0.89 to 2.68; P = .12), ipsilateral breast-relapse-free rate (90% v 86%; HR, 0.932; 95% CI, 0.42 to 2.05; P = .86), or contralateral breast-relapse-free rate (95% v 93%; HR, 0.892; 95% CI, 0.53 to 1.48; P = .66). Although the concurrent use of TAM with RT may theoretically render cancer cells less responsive to RT, this retrospective study suggests that in practical application, concurrent administration of TAM with RT does not compromise local control.

15545665

Impact of concurrent versus sequential tamoxifen with radiation therapy in early-stage breast cancer patients undergoing breast conservation treatment.

To assess the impact of sequencing of tamoxifen and radiation therapy (RT) on outcomes in early-stage breast cancer. This retrospective study evaluates the effect of the sequence of tamoxifen with RT on outcomes in stage I to II breast cancer patients who underwent breast-conservation treatment (BCT) and received adjuvant tamoxifen, with or without adjuvant chemotherapy. Patients were grouped as concurrent (tamoxifen given during RT followed by continued tamoxifen; 174 patients) and sequential (RT followed by tamoxifen; 104 patients). Median follow-up after RT was 8.6 years for both groups. The pathologic T and N stage, race, estrogen and progesterone status, number of positive nodes, and RT were comparable between the two groups (all P >/= .08). More women age 49 years or younger and women who received chemotherapy were in the sequential group than the concurrent group (6% and 25%, respectively; P < .0001). The sequence of tamoxifen therapy did not influence 10-year local recurrence rates (sequential, 7%; concurrent, 3%; P = .52), overall survival (sequential, 86%; concurrent, 81%; P = .64), or relapse-free survival (sequential, 76%; concurrent, 85%; P = .35). When adjusting age and chemotherapy use in the multivariable Cox model, hazard ratios comparing sequential versus concurrent tamoxifen therapy were 1.56 (95% CI, 0.87 to 2.79), 1.23 (95% CI, 0.63 to 2.41), and 1.22 (95% CI, 0.33 to 4.49) for the overall survival, relapse-free survival, and local recurrence, respectively. The therapeutic regimens of tamoxifen given concurrently or sequentially with RT both appear to be reasonable options for patients treated with BCT.

15545664

American Society of Clinical Oncology technology assessment on the use of aromatase inhibitors as adjuvant therapy for postmenopausal women with hormone receptor-positive breast cancer: status report 2004.

To update the 2003 American Society of Clinical Oncology technology assessment on adjuvant use of aromatase inhibitors. Based on results from multiple large randomized trials, adjuvant therapy for postmenopausal women with hormone receptor-positive breast cancer should include an aromatase inhibitor in order to lower the risk of tumor recurrence. Neither the optimal timing nor duration of aromatase inhibitor therapy is established. Aromatase inhibitors are appropriate as initial treatment for women with contraindications to tamoxifen. For all other postmenopausal women, treatment options include 5 years of aromatase inhibitors treatment or sequential therapy consisting of tamoxifen (for either 2 to 3 years or 5 years) followed by aromatase inhibitors for 2 to 3, or 5 years. Patients intolerant of aromatase inhibitors should receive tamoxifen. There are no data on the use of tamoxifen after an aromatase inhibitor in the adjuvant setting. Women with hormone receptor-negative tumors should not receive adjuvant endocrine therapy. The role of other biomarkers such as progesterone receptor and HER2 status in selecting optimal endocrine therapy remains controversial. Aromatase inhibitors are contraindicated in premenopausal women; there are limited data concerning their role in women with treatment-related amenorrhea. The side effect profiles of tamoxifen and aromatase inhibitors differ. The late consequences of aromatase inhibitor therapy, including osteoporosis, are not well characterized. The Panel believes that optimal adjuvant hormonal therapy for a postmenopausal woman with receptor-positive breast cancer includes an aromatase inhibitor as initial therapy or after treatment with tamoxifen. Women with breast cancer and their physicians must weigh the risks and benefits of all therapeutic options.

15545661

Effect of addition of adjuvant paclitaxel on radiotherapy delivery and locoregional control of node-positive breast cancer: cancer and leukemia group B 9344.

We compared radiotherapy (RT) delivery and locoregional control in patients with node-positive breast cancer randomly assigned on Cancer and Leukemia Group B 9344 to receive adjuvant doxorubicin/cyclophosphamide (AC) with patients assigned to receive AC followed by paclitaxel (AC+T). Eligible patients were randomly assigned to receive adjuvant AC versus AC+T chemotherapy. RT was required if breast-conserving surgery was performed but was elective after mastectomy. Information about RT delivery was retrospectively collected. Cumulative incidence of locoregional recurrence (LRR), use of elective RT, and RT delivery were compared between treatment arms. For patients treated with breast-conserving surgery and RT, the 5-year cumulative incidence of isolated LRR was 9.7% in the AC arm and 3.7% in the AC+T arm (P = .04) and of LRR as any component of failure was 12.9% versus 6.1%, respectively (P = .04). Although LRR rates in patients who did not receive postmastectomy RT were lower in the AC+T arm, the difference was not statistically significant. Despite the lack of protocol guidelines, RT use did not differ between arms, nor did RT dose, treatment interruption, or completion. Despite the delay to RT during additional chemotherapy, adjuvant AC+T afforded better local control than AC alone in patients treated with breast-conserving therapy. Addition of paclitaxel did not adversely affect delivery or ability to tolerate RT, as indicated by similar rates of completion of timely, full-dose RT between arms.

15545660

Selection on Glycine beta-1,3-endoglucanase genes differentially inhibited by a Phytophthora glucanase inhibitor protein.

Plant endo-beta-1,3-glucanases (EGases) degrade the cell wall polysaccharides of attacking pathogens and release elicitors of additional plant defenses. Isozymes EGaseA and EGaseB of soybean differ in susceptibility to a glucanase inhibitor protein (GIP1) produced by Phytophthora sojae, a major soybean pathogen. EGaseA, the major elicitor-releasing isozyme, is a high-affinity ligand for GIP1, which completely inhibits it, whereas EGaseB is unaffected by GIP1. We tested for departures from neutral evolution on the basis of partial sequences of EGaseA and EGaseB from 20 widespread accessions of Glycine soja (the wild progenitor of soybean), from 4 other Glycine species, and across dicotyledonous plants. G. soja exhibited little intraspecific variation at either locus. Phylogeny-based codon evolution models detected strong evidence of positive selection on Glycine EGaseA and weaker evidence for selection on dicot EGases and Glycine EGaseB. Positively selected peptide sites were identified and located on a structural model of EGase bound to GIP1. Positively selected sites and highly variable sites were found disproportionately within 4.5 angstroms of bound GIP1. Low variation within G. soja EGases, coupled with positive selection in both Glycine and dicot lineages and the proximity of rapidly evolving sites to GIP1, suggests an arms race involving repeated adaptation to pathogen attack and inhibition.

15545655

Conserved locus-specific silencing functions of Schizosaccharomyces pombe sir2+.

In Schizosaccharomyces pombe, three genes, sir2(+), hst2(+), and hst4(+), encode members of the Sir2 family of conserved NAD(+)-dependent protein deacetylases. The S. pombe sir2(+) gene encodes a nuclear protein that is not essential for viability or for resistance to treatment with UV or a microtubule-destabilizing agent. However, sir2(+) is essential for full transcriptional silencing of centromeres, telomeres, and the cryptic mating-type loci. Chromatin immunoprecipitation results suggest that the Sir2 protein acts directly at these chromosomal regions. Enrichment of Sir2p at silenced regions does not require the HP1 homolog Swi6p; instead, Swi6-GFP localization to telomeres depends in part on Sir2p. The phenotype of sir2 swi6 double mutants supports a model whereby Sir2p functions prior to Swi6p at telomeres and the silent mating-type loci. However, Sir2p does not appear to be essential for the localization of Swi6p to centromeric foci. Cross-complementation experiments showed that the Saccharomyces cerevisiae SIR2 gene can function in place of S. pombe sir2(+), suggesting overlapping deacetylation substrates in both species. These results also suggest that, despite differences in most of the other molecules required, the two distantly related yeast species share a mechanism for targeting Sir2p homologs to silent chromatin.

15545654

Gene genealogy and properties of test statistics of neutrality under population growth.

We consider the Wright-Fisher model with exponential population growth and investigate effects of population growth on the shape of genealogy and the distributions of several test statistics of neutrality. In the limiting case as the population grows rapidly, the rapid-growth-limit genealogy is characterized. We obtained approximate expressions for expectations and variances of test statistics in the rapid-growth-limit genealogy and star genealogy. The distributions in the star genealogy are narrower than those in the cases of the simulated and rapid-growth-limit genealogies. The expectations and variances of the test statistics are monotone decreasing functions of the time length of the expansion, and the higher power of R(2) against population growth is suggested to be due to their smaller variances rather than to change of the expectations. We also investigated by simulation how quickly the distributions of test statistics approach those of the rapid-growth-limit genealogy.

15545653

Patterns of selection on synonymous and nonsynonymous variants in Drosophila miranda.

We have investigated patterns of within-species polymorphism and between-species divergence for synonymous and nonsynonymous variants at a set of autosomal and X-linked loci of Drosophila miranda. D. pseudoobscura and D. affinis were used for the between-species comparisons. The results suggest the action of purifying selection on nonsynonymous, polymorphic variants. Among synonymous polymorphisms, there is a significant excess of synonymous mutations from preferred to unpreferred codons and of GC to AT mutations. There was no excess of GC to AT mutations among polymorphisms at noncoding sites. This suggests that selection is acting to maintain the use of preferred codons. Indirect evidence suggests that biased gene conversion in favor of GC base pairs may also be operating. The joint intensity of selection and biased gene conversion, in terms of the product of effective population size and the sum of the selection and conversion coefficients, was estimated to be approximately 0.65.

15545651

Lig4 and rad54 are required for repair of DNA double-strand breaks induced by P-element excision in Drosophila.

Site-specific double-strand breaks (DSBs) were generated in the white gene located on the X chromosome of Drosophila by excision of the w(hd) P-element. To investigate the role of nonhomologous end joining (NHEJ) and homologous recombination (HR) in the repair of these breaks, the w(hd) P-element was mobilized in flies carrying mutant alleles of either lig4 or rad54. The survival of both lig4- and rad54-deficient males was reduced to 25% in comparison to the wild type, indicating that both NHEJ and HR are involved in the repair P-induced gaps in males. Survival of lig4-deficient females was not affected at all, implying that HR using the homologous chromosome as a template can partially compensate for the impaired NHEJ pathway. In rad54 mutant females survival was reduced to 70% after w(hd) excision. PCR analysis indicated that the undamaged homologous chromosome may compensate for the potential loss of the broken chromosome in rad54 mutant females after excision. Molecular analysis of the repair junctions revealed microhomology (2-8 bp)-dependent DSB repair in most products. In the absence of Lig4, the 8-bp target site duplication is used more frequently for repair. Our data indicate the presence of efficient alternative end-joining mechanisms, which partly depend on the presence of microhomology but do not require Lig4.

15545650

Interaction between the oxa1 and rmp1 genes modulates respiratory complex assembly and life span in Podospora anserina.

A causal link between deficiency of the cytochrome respiratory pathway and life span was previously shown in the filamentous fungus Podospora anserina. To gain more insight into the relationship between mitochondrial function and life span, we have constructed a strain carrying a thermosensitive mutation of the gene oxa1. OXA1 is a membrane protein conserved from bacteria to human. The mitochondrial OXA1 protein is involved in the assembly/insertion of several respiratory complexes. We show here that oxa1 is an essential gene in P. anserina. The oxa1(ts) mutant exhibits severe defects in the respiratory complexes I and IV, which are correlated with an increased life span, a strong induction of the alternative oxidase, and a reduction in ROS production. However, there is no causal link between alternative oxidase level and life span. We also show that in the oxa1(ts) mutant, the extent of the defects in complexes I and IV and the life-span increase depends on the essential gene rmp1. The RMP1 protein, whose function is still unknown, can be localized in the mitochondria and/or the cytosolic compartment, depending on the developmental stage. We propose that the RMP1 protein could be involved in the process of OXA1-dependent protein insertion.

15545649

Protein kinase A regulates constitutive expression of small heat-shock genes in an Msn2/4p-independent and Hsf1p-dependent manner in Saccharomyces cerevisiae.

Hsf1p, the heat-shock transcription factor from Saccharomyces cerevisiae, has a low level of constitutive transcriptional activity and is kept in this state through negative regulation. In an effort to understand this negative regulation, we developed a novel genetic selection that detects altered expression from the HSP26 promoter. Using this reporter strain, we identified mutations and dosage compensators in the Ras/cAMP signaling pathway that decrease cAMP levels and increase expression from the HSP26 promoter. In yeast, low cAMP levels reduce the catalytic activity of the cAMP-dependent kinase PKA. Previous studies had proposed that the stress response transcription factors Msn2p/4p, but not Hsf1p, are repressed by PKA. However, we found that reduction or elimination of PKA activity strongly derepresses transcription of the small heat-shock genes HSP26 and HSP12, even in the absence of MSN2/4. In a strain deleted for MSN2/4 and the PKA catalytic subunits, expression of HSP12 and HSP26 depends on HSF1 expression. Our findings indicate that Hsf1p functions downstream of PKA and suggest that PKA might be involved in negative regulation of Hsf1p activity. These results represent a major change in our understanding of how PKA signaling influences the heat-shock response and heat-shock protein expression.

15545648

Genetic factors that regulate the attenuation of the general stress response of yeast.

The general stress response of yeast involves the induction of approximately 200 genes in response to any one of several stresses. These genes are activated by Msn2 and repressed by the Srb10 kinase, a member of the mediator complex. Normally, Msn2 is exported from the nucleus, and Srb10 represses STRE gene expression. Under stress, Msn2 relocalizes to the nucleus and, with the relief of Srb10 repression, activates transcription. The stress response is rapid, but quickly attenuated. We show here that this attenuation is due to a nuclear-dependent degradation of Msn2. Msn2 rapidly disappeared from cells after heat or osmotic shock. This disappearance was not due to a change in MSN2 RNA levels, which remain constant during stress. Pulse-chase experiments confirmed the stress-dependent Msn2 degradation. The levels of Msn2 were significantly reduced in msn5 deletion cells that have been shown to constitutively retain Msn2 in the nucleus. The degradation was Srb10-dependent; Msn2 was not degraded in an srb10 deletion mutant. An Msn2 internal deletion mutant was insensitive to Srb10 repression, but was degraded by the Srb10-dependent mechanism. Thus, this mutation uncoupled Srb10 repression from degradation.

15545647

The genomes of recombinant inbred lines.

Recombinant inbred lines (RILs) can serve as powerful tools for genetic mapping. Recently, members of the Complex Trait Consortium proposed the development of a large panel of eight-way RILs in the mouse, derived from eight genetically diverse parental strains. Such a panel would be a valuable community resource. The use of such eight-way RILs will require a detailed understanding of the relationship between alleles at linked loci on an RI chromosome. We extend the work of Haldane and Waddington on two-way RILs and describe the map expansion, clustering of breakpoints, and other features of the genomes of multiple-strain RILs as a function of the level of crossover interference in meiosis.

15545646

Meiotic recombination in Drosophila females depends on chromosome continuity between genetically defined boundaries.

In the pairing-site model, specialized regions on each chromosome function to establish meiotic homolog pairing. Analysis of these sites could provide insights into the mechanism used by Drosophila females to form a synaptonemal complex (SC) in the absence of meiotic recombination. These specialized sites were first established on the X chromosome by noting that there were barriers to crossover suppression caused by translocation heterozygotes. These sites were genetically mapped and proposed to be pairing sites. By comparing the cytological breakpoints of third chromosome translocations to their patterns of crossover suppression, we have mapped two sites on chromosome 3R. We have performed experiments to determine if these sites have a role in meiotic homolog pairing and the initiation of recombination. Translocation heterozygotes exhibit reduced gene conversion within the crossover-suppressed region, consistent with an effect on the initiation of meiotic recombination. To determine if homolog pairing is disrupted in translocation heterozygotes, we used fluorescent in situ hybridization to measure the extent of homolog pairing. In wild-type oocytes, homologs are paired along their entire lengths prior to accumulation of the SC protein C(3)G. Surprisingly, translocation heterozygotes exhibited homolog pairing similar to wild type within the crossover-suppressed regions. This result contrasted with our observations of c(3)G mutant females, which were found to be defective in pairing. We propose that each Drosophila chromosome is divided into several domains by specialized sites. These sites are not required for homolog pairing. Instead, the initiation of meiotic recombination requires continuity of the meiotic chromosome structure within each of these domains.

15545645

RNA silencing in Aspergillus nidulans is independent of RNA-dependent RNA polymerases.

The versatility of RNA-dependent RNA polymerases (RDRPs) in eukaryotic gene silencing is perhaps best illustrated in the kingdom Fungi. Biochemical and genetic studies of Schizosaccharomyces pombe and Neurospora crassa show that these types of enzymes are involved in a number of fundamental gene-silencing processes, including heterochromatin regulation and RNA silencing in S. pombe and meiotic silencing and RNA silencing in N. crassa. Here we show that Aspergillus nidulans, another model fungus, does not require an RDRP for inverted repeat transgene (IRT)-induced RNA silencing. However, RDRP requirements may vary within the Aspergillus genus as genomic analysis indicates that A. nidulans, but not A. fumigatus or A. oryzae, has lost a QDE-1 ortholog, an RDRP associated with RNA silencing in N. crassa. We also provide evidence suggesting that 5' --> 3' transitive RNA silencing is not a significant aspect of A. nidulans IRT-RNA silencing. These results indicate a lack of conserved kingdom-wide requirements for RDRPs in fungal RNA silencing.

15545643

An active transposable element, Herves, from the African malaria mosquito Anopheles gambiae.

Transposable elements have proven to be invaluable tools for genetically manipulating a wide variety of plants, animals, and microbes. Some have suggested that they could be used to spread desirable genes, such as refractoriness to Plasmodium infection, through target populations of Anopheles gambiae, thereby disabling the mosquito's ability to transmit malaria. To achieve this, a transposon must remain mobile and intact after the initial introduction into the genome. Endogenous, active class II transposable elements from An. gambiae have not been exploited as gene vectors/drivers because none have been isolated. We report the discovery of an active class II transposable element, Herves, from the mosquito An. gambiae. Herves is a member of a distinct subfamily of hAT elements that includes the hopper-we element from Bactrocera dorsalis and B. cucurbitae. Herves was transpositionally active in mobility assays performed in Drosophila melanogaster S2 cells and developing embryos and was used as a germ-line transformation vector in D. melanogaster. Herves displays an altered target-site preference from the distantly related hAT elements, Hermes and hobo. Herves is also present in An. arabiensis and An. merus with copy numbers similar to that found in An. gambiae. Preliminary data from an East African population are consistent with the element being transpositionally active in mosquitoes.

15545644

Protecting haploid polymorphisms in temporally variable environments.

Analysis of a continuous-time model shows that a protected polymorphism can arise in a haploid population subject to temporal fluctuations in selection. The requirements are that population size is regulated in a density-dependent manner and that an allele's arithmetic mean relative growth rate is greater than one when rare and that its harmonic mean relative growth rate is less than one when common. There is no requirement that relative growth rate be frequency dependent. Comparisons with discrete-time models show that the standard formalism used by population genetics ignores forced changes in generation time as rare advantageous alleles sweep into a population. In temporally variable environments, frequency-dependent changes in generation times tend to counteract these invasions. Such changes can prevent fixation and protect polymorphisms.

15545642

Missense mutations that inactivate the Aspergillus nidulans nrtA gene encoding a high-affinity nitrate transporter.

The transport of nitrate into prokaryotic and eukaryotic cells, of considerable interest to agriculture, ecology, and human health, is carried out by members of a distinct cluster of proteins within the major facilitator superfamily. To obtain structure/function information on this important class of nitrate permeases, a collection of chemically induced mutations in the nrtA gene encoding a 12-transmembrane domain, high-affinity nitrate transporter from the eukaryote Aspergillus nidulans was isolated and characterized. This mutational analysis, coupled with protein alignments, demonstrates the utility of the approach to predicting peptide motifs and individual residues important for the movement of nitrate across the membrane. These include the highly conserved nitrate signature motif (residues 166-173) in Tm 5, the conserved charged residues Arg87 (Tm 2) and Arg368 (Tm 8), as well as the aromatic residue Phe47 (Tm 1), all within transmembrane helices. No mutations were observed in the large central loop (Lp 6/7) between Tm 6 and Tm 7. Finally, the study of a strain with a conversion of Trp481 (Tm 12) to a stop codon suggests that all 12 transmembrane domains and/or the C-terminal tail are required for membrane insertion and/or stability of NrtA.

15545641

Evidence for abundant slightly deleterious polymorphisms in bacterial populations.

The nearly neutral theory of molecular evolution predicts that slightly deleterious mutations subject to purifying selection are widespread in natural populations, particularly those of large effective population size. To test this hypothesis, the standardized difference between pairwise nucleotide difference and number of segregation sites (corrected for number of sequences) was estimated for 149 population data sets from 84 species of bacteria. This quantity (Tajima's D-statistic) was estimated separately for synonymous (D(syn)) and nonsynonymous (D(non)) polymorphisms. D(syn) was positive in 70% of data sets, and the overall median D(syn) (0.873) was positive. By contrast D(non) was negative in 68% of data sets, and the overall median D(non) (-0.656) was negative. The preponderance of negative values of D(non) is evidence that there are widespread rare nonsynonymous polymorphisms in the process of being eliminated by purifying selection, as predicted to occur in populations with large effective size by the nearly neutral theory. The major exceptions to this trend were seen among surface proteins, particularly those of bacteria parasitic on vertebrates, which included a number of cases of polymorphisms apparently maintained by balancing selection.

15545640

Bonus, a Drosophila TIF1 homolog, is a chromatin-associated protein that acts as a modifier of position-effect variegation.

Bonus, a Drosophila TIF1 homolog, is a nuclear receptor cofactor required for viability, molting, and numerous morphological events. Here we establish a role for Bonus in the modulation of chromatin structure. We show that weak loss-of-function alleles of bonus have a more deleterious effect on males than on females. This male-enhanced lethality is not due to a defect in dosage compensation or somatic sex differentiation, but to the presence of the Y chromosome. Additionally, we show that bonus acts as both an enhancer and a suppressor of position-effect variegation. By immunostaining, we demonstrate that Bonus is associated with both interphase and prophase chromosomes and through chromatin immunoprecipitation show that two of these sites correspond to the histone gene cluster and the Stellate locus.

15545639

Hsp70 chaperones as modulators of prion life cycle: novel effects of Ssa and Ssb on the Saccharomyces cerevisiae prion [PSI+].

[PSI(+)] is a prion isoform of the yeast release factor Sup35. In some assays, the cytosolic chaperones Ssa1 and Ssb1/2 of the Hsp70 family were previously shown to exhibit "pro-[PSI(+)]" and "anti-[PSI(+)]" effects, respectively. Here, it is demonstrated for the first time that excess Ssa1 increases de novo formation of [PSI(+)] and that pro-[PSI(+)] effects of Ssa1 are shared by all other Ssa proteins. Experiments with chimeric constructs show that the peptide-binding domain is a major determinant of differences in the effects of Ssa and Ssb proteins on [PSI(+)]. Surprisingly, overproduction of either chaperone increases loss of [PSI(+)] when Sup35 is simultaneously overproduced. Excess Ssa increases both the average size of prion polymers and the proportion of monomeric Sup35 protein. Both in vivo and in vitro experiments uncover direct physical interactions between Sup35 and Hsp70 proteins. The proposed model postulates that Ssa stimulates prion formation and polymer growth by stabilizing misfolded proteins, which serve as substrates for prion conversion. In the case of very large prion aggregates, further increase in size may lead to the loss of prion activity. In contrast, Ssb either stimulates refolding into nonprion conformation or targets misfolded proteins for degradation, in this way counteracting prion formation and propagation.

15545638

A natural meiotic DNA break site in Schizosaccharomyces pombe is a hotspot of gene conversion, highly associated with crossing over.

In Schizosaccharomyces pombe, meiosis-specific DNA breaks that initiate recombination are observed at prominent but widely separated sites. We investigated the relationship between breakage and recombination at one of these sites, the mbs1 locus on chromosome I. Breaks corresponding to 10% of chromatids were mapped to four clusters spread over a 2.1-kb region. Gene conversion of markers within the clusters occurred in 11% of tetrads (3% of meiotic chromatids), making mbs1 a conversion hotspot when compared to other fission yeast markers. Approximately 80% of these conversions were associated with crossing over of flanking markers, suggesting a strong bias in meiotic break repair toward the generation of crossovers. This bias was observed in conversion events at three other loci, ade6, ade7, and ura1. A total of 50-80% of all crossovers seen in a 90-kb region flanking mbs1 occurred in a 4.8-kb interval containing the break sites. Thus, mbs1 is also a hotspot of crossing over, with breakage at mbs1 generating most of the crossovers in the 90-kb interval. Neither Rec12 (Spo11 ortholog) nor I-SceI-induced breakage at mbs1 was significantly associated with crossing over in an apparently break-free interval >25 kb away. Possible mechanisms for generating crossovers in such break-free intervals are discussed.

15545637

RNA interference using boranophosphate siRNAs: structure-activity relationships.

In RNA interference (RNAi), short double-stranded RNA (known as siRNA) inhibits expression from homologous genes. Clinical or pre-clinical use of siRNAs is likely to require stabilizing modifications because of the prevalence of intracellular and extracellular nucleases. In order to examine the effect of modification on siRNA efficacy and stability, we developed a new method for synthesizing stereoregular boranophosphate siRNAs. This work demonstrates that boranophosphate siRNAs are consistently more effective than siRNAs with the widely used phosphorothioate modification. Furthermore, boranophosphate siRNAs are frequently more active than native siRNA if the center of the antisense strand is not modified. Boranophosphate modification also increases siRNA potency. The finding that boranophosphate siRNAs are at least ten times more nuclease resistant than unmodified siRNAs may explain some of the positive effects of boranophosphate modification. The biochemical properties of boranophosphate siRNAs make them promising candidates for an RNAi-based therapeutic.

15545636

Roles of Rad23 protein in yeast nucleotide excision repair.

Nucleotide excision repair (NER) removes many different types of DNA lesions. Most NER proteins are indispensable for repair. In contrast, the yeast Rad23 represents a class of accessory NER proteins, without which NER activity is reduced but not eliminated. In mammals, the complex of HR23B (Rad23 homolog) and XPC (yeast Rad4 homolog) has been suggested to function in the damage recognition step of NER. However, the precise function of Rad23 or HR23B in NER remains unknown. Recently, it was suggested that the primary function of RAD23 protein in NER is its stabilization of XPC protein. Here, we tested the significance of Rad23-mediated Rad4 stabilization in NER, and analyzed the repair and biochemical activities of purified yeast Rad23 protein. Cellular Rad4 was indeed stabilized by Rad23 in the absence of DNA damage. Persistent overexpression of Rad4 in rad23 mutant cells, however, largely failed to complement the ultraviolet sensitivity of the mutant. Consistently, deficient NER in rad23 mutant cell extracts could not be complemented by purified Rad4 protein in vitro. In contrast, partial complementation was observed with purified Rad23 protein. Specific complementation to the level of wild-type repair was achieved by adding purified Rad23 together with small amounts of Rad4 protein to rad23 mutant cell extracts. Purified Rad23 protein was unable to bind to DNA, but stimulated the binding activity of purified Rad4 protein to N-acetyl-2-aminofluorene-damaged DNA. These results support two roles of Rad23 protein in NER: (i) its direct participation in the repair biochemistry, possibly due to its stimulatory activity on Rad4-mediated damage binding/recognition; and (ii) its stabilization of cellular Rad4 protein.

15545635

Vascular endothelial growth factor controls neuronal migration and cooperates with Sema3A to pattern distinct compartments of the facial nerve.

Developing neurons accurately position their somata within the neural tube to make contact with appropriate neighbors and project axons to their preferred targets. Taking advantage of a collection of genetically engineered mouse mutants, we now demonstrate that the behavior of somata and axons of the facial nerve is regulated independently by two secreted ligands for the transmembrane receptor neuropilin 1 (Nrp1), the semaphorin Sema3A and the VEGF164 isoform of Vascular Endothelial Growth Factor. Although Sema3A is known to control the guidance of facial nerve axons, we now show that it is not required for the pathfinding of their somata. Vice versa, we find that VEGF164 is not required for axon guidance of facial motor neurons, but is essential for the correct migration of their somata. These observations demonstrate, for the first time, that VEGF contributes to neuronal patterning in vivo, and that different compartments of one cell can be co-ordinately patterned by structurally distinct ligands for a shared receptor.

15545634

A chaperone network controls the heat shock response in E. coli.

The heat shock response controls levels of chaperones and proteases to ensure a proper cellular environment for protein folding. In Escherichia coli, this response is mediated by the bacterial-specific transcription factor, sigma32. The DnaK chaperone machine regulates both the amount and activity of sigma32, thereby coupling sigma32 function to the cellular protein folding state. In this manuscript, we analyze the ability of other major chaperones in E. coli to regulate sigma32, and we demonstrate that the GroEL/S chaperonin is an additional regulator of sigma32. We show that increasing the level of GroEL/S leads to a decrease in sigma32 activity in vivo and this effect can be eliminated by co-overexpression of a GroEL/S-specific substrate. We also show that depletion of GroEL/S in vivo leads to up-regulation of sigma32 by increasing the level of sigma32. In addition, we show that changing the levels of GroEL/S during stress conditions leads to measurable changes in the heat shock response. Using purified proteins, we show that that GroEL binds to sigma32 and decreases sigma32-dependent transcription in vitro, suggesting that this regulation is direct. We discuss why using a chaperone network to regulate sigma32 results in a more sensitive and accurate detection of the protein folding environment.

15545633

Cold shock and regulation of surface protein trafficking convey sensitization to inducers of stage differentiation in Trypanosoma brucei.

Transmission of a protozoan parasite from a vertebrate to invertebrate host is accompanied by cellular differentiation. The signals from the environment that trigger the process are poorly understood. The model parasite Trypanosoma brucei proliferates in the mammalian bloodstream and in the tsetse fly. On ingestion by the tsetse, the trypanosome undergoes a rapid differentiation that is marked by replacement of the variant surface glycoprotein (VSG) coat with GPI-anchored EP and GPEET procyclins. Here we show that a cold shock of DeltaT > 15 degrees C is sufficient to reversibly induce high-level expression of the insect stage-specific EP gene in the mammalian bloodstream stages of T. brucei. The 3'-UTR of the EP mRNA is necessary and sufficient for the increased expression. During cold shock, EP protein accumulates in the endosomal compartment in the proliferating, slender, bloodstream stage, whereas the EP is present on the plasma membrane in the quiescent, stumpy, bloodstream stage. Thus, there is a novel developmentally regulated cell surface access control mechanism for a GPI-anchored protein. In addition to inducing EP expression, cold shock results in the acquisition of sensitivity to micromolar concentrations of cis-aconitate and citrate by stumpy but not slender bloodstream forms. The cis-aconitate and citrate commit stumpy bloodstream cells to differentiation to the procyclic stage along with rapid initial proliferation. We propose a hierarchical model of three events that regulate differentiation after transmission to the tsetse: sensing the temperature change, surface access of a putative receptor, and sensing of a chemical cue.

15545632

c-Myc controls the balance between hematopoietic stem cell self-renewal and differentiation.

The activity of adult stem cells is essential to replenish mature cells constantly lost due to normal tissue turnover. By a poorly understood mechanism, stem cells are maintained through self-renewal while concomitantly producing differentiated progeny. Here, we provide genetic evidence for an unexpected function of the c-Myc protein in the homeostasis of hematopoietic stem cells (HSCs). Conditional elimination of c-Myc activity in the bone marrow (BM) results in severe cytopenia and accumulation of HSCs in situ. Mutant HSCs self-renew and accumulate due to their failure to initiate normal stem cell differentiation. Impaired differentiation of c-Myc-deficient HSCs is linked to their localization in the differentiation preventative BM niche environment, and correlates with up-regulation of N-cadherin and a number of adhesion receptors, suggesting that release of HSCs from the stem cell niche requires c-Myc activity. Accordingly, enforced c-Myc expression in HSCs represses N-cadherin and integrins leading to loss of self-renewal activity at the expense of differentiation. Endogenous c-Myc is differentially expressed and induced upon differentiation of long-term HSCs. Collectively, our data indicate that c-Myc controls the balance between stem cell self-renewal and differentiation, presumably by regulating the interaction between HSCs and their niche.

15545631

Notch3 is required for arterial identity and maturation of vascular smooth muscle cells.

Formation of a fully functional artery proceeds through a multistep process. Here we show that Notch3 is required to generate functional arteries in mice by regulating arterial differentiation and maturation of vascular smooth muscle cells (vSMC). In adult Notch3-/- mice distal arteries exhibit structural defects and arterial myogenic responses are defective. The postnatal maturation stage of vSMC is deficient in Notch3-/- mice. We further show that Notch3 is required for arterial specification of vSMC but not of endothelial cells. Our data reveal Notch3 to be the first cell-autonomous regulator of arterial differentiation and maturation of vSMC.

15545630

MIM/BEG4, a Sonic hedgehog-responsive gene that potentiates Gli-dependent transcription.

Sonic hedgehog (Shh) signaling plays a critical role during development and carcinogenesis. While Gli family members govern the transcriptional output of Shh signaling, little is known how Gli-mediated transcriptional activity is regulated. Here we identify the actin-binding protein Missing in Metastasis (MIM) as a new Shh-responsive gene. Together, Gli1 and MIM recapitulate Shh-mediated epidermal proliferation and invasion in regenerated human skin. MIM is part of a Gli/Suppressor of Fused complex and potentiates Gli-dependent transcription using domains distinct from those used for monomeric actin binding. These data define MIM as both a Shh-responsive gene and a new member of the pathway that modulates Gli responses during growth and tumorigenesis.

15545629

LEF1-mediated regulation of Delta-like1 links Wnt and Notch signaling in somitogenesis.

Wnt signaling, which is mediated by LEF1/TCF transcription factors, has been placed upstream of the Notch pathway in vertebrate somitogenesis. Here, we examine the molecular basis for this presumed hierarchy and show that a targeted mutation of Lef1, which abrogates LEF1 function and impairs the activity of coexpressed TCF factors, affects the patterning of somites and the expression of components of the Notch pathway. LEF1 was found to bind multiple sites in the Dll1 promoter in vitro and in vivo. Moreover, mutations of LEF1-binding sites in the Dll1 promoter impair expression of a Dll1-LacZ transgene in the presomitic mesoderm. Finally, the induced expression of LEF1-beta-catenin activates the expression of endogenous Dll1 in fibroblastic cells. Thus, Wnt signaling can affect the Notch pathway by a LEF1-mediated regulation of Dll1.

15545628

WNT signaling, in synergy with T/TBX6, controls Notch signaling by regulating Dll1 expression in the presomitic mesoderm of mouse embryos.

Notch signaling in the presomitic mesoderm (psm) is critical for somite formation and patterning. Here, we show that WNT signals regulate transcription of the Notch ligand Dll1 in the tailbud and psm. LEF/TCF factors cooperate with TBX6 to activate transcription from the Dll1 promoter in vitro. Mutating either T or LEF/TCF sites in the Dll1 promoter abolishes reporter gene expression in vitro as well as in the tail bud and psm of transgenic embryos. Our results indicate that WNT activity, in synergy with TBX6, regulates Dll1 transcription and thereby controls Notch activity, somite formation, and patterning.

15545627

Living with or without cyclins and cyclin-dependent kinases.

Entry into, progression through, and exit from the G1 phase of the mammalian cell cycle in response to extracellular mitogenic cues are presumed to be governed by cyclin-dependent kinases (Cdks) regulated by the D-type and E-type cyclins. Studies performed over more than a decade have supported the view that these holoenzymes are important, if not required, for these processes. However, recent experiments in which the genes encoding all three D-type cyclins, the two E-type cyclins, cyclin D-dependent Cdk4 and Cdk6, or cyclin E-dependent Cdk2 have been disrupted in the mouse germ line have revealed that much of fetal development occurs normally in their absence. Thus, none of these genes is strictly essential for cell cycle progression. To what extent is the prevailing dogma incorrect, and how can the recent findings be reconciled with past work?

15545626

The hypoxia-induced paralogs Scylla and Charybdis inhibit growth by down-regulating S6K activity upstream of TSC in Drosophila.

Diverse extrinsic and intrinsic cues must be integrated within a developing organism to ensure appropriate growth at the cellular and organismal level. In Drosophila, the insulin receptor/TOR/S6K signaling network plays a fundamental role in the control of metabolism and cell growth. Here we show that scylla and charybdis, two homologous genes identified as growth suppressors in an EP (enhancer/promoter) overexpression screen, act as negative regulators of growth. The simultaneous loss of both genes generates flies that are more susceptible to reduced oxygen concentrations (hypoxia) and that show mild overgrowth phenotypes. Conversely, scylla or charybdis overactivation reduces growth. Growth inhibition is associated with a reduction in S6K but not PKB/Akt activity. Together, genetic and biochemical analysis places Scylla/Charybdis downstream of PKB and upstream of TSC. Furthermore, we show that scylla and charybdis are induced under hypoxic conditions and that scylla is a target of Drosophila HIF-1 (hypoxia-inducible factor-1) like its mammalian counterpart RTP801/REDD1, thus establishing a potential cross-talk between growth and oxygen sensing.

15545625

Regulation of mTOR function in response to hypoxia by REDD1 and the TSC1/TSC2 tumor suppressor complex.

Mammalian target of rapamycin (mTOR) is a central regulator of protein synthesis whose activity is modulated by a variety of signals. Energy depletion and hypoxia result in mTOR inhibition. While energy depletion inhibits mTOR through a process involving the activation of AMP-activated protein kinase (AMPK) by LKB1 and subsequent phosphorylation of TSC2, the mechanism of mTOR inhibition by hypoxia is not known. Here we show that mTOR inhibition by hypoxia requires the TSC1/TSC2 tumor suppressor complex and the hypoxia-inducible gene REDD1/RTP801. Disruption of the TSC1/TSC2 complex through loss of TSC1 or TSC2 blocks the effects of hypoxia on mTOR, as measured by changes in the mTOR targets S6K and 4E-BP1, and results in abnormal accumulation of Hypoxia-inducible factor (HIF). In contrast to energy depletion, mTOR inhibition by hypoxia does not require AMPK or LKB1. Down-regulation of mTOR activity by hypoxia requires de novo mRNA synthesis and correlates with increased expression of the hypoxia-inducible REDD1 gene. Disruption of REDD1 abrogates the hypoxia-induced inhibition of mTOR, and REDD1 overexpression is sufficient to down-regulate S6K phosphorylation in a TSC1/TSC2-dependent manner. Inhibition of mTOR function by hypoxia is likely to be important for tumor suppression as TSC2-deficient cells maintain abnormally high levels of cell proliferation under hypoxia.

15545624

Identification of a Drosophila Myb-E2F2/RBF transcriptional repressor complex.

The Drosophila Myb complex has roles in both activating and repressing developmentally regulated DNA replication. To further understand biochemically the functions of the Myb complex, we fractionated Drosophila embryo extracts relying upon affinity chromatography. We found that E2F2, DP, RBF1, RBF2, and the Drosophila homolog of LIN-52, a class B synthetic multivulva (synMuv) protein, copurify with the Myb complex components to form the Myb-MuvB complex. In addition, we found that the transcriptional repressor protein, lethal (3) malignant brain tumor protein, L(3)MBT, and the histone deacetylase, Rpd3, associated with the Myb-MuvB complex. Members of the Myb-MuvB complex were localized to promoters and were shown to corepress transcription of developmentally regulated genes. These and other data now link together the Myb and E2F2 complexes in higher-order assembly to specific chromosomal sites for the regulation of transcription.

15545623

JNK potentiates TNF-stimulated necrosis by increasing the production of cytotoxic reactive oxygen species.

The c-Jun NH(2)-terminal kinase (JNK) has been implicated in both cell death and survival responses to different stimuli. Here we reexamine the function of JNK in tumor necrosis factor (TNF)-stimulated cell death using fibroblasts isolated from wild-type, Mkk4(-/-) Mkk7(-/-), and Jnk1(-/-) Jnk2(-/-) mice. We demonstrate that JNK can act to suppress TNF-stimulated apoptosis. However, we find that JNK can also potentiate TNF-stimulated necrosis by increasing the production of reactive oxygen species (ROS). Together, these data indicate that JNK can shift the balance of TNF-stimulated cell death from apoptosis to necrosis. Increased necrosis may represent a contributing factor in stress-induced inflammatory responses mediated by JNK.

15545622

siRNAs targeting an intronic transposon in the regulation of natural flowering behavior in Arabidopsis.

Allelic variation in FLOWERING LOCUS C (FLC), a central repressor of flowering, contributes to natural differences in flowering behavior among Arabidopsis accessions. The weak nature of the FLC allele in the Ler accession is due to low levels of FLC RNA resulting, through an unknown mechanism, from a transposable element inserted in an intron of FLC. Here we show that the transposable element renders FLC-Ler subject to repressive chromatin modifications mediated by short interfering RNAs generated from homologous transposable elements in the genome. Our studies have general implications for the role of transposable elements in eukaryotic gene expression and evolution.

15545621

The natural history of type B Niemann-Pick disease: results from a 10-year longitudinal study.

Type B Niemann-Pick disease (NPD-B) caused by acid sphingomyelinase deficiency is a rare, autosomal recessive, lysosomal storage disorder with a broad range of disease severity. The objectives of this study were to document the natural history of the disease in a large, clinically heterogeneous patient population that was followed for a period of 10 years and to determine how genotype influences phenotype. Twenty-nine patients with NPD-B had serial evaluations at least 9 months apart. Organ volumes, hematologic indices, lipid concentrations, pulmonary function, and hepatic activity were studied, and individual phenotypic severity was compared with genotype. All patients with intact spleens had splenomegaly (mean value: 12.7 multiples of normal [MN]; range: 4.5-27.3 MN), and all but 1 had hepatomegaly (mean volume: 1.91 MN; range: 0.93-3.21 MN). At initial visit, 39% had thrombocytopenia and 3% had leukopenia. At final visit, the percentages increased to 54% and 34%, respectively. Mean annual decreases in platelet count and leukocyte count were 7 x 10(3) and 0.2 x 10(3) per mm3, respectively. The typical atherogenic lipid profile was worse in older patients. A total of 69% of patients had low diffusion capacity for carbon monoxide, and more than one third had low forced expiratory volume in 1 second, forced vital capacity, and forced expiratory volume in 1 second/forced vital capacity at initial visit. All measurements of pulmonary function showed a gradual deterioration over time. Liver dysfunction was characterized by stable elevation of hepatic transaminases and bilirubin. Homozygotes for DeltaR608, P323A, and P330R had milder disease than patients with all other genotypes. The natural history of NPD-B is characterized by hepatosplenomegaly with progressive hypersplenism, worsening atherogenic lipid profile, gradual deterioration in pulmonary function, and stable liver dysfunction.

15545620

Bedside limited echocardiography by the emergency physician is accurate during evaluation of the critically ill patient.

Echocardiography can be a rapid, noninvasive, objective tool in the assessment of ventricular function and preload during resuscitation of a critically ill or injured child. We sought to determine the accuracy of bedside limited echocardiography by the emergency physician (BLEEP) in estimation of (1) left ventricular function (LVF) and (2) inferior vena cava (IVC) volume, as an indirect measure of preload. We conducted a prospective observational study of a convenience sample of patients who were admitted to our intensive care unit. All patients underwent BLEEP followed by an independent formal echocardiogram by an experienced pediatric echocardiography provider (PEP). IVC volume was assessed by measurement of the maximal diameter of the IVC. LVF was determined by calculating shortening fraction (SF) using M-mode measurements on the parasternal short-axis view at the level of the papillary muscle. An independent blinded pediatric cardiologist reviewed all images for accuracy and quality. Estimates of SF obtained on the BLEEP examination were compared with those obtained by the PEP. Thirty-one patients were enrolled. The mean age was 5.1 years (range: 23 days-16 years); 48.4% (15 of 31) were girls; 58.1% (18 of 31) were on mechanical ventilatory support at the time of their study. There was good agreement between the emergency physician (EP) and the PEP for estimation of SF (r = 0.78). The mean difference in the estimate of SF between the providers was 4.4% (95% confidence interval: 1.6%-7.2%). This difference in estimate of SF was statistically significant. Similarly, there was good agreement between the EP and the PEP for estimation of IVC volume (r = 0.8). The mean difference in the estimate of IVC diameter by the PEP and the EP was 0.068 mm (95% confidence interval: -0.16 to 0.025 mm). This difference was not statistically significant. Our study suggests that PEP sonographers are capable of obtaining images that permit accurate assessment of LVF and IVC volume. BLEEP can be performed with focused training and oversight by a pediatric cardiologist.

15545619

HIV infection and zidovudine use in childbearing women.

The risk of vertical HIV transmission from mother to child has been shown to be markedly decreased through HIV education, counseling, testing, and zidovudine (ZDV) use. The US Public Health Service published guidelines in 1994 for the use of ZDV on the basis of results of the AIDS Clinical Trials Group Protocol, a multicenter clinical trial of ZDV. The Public Health Service followed these guidelines with recommendations for routine HIV counseling and testing with informed consent for all pregnant women. New Jersey adopted these guidelines as the standard of care and created a program implementing the standard in all hospitals with maternity units. The purpose of this report was to study the trends in the rate of HIV infection in childbearing women over the past decade and to follow patterns of use of ZDV in the HIV-positive women, as a marker for the success of New Jersey's policy and program to reduce mother-to-child transmission. Since 1988 in New Jersey, blood from heel-stick filter papers has been tested for the presence of HIV antibody through anonymous, unlinked surveys. Excess blood from screening for inborn errors of metabolism for all infants who were born in the state from July through September of each year was tested using a Food and Drug Administration-approved HIV-1 and Western blot test. Age, race, and ethnicity were recorded, as well as the results from the HIV-1 and Western blot tests. Since 1994, specimens confirmed to be HIV-1 positive by Western blot test were tested for the presence of ZDV, and the results were recorded. The number of New Jersey women included in the study for the period 1990 through 2002 numbered 372305. The percentage of childbearing women who tested positive for HIV declined by 55% during the period, although the declines were not uniform in all subgroups. In the early 1990s, women who were <30 years old had higher infection rates than older women, but this has not been a consistent pattern during the period. Younger women again had a higher rate in 2002. When HIV-positive rates are examined by 5-year age groups, the declines are dramatic for younger women. The rate per 100 women 20 through 24 years decreased from 0.46 per 100 tested women in 1990 to 0.29 in 2002 and for women 25 through 29 years from 0.51 per 100 in 1990 to 0.25 in 2002. The rate for women 30 through 34 years of age declined from 0.54 in 1990 to 0.13 in 2002. During the same time period, the rate per 100 tested women 35 though 39 years of age increased from 0.23 to 0.33. Black non-Hispanic women who give birth to live infants have the highest HIV-positive rates, followed by Hispanic women and white non-Hispanic women. In 2002, this rate was 0.74 per 100 in black non-Hispanic women, 0.22 per 100 in Hispanic women, and 0.08 in white non-Hispanic women. Although major disparities continue, the infection rate in black non-Hispanic women demonstrated the greatest decrease during the period, followed by the decline among Hispanic women. The use of ZDV in HIV-positive women increased dramatically during the period, from 13.3% in 1994, when it was first tested in New Jersey, to an all-time high of 88.5% in 2002. Reducing perinatal HIV transmission is a priority for the New Jersey Department of Health and Senior Services. Reducing perinatal transmission can be accomplished by reducing the number of infants who are exposed perinatally or decreasing the percentage of exposed infants for whom transmission occurs or both. The decrease in prevalence of HIV-positive status in childbearing women is in opposition to an overall increasing trend in prevalence rates. This decrease is thought to be attributable in part to the positive impact of numerous education and prevention programs but may also be the result of a voluntary decision on the part of HIV-infected women not to become pregnant or not to carry to term. In addition, the cohort of women who became infected in the early years of the epidemic may be aging out of their childbearing years, may have more advanced disease with a concomitant difficulty with fertility and carrying to term, or may have died. In New Jersey, a greater proportion of women with newly diagnosed HIV disease are past their childbearing years as compared with earlier years. Increased use of ZDV is thought to be attributable to several factors: dissemination of information to health care providers via continuing medical education activities; dissemination of information to the public, in particular to women; outreach via community-based organizations; and New Jersey Department of Health and Senior Service regulations and policies for mandatory counseling and voluntary testing of all pregnant women. A recent addition to New Jersey's comprehensive program to decrease perinatal transmission occurred in 2002 with dissemination to hospitals of the department's standard of care for women who present in labor with unknown HIV status. Physicians, nurses, and hospitals play vital roles in preventing vertical transmission of HIV by providing preconception and postconception counseling, testing with consent of pregnant women, and treatment for HIV-positive mothers, including administration of ZDV. This study not only provides an estimate of the prevalence of HIV infection in the population of childbearing women but also provides a means of examining the vertical transmission of HIV infection from mother to child. Continued research on this subpopulation as well as on other groups will provide additional knowledge to help in the overall goal of reducing HIV prevalence.

15545618

Echocardiographic findings in the PANDAS subgroup.

Sydenham's chorea is the neurologic manifestation of rheumatic fever and is a diagnosis of exclusion requiring only the presence of frank chorea in the absence of another neurologic disorder. Two thirds of children with Sydenham's chorea also have rheumatic carditis (pathologic mitral valve regurgitation). Although there are similar neuropsychiatric symptoms and preceding group A beta-hemolytic streptococcal infection associated with both Sydenham's chorea and the PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections) subgroup, it is unknown whether patients in the PANDAS subgroup have any cardiac involvement. Sixty children meeting the criteria for PANDAS were entered into protocols at National Institute of Mental Health between 1993 and 2002. Doppler and 2-dimensional echocardiograms were performed on these subjects to assess valvular heart disease. Of these 60 children, no echocardiographic evidence of significant mitral or aortic valve regurgitation was found. One patient was found to have mild mitral regurgitation, and all patients had normal left atrial size and normal left ventricular size and function. Follow-up echocardiograms on 20 children showed no significant valvular regurgitation. The evidence of a clear lack of rheumatic carditis in these children supports the hypothesis that PANDAS is a distinct neuropsychiatric diagnosis separate from Sydenham's chorea.

15545617

Treatment of acute Kawasaki disease: aspirin's role in the febrile stage revisited.

To evaluate the effect of treatment without aspirin in the acute phase of Kawasaki disease (KD) and to determine whether it is necessary to expose children to high- or medium-dose aspirin. A total of 162 patients who fulfilled the established criteria of acute KD between 1993 and 2003 were included in this retrospective study. All patients were treated with high-dose intravenous immunoglobulin (IVIG; 2 g/kg) as a single infusion without concomitant aspirin treatment. Low-dose aspirin (3-5 mg/kg per day) was subsequently prescribed when fever subsided. Patients who had defervescence within 3 days after the completion of IVIG treatment were classified as the IVIG-responsive group, and those whose fever persisted for >3 days were classified as the IVIG-nonresponsive group. The 162 patients were divided further into 2 groups: those who were treated with IVIG before illness day 5, and those who were treated after illness day 5. We compared the response rate of IVIG therapy, duration of fever, and incidence of coronary artery abnormalities (CAAs) between these groups. A total of 153 patients were classified into the IVIG-responsive group, and 128 (83.66%) of them had defervescence within 24 hours after completion of IVIG therapy. Nine (5.56%) patients were classified into the IVIG nonresponsive group, and all received additional IVIG (2 g/kg) without aspirin. Six (66.67%) had defervescence within 3 days after additional therapy. Patients in the IVIG-nonresponsive group had a significantly higher incidence of CAAs than those in the IVIG-responsive group (25% vs 2.92%). In the group that was treated before illness day 5 (n = 16), all patients had defervescence within 3 days after IVIG therapy and 13 (81.25%) had defervescence within 24 hours. In the group that was treated after illness day 5 (n = 146), 137 (93.84%) patients had defervescence within 3 days and 115 (78.77%) had defervescence within 24 hours. One (6.67%) patient in the group that was treated before illness day 5 got a new onset of CAAs, as did 5 (3.85%) in the group that was treated after illness day 5. There was no statistically significant difference in the response rate of IVIG therapy, duration of fever, and incidence of CAAs between these 2 groups. The results of our study indicate that the treatment without aspirin in acute stage of KD had no effect on the response rate of IVIG therapy, duration of fever, or incidence of CAAs when children were treated with high-dose (2 g/kg) IVIG as a single infusion, despite treatment before or after day 5 of illness. We conclude that it seems unnecessary to expose children to high- or medium-dose aspirin therapy in acute KD when the available data show no appreciable benefit in preventing the failure of IVIG therapy, formation of CAAs, or shortening the duration of fever.

15545616

Growth, efficacy, and safety of feeding an iron-fortified human milk fortifier.

Survival rates for preterm infants who weigh between 501 and 1500 g at birth have continued to improve over time. In response to this continuing decrease in birth weight of surviving preterm infants, Enfamil Human Milk Fortifier has recently been reformulated to meet the nutritional requirements of these smaller, more rapidly growing infants. It now provides an increased protein level of 1.1 g/58 kJ, a decreased carbohydrate level of 0.2 g/58 kJ, and a combined linoleic and alpha-linolenic fatty acid content of 157 mg/58 kJ. As these very small preterm infants have an increased requirement for dietary iron, the fortifier has been supplemented with 1.44 mg/58 kJ of iron, an amount of iron similar to that provided in a typical iron-fortified term infant formula. An iron-fortified product obviates the need for administration of an iron supplement, a hyperosmolar-inducing intervention. The purpose of this prospective, double-blind, randomized, controlled study was to evaluate growth, safety, and efficacy in a population of very low birth weight (VLBW) preterm infants who received human milk fortified with either the reformulated iron-fortified powdered human milk fortifier test product (HMF-T) or a powdered commercially available human milk fortifier control product (HMF-C). Infants who weighed < or =1500 g, had a gestational age < or =33 weeks postmenstrual age, and had an enteral intake of at least 100 mL/kg per day of unfortified human milk were stratified by gender and birth weight and randomized to receive HMF-T or HMF-C product from study day 1 to study day 28, hospital discharge, or the termination of human milk feedings, whichever came first. Unless medically indicated, investigators were not to administer iron supplements from study days 1 to 14. Infants were assessed serially for growth; enteral and parenteral intake; serum chemistry and hematologic values; clinical histories, including the administration of blood transfusions; feeding tolerance; respiratory outcomes; and morbidities, including adverse events. Of the 181 participating infants in this study, 96 received HMF-T and 85 received HMF-C. At randomization, there were no significant differences in infant characteristics between the fortifier groups. The percentage of participants who remained in the study for 28 days was similar between fortifier groups (57% HMF-T, 46% HMF-C). For both fortifier groups, the most frequent reasons for discontinuing the study before study day 28 were unavailability of human milk and hospital discharge. Rate of weight gain was similar between the fortifier groups (17.5 +/- 0.53 g/kg per day for HMF-T and 17.3 +/- 0.59 g/kg per day for HMF-C). Mean achieved weight, length, and head circumference were comparable between groups across the 28-day study period. Total protein intake from enteral and parenteral nutrition was significantly greater for the HMF-T fortifier group; however, this difference did not result in any difference in growth between the 2 fortifier groups. An analysis of the growth and energy intake data of a subset of the intent-to-treat population who adhered more strictly to the study feeding protocol yielded results similar to those seen for the intent-to-treat population. There were no clinically significant differences in the results of laboratory studies between the groups at study days 0, 14, and 28. Anemia of prematurity was prevalent in both study groups; by study day 28, median hematocrit levels were 27.0% (interquartile range [IQR]: 24.0%-29.6%) for the HMF-T group and 26.0% (IQR: 24.0%-31.0%) for the HMF-C group. Median ferritin levels were 77.0 ng/mL (IQR: 37-155 ng/ml) for HMF-T and 92.0 ng/mL (IQR: 33-110 ng/mL) for HMF-C. There were no significant differences between the study fortifier groups in regard to the receipt of medically indicated iron supplements on or before study day 14 or in the administration of blood transfusions before study day 0 or from study days 0 through 14. However, from study day 15 to study day 28, fewer HMF-T infants (n = 12) required a blood transfusion than did HMF-C infants (n = 20). Although the higher levels of iron in the HMF-T fortifier (1.44 mg vs 0.35 mg for HMF-C per 4 packets of powdered fortifier) did not prevent anemia per se, it did reduce the frequency of one of the most serious outcomes of anemia: the need for a blood transfusion. There was no statistically significant difference between fortifier groups in regard to feeding tolerance. Rates of suspected sepsis (26% HMF-T vs 31% HMF-C) and confirmed sepsis (5% HMF-T, 7% HMF-C) were low as were the rates of suspected necrotizing enterocolitis (NEC; 6% HMF-T and 5% HMF-C) and confirmed Bell's stage 2 or more NEC (1% HMF-T and 1% HMF-C). There were no statistically significant differences between the study fortifier groups in regard to the incidence of confirmed and suspected sepsis and NEC. Both human milk fortifiers studied are safe, are well tolerated, and facilitate comparable good growth; however, using the iron-fortified product may reduce the need for blood transfusions in VLBW infants. The similar low rates of suspected and confirmed NEC and sepsis seen in both fortifier groups in this study refutes the premise that the inclusion of iron in fortifiers will increase the incidence of sepsis and NEC. Indeed, the incidence for NEC and sepsis for both groups in this study was lower than is reported for VLBW infants and similar to that seen for infants who are fed human milk.

15545615

Kikuchi-Fujimoto disease with prolonged fever in children.

We reviewed 12 patients who had Kikuchi-Fujimoto disease (KFD) and presented with prolonged fever and lymphadenopathy. The clinical and laboratory aspects of the patients confirmed by excisional lymph node biopsy were analyzed. The mean age of the children was 11.0 +/- 3.0 years (range: 6-15 years). The male-to-female ratio was 1.4:1. The median duration of fever before admission and the total duration of fever was 13 days (range: 7-65 days) and 19.5 days (range: 9-75 days), respectively. One patient had supraclavicular lymphadenopathy, 10 had cervical involvement, and 1 had axillary lymphadenopathy. All of the histologic findings of the lymph node biopsies showed the characteristic findings consistent with KFD, such as paracortical necrosis with karyorrhexis and an increase in the number of phagocytic histiocytes and atypical lymphocytes. As for the laboratory findings, leukopenia (3600 +/- 900 per mm3), anemia (hemoglobin 11.4 +/- 1.2 g/dL), an elevated erythrocyte sedimentation rate (44 +/- 18 mm/hour), and a relatively low C-reactive protein level (1.3 +/- 1.1 mg/dL) were noted. Eight patients received conservative therapy with antipyretics, and 3 patients were treated with prednisolone. KFD is a rare disease yet should be considered in the differential diagnosis for older children with prolonged fever and lymphadenopathy.

15545614

A prolactin family paralog regulates reproductive adaptations to a physiological stressor.

Successful species develop strategies to optimize their reproductive performance. This optimization likely includes the evolution of genes that specifically permit reproduction in physiologically challenging conditions. The prolactin (PRL) family gene cluster is one of 25 mouse-specific gene clusters, the majority of which are associated with reproduction. A prevailing theme characterizing the PRL family is its connection with pregnancy and mechanisms controlling viviparity. PRL-like protein A (PLP-A) is one of 26 genes located within the PRL family locus. It is a nonclassical member of the PRL family (e.g., PLP-A does not use the PRL receptor) produced by trophoblast cells of the chorioallantoic placenta and acts on uterine natural killer cells. In this report, the biology of PLP-A has been investigated by generating mice with a PLP-A null mutation. Under standardized animal husbandry conditions, PLP-A possesses modest effects on reproductive performance. However, this same gene is critical for reproduction when mice are exposed to a physiological stressor. Wild-type mice exposed to hypobaric hypoxia during gestation readily adapt and maintain their pregnancies, whereas PLP-A null mutant mice fail to adapt, resulting in pregnancy failure. PLP-A contributes to hypoxia-induced adaptations critical to hemochorial placentation and thus nutrient flow to extraembryonic and embryonic tissues. The findings provide insights into species-specific reproductive adaptations.

15545613

TATA box binding protein induces structure in the recombinant glucocorticoid receptor AF1 domain.

A number of transcription factor proteins contain domains that are fully or partially unstructured. The means by which such proteins acquire naturally folded conformations are not well understood. When they encounter their proper binding partner(s), several of these proteins adopt a folded conformation through an induced-fit mechanism. The glucocorticoid receptor (GR) is a ligand-activated transcription factor. Expressed independently as a recombinant peptide, the N-terminal transactivation domain (AF1) of the GR shows little structure and appears to exist as a collection of random coil configurations. The GR AF1 is known to interact with other transcription factors, including a critical component of the general transcription machinery proteins, the TATA box binding protein (TBP). We tested whether this interaction can lead to acquisition of structure in the GR AF1. Our results show that recombinant GR AF1 acquires a significant amount of helical content when it interacts with TBP. These structural changes were monitored by Fourier transform infrared and NMR spectroscopies, and by proteolytic digestions. Our results support a model in which TBP binding interaction with the GR AF1 induces significantly greater helical structure in the AF1 domain. This increased helical content in the GR AF1 appears to come mostly at the expense of random coil conformation. These results are in accordance with the hypothesis that an induced-fit mechanism gives structure to the GR AF1 when it encounters TBP.

15545612

On the evolutionary history of Ephedra: Cretaceous fossils and extant molecules.

Gnetales comprise three unusual genera of seed plants, Ephedra, Gnetum, and Welwitschia. Their extraordinary morphological diversity suggests that they are survivors of an ancient, more diverse group. Gnetalean antiquity is also supported by fossil data. Dispersed "ephedroid" (polyplicate) pollen first appeared in the Permian >250 million years ago (Myr), and a few megafossils document the presence of gnetalean features in the early Cretaceous. The Cretaceous welwitschioid seedling Cratonia cotyledon dates the split between Gnetum and Welwitschia to before 110 Myr. Ages and character evolution of modern diversity are, however, controversial, and, based on molecular data, it has recently been suggested that Ephedra is very young, only 8-32 Myr. Here, we present data on the evolutionary history of Ephedra. Fossil seeds from Buarcos, Portugal, unequivocally link one type of Cretaceous polyplicate pollen to Ephedra and document that plants with unique characters, including the peculiar naked male gametophyte, were established already in the Early Cretaceous. Clades in our molecular phylogeny of extant species correspond to geographical regions, with African species in a basal grade/clade. The study demonstrates extremely low divergence in both molecular and morphological characters in Ephedra. Features observed in the fossils are present in all major extant clades, showing that modern species have retained unique reproductive characters for >110 million years. A recent origin of modern species of Ephedra would imply that the Cretaceous Ephedra fossils discussed here were members of widespread, now extinct sister lineage(s), and that no morphological innovations characterized the second diversification.

15545611

Identification and functional characterization of a sex pheromone receptor in the silkmoth Bombyx mori.

Sex pheromones released by female moths are detected with high specificity and sensitivity in the olfactory sensilla of antennae of conspecific males. Bombykol in the silkmoth Bombyx mori was the first sex pheromone to be identified. Here we identify a male-specific G protein-coupled olfactory receptor gene, B. mori olfactory receptor 1 (BmOR-1), that appears to encode a bombykol receptor. The BmOR-1 gene is located on the Z sex chromosome, has an eight-exon/seven-intron structure, and exhibits male-specific expression in the pheromone receptor neurons of male moth antenna during late pupal and adult stages. Bombykol stimulation of Xenopus laevis oocytes expressing BmOR-1 and BmGalphaq elicited robust dose-dependent inward currents on two-electrode voltage clamp recordings, demonstrating that the binding of bombykol to BmOR-1 leads to the activation of a BmGalphaq-mediated signaling cascade. Antennae of female moths infected with BmOR-1-recombinant baculovirus showed electrophysiological responses to bombykol but not to bombykal. These results provide evidence that BmOR-1 is a G protein-coupled sex pheromone receptor that recognizes bombykol.

15545610

Long-range compaction and flexibility of interphase chromatin in budding yeast analyzed by high-resolution imaging techniques.

Little is known about how chromatin folds in its native state. Using optimized in situ hybridization and live imaging techniques have determined compaction ratios and fiber flexibility for interphase chromatin in budding yeast. Unlike previous studies, ours examines nonrepetitive chromatin at intervals short enough to be meaningful for yeast chromosomes and functional domains in higher eukaryotes. We reconcile high-resolution fluorescence in situ hybridization data from intervals of 14-100 kb along single chromatids with measurements of whole chromosome arms (122-623 kb in length), monitored in intact cells through the targeted binding of bacterial repressors fused to GFP derivatives. The results are interpreted with a flexible polymer model and suggest that interphase chromatin exists in a compact higher-order conformation with a persistence length of 170-220 nm and a mass density of approximately 110-150 bp/nm. These values are equivalent to 7-10 nucleosomes per 11-nm turn within a 30-nm-like fiber structure. Comparison of long and short chromatid arm measurements demonstrates that chromatin fiber extension is also influenced by nuclear geometry. The observation of this surprisingly compact chromatin structure for transcriptionally competent chromatin in living yeast cells suggests that the passage of RNA polymerase II requires a very transient unfolding of higher-order chromatin structure.

15545609

Gestational exposure of Ahr and Arnt hypomorphs to dioxin rescues vascular development.

The aryl hydrocarbon receptor (AHR) is commonly known for its role in the adaptive metabolism of xenobiotics and in the toxic events that follow exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin). Previously, we have demonstrated that the AHR and its heterodimeric partner, the AHR nuclear translocator (ARNT), play a role in the developmental closure of a hepatic vascular shunt known as the ductus venosus (DV). To investigate the mechanism of DV closure, we generated hypomorphic alleles of the Ahr and Arnt loci. Using these models, we then asked whether this vascular defect could be rescued by receptor activation during late development. By manipulating gestational exposure, the patent DV in AHR or ARNT hypomorphs could be efficiently closed by dioxin exposure as early as embryonic day 12.5 and as late as embryonic day 18.5. These findings define the temporal regulation of receptor activation during normal ontogeny and provide evidence to support the idea that receptor activation and AHR-ARNT heterodimerization are essential for normal vascular development. Taken in the broader context, these data demonstrate that similar AHR signaling steps govern all major aspects of AHR biology.

15545608

Medicinal chemistry applied to a synthetic protein: development of highly potent HIV entry inhibitors.

We have used total chemical synthesis to perform high-resolution dissection of the pharmacophore of a potent anti-HIV protein, the aminooxypentane oxime of [glyoxylyl1]RANTES(2-68), known as AOP-RANTES, of which we designed and made 37 analogs. All involved incorporation of one or more rationally chosen nonnatural noncoded structures, for which we found a clear comparative advantage over coded ones. We investigated structure-activity relationships in the pharmacophore by screening the analogs for their ability to block the HIV entry process and produced a derivative, PSC-RANTES [N-nonanoyl, des-Ser1[L-thioproline2, L-cyclohexylglycine3]-RANTES(2-68)], which is 50 times more potent than AOP-RANTES. This promising group of compounds might be optimized yet further as potential prophylactic and therapeutic anti-HIV agents. The remarkable potency of our RANTES analogs probably involves the unusual mechanism of intracellular sequestration of CC-chemokine receptor 5 (CCR5), and it has been suggested that this arises from enhanced affinity for the receptor. We found that inhibitory potency and capacity to induce CCR5 down-modulation do appear to be correlated, but that unexpectedly, inhibitory potency and affinity for CCR5 do not. We believe this study represents the proof of principle for the use of a medicinal chemistry approach, above all one showing the advantage of noncoded structures, to the optimization of the pharmacological properties of a protein. Medicinal chemistry of small molecules is the foundation of modern pharmaceutical practice, and we believe we have shown that techniques have now reached the point at which the approach could also be applied to the many macromolecular drugs now in common use.

15545607

Mitochondrial biogenesis by NO yields functionally active mitochondria in mammals.

We recently found that long-term exposure to nitric oxide (NO) triggers mitochondrial biogenesis in mammalian cells and tissues by activation of guanylate cyclase and generation of cGMP. Here, we report that the NO/cGMP-dependent mitochondrial biogenesis is associated with enhanced coupled respiration and content of ATP in U937, L6, and PC12 cells. The observed increase in ATP content depended entirely on oxidative phosphorylation, because ATP formation by glycolysis was unchanged. Brain, kidney, liver, heart, and gastrocnemius muscle from endothelial NO synthase null mutant mice displayed markedly reduced mitochondrial content associated with significantly lower oxygen consumption and ATP content. In these tissues, ultrastructural analyses revealed significantly smaller mitochondria. Furthermore, a significant reduction in the number of mitochondria was observed in the subsarcolemmal region of the gastrocnemius muscle. We conclude that NO/cGMP stimulates mitochondrial biogenesis, both in vitro and in vivo, and that this stimulation is associated with increased mitochondrial function, resulting in enhanced formation of ATP.

15545606

Climate change impacts are sensitive to the concentration stabilization path.

Analysis of policies to achieve the long-term objective of the United Nations Framework Convention on Climate Change, stabilizing concentrations of greenhouse gases at levels that avoid "dangerous" climate changes, must discriminate among the infinite number of emission and concentration trajectories that yield the same final concentration. Considerable attention has been devoted to path-dependent mitigation costs, generally for CO2 alone, but not to the differential climate change impacts implied by alternative trajectories. Here, we derive pathways leading to stabilization of equivalent CO2 concentration (including radiative forcing effects of all significant trace gases and aerosols) with a range of transient behavior before stabilization, including temporary overshoot of the final value. We compare resulting climate changes to the sensitivity of representative geophysical and ecological systems. Based on the limited available information, some physical and ecological systems appear to be quite sensitive to the details of the approach to stabilization. The likelihood of occurrence of impacts that might be considered dangerous increases under trajectories that delay emissions reduction or overshoot the final concentration.

15545605

Fluorescent indicators of cAMP and Epac activation reveal differential dynamics of cAMP signaling within discrete subcellular compartments.

Second messenger cAMP regulates many cellular functions through its effectors, such as cAMP-dependent protein kinase (PKA) and Epac (exchange proteins directly activated by cAMP). Spatial and temporal control of cAMP signaling is crucial to differential regulation of cellular targets involved in various signaling cascades. To investigate the compartmentalized cAMP signaling, we constructed fluorescent indicators that report intracellular cAMP dynamics and Epac activation by sandwiching the full-length Epac1 between cyan and yellow mutants of GFP. Elevations of cAMP decreased FRET and increased the ratio of cyan-to-yellow emissions by 10-30% in living mammalian cells. This response can be reversed by removing cAMP-elevating agents and abolished by mutating the critical residue responsible for cAMP binding. Targeting of the reporter to the plasma membrane, where cAMP is produced in response to the activation of beta-adrenergic receptor, revealed a faster cAMP response at the membrane than in the cytoplasm and mitochondria. Simultaneous imaging with targeted cAMP indicator and PKA activity reporter allowed the detection of a much delayed PKA response in the nucleus after the rapid accumulation of cAMP at the plasma membrane of the same cell, despite the immediate presence of a pool of cAMP in the nucleus. Thus, cAMP dynamics and the activation of its effectors are precisely controlled spatiotemporally in vivo.

15545604

Integrative analysis of genome-scale data by using pseudoinverse projection predicts novel correlation between DNA replication and RNA transcription.

We describe an integrative data-driven mathematical framework that formulates any number of genome-scale molecular biological data sets in terms of one chosen set of data samples, or of profiles extracted mathematically from data samples, designated the "basis" set. By using pseudoinverse projection, the molecular biological profiles of the data samples are least-squares-approximated as superpositions of the basis profiles. Reconstruction of the data in the basis simulates experimental observation of only the cellular states manifest in the data that correspond to those of the basis. Classification of the data samples according to their reconstruction in the basis, rather than their overall measured profiles, maps the cellular states of the data onto those of the basis and gives a global picture of the correlations and possibly also causal coordination of these two sets of states. We illustrate this framework with an integration of yeast genome-scale proteins' DNA-binding data with cell cycle mRNA expression time course data. Novel correlation between DNA replication initiation and RNA transcription during the yeast cell cycle, which might be due to a previously unknown mechanism of regulation, is predicted.

15545602

A genetic screen to isolate type III effectors translocated into pepper cells during Xanthomonas infection.

The bacterial pathogen Xanthomonas campestris pv. vesicatoria (Xcv) uses a type III secretion system (TTSS) to translocate effector proteins into host plant cells. The TTSS is required for Xcv colonization, yet the identity of many proteins translocated through this apparatus is not known. We used a genetic screen to functionally identify Xcv TTSS effectors. A transposon 5 (Tn5)-based transposon construct including the coding sequence for the Xcv AvrBs2 effector devoid of its TTSS signal was randomly inserted into the Xcv genome. Insertion of the avrBs2 reporter gene into Xcv genes coding for proteins containing a functional TTSS signal peptide resulted in the creation of chimeric TTSS effector::AvrBs2 fusion proteins. Xcv strains containing these fusions translocated the AvrBs2 reporter in a TTSS-dependent manner into resistant BS2 pepper cells during infection, activating the avrBs2-dependent hypersensitive response (HR). We isolated seven chimeric fusion proteins and designated the identified TTSS effectors as Xanthomonas outer proteins (Xops). Translocation of each Xop was confirmed by using the calmodulin-dependent adenylate cydase reporter assay. Three xop genes are Xanthomonas spp.-specific, whereas homologs for the rest are found in other phytopathogenic bacteria. XopF1 and XopF2 define an effector gene family in Xcv. XopN contains a eukaryotic protein fold repeat and is required for full Xcv pathogenicity in pepper and tomato. The translocated effectors identified in this work expand our knowledge of the diversity of proteins that Xcv uses to manipulate its hosts.

15545601

Activation of RasGRP3 by phosphorylation of Thr-133 is required for B cell receptor-mediated Ras activation.

The Ras signaling pathway plays a critical role in B lymphocyte development and activation, but its activation mechanism has not been well understood. At least one mode of Ras regulation in B cells involves a Ras-guanyl nucleotide exchange factor, RasGRP3. We demonstrate here that RasGRP3 undergoes phosphorylation at Thr-133 upon B cell receptor cross-linking, thereby resulting in its activation. Deletion of phospholipase C-gamma2 or pharmacological interference with conventional PKCs resulted in marked reduction in both Thr-133 phosphorylation and Ras activation. Moreover, mutation of Thr-133 in RasGRP3 alone severely impaired its ability to activate Ras in B cell receptor signaling. Hence, our data suggest that PKC, after being activated by diacylglycerol, phosphorylates RasGRP3, thereby contributing to its full activation.

15545600

The role of the glycocalyx in reorganization of the actin cytoskeleton under fluid shear stress: a "bumper-car" model.

We propose a conceptual model for the cytoskeletal organization of endothelial cells (ECs) based on a major dichotomy in structure and function at basal and apical aspects of the cells. Intracellular distributions of filamentous actin (F-actin), vinculin, paxillin, ZO-1, and Cx43 were analyzed from confocal micrographs of rat fat-pad ECs after 5 h of shear stress. With intact glycocalyx, there was severe disruption of the dense peripheral actin bands (DPABs) and migration of vinculin to cell borders under a uniform shear stress (10.5 dyne/cm2; 1 dyne = 10 microN). This behavior was augmented in corner flow regions of the flow chamber where high shear stress gradients were present. In striking contrast, no such reorganization was observed if the glycocalyx was compromised. These results are explained in terms of a "bumper-car" model, in which the actin cortical web and DPAB are only loosely connected to basal attachment sites, allowing for two distinct cellular signaling pathways in response to fluid shear stress, one transmitted by glycocalyx core proteins as a torque that acts on the actin cortical web (ACW) and DPAB, and the other emanating from focal adhesions and stress fibers at the basal and apical membranes of the cell.

15545599

TNF receptor-associated factor (TRAF) domain and Bcl-2 cooperate to induce small B cell lymphoma/chronic lymphocytic leukemia in transgenic mice.

Transgenic mice overexpressing in B lymphocytes either Bcl-2 or a TNF receptor-associated factor (TRAF)2 mutant lacking the N-terminal RING and zinc finger domains located at the N terminus of the molecule (TRAF2DN), which mimics TRAF1, developed lymphadenopathy and splenomegaly due to polyclonal B cell expansion. Remarkably, TRAF2DN/Bcl-2 double-transgenic mice contained B cell populations similar to those observed in TRAF2DN mice. However, over time, they developed severe splenomegaly and lymphadenopathy, and most animals also developed leukemia, pleural effusion, and, in some cases, ascites associated with monoclonal and oligoclonal B cell neoplasms. The life span of TRAF2DN/Bcl-2 mice was markedly reduced compared with Bcl-2 and TRAF2DN single-transgenics or wild-type littermates. The expanded B cell population of TRAF2DN/Bcl-2 double-transgenic mice was primarily comprised of small/medium-size noncycling B220(M)/IgM(H)/IgD(L)/CD21(L)/CD23(NULL)/CD11b(+)/CD5+ cells that were Bcl-6-negative, consistent with a B-1 phenotype. The cells also expressed high levels of CD54 and other adhesion molecules. In vitro, these B cells showed comparable proliferation rates to those of wild-type counterparts but exhibited markedly increased survival and were resistant to apoptosis induced by chemotherapeutic agents and glucocorticoids. Histopathologic features were consistent with mouse small B cell lymphoma progressing to leukemia with many similarities to human chronic lymphocytic leukemia. Given that many human chronic lymphocytic leukemias overexpress TRAF1 and Bcl-2, our findings suggest that cooperation between Bcl-2 and TRAF pathways contributes to the development of this type of leukemia.

15545598

Molecular evolution of dinoflagellate luciferases, enzymes with three catalytic domains in a single polypeptide.

Enzymes with multiple catalytic sites are rare, and their evolutionary significance remains to be established. This study of luciferases from seven dinoflagellate species examines the previously undescribed evolution of such proteins. All these enzymes have the same unique structure: three homologous domains, each with catalytic activity, preceded by an N-terminal region of unknown function. Both pairwise comparison and phylogenetic inference indicate that the similarity of the corresponding individual domains between species is greater than that between the three different domains of each polypeptide. Trees constructed from each of the three individual domains are congruent with the tree of the full-length coding sequence. Luciferase and ribosomal DNA trees both indicate that the Lingulodinium polyedrum luciferase diverged early from the other six. In all species, the amino acid sequence in the central regions of the three domains is strongly conserved, suggesting it as the catalytic site. Synonymous substitution rates also are greatly reduced in the central regions of two species but not in the other five. This lineage-specific difference in synonymous substitution rates in the central region of the domains correlates inversely with the content of GC3, which can be accounted for by the biased usage toward C-ending codons at the degenerate sites. RNA modeling of the central region of the L. polyedrum luciferase domain suggests a function of the constrained synonymous substitutions in the circadian-controlled protein synthesis.

15545590

Subcutaneous emphysema and pneumomediastinum complicating tonsillectomy.

Subcutaneous emphysema and pneumomediastinum are very rare complications of tonsillectomy. To report on a case study and a literature review of these conditions; to discuss the management and controversial causes. Case study and literature review. A case is presented of subcutaneous emphysema and pneumomediastinum complicating tonsillectomy. All published reports of these complications dating back to 1910 are reviewed. An additional 30 cases of subcutaneous emphysema and 9 of pneumomediastinum have been reported following tonsillectomy. This group of 32 patients with emphysematous complications included 17 male patients, 14 female, and a child of unknown sex. The mean age was 18.6 years (range, 2-65 years). All tonsillectomies were performed with a cold technique except our case. Subcutaneous emphysema was first noted intraoperatively in 8 patients (25%) a mean of 3.5 hours (range, 10 minutes to 14 hours) after the procedure in 18 patients (56%) and not until the next morning in 6 patients (19%). Most cases resolved without specific treatment or significant sequelae. Some cases were life-threatening and required urgent intervention. Others were misdiagnosed, which resulted in unnecessary surgery. Subcutaneous emphysema and pneumomediastinum are rare occurrences following tonsillectomy that should alert one to the possibility of more serious complications.

15545589

Intralesional cidofovir for pediatric recurrent respiratory papillomatosis.

To compare disease response among children with recurrent respiratory papillomatosis (RRP) who underwent combined surgical debulking and intralesional cidofovir injections vs repeated surgical debulking only. Retrospective medical record review; follow-up range: 16 to 56 months. Tertiary care children's hospital. Seven children with RRP, including 4 subjects treated with cidofovir and 3 controls matched for age and initial papilloma staging score. Subjects treated with cidofovir underwent combined surgical debulking and intralesional cidofovir injection every 2 months until disease remission. Control subjects underwent repeated surgical debulking at individually determined intervals. Intraoperative endoscopic photographs were retrospectively assigned papilloma staging scores. Cidofovir and control group comparisons were made using nonparametric 2-sample Wilcoxon rank-sum (Mann-Whitney) testing, and comparisons of initial and final papilloma staging scores were made using nonparametric matched-pair Wilcoxon signed-rank testing. The final cidofovir group scores were significantly lower than the control group scores (P < .05). Within-group differences between initial and final scores were not significant (cidofovir group, P = .07; control group, P = .29). Four children with RRP were safely and successfully treated with intralesional cidofovir injection. Consideration should be given to using cidofovir more widely for treatment of pediatric RRP. Larger numbers in the cidofovir and control groups are needed in future studies to determine the true impact of cidofovir on management of this disease.

15545588

The effect on snoring of structural nasal valve dilatation with a butterfly graft.

To evaluate the effect on snoring of structural nasal valve dilatation with butterfly spreader grafts in patients with nasal valve insufficiency. Retrospective medical chart review and telephone follow-up; mean +/- SD follow-up time, 20.7 +/- 11.34 months (range, 3-48 months). Tertiary care referral center. A total of 37 snoring patients with nasal valve insufficiency who underwent nasal valve dilatation with a butterfly spreader graft. The conchal cartilage butterfly graft technique was performed during rhinoplasty through either an external or endonasal approach. To establish through a retrospective review that butterfly graft conchal cartilage nasal reconstruction is effective in reducing snoring. After surgery, 30 patients (81%) had significant improvement in breathing, 5 (14%) had slight improvement, and 2 (5%) had no benefit in breathing. Snoring stopped completely in 11 (30%) of the patients after surgery. The improvement in snoring was significant in 13 patients (35%) and slight in 3 (8%). Twenty-six patients (70%) reported tiredness and grogginess on awakening before the surgery. Surgery significantly improved patients' tiredness and grogginess on awakening in 15 cases (58%), slightly improved them in 5 (19%), and did not change the patients' tiredness and grogginess in 6 cases (23%). The conchal cartilage butterfly graft yields successful results not only in breathing but also in snoring symptoms in patients with nasal valve insufficiency.

15545587

Radiation therapy does not impact local complication rates after free flap reconstruction for head and neck cancer.

To determine whether external beam radiation therapy (XRT), administered either before or after surgery, increases the rate and/or severity of local postoperative complications in patients with head and neck cancer who undergo microvascular free flap reconstruction. Retrospective cohort study. University of Washington Medical Center, Seattle, a tertiary care hospital. A total of 100 consecutive patients underwent fibular free flap reconstruction of the mandible. The study cohort was divided according to radiation treatment status: (1) no XRT (28 patients), (2) preoperative XRT (37 patients), and (3) postoperative XRT (35 patients). The median follow-up after surgery was 11 months (range, 1-89 months). Rate and severity of local postoperative complications. Fifty-four patients (54%) had at least 1 postoperative complication. There were no differences among the 3 XRT subgroups in the overall proportion of patients with complications of any severity (15 [54%] of 28 patients in the no XRT group, 24 [65%] of 37 patients in the preoperative XRT group, and 16 [46%] of 35 patients in the postoperative XRT group; P = .26, chi(2) analysis). There were also no differences seen when mild and severe complication rates were specifically examined (P = .58 and P = .10, respectively). No case of complete flap loss was observed. We noted no significant correlations between the rate of postoperative complications and the following covariates: total radiation dose, size of radiation field, disease stage, exposure to chemotherapy, presence of serious medical comorbidities, patient age, or history of tobacco use. Our experience suggests that XRT can be safely administered before or after surgery to patients undergoing head and neck free flap reconstruction at an experienced surgical referral center. Postoperative complication rates were not significantly affected by administration, timing, dose, or extent of XRT.

15545586

Randomized, controlled, multisite study of intracapsular tonsillectomy using low-temperature plasma excision.

To determine the efficacy of intracapsular tonsillectomy using low-temperature plasma excision for improving the quality of the postoperative experience and for treating obstructive symptoms through 12 months postoperatively. Prospective, randomized, controlled, single-blind study. Multiple private or institutional otolaryngology clinics. Fifty-five children (aged 3-12 years) with obstructive tonsillar hypertrophy. Patients were randomly assigned and blinded to undergo either intracapsular tonsillectomy using low-temperature plasma excision (n = 27) or total tonsillectomy using conventional electrosurgery (n = 28). Operative data, 14-day recovery variables, and obstructive symptoms were prospectively collected through 12 months. During the first 14 days, significantly fewer children in the intracapsular group reported nausea (P = .01) or lost weight (P = .003). The intracapsular group had a significantly faster resolution of pain (P = .01), had an earlier return to a normal diet (P = .004), ceased taking pain medication sooner (P = .002), and returned to normal activity sooner (P = .04). Postoperatively, the intracapsular group had more residual tonsil tissue than the total tonsillectomy group (P = .002 for the 3- and 12-month visits). However, the incidence of recurring obstructive symptoms, pharyngitis, and antibiotic use was similar in both treatment groups during the 12 months. Postoperative morbidity normally associated with traditional (total) tonsillectomy was significantly reduced after intracapsular tonsillectomy using low-temperature plasma excision. The residual tonsillar tissue associated with this technique was of no clinical consequence.

15545585

Tracheal anomalies in Pfeiffer syndrome.

To determine the types and frequency of airway anomalies in patients with Pfeiffer syndrome. Retrospective case series. Academic tertiary care pediatric hospital. Eleven patients with Pfeiffer syndrome, 6 of whom were severely affected, were identified. All were included in the study. Presence of tracheal anomalies, need for tracheotomy, and length of life. The 6 severely affected patients had mutations in genes that code for fibroblast growth factor receptor 2 (S351C [3 patients]; C342S [2 patients]; and W290C [1 patient]). Five of these patients were diagnosed during bronchoscopy or tracheotomy as having a congenital tracheal cartilaginous sleeve. In 1 patient, supportive care was withdrawn at 2 weeks of life, and the patient died. The remaining 5 patients required tracheotomy because of severe upper airway obstruction. Three of these patients died (at ages 9 months and 7 and 15 years). Two are still alive at ages 23 and 18 months. Patients with Pfeiffer syndrome manifest significant airway pathologic conditions. Upper airway obstruction is related to midface hypoplasia and secondary nasal obstruction. Tracheal anomalies have been infrequently reported.

15545584

Lack of association between esophageal biopsy, bronchoalveolar lavage, and endoscopy findings in hoarse children.

To determine the prevalence of esophagitis (based on esophageal biopsy results) and aspiration (based on bronchoalveolar lavage [BAL]) in children with hoarseness. Retrospective medical chart review spanning 24 months of 127 consecutive children (mean age, 6.9 years; range, 1.8-17 years) who presented with hoarseness to 2 attending otolaryngologists. Tertiary care children's hospital.Intervention All subjects underwent direct laryngoscopy, rigid bronchoscopy with BAL, and rigid or flexible esophagoscopy with biopsy. The BAL result was considered positive if the number of lipid-laden macrophages was "moderate" or "large," and the esophageal biopsy result was considered positive if any 2 of the following 3 histologic criteria were present: basal cell hyperplasia, increased papillary height, and intraepithelial inflammatory infiltrate. Comparisons between subjective endoscopic findings and objective test results were made using the t test and contingency table analysis, where appropriate. Of the 127 children, 104 (82%) had vocal nodules; 53 (43%) had endoscopically visualized laryngitis; 36 (28%) had tracheobronchial inflammatory changes; 60 (47%) had abnormal esophagoscopy findings; 47 (37%) had a positive BAL result; and 38 (30%) had a positive esophageal biopsy result. There was no significant correlation between BAL and esophageal biopsy results (P = .11). The odds of having positive BAL or esophageal biopsy results were unaffected by the presence of vocal nodules; endoscopically visualized inflammation of the larynx, trachea, or esophagus; or symptoms or previous clinical history of gastroesophageal reflux disease. Positive esophageal biopsy and BAL results are prevalent among children with hoarseness, regardless of subjective upper aerodigestive tract endoscopic findings.

15545583

A comparison of ocular protective measures during carbon dioxide laser laryngoscopy.

To determine the efficacy of various eye protection measures during carbon dioxide laser laryngoscopy. A standard medical mannequin was equipped with indicator paper over the eyes and subjected to multiple passes of direct carbon dioxide laser beam contact at 400-mm focal length with powers ranging from 2.5 to 4.0 W during simulated laser microlaryngoscopy. Several different eye protection materials, including silk tape, paper tape, cloth tape, occlusive dressing, and eye pads, were used to cover the eyes and tested for their degree of protection against the laser beam. Thermal injuries were quantified and compared among these protective materials. Academic medical center. Degree of eye protection against the laser beam injury on a scale of 0 to 4. The carbon dioxide laser beam at both 2.5 and 4.0 W produced considerable thermal damage to the indicator paper in the absence of any protective barrier. Among the adhesive protective barriers, moistened cloth tape was the most effective adhesive material against laser beam-induced thermal damage (P<.001). Silk tape and paper tape offered poor protection. Moist eye pads, although not adhesive and therefore unable to maintain eye closure, were very effective barriers against the laser beam. Dry paper tape and dry eye pads were imminently flammable. A combination of moistened cloth tape to maintain eye closure coupled with placement of well- moistened cotton-based eye pads over the tape provides excellent eye protection during carbon dioxide laser laryngoscopy.

15545582

Evolutions in the management of congenital intranasal skull base defects.

Congenital skull base defects have traditionally been treated via an intracranial approach. Recent advances in endoscopic management have made minimally invasive extracranial approaches feasible, with less morbidity. To determine the success of endoscopic treatment of congenital cerebrospinal fluid leaks and encephaloceles. Retrospective review of congenital cerebrospinal fluid leaks and encephaloceles treated from January 1, 1992, to December 31, 2003. Data collected include demographic characteristics, presenting signs/symptoms, site of the skull base defect, surgical approach, repair technique, and clinical follow-up. Eight patients were treated via the endoscopic approach for congenital cerebrospinal fluid leaks and encephaloceles. The average age at presentation was 6 years (range, birth to 18 years). Three patients presented with meningitis (average age, 6 years), 4 had cerebrospinal fluid rhinorrhea, and 3 developed a nasal obstruction. Five defects originated at the foramen cecum, and 3 others involved the ethmoid roof/cribriform plate only. Our endoscopic approaches were successful on the first attempt, with a mean follow-up of 19 months. One patient experienced nasal stenosis postoperatively. Continuing progress in the surgical management of congenital skull base defects demonstrates that endoscopic repair is a successful alternative to traditional craniotomy approaches, with less morbidity. This technique requires meticulous preparation and precise grafting of the defect to avoid collateral damage to surrounding structures. While reduction in the risk of meningitis, intracranial complications, and facial growth abnormalities and alleviation of nasal obstruction necessitate the timely repair of these skull base defects, special considerations are discussed regarding the optimal timing of surgical intervention, operative working space, and exposure in a smaller nasal cavity.

15545581

Patient, caregiver, and surgeon perceptions of quality of life following anterior skull base surgery.

To estimate the agreement between surgeons' and lay caregivers' quality-of-life (QOL) perceptions of patients undergoing major skull base surgery. Cohort survey of patients who had undergone anterior skull base tumor excision. University-affiliated medical center. Thirty-five patients and their lay caregivers participated in the study. A triple survey was performed: each patient and his or her caregiver were asked to answer 35 questions related to 6 distinct QOL domains: role of performance, physical function, vitality, pain, specific symptoms, and effect on emotions. The composite health-related QOL of the patients was also rated on an ordinal scale by 3 surgeons who participated in the operation and follow-up. An overall significant agreement was found between patients' and caregivers' scores at the group level (mean scores of each domain) and individual level (patient-caregiver pairs) (r = 0.76, P<.001). There was a minor correlation in the effect on emotions domain and no correlation in the pain domain. We found no correlation between the surgeons' and patients' ratings. The operating surgeons tended to overate their patients' QOL. The study results show that the surgeon's perception of his or her patient's QOL is not sufficiently accurate to correctly estimate patients' QOL status. These judgments should come from the patient or from the caregiver, whose perception can be used in clinical trials as a proxy for estimation of a patient's QOL.

15545580

The economic cost of squamous cell cancer of the head and neck: findings from linked SEER-Medicare data.

To evaluate the excess mortality, resource use, and costs associated with squamous cell carcinoma of the head and neck (SCCHN) among elderly Medicare beneficiaries. Retrospective cohort analysis using data from the Surveillance, Epidemiology, and End Results (SEER) Program of the National Cancer Institute and Medicare claims. Study cohorts included patients aged 65 years and older who were newly diagnosed as having SCCHN in a SEER registry between 1991 and 1993 (N = 4536) and controls matched 1:1 by age and sex. Patients were followed up for 5 years or until death, whichever occurred first. Initial treatment was primarily surgery and/or radiation among patients with early-stage SCCHN, with only modest use of chemotherapy. Patients with SCCHN had significantly (P<.001) higher 5-year mortality (64% vs 25%) and health care costs than controls. Average Medicare payments (1998 US dollars) among patients with SCCHN were $25 542 higher than those of matched comparison patients (P<.001), with monthly payments 3 times as high ($1428 vs $446). Patients diagnosed as having advanced SCCHN had shorter survival times (5-year mortality, 85%, 75%, 47%, and 35% among patients diagnosed as having distant, regional, local, and in situ cancer, respectively) and higher costs (average total Medicare payments, $53 741, $58 387, $42 698, and $37 434, respectively). These results suggest that the health economic burden of SCCHN is substantial, with costs that are comparable with or higher than those of other solid tumors.

15545579

Auditory effects after organ preservation protocol for laryngeal/hypopharyngeal carcinomas.

To investigate the prevalence of hearing loss after concomitant radiochemotherapy in patients enrolled in a larynx preservation protocol. Prospective study. Consecutive patients treated in a tertiary cancer center hospital between 2001 and 2002. Eligible subjects included patients prospectively enrolled in an organ preservation protocol based on concomitant radiotherapy and chemotherapy (cisplatin and paclitaxel). Descriptive analysis of the results of audiologic evaluations, including pure-tone audiometry and immitance audiometry, which were performed prior to and 8 months after treatment. Change in hearing sensitivity was computed relative to baseline measures. Criteria to indicate hearing decrease after the treatment were defined as either a 20-dB decrease at any single test frequency or a 10-dB decrease at any 2 adjacent test frequencies. A total of 11 patients were analyzed. Four patients (36%) had hearing loss after the treatment. Our results suggest that the prevalence of hearing loss after radiochemotherapy in larynx preservation protocols is high (36%); however, it was usually mild and asymptomatic.

15545578

Serum cotinine level and incident hearing loss: a case-control study.

A growing body of literature suggests an association between cigarette smoking and hearing loss. To assess the relation between levels of serum cotinine, a biomarker of exposure to tobacco smoke, and incident hearing loss. A cross-sectional, incident, case-control study of participants selected from a population-based cohort. Testing was conducted at the Beaver Dam Community Hospital, Beaver Dam, Wis. A total of 197 participants with incident hearing loss and 394 control participants, aged 53 to 75 years, selected from the 2800 participants of the 5-year follow-up examination of the population-based Epidemiology of Hearing Loss Study, 1998-2000. Incident hearing loss. The incidence of hearing loss was defined as a pure-tone average of thresholds at 500, 1000, 2000, and 4000 Hz greater than 25-dB hearing level in either ear at follow-up among those without hearing loss at baseline. No significant associations were found between serum cotinine levels and incident hearing loss. These results were not consistent with a previous report, which found cross-sectional associations between prevalent hearing loss and current smoking and environmental tobacco smoke exposure in the home. Longer-term longitudinal studies of smoking and/or serum cotinine levels and the subsequent development of hearing loss may help clarify these associations.

15545574

Trocar site hernia.

To review the relationship between the pathogenesis and clinical manifestations of trocar site hernias seeking to confirm the definition of trocar site hernias by classification. We searched this subject in English on MEDLINE by combining the words "trocar," "port," "hernia, and "laparoscopy."Data Extraction and We limited the main operations to cholecystectomy, colon and rectal surgery, fundoplication, and gastric surgery; finding 44 reports on these procedures. Of these, 19 were case reports, 18 were original articles (setting criteria; the incidence of the trocar site hernia was clarified, and involved >100 patients), and 7 technical notes on "how to do it" were collected. We obtained 19 additional reports using the references of those previously obtained. We, thus, reviewed 63 reports (24 case reports, 27 original articles, 7 technical notes, and 5 review articles). Trocar site hernia was classified into 3 types. The early-onset type that occurred immediately after the operation, with a small-bowel obstruction, especially the Richter hernia, frequently developing. The late-onset type that occurred several months after the operation, mostly with local abdominal bulging with no small-bowel obstruction developing. The special type that occurred indicated the protrusion of the intestine and/or omentum. Trocar site hernias with fascial defects of 10 mm or larger should be closed, including the peritoneum. Opinion varied if a 5-mm trocar site defect should be closed. It is useful to clearly classify trocar site hernias to improve management of laparoscopic procedures.

15545572

Isolated limb perfusion for unresectable melanoma of the extremities.

In patients with truly unresectable melanoma of the extremities, results after isolated limb perfusion (ILP) are absent in the literature. Complete response rates are probably lower than the reported 54% for locoregional recurrent melanoma. In these patients, ILP with melphalan and tumor necrosis factor alpha (TNF-alpha) could be superior to ILP with melphalan alone. Retrospective analysis with a median follow-up period of 21 months (interquartile range, 9-40 months). Two tertiary care cancer centers in the Netherlands. We assessed all 130 consecutive patients who underwent ILP for unresectable melanoma of the extremities, performed between 1978 and 2001. Of these patients, 38% had stage IIIA melanoma and 45% had stage IIIAB melanoma according to criteria of the MD Anderson Cancer Center. Lesions were considered unresectable on the basis of their size, number, or localization. Forty ILPs were performed with melphalan, and 90 were done with TNF-alpha and melphalan. Response rate, disease-free survival, limb salvage rate, and overall survival. In 45% of the patients, a complete response was attained after ILP with melphalan (95% confidence interval, 29%-61%) compared with 59% after ILP with TNF-alpha and melphalan (95% confidence interval, 49%-69%; P = .14). The time to complete response was 3 months (interquartile range, 2-6 months) vs 2 months (interquartile range, 1-3 months; P = .01), respectively. The recurrence rate and median limb recurrence-free survival were not significantly different for both ILP types. The overall limb salvage rate was 96%. Overall 5-year survival was 29% (95% confidence interval, 20%-38%). The ILP type was not an independent prognostic factor for complete response, nor was limb recurrence-free survival, whereas stage IIIA was a favorable prognostic factor (P = .01 and P = .02, respectively). Favorable prognostic factors for improved survival were complete response (P<.001) and a tumor size of 3 cm or less (P = .01). In more than half of the patients with truly unresectable melanoma of the extremities, a complete response was obtained after ILP with melphalan with or without TNF-alpha. The ILP type was not an independent prognostic factor for complete response, limb recurrence-free survival, or overall survival.

15545571

Increased survival among severe trauma patients: the impact of a national trauma system.

The survival of severe trauma patients is affected by the implementation of a national trauma system, which brought about developments both at the hospital and prehospital levels during the past decade. A retrospective cohort study of all severely injured patients (Injury Severity Score >16) recorded in the Israeli National Trauma Registry at all level I trauma centers in Israel from January 1, 1997, to December 31, 2001. Inpatient death rates were examined overall and by subgroups. The National Trauma Registry includes trauma (International Statistical Classification of Diseases, 9th Revision, Clinical Modification diagnosis codes 800-959) hospitalizations, patients who were transferred to or from other hospitals, and those who died in the emergency department. It excludes patients who were dead on arrival, discharged following treatment in the emergency department, and patients who do not fall into the definition of trauma. Main Outcome Measure Inpatient death. Seven thousand four hundred twenty-three severe trauma patients were recorded. Inpatient death rates decreased significantly from 21.6% in 1997 to 14.7% in 2001. The odds ratios of mortality in 1998 through 2001 vs 1997, adjusted for year, age, sex, penetrating injury, and severity of injury (Injury Severity Score >25), were 0.92, 0.89, 0.70, and 0.65, respectively, confirming the downward trend. A steady significant reduction in the inpatient death rate of severe trauma patients hospitalized at all level I trauma centers in Israel between 1997 and 2001 was observed. Although a single factor that explains the reduction was not identified, it is evident that the establishment of the trauma system brought about a significant decrease in mortality. We believe that integrated cooperation of various components of the national trauma system in Israel across the years may explain the reduction.

15545570

Impact of primary resection on the outcome of patients with perforated diverticulitis.

Primary resection has replaced the conventional drainage procedure in the management of patients with generalized peritonitis complicating diverticular disease of the colon. This study investigates the impact of primary resection on operative mortality, identifies predictors of mortality, and compares the results with those of our earlier experience. Primary resection of the perforated diseased segment of the colon is associated with lower mortality rates than the drainage procedure in patients with Hinchey stages 3 and 4 diverticulitis. Retrospective analysis. Tertiary care referral center. We included 138 consecutive patients who underwent emergent operation for generalized peritonitis complicating diverticular disease of the colon (Hinchey stages 3 and 4) during a period of 16 years (January 1983 to May 1999). The 30-day mortality rate was analyzed and predictors of mortality identified. Patients were classified as having spreading purulent peritonitis (n = 44, 31.9%), diffuse peritonitis (n = 64, 46.4%), or fecal peritonitis (n = 30, 21.7%). One hundred thirty-one patients (94.9%) underwent primary resection, 6 patients (4.3%) underwent resection and primary anastomosis, and 1 patient required total colectomy and end ileostomy. Thirteen of the 138 patients in the present group died (1983-1998), representing a perioperative mortality rate of 9%. There was no significant difference in mortality when compared with our earlier study (1972-1982), which had a mortality rate of 12%, considering that more than 25% of the patients in that group were managed by colostomy and drainage alone. Factors identified univariately as predictors of mortality were age of more than 70 years (P = .047), 2 or more comorbid conditions (P<.01), obstipation at initial examination (P = .02), use of steroids (P = .01), and perioperative sepsis (P<.001). Primary resection has become the standard practice for patients with generalized peritonitis complicating diverticulitis. Mortality rates have not significantly declined despite more aggressive surgical management of the septic source. Because advanced age, comorbid conditions, and perioperative sepsis predict mortality, it is suggested that further reduction in mortality will require improvement in medical management of perioperative sepsis and comorbid conditions.

15545569

Identification and categorization of technical errors by Observational Clinical Human Reliability Assessment (OCHRA) during laparoscopic cholecystectomy.

Surgical operative performance benefits from analysis of the mechanisms underlying technical errors committed during surgery. Prospective study using the Observational Clinical Human Reliability Assessment (OCHRA) system and complete unedited videotapes of the operations. Three National Health Service hospitals within the United Kingdom. Two hundred consecutive patients with symptomatic gallstone disease. Elective laparoscopic cholecystectomy for symptomatic gallstone disease by surgeons, who were blind to the nature and objectives of the study, using their usual operative technique. Surgical consequential and inconsequential operative errors. The analysis of 38 062 steps of the 200 laparoscopic cholecystectomies performed by 26 surgeons identified 2242 errors. The mean +/- SD total, inconsequential, and consequential errors per surgical procedure were 11.0 +/- 8.0, 8.0 +/- 6.0, and 4.0 +/- 3.0, respectively. Dissection of the Calot triangle (second task zone of the operation) incurred more total errors (6.5 +/- 5.4) compared with the first (2.9 +/- 2.8, P<.001) and third (5.1 +/- 3.9, P<.05) task zones. This translated to a higher error probability (6.9% vs 3.5% for the first and 5.5% for third task zones). The combined sharp and blunt dissection method had fewer errors than the blunt/teasing dissection technique (9.45 +/- 7.6 vs 13.9 +/- 7.3, P<.001) although different surgeons were involved. The most serious consequences were encountered during dissection with the electrosurgical hook knife. This study has confirmed that the Observational Clinical Human Reliability Assessment system provides a comprehensive objective assessment of the quality of surgical operative performance by documenting the errors, the stage of the operation in which errors are enacted most frequently, and where these errors have serious consequences (hazard zones).

15545568

Professional satisfaction of women in surgery: results of a national study.

Individual, group, and organizational factors influence the professional satisfaction of women surgeons in Austria. Survey on professional and private issues sent out by mail in 2000 and 2001. Women surgeons working in hospitals and/or in private practices and those who were retired or on maternity leave. All 351 Austrian women surgeons of all core surgical specialties (general, trauma, pediatric, plastic, thoracic, and cardiovascular), certified or in training, were addressed. Proportional odds regression models were used to correlate professional satisfaction with objectively measurable prognostic factors such as age, surgical subspecialty, status of training, type of hospital, location of work (federal states vs the capital), status of activity (active vs on maternity leave), profession of private partner, number of children, and subjectively assessed prognostic factors such as operative volume and departmental organization. The response rate was 58.7% (206/351). One hundred eighty-seven surgeons-active or on maternity leave-were included in the analysis. Higher satisfaction was reported by active surgeons in subspecialties, certified surgeons, comparatively younger and older surgeons, surgeons working in hospitals outside the capital, and surgeons with a physician as a partner. When entering subjectively assessed variables into the model, the quality of departmental organization and operative volume (P<.001), as well as the status of activity (P<.001), had the strongest effect. Women surgeons' professional satisfaction highly depends on departmental organization and status of activity. Inadequate leadership, low operative volume, and being on maternity leave have a negative effect on job satisfaction. Private factors seem to be of little influence. Optimal departmental organization would help women to reconcile their professional and their private lives.

15545567

Hypothyroidism and adrenal insufficiency in sepsis and hemorrhagic shock.

We hypothesized that hypothyroidism and adrenal insufficiency frequently occur together in critically ill patients. A prospective observational study. Surgical intensive care unit of a university-affiliated tertiary referral center. Sixty-six consecutive patients with severe sepsis, septic shock, and hemorrhagic shock who required pulmonary artery catheterization for resuscitation were studied. Thyrotropin and baseline cortisol levels were obtained at 3 am followed by intravenous injection of 250 mug of cosyntropin, a synthetic adrenocorticotropic hormone derivative. A second measurement of the cortisol level was performed 1 hour later. Incidence of hypothyroidism and adrenal insufficiency and mortality. Mean (SD) age was 62 (19) years. The mean (SD) Acute Physiology and Chronic Health Evaluation II score was 21 (5). Twenty-seven patients (40.9%) had severe sepsis, 31 (46.9%) had septic shock, and 8 (12.1%) had hemorrhagic shock. Five patients (7.6%) had hypothyroidism alone and 35 (53.0%) had only adrenal insufficiency. Eight patients (12.1%) had both hypothyroidism and adrenal insufficiency. All patients with endocrine abnormalities were treated. Mortality for the total group was 15 (22.7%) of 66 patients. There is a 12% incidence of simultaneous hypothyroidism and adrenal insufficiency in our study and the routine testing for both may be indicated in this population of critically ill patients.

15545566

Improving team structure and communication: a key to hospital efficiency.

Improving team structure and heightening communication will help provide cost-effective and high-quality patient care for general surgery patients. This study surveys teamwork initiatives and their effects on specific variables related to patient care. The study population comprised all patients admitted to the hospital's general surgery teams during 5 years 3 months. Tertiary care hospital. A complete restructuring of the patient care team for general surgery patients admitted to the hospital. The intervention occurred midway through the study period. Mean length of stay for general surgery patients as a marker of team efficiency and a standardized patient satisfaction survey. The mean length of stay after initiation of the restructured care team was significantly shorter than before initiation. The significance was present despite a consistent patient acuity measure and was associated with a high patient satisfaction level. Restructuring the patient care team yielded a decreased mean length of stay while maintaining a high level of patient satisfaction. This analysis helps validate a hospital-wide initiative to maintain a high level of patient care while increasing patient volume.

15545565

Simultaneous liver-kidney transplantation for adult recipients with irreversible end-stage renal disease.

Combined liver-kidney transplantation is safe (low morbidity and acceptable mortality) and effective in patients with end-stage liver disease. Although refinements in surgical technique have resulted in better patient and allograft outcomes, the negative impact of renal insufficiency on survival in patients undergoing liver transplantation has been widely reported, although some aspects are controversial. Analysis of the clinical characteristics and outcome in the management of patients undergoing combined liver-kidney transplantation. The end points were operative mortality, morbidity, and long-term survival. University Hospital 12 de Octubre. Between May 1986 and December 2001, 820 liver transplantations were performed. There were 16 cases (1.96%) of combined liver-kidney transplantations, which represent the sample of this study. Mean +/- SD follow-up of 42.2 +/- 29 months: 6 patients died (37.5% mortality rate). There were 4 (25%) hospital deaths within 6 months following surgery and 2 after 6 months (4 sepsis, 1 refractory heart failure, and 1 recurrent hepatitis C virus disease). Univariate analysis related to mortality included age, sex, etiology, preoperative creatinine level, United Network for Organ Sharing status, Child-Pugh score, type of hepatectomy (piggyback), intraoperative blood product administration, and the presence of postoperative complications. The only 2 significant factors were the presence of postoperative complications (P = .01) and the United Network for Organ Sharing status (P = .02). Crude survival rate was 62.5%. Actuarial survival rates were 80%, 71%, and 60% at 1, 3, and 5 years, respectively. Because end-stage renal disease is not a formal contraindication for liver transplantation, a combined liver-kidney transplantation for adults with end-stage renal disease can be done safely and effectively.

15545564

Complication of thyroidectomy in patients with radiation-induced thyroid neoplasms.

The complication rate for thyroidectomy is the same in patients with and without a history of radiation exposure. Retrospective medical record review of 171 consecutive patients who had a previous history of radiation treatment and had undergone a thyroid operation from 1961 to 1999. University of California, San Francisco, Medical Center and affiliated hospitals. We selected 107 patients with a history of radiation exposure who had undergone thyroid operations (81 total thyroidectomies) and 107 control patients who underwent comparable operations but had no history of radiation exposure. Among patients with a history of radiation exposure (mean age, 47.2 years), there was 1 recurrent nerve injury, 1 external nerve injury, 20 patients with transient hypocalcemia, and 1 patient with a hematoma. Among patients without a history of radiation exposure (mean age, 47.5 years), there were 2 recurrent nerve injuries, 18 patients with transient hypocalcemia, and 1 patient with a hematoma. All cases of hypocalcemia and recurrent nerve injury in both groups were transient. One patient had a permanent superior laryngeal nerve injury. In patients who underwent operations since January 1, 1990, duration of hospitalization was 1.2 days in patients with a history of radiation exposure (65 patients) and 1.1 days in patients without (101 patients). Our data document that the risk of transient and permanent complications after total thyroidectomy is similar in patients with and without a history of radiation exposure. The relatively low long-term complication rate supports prophylactic total thyroidectomy for patients with thyroid nodules and a history of radiation exposure.

15545563

1% lymphazurin vs 10% fluorescein for sentinel node mapping in colorectal tumors.

Ten percent fluorescein may be successfully used as an alternative to 1% Lymphazurin (1% isosulfan blue; US Surgical Corp, North Haven, Conn) in sentinel lymph node (SLN) mapping for the accurate staging of colorectal tumors. Review of prospectively gathered data. University-affiliated regional medical center. Sentinel lymph node mapping was performed in 120 consecutive patients with colorectal malignancies. The first 1 to 4 blue nodes detected within 5 minutes were designated as Lymphazurin-detected SLNs. The first 1 to 4 fluorescent nodes seen under the Wood light were designated as fluorescein-detected SLNs. Multilevel serial sections for hematoxylin-eosin and immunohistochemistry studies for cytokeratin were performed on all SLNs. Successful mapping, accuracy, skip metastasis, adverse reactions, occult micrometastases detection, and cost. Mapping was successful using Lymphazurin in 99% of the patients vs 97% of the patients using fluorescein (P =.89). The accuracy of predicting nodal metastases with each tracer was 95.8% vs 93.1%, respectively (P =.82). The skip metastases rate was 4.2% for Lymphazurin vs 6.9% for fluorescein (P =.37). The 5 patients in whom nodal disease was only identified as occult micrometastasis in the SLNs had a total of 5 SLNs, all of which were identified by both tracers. No adverse reactions occurred. The cost for Lymphazurin was $99.00, while the cost for fluorescein was $2.10. With the exception of cost, there were no statistically significant differences between the 2 dyes. While easy availability and lower cost remain distinct advantages of fluorescein, Lymphazurin remains the gold standard. In patients with known hypersensitivity to Lymphazurin and when availability and cost are an issue, fluorescein may be used effectively for SLN mapping in colorectal tumors.

15545562

Clinicopathologic and radiopharmacokinetic factors affecting gamma probe-guided parathyroidectomy.

The kinetics of technetium Tc 99m sestamibi (MIBI) in primary hyperparathyroidism are variable and affected by the cellular size of the abnormal glands, the parathyroid hormone levels, and the functional expression of P-glycoprotein (Pgp). The success of gamma probe-guided parathyroidectomy is closely related to the parathyroid-to-thyroid activity ratio at the time of surgery. Preoperative determination of maximum uptake ratio may improve the surgical outcome. Thirty-one patients with primary hyperparathyroidism attributed to a solitary parathyroid adenoma (27 patients) or multiglandular hyperplasia (4 patients) underwent dynamic MIBI imaging preoperatively. Maximum MIBI activity and activity elimination half-life in the abnormal parathyroid glands and thyroid glands were measured, and the maximum uptake ratio was calculated. After a second MIBI injection on the day of surgery, all patients underwent gamma probe-guided parathyroidectomy and cervical exploration. Timing of surgery after MIBI injection was individualized according to the optimal time to surgery (time to maximum uptake ratio), which was determined by preoperative scintigraphy. During surgery, the gamma probe was used to measure ex vivo counts of excised lesions and adjacent postexcision normal tissue (background). Image characteristics, MIBI kinetics, and gamma probe findings were correlated with gland volume, oxyphil cell content, Pgp expression, and serum parathyroid hormone levels. Probe localization of abnormal glands at maximum uptake ratio was successful in all patients. The volume of the parathyroid lesion ranged from 0.03 to 9.8 mL (median, 0.7 mL). Parathyroid maximum MIBI activity correlated with the volume of the gland (r = 0.54, P = .002) and serum parathyroid hormone level (r = 0.58, P = .001). No correlation between maximum MIBI activity and oxyphil cell content or Pgp expression could be demonstrated. Elimination half-life of MIBI from parathyroid inversely correlated with Pgp (r = -0.36, P = .05). The ex vivo lesion-background count ratio positively correlated with volume of the gland (r = 0.66, P = .001) and parathyroid hormone level (r = 0.48, P = .006). Ex vivo lesion counts and Pgp expression were negatively correlated (r = -0.37, P = .04). A strong relationship between volume of the parathyroid gland, serum parathyroid hormone levels, and MIBI uptake exists in primary hyperparathyroidism. Gamma probe-guided localization of abnormal gland(s) can be more successful if surgery is undertaken at maximum uptake ratio. High Pgp expression increases MIBI parathyroid clearance rate, decreases gamma probe counts, and may significantly alter the optimal time to surgery.

15545561

The clinical risk score: emerging as a reliable preoperative prognostic index in hepatectomy for colorectal metastases.

The purpose of this study was to examine the validity of the clinical risk score (CRS), a prognostic tool developed by Fong et al, when translated to another center. This study assesses 5 independent preoperative prognostic criteria, nodal status of the primary lesion, disease-free interval, number of hepatic metastases, size of the largest metastasis, and preoperative carcinoembryonic antigen level, to determine a preoperative CRS for each patient included in the study. The hepatobiliary unit of The Queen Elizabeth Hospital, Adelaide, South Australia. Medical records of patients admitted to The Queen Elizabeth Hospital undergoing potentially curative hepatic resection for colorectal metastases during the period of July 1993 to April 2003 were included in the study. Main Outcome Measure The primary outcome measure of the study was survival. The calculated CRS was analyzed with respect to patient postoperative survival. During the 10-year period, 77 patients underwent hepatic resection. Overall survival rates for 1, 3, and 5 years were found to be 80.9%, 57.5%, and 42.3%, respectively. One- and 5-year survival rates for CRSs of 0 and 1 were found to be 93.8% and 72.5%, respectively; for scores of 2 and 3, 76.6% and 31.2%, respectively;and for scores of 4 and 5, 75% and 0%, respectively. No patient with a CRS greater than 3 survived more than 2 years. This study validates the CRS, finding it to be highly predictive of patient outcome and survival.

15545560

Multicenter, prospective, double-blind, randomized trial of laparoscopic nissen vs anterior 90 degrees partial fundoplication.

Laparoscopic anterior 90 degrees partial fundoplication for gastroesophageal reflux is associated with a lower incidence of postoperative dysphagia and other adverse effects compared with laparoscopic Nissen fundoplication. A multicenter, prospective, double-blind, randomized controlled trial. Nine university teaching hospitals in 6 major cities in Australia and New Zealand. One hundred twelve patients with proven gastroesophageal reflux disease presenting for laparoscopic fundoplication were randomized to undergo either a Nissen (52 patients) or an anterior 90 degrees partial procedure (60 patients). Patients with esophageal motility disorders, patients requiring a concurrent abdominal procedure, and patients who had undergone previous antireflux surgery were excluded from this study. Laparoscopic Nissen fundoplication with division of the short gastric vessels or laparoscopic anterior 90 degrees partial fundoplication. Independent assessment of dysphagia, heartburn, and overall satisfaction 1, 3, and 6 months after surgery using multiple clinical grading systems. Objective measurement of esophageal manometric parameters, esophageal acid exposure, and endoscopic assessment. Postoperative dysphagia, and wind-related adverse effects were less common after a laparoscopic anterior 90 degrees partial fundoplication. Relief of heartburn was better following laparoscopic Nissen fundoplication. Overall satisfaction was better after anterior 90 degrees partial fundoplication. Lower esophageal sphincter pressure, acid exposure, and endoscopy findings were similar for both procedures. At the 6-month follow-up, laparoscopic anterior 90 degrees culine partial fundoplication is followed by fewer adverse effects than laparoscopic Nissen fundoplication with full fundal mobilization, and it achieves a higher rate of satisfaction with the overall outcome. However, this is offset to some extent by a greater likelihood of recurrent gastroesophageal reflux symptoms.