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+{"text": "Cigarette smoking and coal burning are the primary sources of indoor air pollution in Chinese households. However, effects of these exposures on Chinese children's respiratory health are not well characterized.Seventh grade students (N = 5051) from 22 randomly selected schools in the greater metropolitan area of Wuhan, China, completed an in-class self-administered questionnaire on their respiratory health and home environment.Coal burning for cooking and/or heating increased odds of wheezing with colds  and without colds . For smoking in the home, the strongest associations were seen for cough  and phlegm production  without colds among children who lived with two or more smokers.Chinese children living with smokers or in coal-burning homes are at increased risk for respiratory impairment. While economic development in China may decrease coal burning by providing cleaner fuels for household energy use, the increasing prevalence of cigarette smoking is a growing public health concern due to its effects on children. Adverse effects of tobacco smoke exposure were seen despite the low rates of maternal smoking (3.6%) in this population. Residential coal burning and cigarette smoking are the most common sources of indoor air pollution in Chinese households -3. Altho2), carbon monoxide (CO), particulate matter, and other pollutants [Coal has been widely used for cooking and heating in China ,9. Domesllutants ,10,11. SAn increasing proportion of Chinese children are exposed to tobacco smoke because smoking prevalence in China has increased rapidly, especially among men, during the last decades ,12. OverWe examined the associations between respiratory symptoms and exposure to residential coal burning and environmental tobacco smoke in a cross-sectional study of seventh grade students in the greater metropolitan area of Wuhan, China.In the spring of 1999, 5231 seventh grade students at 22 public schools in and around Wuhan, China were invited to complete a self-administered questionnaire on respiratory symptoms and home environment. Two schools were randomly selected from each of the 11 administrative units governed by the city of Wuhan, the capital of Hubei Province. The resulting schools included 14 schools from urban (53.0% of the students), three schools from suburban (25.1%), and five schools from rural areas (21.9%). Of the 5231 students, 5051 (97 %) provided parental consent and completed the questionnaire in class with study staff in attendance. Before completing the questions students viewed a video demonstrating wheezing . The stuThe questionnaire included questions on respiratory health and potential risk factors such as exposure to cooking and heating fuels, smokers in the home, and personal smoking. We incorporated respiratory health items from a standardized questionnaire (ATS-DLD-78-C) translated into Chinese for a previous study in Wuhan and other Chinese cities . Our priWe assessed exposure to residential coal burning by questions defining the types of fuels used for cooking and heating in the child's home. We combined information on cooking and heating with coal into a single variable with the following categories: no coal stove, coal stove used only for heating, coal stove used only for cooking, and coal stove used for both cooking and heating. To assess environmental tobacco smoke exposure, we asked the child to list all household members and indicate whether each person currently smoked. We created three exposure categories: no smokers in the home, one smoker (1) in the home, and two or more smokers (2 +) in the home. Personal smoking was not considered in the analysis because of very low prevalence (0.6%).We calculated prevalence odds ratios  for each of the six outcome measures  by unconditional logistic regression . Although the odds ratio is the most common measure of association in cross-sectional studies , divergeWe excluded 521 subjects with missing data on any of the outcome or exposure variables leaving 4,530 subjects for the analysis. The following covariates were considered as potential confounders or modifying factors within the logistic models: child's gender, presence of animals in the household, presence of pests , crowding in the household, presence of older siblings, parental asthma, physical activity, living area (school district), and time spent indoors and outdoors. To account for variation due to the type of neighborhood the children lived in, we included school district (22 districts) in the models using CLASS and REPEATED statements within Proc Genmod in SAS. The models reported here are adjusted for coal use, smokers in the home, school district, and child's sex because inclusion of the other variables did not appreciably change the associations.Characteristics of the study population are presented in Table After adjusting for gender, ETS, and living area, residential coal burning was primarily associated with wheezing in the past 12 months  in this population. The majority of the diagnosed asthmatics (76.4%) lived in urban areas, where prevalence of coal use was lower than in non-urban areas. The diagnostic ascertainment of asthma most likely was greater in the urban than in the rural areas.In utero exposure, via maternal smoking, that is believed to contribute to adverse effects of ETS in children [The harmful effects of ETS in children, primarily from living with smokers, have been widely studied ,28-31. Ichildren ,33 is unOur results indicated an exposure dependent response to ETS; having two or more smokers in the household increased the odds of cough and phlegm production compared to having only one smoker in the household. We did not find strong evidence suggesting modifying effects by gender, although the effect of ETS on persistent cough without colds was more pronounced among boys than girls (data not shown). Exposure levels may be influenced by time-activity patterns that can differ by gender. Boys may be more likely to spend time in close proximity with their smoking fathers or male relatives than girls.Mechanisms responsible for the respiratory effects of ETS have been proposed in the literature . In addiIn general, our findings agree with available data on Chinese children's respiratory health ,23. HoweExposure to indoor air pollutants is not only influenced by the source strength and other emission characteristics, but also by air exchange rates. A recent study showed that ventilation could modify effects between respiratory health outcomes and indoor air pollutants . In thatThe composition of pollutants produced by residential coal burning and smoking can be highly variable, but both exposures contribute substantially to inhalable and respirable particulate matter in indoor environments ,3,41. ExOur outcome and exposure measures were determined by questionnaire alone, which is one of the major limitations of the study. Nonetheless, large epidemiological studies of respiratory health often rely on reports on recent symptom history because self-reported measures are cost efficient, practical and their repeatability is good ,43. Gene2, often exceed the levels outdoors [3) and SO2 (173 \u03bcg/m3) can reach high levels indoors [2.5, PM10, and SO2 in urban areas were 73 \u03bcg/m3, 129 \u03bcg/m3, and 73 \u03bcg/m3, respectively [Although urban air pollution has long been a major environmental concern in China, we do not believe that outdoor air pollution alone could explain the observed associations. Exposures to indoor air pollutants are likely to dominate the total exposure burden , especiaoutdoors . In Wuhaectively . Because and SO2 73 \u03bcg/m3 The major strength of this study is that the public school system ensured a large and representative sample of rural, suburban, and urban populations in the Wuhan area. Our study is one of the few studies that have examined effects of major indoor pollutants in relation to children's respiratory health in mainland China ,23-25.Coal burning and living with smokers contributed to persistent respiratory symptoms in this cohort of Chinese adolescents. Adverse effects of tobacco smoke in the home were seen despite the very low prevalence of maternal smoking. Even if exposure to residential coal burning declines in response to economic changes in China, the increasing prevalence in smoking augur an increase in children's exposure to environmental tobacco smoke. Because many men initiate smoking during adulthood, and the rate of quitting and desire to quit smoking are low , future CI = confidence intervalCO = carbon monoxideETS = environmental tobacco smokeOR = odds ratio10 = particulate matter with an aerodynamic diameter less or equal to 10 \u03bcmPM2.5 = particulate matter with an aerodynamic diameter less or equal to 2.5 \u03bcmPM2 = sulfur dioxideSOThe authors declare that they have no competing interests.Contributors: PMS analyzed the data and wrote the manuscript with input from all investigators. YL, JX and CL are key investigators for the data collection. ELA assisted with data collection, and GK assisted with data analysis. CAJ was involved in design of the study. SJL is the principal investigator and guarantor of the manuscript."}
+{"text": "The objective of this study was to evaluate how indoor pollution from tobaccoand home heating may adversely affect respiratory health in youngchildren.A birth cohort was followed longitudinally for 3 years to determine incidenceof lower respiratory illness (LRI).A total of 452 children born 1994\u20131996 in two districts in theCzech Republic participated.Indoor combustion exposures were home heating and cooking fuel, mother\u2019ssmoking during pregnancy, and other adult smokers in the household. Diagnosesof LRI (primarily acute bronchitis) from birth to 3 yearsof age were abstracted from pediatric records. Questionnaires completedat delivery and at 3-year follow-up provided covariate information. LRIincidence rates were modeled with generalized linear modelsadjusting for repeated measures and for numerous potential confounders.LRI diagnoses occurred more frequently in children from homes heated bycoal . Maternal prenatal smoking and other adult smokers alsoincreased LRI rates . Cookingfuels  werenot associated with LRI incidence. For children never breast-fed, coalhome heating and mother\u2019s smoking conferred substantiallygreater risks: RR = 2.77  andRR = 2.52 , respectively.Maternal smoking and coal home heating increased risk for LRI in the first 3 yearsof life, particularly in children not breast-fed.Few studies have described effects of coal heating fuel on children\u2019shealth in a Western country. Breast-feeding may attenuate adverseeffects of prenatal and childhood exposures to combustion products. Children < 3 years of age, and especially in their first year, are atgreatest risk of serious respiratory illnesses . StudiesRecently, increasing attention has focused on effects of indoor air exposureson respiratory health of children . These eHeightened susceptibility of infants and young children to environmentaltoxicants has been suggested. Inhaled pollutant dose per unit body weightis likely to be greater, because their body weight is smaller andtheir respiratory rate higher than in adults . InfantsThis study, part of a large program of research on air pollution healtheffects in the Czech Republic (Teplice Program), focused on respiratoryillnesses in young children in relation to indoor combustion of cigarettesand of coal, wood, natural gas, and propane for heating or cooking. Ahigh proportion of adults smoke in the Czech Republic, and coalis still used for heating of some homes; hence, this setting is advantageousfor studying indoor air exposures.We followed a birth cohort to 3 years of age in two districts in the CzechRepublic: Teplice in the northwest, and Prachatice in the southwest. TheTeplice district, with a population of about 130,000, is infamousfor high air pollution due to power plants and home heating with coal. ThePrachatice district has a population of about 50,000 and no powerplants. Children born between May 1994 and December 1996 in the twodistricts were eligible for this study.n = 615) (The evolution of our study sample is shown in n = 615) . InitialFor this investigation, we recontacted the IBS participants at 3 yearsof age. Of the 615 IBS children, 50 were ineligible for follow-up . Of the remaining 565, pediatric datawere abstracted for 548 (97%): Nine families were not locatedfor follow-up, and eight mothers denied permission to review medicalrecords. Of the 548 with pediatric data available, 452 (82.5%) ofthe mothers completed a 3-year follow-up questionnaire. Their childrenwere the subjects of this analysis. This study was approved bythe institutional committees on human subjects at the Institute for ExperimentalMedicine in Prague, the University of North Carolina, ChapelHill, and the University of California, Davis.Trained nurses administered questionnaires at the birth hospital within 3 daysof delivery and elicited reproductive and medical history; medications; demographicinformation; smoking, alcohol, and other lifestylefactors; and work histories and occupational exposures. For the 3-yearfollow-up, pediatric nurses contacted families to invite their participationand provided them a new questionnaire regarding breast-feeding, childcare attendance, child\u2019s and family members\u2019 allergies, indoorheating and cooking fuel sources, and informationabout household members\u2019 age, relation to child, and smoking behaviors. Informedconsent was administered at birth and again at follow-up, beforecollection of data.International Classification of Diseases, 10th Revision (ICD-10) codes  codes , and anyn = 70) were excluded from this analysis. The date of pediatrician\u2019sdiagnosis served as a proxy for date of illness occurrence. Toavoid counting the same illness twice, we did not include diagnosesoccurring within 30 days after a previous LRI diagnosis, leaving 893 incidentLRI events.The 452 children in our study sample experienced 1,049 LRI events basedon ICD-10 codes J20, J21, J40, and J44, of which 98% were acutebronchitis (J20). Pneumonia diagnoses  in the home. Because most fuelsfor heating conferred no increase in LRI rates or were used by veryfew households, in final models we compared coal home heating with allother heating sources. Direct measurements of organic carbon, elementalcarbon, or other elements were not made in homes.Information on ETS exposure was available from both the birth and 3-yearquestionnaires. We assessed mother\u2019s smoking during pregnancy (yes/no) andadult smokers in the household at 3-year follow-up (yes/no).a priori based on a review of the literature and preliminary bivariate analyses. Time-independentcovariates included district of residence (Teplicevs. Prachatice), child\u2019s sex, maternal ethnicity [eitherCentral European or Rom (\u201cGypsy\u201d) based on self-report], andmaternal education .Covariates of interest were selected On the 3-year follow-up questionnaire, mothers were asked until what agetheir child was \u201cfully\u201d and \u201cpartially\u201d breast-fed; weused the maximum of these two responses to constructthree time-varying categories: currently breast-feeding, ever breast-fed, andnever breast-fed. We also constructed categories for time sincecessation of breast-feeding, to capture possible protection beyondthe weaning period: currently breast-feeding, breast-fed in the lastmonth, last breast-fed 1\u20136 months ago, last breast-fed > 6 monthsago, and never breast-fed. For analyses of breast-feeding as amodifier of the effects of indoor air exposures, we dichotomized childrenas ever or never breast-fed.Other time-dependent variables included child\u2019s age in years, currentchild care attendance , numberof other household members \u2264 14 years of age , andhousehold density (number of adults and children livingin the household divided by the number of rooms). The seasons were winter (December\u2013February), spring (March\u2013May), summer (June\u2013August), andautumn (September\u2013November). This categorizationcaptured school vacation (June\u2013August) as a distinctperiod. Days of the week were grouped based on similarity in LRI rates: Saturday\u2013Sunday, Monday, and Tuesday\u2013Friday.We examined temperature and relative humidity using 24-hr means from whichwe calculated moving averages for 3, 7, 14, and 30 days before (andincluding) the day of diagnosis. Of these averaging periods, 14-dayaverage temperature showed the strongest association with LRI and thereforewas selected. The log-odds of LRI decreased linearly with temperature, permittinga continuous term for temperature. Relative humiditywas not associated with LRI diagnosis.n observation-days, where n is the number of LRI events separated by at least 30 days. On a givenday of life, occurrence of an LRI was coded 1; nonevent days were coded 0.We used generalized estimating equations (GEEs) to model the associationswith LRI diagnoses . This mee\u03b2 for each variable, or category, and corresponding 95% confidenceintervals (CIs) were calculated.We used a logit link, that is, a logistic model, of the binary outcome (eventday: yes/ no) and specified an exchangeable correlation structureto account for greater within-child than between-children homogeneity. Theprobability (rate per child-day) of an LRI event occurring onany given child-day was small (0.002). Odds of an event are thereforearithmetically very close to the per child-day rate. The rate ratio (RR) wasestimated by Because the children in this follow-up study were not a simple random sampleof births in Teplice and Prachatice districts, we also accountedfor the unequal sampling probabilities of normal-birth-weight/full-termand low-birth-weight/preterm infants, which differed by district andby year of study. Inverse sampling probabilities were used as weightswith a design of stratified sampling without replacement. Model fittingwas conducted using SUDAAN software .p-value of < 0.15) with LRI in bivariable analyses were entered intoa full model, and less influential covariates were removed, one by one, beginningwith the least influential . Covariates for which removal changed the point estimates for coalheat or ETS by \u2265 10% were retained as confounders. Finalmodels included coal heating, mother\u2019s smoking during pregnancy, presenceof other adult smokers in the household, mother\u2019sage and education, child\u2019s sex and year of life, breast-feeding, childcare attendance, number of other children living in thehome, season, day of the week, and 14-day moving-average temperature.Initially, we determined \u201ccrude\u201d rates of LRI (LRI eventcounts per child-time at risk) within categories of predictor variables. RRsand 95% CIs were then estimated using GEE models adjustedfor sampling design, but not for other covariates. Next, covariatesassociated .Fifty percent of homes were heated primarily by distant heating (heat froma remote source outside the home), 23% by natural gas, 13% bycoal, and 6% by wood . NaturalEight percent of mothers were of Rom ethnicity, and 87% breast-fedthe children, 23% for > 6 months. Twenty-one percent ofchildren attended child care with other children at some time duringthe first 3 years of life, and about three-fifths lived in householdswith another child.Coal heat, mother\u2019s smoking during pregnancy, and ETS during thefirst 3 years of life were all associated with greater incidence ofLRI . Use of The observed associations between indoor combustion exposures and LRI incidencepersisted after multivariate control for mother\u2019s ageand education, child\u2019s sex and year of life, breast-feeding, currentattendance at child care, other children living in the household, season, dayof the week, and 14-day moving-average temperature . ChildreWe found that children exposed to indoor coal combustion experience a greaterincidence of pediatrician-diagnosed LRI during the first 3 yearsof life. We did not observe associations between LRI incidence and useof natural gas or propane as heating fuel. The coefficient for woodas a heating source was elevated, but the CIs were wide, largely becausewood was used by very few households. Coal, wood, and propane forcooking showed elevated but imprecise relative risks, and both coal andwood were rarely used for cooking. Fewer than half of families withgas heating had a furnace inside the flat, suggesting that exposures suchas nitric oxide and nitrogen dioxide would occur only in some of thehomes. Nevertheless, previous studies of infants also indicate no increasesin LRI from exposures to nitrogen dioxide . Thus, oa]pyrene to lead in ambient outdoor air increased 5\u201315 timesover the ratio observed in summer months, which was attributed toemissions from residential home heating by coal combustion . These errors seem unlikely to differ by exposure, butcould possibly mask covariate\u2013respiratory illness associations, whichmay limit the ability to control for these covariatesas potential confounders of pollutant\u2013respiratory illness associations.Another limitation was the lack of information on changes in some homeenvironmental characteristics between birth and 3 years of age. At thetime of our study, a national policy was in effect to replace coal inhome heating with natural gas, with the goal to decrease outdoor airpollution. Despite this, about 13% of the study children\u2019shomes were still heated primarily by coal at follow-up (1997\u20131999). Afamily that switched from coal to gas would have reporteduse of gas at 3-year follow-up, resulting in misclassification of heatingtype for some part of the 3-year follow-up. Generally, this mighthave attenuated the results, with some of the \u201cunexposed\u201d childrenactually exposed to coal. If switching were related to otherfactors, the direction of bias would be difficult to predict.This study used pediatrician-reported LRI. Capture of an LRI diagnosisdepends on health service utilization, as well as the ability of the pediatricianto reliably record the type of illness. Our data demonstrateextensive utilization of health care services by this population ofCzech mothers. Despite some changes in the provision of health care inthe Czech Republic over the past decade, including the emergence ofprivate health insurance, mostly provided by employers, all residentsare entitled to health care, and consumer cost is relatively low. An indicationof the high utilization of physicians is the completeness ofthe legally mandated 18-and 36-month pediatric well-child visits, whichin our study sample was > 96% and > 98%, respectively. Ratesof complete series of immunizations for diphtheria, pertussis, andtetanus (DPT); polio; and measles were high, far exceedingU.S. 1997 rates for children of the same age . For exaIn summary, we found exposure to coal home heating and ETS increase youngchildren\u2019s LRI rates during the first 3 years of life. Theseeffects were attenuated by breast-feeding. Although ETS has been wellstudied, residential coal combustion in economically developed countrieshas not; these findings demonstrate that both sources of indoor aircombustion pollutants present a hazard to respiratory health in infancyand early childhood. Efforts to reduce these emissions would benefitinfants and young children perhaps especially in the Czech setting, wherecoal is still commonly used in home heating and smoking ratesare relatively high."}
+{"text": "The burning of biomass in the developing world for heating and cooking results in high indoor particle concentrations. Long-term exposure to airborne particulate matter (PM) has been associated with increased rates of acute respiratory infections, chronic obstructive lung disease and cancer. In this study we determined the oxidative activity of combustion particles derived from the biomass fuel dung cake by examining their capacity to deplete antioxidants from a model human respiratory tract lining fluid (RTLF). For comparison, the observed oxidative activity was compared with that of particles derived from industrial and vehicular sources.Incubation of the dung cake particle suspensions in the RTLF for 4 h resulted in a mean loss of ascorbate of 72.1 \u00b1 0.7 and 89.7 \u00b1 2.5% at 50 and 100 \u03bcg/ml, respectively. Reduced glutathione was depleted by 49.6 \u00b1 4.3 and 63.5 \u00b1 22.4% under the same conditions. The capacity of these samples to deplete ascorbate was in excess of that observed with diesel or gasoline particles, but comparable to that seen with residual oil fly ash and considerably in excess of all three control particles in terms of glutathione depletion. Co-incubation with the metal chelator diethylenetriaminepentaacetate inhibited these losses, whilst minimal inhibition was seen with superoxide dismutase and catalase treatment. The majority of the activity observed appeared to be contained within aqueous particle extracts.These data demonstrate that biomass derived particles have considerable oxidative activity, largely attributable to their transition metal content. The btilation . These c average .10 [An increasing body of evidence has linked exposure to indoor pollutants with increased rates of respiratory morbidity and mortality. Women and children exposed to high indoor PM concentrations have significantly increased rates of acute respiratory infections (ARI) ,8-10, an10 . Whilst 10 , as well10 ,16 have The capacity of inhaled PM to elicit damaging oxidation reactions in the lung and systemic circulation may account for many of the toxic responses observed in exposed individuals . CertainIn this study we evaluated the oxidative activity of PM samples derived from the controlled burning of cow-dung cake using a traditional Indian cooking stove. These assessments were based on the capacity of dung cake particulate suspensions to deplete physiologically relevant antioxidants, ascorbate (AA), urate (UA) and reduced glutathione (GSH) from a synthetic model of human respiratory tract lining fluid. Previously we have used this model to investigate the nature of gaseous and particulate pollutant-antioxidant interactions at the air lung interface ,22. In a2.5. Particle suspensions prepared from each of the three filters depleted AA from the synthetic RTLF in a dose dependent manner with a mean loss of 72.1 \u00b1 0.72 and 89.7 \u00b1 3.7% AA at 50 and 100 \u03bcg/m3 respectively relative to the 4 h particle free control concentration. This loss of AA was significantly greater than that associated with an equal mass (50 \u03bcg/m3) of diesel (6.4%), gasoline (5.2%) or residual oil fly ash (ROFA) (53.9%) particles , 1.20 (DC2) and 1.14 mg (DC3) of PM, 1.20  and 15.7% (gasoline) , 13.5 \u00b1 0.49 (DC2) and 13.3 \u00b1 0.19 nM/s (DC3). Co-incubation with the metal chelator DTPA (100 \u03bcM) significantly reduced the rate of AA oxidation: 6.5 \u00b1 0.36 versus 13.5 \u00b1 0.27 nM/s for all three samples  of the oxidative activity was found to be associated with water-soluble components Figure . The resIndoor air pollution poses a significant health risk worldwide. WHO estimates suggest that up to 6.5% of the annual disease burden in developing nations is attributable to the burning of solid fuels in the indoor environment ,25. Smokin vivo into quinones species through the action of the cytochrome P450, epoxide hydrolase and dihydrodial dehydrogenase detoxification pathway [It has been proposed that the capacity of inhaled particles to elicit inflammation and injury in the lung, as well as systemically, may be related to their capacity to cause oxidative stress . In this pathway  and fina pathway , futile  pathway , or by t pathway . The intin vitro screening procedure that assessed the capacity of PM associated pro-oxidant components  to deplete physiologically relevant antioxidants, ascorbate, urate and reduced glutathione from a synthetic model of the RTLF [In this study we measured PM oxidative activity using an the RTLF ,22. Thisthe RTLF ,30 suggethe RTLF .3+ and Cu2+.The depletion of both AA and GSH from the model was prevented by co-incubation with the metal chelator diethylenetriaminepentaacetate (DTPA) indicating that the losses observed were driven by redox active metals such as Fe, Cu, Ni, and Cr. DTPA has five acetate groups linked to a molecular backbone that permits it to form tight complexes with a broad range of metals, preventing them from catalysing damaging oxidation reactions. Desferoxamine (DFO) was not used in these studies as it has been reported to reduce chelated Cu that would have resulted in interpretive difficulties when examining its protective role in mixtures of soluble metals . The con3+ to Fe2+ and Cu2+ to Cu+. The superoxide formed by the subsequent oxidation of ferric and cupric ions could undergo dismutation to hydrogen peroxide, reduce Fe3+ and Cu2+, or oxidise ascorbate, urate or glutathione within the synthetic RTLF. As the reaction rate between Fe3+ and superoxide (1.5 \u00d7 108 M-1s-1) greatly exceeds that its dismutation at physiological pH  it seems likely that the former reaction predominated, especially as little loss of ascorbate or glutathione could be attributed to superoxide or hydrogen peroxide production. Interestingly, we saw no evidence of urate depletion, despite the importance of this antioxidant in protecting the airway against oxidant gases [9 M-1s-1, pH 6\u20137) is broadly similar to that of both ascorbate  and glutathione . Thus the absence of UA depletion in this model supported the contention that superoxide dismutation to hydrogen peroxide was not occurring to any great extent, with little evidence of hydroxyl radical generation. Whilst the redox potentials of UA and AA at pH7 (E\u00b0' = 590 and 282 mV respectively) [Thus we conclude that AA and GSH oxidation occurred predominately by their direct reduction of Fent gases , peroxynnt gases  and hydrnt gases . The ratctively)  may suggWe also observed that the capacity of DTPA to inhibit ascorbate oxidation was significantly reduced in the ascorbate only incubation experiment: only 100 \u03bcM being required for full inhibition in the complete synthetic RTLF as opposed to 200 \u03bcM in the ascorbate only RTLF model. This finding may imply that either GSH or UA is limiting the bioavailability of Fe, either through chelation, as has been proposed for UA , or by i2.5), used in the current study have been described previously [As these findings implicated redox active metals in the oxidation process we measured the bioavailable Fe and Cu content of the DC particles. This measurement included reduced and oxidised forms of these metals, both water-soluble and surface mobilisable through ligation to the chromogenic chelators bathophenantroline-disulphonate (BPS)  and batheviously ,40 usingeviously , which cThe high metal content of the dung cake may reflect both the presence of biological metals; especially Fe and Cu in dung , as wellThese data demonstrate that fine particles derived from the burning of the biomass fuel dung cake are highly oxidising. This activity appears to be largely related to their content of redox active metals, with the caveats mention above. As oxidative activity is a biologically meaningful index, the high activity associated with dung cake fine particles allied to their high concentrations in homes using dung cake fuel emphasises their potential negative health impact.Dung-cake was obtained from Eksaal, near Mumbai. Dung cake has a high ash content, 25\u201330% and is made by mixing dung with mud/clay and straw to improve its mechanical strength. It is then patted down, moulded and sun-dried for 24 h, which achieves moisture content of 7\u20138%. As dung-cake combustion results in copious particle emissions a sampling rate of 3\u20134 L per minute was used in the particle sampler, and a burn time of 12 min, to ensure no clogging of the Teflon filter substrates used for particle collection. The particle sampler employed had a cyclone inlet to exclude particles larger than 2.6 \u03bcm diameter . All subFor the initial experiments dung cake extracts were diluted to either 100 or 50 \u03bcg/ml and incubated at 37\u00b0C in synthetic RTLF  for a period of 4 h. After incubation, samples were either acidified with metaphosphoric acid to a final concentration of 5% (w/v) for UA and AA measurement or diluted into 100 mM phosphate buffer for determination of GSH. Antioxidant determinations have been described previously . Subsequ1 ml of each dung cake particle suspension at 55.6 \u03bcg/ml was vortexed hard for 2 minutes with an equal volume of HPLC grade hexane. At the end of this mixing period the organic and aqueous phases were separated, and the former dried under nitrogen at room temperature. The hexane extract was then resuspended according to the standard protocol in an equal volume of Chelex-treated water containing 5% methanol.2+-specfic chromogenic chelator, bathophenantroline disulphonate (BPS) [Total iron concentrations in the particle suspensions were determined using the Fete (BPS) . DC partte (BPS) , with thAll data are expressed throughout as means with SD. Specific experimental details are summarised the each Figure legend. Experimental group comparisons were performed using two way repeated measures ANOVA. Post-hoc comparisons of group means were performed using the Student-Newman-Kuels test. All statistical analyses were performed using SPSS for windows, version 11.5 or the Unistat Exel plug-in, version 4.53.AOX AntioxidantAA AscorbateUA UrateGSH Reduced glutathioneGSSG Glutathione disulphideRTLF Respiratory tract lining fluidDC Dung cakeDFO DesferoxamineBPS Bathophenantroline-disulphonateBCS Bathocuproine-disulphonateDPTA Diethylene triamine pentaacetateSOD Superoxide dismutasehiSOD Heat inactivated superoxide dismutaseCAT CatalasePAH Polyaromatic hydrocarbonsPM Particulate matterWHO World Health OrganisationIan S Mudway: NoneSean T Duggan: NoneChandra Venkataraman: NoneFrank J Kelly: NoneJonathon Grigg: NoneIan S Mudway: Study design and experimental work, data analysis, preparation of manuscriptSean T Duggan: Study design and experimental analysisChandra Venkataraman: Collection of particle samples from dung cake combustionGazala Habib: Collection of particle samples from dung cake combustionFrank J Kelly: Study conception and design, manuscript reviewJonathon Grigg: Study conception and design"}
+{"text": "Cigarette smuggling is a major public health issue, stimulating increased tobacco consumption and undermining tobacco control measures. China is the ultimate prize among tobacco's emerging markets, and is also believed to have the world's largest cigarette smuggling problem. Previous work has demonstrated the complicity of British American Tobacco (BAT) in this illicit trade within Asia and the former Soviet Union.This paper analyses internal documents of BAT available on site from the Guildford Depository and online from the BAT Document Archive. Documents dating from the early 1900s to 2003 were searched and indexed on a specially designed project database to enable the construction of an historical narrative. Document analysis incorporated several validation techniques within a hermeneutic process. This paper describes the huge scale of this illicit trade in China, amounting to billions of (United States) dollars in sales, and the key supply routes by which it has been conducted. It examines BAT's efforts to optimise earnings by restructuring operations, and controlling the supply chain and pricing of smuggled cigarettes.Our research shows that smuggling has been strategically critical to BAT's ongoing efforts to penetrate the Chinese market, and to its overall goal to become the leading company within an increasingly global industry. These findings support the need for concerted efforts to strengthen global collaboration to combat cigarette smuggling. Smoking tobacco kills about 5 million people around the world every year, and according to the World Health Organization, half of the people who smoke will eventually die as a result. The habit is in decline in most of the world's richer nations, but it is growing in developing countries. Governments can control consumption, and raise substantial tax revenues, by levying high taxes on tobacco. However, this is undermined when tobacco is smuggled into the country and sold illegally. Smuggling makes cigarettes more affordable, thus stimulating consumption, and undermines efforts to reduce smoking rates.\u2003\u2003British American Tobacco (BAT) is one of the largest international tobacco companies. Following a legal case in the United States, millions of pages of BAT internal documents (dating back to the early 1900s) have been made public. They are held in a depository in the United Kingdom, and most can now be seen online. Researchers have already undertaken wide-ranging studies of these documents and have drawn conclusions about the way BAT has sought to enter emerging markets throughout the world.Previously published studies have concluded that BAT has been \u201ccomplicit\u201d in large-scale cigarette smuggling, described in documents by the euphemisms \u201ctransit,\u201d \u201cgeneral trade\u201d (\u201cGT\u201d), \u201cduty not paid\u201d (\u201cDNP\u201d), and \u201cfree market.\u201d This study focuses on such activities in China, which is believed to have the world's biggest tobacco smuggling problem. China is a key market for BAT and other tobacco companies because of the size of its population, high smoking prevalence, and rapidly growing economy. The development of effective tobacco control policy in China constitutes one of the greatest opportunities to advance global health. The State Tobacco Monopoly Administration is the only agency legally allowed to trade in tobacco in China.The authors have put together an account of the company's trading in China in the period from 1986 to 1999. They begin by describing the scale on which tobacco was smuggled into China and the main supply routes through which tobacco entered the country. They found evidence that smuggling increased considerably during the period.\u2003\u2003The researchers found that BAT's own documents describe the company's efforts to improve its earnings from China by restructuring operations and controlling the supply chain\u2014that is, how cigarettes are manufactured and legally exported, are handled by various \u201cagents\u201d or middlemen, and then are sold illicitly as contraband. The title of this article is taken from one BAT document that states that exports from Hong Kong to China are the \u201ckey to the future\u201d of the company's planned growth. The researchers say that this can be taken to mean smuggling, and that BAT's documents indicate that the company benefited from, encouraged, and attempted to control the smuggling of tobacco into China.BAT has stated publicly that it does not approve of smuggling. However, from the researchers' analysis of the information in the internal papers, they conclude that \u201cthe documents demonstrate that contraband has been a hugely profitable and integral part of BAT operations in China over the past two decades.\u201d They estimate that the value of the illicit trade amounts to billions of (United States) dollars and go on to say that \u201cfor the Chinese government, this illicit trade undermined restrictions on imports, represented an enormous loss of tax revenue, and stimulated demand for premium brand cigarettes.\u201d The researchers argue that action should be taken internationally to reduce tobacco smuggling.http://dx.doi.org/10.1371/journal.pmed.0030228.Please access these Web sites via the online version of this summary atBritish American Tobacco Documents Archive created by public health researchers \u2022\u2009Most of the documents used in this paper can be found at theTobacco Free Initiative, based at the World Health Organization, provides information on the global scale of the tobacco problem, and about efforts to combat the situation\u2022\u2009Thenational anti-smoking organizations; the United Kingdom charity Action on Smoking and Health has a useful list of these\u2022\u2009Many countries haveAmerican Academy of Family Physicians andNHS Direct \u2022\u2009Information about the dangers of smoking to personal health and advice on giving up the habit are available from the Web sites of the Internal documents from British American Tobacco demonstrate the scale of tobacco smuggling into China and show that this illicit trade has been a critical factor in BAT's expansion in this market. Cigarette smuggling has emerged as a critical public health issue. As well as being the source of huge losses in government revenues, smuggling makes cigarettes more affordable, thus stimulating consumption and undermining control measures, most notably among youth and low-income consumers ,2. The Tobacco industry documents, released following litigation in the United States, have demonstrated the complicity of British American Tobacco (BAT) in large-scale smuggling . Whereas12) stick market. However, up to 50 billion (5 \u00d7 1010) foreign cigarettes were believed to be entering the country illegally each year by the late 1990s  business\u2026representing approximately 92% of the total\u201d . The exiThere is inadequate segregation of the management of the various markets currently served by BAT HK, marketing activities are inadequately co-ordinated and the structure is insufficiently flexible to respond to changes which may occur in the lead up to 1997 .Reorganisation was seen as necessary to facilitate further expansion of the contraband trade across Asia. As Roy Salter  wrote, \u201cOpportunities will emerge for sales to traders dealing in other markets, e.g. Thailand, possibly in conflict with established BATUKE [BAT United Kingdom and Export] and B&W [Brown and Williamson] agents, such as SUTL [Singapura United Tobacco Limited]\u201d .In 1990, a report was commissioned from consultancy firm Griffiths Management Limited to develop management and corporate structures that would provide BAT \u201cwith a competitive advantage over other international manufacturers\u201d  in ChinaThe proposed restructuring would be completed with the creation of \u201can offshore company\u2026domiciled in the UK\u201d to handle \u201cselling to trading organisations supplying China and other adjacent markets\u201d. The offshore company was provisionally named BAT Distribution, and later referred to as International Tobacco (Overseas) Limited, and the report was explicit in the description of its proposed remit:The transit trade for China, Taiwan and elsewhere in Asia should be undertaken by BAT Distribution Limited, incorporated in a tax haven of choice, probably UK. It could be owned by BAT Industries or whatever other company BAT Group prefers.BAT Distribution should be little more than a brassplate company with very low overhead and the flexibility to establish branch offices wherever the transit traders move .The primary objective of this complex model was to provide an operational divide between legal and illegal trade while, critically, ensuring clear oversight and control:BAT Distribution is set up to enable BATHK to effect a formal separation of its transit from its official trade by transferring the transit trade to this company. BAT Distribution activities remain fully controlled by the BAT China Group (and not any of its subsidiaries) but structurally and formally, BAT Distribution is owned elsewhere in the BAT Group in a tax haven .The strategic need for this separation is reaffirmed by BAT executives in discussing procedures for handling orders, invoicing, payment, and supply . The neeIt has also been suggested that the Philip Morris model be adopted which places an intermediary between Philip Morris Asia and the transit traders. We have two objections. First the transit trade still originates with Philip Morris Asia and is controlled by that company. That is similar to the present situation with BATHK except for the intermediary. The second objection is that the intermediary adds another level of profit absorption and is harder to control than a dedicated group company. The apparent distancing has little effect since the business is transit trade. All the customers BATHK deals with are legitimate, properly incorporated businesses. Provided this remains the case the BAT Group can pretend ignorance that its cigarettes are being distributed through the transit trade as much and as justifiably as Philip Morris can .This proposed restructuring was submitted to BAT Chairman Sir Patrick Sheehy in March 1991 by former Chairman Barry Bramley, with the reported support of future Chairman Martin Broughton. The paper recommended that BAT Distribution be established with the following responsibilities:(a) To manage and to exploit opportunities for the sale of BAT Group products to private traders selling into the China, and adjacent general trade markets, in a regulated manner, (effectively replacing the current role of BAT(HK)).(b) Identification of new trading organisations with the potential to supply China and adjacent markets.(c) To ensure the efficient distribution within the China markets of duty free BAT products .Sheehy and fellow directors approved the reorganisation in April 1991, with BAT Distribution more discretely referred to as \u201cresponsible for sales to trading organisations supplying China and other adjacent territories\u201d .The planned reorganisation was then reviewed by in-house staff and Price Waterhouse in terms of the complex tax implications for BAT. Senior management sought to identify the optimal location of the three proposed companies to minimise tax liability, noting the \u201crisk that the [UK] Inland Revenue may examine the operation carefully as a result of the overall reduced taxable profits\u201d .Given such considerations, a revised restructuring proposal, written by project co-ordinator David Leach and supported by Bramley and BAT Far East Director (now Chief Executive) Paul Adams, was submitted in November 1991 . This prFESU, a Division of BATUKE, set up in the UK to co-ordinate the export business currently serviced by BAT HK, and to administer product invoicing and supply from the manufacturers to BAT China, BAT HK and independent distributors. This unit, which will be managed as a profit centre, will be responsible for co-ordinating sales forecasts, arranging contract manufacture and ensuring shipments are made in accordance with schedules .This arrangement would enable the group to take advantage of the UK's lower corporate tax rates , and facIn seeking to further \u201cdisassociate BAT China from the export business\u201d and establish a \u201cstructure appropriate for the return of Hong Kong to Chinese control in 1997\u201d , the conThis is because we hope to be able to move the distribution of product currently being sold through Hong Kong exports to the Singapore warehouse which will act as a buffer warehouse in the supply chain. We can minimise costs if we can use ship-to-ship or container-to-container transhipment through the Free Trade Zone in Singapore .In July 1993, the warehouse facility opened as \u201cthe export supply point for approximately 40% of the combined export sales volume into China\u201d, producing tax savings of \u00a31.5 million  in 1993  :We could push as many cigarettes as possible, but to maintain a consistent growth in the long term would require a more strategic approach. We feel that we may be milking the cow, and not feeding it enough to maintain its premium positioning\u2026 in China might be counterproductive:On China, the current view was that the return from exports (including some transit via the USSR) was much higher than from investment. Therefore, a visit by the Chairman [Patrick Sheehy] to Beijing in October  might actually be counterproductive if he was under pressure to commit to investment .The company thus approached JV negotiations with relative caution . The strWhile Hong Kong exports were seen as the \u201ckey to the future [of BAT]\u201d, BAT Head of Corporate Planning Graham Burgess recognised the trade's vulnerability given the \u201cdanger of serious action by the authorities\u201d . In reviIt is clear that we have relied (and I suspect will continue to rely) on Hong Kong Exports/FESU to meet budget and drive BATCo. growth. I understand and accept the realities of this.However, it occurs to me that our ability to \u201cmake our numbers\u201d through Hong Kong Exports may have caused us to ease the performance pressures off other markets\u2026.Progress by competitors seeking JVs in China was also a concern. When PM reached agreement in 1993 for Marlboro to be produced locally, a memo from BAT China's Susan Osborne warned of \u201cserious implications for our GT [general trade] business in China in the medium to longer term\u201d , while AReliance on an increasingly vulnerable contraband trade in the region shaped the extensive reorganisation of BAT operations in 1993 under Projects Rubicon and Battalion. Given \u201cthe increasing \u2018globality' of the tobacco business\u201d, the need for structural change was recognised \u201cif BATCo is to fulfil its prime objectives of: (i) growing the value of BAT's brand assets and; (ii) maximising the income stream from each region\u201d . The reoAs long as free market sales remain dominant, alternative routes of distribution of unofficial imports need to be examined, evaluated and, if appropriate, maximised. It is recognised that distribution of our product in China is key to BATCo's long term success and a key objective within BAT China's JV initiative is to achieve widespread distribution networks\u2026.It is important that BAT do not get left behind\u2026BAT would benefit through a wider spread of distribution, particularly in secondary and tertiary cities, ahead of competition\u2026It is envisaged that this initiative would be a short to medium term strategy prior to establishment of any JV operation and naturally any action taken would need to run in parallel with our JV plans\u2026.As part of BAT's ultimate goal of becoming \u201cthe Premier Tobacco Company in the World\u201d , the comIn common with other TTCs, BAT has depicted smuggling as an inevitable consequence of \u201ctax differentials, weak border controls, and import restrictions and bans\u201d . In his Governments must consider whether they want the industry to slip into the hands of traffickers and criminals, or to be run by legitimate, tax-paying companies working to manage a risky product responsibly. Many governments recognize that \u2018Big Tobacco' is \u2018Responsible Tobacco' .The documents presented in this paper demonstrate that the separation between \u201cBig Tobacco\u201d and \u201ctraffickers and criminals\u201d has been less stark than such posturing would suggest. While public statements by BAT have portrayed smuggling as \u201cinimical to our long-term business interests\u201d , internaThe documents demonstrate that contraband has been a hugely profitable and integral part of BAT operations in China over the past two decades. Initially a means of circumventing restricted access to the Chinese market, it became a hugely profitable income stream. Contraband was then used to build market presence, in competition with other international brands, with supply and price carefully managed. By the early 1990s, smuggling into China represented \u201cthe largest BATCo market\u201d , integraAlongside this lucrative trade, BAT China, deliberately insulated from such operations, sought to build relations with Chinese officials . Paul BiDemand for our international brands outstrips the supply via the CNTC Friendship Stores and Duty Free shops. This created a black market. Substantial domestic manufacture by a j.v. would provide China with a share in the profits hitherto taken by smugglers\u2026.China had much more to gain with us than without us Click here for additional data file.Text S1(443 KB PDF)Click here for additional data file.Text S2(3.3 MB PDF)Click here for additional data file.Text S3(341 KB PDF)Click here for additional data file.Text S4(450 KB PDF)Click here for additional data file.Text S5(1 MB PDF)Click here for additional data file.Text S6(1.9 MB PDF)Click here for additional data file.Text S7(2.8 MB PDF)Click here for additional data file.Text S8(2.3 MB PDF)Click here for additional data file.Text S9(1.1 MB PDF)Click here for additional data file.Text S10(1.2 MB PDF)Click here for additional data file.Text S11(812 KB PDF)Click here for additional data file.Text S12(756 KB PDF)Click here for additional data file.Protocol S1(739 KB DOC)Click here for additional data file."}
+{"text": "Nearly all China\u2019s rural residents and a shrinking fraction of urban residents use solid fuels  for household cooking and/or heating. Consequently, global meta-analyses of epidemiologic studies indicate that indoor air pollution from solid fuel use in China is responsible for approximately 420,000 premature deaths annually, more than the approximately 300,000 attributed to urban outdoor air pollution in the country. Our objective in this review was to help elucidate the extent of this indoor air pollution health hazard.We reviewed approximately 200 publications in both Chinese- and English-language journals that reported health effects, exposure characteristics, and fuel/stove intervention options.Observed health effects include respiratory illnesses, lung cancer, chronic obstructive pulmonary disease, weakening of the immune system, and reduction in lung function. Arsenic poisoning and fluorosis resulting from the use of \u201cpoisonous\u201d coal have been observed in certain regions of China. Although attempts have been made in a few studies to identify specific coal smoke constituents responsible for specific adverse health effects, the majority of indoor air measurements include those of only particulate matter, carbon monoxide, sulfur dioxide, and/or nitrogen dioxide. These measurements indicate that pollution levels in households using solid fuel generally exceed China\u2019s indoor air quality standards. Intervention technologies ranging from simply adding a chimney to the more complex modernized bioenergy program are available, but they can be viable only with coordinated support from the government and the commercial sector. Although some areas of China are becoming more urban, more than 60% of the population is still rural, most of which still uses biomass (mainly wood and crop residues) and coal fuels that produce substantial pollution in simple stoves. In 2003 approximately 80% of the energy consumed by rural households was in the form of biomass and almost 10% as coal. Furthermore, although most Chinese cities have plans to eliminate coal for households, many urban communities continue to rely on coal. The combustion of biomass and coal  is the dominant source of indoor air pollution (IAP) in the country and contributes significantly to the total burden of ill health.In the most recent global analysis of the health effects of major risk factors, the World Health Organization (WHO) estimated that solid fuels used in Chinese households cause approximately 420,000 premature deaths annually; this is 40% more than the approximately 300,000 premature deaths attributed to outdoor air pollution in Chinese cities with populations of more than 100,000 . HousehoIn the 1980s China conducted more IAP measurements focused on household combustion than all other developing countries combined. Indeed, by the early 1990s, the results of more than 100 published studies were combined into a WHO database . In conta) What toxic constituents have been found in the emissions of solid fuel combustion? b) What are the reported human exposure characteristics? c) What health effects have been documented? and d) What technologies exist or are possible for reducing this IAP exposure?In this review we address the following questions in order to put Chinese household IAP from solid fuel use into perspective, nationally and internationally, and highlight research gaps: We conducted computer searches covering literature from 1980\u20132006 of the following bibliographic databases in English: Solid fuels are difficult to burn in simple combustion devices such as household cooking and heating stoves without substantial emissions of pollutants, principally because of the difficulty of completely premixing the fuel and air during burning, which is done easily with liquid and gaseous fuels . Consequ2), and particulate matter (PM). In studies characterizing PIC emissions from 28 fuel/stove combinations commonly found in China in the early 1990s, more than 60 hydrocarbons and 17 aldehydes and ketones were measured in larger quantities in the flue gases of solid fuel\u2013burning stoves than in the flue gases of stoves burning liquid or gaseous fuel (PICs released from solid fuel combustion are a complex mixture of particulate and gaseous species. Some of the PICs are commonly regulated air pollutants such as carbon monoxide (CO), nitrogen dioxide , a 5-ring PAH of high cancer potency, are predominantly present in the particle phase, particles emitted from household coal combustion have been subjected to compositional analysis of PAHs and PAH derivatives. These analyses provide much needed information on the carcinogenicity and mutagenicity of coal smoke. For example, carcinogenic PAHs, methylated PAHs, and nitrogen-containing heterocyclic aromatic compounds were found in large abundance in the particles emitted from bituminous (smoky) coal combustion. Because carcinogenic PAHs, especially (B[a]P), a 5-ring PAH of high cancer potency, are predominantly present in the particle phase, particles emitted from household coal combustion have been subjected to compositional analysis of PAHs and PAH derivatives. These analyses provide much needed information on the carcinogenicity and mutagenicity of coal smoke. For example, carcinogenic PAHs, methylated PAHs, and nitrogen-containing heterocyclic aromatic compounds were found in large abundance in the particles emitted from bituminous (smoky) coal combustion, as is typically found in numerous households in Xuanwei (It is well known that polycyclic aromatic hydrocarbons (PAHs) are formed during incomplete combustion of all carbon-based fuels, including wood and coal. Lower molecular-weight PAHs (with two to four aromatic rings) are present predominantly in the gas phase, whereas higher molecular-weight PAHs are present predominantly in the particle phase. Because carcinogenic PAHs, especially benzo or respirable particles [PM with an aerodynamic diameter \u22642.5 \u03bcm (PM2.5)]. Although we could find no published studies on ultrafine particles [PM with an aerodynamic diameter \u2264 0.1 \u03bcm], it is expected that indoor levels of ultrafine particles would be high.Although PM generated from the fuel combustion itself is fine and ultrafine in size, the smoke may contain larger particles resulting from suspension of ash and solid fuel debris. Because particle size determines how deep the particles can travel within and beyond the respiratory tract, determining size distribution is important in assessing health impacts. For this reason, in most recent studies there has been a switch to measuring inhalable particles  and SO2 are highest in the kitchens of households using coal. This \u201ckitchen effect\u201d is, however, less profound and less consistent for NO2 and CO, perhaps because of the confounding from coal heating and tobacco smoking. Presumably, coal combustion for heating occurs at more steady burning conditions than for cooking. Peak emissions of particles occur during unsteady combustion stages such as at the beginning and at the end of the fire. These types of heterogeneous spatial and temporal patterns of pollutant concentration have important exposure implications for individual household members. A recent winter study in Jilin Province shows that households burning solid fuel had mean 24-hr levels of PM and CO that correlated with but were about 6 times lower than peak (1-hr) levels and that indoor levels were dominated by heating not cooking sources  in kitchens and living rooms over  would overlook large house-to-house differences in pollutant concentrations and exposures. It should be noted that although these studies employed a systematic measurement approach for all the measured households, the household selection procedure does not appear to be population-based or random; hence, the reported pollutant concentrations may not be generalizable to the general households in the four provinces. However, the spatial and temporal patterns observed in the study are expected to be typical for similar houses in the same regions -null genotype  classified emissions of indoor combustion of coal as carcinogenic to humans (Group 1) on the basis of sufficient evidence both in humans and in animals . The rolgenotype . There igenotype  and cancgenotype . This magenotype .2 and PM10 , monotonic and positive exposure\u2013response relationships were observed for OR estimates of phlegm, cough with phlegm, and bronchitis. In addition, OR estimates for cough, wheeze, and asthma were all > 1 in the exposed groups relative to the no-exposure group .1), or peak expiratory flow rate (PEFR). These reductions were associated with the use of coal for cooking/heating compared with the use of natural gas or LPG  and Shanghai, measurements made in winter showed reductions of 1.5\u201310.7% in forced vital capacity (FVC), forced expiratory volume in 1 sec  that have been deemed \u201cendemic\u201d because local coal deposits have high contents of toxic elements . The mosIt is known that high arsenic exposure via drinking water causes bladder, lung, and skin cancers . HoweverThere have been numerous reported acute poisonings, including fatal cases, especially during heating seasons, resulting from indoor coal combustion under poor ventilation conditions. Under normal combustion and ventilation conditions, Assignment of causality is difficult on the basis of observational studies alone, the interpretation of which is limited by potential unmeasured confounders, for example, socioeconomic status, which is often associated with disease outcome as well as use of dirty household fuels. No randomized intervention trials providing stronger evidence have yet to be published for household solid fuel use in China and would be difficult to conduct for chronic diseases such as COPD and lung cancer that are the results of many years of exposure. Using the rich database of studies in Xuanwei, however, two intervention studies  have beeIn a retrospective study of 21,232 farmers between 1972 and 1992, during which 17,184 shifted to improved stoves, hospital records indicated 1,384 cases of lung cancer . Cox-modThe 1976\u20131992 COPD histories of 20,453 farmers were determined by asking whether they had ever been diagnosed with chronic bronchitis or emphysema and by death records . Of thes10, approximately 700 \u03bcg/m3, were still high  or greater (for COPD) than for women who are usually thought to have higher exposures from stove emissions. For the lower COPD effect, we offer the explanation that cooking exposures start early in life, and thus women may have been compromised before the intervention. We also point out that even after intervention, indoor levels of PMill high . If one The infamous smog episode killing thousands of people in the winter of 1952 eventually led to the ban of household use of coal fireplaces in London. Today, in the United Kingdom and other developed countries, household use of coal in cities is almost nonexistent. With the rapid economic growth in China, coal stoves are becoming less common in cities, as they are being replaced with gas stoves and with space heating methods other than direct coal combustion. Despite the declining trend, however, household coal use is still common in urban communities across China. In rural China, coal use seems to be increasing as coal substitutes for biomass, and there have been proposals by national and international agencies to promote household coal use, usually in the form of \u201cclean coal,\u201d to reduce the growing use of petroleum-based fuels and to relieve the pressure on biomass resources. Although household use of piped gas is increasing in cities and LPG use is increasing among affluent rural households, widespread use of gas fuels in rural households is unlikely to occur soon because of the cost and the unreliability of supplies. Hence, interventions to make solid fuels less polluting continue to be important for public health.Because pollutant emissions depend on both fuel quality and stove design, interventions can focus on fuel, stove, or both. To date, stove interventions have focused predominantly on households using biomass, and fuel interventions appear to have focused mainly on coal, although the reverse trends are also becoming apparent.The most impressive organized rural energy intervention in human history was China\u2019s NISP, through which China accomplished more than all other developing countries combined to improve household energy use by introducing more than 180 million improved stoves since the early 1980s. All introduced stoves had chimneys and some had manual or electric blowers to promote more efficient combustion. Unfortunately, the program ended in the mid-1990s, and now there is relatively little action to improve the present rural energy situation. The review of NISP by 2 . For comparison, total coal use in China was 39 EJ in 2004 in all sectors . It has Recently, new pollution problems are arising from crop residues because in more economically developed parts of the country, farmers are becoming reluctant to gather biomass residues from the field and store them for use throughout the year, preferring easier-to-handle fuels such as coal briquettes or LPG. This shift to modern fuels creates an excess of crop residues that are commonly burned in the field, leading to widespread ambient pollution in some seasons. A number of village-scale gasifiers have been built to make more efficient and cleaner use of these residues by distributing the gas to individual households. A Bio-energy Modernization Demonstration Project, carried out in Jilin Province, is designed to develop combined heat, electricity, and cooking fuel production (trigeneration) from corn stalks. The technology seems to be workable, but its widespread use, however, is limited by the lack of commercial viability and potential risks due to acute CO poisoning .2 emission , a fuel with characteristics similar to those of LPG. DME, sold as a substitute for LPG, is currently made from natural gas but can only take on a large role if made from crop residues or coal. Finding ways to convert coal, the country\u2019s most abundant energy resource, to DME has been attractive to researchers and the Chinese government . With fu2 as well as significant reductions in cardiovascular and respiratory death rates (In large Chinese cities, policies of banning household coal use have been in place with the main goal of reducing outdoor air pollution but with the side benefit of reducing IAP levels. Although health benefits have not been directly reported or measured in Chinese cities, the findings from cities in other countries; for example, studies of the ban on coal sales in Dublin, Ireland, documented sudden decreases in atmospheric concentrations of PM and SOth rates .2, implying that biomass stoves can have net global warming potentials, even with a renewable fuel cycle (Simple stove improvement is not expected to reduce total pollutant emissions because chimneys divert only a fraction of the emitted pollutants from the inside of households to the outside, rather, it worsens neighborhood and local air quality. A more permanent solution is to move to clean combustion through changes in fuel and/or stoves, which actually reduce emissions. An added benefit from clean combustion is improved atmospheric visibility and lower ambient pollution as well as potentially lower greenhouse emissions. PICs from low-efficiency biomass combustion in household stoves, for example, contain a relatively large fraction of methane many times more potent in radiative forcing than COel cycle .Solid fuels are still the dominant source of energy in Chinese households, leading to pollutant levels generally exceeding China\u2019s IAQ standards and contributing significantly to the national burden of ill health. Evidence for adverse health outcomes is strong, including lung cancer, respiratory illnesses, acute respiratory infection, and COPD. There is also evidence in China of impacts on lung function and immune system impairment. Therefore, improving IAQ in households using solid fuel should be an urgent and high-priority task on China\u2019s public health agenda. A range of intervention technologies, from one as simple as adding a chimney to the more complex modernized bioenergy program, is available, but these technologies can be viable only with a coordinated support from the government and interested private parties in the commercial sector. Substituting cleaner fuels for the poisonous coals being used in millions of households should have an especially high priority. This was one of the principal recommendations from a Chinese and international scientific and policy workshop convened in early 2005 to review the status of improved stoves in China. The full set of recommendations and related materials can be found in"}
+{"text": "Each year, 540 million Chinese are exposed to secondhand smoke (SHS), resulting in more than 100,000 deaths. Smoke-free policies have been demonstrated to decrease overall cigarette consumption, encourage smokers to quit, and protect the health of nonsmokers. However, restrictions on smoking in China remain limited and ineffective. Internal tobacco industry documents show that transnational tobacco companies (TTCs) have pursued a multifaceted strategy for undermining the adoption of restrictions on smoking in many countries.To understand company activities in China related to SHS, we analyzed British American Tobacco's (BAT's) internal corporate documents produced in response to litigation against the major cigarette manufacturers to understand company activities in China related to SHS. BAT has carried out an extensive strategy to undermine the health policy agenda on SHS in China by attempting to divert public attention from SHS issues towards liver disease prevention, pushing the so-called \u201cresocialisation of smoking\u201d accommodation principles, and providing \u201ctraining\u201d for industry, public officials, and the media based on BAT's corporate agenda that SHS is an insignificant contributor to the larger issue of air pollution.The public health community in China should be aware of the tactics previously used by TTCs, including efforts by the tobacco industry to co-opt prominent Chinese benevolent organizations, when seeking to enact stronger restrictions on smoking in public places. Monique Muggli and colleagues study British American Tobacco (BAT) internal documents and find that from the mid 1990s BAT pursued a strategy aimed at influencing the public debate on secondhand smoke in China. Each year, about one million people die in China from tobacco-caused diseases, including cancer, heart disease, and lung disease. Although most of these deaths occur among smokers\u2014300 million people smoke in China, accounting for one-third of the global \u201cconsumption\u201d of cigarettes\u2014more than 100,000 deaths from tobacco-related causes occur annually among the 540 million Chinese people who are exposed to secondhand smoke. Tobacco smoke contains 4,000 known chemicals, 69 of which are known or probable carcinogens, and, when it is produced in enclosed spaces, both smokers and nonsmokers are exposed to its harmful effects. The only effective way to reduce tobacco smoke exposure indoors to acceptable levels is to implement 100% smoke-free environments\u2014ventilation, filtration, and the provision of segregated areas for smokers and nonsmokers are insufficient. Importantly, as well as protecting nonsmokers from secondhand smoke, the implementation of smoke-free public places also reduces the number of cigarettes smoked among continuing smokers, increases the likelihood of smokers quitting, and reduces the chances of young people taking up smoking.Article 8 of the World Health Organization's Framework Convention on Tobacco Control  calls on countries party to the treaty to protect their citizens from secondhand smoke exposure. China became a party to the FCTC in 2005 but restrictions on smoking in public places in China remain limited and ineffective. Previous analyses of internal tobacco industry documents have revealed that transnational tobacco companies (TTCs) have used a multifaceted approach to undermine the adoption of restrictions on smoking in many countries. TTCs have been shown to influence media coverage of secondhand smoke issues and to promote ineffective ventilation and separate smoking and nonsmoking areas in restaurants, bars, and hotels  with the aim of undermining smoke-free legislation. In addition, TTCs have created organizations interested in non-tobacco-related diseases to draw attention away from the public-health implications of secondhand smoke. In this study, the researchers ask whether TTCs have used a similar approach to undermine the adoption of restrictions on smoking in China, one of the most coveted cigarette markets in the world by the major TTCs.The researchers analyzed internal corporate documents produced by British American Tobacco  in response to litigation against major cigarette manufacturers stored in document depositories in Minnesota, USA and Guildford, UK. Among these documents, they found evidence that BAT had attempted to divert attention from secondhand smoke issues toward liver disease prevention by funding the Beijing Liver Foundation (BFL) from its inception in 1997 until at least 2002 (the most recent year that BAT's corporate records are available for public review). The researchers also found evidence that BAT had promoted \u201cresocialization of smoking\u201d accommodation principles as a \u201croute to avoid smoking bans\u201d and pushed ventilation and air filtration in airports and in establishments serving food and drink. Finally, the researchers found evidence that BAT had sought to \u201cpresent the message that \u2018tobacco smoke is just one of the sources of air polution [sic] and a very insignificant one compared with other pollutants'\u201d through presentations given to the Chinese tobacco industry and media seminars aimed at Chinese journalists.These findings indicate that, beginning in the mid 1990s and continuing until at least 2002, BAT has followed an intensive, multi-pronged strategy designed to undermine the health policy agenda on secondhand smoke in China. Given their findings, the researchers suggest that BFL and other charitable organizations in China must be wary of accepting tobacco money and that measures must be taken to improve the transparency and accountability of these and other public organizations. To meet FCTC obligations under Article 5.3 (industry interference), policy makers in China, they suggest, must be made aware of how BAT and other TTCs have repeatedly sought to influence health policy in China by focusing attention toward the adoption of ineffective air filtration and ventilation systems in hospitality venues rather than the implementation of 100% smoke-free environments. Finally, Chinese policy makers and the media need to be better informed about BAT's long-standing attempts to communicate misleading messages to them about the health effects of secondhand smoke.http://dx.doi.org/10.1371/journal.pmed.0050251.Please access these Web sites via the online version of this summary at smoking statistics for China and other countries in the regionThe World Health Organization's Regional Office for the Western Pacific provides the health problems associated with secondhand smoke, about its Tobacco Free Initiative , and about the Framework Convention on Tobacco Control The World Health Organization provides information on secondhand smoke (available in English and Spanish)MedlinePlus provides links to information about the dangers of Smokefree Web site provides information about the advantages of giving up smoking, how to give up smoking, and the dangers associated with secondhand smokeThe UK National Health Service British American Tobacco Documents ArchiveBritish American Tobacco documents stored in the Minnesota and Guildford Depositories, including those analyzed in this study, can be searched through the  With one-third of the world's smokers, China has remained one of the most coveted cigarette markets in the world ,2. ApproThe implementation of smoke-free public places has been shown to reduce the number of cigarettes smoked per day among continuing smokers, increase the likelihood that smokers will quit smoking, reduce the chances of a young person initiating smoking, and decrease health risks posed to nonsmokers \u201311. The Previous analyses of internal tobacco industry documents have described the global strategy of transnational tobacco companies (TTCs) to undermine scientific evidence demonstrating the harmful health effects of SHS through covertly recruiting scientists in China \u201320 and gIn 2005, China became a party to the WHO Framework Convention on Tobacco Control (FCTC), the world's first international public health treaty, which seeks to reduce tobacco-caused death and disease . ArticleThis paper examines the efforts of British American Tobacco (BAT), the predominant TTC in China , to undePrevious research has described diversionary tactics by BAT and other TTCs aimed at undermining the 8th World Conference on Tobacco or Health (WCTOH) held in Buenos Aires in 1992. TTCs sought to distract media attention away from the 8th WCTOH and toward staged events such as music concerts and soccer matches promoting childhood immunization and HIV/AIDS awareness ,35. Prevhttp://bat.library.ucsf.edu). Documents were also searched on-site at the Guildford Depository in the United Kingdom over multiple visits from 2000 to 2006.This paper analyzes internal corporate documents produced in response to litigation involving the major tobacco manufacturers. The history and public availability of these records have been described previously \u201341. OnliOn-line searches followed an iterative model, initially combining terms such as \u201cChina\u201d, \u201cBeijing\u201d, \u201chealth promotion\u201d, \u201chepatitis\u201d, and \u201cliver disease\u201d with Boolean operators. This was followed by more specific searches using names of BAT projects and personnel. Additionally, files belonging to individuals directly communicating with BAT's subsidiary, BAT China, were searched. All relevant documents were analyzed to create a historical and thematic narrative. Industry publications, newspapers, academic journals, policy documents, and legislation were used to contextualize and triangulate findings.The limitations of using internal tobacco company records in research have been described previously ,41. LimiBAT China funded the BLF  from its inception in 1997 to at least 2002   and Soong Ching Ling Foundation, the two most active antis, to tackle the No. 1 infectious disease in China and to divert the public attention from smoking and health issues to liver diseases\u201d  Health to maintain a perspective on health issues; hepatitis is the number one killer disease in China\u201d . BAT spoTo further foster a relationship with the MPH through BLF, several Chinese regulators were invited as consultants to the BLF, including the vice minister of the MPH and the deputy chairman of SCLF . MoreoveDuring 1997\u20132002, BLF undertook several hepatitis-focused health promotion and disease prevention initiatives throughout China . AlthougBLF launched several educational programs using different approaches to generate public awareness of the causes, symptoms, diagnosis, and treatment of liver diseases . These iIn 2000, another large exhibition on hepatitis prevention and treatment, held in Kunming, in Yunnan province, was used by BAT China as a publicity opportunity: \u201cSenior officials from the provincial and municipal governments, local legislators, leading members of the provincial healthcare administration, representatives from the Madam Song Foundation, the Disease Control Division of the Ministry of Health, the Beijing Health Promotion Council and BAT China attended the ribbon-cutting ceremony. Ms. Li Xiaofen, Representative of BAT China, presented 50,000 cartoon books on the elementary knowledge about hepatitis to the provincial health administration on behalf of the organizing committee of the show\u201d .www.wellness.com.cn), designed for medical consultation for liver diseases he density analysis of indoor ETS content; physical, chemical and physiological analyses of ETS; epidemiological data on the impact of ETS on the health of different age groups; and evaluation of lung cancer risks to non-smokers exposed to ETS\u201d .Their findings were presented at an \u201cIndoor Air Quality Forum\u201d in Beijing and reported by \u201cmajor media including CETV\u201d . The panThe second project, entitled \u201cStudy on factors related with lung cancer risks to non-smoker women in China,\u201d sought to conduct a case-control study of 3,000 nonsmoking Chinese women with the aim to \u201c[H]elp the public [become] better acquainted with the genuine elements leading to increased female lung cancers [sic] cases and enhance their awareness of the danger that environmental pollutants may cause to residents, women residents in particular\u201d .Although several meetings between officials and medical experts from the SCLF, China Preventive Medicine Academy, and BLF had taken place after 1999, the report noted that because of restructuring of the Chinese health care system the project leaders delayed implementation and the project was expected to finish at the end of 2002 . We wereDocuments reviewed also describe additional strategies aimed at weakening secondhand smoke policies in China. Similar to previous efforts in the UK  and otheBAT's plan for \u201cresocialising\u201d smoking in China included pushing ventilation and air filtration in airports and hotel, restaurant, and casino (\u201cHORECA\u201d) outlets . BAT theThe air filtration systems installed in China were manufactured by Colt International and were used by BAT to serve the dual purpose of circumventing restrictions on smoking and allowing the company direct communications with customers through branding opportunities in a restrictive advertising environment ,70. PrevThrough presentations in the mid 1990s to the Chinese tobacco industry and media seminars aimed at Chinese journalists, BAT also sought to \u201cpresent the message that \u2018tobacco smoke is just one of the sources of air polution [sic] and a very insignificant one compared with other pollutants'\u201d . HoweverAccording to BAT China management, employees \u201cdid not know any more about these issues than from what they read in the newspapers\u201d . BAT ChiBAT also sought to train representatives of the Chinese State Tobacco Monopoly Administration (STMA) given its control of 98% of the Chinese market. BAT and BAT China engaged in a systematic \u201cknowledge transfer\u201d to the STMA as a way to communicate to media and policy makers that it could not directly reach ,79: \u201cBriBy 2000, at least six \u201csmoking and health seminars\u201d directed at STMA were held by BAT . BAT's fBAT concluded after its May 2000 seminar that its efforts aided in building a stronger relationship with the Chinese tobacco industry and regulators: \u201cThe seminar has strengthened communications between BAT and the Chinese tobacco industry. It has also strengthened communications between the tobacco industry and regulators. The seminar has helped the Chinese tobacco industry and regulators to better understand the latest development and activities of major antis in the world, which are of key importance for them to work out countering strategies\u201d .real indoor air quality problems facing the Asia region, and to suggest solutions which are appropriate and practical\u201d  media's response to the subject matter, of obtaining balanced coverage and to contain the impact\u201d of the 10th WCTOH . The semactical\u201d . (EmphasRenmin Ribao (China Daily) in December 1998 entitled \u201cStudy finds serious indoor air problem\u201d stated that tests carried out by the government, BAT, and Healthy Buildings International showed that the \u201cthe main causes for typical indoor air problems in these buildings were inadequate ventilation and filtration, and insufficient maintenance of air-purification systems\u201d and that \u201csmoking was reported not an important factor influencing air quality\u201d [Media coverage in China at this time suggests that BAT had some success at framing SHS in terms of poor indoor air quality. For example, an article published in quality\u201d . The artquality\u201d .BAT continued to organize media SHS seminars to generate \u201cpositive and balanced coverage in major media\u201d and to \u201cpost BAT views on the STMA website\u201d until at least 2002 . DocumenThis review of internal documents finds that, beginning in the mid-1990s, BAT pursued a multi-faceted strategy aimed at influencing and undermining the public debate on SHS issues in China. BAT sought to shift policy attention from SHS issues to liver disease prevention through the funding and promotion of the Beijing Liver Foundation. Several aspects of BAT's efforts to create and fund the BLF are notable. First, heart disease, cancer, and cerebrovascular disease account for two-thirds of deaths in China in the population \u2265 40 y of age, for which tobacco is the leading preventable risk factor . In factThis analysis of internal documents also shows that BAT promoted air filtration and ventilation technologies as part of a \u201cresocialisation of smoking\u201d effort in anticipation of potential restrictions on smoking. BAT achieved this by engaging in a \u201cknowledge transfer\u201d of the industry-based message to the Chinese tobacco industry and media that SHS is an insignificant contributor to the larger issue of air pollution. In doing so, BAT advocated the position that SHS exposure in hospitality venues did not warrant regulation. Instead, because SHS was, according to BAT, an insignificant source of pollution, the issue could be addressed by so-called \u201caccommodation\u201d efforts.These findings raise several policy implications. Because BAT was able to incorporate its messages on SHS into the BLF's programs targeted at liver disease prevention, charitable organizations in China must be wary of accepting tobacco industry funding. While funding may appear to be for scientific or benevolent purposes and seemingly unrelated to tobacco use, documents reviewed here reveal that industry motives can be well hidden. Measures to improve transparency and accountability of public organizations, notably their association with the tobacco industry, should continually be improved worldwide. Disclosure of this kind will lessen the capacity of the tobacco industry to covertly use local institutions to pursue its interests. In doing so, parties to the FCTC may act to fulfil their obligations under Article 5.3 of the FCTC to protect implemented public health policies from industry influence .Additionally, as policy makers consider the adoption of smoke-free public places, such as the Beijing smoke-free initiative , ChineseFinally, Chinese policy makers and the media need to be better informed of BAT's decade-long initiative to communicate misleading messages on the health effects of SHS. Public statements in recent years by the Chinese tobacco industry suggest that these messages are being disseminated in China. In 2006, despite over two decades of international scientific consensus on the harmful health effects of SHS , the STMPublic health advocates in China should expose such misinformation as part of their efforts to address the health effects of SHS."}
+{"text": "In Xuanwei County, China, unvented indoor coal burning is strongly associated with increased risk of lung cancer and chronic obstructive pulmonary disease. However, the impact of coal burning and stove improvement on risk of pneumonia is not clear.We conducted a retrospective cohort study among all farmers born 1917 through 1951 and living in Xuanwei as of 1 January 1976. The analysis included a total of 42,422 cohort members. Follow-up identified all deaths in the cohort from 1976 through 1996. Ages at entry into and at exit from follow-up ranged from 24 to 59 years and from 25 to 80 years, respectively. The record search detected 225 deaths from pneumonia, and 32,332 (76%) were alive as of 31 December 1996. We constructed multivariable Cox models (time variable = age) to estimate hazard ratios (HRs) and 95% confidence intervals (CIs).Use of coal, especially smokeless coal, was positively associated with pneumonia mortality. Annual tonnage and lifetime duration of smoky and smokeless coal use were positively associated with pneumonia mortality. Stove improvement was associated with a 50% reduction in pneumonia deaths .Our analysis is the first to suggest that indoor air pollution from unvented coal burning is an important risk factor for pneumonia death in adults and that improving ventilation by installing a chimney is an effective measure to decrease it. Nearly half of the world\u2019s households use unprocessed biomass fuels and coal for cooking and heating . This isXuanwei County is well known for its high incidence of lung cancer, attributed mainly to the wide use of smoky coal mined locally and the use of unvented fire-pit stoves . It accoTo control the high incidence of lung cancer, the Xuanwei County government implemented a program in the late 1970s to change the traditional unvented fire-pit stove to a stove with chimney to improve ventilation and reduce exposures. To quantitatively assess the effect of coal use and stove improvement on morbidity and mortality in adults, we conducted a retrospective cohort study in Xuanwei County from 1992 to 1996. We previously reported that the installation of chimneys was strongly associated with reduced risk of incident lung cancer and chronic obstructive pulmonary disease (COPD) . PreviouIn 1992, we searched local administrative records to identify all farmers born from 1917 through 1951 and living in four of Xuanwei\u2019s communes  as of 1 January 1976. In total, we identified 44,580 records. A total of 42,422 farmers were available for study after excluding 2,108 subjects who had moved out of the study area and 50 subjects with insufficient information for analysis.In three of the four communes, most residents used smoky coal (mainly from Lai Bin). In the other commune, most residents used smokeless coal (mainly from Re Shui). In Xuanwei, smoky and smokeless coals correspond to bituminous coal and anthracite, respectively. The terms \u201csmoky\u201d and \u201csmokeless\u201d coal have been used in many published reports from studies in this area , and theIn 1992, trained interviewers administered a standardized questionnaire for all cohort members. They interviewed subjects directly, when feasible, or surrogate respondents for subjects deceased or not present (41%). This study was approved by the Institutional Review Board of the Chinese Academy of Preventive Medicine. Interviewers obtained written informed consent from all literate questionnaire respondents and verbal consent from others. The questionnaire elicited information on demographic factors, residential history, lifetime use of household stove and fuel type, occupational history, tobacco smoking, cooking food, time spent indoors and outdoors, and medical history. The questionnaire interview typically took about 40 min.International Classification of Diseases, 9th Revision  and the Yunnan Province Hospital to identify patients who died outside Xuanwei. The diagnosis of pneumonia during the time period of cohort follow-up was based primarily on symptoms, clinical exam, and a chest X-ray. Empirical treatment with antibiotics was used in general. We excluded tuberculosis and COPD when pneumonia was diagnosed as the cause of death. If a patient died from pneumonia, a physician completed a death certificate, which is how death from pneumonia in Xuanwei is documented. A death certificate is required for official documentation in hospitals, public security bureaus, and public health bureaus. Information abstracted from the death records included name, sex, date of birth, residence, date of death, and cause of death. We used name, sex, date of birth, and residence information to identify unique study subjects in the cohort. We included a total of 787,814 person-years during follow-up, with an average of 18.6 person-years per subject. We coded the cause of death using the n ICD-9; .We included 42,422 subjects  in the analysis. For each subject, we calculated the age at entry into cohort follow-up as the number of years from the subject\u2019s birth date to 1 January 1976. After entry, subjects could exit from follow-up in one of the following three ways: death from pneumonia, death from other causes before the study ended in 1996, or living as of 31 December 1996. Ages at entry into and at exit from follow-up ranged from 24 to 59 years and from 25 to 80 years, respectively.In this report, the term \u201cstove improvement\u201d refers to changing permanently from fire-pit or stove without a chimney to stove with a chimney. We constructed multivariable Cox models using the SAS PHREG procedure  to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). The time variable (time axis) was age, starting with age as of 1 January 1976 and ending with age at exit from follow-up. The outcome event was death from pneumonia . We considered individuals who died from other causes before the study ended in 1996, or who remained alive as of 31 December 1996, as censored (competing-risks approach). In the models, we assessed the effects of coal use, stove improvement, years of smoking, and years of cooking as time-dependent continuous or categorical variables based on the starting age for each of these activities. To adjust for potential birth cohort effects, models were stratified into seven birth cohort strata: 1917\u20131921, 1922\u20131926, 1927\u20131931, 1932\u20131936, 1937\u20131941, 1942\u20131946, and 1947\u20131951.We analyzed the data in four Cox models: two separate models for men and women, and two separate models for smoky coal and smokeless coal users. Nearly all subjects used either smoky or smokeless coal, so we included only the variable \u201cuse of smokeless coal\u201d (yes or no) in the sex-specific models. We calculated the time-dependent variables tonnage per year and duration in years for all coals combined and for smoky coal and smokeless coal separately, categorized into tertiles. We used two time-dependent variables for stove improvement: a binary variable for the overall effect of stove improvement (0 before and 1 after the age at stove improvement) and a categorical variable indicating time since stove improvement . We analyzed the time-dependent variable for duration of tobacco smoking (20\u201340 years or \u2265 40 years), relative to a reference category of subjects who had not smoked or smoked < 20 years, in the men\u2019s model. We used another time-dependent binary variable of tobacco smoking in coal type\u2013specific models, which switched from 0 to 1 at the age at first smoking. Similarly, we used a time-dependent binary variable for cooking (yes/no) in the men\u2019s model and coal type\u2013specific models; we included two time-dependent variables for different durations (20\u201340 years or \u2265 40 years) of cooking food, relative to a reference category of subjects who had not cooked food or cooked for < 20 years, in the women\u2019s model.a) sex in the coal type\u2013specific models; b) having any education, to adjust for socioeconomic status; c) average number of rooms in the home over the lifetime, to adjust for socioeconomic status and for potential effects of coal smoke dilution in the home; d) average number of people in the home over the lifetime, to adjust for crowding pattern; e) spending \u2265 7 daily waking hours indoors through 20 years of age, to adjust for elevated exposure to indoor coal smoke early in life; f) reporting diagnosed chronic bronchitis, emphysema, or asthma (often observed to be pneumonia risk factors); and g) a dummy variable for ever being a coal miner in the men\u2019s model .In addition to the variables mentioned above, we included the following variables in the Cox models: Lung cancer was the major cause of death with 2,459 deaths during follow-up. There were 225 deaths from pneumonia. The lung cancer mortality rates in the smoky coal\u2013using communes were about 17 times higher in men and 39 times higher in women compared with national rates in China . This isThe mean age at death due to pneumonia was 63.2 years. When the follow-up ended in 1996, 32,332 (76%) subjects were alive. Age-adjusted mortality rates of pneumonia were higher in smokeless coal users  and among people who had not improved their stove . Survival rates were lower among smoky coal users compared with smokeless coal users (74% vs. 81%) and among people living in houses without chimneys than among those in houses with chimneys (70% vs. 80%).About half of men (46%), but only 23% of women, had some education. About 5% and 6% of men had been coal miners and construction workers, respectively. More people who used smoky coal and experienced stove improvement had a nonfarming job.Men spent less time in the home than did women. About 10% of subjects had been diagnosed with chronic bronchitis, asthma, or emphysema. Most men (92%) had smoked, and few had quit; few women smoked (< 1%). Almost all women (98%) cooked food for the family from early in life, and only 8% of men ever cooked.Seventy percent of people used smoky coal. Most families burned about 3 tons of coal each year. Around 60% of families improved stoves with chimneys before follow-up ended. More people using smoky coal improved their stoves than did people using smokeless coal (75% vs. 19%) because the government sponsored a stove improvement campaign in smoky coal\u2013using areas in the 1970s to control the epidemic of lung cancer.p = 0.005) (data not shown). The annual tonnage of coal used and the duration of coal use in years were associated with significantly increased risk at the highest tertiles. The HRs for tonnage and duration of coal use were a bit larger among women than among men. The number of people in the home was associated with a borderline increase of risk of pneumonia death in women . History of non-malignant pulmonary diseases was a major risk factor for pneumonia death . Smoking for \u2265 40 years among men and cooking for \u2265 20 years among women were associated with a nonsignificantly increased risk of pneumonia death. Educational attainment was associated with a reduced risk of pneumonia death in men only .In the model for smoky coal users , the proIn the model for smokeless coal users , higher In the analysis, we found that use of coal, especially smokeless coal, and stove improvement that decreases coal smoke level indoors were important risk and protective factors, respectively, for pneumonia death in Xuanwei. The fatality rate of pneumonia would be very high if patients were not treated immediately with antibiotics, which often occurred in rural areas of China. Many studies have found that solid fuel use was associated with high incidence of ARIs in children . Smith eCoal smoke contains many chemicals  and particles that irritate respiratory tracts and lung, adversely affect the host\u2019s defense systems against pathogens, and elevate the risk of respiratory tract infections . Coal sm3 and total suspended particulate matter (PM) from 5.64 to 1.21 mg/m3, based on a 5-day measurement (10) with unvented burning of coal were almost three times higher than for burning with open chimneys (2.08 vs. 0.71 mg/m3) . Another study in Burkina Faso (Africa) found that good ventilation conditions considerably reduced the fraction of child ARI attributable to smoke from biomass solid fuel  . In addilid fuel . Ezzati We found that, among women, protection was stronger soon after stove improvement and vanished in later periods. This may be attributable to decreased effectiveness of ventilation with passage of time after improvement. Our researchers observed that some families could not maintain the stove in good condition. However, we did not find a significant interaction between stove improvement and sex. Such heterogeneity of risk reduction between sexes may be due to statistical variation.Unlike lung cancer, for which smoky coal use was the dominant risk factor , in the Coal emissions have additional components that can irritate the respiratory system and plausibly increase the risk of pneumonia death, including ozone, inhalable particles, heavy metals, and dioxins. We found the HRs were proportional to the amount and the duration of coal used per family for both smoky coal and smokeless coal. The observed exposure\u2013response associations lend weight to causal inference.Occurrence of chronic nonmalignant pulmonary diseases, an acknowledged risk factor for pneumonia, was a strong risk factor for pneumonia death in smokeless coal users. The occurrence of these pulmonary diseases increased in Xuanwei because of indoor air pollution . It alsoWe found a nonsignificant effect for smoking among men and for cooking among women on pneumonia mortality in Xuanwei, which may reflect the strong effect of coal use, as previously observed for lung cancer . It may Compared with previous reports from this cohort study , the preThere is a possibility of misclassification of the diagnosis of pneumonia with other respiratory diseases . In addition, we cannot exclude the possibility that some pneumonia cases may also have had undiagnosed lung cancer. However, pneumonia diagnosis was made by government hospitals and was mainly based on symptoms, clinical exam, and a chest X-ray. Doctors throughout Xuanwei County used the same procedures and criteria to diagnose pneumonia and document cause of death. If there is some minimal error in the diagnosis of pneumonia in this population, it is highly unlikely that the error would be related to whether or not a patient had a home with or without improved ventilation. Thus any error in diagnosis would not be expected to inflate the effect of improved ventilation.In conclusion, our results strongly support the hypothesis that unvented household coal burning is an important risk factor for pneumonia death in adults. Stove improvement was followed by reduced risk of death from pneumonia in coal users. Indoor air pollution due to solid fuel burning is a serious health hazard in developing countries. Coal smoke exposure is associated with multiple medical conditions, mainly lung-related diseases. The population at risk for illness mediated by indoor air pollution numbers billions worldwide . There w"}
+{"text": "To provide an overview of the complicity of British American Tobacco (BAT) in the illicit trade of cigarettes across the African continent in terms of rationale, supply routes and scale.Analysis of internal BAT documents and industry publications.BAT has relied on illegal channels to supply markets across Africa since the 1980s. Available documents suggest smuggling has been an important component of BAT\u2019s market entry strategy in order to gain leverage in negotiating with governments for tax concessions, compete with other transnational tobacco companies, circumvent local import restrictions and unstable political and economic conditions and gain a market presence. BAT worked through distributors and local agents to exploit weak government capacity to gain substantial market share in major countries.Documents demonstrate that the complicity of BAT in cigarette smuggling extends to Africa, which includes many of the poorest countries in the world. This is in direct conflict with offers by the company to contribute to stronger international cooperation to tackle the illicit tobacco trade. It has been estimated that 10.7% of global cigarette sales were attributed to illicit trade in 2006.5\u20138\u201315Contraband is a key component of illicit trade, and previous analyses have detailed the complicity of transnational tobacco companies (TTCs) in cigarette smuggling in Europe,6Understanding cigarette smuggling in Africa is important for two reasons. First, smuggling is substantial and, according to BAT\u2019s estimates, is growing,This paper focuses on BAT, which historically has had a large market presence in Africa; at times, having a market share of over 90% in 11 sub-Saharan African countries and an overall market share of about 15% across the continent.\u2013This paper is based on internal documents from the Guildford Depository and BATDA. The obstacles to accessing, and the limitations of working with, BAT documents have been previously described.22\u201cAlternative routes\u2026to keep the franchise alive, meet targets and fend off competition.\u201d33Documents suggest that the contraband trade accomplished several goals for TTCs in Africa. First, cigarette smuggling allowed TTCs to gain leverage in negotiations, similar to that used with governments for improved market access and foreign investment similar to efforts in Vietnam,PMI [Philip Morris International] wish[es] to propose an industry presentation to the Nigerian Government for a specific import duty to replace ad valorem rate. The objective would be to legalise \u2018profitable\u2019 imports thus providing the Nigerian Government with revenue currently lost by the proliferation of GT [General Trade].35Similarly, minimising taxation was described as a motive to engage in contraband tobacco trade by BAT in Nigeria.36Second, cigarette smuggling has been a response to intense regional competition among TTCs for establishing and growing market share. BAT, for example, observed the growing contraband trade, and perceived the need to do the same to remain competitive.39Third, contraband has acted to circumvent barriers to market access posed by government restrictions or local conditions. In the late 1990s, BAT recognised that trade barriers were substantial in Africa and noted that \u201cinstability will continue to characterise the African political scene\u201d.Resurgence of the civil war since October 1992 has had its toll on the economy of an already battered country \u2026 pushing most businesses towards the use of the parallel and the black market, which is \u201cillegal\u201d. In not using the black market ETA is at a significant disadvantage to other companies.41In some countries, where cigarette imports were not permitted, contraband was seen as the only supply route:Zaire \u2026 is changing into a GT [General Trade] market and there is no reliable information regarding the current levels of imported brands and the shares \u2026 Imports into Malawi, and Uganda are specifically banned and the only route in will be by GT.42In other cases, smuggling was supported even where legal imports were permitted. In a 1991 memo, BAT marketing executive, Joe Green considered the distribution strategies for Cameroon in terms of two scenarios where (1) legal imports were permitted and (2) legal imports were prohibited. Even if legal imports were allowed, Green stated, \u201cGT shipments will remain the mainstay of our activity ... . The Malabo distribution channel will have to be maintained \u2026 . Maintain a minimum cover level of BHSF [Benson and Hedges brand] via legal imports\u201d.43Although comprehensive data of smuggled product by volume or value remain elusive owing to the illegal nature of the trade, BAT\u2019s sales figures for certain years estimate that contraband represents a high proportion of the total market in some African countries. In West Africa, the flow and amount of contraband across countries is described for 1987 in 47Documents describe how BAT worked through distributors that, acting as middlemen, purchased cigarettes from BATUKE and then supplied them to \u201ctransiteers\u201d52A key function of distributors was to insulate BAT from direct contact with transiteers, thus reducing the risk of detection and prosecution. Soropex, for example, \u201cprovide[d] \u2018cover\u2019, albeit increasingly flimsy, for BAT in some fairly shady business\u201d.It was agreed that Michel Chevaly [of Sorepex] was in an exposed position and in [the] future JMT  should not travel with him in Cameroon. One of the main functions of SOREPEX was to allow BAT to keep at arm\u2019s length from transit customers\u2014particularly in Cameroon.48Documents show that distributors offered BAT detailed knowledge of major entry points to the African continent, including Port Said, Egypt, for North Africa, Djibouti for East Africa and Malabo, Equatorial Guinea, for West Africa.54Once contraband reached the continent, distributors\u2019 knowledge of specific border crossings for onward supply to individual countries was critical. In 1987, Sorepex reported:In August there will be a shipment by sea. If this shipment goes smoothly then future shipments will be by sea for cost saving purposes\u2026.Zone II\u2014others\u2026 a particular customer \u2026 has opened two new routes to Sudan:i. From Chad: N\u2019Jamena to Abeche and Adre (Sudan border)ii. From RCA: Bangui to Biaro (Sudan border). It is anticipated that 800 cases of BHSF [Benson and Hedges brand] will go via these routes in July.55Plans to conceal cigarettes among other merchandise and falsify documents on the origin of the stock were made known to BAT:Sales departing from Malabo to North Cameroun and Chad. Via three transporters: Sodisa, Mouchili and Bogno \u2026(1) For the first time, Bogno will buy 300 cartons: this is the capacity of his truck, taking into account the other merchandise which will \u2018hide\u2019 the cigarettes(2) Each time, I will ask M & B [Mouchili and Bogno] to sign a document testifying that the cigarettes have really been exported from the North. I will countersign this document which will enable BAT to pay them\u2014via Sorepex \u2026[translated from original French]56Similarly, the supply routes for State Express Filter King cigarettes to Sierra Leone were described in 1985 as follows:In the three weeks, Bah [smuggler] had sold 300 of the 420 cases. Next SEFK will be sent (via) Conakry, instead of via Dakar (where two borders had to be crossed). Transit Conakry/Freetown would be by lorry \u2026 . Later they want BHSF, which would be stuffed in same container, but documents/cases, would have to be marked \u2018in transit\u2019.57Similarly, to avoid detection between Niger and Nigeria, Sorepex reported that \u201c[D]irect imports to Nigeria [would be] through Mr Adji\u2026[who] would disguise the cigarette importations by calling the shipment something else, e.g. matches \u2026\u201d.Point 38 is an area of land on the Zaire/Sudan border near Uganda, it is somewhat of a no-man\u2019s land and is a major centre for barter. Because of the dangers in the area and the customs situation in Kenya the lorries in convoys of 30\u201350 trucks are escorted through Kenya and Uganda by the army....Dr Kaboash would like to supply Benson & Hedges through this route and feels that 500 cases every 2 months would suffice with shipment by sea to Mombasa [sic] and that this business should start in April. This appeared to be an interesting opportunity which we should follow up.59Another consideration in identifying border crossings was the volume of potential trade to be achieved. In Sudan, Sorepex reported that an additional smuggling route might not only appease transiteers, but also would ensure larger distribution:Re. Sodisa: I asked Daher to create a second sales circuit, parallel to his customary route, and to negotiate directly with the Sudanese who sell their gum Arabic [adhesive for cigarette papers] in Bangui (bypassing Ousta as intermediary). This will enable him to crack the monopoly of the Birao [city in Central African Republic] dealers, whilst keeping them on his side  because these people are indispensable in negotiating border crossings; Daher will use this new circuit to sell 300 cartons which we will send him at the end of March. [Translated from original French]60For many countries, flexibility in the mode of transport  was another important consideration. In Guinea, military aircraft were used for air transport during the wet season.CIGARETTES BY AIR Cigarettes are arriving at Conakry Airport on a fairly regular basis. Air transport is obviously more expensive than traditional methods but it is quicker and customs at Conakry airport are certainly less strict and organised than at port.62Where one mode of transport became unavailable, the distributor investigated alternatives:The Djibouti Government has again closed the port for dhow [Arab sailing vessel] trade thereby preventing re-exports to Somalia by this route. As on the previous occasion, our distributor in Djibouti is investigating methods of supplying Somalia by overland route.63The illegal nature of the business, and often unstable market conditions, at times, led to tensions among BAT, Soropex and local agents, which may have played a part in BAT\u2019s efforts to have more control over its transit business. For example, in Djibouti, the failure of prepaid contraband to arrive raised the question of which party should incur the loss, with transiteers apparently threatening to cease moving BAT contraband.One of Bobo\u2019s key distributors, Hadi, the agent in Lab\u00e9, has just agreed to act as a transiteer for Marlboro. This is obviously in direct conflict with his role in Bobo\u2019s organisation and in the view of the BAT representative is totally unacceptable.62These concerns followed earlier suspicions that a BAT employee based in Guinea was also smuggling Marlboro cigarettes:Enlarging previous info, RG [BAT executive Rob Galgut] said that PR [B&W executive Paul Richardson] had been known to boast that he made more selling containers of [sic] Marlboro than he got from BAT/B&W\u2026 PR worked for BATUKE at one time, was well connected with transiteers, particularly in Conakry.66BAT may have also sought more control of the business owing to a perceived loss of income from transit sales because of unstable market conditions in Africa. In 1987, BATUKE sought to reduce reliance on Sorepex, as a cost-saving measure, but also because of the distributor\u2019s failure to execute plans as expected.64In West Africa, BAT established its own staff on the ground to effectively bypass the distributor. At the same time, it was important for the company that Sorepex remained to be seen as in command within \u201csensitive markets\u201d. For example, in business agreements intended to govern the relationship between BAT and Sorepex into the 1990s, it was reported that:BAT has \u201cstaffed up\u201d in West Africa to the point where there is duplication of efforts between SOREPEX and BAT (UK&E). Our objective now is to preserve the \u201cfa\u00e7ade\u201d that SOREPEX represents between us and the sensitive markets of Togo, Benin, Niger (Unit I) and Equatorial Africa (Unit II), but at the same time, enable the BAT field force to take over the management of this important business.67A 1988 BATUKE document suggests that the company subsequently incorporated the role of the distributor, in developing and maintaining supply routes for contraband, into the role of the company\u2019s own \u201cRed Sea/Central\u201d area manager:Transit\u2014as a result of the hard currency shortages and internal chaos and corruption in most markets there is a growing demand for transit. The Job Holder is required to evaluate routes, prices and risks, then to recommend action to the Area Manager. He is also required to exercise judgement over sales/shipments when markets are supplied by more than one source. Constant vigilance is necessary in pricing because of the competitiveness of the area and the problem of parallel shipments between various territories within the Middle East and West Africa.68BAT staff, such as Paul Richardson , also appeared to take a more active role tracking the movement of contraband:I would however like to take the opportunity  to visit Sierra Leone en route primarily to assess the volume of 555 transit and to a lesser extent that of Lucky Strike. I would also be interested in finding out if any of the Gladstone brought through Conakry port \u201cin transit for Sierra Leone\u201d is actually being consumed there.69In 1986, BATUKE recognised that legal sales of certain brands remained low in volume in West Africa.When the issue\u2026was discussed where BATUKE wish to appoint a domestic importer enabling us to provide cover for advertising and GT business, Sir Patrick  felt that it was perfectly acceptable for BAT Cameroon to recommend a domestic importer for BHS [Benson & Hedges Specials].71Another 1991 memo, regarding the launch of a new brand in West Africa, suggests similar intentions to use newly introduced legal cigarettes as cover for contraband sales:The reasons why we were so enthusiastic about Lambert & Butler were: ... the possibility of GT exploitation were considered to be good  ... this is highly political and the best interests of the who[le] BAT group may not be the deciding factor \u2026 I accept that in retrospect we may have been over optimistic about its [L&B] potential in Togo, Benin, Niger, but the main reason for its launch in those markets was not to exploit domestic markets but for GT opportunities.72Using small legal sales as cover seems to have been standard practice for BAT in Africa. In Zaire the company evaluated various options for undertaking this process:There are two questions key to Sorepex\u2019s future business in Zaire.official importer who will pay taxes to act as cover for transit business? ...(a) Can Sorepex with BAT Z\u2019s [BAT\u2019s subsidiary in Zaire] permission appoint another (b) If it is not feasible, would BAT Z consider selling official imports to Sorepex client directly and so enabling cover?73In Sierra Leone, it was recognised that the instigation of legal cover could help \u201cfend off the competition\u201d.Obviously it is practically impossible to develop a pure GT [general trade] brand, thus a home base is necessary. It would therefore seem logical to import legally some quantity, allowing also for an advertising campaign to take place \u202675Similarly, Paul Richardson asserted that legal imports would serve as a cover for smuggling Kool cigarettes and for enabling the brand\u2019s local promotion:Kool is considered to be the best B&W product offering for the Nigerian market. ... Both legal and transit importing would be required to properly\u2014and profitably\u2014develop the brand \u2026 Legal imports would be loss making and significantly under invoiced because of Nigeria\u2019s high duty rates. Legal [sic] imports would however establish a legitimate presence and enable B&W to promote the brand.76Internal BAT documents demonstrate that the use of cigarette smuggling to gain market access to emerging markets extended to the African continent. Documents suggest that the contraband trade had helped BAT gain leverage in negotiating with governments, compete for market share, circumvent local import restrictions and overcome local political and economic instability and gain a market presence. Although senior executives have denied company involvement,An understanding of the organisation and logistics of cigarette smuggling in Africa provides essential insights for designing effective legislative and administrative responses. Contraband tobacco trade is exceedingly dynamic in terms of supply routes and modes of transport. It is not a consequence of price differentials. Contraband trade has been an integral element of BAT\u2019s market entry strategy, which is not driven by short term financial gain, but rather efforts are undertaken to advance longer-term corporate objectives. Therefore, policy responses cannot focus solely on current mechanisms of smuggling, especially those reliant on controls at the border, because these policy strategies are likely to be circumvented. Legislation must be expansive, and the process for amendment sufficiently flexible to ensure that smuggling means not yet exposed are either captured or can be quickly added to the regulatory response. Given that smuggling abetted by TTCs in country X most often occurs between countries Y and Z, effective legislative controls should include conspiracy to smuggle in another country.Similarly, measures that seek only to deter contraband by removing the immediate economic gain will fail to address how the industry uses smuggling tactically, often as a loss leader, either to undermine competition or drive governments into making policy decisions that best suit TTCs\u2019 needs.In addition to sufficiently large fines applied at the corporate level, actively enforced criminal penalties that include incarceration for corporate officials is warranted. The potential for lengthy prison sentence imposed on senior tobacco company officials will probably create a far greater deterrent effect than confiscation of product and profits on those instances where contraband is intercepted. Perhaps most important of all, sharing of information and reciprocal enforcement of judgments will be essential. Transborder movement of information and judgments is necessary to combat transborder smuggling of cigarettes.The documents reviewed in this paper reiterate the importance of addressing the contraband trade from a regional and global perspective. The illicit supply of BAT cigarettes to Africa has been carried out, via distributors and transiteers, with little regard for national borders. Furthermore, the limited capacity of customs and excise in many African countries has been unable to prevent the establishment of regional supply routes across the continent. These findings support the conclusion of the First Session of the Intergovernmental Negotiating Body on a Protocol on Illicit Trade in Tobacco Products, of the WHO FCTC, for \u201cincorporating strong provisions on international cooperation\u201d.5Effective deterrents, including criminal sanctions as advocated here, will only happen if manufacturers remain legally responsible for their products as they move through the supply chain. This will require a systematically collected evidentiary base at all stages. That evidence can be most readily derived using a licensing system throughout the supply chain and the sort of closely monitored tracking and tracing system suggested by others.78Of all WHO regions, Africa represents the region with the greatest need for technical assistance to gather and analyse data, develop and maintain tracking and tracing systems, and investigate and prosecute alleged offenders. Africa would benefit from the sharing of experiences of other regions, and the establishment of a regional resource centre for \u201cenriching the skills sets of enforcement agencies\u201d.79This paper affirms the need to exclude TTCs from involvement in the development of policies to combat tobacco smuggling. The industry has adopted a public position of promoting actions to tackle contraband as part of \u201cbalanced regulation\u201d framed within claims of corporate social responsibility.82The findings of this paper suggest that BAT\u2019s complicity in contraband trade extended across the African continent, and that its efforts to contribute to international cooperation to address this problem should be understood in this context. In reality, BAT\u2019s proposals seek to advance corporate interests and undermine an effective global response to tobacco smuggling. For example, BAT has prioritised the problem of counterfeit cigarettes, for which protection and redress already exists via trade agreements.This paper adds to the existing evidence of the transnational tobacco companies\u2019 (TTCs) knowledge of and complicity in cigarette smuggling and is the first detailed and comprehensive analysis of internal tobacco industry documents regarding the TTCs\u2019 smuggling activities in Africa.Documents show that BAT has advanced its corporate interests by systematically exploiting strategic opportunities to supply the contraband tobacco trade throughout Africa. To date, BAT and its senior directors have remained unaccountable via litigation or public inquiry for these activities, which was epitomised by the abandoned investigation by the UK Department of Trade and Industry in 2004 amid reports of political pressure.Information from customs and law enforcement bodies indicate that smuggling is a major and growing problem in Africa, based on frequency and size of seizures, although there remains no systematic collection of data."}
+{"text": "Blood sampling was carried out on day 1 of the first cycle. The 5-fluorouracil area under the curve0\u21923h in the index patient was 24.1\u2009mg\u2009h\u2009l\u22121 compared to 9.8\u00b13.6 (range 5.4\u201315.3) mg\u2009h\u2009l\u22121 in control patients. The 5-fluorouracil clearance was 520\u2009ml\u2009min\u22121vs 1293\u00b1302 (range 980\u20131780) ml\u2009min\u22121 in controls. The activity of dihydropyrimidine dehydrogenase in mononuclear cells was lower in the index patient (5.5\u2009nmol\u2009mg\u2009h\u22121) compared to the six controls . Sequence analysis of the dihydropyrimidine dehydrogenase gene revealed that the index patient was heterozygous for a IVS14+1G>A point mutation. Our results indicate that the inactivation of one dihydropyrimidine dehydrogenase allele can result in a strong reduction in 5-fluorouracil clearance, causing severe 5-fluorouracil induced toxicity.5-fluorouracil pharmacokinetics, dihydropyrimidine dehydrogenase-activity and DNA sequence analysis were compared between a patient with extreme 5-fluorouracil induced toxicity and six control patients with normal 5-fluorouracil related symptoms. Patients were treated for colorectal cancer and received chemotherapy consisting of leucovorin 20\u2009mg\u2009mBritish Journal of Cancer (2002) 86, 1028\u20131033. DOI: 10.1038/sj/bjc/6600199www.bjcancer.comCancer Research UK\u00a9 2002  Fluorouracil (5-FU) is widely used in chemotherapeutic regimens for the treatment of breast-, colorectal- and head- and neck cancer. The cytotoxic mechanism of 5-FU is complex, requiring intracellular bioconversion of 5-FU into cytotoxic nucleotides  was obtained from Moravek Biochemicals  and Lymphoprep  was from Nycomed Pharma AS . Leucosep tubes were supplied by Greiner . All other chemicals were of analytical grade.5-Fluorouracil was obtained from Sigma Chemical Co. . 5,6-Dihydro-5-fluorouracil was kindly provided by Roche Laboratories . AmpliTaq Taq polymerase and BigDye-Terminator-Cycle-Sequencing-Ready-Reaction kit were supplied by Perkin Elmer . A Quaquik Gel Extraction kit was obtained from Qiagen . Human heparinised plasma was obtained from the Red Cross Blood Bank . [4-\u22122 combined with 5-FU 425\u2009mg\u2009m\u22122, both on 5 successive days, in a 28-day cycle. Blood sampling was carried out on the first day of the first chemotherapy cycle immediately following the 5-FU dose, administered as bolus intravenous injection over 2\u2009min. Leucovorin was infused after the end of blood sampling. On the following 4 days, the same 5-FU dose was administered as short time infusion, after leucovorin administration. One patient experienced severe toxicity during the first chemotherapy cycle and, therefore, a screening on DPD deficiency was initiated. Data from six patients who participated in the same study protocol and who showed no signs of severe toxicity were randomly selected for reference pharmacokinetics, DPYD genotyping and DPD enzyme activity. These patients served as controls.All patients were treated for Dukes C colorectal cancer and participated in a protocol that had been designed to study 5-FU and DHFU pharmacokinetics. The protocol was approved by the Medical Ethics Review Committee of the Martini Hospital Groningen and written informed consent was obtained from all patients. All patients who entered this protocol were chemotherapy naive. Chemotherapy consisted of leucovorin 20\u2009mg\u2009mg for 10\u2009min. The plasma samples were analysed for 5-FU and DHFU concentrations by high-performance liquid chromatography (HPLC) on the day of collection. Blood samples for DPD analysis were collected 5 to 23 months after blood sampling for 5-FU pharmacokinetics, which corresponds to intervals ranging from 2 to 17 months after the last 5-FU dose. None of the patients received chemotherapy at that moment.For pharmacokinetic sampling, a canule was placed in the arm of the patient contralateral from drug administration. Blood samples (5\u2009ml) were collected in heparinised tubes just before, and 2, 5, 10, 20, 30, 45, 60, 80, 100, 120, 150 and 180\u2009min postinjection. Collected samples were immediately placed on ice and subsequently centrifuged at 2500 \u22121 in water) was added to 1\u2009ml plasma sample, and this mixture was vortexed and subsequently deproteinated with 50\u2009\u03bcl of a 50% (w\u2009v\u22121) trichloracetic acid solution. After centrifugation at 8000\u2009g for 2\u2009min the supernatant was transferred into a 20\u2009ml centrifuge tube and neutralised with 1\u2009ml 1\u2009M sodium acetate solution. Then 5\u2009ml ethylacetate was added and the mixture was vortexed during 10\u2009min. After separation of the organic and aqueous layers by centrifugation at 5000 g for 5\u2009min, the ethylacetate layer was transferred into a 10\u2009ml tube and evaporated under a stream of nitrogen at 25\u00b0C. The residue was dissolved in 100 \u03bcl ultrapure water and 20 \u03bcl was injected. 5-Fluorouracil and DHFU standards ranging from 0.5 to 20\u2009mg\u2009l\u22121 were prepared in human plasma. The chromatographic system consisted of a Waters 616 pump equipped with a Waters 717+ autosampler. The separation of 5-FU and DHFU was accomplished by gradient elution at ambient temperature on a Phenomenex Prodigy ODS 3 column  equipped with a guard column (30\u00d74.6\u2009mm) of the same material. Mobile phase A consisted of 1.5\u2009mM K3PO4 and 1% (v\u2009v\u22121) methanol (pH=6.0) and mobile phase B of 1.5\u2009mM K3PO4 and 5% (v v\u22121) methanol (pH=6.0).5-Fluorouracil and DHFU concentrations were measured by HPLC analysis using a modification of the method described by The gradient was programmed as follows: 100% A during 2\u2009min; 100% A\u2192100% B in 0.5\u2009min; 100% B during 7\u2009min; 100% B\u2192100% A in 0.5\u2009min; 100% A during 10\u2009min. Detection was performed using a Waters 996 Photo Diode Array UV detector interfaced with a Millenium 2010 Chromatography Manager Workstation. Spectra were acquired in the 201\u2013300\u2009nm range. 5-FU was monitored at 266\u2009nm and DHFU at 205\u2009nm. The internal standard chlorouracil was monitored at both wavelengths.The pharmacokinetic analyses were performed in the ADAPT II computer program . The pharmacokinetic data of both the index patient and the six control patients were tested in eight different models. In each model the patient's data were fitted individually and for each data set the Akaike Information Criterion (AIC) was calculated. The model with the lowest summarised AIC value was selected as the better one (data not shown). The model used for calculating 5-Fluorouracil pharmacokinetics is a two-compartment model with Michaelis\u2013Menten elimination from the first compartment and is described by two differential equations:X1 and X2 indicate the amount of drug in each compartment, respectively. The k-values represent linear distribution- and elimination rate constants, and the Vmax and Km values represent Michaelis\u2013Menten constants for non-linear elimination from the first compartment. Rinf represents the infusion rate of 5-FU.0\u21923h).The area under the curve of 5-FU and DHFU was calculated using the trapezoid rule. The average systemic clearance of 5-FU was calculated by dividing the administered dose by the area under the curve  perchloric acid. The reaction mixture was centrifuged at 11\u200a000\u2009g for 5\u2009min to remove protein. The separation of radiolabelled thymine and the reaction products was performed by reversed phase HPLC. Protein concentrations were determined with a copper-reduction method using bicinchoninic acid, as described by To investigate whether the 5-FU toxicity might have been caused by a partial deficiency of DPD, we determined the activity of DPD in peripheral blood mononuclear (PBM) cells. Therefore PBM cells were isolated from 15\u2009ml EDTA anticoagulated blood and the activity of DPD was determined according previously described methods . We did not match our control patients for age and gender.Each value, measured in the index patient, was compared to the mean\u00b12 s.d. range of the corresponding parameter in the control group. Values outside this range were considered abnormal  and thrombocytopenia (26\u00d7109 platelets l\u22121) developed along with nausea, diarrhoea, stomatitis, fever and hair loss. The next day leukocytes and platelets decreased to 0.5\u00d7109\u2009l\u22121 and 12\u00d7109\u2009l\u22121 . During this period the patient developed leukopenic fever (40\u00b0C) for which antibiotics were administered. Until day 20 the leucocytes and platelets remained low (1\u00d7109\u2009l\u22121 and 13\u00d7109\u2009l\u22121 respectively). During the subsequent week the clinical picture and hematological parameters gradually improved and normalised. On day 34 the patient was discharged from the hospital.Patient characteristics from the index and control patients, as measured before 5-FU administration on the first day of the first chemotherapy cycle, are listed in Table 1n=4), vomiting (n=2) and CTC grade 1 stomatitis (n=1).The toxicity observed in the six control patients was limited to mild nausea , revealed a 16\u00b14% increase of the 5-FU AUC.Simulation of anuria in the control group (K\u22121\u2009h\u22121) compared to the six control patients  and comparable to obligate heterozygotes  cells was lower in the patient experiencing severe toxicity , leading to the skipping of exon 14 directly upstream of the mutated splice donor site during DPD pre-mRNA splicing.Sequence analysis of exon 14 of DPYD of the six control patients revealed no mutations.\u22122 5-FU to a patient with non-detectable DPD-activity in PBM cells and found a very low 5-FU clearance rate. This patient was probably homozygous for a mutant DPD allele, although the genetic cause was never elucidated. \u22122 as 2\u2009h intravenous infusion plus 125\u2009mg orally, followed by 5-FU 2 g m\u22122 as a 24\u2009h continuous infusion. They found a 5-FU plasma level of 0.3\u2009mg\u2009l\u22121 on day 15 after administration, which implies a dramatic overexposure to 5-FU. This patient could not have been homozygous deficient because the DPD activity in lymphocytes was within the normal range. The role of PBM\u2013DPD activity as an indicator for 5-FU clearance is, however, questionable. 5-Fluorouracil remains the major drug in the treatment of advanced colorectal cancer. Dihydropyrimide dehydrogenase is the key metabolic enzyme in 5-FU degradation and since more than 80% of the dose is metabolised by this enzyme, DPD activity is one of the main factors determining drug exposure  in previous work . This might indicate that not only obligate heterozygotes, but also low normal homozygotes are at risk for developing severe toxicity when treated with 5-FU. It is however as yet unclear whether the pharmacokinetic profile of 5-FU is identical in both groups.In this paper, we report a combined pharmacokinetic and genetic analysis of the DPYD gene and demonstrate that a single G\u2192A point mutation in the invariant splice donor site IVS14+1 of the DPYD gene has profound impact on the clearance of 5-FU. We also found a lower PBM\u2013DPD activity in the index patient compared to six controls. The DPD activity was comparable to the activity observed in obligate heterozygotes assay is available yet, and therefore in most hospitals therapeutic drug monitoring of 5-FU is not yet feasible."}
+{"text": "Gingko biloba leaves, polymeric proanthocyanidins were eluted after the fractions of flavonol glycosides and biflavone glycosides. A purified proanthocyanidin polymer accounted for 86.6% of the total proanthocyanidins, and for 37.7% of the total antioxidant activity of this leaf extract. For structure elucidation, the polymer was submitted to acidic depolymerization in the presence of phloroglucinol. The structures of the resulting flavan-3-ols and phloroglucinol adducts were determined on the basis of 1D-and reverse 2D-NMR  spectra of their peracetylated derivatives, MALDI-TOF-MS and CD-spectroscopy. The observations resulting from the degradation with phloroglucinol were confirmed by 13C-NMR spectroscopy of the polymer. The mean molecular weight of the polymeric fraction was estimated to be 9\u201310 flavan-3-ol-units.By Sephadex LH-20 gel chromatography of an extract from  Ginkgo biloba L.  are widely used in herbal medicine for the treatment of mild to moderate cognitive disorders, concentration problems, tinnitus and dementia + in the MALDI-TOF-MS of its peracetate (compound 1a), which suggests a B-type diflavanoid constitution composed of two (epi)gallocatechin units and one additional phloroglucinol ring. The 1H-NMR spectrum at ambient temperature showed complex signal duplication due to rotational isomerism. The assignment of signals and the point of interflavan-linkage were achieved by extensive 2D-NMR experiments. The spectral pattern of the heterocyclic region of compound 1a was almost identical with the corresponding procyanidin derivative epicatechin-(4\u03b2\u21928)-epicatechin-(4\u03b2\u21922)-phloroglucinol + of its peracetate (2a), indicative of a dimeric proanthcyanidin derivative composed of two gallocatechin/epigallocatechin and one phloroglucinol ring. The 1H-NMR of 2a in CDCl3 (600 MHz) gave two sharp two-proton singlets at \u03b4 7.16 and 7.29 ppm typical for pyrogallol-type-B rings of the constituent flavan-3-ol units. The location of the inter-flavanoid linkage was recognized for 2a by long-range correlation (HMBC) of H-4 (C) with C-5 (D) 20 = +99.3\u00b0 . 60 mg were acetylated to give 1a: MALDI-TOF-MS: [M+Na]+m/z 1384. 1H NMR : \u03b4 1.21\u20132.41 , 4.38 , 4.43 , 4.47 , 4.70 , 4.85 , 4.91 , 5.03 , 5.30 , 5.33 , 5.44 , 5.49 , 5.68 , 6.03, 6.26, 6.67, 6.74 , 6.65 , 6.85 , 6.83, 6.87, 6.94, 7.04 , 6.81, 6.89 , 6.92, 7.08 . 13C NMR : 32.9 (C-4 C), 34.8 (Cr-4 C), 35.3 (C-4 F), 35.8 (Cr-4 F), 70.2 (Cr-3 C), 70.9 (C-3 C), 71.4 (C-3 F)), 71.4 (Cr-3 F), 73.4 (C-2 C), 74.3 (Cr-2 F), 74.7 (Cr-2 C), 75.4 (C-2 F), 107.4, 107.5, 108.4, 109.3 , 110.5, 110.8 (C-6 D and Cr-6 D), 111.4 (C-4a D), 111.8 (C-4a A), 114.2, 114.4, 114.6, 114.8 , 118.9, 119.2, 119.9, 120.2 , 151.8 (C-8a D), 152.1 (C-8a A). After the reaction of compound 1 (20 mg) in 0.1 N ethanolic HCl (1 mL) 20 = +179\u00b0 . 20 mg were acetylated to give 2a: MALDI-TOF-MS: [M+Na]+m/z 1384. 1H NMR : \u03b4 1.42\u20132.43 , 4.25 , 4.45 , 4.84 , 4.95 , 5.03 , 5.40 , 5.68 , 6.60 , 6.62 , 6.80 , 6.76 , 6.88 , 7.16 , 7.29 . 13C NMR : 32.7 (C-4 (C), 33.0 (C-4 F), 70.6 (C-3 F), 71.2 (C-3 C), 73.4 (C-2 (F), 73.6 (C-2 C), 107.5 (C-6 A), 107.7 (C-8 A), 109.7 (C-6 D), 111.6 (C-4a D), 111.9 (C-4a A), 114.4 (C-4 G or C-6 G), 114.6 (C-6 G or C-4 G), 119.2 (C-2\u2019/C-6\u2019 E), 119.6 (C-2\u2019/C-6\u2019 B) 151.6 (C-8a D), 152.3 (C-8a A). After the reaction of compound 2 (10 mg) in 0.1 N ethanolic HCl (1 mL) [2 to dryness and purified on preparative TLC in system A. The main degradation products were further purified on preparative TLC on cellulose  to yield epigallocatechin-(4\u03b2\u21922)-phloroglucinol (2.4 mg) and phloroglucinol (4.1 mg).Fraction10  obtained from the Sephadex LH-20 column as described above was subjected to chromatography on MCI-gel CHP 20P (25 \u00d7 450 mm) with a 10\u201350% MeOH linear gradient (17 mL/subfraction) to yield an amorphous powder  l (1 mL) , the mix"}
+{"text": "The consecutive radical/ionic reaction consisting of radical formylation of alkyl bromides and nucleophilic addition of a cyanide ion was investigated, which gave moderate to good yields of cyanohydrin derivatives in one-pot. In 19nitiator \u201317. Recenitiator , hydroxymediator , which lCyanohydrins are important subunits frequently found in biologically active compounds and are also versatile building blocks for further synthetic transformations \u201321. The 1a) with Bu3SnH under 80 atm of CO pressure in the presence of a cyanide source (2a') was slow, which gave 16% of 3a' and 51% of nonanal. The use of AcCN (2a'') also gave 3a'' but only in 12% yield. However, when ethyl cyanoformate (2a) was used together with Et3N [3a was obtained in 62% yield. When we used higher CO pressure such as 120 atm, the yield of 3a increased to 79%.We examined AIBN-induced radical formylation of 1-bromooctane  afforded the lowest yield of cyanohydrin 3j, which possessed an olefin structure arising from the ring-opening of a cyclopropylcarbinyl radical  in the presence of Bu3SnH, AIBN, and Et3N, which gave moderate to good yields of cyanohydrin derivatives 3. This protocol represents a one-pot method [In summary, we have demonstrated a three-component coupling reaction comprising alkyl bromides t method \u201333 based3a) [1a, 96.6 mg, 0.5 mmol), ethyl cyanoformate , tributyltin hydride , triethylamine , and AIBN  in C6H6 (17 mL) were placed in a 100 mL stainless steel autoclave. The reaction mixture was degassed 3 times with 10 atm of CO and charged with 90 atm of CO at \u221240 \u00b0C (MeCN\u2013dry ice bath). Then the autoclave was allowed to warm to room temperature, which caused the pressure gauge to indicate 120 atm. Then the reaction was conducted at 80 \u00b0C for 3 h. After cooling to room temperature, the reaction mixture was concentrated and purified by silica gel flash chromatography (hexane/EtOAc 97:3) to afford 3a . 1H NMR  \u03b4 5.18 , 4.4\u20134.2 , 2.0\u20131.9 , 1.6\u20131.5 , 1.4\u20131.2 , 0.88 ; 13C NMR  \u03b4 153.56, 116.51, 65.27, 64.66, 32.31, 31.68, 29.12, 28.99, 28.71, 24.34, 22.53, 14.05, 13.93.Typical procedure for radical/ionic three-component coupling reaction leading to cyanohydrin derivatives 1-cyanononyl ethyl carbonate (3a)  Table 13a [Table"}
+{"text": "Dr. Mary F. Lopez is incorrectly listed as an author on this article. The authors apologize for this error. The correct author list is as follows:Paul E. Oran, Olgica Trenchevska, Dobrin Nedelkov, Chad R. Borges, Matthew R. Schaab, Douglas S. Rehder, Jason W. Jarvis, Nisha D. Sherma, Luhui Shen, Bryan Krastins, Dawn C. Schwenke, Peter D. Reaven, Randall W. NelsonThe legends for Additionally, the following sentence should be added to the Acknowledgements section:\u201cThis work was supported in part with resources and of facilities at the Phoenix VA Health Care System. The contents do not represent the views of the Department of Veterans Affairs or the United States Government.\u201dThe complete Acknowledgements read:\u201cWe would also like to express gratitude and acknowledge the following ACT NOW study investigators for their assistance with sample collection: Ralph A. DeFronzo, MD. MaryAnn Banerji, MD, FACP, George A. Bray, MD, Thomas A. Buchanan, MD, Stephen C. Clement, MD, Robert R. Henry, MD, Abbas E. Kitabchi, Ph.D., MD, FACP, FACE, Sunder Mudaliar, MD, Robert E. Ratner, MD, FACP, Frankie B. Stentz, MS, PhD, Nicolas Musi, MD. This work was supported in part with resources and of facilities at the Phoenix VA Health Care System. The contents do not represent the views of the Department of Veterans Affairs or the United States Government.\u201d"}
+{"text": "Cardiopulmonary bypass (CPB) provokes inflammation culminating in organ dysfunction and increased mortality. Recently, neutrophil extracellular traps (NETs) have been found to be involved in a variety of cardiovascular diseases promoting tissue and organ injury. Here, we aimed to elaborate the proinflammatory potential of circulating cell-free (cf)DNA in patients undergoing cardiac surgery with CPB. Plasma was collected pre- and postoperatively as well as at d1, d3, d5 and d8 after surgery. At d1, we found circulating cfDNA levels to be significantly increased in patients with prolonged CPB duration (>100\u2009min) when compared to those with shorter CPB times (CPB\u2009<\u2009100\u2009min). Increased CPB duration yielded in higher levels of circulating mitochondrial (mt)DNA, soluble thrombomodulin (sCD141) and ICAM-1, reflecting endothelial damage. Positive correlation between cfDNA and sCD141 was demonstrated at all time points. Plasma and cfDNA from patients with CPB\u2009>\u2009100\u2009min induced NETs release by neutrophils from healthy donors which was not suppressed by inhibitors of intracellular toll-like receptor (TLR)9. DNA binding to neutrophils\u2019 surface (s)TLR9 has been evidenced. Altogether, we demonstrate that elevated plasma cfDNA might be useful to assess CPB-mediated detrimental effects, including endothelial damage, in cardiac surgical patients with prolonged CPB duration. cfDNA-triggered NETosis is independent of classical TLR9 signaling. In this regard, many studies demonstrated increased inflammatory markers, such as TNF-\u03b1, IL-6, IL-8 after cardiac surgery with CPB3. Massive activation of leukocytes, e.g. neutrophils, and different biochemical pathways may result in microthrombosis, microemboli and depletion of coagulation factors. Neutrophil-derived enzymes, such as elastase and myeloperoxidase (MPO) and reactive oxygen species (ROS) contribute to tissue injury and endothelial dysfunction, predisposing patients to organ injury. Further on, activated neutrophils also directly activate endothelial cells thereby increasing perivascular edema and leukocyte transmigration into extracellular matrix4. Recently, the release of neutrophil extracellular traps (NETs)/cell-free DNA (cfDNA), by a process termed NETosis, and their potent proinflammatory and cytotoxic effects have gained much attention as risk factors for cardiovascular diseases as well as the development of postoperative complications7. NETs are web-like structures composed of decondensed chromatin and antimicrobial proteins that can entrap pathogens but also contribute to the pathophysiology of multiple inflammatory diseases such as myocardial ischemia/reperfusion injury and stroke8. Many physiological inducers of NETosis have been reported, including microorganisms9, activated platelets10, activated endothelial cells11 and proinflammatory cytokines12. However, inappropriate NETs release may cause tissue damage and inflammation. Previous studies have shown, that MPO and histones are responsible for NETs-mediated endothelial and epithelial cell cytotoxicity13. Additionally, NETs ingredients might degrade inhibitors of coagulation favoring intravascular thrombus formation14. Notably, marked increase in NETs formation in patients undergoing elective cardiac surgery and correlation with perioperative renal dysfunction was reported15. However, NETosis does not mandatory require neutrophil death and few years ago NETs release by viable neutrophils has been demonstrated, whereby these structures are formed from pure mitochondrial DNA (mtDNA)16. In addition, release of nuclear DNA and mtDNA upon neutrophil stimulation with PMA and NO has also been demonstrated17.Cardiac surgery with cardiopulmonary bypass (CPB) support initiates a systemic inflammatory response (SIRS), presumably caused by contact of blood components with the artificial surface of the extracorporeal circuit, that is associated with postoperative morbidity and mortality18 and during sterile SIRS19. mtDNA fragments participate in different kinds of innate immune modulation by activating pattern recognition receptors, of which toll-like receptors (TLRs) are the most prominent one. Proinflammatory mtDNA mediates inflammatory responses through CpG/TLR9 interactions, supporting neutrophil activation and TLR9 inhibition significantly attenuates mtDNA-induced systemic inflammation in mice20. Recently, a study based on multiple cohorts showed that mtDNA can improve risk prediction and there is a tight relationship between elevated plasma mtDNA level and 28-day mortality21. Postoperative inflammatory responses are highly related to the prognosis of cardiac surgery. However, the impact of CPB on neutrophil TLR9 expression and circulating cfDNA as well as the potential relevance of cfDNA for patients\u2019 outcome has not been reported until now. Here, we hypothesize that circulating cfDNA might reflect the onset of CPB-induced systemic inflammation in patients undergoing cardiac surgery. We further sought to evaluate how cfDNA might amplify neutrophil-mediated inflammatory reactions and to further elucidate the significance of the classical DNA receptor TLR9 in this process.Human mitochondrial DNA (mtDNA) consists of an approximately 16.5\u2009kb circular, double-stranded extrachromosomal DNA and might contain high amounts of unmethylathed CpG. Recent research has implicated mtDNA as a damage-associated molecular pattern (DAMP) and marked increase in extracellular mtDNA was already found in different pathological disorders, e.g after cardiac surgeryPatients\u2019 baseline demographics, surgery information as well as physiologic parameters are summarized in Table\u00a0Table\u00a0DNase I activity as no alternations in DNase I activity were detected over time  and CPB\u2009>\u2009100\u2009min (long-CPB), suggesting activation of proinflammatory pathways by the extracorporeal circuit Fig.\u00a0. In patiime Fig.\u00a0. Thus, e17 and its proinflammatory property in DAMP-associated inflammation is well documented20. We therefore questioned if mtDNA might also be detected in circulating cfDNA in patients\u2019 plasma. Real time PCR analyses of DNA isolated from plasma samples revealed that mtDNA levels were significantly increased in patients with long-CPB vs. those with short-CPB at admission, indicating that patients included in the long-CPB group display a more severe disease and are more prone for systemic inflammation  might represent an ingredient of NETs structures among othersion Fig.\u00a0. CirculaTaken into account that cfDNA might include large amounts of NETs which represent potential inducers of endothelial damage, we further assessed plasma concentrations of soluble thrombomodulin (sCD141) and ICAM-1 which represent common molecular markers of endothelial cell injury Fig.\u00a0. Whereas24 and circulating mtDNA was found to amplify inflammation19 by inducing NETs release via TLR9 activation amongst others25. Neutrophils isolated from healthy volunteers, which were already reported to be unstimulated26, were activated in vitro with pooled plasma from volunteers as well as from cardiac surgery patients (five patients of each group), and cfDNA/NETs were quantified in culture supernatants . By performing Real time PCR the following mean mtDNA contents were detected: 0.0026 in samples from healthy volunteers, 8.53 in plasma from CPB\u2009<\u2009100\u2009min and 88.33 in plasma from CPB\u2009>\u2009100\u2009min. As shown in Fig.\u00a0DNase I markedly impaired the onset of plasma-induced NETosis  for patients\u2019 plasma-triggered NETosis. Neutrophil sTLR9 expression was evidenced by immunofluorescence . Although patients with CPB\u2009>\u2009100\u2009min displayed rapid TLR9 upregulation after surgery, it has to be taken into account that we did not differentiate between endosomal and sTLR9 expression representing a further major limitation or our study. Thus, in vivo sTLR9 regulation, especially after prolonged bypass, cannot be surely excluded. On the other hand, negative correlation of total TLR9 and sCD141 might indicate TLR9-linked endothelial protection. On the basis of our data, we speculate that increased NETosis in patients with prolonged CPB duration is rather due to an upregulation of neutrophil sTLR9 whereby only a minor implication of endosomal TLR9 can be assumed.It has been found that mtDNA upregulates TLR9 expression in macrophages and also neutrophilsIn summary, cfDNA represents an early biomarker for CPB-induced inflammation and a potential mediator of endothelial damage after cardiac surgery with prolonged bypass duration. To our knowledge, our study demonstrates for the first time that cfDNA boosts inflammation, amplifying NETosis by a novel mechanism which is independent on endosomal TLR9 and ROS and at least partially depends on sTLR9. However, the therapeutic value of cfDNA-targeting molecules/enzymes and the functional role of neutrophil sTLR9 in inflammation should be validated in future clinical and experimental studies using animal CPB models.41. Additionally, the ICU scores SAPS II and TISS43 were calculated for each patient. Included patients were stratified according to the time on CPB (cut-off: 100\u2009min)44. In addition, fifteen patients undergoing off-pump coronary artery bypass (OPCAB) surgery were included (#13\u2013230).This prospective pilot study was approved by the Ethics Committee of the Medical Faculty of the University of Cologne (#15\u2013393). Written informed consent was obtained from all participants at the time of admission. Forty-eight patients undergoing any kind of on-pump cardiac surgery between April 2016 and March 2017, aged 18 years or older were enrolled in this study. Exclusion criteria were withdrawal of patients\u2019 consent, infectious diseases and malignancy. In addition, patients with known preexisting acute or chronic immunological disorders or systemic immunosuppression were excluded from the study. The preoperative risk for operative mortality was evaluated by means of the additive and logistic EuroSCOREFourteen healthy volunteer blood donors  aged between 21 and 60 years served as controls. Exclusion criteria were age under 18 years, recent or ongoing infection, cardiovascular disorders, immunosuppressive therapy or pregnancy.g) and stored at \u221280\u2009\u00b0C until further processing.Blood samples were collected into tubes containing lithium heparin  at the day of admission, immediately after surgery as well as postoperatively at day 1, day 3, day 5 and day 8. In some cases, no blood samples were collected from patients or patients were released from the hospital before day 8 after surgery, respectively. Blood samples were immediately used for neutrophil isolation and subsequent analyses. Plasma samples were harvested by centrifugation  at 4\u2009\u00b0C for 10\u2009min. The purity and viability of neutrophils were >95% as examined by flow cytometry analysis and trypan blue exclusion, respectively.Human neutrophils were separated from whole blood by discontinuous density-gradient centrifugation on Percoll (Millipore)6/ml) were pre-cultured with chloroquine , DPI , or ODN TTAGGG  in medium supplemented with 2% FCS for 30\u2009min. Then, 20% of pooled plasma from patients, volunteers, or FCS was added. After 3.5\u2009hours, cell culture supernatants were harvested and stored at \u221280\u2009\u00b0C.For NETs induction, human neutrophils , permeabilized in methanol and stained with PE-labelled rat anti-human TLR9 antibody  in 0.1% Triton X-100 and 3% FCS in cell wash buffer (BD) for 30\u2009min in the dark. After washing two times with 0.1% Triton X-100 and 3% FCS in cell wash buffer (BD), cells were analyzed by flow cytometry using a MACSQuant Analyzer (Miltenyi Biotec) or FACSCalibur (BD). For this, neutrophils were gated using forward and side scatter. For surface TLR9 staining, non-permeabilized cells were incubated with the same antibody in cell wash buffer with 3% FCS for 30\u2009min on ice. Cells were washed twice and immediately analyzed by flow cytometry.To assess TLR9 protein expression in neutrophils, 1\u2009\u00d7\u20091046. For the quantification of NETs in cultured neutrophils, cells were suspended and centrifuged at 300\u2009\u00d7\u2009g for 5\u2009min at 4\u2009\u00b0C. Supernatants containing NETs were collected and quantified by Pico Green staining. The fluorescence intensity reflects the amounts of DNA and was measured at excitation and emission wavelengths of 485\u2009nm and 530\u2009nm, respectively in a microplate reader . A standard calibration curve by means of defined calf thymus DNA (Sigma Aldrich) amounts ranging from 0 to 2\u2009\u00b5g/ml has been used in all analyses.Plasma levels of cfDNA were quantified by Quant-iT Pico Green dsDNA assay by following the manufacturer\u2019s instructions DNase I activity in patients\u2019 plasma was measured by the method previously described with some modifications47. Briefly, plasma samples were diluted 1:10 with PBS\u2009+\u200910\u2009mM MgSO4 supplemented with 1\u2009\u00b5g/ml of calf thymus DNA (Sigma-Aldrich) and stained with Pico Green (Invitrogen). To allow digestion of DNA by plasma DNase I, samples were incubated for 2\u2009h at 37\u2009\u00b0C. The intensity of Pico Green fluorescence was measured at time points 0\u2009h and 2\u2009h using a microplate reader . Relative DNase I activity was expressed as the ratio of relative fluorescence units measured at time point 0\u2009h and 2\u2009h, whereby high values may reflect high DNase I activity.Plasma IL-6 and ICAM-1 were determined by using the Human IL-6 DuoSet Elisa  and the Human ICAM-1/CD54 DuoSet Elisa  according to the manufacturer\u2019s instructions. For the quantification of soluble thrombomodulin (sCD141) levels, the CD141/Thrombomodulin Elisa  has been used.2O.200\u2009\u00b5l of plasma in a total volume of 500\u2009\u00b5l were mixed with 5\u2009\u00b5l Triton X-100 and heat denaturated at 98\u2009\u00b0C for 5\u2009min. Samples were placed on ice for 5\u2009min and DNA was extracted with an equal volume of phenol chloroform isoamyl alcohol  and centrifuged for 10\u2009min at 4\u2009\u00b0C. The aqueous phase was precipitated overnight with 1/10 volume of 3\u2009M NaOAc and 2.5 volume 100% ethanol at \u221220\u2009\u00b0C. The DNA pellet was washed twice with ethanol, air-dried and resuspended in 20\u201330\u2009\u00b5l of ddH48. In brief, heparinase I (50\u2009U) was dissolved in 50\u2009mM Tris (pH 7.5) and 1\u2009mM CaCl2, and stored at \u221220\u2009\u00b0C. 5\u2009\u00b5l of freshly isolated DNA was added to 19\u2009\u00b5l of heparinase I and samples were incubated for 2\u2009hours at 25\u2009\u00b0C. 5\u2009\u00b5l of the treated DNA solution was used as template in subsequent real time PCR reactions.Digestion of plasma samples using heparinase I (Sigma Aldrich) was performed as previously described49. PCR was performed in 12.5\u2009\u00b5l of total reaction volume containing 5\u2009\u00b5l treated DNA (10\u2009ng-20\u2009ng), 6.25\u2009\u00b5l 2\u2009\u00d7\u2009SYBR Green PCR master mix (Applied Biosystems) and 800\u2009nM forward and reverse primers. The following thermal cycling conditions were used: denaturation step at 95\u2009\u00b0C for 10\u2009min, 40 cycles of 60\u2009sec at 60\u2009\u00b0C and 15\u2009sec at 95\u2009\u00b0C. All samples were analyzed in duplicate. The average threshold cycle number (Ct) values were determined in the same quantitative PCR run. The threshold was set in the same level for each run. The level of mtDNA was calculated relative to GAPDH as followed: relative copy number 2\u2206Ct, whereby \u2206Ct\u2009=\u2009Ctnuc \u2212 Ctmit.Relative mitochondrial DNA (mtDNA) copy numbers were quantified by Real time PCR and normalized by simultaneous detection of nuclear DNA (nucDNA). The ABI StepOne Plus Real-Time PCR System (Applied Biosystems) was used to amplify GAPDH, the nuclear gene, and the mtDNA-encoded ATPase 8 gene by using previously published primer sequences6) were seeded on poly-D-lysine coated coverslips and incubated in the presence of 10\u2009\u00b5M DPI or 10\u2009\u00b5g/ml chloroquine for 30\u2009min. Then, cells were stimulated with serum or phorbol 12-myristate 13-acetate  for 3.5\u2009hours. After that, samples were fixed with 4% PFA, blocked and incubated with a polyclonal rabbit anti-myeloperoxidase antibody  and a secondary goat anti-rabbit Alexa Fluor 488-conjugated antibody . To demonstrate binding of DNA to neutrophils\u2019 surface, PMN were incubated with FITC-conjugated CpG ODN 2006 . Plasma membrane was stained with Rhodamine Wheat Germ Agglutinin . For colocalization studies of DNA and surface TLR9, neutrophils were fixed and incubated with PE-conjugated anti-TLR9 antibody . For intracellular TLR9 staining, cells were permeabilized with PBS\u2009+\u20091% FCS\u2009+\u20090.3% TritonX-100 prior incubation with anti-TLR9 antibody. Isotype control was used as control. All cells were counterstained with DAPI  and specimens were mounted in Dako fluorescent mounting medium (Dako). Cells were finally visualized by immunofluorescence microscopy  using the NIS Elements BR 3.10 software package. Neutrophil sTLR9 surface expression and colocalization with FITC-CpG-ODN 2006 were confirmed by confocal microscopy using the Zeiss LSM 710 Inverted Meta confocal microscope (Carl Zeiss).For NETs staining, freshly isolated neutrophils . The difference among non-normally distributed data was determined by the Kruskal-Wallis approach with Dunn\u2019s post hoc. Unpaired data of two groups were analyzed using the Mann-Whitney test. Spearman correlation coefficient (r) was used to determine the presence or absence of a correlation between variables. Normally distributed unpaired data of multiple groups were analyzed with ANOVA and Newman Keuls post-hoc test. P-value less than 0.05 was considered as statistically significant.Data were analyzed with GraphPad Prism 5 software. Patient data are presented as box plots representing the median (heavy line in boxes) and the 25All data generated or analyzed in this study are available from the corresponding author on reasonable request.All experiments were performed in accordance with relevant guidelines and regulations.Supplementary Information"}
+{"text": "Eventually, a three-dimensional (3D) FE model in small-hole drilling of Al6063/SiCp composites, using multi-objective identified constitutive formalism, is developed. Comparison with the experimental observations in thrust force, torque, and chip morphology provides valid evidence on the applicability of the developed multi-objective identification strategy in identifying constitutive parameters.Discrepancies in capturing material behavior of some materials, such as Particulate Reinforced Metal Matrix Composites, by using conventional ad hoc strategy make the applicability of Johnson-Cook constitutive model challenged. Despites applicable efforts, its extended formalism with more fitting parameters would increase the difficulty in identifying constitutive parameters. A weighted multi-objective strategy for identifying any constitutive formalism is developed to predict mechanical behavior in static and dynamic loading conditions equally well. These varying weighting is based on the Gaussian-distributed noise evaluation of experimentally obtained stress-strain data in quasi-static or dynamic mode. This universal method can be used to determine fast and directly whether the constitutive formalism is suitable to describe the material constitutive behavior by measuring goodness-of-fit. A quantitative comparison of different fitting strategies on identifying Al6063/SiC Particulate Reinforced Metal Matrix Composites (PRMMCs) present low-temp properties, high strength-to-weight ratio, good wear resistance, and low thermal expansion coefficient, but poor machinability ,2,3. DueMost engineering materials exhibit varying deformation behavior at low (quasi-static) and high (dynamic) deformation rates or temperatures. This brings great challenges to the establishment and identification of the material constitutive model that can describe the mechanical response under different loading conditions. The Johnson-Cook (J-C) constitutive model is one of the most commonly used semi-empirical phenomenological ones for describing the plastic deformation behaviors at high strain, high strain rate, and high temperate, especially suitable for the simulation of machining processes . Its disIn the J-C constitutive equation, the flow stress and Cook .(1)\u03c3= are investigated to focus on the mechanical responses in quasi-static compression and SHPB tests. p/65p composites, with uniformly distributed SiC particles in the absence of obvious particulate clustering and preferred orientation. At low strain rates, the temperature rise caused by plastic strain at room temperature is considered to be negligible, while at high strain rates, there exists coupling between strain rate and deformation temperature since a great amount of heat is not timely transferred. To deduce the uncoupled relationship between temperature and strain rate, the isothermal mechanical behavior at elevated temperature is to be considered in the dynamic tests at high strain rates [Al6063 matrix composites reinforced with 65% volume fraction of SiC particulate . The specimens were machined into the cylinders of \u03a66 mm given in . In SHPBp/65p composites in quasi-static and dynamic SHPB compression tests, respectively. It is to note that in quasi-static mode, Al6063/SiCp/65p composites show rate dependence to stress response, which differs from pure Al6063 alloy with no rate-dependent mechanical behavior in quasi-static compression [\u22124 to 10\u22122 is not significant, so the reference strain rate in fitting constitutive model for Al6063/SiCp/65p composites is identified as 10\u22122. As illustrated in p/65p composites is shown to be of significant rate- and temperature-dependence. The higher the deformation rate is, the greater the data fluctuation of the measured stress-strain curves is; the higher the deformation temperature is, the less the fluctuation is. Therefore, the measurement noise at high strain rates and low temperatures become more considerable in dynamic mode, which influences the reliability and accuracy of fitted data in constitutive identification.pression . This ispression . The str\u03c3(\u03b5p = 0.002) is within a range of 10%, The J-C model in Equation (1) involves a set of five parameters  and (11), an overall objective function is represented as additive relationship with different weighting factors by Equation (12).The fluctuation in some flow stress curves corresponding to high strain-rate loading conditions observed in Since the measure error covariance Similarly,So, based on the definition of weighting matrix, this seemingly bi-objective optimization actually in practice is a multi-objective minimization problem relying on the number of loading condition combinations. Thus, the following aim is to minimize the bi-objective reduced Chi-squared error summation for the flow stress data experimentally determined from different loading conditions. An updated Levenberg-Marquardt formula based on the gradient scaling is employed in solving Equation (12) .(19)[JTInitialization and update of damping factor p/65p material model identification are summarized in R2). This indicates that it is not always necessary to modify such a classic constitutive model under scientific hypothesis that the parameter coefficient A is a variable to be optimized rather than offset yield point. Besides, the overall fit standard error of 21.88 MPa indicates the model fitting errors are of the same order as the measured ones. That is to say, the multi-objective optimization model can be relatively capable of fitting experimental noise.Based on the relationship of \u22121 and 2200 s\u22121 is mainly attributed to the experimental measurement resulting from poor signal-to-noise ratio, which easily occurs in the SHPB tests for particulate reinforced metal matrix composites. Hence, the important significance of introducing the varying weighting factors under different loading conditions into formulating multi-objective function is manifested in this aspect. When compared to the ad hoc strategy, the multi-objective optimization one enables the identified J-C model to fit the observations under high temperatures and high strain rates more accurately, in An accurate and reliable set of J-C material parameters under various combination of strain rates and temperatures could give rise to similar chip morphologies and cutting forces to experimental observations in cutting ,29,30. Tp/65p composites is given in The J-C material model for Al6063/SiCIn terms of macro- and microscopic strain-stress relationships under homogeneous strain boundary conditions shown in fined by .(22)\u03b5\u203e=p/65p composites are roughly considered macroscopically isotropic. The plastic strain part on both sides for Equation (22) should be equal, and SiC particulate is elastically deformed, and therefore the failure criterion for Al6063 matrix composites can be approximately deduced using Equations (21) and (22) asIn macro scale, due to the polycrystalline aggregates characteristic and randomly oriented distribution of SiC particle in as-cast Al6063 composites, the mechanical responses of Al6063/SiCEquation (23) is normalized to obtain the failure parameters.p/65p composites are deduced by Equation (25), as shown in The Johnson-Cook damage parameters given in . The JohThe shear criterion is a phenomenological one for describing shear band localization, with the following form (26)\u03b5pS, haveWhen the damage factor Provided that material damage is initiated, the stress-strain law originally based on J-C material model no longer describes the material deformation behavior accurately . To avoiThe overall damage variable A linear softening law of flow stress with equivalent plastic displacement After damage initiation, the effective flow stress Therefore, when the value of D reaches 1, the material failure occurs, accompanied by element deletion.p/65p composites involving \u03b1-SiC hard phase and Al6063 matrix, the specific heat capacity can be estimated according to the rule of mixtures by Equation (35).p/65p composites for material modeling are listed in For Al6063/SiCp/65p composites. For workpiece modelling, the workpiece part neighbored to the tool tip are accounted for in drilling model, and a cone-like concave machined surface is prefabricated on the workpiece surface, such that stable drilling can be arrived at as soon as possible, as shown in A three-dimensional (3D) drilling model based on FE-based appoach has been built for simulating small-hole manufacturing process of Al6063/SiCIt is worth noting that at least five-layer locally meshed elements along the feed direction are allocated for feed per revolution, only by such meshing strategy can the chip be formed, as shown in The stick/slip contact model across tool-chip interface is adopted, as suggested by Zorev , for toop/65p composites based on the constitutive formalism determined using multi-objective optimization strategy. The thrust force is the applied resultant force on cutting tool along the z direction.The cutting forces and chip morphology were obtained numerically by running FE model of drilling Al6063/SiCp/65p composites, the drilling experiments of Al6063/SiCp/65p composites were performed on a CNC machining center DMU 80 monoBLOCK , as shown in To validate the built constitutive equation and drilling model for Al6063/SiCDue to semi-enclosed characteristics and the deformed complexity of drilling operation, the contact between the newly-formed chips by element deletion and machined surface in 3D drilling is difficult to define, so the chips flow away from the workpiece side wall, and do not impede the tool operation. This is in contradiction to the fact that in realistic drilling the newly-formed chips is inconvenient to remove, thus increasing cutting forces. To better approximate the simulation conditions, a peck drilling operation (with 0.05 mm step size) that facilitates the chip removal is employed to reduce the interference from the chips.Based on special consideration of drilling modelling, only the force signals at stable cutting are extracted for comparison. To eliminate the interference when the forcing frequencies approach natural frequencies of piezoelectric dynamometer a, the TyBased on our drilling modelling strategy, the simulation results during stable cutting is take into account for comparison with the experimental data. When compared to the experimental observations in The mechanical behaviors of the workpiece play a significant role in mechanics of chip formation and evolution where the workpiece material is subjected to plastic deformation and shear along the primary shear zone by dint of cutting tool . The detp/65p composites in quasi-static compression and SHPB tests are investigated to obtain the stress-strain responses for identifying J-C constitutive constants. Aimed at hard identification of material parameters using conventional ad hoc method, a weighted multi-objective optimization strategy for identifying J-C constitutive parameters is proposed, coupled with updated Levenberg-Marquardt implementation algorithm. When compared to ad hoc optimization strategy, multi-objective strategy applied for J-C parameters optimization leads to a higher predictability of experimental observations and better capability of fitting experimental noise. It is found that it is not always necessary to modify such a classic J-C constitutive model under scientific hypothesis that parameter the coefficient A is a variable to be optimized rather than offset yield point.In this paper, the mechanical behaviors of Al6063/SiCp/65p composites was implemented, based on the constitutive model determined using weighted multi-objective strategy and failure criteria in a maximum sense. The maximum prediction error of no more than 11.00% for thrust forces indicates the reliability and accuracy of the proposed drilling model. It can be found from numerical and experimental results that there are the better machining conditions at rotational speed of 2000 rpm and feed of 75 mm/min. The developed FE model allows for presenting the thrust force dependence on the drilling conditions in the much more visual and usable form. Accurate prediction of thrust force is of great importance for investigating drilling induced edge defects at hole exit and entrance in PRMMCs or delamination defects in laminated composites. Hence, this proposed FE model can be used to optimize cutting processes to improve drilling performance and hole quality, based on the cutting forces effect on drilling induced defect. Accurate prediction of chip formation contributes to the disclosure of material removal mechanism. Comparative results of thrust force, torque, and chip morphology also verify the feasibility of the developed multi-objective constitutive identification strategy with varying weighted consideration to each combination of deformation rates and temperatures.A 3D FE simulation in small hole drilling of Al6063/SiC"}
+{"text": "Pterostilbene has been reported as a potential drug to inhibit oxidative stress and inflammation. However, the effect of pterostilbene on chondrocytes and osteoarthritis remains to be elucidated. We sought to investigate whether pterostilbene could protect chondrocytes from inflammation and ROS production through factor erythroid 2-related factor 2 (Nrf2) activation. The pterostilbene toxicity on chondrocytes collected from cartilages of Sprague-Dawley rats was assessed by CCK-8 test. Immunofluorescence and Western blotting explored the nuclear translocation of Nrf2. Nrf2 expression was silenced by siRNA to evaluate the involvement of Nrf2 in the effect of pterostilbene on chondrocytes. Finally, osteoarthritis model was established by the transection of anterior cruciate ligament and partial medial meniscectomy in rats, and then these rats received pterostilbene 30 mg/kg, daily, p.o. for 8 weeks. Histology and immunohistochemistry were used to assess histopathological change and Nrf2 expression in cartilage. Nuclear translocation of Nrf2 was stimulated by pterostilbene without cellular toxicity. Pterostilbene inhibited the level of COX-2, iNOS, PGE2, and NO, as well as the mitochondrial and total intracellular ROS production induced by IL-1\u03b2 in chondrocytes, partially reversed by the Nrf2 silencing. Pterostilbene prevented cartilage degeneration and promoted the nuclear translocation of Nrf2 in cartilage. These results suggest that pterostilbene could inhibit the IL-1\u03b2-induced inflammation and ROS production in chondrocytes by stimulating the nuclear translocation of Nrf2. Osteoarthritis (OA), a most common joint disease, causes the joint deformity and pain, contributing to work disability and sick leave . The eroSeveral compelling pieces of evidences have demonstrated the inflammation-associated changes in the OA cartilage , 4. InflThe main drugs against inflammation and pain in OA are the non-steroidal anti-inflammatory drugs (NSAIDs) with some side effects and high economic burden . Howeverin vivo. The OA model of SD rats were used in the present study to investigate the role of PTE on the cartilage degeneration and the nuclear translocation of Nrf2 in cartilages.Nuclear factor erythroid 2-related factor2 (Nrf2) is a basic leucine zipper transcription factor in an intracellular adaptive mechanism against the hostile stimulations of oxidative stress by managing the level of phase II detoxifying enzymes andproteins . Upon stP < 0.05). A significant difference was not observed between 0\u201340 \u03bcM PTE-treated and control chondrocytes. 20 \u03bcM PTE treatment for 0\u201336 hours did not decrease cell viability , suggesting that the PTE treatment for less than 48 h was not detrimental for rat chondrocytes. Therefore, 20 \u03bcM PTE treatment for 24 h was used in the following experiments.Cell viability after PTE treatment was first analyzed in our study. After 24 h treatment, only the chondrocytes treated with 60 \u03bcM and 80 \u03bcM PTE showed decreased viability . As anticipated, IL-1\u03b2 significantly increased the expression of COX-2 and iNOS mRNA observed by real-time PCR. IL-1\u03b2 significantly elevated the release of NO and PGE-2 investigated by nitrite assay and ELISA . However, PTE could partly attenuate the increase of these inflammatory mediators in the IL-1\u03b2-treated chondrocytes (P < 0.05), indicating the anti-inflammatory role of PTE. Compared to the cells treated with IL-1\u03b2 and PTE, Nrf2 inhibition could abolish the inhibitory effect of PTE on the expression of COX-2 and iNOS mRNA and the release of NO and PGE-2 in chondrocytes, suggesting that Nrf2 pathway might mediate the protective role of PTE on chondrocytes under inflammatory condition.RNAi against Nrf2 was utilized to analyze the involvement of Nrf2 in the effect of PTE on IL-1\u03b2-induced inflammation. Western blotting showed that the nuclear Nrf2 expression was inhibited significantly in both the control and PTE-treated chondrocytes after Nrf2 siRNA transfection. Conversely, Nrf2 silencing could reverse the inhibitory role of PTE on the generation of mitochondrial superoxide, indicating that Nrf2 was a potential mediator of the protective effect  staining for analyzing the production of mitochondrial superoxide and intracellular ROS. Unsurprisingly, IL-1\u03b2 markedly enhanced the red fluorescence density of MitoSOX-Red staining, which was partially attenuated by the pre-treatment of PTE, indicating the inhibitory effect of PTE on the generation of mitochondrial superoxide  and liver of SAMP8 mice with a systemic accumulation of oxidative stress [Although the mechanisms mediating the protective role of PTE included several signaling pathways such as the regulation of Sirt1, extracellular regulated kinase (ERK) and NF-\u0138B pathways , the acte stress , 31. In Under physiological conditions, Nrf2 binds to Kelch-like ECH-associated protein-1 (Keap1), therefore, the dissociation of Nrf2 from Keap1 is the first step of Nrf2 nuclear translocation . RamkumaIn conclusion, these results indicate that PTE stimulates the nuclear translocation of Nrf2 and inhibits inflammation and ROS production in the IL-1\u03b2-induced chondrocytes. The activation of Nrf2 pathway promotes the transcription of antioxidant genes and inhibits ROS production, as well as the inflammation. PTE could prevent the cartilage degeneration and activate Nrf2 in the OA cartilage.The experimental protocols for animals were approved by Wenzhou Medical University Animal Care and Use Committee (Ethics Reference NO. L-2016-07) .PTE, hematoxylin, eosin, safranin O, fast green, and type II collagenase were purchased from Sigma-Aldrich . The primary antibodies for Nrf2, \u03b2-actin, and Lamin B and ELISA kits for Prostaglandin E2 (PGE2) were acquired from Abcam . The secondary antibodies for immunohistochemistry and Western blotting, DAPI, cytoplasmic and nuclear protein extraction reagents, DCFDA assay kits were from Beyotime . The cell culture reagents including Dulbecco modified Eagle medium (DMEM), fetal bovine serum (FBS), and 0.25% trypsin were bought from Gibco . Cell Counting Kit-8 (CCK-8) was obtained from Dojindo . Real-time PCR reagents including TRIzol and SYBR Premix Ex Taq mixture were procured from Takara . The cDNA synthesis kit was purchased from MBI Fermentas . MitoSOX Red staining kit was obtained from Molecular Probes .2 at 37\u00b0C. In order to eliminate the influence of de-differentiation in chondrocytes, the second passage cells were used in the downstream experiments.The chondrocyte extraction and culture were carried out according to the previous reports . 5 male Primary rat chondrocytes were isolated six times, and the following experiments were repeated six times using the isolated chondrocytes each time. First, the cellular toxicity of PTE was examined by CCK-8 assay. Then the effect of PTE on Nrf2 activation was explored, followed by the investigation of its protective role against inflammatory and ROS production, as well as the involvement of Nrf2. 10 ng/mL IL-1\u03b2 was used to promote inflammation and ROS production in the chondrocytes. PTE were added to the medium 2 h prior to the IL-1\u03b2. RNAi was used for the silence of Nrf2. 48 h post transfection, the PTE and IL-1\u03b2 were added successively.3 chondrocytes in a 96-well plate. 10 \u03bcL reagent was then added to the medium for 1 h at 37\u00b0C. The absorbance was measured at 450 nm in the microplate reader .Different concentrations of PTE (0\u201380 \u03bcM) for 24 h and 20 \u03bcM PTE with 0\u201348 h treatments were used to treat the 2 \u00d7 104 chondrocytes cultured in 48-well plate were treated with 10 and 20 \u03bcM PTE for 24 h. The chondrocytes were fixed with 4% paraformaldehyde for 10 min at 4\u00b0C. Subsequently, the cellular membrane was permeabilized with 0.2%Triton X-100 for 15 min and blocked with 5% BSA for 30 min. The chondrocytes were incubated with the Nrf2 primary antibody (1:100) at 4\u00b0C for 12\u201316 h, followed by incubation with FITC conjugated secondary antibodies. The nuclei were counterstained with DAPI. Finally, the chondrocytes were observed under a confocal microscope .2 \u00d7 10The nuclear and cytoplasmic protein from the chondrocytes was isolated as recommended by the manufacturer using the extraction kits . The nuclear and cytoplasmic protein concentration was determined using an Enhanced BCA Protein Assay Kit. The total protein was resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto the polyvinylidene difluoride (PVDF) membrane (BIO-RAD USA). After blocking with 5% nonfat milk for 2 h, the membrane was incubated with the Nrf2 primary antibody (1:1000) and HRP-conjugated secondary antibodies. ECL Plus reagent was used to expose the membrane in an enhanced chemiluminescence detection system . Semi-quantitative analysis of band intensity was analyzed byAlphaEaseFC 4.0 software.5 cells/well). siRNA was delivered into chondrocytes using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. 48 h post-transfection, the infected cells were treated as described in the experimental design. The non-specific non-targeting siRNA (scrambled) was used as a control. Western blotting consequently examined the inhibitory efficiency of specific silencing using the nuclear extracts.The sequences of small interfering RNAs (siRNA), shown in Table \u2212\u0394\u0394Ct method.After treatments as the experimental design described, the total RNA of chondrocytes was extracted using TRIzol reagent, followed by the synthesis of cNDA using 1\u03bcg total RNA. The RNA was quantified with a spectrophotometer  by controlling the range of ratio OD 260/280 between 1.8 and 2.0. The RT-PCR 20 \u03bcL reaction included 2 \u03bcL of 2-fold diluted cDNA, 10 \u03bcL of 2 \u00d7 SYBR Premix Ex Taq mixture, 0.2 \u03bcmol/L of each primer and sterile distilled water. The 8-strip PCR tubes and placed in a LightCycler  for detection. The thermocycler parameters were 50\u00b0C for 2 min, 95\u00b0C for 30s, 40 cycles at 95\u00b0C for 5 s, and 60\u00b0C for 34 s . DNA conThe nitrite level in the supernatant of the medium was assessed by the classic Griess method . After tAfter treatment with PTE and IL-1\u03b2 for 24 h, the culture supernatants were obtained. The concentration of PGE-2 was measured by ELISA according to the manufacturer's instructions. The sensitivity of PGE-2 ELISA assay was 13.4pg/mL.2-). First, 50\u03bcg MitoSOX dye dissolved in 13 \u03bcL DMSO was added into a serum-free medium for a 5 mM stock concentration. 5 \u00d7 104 chondrocytes cultured in glass dishes were incubated with the 1 mL 5 \u03bcM working solution for 15 min at 37\u00b0C, followed by DAPI staining for 5 min at 37\u00b0C. After washing with PBS three times, the cells cultured in fresh DMEM were observed under the confocal microscope .MitoSOX Red is a novel dye targeting the mitochondria and emitting red fluorescence when oxidized by the mitochondrial Superoxide Anion  rat model was established as previously described . BrieflyThe whole knee joints of each rat were fixed in 10% neutral formalin and decalcified with10% EDTA for 2 months, and then paraffin-embedded for midsagittal serial sectioning. The sections were HE stained to assess the destruction of cartilage. In order to investigate the matrix proteoglycans expression, Safranin O/Fast Green staining was performed on the sections. Cartilage degradation was quantified using the Osteoarthritis Research Society International (OARSI) cartilage grading system . Three i2O2 for 10 min. The antigen was retrieved by the conventional heat method, followed by blocking with 5% BSA and permeabilization with 1% Tween-20 in PBS for 30 min. The sections were incubated with the primary antibodies against Nrf2 (1:200) and PBS as negative controls for 12\u201316 h at 4\u00b0C, followed by HRP-conjugated secondary antibodies and counterstained with hematoxylin. Finally, the sections were microscopically examined. To assure the accuracy, three sections from the 6 knee joints were utilized to observe the expression of Nrf2 in the articular cartilage. In order to analyze the percentage of cells with positive Nrf2 in nuclei, three pictures were taken under 40\u00d7 magnification in cartilage sections from knees joints, representing the centre, medial and lateral tibial surface. The number of cells with brown staining in the nuclei and the total cell number in each section was counted.The above sections were used to perform the immunohistochemistry. After deparaffinization and graded rehydration, the endogenous peroxidase activity was blocked with 3% HP < 0.05 was statistically significant.The differences between the groups were evaluated by ANOVA using the SPSS15 package . If the result of ANOVA had significance, the Bonferroni test was used to explore the differences between the two groups."}
+{"text": "Distal cholangiocarcinoma (DCC) is a rare but over the last decade increasing malignancy and is associated with poor prognosis. According to the present knowledge curative surgery is the only chance for long term survival. This study was performed to evaluate prognostic factors for the outcome of patients undergoing curative surgery for distal cholangiocarcinoma.75 patients who underwent surgery between January 2000 and December 2014 for DCC in curative intention were analysed retrospectively. Potential prognostic factors for survival were investigated including the extent of surgery using purposeful selection of covariates in multivariable Cox regression modeling.p\u2009=\u20090.020), the extent of surgery in case of positive histological venous invasion , lymph node staging , perineural invasion  and postoperative complications graded in points according to Clavien-Dindo  were indentified as independent significant risk factors for survival. Patients receiving preoperative biliary stenting showed prolonged duration between onset of symptoms and date of operation (p\u2009=\u20090.048).Preoperative biliary stenting (Hazard ratio (HR): 2.530; 95%-CI: 1.146\u20136.464, Preoperative biliary stenting reduces survival possibly due to delayed surgery. The extent of surgery is not an independent risk factor for survival except for patients with concomitant histological venous invasion. Oncological factors and postoperative surgical complications are independent prognostic factors for survival. Cholangiocarcinoma is a malignant disease with poor prognosis and occurs with an incidence of 1.3 to 3.4 cases per 100,000 in Western countries [The primary location of the carcinoma in reference to the biliary tract influences clinical manifestation and therapy. Therefore they are classified into intrahepatic, proximal extrahepatic and distal extrahepatic cholangiocarcinomas (DCC) .DCC constitute up to 20\u201330% of all cholangiocarcinomas and are located between the cystic duct entry into the main hepatic duct and the Ampulla of Vater. Clinical symptoms comprise painless jaundice and/or weight loss [Curative surgery is the only chance for long-term survival \u20139. PalliApart from negative lymph node status, a tumor-free resection margin has been reported to have crucial impact on disease free survival , 11, 12.Most published data describe inhomogeneous patient groups with respect to the primary location of biliary tract carcinoma \u20139. The oThis is a retrospective analysis of 75 patients with DCC undergoing surgical resection at the Department of General, Visceral and Transplant Surgery, Hannover Medical School, Germany between January 2000 and December 2014. Median overall survival follow-up was 19\u00a0months (range: 0\u2013178\u00a0months). The clinical and histopathological variables of the study cohort are summarized in Table\u00a0n\u2009=\u200975) in patients older than 18\u00a0years of age. No exclusion criteria were defined.Included were all resections for histologically confirmed DCC , partial pancreaticoduodenectomy or pancreatic resection with additional partial resection of perihilar bile ducts, portal vein or liver. Oncological lymphadenectomy along the hepatic artery and down to the celiac trunk was performed in all patients receiving pancreatic resections. The extent of surgery was then graded in points as displayed in Table DCC was histopathologically confirmed by paraffin embeddeded and haematoxylin and eosin (HE), Periodic acid\u2013Schiff (PAS) and elastic Van Gieson\u2019s (EVG) stained slides. In some cases additional immunohistochemical staining for IgG4 was performed. Tumors were classified according to the classification system proposed by the International Union Against Cancer .The primary study endpoints were overall survival (OS) and postoperative complications graded according to the Clavien-Dindo Classification of Surgical Complications . SecondaThe influence of nominal and ordinal variables on binary study endpoints were analysed with chi-squared test and Fisher\u2019s exact test while the influence of continuous variables on these endpoints was analysed with univariable logistic regression. Median and mean values between groups were compared with the Mann-Whitney U test. Ordinal regression and Kaplan-Meier analyses with the Log-rank test were performed where appropriate.Risk factors for patient survival were initially analysed with univariable Cox regression analysis.Identification of independent risk factors influencing overall patient survival was achieved by developing a multivariable Cox regression model including potential multiplicative or additional factor interactions based on backwards likelihood elimination followed by forward likelihood inclusion of variables as has been proposed by Hosmer et al. and has been applied and published recently by our workgroup , 19.The collected data was implemented and analysed using SPSS statistical software  and JMP statistical software .n\u2009=\u200962) of the patients were male (Table The median age of the study population at time of the operation was 67\u00a0years (33\u201387\u00a0years) and 82.7%  received by 88.0% of the patients, ultrasound (78.7%) and computed tomography (CT) (74.7%) or magnetic resonance imaging (17.3%). Location of the tumor was primarily defined by ERCP. The extent of tumor was anticipated mainly according to CT, however CT scans could not identify any tumor mass in 22 patients. Five of these patients underwent additional MR tomography for verification of tumor extent with successful detection of the lesions in two patients.Biopsies were performed in 33 patients (44.0%) whereas brush cytologies were performed in 39 patients (52.0%). Hereby the diagnosis was verified in 49.3% of the patients.n\u2009=\u200958). Percutaneous Transhepatic Cholangio-Drainage (PTCD) was not required in our patient collective. Patients receiving biliary stenting prior to operation showed significantly prolonged duration between onset of symptoms and operation (p\u2009=\u20090.048). Preoperative biliary stenting was found to be an independent risk factor for worse overall survival in multivariable Cox regression analysis   as a consequence of relevant cholestasis was performed in 77.3% of the patients (0) Table\u00a0.Table 2SThe median time interval between onset of symptoms and operation was 34\u00a0days Table .In ten patients local excisions of the extrahepatic bile ducts were performed which included lymphadenectomy of the hepato-duodenal ligament representing a collective undergoing rather minor extent of surgery.26 patients underwent pylorus-preserving partial pancreaticoduodenectomy and 28 patients underwent classical partial pancreaticoduodenectomy without preservation of the pylorus.Six patients underwent additional partial portal vein resection and three patients underwent additional resection of proximal/central extrahepatic bile ducts a in addition to pancreaticoduodenectomy. One patient underwent right trisectorectomy of the liver  and perihilar bile duct resection in addition to classical partial pancreaticoduodenectomy.p\u2009=\u20090.032) .Since resection of the perihilar bile duct bifurcation, due to proximal tumor infiltration of the resection margin, was performed in seven patients we separately analysed postoperative outcome of these cases. Tumor-free resection margins were diagnosed in final histology in four of these seven patients and none developed anastomotic leakage. Median overall survival (28.0\u00a0months) was longer as compared to all other patients (16.5), however Cox regression analysis revealed no significant influence on survival (p\u2009=\u20090.129).Segmental portal vein resection due to tumor infiltration, representing a rather extended approach of surgical resection, was performed in six patients in total (8%). Vein reconstruction was achieved by primary suture in five and by insertion of a ringed polytetrafluoroethylene (PTFE) graft in one case. Portal vein thrombosis occurred in one patient and was treated by thrombectomy. Following additional portal vein resection all patients died during follow-up with a lower median overall survival (13.5 versus 21.0\u00a0months). Accordingly, Cox regression analysis revealed a negative impact of portal vein infiltration, although not statistically significant (Negative resection margins (R0) in final histology were achieved in 86.7% of the patients. Residual tumor did not significantly influence overall survival in uni- or multivariable analysis as displayed in Table Initial intraoperative resection margins revealed residual tumor in 24 patients (32.0%). In eleven of these patients tumor free resection margins could be achieved by further local resection of adjacent tissue and in three patients by an extension of the surgical approach, i.e. pancreatic and hepatic resection respectively.In the remaining patients further resection was not possible due to infiltration of superior mesenteric artery and/or due to the patients reduced general health condition respectively.Seven of the ten patients undergoing local excision of the bile duct showed residual tumor in the initial intraoperative resection margins. Residual tumor was localized at the distal resection margin in six patients with simultaneous infiltration of the proximal resection margin in one patient. In one patient residual tumor was detected at the proximal resection margin. Further local resection ultimately led to tumor free resection margins in all but three patients.p\u2009=\u20090.092, p\u2009=\u20090.903 respectively).Patients with tumor free resection margins in the initial intraoperative frozen sections did not show significantly better outcome regarding recurrence or survival when compared to patients undergoing further local or extended resection in order to achieve tumor free resection margins in final histopathological analysis . However, delayed surgery did not significantly influence tumor stage according to UICC  or the success of the operation defined as tumor-free resection margins (Odds ratio (OR): 0.995; 0.974\u20131.017; p\u2009=\u20090.643, logistic regression).Low T-staging depended significantly on a short time period between first symptoms and operation (p\u2009=\u20090.020) and multivariable analysis  , detected in 41 patients (54.7%), was a significant risk factor for worse overall survival in univariable (06) Fig. .p\u2009=\u20090.019) and multivariable analysis  identified perineural invasion as significant risk factor for overall survival  histopathological analysis revealed perineural invasion. Univariable   with significant influence on worse overall survival in univariable analysis (48) Fig. . Further1.6% withMedian postoperative hospital stay was 25\u00a0days including a median of 3\u00a0days in the ICU. Extended surgery did not significantly prolong postoperative hospital stay nor time spent in the ICU.p\u2009=\u20090.020) and multivariable analysis  identified the grade of postoperative complications as significant risk factor for worse overall survival  had complications with varying degrees of severity graded according to Clavien-Dindo Tables  and 3. Uval Fig. .Table 3CPatients undergoing different extents of surgery as stated above did not experience statistically significant differences regarding rate and severity of postoperative complications.The most common complications were wound infections with a rate of 21.3%.Type A pancreatic fistulas were not documented due to missing data on enzyme measurements from abdominal drains. Type B pancreatic fistulas were observed in ten patients (13.3%). Type C pancreatic fistulas were diagnosed in seven patients (9.3%) and total pancreatectomy was performed in six individuals (8.0%).Eight patients (10.7%) suffered from insufficiency of the biliodigestive anastomosis.n\u2009=\u20095). Causes of death were cardiac arrest due to cardiac infarction in one patient, sepsis and organ failure after total pancreatectomy due to insufficiency of the pancreatic anastomosis and arterial bleeding in two patients, and organ failure due to severe pneumonia in two further patients.Early mortality, defined as death in the first 30 post-operative days was 6.7% (n\u2009=\u20095), to lymph node, liver, bone and peritoneal metastases (n\u2009=\u200916). Three of eleven patients after extend pancreatic surgery suffered from local recurrence whereas two patients developed distant metastases in liver and peritoneum.After local excision of the extrahepatic bile duct three of ten patients suffered from local tumor recurrence while another patient developed liver metastasis in the observation period. Patients undergoing standard pancreatic resection developed tumor recurrence in 21 documented cases ranging from local recurrence . Fifty-two (72.0%), 18 (30.0%) and 11 (22.0%) patients survived more than 1, 3 and 5\u00a0years, respectively. Thirteen patients (17.3%) were still alive at the time of data analysis.Due to the aforementioned comparatively low incidence of DCC among the general population, especially from Western countries, reports on outcome after surgery, of large patient collectives, are scarce. This data from a single high-volume center was analysed to identify prognostic factors for unfavourable patient outcome.Interestingly no published data investigated preoperative stenting in patients with DCC and its impact on survival yet. In multivariable analysis we identified preoperative stenting as strongest risk factor for survival. A significantly longer duration between first symptoms and surgery in these patients could provide a clinically plausible explanation for the inferior outcome Table . A prospSurvival after surgical treatment in the current study was similar to several previous studies \u201328.Reported outcomes of patients with DCC after extended pancreatic surgery are extremely scarce. Solely Courtin-Tanguy et al. investigated extended surgery for treatment of DCC and found that combined organ resection was an independent risk factor for worse survival . InteresPartial portal vein resection due to macroscopic tumor invasion had no statistically significant influence on survival in Cox regression analysis Table  in this Intraoperative frozen sections were crucial for flexible intraoperative decision making on the extent of resection, achieving tumor-free resection margins and partially led to an extension of the surgical strategy including partial portal vein, additional liver or perhilar bile duct resection. This is emphasized by the fact that the preoperative CT scans in the displayed cohort were not a reliant predictor of tumor extent. CT scans of 22 patients identified no measurable tumor mass although final histology revealed tumor sizes between T stage 1 and 4 in these patients. Even additional MR tomography did not provide significantly better results in preoperative detection of tumor extent. Therefore, in some cases endosonography might help for identification of tumor extent.Finally patients with stricture of the distal bile duct suspicious of a malignant tumor should be explored surgically even if histology was negative, since 49.3% of preoperative histologies were false negative.The impact of positive tumor infiltration at the surgical resection margin (R1/2 resection) on postoperative survival following curative resection of DCCs is widely discussed. We found no significant impact of R1/2 resection on overall survival in Cox regression analysis (Table The presence of tumor positive lymph nodes was identified as a significant prognostic factor in multivariable analysis Table  as was rPostoperative complications graded in points according to Clavien-Dindo had a significant impact on patient outcome Table . The infPreoperative biliary stenting, positive lymph node staging, perineural invasion as well as extended surgery in patients with concomitant venous invasion and postoperative complications should be considered within the perioperative therapeutic strategy. It is important to state that it is difficult to draw definite conclusions from this retrospective study considering the small patient collective undergoing extended surgery, respectively. Therefore it would certainly be beneficial performing multi-center studies with prospective analysis to determine which surgical approach is justifiable regarding optimal patient outcome. In addition the effects of preoperative biliary drainage in patients with DCC and relevant cholestasis on survival should be thoroughly examined in respect of our presented data."}
+{"text": "We previously reported that lower systemic antioxidant capacity is involved in primary open-angle glaucoma (POAG) and exfoliation syndrome pathogeneses as measured by ferric-reducing activity. In the present study, we measured hydroxylinoleate (HODE) and hydroxyarachidonate (HETE) isomer serum levels after sample reduction and saponification to investigate POAG pathogenesis. POAG patients (n\u2009=\u2009198) were recruited and divided into normal- and high-tension glaucoma groups  depending on intraocular pressure. Total HODE (/linoleic acid) and HETE (/arachidonic acid) serum levels were significantly higher in the POAG group  than in controls . The associations between HODEs/HETEs and glaucoma were further confirmed by multivariate analyses after adjusting for differences in demographic parameters. Among the HODE isomers, the levels of 9- and 13--HODEs (p\u2009=\u20090.0014) and singlet oxygen-specific products -HODEs, p\u2009=\u20090.0345) were higher in the POAG group than in controls, while free radical-mediated oxidation-specific products -HODEs, p\u2009=\u20090.0557) demonstrated a marginal difference. Enzymatic and singlet oxygen-mediated fatty acid oxidation may be major pathways of oxidation process in glaucoma subjects. Furthermore, they can provide information on the effectiveness of functional factors in foods for the prevention of diseases. The advantage of measuring oxidation products of linoleates is that they are the most abundant polyunsaturated fatty acids in vivo and their oxidation proceeds by a straightforward mechanism that yields much simpler products than arachidonates and more highly unsaturated fatty acids such as docosahexaenoates5. Hydroperoxy octadecadienoates (HPODEs), derived from linoleates, are primary products obtained by simple mechanisms from abundant parent lipids in vivo. The absolute concentrations of lipid hydroperoxides in vivo are considered minimal since they are substrates of many enzymes such as glutathione peroxidases and phospholipases. In such cases, the stable oxidation products are HODEs. Our method for measuring totally assessed HODE is characterized by the reduction and saponification of samples beforehand. By this method, we can measure the ester and free forms of hydroperoxides and hydroxides as HODE. H(P)ODEs are formed by a radical-mediated oxidation mechanism that consists of four isomers -H(P)ODE are specific products of radical-mediated oxidation. On the other hand, singlet oxygen oxidizes linoleic acids (LAs) by non-radical oxidation to form 13--H(P)ODE, 10--H(P)ODE, 12--H(P)ODE, and 9--H(P)ODE. In this case, 10- and 12--H(P)ODEs are specific oxidation products of singlet oxygen7. There are some reports that describe the formation of singlet oxygen in vivo. For example, singlet oxygen is formed by the Russell mechanism, the coupling of peroxyl radicals, and from the reaction of hydroperoxides with metal ions, HOCl, peroxynitrite, and cytochrome c8. Other works showed Type I and Type II photooxidation mechanisms in the presence of photosensitizers13. The implications of using HODEs and hydroxyarachidonates (HETEs) as biomarkers for detecting early-stage diseases were reviewed previously6. Among them, the usefulness for detecting the risk of diabetes is striking14.We proposed totally assessed hydroxylinoleates (HODEs) from biological samples as biomarkers for investigations of pathogenesis, disease progression, and prognosisers Fig.\u00a0: 13- and RGC axon loss cause glaucomatous optic neuropathy, in which elevated intraocular pressure (IOP) is the primary risk factor15. The IOP in patients with primary open-angle glaucoma (POAG) increases because of reduced aqueous humour outflow at the trabecular meshwork (TM)18. Treatment using hydrogen peroxide affects the cytoskeletal structure and cell-matrix interactions in TM cells19; depletion of glutathione and hydrogen peroxide treatment decrease TM outflow facility20. Various oxidative stresses have been reported to induce RGC death in experimental studies22; free-radical scavengers prevent glaucomatous tissue injury, specifically, glutamate- and IOP-induced RGC death24. Evidence suggests that oxidative stress is involved in IOP elevation and RGC loss in POAG and in POAG without marked IOP elevation such as in normal-tension glaucoma (NTG). We previously reported lower systemic antioxidant capacity measured by ferric-reducing activity in patients with POAG and glaucoma secondary to exfoliation syndrome compared with controls17. We also have shown that lower systemic antioxidant capacity measured by ferric-reducing activity was associated with higher IOP and more severe visual field damage in open-angle glaucoma in a comprehensive assessment26. In the present study, we investigated the involvement of oxidative stress in this disease by using our own proposed oxidative stress marker HODEs and HETEs quantitatively.Glaucoma is characterized as progressive glaucomatous optic neuropathy leading to visual field loss. It is a cause of irreversible blindness worldwideTable\u00a0We measured serum levels of oxidative stress markers, HODEs, and HETEs together with their parent molecules, LAs and arachidonic acids (AAs), respectively, especially focusing on the isomer distributions. All results are summarized in Table\u00a0We further analysed correlations between oxidation markers and clinical parameters to specify oxidation mechanisms. Among the various demographic parameters, we selected age, sex, diabetes status, DBP, and IOPs (IOP on the blood sampling day and the highest IOP recorded) for analyses since these items were different between the CT and glaucoma groups Table\u00a0. In the 6. Singlet oxygen also produces four isomers of 9-, 10-, 12-, and 13--HODEs by the reduction of the corresponding HPODEs. Only enzymes such as 15-lipoxygenase are known to yield 9- and 13--HPODEs in in vivo enzymatic oxidation leading to the corresponding 9- and 13--HODEs. Accordingly, 10- and 12--HODEs are specific products from singlet oxygen while 9- and 13--HODEs are free radical-specific ones. The amounts from the subtraction of 9- and 13--HODEs and 10- and 12--HODEs from 9- and 13--HODEs are attributed to enzymatic oxidation in vivo. This study  after saponification and reduction of the biological samples. Using this protocol, large amounts of oxidation products from linoleates are quantitatively assessed. The advantage of measuring HODEs as oxidative stress markers is that we can understand the oxidative mechanism in diseases by the distribution of their isomers. The standardization of their absolute values by their parent molecules\u2019 linoleates is another advantage for comparisons among individuals. As shown in Fig.\u00a027. Collectively, systemic changes in the oxidation/reduction balance were additionally confirmed in patients with glaucoma. The most important observation we made was that the serum levels of 9- and 13--HODEs/LA in the POAG group were much higher than those in the CT group whereas the levels of 9- and 13--HODEs did not show the same results. These observations regarding the associations between 9- and 13--HODEs and glaucoma were further confirmed in multivariate analyses -HODEs/LA in the POAG group in our study. In our previous comparative study between CT and POAG groups, we did not find any significant difference in serum levels of lipid peroxides that were measured by diacron reactive oxygen metabolites17, suggesting the possible specific involvement of these fatty acid metabolites  in the pathogenesis of glaucoma.We found that several HODEs/HETE were higher in the POAG group than in the CT group. We and other investigators previously reported significantly lower systemic antioxidant capacity in blood samples from patients with POAG compared with controlses Table\u00a0. These o24. We previously reported a significant correlation between lower systemic antioxidant levels and higher IOPs in subjects with glaucoma27. Collectively, the current results suggest that increased systemic oxidative stress markers are related more closely to IOP elevation than to neuronal damage. Interestingly, the serum levels of 10- and 12--HODEs/LA were strongly correlated with the IOP -HODEs in POAG disappeared in multivariate analyses , isoP-d4, 5-hydroxyeicosa-6E,8Z,11Z,14Z-tetraenoic acid (5-HETE), 12-hydroxyeicosa-5Z,8Z,10E,14Z-tetraenoic acid (12-HETE), and 15- hydroxyeicosa-5Z,8Z,11Z,13E-tetraenoic acid (15-HETE) were obtained from Cayman Chemical Company ; 13-hydroxy-9Z,11E-octadecadienoic acid , 9-hydroxy-10E,12Z-octadecadienoic acid , 13-hydroxy-9E,11E-octadecadienoic acid , 9-hydroxy-10E,12E-octadecadienoic acid , 10-hydroxy-8E,12Z-octadecadienoic acid , 12-hydroxy-9Z,13E-octadecadienoic acid , and 13S-hydroxy-10E,12Z-octadecadienoic-9,10,12,13-d4 acid (13-HODE-d4) were obtained from Larodan Fine Chemicals ; and LA and AA were obtained from Sigma-Aldrich . Other materials were of the highest commercially available grade.Lipid-peroxidation products such as 8-iso-prostaglandin F27. All measurements of oxidative stress markers were conducted at the National Institute of Advanced Industrial Science and Technology. The current study adhered to the tenets of the Declaration of Helsinki. The institutional review boards of Shimane University Hospital, Iinan Hospital, and the National Institute of Advanced Industrial Science and Technology reviewed and approved the research. All subjects provided written informed consent. All subjects underwent ophthalmologic examinations including measurements of best-corrected visual acuity (VA) and IOP by Goldmann applanation tonometry and slit-lamp, gonioscopic, and funduscopic examinations under pupillary dilatation. POAG was diagnosed based on the presence of an open iridocorneal angle bilaterally, the characteristic appearance of glaucomatous optic neuropathy such as enlargement of the optic disc cup or focal thinning of the neuroretinal rim, corresponding visual field defects identified using the Humphrey Visual Field Analyzer  in at least one eye, and no evidence of secondary glaucoma bilaterally. In the POAG group, 114 subjects with a history of an untreated IOP of 21\u2009mmHg or higher in at least one eye were considered to have HTG and the other 84 subjects who did not have a history of an untreated IOP of 21\u2009mmHg or higher were considered to have NTG. The CT subjects had a corrected VA of 0.7 or better measured in both eyes using a decimal VA chart, and no glaucomatous optic neuropathy or history of an IOP of 21\u2009mmHg or higher. Except for a cataract and/or glaucoma, no subjects had ocular pathologies such as clinically detectable ocular inflammation, infection, neuropathies, retinopathies, or maculopathies.A total of 317 Japanese subjects comprised the POAG (n\u2009=\u2009198) and non-glaucomatous CT (n\u2009=\u2009119) groups. These subjects were recruited consecutively at Shimane University Hospital and Iinan Hospital, Shimane, Japan as described previouslyTo avoid the possible confounding effect of systemic diseases, the subjects were questioned about their histories of severe systemic diseases during interviews before enrolment in the study. These diseases included acute brain infarction and haemorrhage, systemic neurologic diseases, cardiac diseases requiring catheter placement or surgery, cardiac failure and other systemic diseases affecting the subjects\u2019 physical activity, lung diseases causing dyspnoea, chronic and acute hepatitis requiring interferon therapy, liver cirrhosis, renal failure requiring haemodialysis, autoimmune diseases requiring systemic steroids and other immunosuppressive therapies, severe anaemia requiring blood transfusions, major visceral surgery, malignancies, severe hypertension causing cardiac and kidney failure, and severe diabetes requiring insulin therapy. In addition to a history of severe systemic diseases, to adjust for the possible confounding effects of other factors such as differences in nutrition, blood pressure, blood glucose, and smoking habits, the presence or absence of diabetes, current smoking status, and time since the last meal, systolic blood pressure (SBP), DBP, and pulse rate were recorded before blood samples were collected. The highest known IOP measured on the day of sample collection or the highest previously measured IOP recorded in the medical charts also was collected. Venous blood specimens were collected from the antecubital vein into evacuated tubes. Serum samples obtained by centrifugation of the collected venous blood were stored at \u221280\u2009\u00b0C until analysis. During all handling procedures, including transportation from the clinical setting to the laboratory and centrifugation, the temperature was maintained at 4\u2009\u00b0C.3 with slight modifications. The parent molecules\u2014i.e., LAs and AAs\u2014were detected using the same protocol. Briefly, 200\u2009\u03bcL of plasma was mixed with 300\u2009\u03bcL of saline and 500\u2009\u03bcL of methanol containing the internal standards 8-iso-PGF2\u03b1-d4 and 13-HODE-d4, and 100\u2009\u03bcM of butylated hydroxytoluene was added to the samples. This was followed by hydroperoxide reduction using an excess of triphenylphosphine and saponification with 1\u2009M KOH in methanol. The mixture was acidified with 10% acetic acid in water and extracted with chloroform and ethyl acetate. An aliquot of the sample was analysed by liquid chromatography/tandem mass spectrometry (LC-MS/MS) on a TSQ Quantum Access Max system . The Dionex Ultimate 3000 system (Thermo Fisher Scientific) used for HPLC consisted of an HPG-3400 RS pump, WPS-3000 TPL RS Well Plate autosampler, and TCC-3000 RS column compartment equipped with a Hypersil Gold ODS column  set at 40\u2009\u00b0C. Elution was performed using a gradient of solvent A (2\u2009mM ammonium acetate in water) and solvent B (methanol:acetonitrile\u2009=\u20095:95) at a flow rate of 0.2\u2009mL/min. The initial gradient composition was 80% A and 20% B. The composition was changed to 79% A and 21% B for 10\u2009min and then to 0% A and 100% B for 45\u2009min. Electrospray ionization was performed at a needle voltage of 3.0\u2009kV. Nitrogen was used as the sheath gas (50\u2009psi) and auxiliary gas (10 units). The capillary was heated to 300\u2009\u00b0C and the mass spectrometer was optimized for maximum sensitivity. A specific precursor-to-product ion transition was achieved by monitoring selected reactions after collision-induced dissociation in negative mode. Argon was used as the collision gas, and the collision cell pressure was 1.5 mTorr. The precursor, product ions, and collision energy were determined after MS/MS optimization as follows: m/z\u2009=\u2009353.1 and 193.1 at 26\u2009eV for 8-iso-PGF2\u03b1; m/z\u2009=\u2009357.1 and 197 at 26\u2009eV for 8-iso-PGF2\u03b1-d4; m/z\u2009=\u2009295.0 and 195 at 18\u2009eV for both 13-Z,E-HODE and 13-E,E-HODE; m/z\u2009=\u2009295.0 and 171 at 18\u2009eV for both 9-E,Z-HODE and 9-E,E-HODE; m/z\u2009=\u2009295.0 and 183 at 18\u2009eV for both 10-Z,E-HODE and 12-Z,E-HODE; m/z\u2009=\u2009299.0 and 198 at 18\u2009eV for 13-HODE-d4; m/z\u2009=\u2009319.0 and 115 at 14\u2009eV for 5-HETE; m/z\u2009=\u2009319.0 and 163 at 14\u2009eV for 12-HETE; m/z\u2009=\u2009319.0 and 203 at 14\u2009eV for 15-HETE; m/z\u2009=\u2009279 at 5\u2009eV for LA; and m/z\u2009=\u2009303 and 259 at 12\u2009eV for AA.The levels of HODEs, HETEs, and isoP were measured as described previouslyAll statistical analyses were performed using IBM SPSS Statistics version 21.0. . All continuous data are expressed as the mean\u2009\u00b1\u2009standard deviation. For comparisons between the CT and POAG groups, the Mann-Whitney U test was used for continuous variables including age, SBP, DBP, pulse rate, time from the last meal, IOP on the blood sampling day, highest IOP recorded, and various oxidative stress makers . Fisher\u2019s exact probability test was used for categorical variables including sex, diabetes status, and current smoking habits. For comparisons between the CT, NTG, and HTG groups, the Kruskal-Wallis test followed by the post hoc Mann-Whitney U test were used for continuous variables, and the G-test followed by the post hoc Fisher\u2019s exact probability test were used for categorical variables. Possible correlations between demographic data and various oxidative stress markers were assessed by Spearman\u2019s rank correlation test. To adjust for differences in demographic data between the groups, associations between glaucoma and oxidative stress markers were tested using multiple logistic regression analyses with CT/POAG set as dependent variables and six demographic factors  and each of the oxidative stress makers  were set as independent variables. P-values of 0.0167 and 0.0033 were considered significant at levels of 5% and 1%, respectively, by the Bonferroni correction for three-group comparisons; otherwise, a p-value of 0.05 was considered statistically significant."}
+{"text": "The blood-brain barrier (BBB) poses a major obstacle by preventing potential therapeutic agents from reaching their intended brain targets at sufficient concentrations. While transient disruption of the BBB has been used to enhance chemotherapeutic efficacy in treating brain tumors, limitations in terms of magnitude and duration of BBB disruption exist. In the present study, the preliminary safety and efficacy profile of HAV6, a peptide that binds to the external domains of cadherin, to transiently open the BBB and improve the delivery of a therapeutic agent, was evaluated in a murine brain tumor model. Transient opening of the BBB in response to HAV6 peptide administration was quantitatively characterized using both a gadolinium magnetic resonance imaging (MRI) contrast agent and adenanthin (Ade), the intended therapeutic agent. The effects of HAV6 peptide on BBB integrity and the efficacy of concurrent administration of HAV6 peptide and the small molecule inhibitor, Ade, in the growth and progression of an orthotopic medulloblastoma mouse model using human D425 tumor cells was examined. Systemic administration of HAV6 peptide caused transient, reversible disruption of BBB in mice. Increases in BBB permeability produced by HAV6 were rapid in onset and observed in all regions of the brain examined. Concurrent administration of HAV6 peptide with Ade, a BBB impermeable inhibitor of Peroxiredoxin-1, caused reduced tumor growth and increased survival in mice bearing medulloblastoma. The rapid onset and transient nature of the BBB modulation produced with the HAV6 peptide along with its uniform disruption and biocompatibility is well-suited for CNS drug delivery applications, especially in the treatment of brain tumors. Chemotherapy and more recently targeted therapy directed at specific tumor targets, are important modalities for treatment of pediatric brain tumors. Numerous chemotherapeutic drugs and small molecule inhibitors that demonstrated significant antitumor activity in preclinical studies have failed in human clinical trials . These dThe cadherin proteins found within the adherens junction have an important role in the BBB for establishing cell\u2013cell contact and contributing to the tight junction complex. The formation of homodimer complexes of cadherin proteins on adjacent brain microvessel endothelial cells act to physically restrict the paracellular passage of solutes from the blood to the brain extracellular environment ,7. MolecIsodon adenantha inhibits the peroxidase activity of PRDX 1 and 2  of Ade with the transition 513.2 > 453.1 m/z using a collision energy of 30 eV. The desolvation line temperature was 250 \u00b0C; the heating block temperature was 400 \u00b0C; the nebulizing gas flow was 2 L/min; and drying gas flowed at 15 L/min. An XTerra\u00ae MS C18   column was used with 0.3 mL/min flow rate at 30 \u00b0C. The mobile phases A (50% aqueous methanol with 0.1% formic acid) and B (85% aqueous methanol), and 0.1% formic acid were used in a step gradient starting at 100% A for 1 min, then stepping to 100% B for 3 min and stepping back to 100% A for 2 min before the next injection.A Shimadzu Nexera ultra high performance liquid chromatography system connected to an 8040 triple-quadrupole mass spectrometer (LC-MS/MS)  was used to measure Ade. A dual Electrospray ionization (ESI) / atmospheric pressure chemical ionization (APCI) ion source was used in the positive mode multiple reaction monitoring (MRM) for the sodium adduct ..www.moliConsistent with the initial in silico assessment, in vivo analysis in mice revealed that Ade, when administered alone, had limited BBB permeability, with brain concentrations below the quantitative limit  B. HoweveTo functionally demonstrate that the HAV6 peptide can transiently disrupt the BBB and facilitate delivery of Ade to the brain, and therapeutically target PRDX1 we treated mice bearing Group 3 D425-Med-Luc tumors with either Ade alone or Ade in combination with HAV6 peptide. The mice receiving placebo or Ade alone had a median survival of 20 and 19 days post tumor cell injection respectively C. FurtheAt the cellular level, tumor cells were positive for PRDX1, an ROS scavenging enzyme that is the molecular target of Ade . Mice reThe development of drugs for the treatment of many CNS disorders has long been a difficult process, in part due to the inability of the drugs to reach relevant therapeutic concentration in the brain and the spinal cord. This limited access to the brain and spinal cord for many drugs is a direct result of the protective properties of the BBB and blood cerebral spinal fluid barrier (BCSFB). These barriers are composed of epithelial (BCSFB) or endothelial (BBB) cells with tight junctions and active efflux transporters that together restrict both the paracellular and transcellular passages of solutes into the brain ,29. WhilApproaches such as osmotic disruption and focused, high-intensity ultrasound, have shown promise in preclinical studies and have been used in the clinic to enhance chemotherapeutic drug delivery to brain tumors. However, the long duration of BBB disruption (up to 8 h or more) can lead to neurotoxicity and inflammation ,38. A loSynthetic HAV6 peptide disrupts the BBB by inhibiting homodimer interactions between E-cadherin, an essential protein that forms the adherens junctions of the BBB ,40. BaseThe initial characterization of HAV6 peptide mediated BBB disruption in nude mice was examined using Gd-DTPA and MRI. Following HAV6 peptide administration, there was an approximately two to four-fold enhancement in Gd-DTPA contrast throughout all areas of the brain examined. Both the magnitude and time frame of BBB disruption mediated by HAV6 peptide in the current study were consistent with our previous findings . The abiThe anticancer properties of Ade were originally reported by Liu et al.  in studiAdenanthin had little to no permeability through the BBB when administered alone; however, co-administration of Ade with HAV6 peptide significantly enhanced its accumulation in the brain. The concentration of Ade in the brain following HAV6 administration was similar for all doses of Ade examined. This could be due to the saturation of the influx of Ade into the brain following the disruption with HAV6 peptide. Of importance for the present study is the observation that concurrent administration of Ade (10 mg/kg dose) and HAV6 peptide resulted in brain concentrations of Ade (>10 \u03bcM) that would be expected to be in the therapeutic range required for anti-tumor activity. In addition, it should be noted that despite the large increases in Ade accumulation in the brain, there were no overt clinical signs of toxicity . Furthermore, examination of GFAP and Iba1 expression in the brain showed no enhancement in these neuroinflammatory markers as a result of HAV6 either alone or in combination with Ade. This is an important finding, as increased GFAP and Iba1 expression, markers of reactive astrocytosis and activated microglia, respectively, have been reported with both osmotic and focused ultrasound BBB modulation ,44.We had three cohorts of mice  to evaluate the in vivo effect of concurrent administration of Ade and HAV6 peptide in an orthotopic Group-3 MBL model. The HAV6 peptide was not included as a cohort, as the peptide had no effect on Group-3 MBL cells C. The avIn summary, we have shown that HAV6 peptide reversibly modifies BBB permeability allowing for effective delivery of Ade for the treatment of brain tumors. The transient modulation of BBB permeability produced by HAV6 cadherin peptide appeared to be well tolerated. While additional studies are required to validate both the effectiveness of adenanthin and our drug delivery approach in treating Group 3 MBL, the current studies suggest that HAV cadherin peptide can be used to enhance the brain delivery and effectiveness of poorly BBB permeable therapeutic agents. The effects of cadherin peptide based BBB modulation in terms of both the duration and magnitude of BBB opening, coupled with its effectiveness in repeated use settings could prove advantageous for treatment of brain tumors."}
+{"text": "Diabetes is a metabolic disorder leading to many complications. The treatment of diabetes mainly depends on hypoglycemic drugs, often with side effects, which drive us to develop novel agents. AWRK6 was a peptide developed from the antimicrobial peptide Dybowskin-2CDYa in our previous study, and the availability of AWRK6 on diabetes intervention was unknown. Here, in vivo and in vitro experiments were carried out to investigate the effects of AWRK6 against diabetes. In diabetic mice, induced by high-fat diet followed by streptozocin (STZ) administration, the daily administration of AWRK6 presented acute and sustained hypoglycemic effects. The plasma insulin was significantly elevated by AWRK6 during an oral glucose tolerance test (OGTT). The relative \u03b2 cell mass in diabetic mice was increased by AWRK6 treatment. The body weight and food intake were remarkably reduced by AWRK6 administration. In the mouse pancreatic \u03b2 cell line Min6 cells, the intracellular calcium concentration was found to be enhanced under the treatment with AWRK6, and protein kinase A (PKA) inhibitor H-89 and Epac2 inhibitor HJC0350 represented inhibitory effects of the insulinotropic function of AWRK6. By FITC-AWRK6 incubation and GLP-1 receptor (GLP-1R) knockdown, AWRK6 proved to be a novel GLP-1R agonist. In addition, AWRK6 showed no toxicity in cell viability and membrane integrity in Min6 cells, and no hypoglycemia risk and no lethal toxicity in mice. In summary, AWRK6 was found as a novel agonist of GLP-1R, which could stimulate insulin secretion to regulate blood glucose and energy metabolism, via cAMP-calcium signaling pathway, without significant toxicity. The peptide AWRK6 might become a novel candidate for diabetes treatment. Diabetes mellitus  is a metabolic disorder with enhanced blood sugar, leading to many complications like cardiovascular disease, kidney disease, foot ulcers, and eye damage ,2. The iGLP-1 receptor (GLP-1R), a G protein-coupled receptor expressed in various tissues such as islets, liver, and the nervous system, is involved in the control of blood sugar by binding GLP-1 and glucagon . Thus, GIn this study, we aimed to investigate the protective effects of AWRK6 against diabetes in mice, as well as the underlying mechanisms, which might offer a novel strategy for diabetes therapy.N-1-deoxy fructosyl component of hemoglobin) levels  followed by streptozocin (STZ) administration. The daily administration of AWRK6  was carried out intraperitoneally over 4 weeks and the fasting blood glucose (FBG) was detected every 7 days. As shown in ) levels G. Furthe) levels C,D. Mean) levels E,F. ThesIn the diabetic mice model, STZ could cause \u03b2 cell damage by triggering immune responses. To investigate the protective effects of AWRK6 against islet injury, the pancreas tissues of the mice were collected and fixed after the treatment with AWRK6 (100 nmol/kg) for 4 weeks. Paraffin sections were made and immunohistochemistry (IHC) analysis using an anti-insulin antibody was carried out. The morphology of pancreas was observed under a microscope and the relative \u03b2 cell mass was analyzed using ImageJ software. As shown in Considering that obesity is generally in close relationship with diabetes, the body weight and food intake were monitored during the treatment with AWRK6. For diabetic mice induced by high-fat feeding and STZ, body weight was significantly elevated and the daily treatment with AWRK6 over 4 weeks significantly decreased the body weight A,B. FurtTo assess potential signaling bias, the mouse pancreatic \u03b2 cell line Min6 cells were treated with AWRK6. The insulin in the culture medium was detected by ELISA, as shown in After incubating with FITC-conjugated AWRK6, the Min6 cells were found to be stained with fluorescent dye on the cell surface, suggesting that AWRK6 might interact with receptors on the cell membrane A. Then, For the potential application of AWRK6 against diabetes, it is essential to assess its toxicity in vitro and in vivo. The cell viability of Min6 cells treated with AWRK6 was determined by CCK-8 assay, and there was no significant reduction in the AWRK6-treated cells vs control group A. ConsidWith the increasing incidence of diabetes, strict glycemic control and treatment of complications are facing severe challenges. The strategies promoting insulin secretion have been the mainstay of the treatment of diabetes. In recent years, several peptides from frog skin, firstly identified by their antimicrobial or immunomodulatory activities, were proven to have the function of stimulating insulin release in vitro and in vivo, thus showing the potential of development into agents against diabetes . In thisGenerally, the secretion of insulin was regulated by the intracellular calcium concentration in \u03b2 cells, which was under the control of ATP sensitive potassium channels, calcium channels, and intracellular calcium pools on the plasma membrane ,20,21,22In previous studies, GLP-1R agonists such as GLP-1, Exenatide, and Liraglutide, present multiple biological actions in type 2 diabetes, including restoration glucose-dependent stimulation of insulin secretion, more adequate insulin secretory response after meals, suppression of glucagon secretion, and enhancement of glucagon secretion when plasma glucose is low . Here, AIn this study, we have found the peptide AWRK6 as a novel agonist of GLP-1R, which could stimulate insulin secretion to regulate blood glucose and energy metabolism, via cAMP-calcium signaling pathway, with no significant toxicity. The peptide AWRK6 might become a novel candidate for diabetes treatment.The synthetic peptide AWRK6 and FITC-conjugated AWRK6 were obtained from GL Biochem Corporation , which were purified by RP-HPLC for a high purity of 99% . ExendinMale Kunming mice (18\u201322 g) were obtained from Changsheng Biotechnology  and housed in a temperature-controlled environment with free access to food and water. To generate diabetic model mice, the mice were fed a high-fat diet with 60% fat (Changsheng Biotechnology) for 3 weeks, then administrated with STZ  by intraperitoneal injection daily for 5 days. After the high-fat diet followed by STZ administration, the mice were treated with AWRK6 through intraperitoneal injection once per day. Exendin-4 was used as a positive control and physiological saline was used as a blank control. The body weight and food intake were monitored weekly. Nine mice were used for each group. The animal experiments were approved by the Ethics Committee of Liaoning University  and conducted according to the ethical guidelines for the Care and Use of Laboratory Animals.Blood samples were collected from a tail vein and the blood glucose was estimated using ACCU-CHEK glucometer . The insulin (in plasma and culture medium) and HbA1c were determined using ELISA kit according to the manufacturer\u2019s instructions. The insulin kit was purchased from Anoric Bio-technology . The HbA1c kit was purchased from Jiancheng Bioengineering Institute . The absorbance at 450 nm was detected on iMARK microplate reader .The pancreas tissues were collected and fixed with 10% formalin. Then the tissues were embedded in paraffin and 5 \u03bcm sections were made. The sections were stained with the rabbit anti-insulin antibody  and visualized using UltraSensitive SP IHC Kit  according to the manufacturer\u2019s instructions. The sections were observed under an Olympus CK31 microscope and the images were analyzed using ImageJ software . For each group, 15 pancreatic histological slides (3 200 \u03bcm-apart sections for each sample) including at least 100 islets were analyzed. The relative \u03b2 cell mass was calculated as the area of insulin-positive cells/total pancreatic area .2. To induce insulin secretion, Min6 cells were cultured in serum-free medium for 2 h, washed and incubated in KRB (Krebs-Ringer bicarbonate buffer) with 2 mM glucose for 30 min. Then the cells were stimulated with glucose (25 and 30.6 mM), AWRK6 (50 nM), and exendin-4 (20 nM) in KRB for 4 h, and the subsequent experiments were carried out as described. To evaluate the involvement of cAMP pathway in AWRK6-induced insulin secretion, PKA inhibitor H-89  and Epac2 inhibitor HJC0350  were used for the pretreatment of Min6 cells at 30 min before glucose stimulation.The mouse pancreatic \u03b2 cell line Min6 cells were obtained from Bogoo Biotech . The cells were cultured in DMEM  supplemented with 15% fetal bovine serum , 0.5% 2-Mercaptoethanol  and 1% penicillin streptomycin (Hyclone) at 37 \u00b0C in a humidified atmosphere containing 5% COThe intracellular calcium of Min6 cells was determined by Fluo 3-AM . Briefly, Min6 cells were seeded in confocal dishes, pretreated with verapamil , EGTA  and stimulated with glucose as indicated. The cells were washed with KRB and incubated with Fluo 3-AM working solution (5 \u03bcM in KRB) for 45 min at 37 \u00b0C in the dark. The cells were washed again and incubated for 30 min, then observed and imaged under FV1000 confocal laser scanning microscope .GLP-1R Silencing was carried out using lentiviruses which were prepared by Micro Helix company . Three shRNAs against mouse GLP-1R and scrambled control were used. The Min6 cells were seeded in 6-well plates and infected with these lentiviruses, the subsequent experiments were carried out at 48 h after infection.The membrane protein was prepared using Mem-PER Plus Membrane Protein Extraction Kit . Equal amounts of protein were separated by SDS-PAGE and transferred to NC membranes. The membranes were incubated overnight at 4 \u00b0C with antibodies against mouse GLP-1R , Epac2  and GAPDH , followed by incubation with HRP-conjugated secondary antibodies . Protein bands were visualized by ECL Plus Reagent  and imaged on MicroChemi chemiluminescence detection system . The protein bands were analyzed using ImageJ software .The cell viability was assayed using CCK-8 (Dojindo). The cells were plated into 96-well plates and incubated overnight. After indicated treatments for 24 h, 10 \u03bcL CCK-8 reagent was added to each well and incubated for 2 h. The absorbance at 450 nm was detected on iMARK microplate reader .Lactate dehydrogenase assay kit  was applied for LDH release analysis. The Min6 cells were seeded in 96-well plates and incubated overnight. The cells were treated as indicated for 24 h and the supernatant was transferred to another 96-well plates for detection according to the manufacturer\u2019s instructions. The absorbance at 450 nm was detected using iMARK microplate reader.t test or one-way ANOVA followed by multiple comparisons. p < 0.05 was considered statistically significant.The survival rates between groups were analyzed using the Mantel-Cox Log-rank test. Other data from three replicas were presented as mean \u00b1 SD and analyzed by student\u2019s"}
+{"text": "SHOX represent the most frequent monogenic cause of short stature and related syndromes. The genetic alterations include point mutations and deletions/duplications spanning both SHOX and its regulatory regions, although microrearrangements are confined to either the downstream or upstream enhancers in many patients. Mutations in the heterozygous state have been identified in up to 60\u201380% of Leri-Weill Dyschondrosteosis  and approximately 4\u20135% of Idiopathic Short Stature  patients. Homozygous or compound heterozygous mutations as well as biallelic deletions of SHOX and/or the enhancer regions result in a more severe phenotype, which is known as Langer Mesomelic Dysplasia .Mutations of SHOX coding sequence and involving its regulatory regions. An additional duplication of 1.6\u20132.5\u2009Mb on 15q25.2 that included 13 genes was also identified. The girl was adopted and the parent\u2019s DNA was not available to establish the origin of the chromosome imbalances.A 17\u2009year old girl, presented with severe short stature, growth hormone deficiency (GHD), precocious puberty, dorsal scoliosis, dysmorphisms and urogenital malformations. She was born with agenesis of the right tibia and fibula, as well as with a supernumerary digit on the left foot. Array comparative genomic hybridization (aCGH) analysis detected the presence of two distinct duplications on Xp22.1 flanking the SHOX region.The complex phenotype observed in our patient is probably the result of the co-occurrence of rearrangements on chromosomes Xp22.1 and 15q25.2. The duplicated region on 15q25.2 region is likely to contain dosage-sensitive genes responsible for some of the clinical features observed in this patient, whereas the extreme short stature and the skeletal anomalies are likely attributable to the comorbidity of GHD and copy number variants in the The online version of this article (10.1186/s12920-018-0445-8) contains supplementary material, which is available to authorized users. SHOX  region and a duplication on 15q25.2 that likely contributes to the the determination of her clinical features.The presence of multiple independent genomic imbalances in the same patient might result in complex phenotypes due to either an additive or interactive effect of single rearrangements. Here, we report the case of a patient carrying two apparently distinct duplications on Xp22.1 that, involve the SHOX, which is located on pseudoautosomal region 1 (PAR1), include intragenic mutations, complete or partial deletions/duplications of the coding sequence and/or the enhancer regions [SHOX gene can result in Ler\u00ec-Weill Dyschondrosteosis  with disproportionate short stature, mesomelia and Madelung deformity of the wrist as well as tibial bowing. Loss of both copies of SHOX cause Langer mesomelic Dysplasia , which is a condition with extreme short stature, severe shortening or aplasia of the ulna and fibula, as well as boththickening and curvature of the radius and tibia. SHOX mutations are also present in approximately 2\u201310% of Idiopathic Short Stature (ISS) patients, depending on which study cohort is considered [Molecular defects of https://decipher.sanger.ac.uk/).Genomic rearrangements involving chromosome 15q25.2 constitute a rare finding. Nevertheless, there have been some reports of copy number variations (CNVs) within this region associated with recognizable phenotypes. In most cases, these mutations were represented by deletions, whereas 15q25.2 microduplications have been described in only three previous cases , 4 and aThe patient was referred to our third-level Paediatric Endocrinology Unit when she was 9\u2009years old for short stature and suspected precocious puberty. She was of Russian origin and was adopted at the age of 2\u2009years. Limited data were available about her family and perinatal history. The biological mother was affected by insulin-dependent diabetes mellitus and arterial hypertension. Nothing was known about the biological father. No data were available about gestational age, neonatal weight and length as well as general conditions at birth. She was born with agenesis of the right tibia and fibula, and underwent mid-thigh amputation when she was 5\u2009years old. She also presented a supernumerary digit of the left foot, which was excised when she was 8\u2009years old.http://www.cdc.gov/growthcharts) with a body mass index in the overweight range according to International Obesity Task Force cut-offs [At the age of 9\u2009years, she was referred to our Paediatric Endocrinology Unit for short stature and with potential precocious puberty. Her height was <3rd percentile on CDC growth charts for sex and age , her weight was in the 25th percentile  analysis was performed using the standard Agilent Human Genome G3 SurePrint 8x60K Microarray . Two distinct duplications flanking the CPEB1 gene. The 15q25.2 duplicated region was examined using the Human resource websites (https://www.ncbi.nlm.nih.gov/projects/genome/guide/human/) at NCBI and the Archive EnsEMBL (https://www.ensembl.org/info/website/archives/index.html). This region contains 13 genes, some of them with a known function  with a probe specifically designed within the Unfortunately, the biological parents were not available to establish the origin of the duplications.https://decipher.sanger.ac.uk/) and identified 4 patients carrying similar micro-duplications  and autosomal dominant hearing loss, respectively. DBA4 is a recessive condition caused by biallelic loss of function mutations, whereas HOMER2 was found to be mutated only once in a family in which deafness co-segregated with a missense mutation [The duplicated 15q25.2 region of 1.6\u20132.5\u2009Mb includes 13 OMIM genes  that function as enhancers of SHOX transcription. In the present patient, the aCGH identified two gains flanking SHOX, including all the upstream and downstream CNEs  (OMIM 249700), which is a rare recessive condition characterized by severe short stature, mesomelic and rhizomelic dysgenesis of the limbs involving hypoplasia or aplasia of the ulna and fibula. Our patients did not exhibit a typical LMD phenotype; however, in addition to the severe short stature, she was born with agenesis of the right tibia and fibula. Hypoplastic or absent proximal half of the fibula has been described among the principal clinical features of LMD [SHOX alleles was altered.The extreme short stature might be the result of the co-occurrence of two independent causes: GHD and rearrangements in the NEs Fig.  as well SHOX regulatory region exacerbated by GH deficiency. The duplication on 15q25.2 might have contributed to some of the clinical features through the action of unidentified dose-sensitive gene/s.In conclusion, the complex phenotype of our patient, including severe short stature and skeletal anomalies, was probably the result of rearrangements in the Additional file 1:Table S1. Genes with a known function included in the 15q25.2 duplication. (DOCX 18 kb)"}
+{"text": "Healthy weight women were advised to meet estimated energy requirements, while overweight/obese women were advised to follow a 25% energy restriction to achieve weight loss.Our programme aimed to prevent weight gain in healthy weight women and reduce weight in women who were overweight/obese since both may improve outcome and well-being after a diagnosis of breast cancer (BC).3 However, our programme was framed as a weight loss/weight gain prevention lifestyle programme to improve future health and quality of life after a diagnosis of BC. There was no evidence of disengagement among heavier women in our study. Mean attendance for group sessions among healthy weight, overweight and obese participants was 64%, 66% and 62%, respectively. In-depth interviews cited advantages in meeting others who were experiencing similar cancer-related problems. Thus, highlighting the importance of the BC diagnosis rather than their weight to the salience of their current situation.We acknowledge the concern that heavier women may not feel comfortable in mixed BMI groups, as previously reported in weight loss focussed programmes.4 Similarly, cardiac rehabilitation programmes include individuals who are a healthy weight (24%), overweight (37.3%) and obese (38%).5Lifestyle behaviour change programmes in other health settings commonly include subjects of mixed BMI, for example, the UK Diabetes Prevention Programmes includes individuals with haemoglobin A1c > 42\u2009mmol/mol, of whom 27% are a healthy weight, 39% overweight and 33% obese.6We did not use multiple imputation since this usually assumes data is missing at random (MAR). MAR was unlikely in the B-AHEAD study, as non-attendance at study appointments was more likely for those who had gained weight. We accept that single imputation approaches are limited and advised generally against for missing data. Analysis of longitudinal studies should consider missing data at each stage of the research and include appropriate sensitivity analysis of the missing data mechanism as highlighted recently by Bell et al.The combined weight change outcome was pragmatic to provide an overall estimate of the programme for all early BC patients. We have also presented changes for the healthy and overweight/obese sub-groups. The 6-month assessment was timed to occur after the end of adjuvant treatments to maximise attendance. It also offers a better reflection of sustained behaviour change beyond the intensive intervention phase.Our study tackled weight control soon after diagnosis and during adjuvant treatment. There is debate, but few data on the best time to initiate lifestyle behaviour change.Optimum timing is based on a number of key criteria. First, when are weight loss/dietary interventions most clinically or biologically relevant? When are patients most likely to engage with programmes? Are there times when patients have difficulties to change behaviours because of the challenges of treatment or other issues? Is intervention timing key to long-term maintained behaviours?8 question whether our uptake could have been greater if we had approached patients after treatment, citing higher uptakes for weight loss studies, which recruited post treatment. Comparing uptakes across studies is difficult, since this depends on the initial mode of patient identification. The LISA trial reported that 80% of women identified in their oncology centres consented to trial screening.7 However, it is not clear whether they approached all eligible women, or used a selective approach thus yielding superior recruitment. The Energy Study mailed 14,051 women to recruit 697 women, hence a 5% uptake.8 No studies have compared uptake at diagnosis vs. post treatment. The individual nature of cancer patients necessitates a range of timings and approaches to maximise engagement with lifestyle behaviour change.Our intervention at diagnosis aimed to offset deleterious gains in fat and loss of lean body mass, which can occur during adjuvant treatments. Bail and colleagues9 which is consistent with the poorer weight control success among B-AHEAD participants receiving adjuvant chemotherapy.Physical activity interventions appear to be effective both during and after active cancer treatments. While dietary interventions have a lower success during active treatment,The B-AHEAD study does not indicate that time of diagnosis is the optimal teachable moment for patients with early BC. However, it highlights the feasibility of engaging significant numbers of patients at this often overlooked/avoided time point, regardless of BMI or adjuvant therapies.10Our programme was successful for preventing weight gain among healthy weight women and for achieving weight loss among those with overweight/obesity not receiving adjuvant chemotherapy. Weight control and energy restriction may be specifically important among patients receiving adjuvant chemotherapy. Our B-AHEAD 2 and other ongoing studies are testing whether more achievable approaches such as intermittent energy restriction are effective among these patients."}
+{"text": "Chronic pain is a common condition in later life that is related to high levels of anxiety and depression. One reason why chronic pain is related to affective distress is that this condition may prevent people from deriving the same positive emotions from enjoyable activities. Few studies, however, have examined how exposure and reactivity to daily events differ by chronic pain status. We hypothesized that those with chronic pain will have less exposure and less positive affect reactivity to positive daily events compared to those without chronic pain. Participants from the diary substudy of MIDUS  completed eight interview days. Chronic pain status was unrelated to the frequency of positive events. Multi-level models revealed that although people with chronic pain had lower levels of daily positive affect, they reacted more positively to daily events . As a result, levels of daily positive affect on days when people experienced a positive event did not vary by pain status . People with chronic pain averaged higher levels of daily negative affect compared to people without chronic pain , but, on days when they experience a positive event, those with chronic pain had a greater decrease in their negative affect. Findings suggest that positive events impact those with chronic pain more than they do individuals without chronic pain."}
+{"text": "SARS-CoV-2 is a coronavirus with high infectivity and has caused dramatic pressure on health systems all over the world. Appropriate personal protection for medical staffs is critical. For ocular protection, there is ongoing hot debate and concern for potential ocular transmission of SARS-CoV-2. Ocular manifestations and positive detection of viral RNA in ocular samples were only reported in very small number of patients infected with SARS-CoV-2. However, health care workers need to face patients more closely and have higher risk of aerosol contamination. Thus, appropriate ocular protection for medical workers is still recommended by organizations such as WHO and American Academy of Ophthalmology. Although eye goggles provide excellent protection and are mandatory for medical practitioners with high risk of exposure, they are not ideal for common clinical practice, because they can disturb vision due to extensive formation of water droplets and frequently cause moderate to severe discomfort after longtime wearing, which have been reported to interfere with working status. For the majority of medical workers who don't deal with high risk patients, they are not advised to wear goggles in daily practice. However, they also face the risk of infection due to the presence of asymptomatic carriers. Especially in situations with high risk of ocular exposure, such as close physical examination, eye surgery, dental clinics and surgery, ocular protection may be needed. Griffithsin has been shown to directly bind to spike proteins and has anti-viral activity against a broad spectrum of viruses, including coronavirus. Griffithsin is found to inhibit the entry of SARS-CoV at relatively low concentration and is stable and non-toxic. SARS-CoV-2 and SARS-CoV share the same entry receptors and their spike proteins are similar in conformation. We hypothesize that contact lenses containing nanoparticles loaded with griffithsin may provide sufficient ocular protection for medical staffs without high risk of exposure during the outbreak period of SARS-CoV-2. If proven effective, griffithsin-loaded contact lens can be considered as a supplementary ocular protective equipment for medical workers who can tolerate well. The daily disposable contact lens should be applied as needed and refrain from extended wearing in order to reduce potential side effects. The novel coronavirus \u201cSARS-CoV-2\u201d is now causing global pandemic and has claimed more than 800,000 lives until July, 2020. Although SARS-CoV-2 is principally a respiratory virus, there is concern that the ocular surface may serve as potential route of SARS-CoV-2 transmission.via the nasolacrimal duct. The nasolacrimal duct drains tear in the conjunctival sac continuously into the inferior nasal meatus and is thought to play important roles in the spreading of ocular virus into the respiratory system. Thus, the eye theoretically possesses dual routes for virus spread: lacrimal drainage-based spread and direct infection via ocular cell receptors  with host surface receptors (ACE2 or CD147) , 4. Thereceptors . Althougeceptors . Besidesbranches . In one branches . Thus, tHowever, according to clinical data and RT-PCR tests of ocular samples, ocular involvement and positive isolation of viral RNA were only reported in a very small number of patients infected with SARS-CoV-2 , 2. The etc.(It has been well-recognized that the ocular surface possesses a variety of mechanisms to protect from viral infection, which may explain the low rate of ocular involvement and RNA detection. Many mechanical activities, like tearing, blinking and barrier function of eyelid and lashes may all prevent landing of virus-containing droplets on ocular surface . In one etc..Although ocular involvement is infrequent in patients infected with SARS-CoV-2, there is evidence for higher risk of ocular transmission for first-line medical workers and the need for ocular protection during high risk procedures. Many procedures such as tracheal intubation, dental surgery and electrocautery generate high concentration of aerosols which may contain the virus and increase the possibility of ocular landing and transmission . For ophGriffithsia spp. (in vitro study, griffithsin was found to prevent cell fusion and cell-to-cell transmission of HIV at a concentration of <1 nM by binding to its envelop protein gp120 (50 value to block the activity of SARS-CoV is 0.28\u20130.96 \u03bcM (In vitro and in vivo toxicology studies demonstrate that griffithsin has no cytotoxicity (Griffithsin is a small lectin consisting of 121 amino acids and is derived from sia spp. . Grifithsia spp. \u201332. In tsia spp. . In an iin gp120 . In micein gp120 . Griffitin gp120 . On crysin gp120 . Thus gr\u20130.96 \u03bcM . On mice\u20130.96 \u03bcM \u201339. More\u20130.96 \u03bcM . In vitrtoxicity . In summtoxicity , 43.As the ocular surface is continuously exposed to the environment, a prolonged eye protection is needed. Traditional eye drops may not provide sufficient protection due to blinking and drainage by nasolacrimal duct. It is estimated that drugs administrated via eye drops only reside in tears for 1\u20133 min and have very low bioavailability . Thus, sThe ocular surface is a possible transmission route of SARS-CoV-2, especially for medical staffs who work in close contact with infected patients. Theoretically, griffithsin can bind to S protein on virus envelop and inhibit the entry of SARS-CoV-2. Contact lenses with nanoparticles releasing griffithsin may be a way to protect the ocular surface from SARS-CoV-2 infection and provide a supplementary protection method for health care workers in daily practice.via close contact and droplets. There is ongoing hot debate on the potential role of ocular surface in the transmission of SARS-CoV-2, and some clinical and laboratory findings support that ocular involvement was observed in a minority of patients. For medical workers with high risk of aerosol exposure and close contact with patients, ocular surface may be a potential and overlooked site of contamination. WHO has alarmed medical stuffs to wear protective goggles during the whole contact period with patients who were suspected or confirmed to be infected (The global pandemic of SARS-CoV-2 in 2020 has caused tremendous pressure on the health systems of almost every country in the world. Due to inappropriate protection and shortage of medical supplies, many medical staffs got infected . SARS-Coinfected .However, for daily medical practice in ordinary clinics, wearing eye goggles is not mandatory or always practical. Although eye goggles seem to provide the best protection and not harmful to ocular surface, they have several disadvantages. First of all, goggles are generally uncomfortable to use, and very likely to disturb vision due to extensive formation of water droplets. Thus protective goggles are very inconvenient for doctors who require precise vision, including ophthalmologists, dentists, surgeons and so on. Besides, long-term use of eye goggles is reported to disturb working status and may lead to increased medical errors. In a recent survey conducted during SARS-CoV-2 outbreak on 231 nurses in China, use of eye goggles caused headache, skin pressure injury and dizziness in 79%, 66%, and 49% of nurses, respectively. 82.7% of nurses subjectively reported that use of eye goggles negatively impacted their working status, and events of medical errors were reported in 19.5% of nurses wearing goggles . Due to E. coli, which could generate more than 20 tons of griffithsin per year at the cost of below 3,500$ (Based on the broad spectrum antiviral activity of griffithsin, we proposed a theoretical device of contact lenses with griffithsin nanoparticles as a potential alternative personal protective equipment against SARS-CoV-2. Although no previous data of the antiviral efficacy of griffithsin on SARS-CoV-2 is available, we made the hypothesis based on the efficacy study of griffithsin on other common viruses, including MERS-CoV, SARS-CoV, HIV, HCV, and so on \u201332. Grifw 3,500$ . This woDespite the potential benefit for griffithsin-loaded contact lens to act against ocular transmission of SARS-CoV-2, special attention should be paid to the safety concerns associated with contact lens wear. Incidence of infectious keratitis, Acanthamoeba and fungal infections related to contact lens use is on the rise in recent years . AccordiAs therapeutic contact lens can only cover the corneal portion of the eye, there is potential risk of uncovered part to be infected. However, griffithsin can dissolve into tear film and spread over the ocular surface. This will expand its protection area beyond the covered part. Besides, as shown in previous studies, griffithsin is a highly potent antiviral agent and is effective at very low concentration, which indicates that griffithsin dissolved in tear film may also have antiviral activity , 36. As in vivo (in vitro study showed that compared with other anti-viral lectins, application of griffithsin showed minimal effects of toxicity, T cell activation and alteration of gene expressions, which indicated excellent safety profile (in vitro and in vivo preclinical studies on the ocular safety of different doses of griffithsin are preliminarily required. The safety issues regarding long-term ocular application of griffithsin via contact lens need to be verified and the concentration of griffithsin need to be set at minimal inhibition concentration in order to avoid supratherapeutic toxicity. The protection benefits and potential adverse effects of griffithsin-loaded contact lens should be balanced and considered before applying for use in clinics.Another potential limitation of griffithsin-loaded contact lens is its potential ocular toxicity associated with the medication. Although lectin is commonly used in ocular formulation to improve drug retention time, currently no ocular formulation and safety profile of griffithsin on ocular tissues is available. As indicated in the inhibition study of SARS-CoV, griffithsin is multivalent and can effectively inhibit the virus at low concentration of 0.28\u20130.96 \u03bcM. A previous safety study showed that mucosal or systemic administration of 2 mg/kg griffithsin on mice should no systemic toxicity in vivo . An in v profile . To dateOverall, griffithsin-loaded contact lens can be considered as a supplementary choice for ocular protection besides eye goggles for health care workers during SARS-CoV-2 outbreak.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.LW conducted literature search. YD provided guidance and approved the final manuscript. Both authors proposed and discussed about the idea of the hypothesis and contributed to manuscript writing and editing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Chorioamnionitis has been linked to spontaneous preterm labor and complications such as neonatal sepsis. We hypothesized that microbial cell-free (cf) DNA would be detectable in maternal plasma in patients with chorioamnionitis and could be the basis for a non-invasive method to detect fetal exposure to microorganisms.The purpose of this study was to determine whether next generation sequencing could detect microbial cfDNA in maternal plasma in patients with chorioamnionitis.Maternal plasma (n = 94) and umbilical cord plasma (n = 120) were collected during delivery at gestational age 28\u201341 weeks. cfDNA was extracted and sequenced. Umbilical cord plasma samples with evidence of contamination were excluded. The prevalence of microorganisms previously implicated in choriomanionitis, neonatal sepsis and intra-amniotic infections, as described in the literature, were examined to determine if there was enrichment of these microorganisms in this cohort. Specific microbial cfDNA associated with chorioamnionitis was first detected in umbilical cord plasma and confirmed in the matched maternal plasma samples (n = 77 matched pairs) among 14 cases of histologically confirmed chorioamnionitis and one case of clinical chorioamnionitis; 63 paired samples were used as controls. A correlation of rank of a given microorganism across maternal plasma and matched umbilical cord plasma was used to assess whether signals found in umbilical cord plasma were also present in maternal plasma.Streptococcus mitis, Ureaplasma spp., and Mycoplasma spp. in cases of suspected chorioamnionitis. This assay also detected cfDNA from Lactobacillus spp. in controls. Comparison between maternal plasma and umbilical cord plasma confirmed these signatures were also present in maternal plasma. Unbiased analysis of microorganisms with significantly correlated signal between matched maternal plasma and umbilical cord plasma identified the above listed 3 microorganisms, all of which have previously been implicated in patients with chorioamnionitis . These data show that the pathogen signal relevant for chorioamnionitis can be identified in both maternal and umbilical cord plasma.Microbial DNA sequences associated with clinical and/or histological chorioamnionitis were enriched in maternal plasma in cases with suspected chorioamnionitis when compared to controls . Analysis of the microbial cfDNA in umbilical cord plasma among the 1,251 microorganisms detectable with this assay identified This is the first report showing the detection of relevant microbial cell-free cfDNA in maternal plasma and umbilical cord plasma in patients with clinical and/or histological chorioamnionitis. These results may lead to the development of a specific assay to detect perinatal infections for targeted therapy to reduce early neonatal sepsis complications. Chorioamnionitis is a common complication of pregnancy and affected patients are at risk for maternal and adverse neonatal outcomes  \u20135. AcuteIn cases of clinical chorioamnionitis, cultures from the mother and offspring are often obtained but commonly do not result pathogen identification nor affect the duration of broad-spectrum antibiotic therapy \u201317. Subc\u00ae Test, and showed that DNA sequencing of cell-free plasma can accurately detect microbial cfDNA in bloodstream infections and deep seated infections  \u201323 21\u201323fections , 30.The current study investigates whether microbial cfDNA can be identified in the maternal plasma and the umbilical cord plasma in patients with clinical or histological chorioamnionitis. Using a research use only (RUO) assay developed by Karius Inc., that includes a modification to the validated clinical Karius test to increase sensitivity to microbial signal, we could detect the presence of microbial cfDNA in maternal plasma and umbilical cord plasma in cases of chorioamnionitis.th, 2010 to December 8th, 2016. The UCSF and Wayne State University Institutional Review Boards approved the studies. Inclusion criteria for the observational studies were: any viable pregnancy, maternal age >18 years, ability to sign informed consent, and intent to deliver and receive antenatal care at either the UCSF or Hutzel Women\u2019s Hospital. Exclusion criteria included: nonviable pregnancies or inability to consent. Patients were followed until delivery. Clinical chorioamnionitis was defined by the presence of maternal fever, and either maternal leukocytosis, uterine fundal tenderness, maternal tachycardia (>100/min), fetal tachycardia (>160/min) and purulent or foul amniotic fluid; and histological chorioamnionitis was defined by neutrophilic infiltration of the chorion.Samples for next generation sequencing (NGS) were selected among residual plasma samples with an available volume of at least 50\u03bcl, collected from two prospective observational studies at the University of California, San Francisco, CA (UCSF) and Hutzel Women\u2019s Hospital in Detroit, MI, part of the Detroit Medical Center, and a major affiliate of the Wayne State University School of Medicine. Samples for NGS were selected among samples banked between January 15\u00ae , processed to plasma by centrifugation (1500 rpm for 10 minutes at room temperature), within 72 hours of collection and stored at -80\u00b0C until shipment to Karius, Inc.  for processing and analysis.Whole blood was collected from patients prior to or shortly after delivery in an EDTA-coated Vacutainer\u00ae 500. Negative controls and positive controls were processed alongside patient samples in every batch. Sequencing reads identified as human were removed, and the remaining sequences were aligned to a curated pathogen database. Abundances for over 1,000 organisms in the Karius clinical reportable range were computed. For detailed methods, see cfDNA was extracted from plasma, DNA libraries constructed and sequenced on an Illumina NextSeqTwo filters were applied to the data before visualization as a heatmap. First, we removed any samples that were dominated by vaginal or gut commensal microorganisms that likely entered the umbilical cord blood sample during collection. Specifically, we removed samples for which 5 or more microorganisms known to be present in normal gut and/or vaginal flora were seen significantly (with p< = 1e-35) above baseline levels determined by negative samples. The threshold of 5 was identified by comparing the distribution of significance levels for all microorganisms in each sample.To account for the background level of each microorganism, originating from either the patients or potential environmental contamination, we converted the estimated concentration of each microorganism in each sample into a z-score where the population is considered to be the set of concentrations of that microorganism across all samples. The heatmap presents microorganism concentrations with z-scores greater than 2, i.e. those that are 2 standard deviations above the cohort mean.In the heatmap, the microorganisms are ordered as consecutive leaves of a tree built from standard taxonomy and the samples were ordered, within preterm and at term births, using Unweighted Pair Group Method with Arithmetic Mean hierarchical clustering using the Pearson\u2019s distance. A black box denotes the microorganism concentrations for which the null hypothesis of having originated from environmental contamination is rejected at a significance threshold of 1e-35.We expect microorganisms that are the source of infection to be elevated in the maternal plasma as well as the umbilical cord plasma, whereas the abundance of contaminants should be independent from one sample to another. Therefore, to identify microorganisms causing infection in this set of patients, we calculated for each microorganism the correlation of the abundances in the maternal plasma samples with their paired umbilical cord plasma samples. The measure used for this correlation was a p-value representing the significance of the enrichment of the microorganism above the expected background. The assumption was that the microorganisms driving the infection would be observed with abundances that are significantly above background in both matching samples. To account for the fact that the signal may be weaker in the maternal plasma than the umbilical cord plasma, we used a Spearman rank correlation for this analysis. P-values for the Spearman correlation were ranked and displayed in comparison to a uniform null distribution. A Benjamini-Hochberg correction was applied and thresholds for a false discovery rate of 0.05 and 0.1 were displayed on the plot.Microorganisms for which the abundance in negative control samples changed significantly due to a reagent lot change were removed from the Spearman correlation analysis. If the mean abundance changed by a factor of at least 2 and a difference of at least 10 molecules per microliter after a lot change, the microorganism was removed from the analysis.120 umbilical cord plasma and 94 maternal plasma samples were sequenced, among which 111 and 89 passed quality control, respectively . We subsStreptococcus mitis, Ureaplasma spp., and Mycoplasma spp. were enriched. Two out of three cases with confirmed histological chorioamnionitis and high abundance of Streptococcus mitis also had clinical chorioamnionitis and neonatal sepsis. Neonates with Streptococcus mitis were born prior to 32 weeks of gestation. In patients without evidence of chorioamnionitis (n = 63), NGS detected a signal for multiple Lactobacillus and Staphylococcus spp. A list of all the microorganisms identified at levels considered significant by the clinical validated assay can be found in Because the signal of cfDNA is strongest near the site of infection and likely more prevalent within the umbilical cord plasma than maternal plasma, we first assessed the microbial signature of chorioamnionitis in the umbilical cord plasma. The microbial abundances of organisms detectable with this RUO assay were compared and microorganisms at levels above baseline of negative control samples are shown in a heatmap . 27/90 pStreptococcus mitis, the mean abundance was over 3 orders of magnitude higher in the chorioamnionitis group  maternal plasma samples had a positive result, with the presence of microorganisms at a significant level . Among tComamonas testosteroni, Ralstonia pickettii, and Cupriavidus metallidurans. The abundance over all negative control samples is plotted in We next analyzed correlations between the maternal and umbilical cord plasma. We calculated the p-value of the Spearman correlation of the abundance of each microorganism across the maternal samples and its abundance in the paired umbilical cord plasma samples. Exact abundance may vary across samples due to background levels of environmental microbial DNA. Hence, the measure we used for this correlation was a p-value representing the significance of the enrichment of the microorganism above the expected background. We first confirmed that each microorganism was not affected by the lot change and, therefore, was not a false positive see . This meStreptococcus mitis, Ureaplasma parvum, and Mycoplasma hominis that show a strongly correlated signal in maternal and cord plasma. M. hominis and U. parvum are significant after Benjamini-Hochberg correction for multiple testing hypothesis with a false discovery rate (FDR) of 0.05, and S. mitis is significant only with an FDR of 0.1. This unbiased analysis suggests these organisms are likely causing chorioamnionitis in the cases in which they are present.In cases of histologic chorioamnionitis, the plot suggests a general correlation between maternal plasma and umbilical cord plasma and identifies three microorganisms, Corynebacterium massiliense and Staphylococcus saprophyticus. Staphylococcus saprophyticus did not change abundance in the negative control samples, suggesting this could be a real signal. This pathogen is a known cause of urinary tract infections [In the patients that had negative histology results, most microorganisms are only correlated as much as expected by chance and therefore match the null distribution. However, there were two microorganisms that showed stronger than expected correlations: fections , 35, altWe demonstrate that NGS can detect microbial cfDNA in the maternal plasma of women with clinical and/or histological chorioamnionitis. There were numerous differences noted between preterm and term pregnancies with certain organisms seen abundantly in preterm patients and not in at term healthy patients. Lactobacillus spp. were found to be more abundant only in at term pregnancies . AdditioThis is the first study that utilized NGS to analyze maternal and umbilical cord plasma for microbial cfDNA. The majority of studies previously have relied on amniotic fluid cultures or molecular diagnostics of the amniotic fluid . HoweverPlasma NGS of microbial cfDNA is a rapid and non-invasive test that is achievable with small quantities of plasma. Future utility of this method will depend on improvements in detecting clinically relevant infections in the maternal plasma samples. The ultimate goal of this method is to diagnose infections during pregnancy from maternal plasma. This would allow for targeted antibiotic therapy before and after delivery and improve upon current practices in which broad-spectrum antibiotics are utilized that disrupt the neonatal microbiome and have known toxic side effects. While this first study in cases of chorioamnionitis show a relevant biological signal of microbial cfDNA in maternal plasma, additional developments are required to evolve this RUO technology into a prenatal diagnostic test.The strengths of this study include having a large matched set of maternal and umbilical cord plasma with well-documented clinical characteristics. Others strengths were the utilization of cfDNA rather than detection of whole organisms and the ability to use samples that were up to 8 years old.The main limitation of this study is that samples were collected within larger observational studies that were not designed to measure performance of NGS, and specifically of this RUO test, in this population. Specifically, samples analyzed in this study did not have matched orthogonal confirmatory tests for IAI, such as culture and PCR of the amniotic fluid. Therefore, the main set of analyses we performed were aimed at finding concordance between maternal plasma and umbilical cord blood plasma and enrichment of previously known chorioamnionitis-causing microorganisms. This work identifies a biological signal of IAI but does not yet function as a diagnostic.A major limitation with our study was the overall small number of cases of clinical or histological chorioamnionitis. Other limitations included the variability in sample collection inherent to observational studies, especially relative to antibiotic initiation, steroid therapy and latency until delivery. Additionally, abundance thresholds for clinical significance of each microorganism have yet to be established in this study population . MicrobIn conclusion, we report the first study demonstrating that NGS can detect microbial cfDNA in maternal plasma and umbilical cord plasma in patients with clinical or histological chorioamnionitis. These findings confirm the previously reported microorganisms in patients with clinical chorioamnionitis, IAI or neonatal sepsis. We also report a correlation between the abundance of microbial cfDNA in matched maternal and umbilical cord plasma. Prospective studies are needed to determine the utility of this test in the diagnosis and management of IAI.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 Appendix(DOCX)Click here for additional data file.S1 FigTo identify such taxa, we plotted and compared the mean abundance of each taxon before and after the lot change. If the mean abundance changed by a factor of at least 2 and a difference of at least 10 molecules per microliter, we removed the taxon from the analysis. This method identified microorganisms for removal that are plotted as shown.(TIF)Click here for additional data file."}
+{"text": "The receptor for advanced glycation end-products (RAGE) and the G protein-coupled angiotensin II (AngII) type I receptor (AT1) play a central role in cardiovascular diseases. It was recently reported that RAGE modifies AngII-mediated AT1 activation via the membrane oligomeric complex of the two receptors. In this study, we investigated the presence of the different directional crosstalk in this phenomenon, that is, the RAGE/AT1 complex plays a role in the signal transduction pathway of RAGE ligands. We generated Chinese hamster ovary (CHO) cells stably expressing RAGE and AT1, mutated AT1, or AT2 receptor. The activation of two types of G protein \u03b1-subunit, Gq and Gi, was estimated through the accumulation of inositol monophosphate and the inhibition of forskolin-induced cAMP production, respectively. Rat kidney epithelial cells were used to assess RAGE ligand-induced cellular responses. We determined that RAGE ligands activated Gi, but not Gq, only in cells expressing RAGE and wildtype AT1. The activation was inhibited by an AT1 blocker (ARB) as well as a RAGE inhibitor. ARBs inhibited RAGE ligand-induced ERK phosphorylation, NF-\u03baB activation, and epithelial\u2013mesenchymal transition of rat renal epithelial cells. Our findings suggest that the activation of AT1 plays a central role in RAGE-mediated cellular responses and elucidate the role of a novel molecular mechanism in the development of cardiovascular diseases. The receptor for AGE (RAGE)\u2014a type I transmembrane glycoprotein of the immunoglobulin superfamily\u2014is a multiligand signal transduction receptor that can interact with multiple ligands, including AGEs, high-mobility group box-1 (HMGB1), and certain members of the S100/calgranulin family, to activate the ERK1/2 (p44/p42 MAP kinase) and JAK/STAT signaling pathways and promote downstream activation of NF-\u03baB3. Previous studies have revealed that mammalian Diaphanous homolog 1 (mDia1)\u2014a member of the formin family of Rho GTPase effector proteins\u2014 plays a key role in regulating RAGE-mediated intracellular signaling. The cytoplasmic domain of RAGE interacts with mDia1 to promote RAGE-mediated intracellular signaling and cellular migration5.Advanced glycation end-products (AGEs) are glycated proteins or lipids produced by non-enzymatic reactions between proteins and sugars, and are upregulated under pathophysiological conditions such as hyperglycemia in diabetic patients6. Previous studies have revealed several novel mechanisms for AT1 activation, including heterodimeric formation with other GPCRs11 and biased activation mechanisms by allosteric ligands14. Interestingly, Pickering et al. reported that AT1 forms a heteromeric complex with RAGE, and that AngII-induced inflammation and atherogenesis are mediated by the transactivation of RAGE15. However, it remains unknown whether there is a different directional crosstalk in this complex, whereby AT1, independent of mDia-1, is involved in RAGE-induced cellular signaling. We previously demonstrated that AT1 forms a complex with the lectin-like oxidized LDL (oxLDL) receptors (LOX-1) on the cell membrane, whereby oxLDL activates AT1 and causes vascular endothelial dysfunction in mice16. Given these findings, we hypothesized that RAGE ligands can initiate cell signaling by interacting with the RAGE/AT1 complex on the cell membrane.Angiotensin II (AngII) type I receptor (AT1)\u2014a member of the G-protein coupled receptor (GPCR) family\u2014in the renin\u2013angiotensin system (RAS) plays an important role in the pathogenesis of cardiovascular diseases and diabetesHerein, we investigated whether RAGE ligands can activate G proteins and their downstream signaling pathways in an AT1-dependent manner. We also investigated whether RAGE ligand-mediated cellular responses are abolished by the inhibition of AT1 in renal epithelial cells.NRK52E cells  or transgenic CHO cells were cultured in DMEM  supplemented with 5% fetal bovine serum  or F12 nutrient mixture with GlutaMAX-I  containing 10% FBS and appropriate selection reagents, respectively. Gene transcription in CHO cells was induced by adding 100\u00a0ng/mL doxycycline  . All cells were serum-starved for 24\u00a0h before stimulation.16. The Tet-On mutant AT1 vector was constructed using the PrimeSTAR mutagenesis basal kit  by deleting amino acids 221 and 222 from pTRE2hyg-HA-FLAG-hAT1 (pTRE2hyg-HA-FLAG-hAT1mt). The Tet-On human AT2 vector tagged with signal peptide-HA-FLAG at the N-terminus was constructed by subcloning full-length human AT2, according to the previous study (pTRE2hyg-HA-FLAG-hAT2)16. The Tet-On human mDiapahnous-1(mDia-1) was constructed by subcloning full-length human mDia-1 into pTRE2hyg (pTRE2hyg-hmDia-1).The V5-tagged human RAGE Tet-On vector was constructed by serially subcloning full-length human RAGE into pcDNA6.2/V5/GW/D-TOPO  and pTRE2hyg  (pTREhyg-V5-hRAGE). The Tet-On human AT1 vector tagged with signal peptide-HA-FLAG at the N-terminus was created in a previous study (pTRE2hyg-HA-FLAG-hAT1)16. CHO-RAGE-AT1, CHO-RAGE-AT1mt, and CHO-RAGE-AT2 were generated by the co-transfection of pTRE2hyg-HA-FLAG-hAT1, pTRE2hyg-HA-FLAG-hAT1mt, and pTRE2hyg-HA-FLAG-hAT2 and pSV2bsr vector into CHO-RAGE cells, respectively16. CHO-RAGE transfected with mDia-1 was also generated by the co-transfection of pTRE2hyg-hmDia-1 and pSV2bsr vector into CHO-RAGE cells. Stable transformants were selected with 400\u00a0\u03bcg/mL hygromycin B  and 10\u00a0\u03bcg/mL blasticidin S . Resistant clones expressing the respective genes in response to doxycycline were selected for use in experiments.CHO-RAGE cells were generated by the stable transfection of CHO-K1 Tet-On cells  with pTRE2hyg-V5-RAGE, according to a previous studyTagged RAGE and AT1 / AT1mt / AT2 in genetically engineered CHO cells were detected using mouse anti-V5  and rat anti-FLAG  antibodies, in combination with rabbit Alexa488-conjugated anti-rat IgG and goat Alexa594-conjugated anti-mouse IgG , respectively. 4\u2032,6-diamidino-2-phenylindole (DAPI)  was used to counterstain the nuclei. Images were acquired with a fluorescence microscope .Cells were seeded at a density of 150,000 cells per well onto 96-well transparent cell-culture plates and incubated overnight at 37\u00a0\u00b0C. The following day, the cultures were transferred to serum-free conditions and the cells were further incubated for 24\u00a0h. Thereafter, the cells were fixed by 4% paraformaldehyde without permeabilization, incubated with mouse anti-V5 or rat anti-FLAG antibodies, and then incubated with HRP-conjugated mouse or rat secondary antibodies, respectively. TMB reagents  were then added to each well and the colorimetric reaction was neutralized using a stopping solution . OD 450 values were measured using Multiskan Go . Measurement value of each sample was adjusted by subtracting that of negative control incubated with the corresponding secondary antibody in the absence of the first antibody.Membrane proteins were extracted using the transmembrane protein extraction kit . Immunoprecipitation was performed using the anti-FLAG-M2 affinity gel . FLAG-fusion proteins were eluted with 3\u2009\u00d7\u2009FLAG peptide . Subsequently, purified proteins were separated by SDS-PAGE under reducing or non-reducing conditions, transferred to PVDF membranes, and subjected to western blotting using antibodies against FLAG and V5.The cells were washed twice with PBS, and lysed using M-PER mammalian protein extraction reagent  with protease  and phosphatase  inhibitors. For western blot analysis, proteins were separated by SDS-PAGE and transferred onto PolyVinylidene DiFluoride (PVDF) membranes. The membranes were blocked with 5% non-fat dry milk and incubated overnight at 4\u00a0\u00b0C using the following primary antibodies: mouse V5 , rat FLAG , rabbit ERK1/2, rabbit phospho-ERK1/2, mouse \u03b1-Tubulin , or mouse \u03b1 smooth muscle actin (SMA) . Bands were detected using Chemi-Lumi One Super  and Chemiluminescence detection system LAS-4000 Mini .16. Images were acquired using the BZ-X700 fluorescence microscope. Quantitative fluorescence cell image analysis was performed using the BZ-X analyzer system .The in situ proximity ligation assay (PLA) was conducted using the Duolink kit  to determine the proximity of membrane receptors using mouse anti-V5 antibody  and rabbit anti-FLAG antibody , as reported previouslyCells were seeded at a density of 8000 cells per well onto 96-well transparent cell culture plates and incubated overnight at 37\u00a0\u00b0C. The following day, cultures were transferred to serum-free conditions and further incubated for 24\u00a0h. Thereafter, the cells were treated for 1\u00a0h with IP1 stimulation buffer mixed with the same amount of DMEM without phenol including vehicle, RAGE-BSA, HMGB-1, and angiotensin II at an indicated concentration at 37\u00a0\u00b0C, 5% CO2. Triton X was then added at a final concentration of 1%, and cell lysates were prepared after shaking the plates for 30\u00a0min. Finally, the cell lysates were transferred to 384-well white plates and IP1 levels were measured using the IP-One assay kit .The emission signals were measured at 620 and 665\u00a0nm after excitation at 320\u00a0nm, using the ARTEMIS plate reader.Cells were seeded at a density of 8000 cells per well into 96-well transparent cell-culture plates and incubated overnight at 37\u00a0\u00b0C. The following day, the cultures were transferred to serum-free conditions and the cells were further incubated for 24\u00a0h. Treatment with 10\u00a0\u00b5M losartan  or 10\u00a0\u00b5M FPS-ZM1  started 24\u00a0h or 30\u00a0min prior to the stimulation, respectively. siRNA for RAGE or AT1a was performed 24\u00a0h prior to the stimulation. Thereafter, the cells were treated for 1\u00a0h with DMEM without phenol, 1\u00a0mM IBMX, and 1\u00a0\u00b5M Forskolin including each reagent at the indicated concentration at 37\u00a0\u00b0C, 5% CO2. Triton X was then added at a final concentration of 1%, and cell lysates were prepared after shaking the plates for 30\u00a0min. Finally, the cell lysates were transferred to 384-well white plates and cAMP levels were measured using the cAMP dynamic 2 kit . The emission signals were measured at 620 and 665\u00a0nm after excitation at 320\u00a0nm, using the ARTEMIS plate reader.Silencer Select siRNA for RAGE (s135582 and s135583) or AT1a (s127417)  was transfected into NRK52E cells plated the previous day in a serum- and antibiotic-free medium using lipofectamine RNAiMAX , according to the manufacturer\u2019s instructions. The cells were treated with BSA or AGE-BSA 24\u00a0h post-transfection.AT1a (Rn02758772_s1 with the reference sequence in NM_030985.4) . We also performed SYBR Green real-time PCR for RAGE (5\u2032-TGAAGGTGGAACAGTCGCTC-3\u2032 and 5\u2032-ATCACCGGTTTCTGTGACCC-3\u2032) and GAPDH (5\u2032-GCCATCAATGACCCCTTCATT-3\u2032 and 5\u2032-TCTCGCTCCTGGAAGATGG-3\u2032). GAPDH was used as an internal control to normalize the expression level of each gene using the \u0394\u0394Ct method.Total RNA was extracted using the RNeasy Mini Kit  and transcribed into cDNA using the ReverTraAce qPCR RT kit . Quantitative real-time PCR was performed with a model 7900 sequence detector system  using TaqMan gene-expression assays for NRK-52E cells seeded in a 24-well plate were transfected with a plasmid containing luciferase driven by the NF-\u03baB binding site . The cells were cultured in medium containing 0.1% FBS for 24\u00a0h with or without an ARB, olmesartan . The cells were then pre-treated with or without a RAGE inhibitor, FPS-ZM1 , for 30\u00a0min and stimulated with 50\u00a0\u03bcg/mL BSA  or 50\u00a0\u03bcg/mL AGE-BSA  for 2\u00a0h. Luciferase activity was measured using the luciferase reporter assay system , according to the manufacturer\u2019s protocol. Measured values were normalized according to the protein concentration in each well.NRK-52E cells were pre-treated overnight with or without irbesartan or losartan , and subsequently stimulated with 100\u00a0\u00b5g/mL BSA , 100\u00a0\u00b5g/mL AGE-BSA , 3\u00a0\u00b5g/mL bovine HMGB1 , or 20\u00a0ng/mL TGF\u03b2  for 72\u00a0h in media supplemented with 0.1% FBS.t-test or one-way ANOVA with Bonferroni testing, respectively.All data are presented as the mean\u2009\u00b1\u2009SEM. The statistical difference between two treatments or among multiple treatments was determined using the Student\u2019s 17, or FLAG-tagged AT2\u2013the AT1 isoform . The immunofluorescence images of these cell lines are shown in Fig.\u00a015. Losartan and irbesartan did not alter the proximity between RAGE and AT1, indicating that the heterodimerized RAGE/AT1 complex is not uncoupled by ARBs 12. Herein, we assessed the activation of Gq and Gi by measuring inositol monophosphate (IP1) accumulation and the inhibition of forskolin-induced cyclic adenosine monophosphate (cAMP) production, respectively. AngII increased inositol monophosphate (IP1) levels in CHO-RAGE-AT1, but not in CHO-RAGE-AT1mt , G\u03b1i/0 (Gi), and G\u03b112/13We then used NRK52E cells, rat renal proximal tubular epithelial cells, to clarify if the observed machinery is relevant in primary cells that express endogenous AT1 and RAGE. AGE-BSA increased ERK1/2 phosphorylation, and the increase at 5 or 30\u00a0min was inhibited by the treatment of olmesartan  , a molecular marker of mesenchymal cellsMT) Fig.\u00a0f. TreatmMT) Fig.\u00a0g.23. The blockades of RAS downregulate TNF\u03b1-induced RAGE expression20 and attenuate RAGE-mediated signaling pathways such as AGE-induced NADPH oxidase signaling22. Pickering et al. recently reported that a heteromeric complex is formed between AT1 and RAGE, and that AngII transactivates RAGE to initiate RAGE-mediated signaling15. In the present study, we reported that the physical interaction between RAGE and AT1 on the cell membrane facilitates the signal transduction pathway from ligand binding with RAGE to the activation of AT1. These findings suggest that the crosstalk between RAGE and AT1 is bidirectional and far more direct than previously supposed.Previous studies have reported the interaction between RAGE and the RAS primarily at the transcriptional level5. Hence, further studies are required to understand the role of mDia1 in RAGE signaling with emphasis on its interaction with AT1. Importantly, our data suggest that AT1 activation induced upon the binding of RAGE ligands to RAGE has distinct characteristics from that induced upon AngII binding. AngII activates Gi and Gq through AT1; conversely, Gi, but not Gq, is activated by RAGE ligands via the AT1/RAGE complex. This can be explained by the recently proposed machinery of biased AT1 activation. Recent structural analyses have revealed that \u03b2-arrestin-biased agonists induce a less \u201copen\u2019\u2019 conformational change of AT1 compared to that induced by Ang II or other agonists with enhanced Gq coupling activity14. It was also shown that \u03b2-arrestin-biased AngII analogs preferentially activate Gi over Gq12. Future structural analysis will be required to clarify how the ligand\u2013RAGE interaction induces conformational change of AT1, but the mechanism of biased activation may explain why RAGE ligands and AngII exert distinct physiological and pathophysiological effects. For example, RAGE ligands do not increase blood pressure as much as AngII because the pressor function of AngII relies on Gq-dependent AT1 signaling. Among the G protein activation induced by AngII binding to AT1, we did not assess the G12/13 pathway that stimulates the activation of Rho kinases. It was reported that biased AngII analogs preferentially activate G12/13 as well as Gi over Gq12. Further studies are required to clarify if RAGE ligands activate G12/13 via an AT1-dependent fashion.Notably, our data suggest that mDia1 has no effect on RAGE ligand-mediated signaling in CHO cells lacking AT1. mDia1 is a key component in RAGE signaling due to its interaction with the RAGE cytoplasmic domain25. While the protective role of ARBs in the development of diabetic nephropathy has been extensively studied, certain inherent mechanisms have been presumed to exist that modulate the direct inhibition of Ang II-mediated AT1 activation. Our current findings provide a novel molecular basis to support the role of ARBs in diabetic nephropathy by directly inhibiting the signaling pathway of RAGE. Interestingly, in line with our findings, it was reported that an ARB, valsartan, inhibited the renal injury induced by the infusion of AGE-modified rat serum albumin, while the AGE inhibitor pyridoxamine inhibited the renal injury induced by Ang II infusion26. Nevertheless, given that ACE inhibitors are also shown to prevent the development of diabetic nephropathy, further investigation will be required to elucidate if, and to what extent, the pharmacological inhibition of AT1 has an additive class effect on the inhibition of diabetic complications other than the blockade of Ang II signaling.We found that the pharmacological and genetic inhibition of AT1 abolished RAGE ligand-induced cellular responses in renal tubular epithelial cells. ARBs were shown to attenuate RAGE ligand-induced EMT, a crucial cellular response in the development of diabetic nephropathyIn conclusion, the present study demonstrates that the cell-surface complex of RAGE and AT1 mediates the selective activation of AT1 induced by RAGE ligands. This phenomenon is relevant in RAGE ligand-induced intracellular signal transduction and cell phenotype change in kidney proximal tubular cells. These findings provide novel insight into the crosstalk between RAGE and AT1, both of which have critical roles in the development of cardiovascular diseases.Supplementary Figures"}
+{"text": "Vulcanolepas fijiensis. The mitogenome was 17,374\u2009bp long, with 76.6% AT content. Its protein-coding gene organization was identical to that of the deep-sea scalpellid Arcoscalpellum epeeum. On the mitogenomic tree, two scalpellomorphan families  were monophyletic while the other scalpellomorphan family Pollicipedidae did not form the monophyletic group with them. Further mitogenomic analysis of undetermined taxa in hydrothermal vents is required to deepen our understanding of their phylogenetic relationships.The family Eolepadidae is the only stalked barnacle in hydrothermal vent regions. Here, we determined the mitogenome of the eolepadid  Eochionelasmus clade . Genomic DNA extraction, sequencing, gene annotation, and phylogenetic analyses followed the methods of Kim et\u00a0al.  by Sanger sequencing. The specimens have been deposited in the National Institute for Biological Resources, Korea (NIBR-IV0000830370) and the Biodiversity Research Museum of Academia Sinica, Taiwan (ASIZCR-000411).On 5 December 2016, m et\u00a0al. , 2018. AV. fijiensis, which was determined with the same individual obtained the CO1 sequence on GenBank submitted by the original description . Their tRNAs rearranged between CYTB and ND1 and between 12S rRNA and ND2.The complete mitogenome of Trp.All of the PCGs had an ATN start codon while ND1 was inferred to begin with GTG and COX1 was not determined. Most of the PCGs terminated with a complete stop codon (TAA or TAG), although COX1, ND3, and ND4 had an incomplete stop codon (T\u2013\u2013). The 16S and 12S rRNAs were 1316\u2009bp (73.4% AT content) and 730\u2009bp (68.1% AT content), respectively. The tRNA genes ranged from 56 to 70\u2009bp in size. A 890-bp-long (76.6% AT content) non-coding region was located between the 12S rRNA and tRNAV. fijiensis and the scalpellid A. epeeum formed a monophyletic clade with high support values of 90% BP and 1.00 BPP. While the pollicipedid Pollicipes polymerus in the intertidal zone did not form the monophyletic group with V. fijiensis and A. epeeum.Phylogenetic trees were constructed with the PCGs of 18 barnacles using maximum-likelihood (ML) and Bayesian inference . The treFurther mitogenomic analysis of undetermined taxa in the major vent clade, Neobrachylepadidae, Neoverrucidae, and other eolepadid genera, is required to deepen our understanding of their phylogenetic relationships."}
+{"text": "M. tuberculosis in the cytoplasm of ciliated bronchiolar epithelium and alveolar pneumocytes in association with elements of the programmed death ligand 1 (PD-L1), cyclo-oxygenase (COX)-2, and fatty acid synthase (FAS) pathways in the early lesion. This suggests that M. tuberculosis uses its secreted antigens to coordinate prolonged subclinical development of the early lesions in preparation for a necrotizing reaction sufficient to produce a cavity, post-primary granulomas, and fibrocaseous disease.The characteristic lesion of primary tuberculosis is the granuloma as is widely studied in human tissues and animal models. Post-primary tuberculosis is different. It develops only in human lungs and begins as a prolonged subclinical obstructive lobular pneumonia that slowly accumulates mycobacterial antigens and host lipids in alveolar macrophages with nearby highly sensitized T cells. After several months, the lesions undergo necrosis to produce a mass of caseous pneumonia large enough to fragment and be coughed out to produce a cavity or be retained as the focus of a post-primary granuloma. Bacteria grow massively on the cavity wall where they can be coughed out to infect new people. Here we extend these findings with the demonstration of secreted mycobacterial antigens, but not acid fast bacilli (AFB) of  Many investigators today believe that cavities form by erosion of caseating granulomas into bronchi. Pathologists in the pre-antibiotic era knew that this is wrong. Cavities form by dissolution of caseous pneumonia, not by erosion of granulomas . EarlierThe early lesions of PPTB do not begin until after establishment of sufficient immunity to prevent disease in all extra pulmonary and most pulmonary sites . PPTB bePost-primary granulomas are easily identified because they form to surround preexisting foci of caseous pneumonia and therefore contain ghosts of alveoli rather than the homogeneous caseum of primary granulomas . PrimaryHere we report finding M. tuberculosis (MTB) antigens, but not acid-fast bacilli (AFB), in alveolar lining cells (pneumocytes) and ciliated bronchial cells together with elements of the programmed death ligand 1 (PD-L1), cyclo-oxygenase (COX)-2, and fatty acid synthase (FAS) pathways in developing lesions of PPTB. This suggests that MTB use secreted antigens to orchestrate an extended sequence of subclinical lesions that prepare for a sudden massive necrotizing pneumonia sufficient to produce cavities from which the organisms can escape to new hosts. Post-primary granulomas arise to surround foci of caseous pneumonia that are not coughed out to form cavities. They persist to become fibrocaseous disease. Most nascent post-primary lesions regress spontaneously leaving apical scars. If we understood why they regress, it might be possible to make them all regress and thereby drive MTB to extinction.Histologic slides from over 50 autopsies of people who died of untreated TB were examined to identify tissue sections with the early lesion of PPTB as previously described ,7. BriefThe early lesion of PPTB is a post-obstructive lobular lipid pneumonia that spreads via bronchi for months prior to caseation ,6,8 Fig. SecreteSince MTB is an obligate human parasite, everything that it does has been selected to ensure survival and transmission among people. MTB secretes many protein antigens that are required for virulence in humans but are not necessary for growth in culture . The funThe early lesion of PPTB is an asymptomatic obstructive lobular pneumonia that develops for 1\u20132 years before onset of symptoms . It has Bronchial obstruction traps alveolar macrophages to produce post-obstructive lipid pneumonia .Alveolar lining cells use FAS to produce lipids.Alveolar macrophages with M2 phenotype (CD163 Staining) become foamy by accumulating host lipids and secreted mycobacterial antigens.Sensitized tissue resident T cells (TRM) accumulate in alveolar walls.+ T cell activity.PD-L1 expression on alveolar macrophages and alveolar pneumocytes suppress PD-1The finding of MTB antigens in multiple types of cells of the early lesion suggests that MTB use these antigens to coordinate diverse components of the host response to subclinically produce the materials necessary for a necrotizing reaction sufficient to form a cavity large enough to support transmission of infection. The components of the early lesion include:These lesions spread via bronchi as an obstructive lobular pneumonia, recognized by CT scans as the \u201ctree-in-bud\u201d sign, until they either regress or undergo necrosis to become caseous pneumonia that is either coughed out to form cavities or retained to become the focus of post-primary granulomas .Our findings include (1) demonstration of mycobacterial antigen within both the type 1 and type 2 alveolar pneumocytes and ciliated bronchiolar lining cells in addition to alveolar monocytes/macrophages; (2) COX-2 expression in reactive alveolar pneumocytes and ciliated bronchiolar epithelium; (3) expression of FAS that could contribute fatty acids to the foamy alveolar macrophages; (4) expression of programmed death ligand (PD-L1) on the alveolar monocytes/macrophages, pneumocytes, and bronchiolar epithelial cells; and (5) CD163 expression on monocytes/macrophages in alveoli.+ cells were present, but abundant CD8+, PD-1+ cells were in the alveolar walls. Foamy macrophages stained strongly with CD68 and frequently also with the dendritic cell marker DEC-205. T-regulatory cells were also observed in the early lesion [In previous studies, we reported that the early lesion of PPTB contains foamy alveolar macrophages with the M2 phenotype that marked with CD163, PD-L1, phosphorylated mTOR, insulin-like growth factor-1 receptor (IGF-1R), and human cyclooxygenase 2 (COX-2) . Relativy lesion . These fy lesion .The early lesion of PPTB appears to be an attractive target for host directed therapies ,18. MiceSeveral lines of evidence also suggest that the early lesion might also be an attractive target for vaccines. First, many nascent lesions regress spontaneously. If we knew why, it might be possible to induce all to regress. Second, slowly progressive pulmonary tuberculosis in the mouse is a model of the early lesion of PPTB . SeveralFormalin-fixed and paraffin-embedded tissue blocks of human tuberculous lung samples were obtained during regular autopsy practice and after the completion of all medical, legal, and ethical requirements and were deidentified. Information provided included the age, sex, pulmonary TB as the primary cause of death, and negative HIV. Drug sensitivities were not known. Furthermore, this study was conducted according to the principles expressed in the Declaration of Helsinki. Hematoxylin and eosin (H&E) staining was used to identify blocks with the early lesion of PPTB . GrosslyImmunohistochemistry was performed on five micrometer sections that were deparaffinized and stained with monoclonal antibodies conjugated with 3,3\u2032-diaminobenzidine (DAB) for detection by immunohistochemistry. The antibodies used were MTB antigen -2 , programmed death-ligand 1 , programmed death-1 (PD-1), , and fatty acid synthase . Positive and negative controls were run concurrently. The immunohistochemical reactions/expressions were assessed as weak or strong by visual inspection in comparison with positive and negative controls. All procedures were conducted in a fully accredited clinical laboratory with all relevant positive and negative controls. None of the markers studied are present in appreciable concentrations in normal lung tissue G.We previously reported that the early lesion of post-primary TB is a distinct disease entity . The preM. tuberculosis needs both to survive. It must simultaneously protect its host and develop means to escape to new hosts. If protection by granulomas fails, the host dies of disseminated infection and the organisms die with it. M. tuberculosis only survives if post-primary lesions induce a cavity from which it can escape to find new hosts and the host remains healthy enough to circulate in the community.The early lesions produce cavities from which the organisms can escape to find new hosts, while granulomas protect the host from disseminated infection. As an obligate human parasite, The study of post-primary lesions will remain challenging because of the paucity of informative human tissues that can be used both to study the disease and to validate animal models. Fortunately, new multiplex technologies can measure proteins and nucleic acids on slides with a depth and precision undreamed of a decade ago . Such te"}
+{"text": "Nylon/saponite nanocomposites were synthesized and characterized. The nanocomposites were prepared by means of a fast, efficient, low cost, and environmentally friendly method. All of the tested preparations resulted in the pre-designed nanocomposites. To this end, delaminated saponites were directly synthesized to be used as a filler in a polymer matrix formed by nylon-6 by the in situ intercalation polymerisation of an \u03b5-caprolactam monomer without the use of surfactants or other organic reagents to organophilise the clay, thus avoiding the drawbacks of contamination. The efficiency of the preparation method increased, and significant savings were achieved in terms of both energy reaction time, savings of 60% and 70%, respectively, by using microwave radiation as an energy source during the synthesis of the nanocomposites. In addition, given that the method that was followed avoids the use of contaminating organophilic agents, it is important to highlight the savings in reagents and the fact that there was zero environmental contamination. In situ intercalation polymerisation was the first method that was used to prepare nylon-6/clay nanocomposites by researchers at Toyota from the monomer \u03b5-caprolactam ,2,3 and There has been a large number of studies on the efficiency of well-exfoliated polymer-clay nanocomposites compared to conventional composites as well as on their synthesis methods, which have been discussed in several papers and literature reviews ,6,7,8,9.The aim of this work to synthesise and characterise nanocomposites that have been prepared by means of a fast, efficient, low cost, and environmentally friendly method. To this end, delaminated saponites were directly synthesized following the method described by Trujillano et al. ,19,20, aThis objective seeks to increase the efficiency of clay synthesis preparation methods while avoiding the use of organic intercalants that should be removed by washing or calcination after the synthesis of the exfoliated clay, thus polluting water and/or the atmosphere. Other advantages that are sought are energy savings and decreasing the reaction time by using microwave radiation during the synthesis of the nanocomposites.3, Na2SiO3 solution (1.39 g/mL and 27% SiO2) MgCl2.6H2O (98\u2013100%), AlCl3.6H2O (95\u2013100%), and C6H11NO (\u03b5-caprolactam 99%) were used as reagents, and all of them were obtained from Panreac; N2 (99.95%), O2 (99.995%), and He (99.995%) were obtained from L\u2019Air Liquide Spain, S.A.NaOH, NaHCOSaponites were prepared following the method proposed by Kawi and Yao  and that2SiO3) aqueous solution was added to a solution (pH = 12\u201314) containing 10.86 g (0.27 mol) of NaOH and 19.78 g (0.23 mol) of NaHCO3 dissolved in 150 mL of distilled water (solution A). Another aqueous solution (B) containing 3.66 g (0.015 mol) of AlCl3.6H2O and 18.67 g (0.092 mol) of MgCl2.6H2O in 15 mL of distilled water was slowly added to solution A under continuous and vigorous stirring conditions. Once the addition was finished, the gelatinous white suspension that was obtained was magnetically stirred for one hour. All of these steps were carried out at room temperature. Then, the formed gel was sealed in a 100 mL Teflon reactor and was hydrothermally treated in a Milestone Ethos Plus microwave oven at 180 \u00b0C for 2 h while the gel was continuously stirred to avoid the platelets from stacking as much as possible. The suspension that was thus obtained was washed with distilled water until the chloride ions had been totally removed (checked by precipitation with AgNO3), and the suspension was then dried in an oven at 75 \u00b0C in air. This sample was labelled as SCA.A portion  of sodium silicate (NaThe method that was followed to prepare the clay/monomer mixture that was used as a precursor for the nanocomposites was based on the method reported by Fukushima and Inagaki : a mixtuPure nylon-6 (N6) was used as a reference, and the nylon-6 nanocomposites N6S5-X  were obtained by heating the precursors in sealed glass ampoules in a conventional muffle furnace.To prepare the N6 samples, 0.5 g of \u03b5-caprolactam was placed in a glass ampoule, and two drops (approximately 0.1 mL) of distilled water were added. The ampoule was then closed and was introduced into the muffle furnace, where the polymerisation reaction was conducted upon heating at 250 \u00b0C for 6 h. The nanocomposites N6S5-X were obtained following the same method as the one referred to for N6, but not water was added since the water that was retained by the clay together with the protons of the surface of the layers would hopefully result in the \u03b5-caprolactam ring opening, thus triggering polymerisation. As such, 0.5 g of SCACapro was placed in a glass ampoule with \u03b5-caprolactam and was then placed in the muffle, where it was heated at 250 \u00b0C for 6 h to produce the polymerisation reaction. The solid that was obtained was labelled as N6S5-6. To obtain the nanocomposites N6S5-24 and N6S5-48, the same procedure was followed, the only difference being the heating time .To reduce the working time and the amount of energy spent, the procedure that was described in the previous paragraph was repeated in order to obtain a new series of nanocomposites, which were designated as N6S5MW-X, where X represents for the reaction time , by using microwave radiation to carry out the polymerisation reaction, aiming to reduce the amounts of energy and the time needed.\u22121 by the KBr technique in a Perkin Elmer FT-IR-1600 spectrometer; a total of 12 scans were recorded to improve the signal-to-noise ratio at a nominal resolution of 4 cm\u22121. Specific surface area and porosity analysis were determined from the nitrogen adsorption\u2013desorption isotherms at \u2212196 \u00b0C and were recorded using a Gemini instrument from Micromeritics after the samples had been degassed for 2 h at 110 \u00b0C in a Flowprep 060 apparatus, which was also from Micromeritics. Differential thermal analyses (DTA) and thermogravimetric (TG) analyses were carried out simultaneously using SDT Q600 equipment from TA instrument, under a dynamic oxygen (50 mL/min) atmosphere at a heating rate of 10 \u00b0C/min.Element chemical analyses were carried out by atomic absorption in a Perkin Elmer Elan 6000 ICP Mass Spectra apparatus at Servicio General de An\u00e1lisis Qu\u00edmico Aplicado . The powder X-ray diffraction (PXRD) patterns were recorded in a Siemens D-500 diffractometer equipped with Diffract-AT software and a DACO-MP microprocessor, a copper (\u03bb = 1.5405 \u00c5) anode, and a graphite filter. The FT-IR spectra were recorded in the range of 4000\u2013400 cm8-xAlx][Mg6]O20(OH)4[M+]x\u22c5nH2O, and in our case, considering the Si/Al/Mg ratio in the reacting mixture, the targeted formula is [Si7Al1][Mg6]O20(OH)4[Na1]\u22c5nH2O. From the chemical analysis data of the solid, and the water content that was determined from TG (vide infra), the formula [Si6.85Al1.15][Mg6.00]O20(OH)4[Na1.54]\u00b79H2O\u00b72.81Mg(OH)2\u00b70.05Al(OH)3 was obtained. A relatively high excess of Mg was observed with respect to the available octahedral positions (6), strongly suggesting its presence as an extra-framework phase and that it had been tentatively formulated out of the formula of saponite as Mg(OH)2. The substitution in the tetrahedral layer is close, although slightly higher, than that of the targeted one. The value of the sum (Si + Al) is 8.05, while the layer contains 8.00 positions, so a small amount of silicon or aluminum may also be present as an extra-framework phase that was formulated out of the saponite formula as Al(OH)3. Thus, the prepared saponite has a high charge, about 1.15 units per half-cell, that is balanced by the interlayer Na+ cations and with a small excess of this element . All of the extra framework phases, which may include simple or mixed oxides that comprise the three elements that are in excess, should be amorphous, as only saponite was identified by powder X-ray diffraction  for the SCA sample is 1.54 \u00c5, a value that very close to that reported by Suquet et al. [A (001) diffraction peak due to saponite should be expected at around 15 \u00c5 in a well-ordered saponite, but it is not observed at all. The positions of the diffraction peaks of the sample SCA are close to those observed by Suquet et al.  in a Kozt et al.  that is t et al. . This re\u22121 is due to the stretching mode of the OH groups from the layers and from the H2O molecules; the broadness of this band is due to the hydrogen bonds between all of the responsible species (OH and H2O). The marked shoulder at 3686 cm\u22121 is due to the O\u2013H stretching mode of the Mg\u2013OH units, thus confirming the presence of a Mg-containing amorphous  [A steady mass loss amounting to approximately 5% of the initial sample mass due to removal of the hydroxyl groups from the clay structure is observed between 200 and 750 \u00b0C. At higher temperatures, between 750 and 850 \u00b0C, the DTA curve shows a very weak endothermic effect that is centred at 765 \u00b0C and that coincides with a slight (1.5%) mass loss that is immediately followed by two clearly recorded exothermic effects at 772 and 841 \u00b0C, which take place with no mass loss and that should correspond to crystallisation or to phase change processes. The first, endothermic effect at 765 \u00b0C is associated with the removal of the remaining hydroxyl groups, that is, the complete dehydroxylation of the clay layers, which leads to a recrystallization of the solid, giving rise to a new phase, a process that is associated with the first exothermic peak at 772 \u00b0C. This new phase corresponds to enstatite, a simple magnesium silicate with the formula MgSiO6Si2O13) ,29.2 adsorption and desorption ability at \u2212196 \u00b0C. The isotherm that was recorded for the SCA sample  diagrams of the starting sample (SCACapro) and of commercial \u03b5-caprolactam are included in Both diagrams are very similar concerning the positions of the peaks that were recorded; however, their relative intensities differ very much from one sample to the other. A very weak peak is recorded in the PXRD diagram of sample SCACapro at 4.35\u00b0 (2\u03b8), corresponding to a spacing of 20.30 \u00c5. No diffraction signal was recorded in or close to this position in the PXRD diagram of the SCA sample, Upon the addition of molecular sized \u03b5-caprolactam, around 7 \u00c5,  to the sAs mentioned above, in order to obtain a reference polymer, \u03b5-caprolactam was submitted to the same treatment  that was applied to prepare the nanocomposite samples; the sample that was thus obtained was called N6. The PXRD diagram of this polymer  shows th2) [1) is expected at 2\u03b8 = 20.05\u00b0, but its results are obscured by the much stronger diffraction peak of the monomer.Most of the maxima that were recorded in the PXRD diagram of sample N6S5-6 correspond to diffraction by \u03b5-caprolactam, and, in addition, the somewhat broader peak at 24.15\u00b0 (2\u03b8) corresponds to the second diffraction peak of the crystalline phase \u03b1 of nylon-6 (\u03b12) . The fir1 and \u03b12, respectively, of the \u03b1-phase of nylon-6. Moreover, the complete absence of any diffraction maxima due to the \u03b5-caprolactam monomer confirmed that 48 h is a long enough reaction time to achieve the complete polymerisation of \u03b5-caprolactam in order to produce a polymeric matrix.The fact that the diffraction maxima corresponding to free \u03b5-caprolactam molecules are still recorded in the PXRD diagram of sample N6S5-6 indicates that calcination for 6 h at 250 \u00b0C is not enough to produce the complete polymerisation of \u03b5-caprolactam. To complete the reaction, the temperature or the reaction time could be increased, and we opted to increase the reaction time. A new sample was prepared following the same process, but in this case, the reaction time was extended up to 48 h, as described in previous studies . The PXR1 and \u03b12) at of 20.45\u00b0 and 24.05\u00b0 (2\u03b8), respectively, in addition to a weak diffraction peak at 21.80\u00b0 (2\u03b8) due to the \u03b3 phase of the polymer [Once we checked that complete polymerisation had not occurred at 6 h but that it could be achieved after a 48 h reaction, we tried to optimize the procedure by using an intermediate reaction time, specifically 24 h, thus obtaining sample N6S5-24. The PXRD diagram of this sample  is ident polymer .It is worthwhile to mention the complete absence of any diffraction peak of the \u03b5-caprolactam monomer, thus concluding that heating for 24 h is enough for its complete polymerisation. Other experiments (not shown here) have confirmed that shorter reaction times (between 6 and 24 h) lead to incomplete polymerisation.The PXRD diagram for sample N6S5-24 in In order to accelerate the reaction and to minimize the use of energy, several tests were carried out using microwave radiation (MW) instead of conventional heating in an oven as a heat source. The SCACapro precursor was placed in a glass ampoule, closed, and submitted to microwave treatment at 250 \u00b0C for 6, 8, and 10 h to obtain the samples N6S5MW-6, N6S5MW-8, and N6S5MW-10, respectively. As with the previous set of samples, a short (6 h) reaction time was used first and then a long (10 h) reaction time, and once we determined that complete polymerisation had been achieved, we tested for intermediate reaction times (8 h). The PXRD diagrams of the three samples are included in 1 and \u03b12 diffraction peaks corresponding to the \u03b1-phase of nylon-6 at 20.15\u00b0 and 24.10\u00b0 (2\u03b8), respectively, in addition to rather weak diffraction peaks that are characteristic of the crystalline phase of \u03b5-caprolactam. As with the previous set of samples, which were prepared by conventional heating, the 6 h reaction was not long enough to produce complete \u03b5-caprolactam polymerisation, i.e., the polymerisation reaction took place but not completely.The diagram for sample N6S5MW-6 shows the \u03b1In a second step, the glass ampoule was submitted to microwave irradiation at 250 \u00b0C for 10 h, leading to sample N6S5MW-10. In this case, which can be seen Tests using shorter reaction times showed that 8 h  are included in \u22121, seen is \u22121 in the spectrum of sample N6S5MW-8 is due to the combination of the free N\u2013H vibrational mode and the vibrational mode of the O\u2013H bond in the water molecules. This band is recorded in the spectra of both samples: N6 and N6S5MW-8. A shoulder due to the N\u2013H bonds that are involved in the formation of the hydrogen bonds [\u22121 in the spectrum of sample N6S5MW-8; however, in the spectrum of reference sample N6, this shoulder is observed as a well-defined band at 3299 cm\u22121, confirming the existence of structural differences between both solids due to the introduction of the clay sheets in the polymer matrix. The band that is recorded at 3085 and 3088 cm\u22121 for samples N6 and N6S5MW-8, respectively, is due to an overtone in the amide group band.An extremely weak band at 3684 cm\u22121 in the FT-IR spectrum of sample N6 are due to the antisymmetric and symmetric vibration modes of the C\u2013H units in the methylene groups, respectively [\u22121 in the FT-IR spectrum of sample N6S5MW-8; the shift to lower wave numbers may be due to conformational changes in the lateral interactions between the polymer chains as a consequence of the presence of the clay sheets [\u22121, the first one due to the C=O stretching mode of the carbonyl groups and the second one to the N\u2013C stretching mode. Two bands that can be ascribed to the amide group vibrations in the \u03b1-phase of nylon-6 (amide III bands) are recorded at around 1260 and 1200 cm\u22121 in both spectra, together with a series of bands at 1120, 1129, and 960 cm\u22121, due to the stretching and bending vibrations of the structural methylene groups. The band at 928 cm\u22121 is due to the C\u2013C bond vibrations between the carbonyl carbon atom of the amide group and the carbon atom of the methylene group that are bound to the amide groups.The bands that are recorded at 2943 and 2869 cmectively . These by sheets . Two ban\u22121 is observed in both spectra and corresponds to the rocking vibration of the methylene groups of the polymer chains. The band at 690 cm\u22121 is due to the vibration of the N\u2013H bond (amide V). Finally, the bands that are recorded at 620 and 576 cm\u22121 are due to the vibrations of the carbonyl group in the \u03b3 and \u03b1 phase of nylon-6, respectively. The band that is attributed to the \u03b3 phase is more intense in the spectrum of sample N6S5MW-8 than it is in the spectrum of sample N6, whereas the opposite occurs for the band that can be attributed to the \u03b1 phase; this suggests that the introduction of a small amount of clay layers dispersed in the polymer structure favours the formation of the \u03b3-phase of nylon-6 versus the predominant \u03b1-phase in pure nylon-6 [In the lower wavenumber range, a band at 730 cm nylon-6 .The TG and DTA curves for samples N6S5MW-8 and N6 (for comparison) are included in A sudden mass loss starts at around 350 \u00b0C and extends up to 520 \u00b0C, corresponding to approximately 70% of the initial sample mass; this loss is due to combustion of the polymer, as confirmed by the very strong exothermic effect that is recorded in the DTA curve at around 475 \u00b0C. These thermal effects are also recorded in the curve for sample N6, but these are shifted towards somewhat lower temperatures, indicating that the polymer is thermally more stable when forming the nanocomposite than by itself. . No therFinally, Another noticeable difference that indicates the improvement in the thermal properties of the polymer thanks to the presence of these clay layers is that the decomposition of pure nylon-6 stars at 160 \u00b0C, whereas in the nanocomposite, it begins at 199 \u00b0C. This onset of decomposition is marked by the same endothermic effect in the DTA curves  of both w/w) was achieved by heating the samples in a furnace in the absence of water, with the reaction being initiated by the water molecules and hydroxyl groups of the clay surface. Complete polymerisation to nylon-6 was achieved after 24 h at 250 \u00b0C, leading to swelling of the saponite layers and the almost complete delamination of the saponite layers in the organic matrix. However, heating under microwave irradiation at 250 \u00b0C for 8 h was enough to achieve a complete polymerisation as well as total the disordering of the saponite layers in the organic matrix of the nanocomposite. The use of microwave irradiation strongly decreases the reaction time required for this process (8 h) when compared to conventional hydrothermal treatment (24 h). It should be also noted that in both cases, no water was needed to trigger the reaction, which was initiated by the water molecules and the hydroxyl groups of the saponite surface.Exfoliated synthetic saponites were obtained by a hydrothermal method under microwave irradiation. The \u03b5-caprolactam monomer was intercalated in the interlayer space of saponite in an aqueous suspension without the use of any organic compound to decrease the hydrophilicity of the clay surface. The polymerisation of the lactam in the nanocomposite (5%"}
+{"text": "M\u2009=\u200915.52; SD\u2009=\u20091.43). They were divided into two groups according to the score obtained in the South Oaks Gambling Screen-Revised for Adolescents (SOGS-RA). Thus, 84 were included in the group without risk of gambling problems and 122 in the group at risk of gambling problems. The results obtained revealed higher scores in negative affect and pathological gambling in the group at risk of gambling problems. Likewise, positive relationships between alexithymia, maladaptive emotion regulation strategies (MERS), and affect were found. Mediation analyses showed that difficulties in identifying feelings were indirectly related to greater use of dysfunctional ERS through their relationship with negative affect in at-risk gamblers.Alexithymia, difficulties in emotion regulation, and negative affect play an important role in adolescents who present pathological gambling. Therefore, the objectives of the present study were, firstly, to analyze the differences between alexithymia, difficulties in emotion regulation, and positive and negative affect in adolescents with and without risk of gambling problems. Secondly, the relationships between all the variables of the study in adolescents with and without risk of problem gambling were analyzed separately. Thirdly, we analyzed the mediating role of positive and negative affect in the relationship between alexithymia and dysfunctional emotion regulation strategies (ERS) in adolescents at risk of gambling problems. The sample was composed of 206 adolescents with ages ranging from 12 to 18\u00a0years ( Gambling disorder, which has been included as an addictive disorder in the last DSM edition , consists of persistent and recurrent gambling behavior despite its negative consequences. Gambling has become one of the most frequent addictive behaviors in adolescents and young adults , and positive and negative affect among gamblers with and without problematic gambling behavior; second, to analyze the relationship among alexithymia, MERS, and positive and negative affect separately between both groups; and third, to study the mediating role of positive and negative affect in the relationship between alexithymia and MERS.M\u2009=\u200915.52, SD\u2009=\u20091.43) participated in this study. The sample was comprised of 96.5% of students, 2% of workers, and 1.5% who both studied and worked. Regarding the students, 41.5% were primary school students, and 49% were high school students, and the rest were university or professional career students (8.5%) or school drop-outs (1%).A total sample of 206 adolescents  with an age range of 12\u201318 (This sample was divided into two groups (with and without at-risk gambling profiles).The group of gamblers without at-risk gambling profile (84 participants) comprised a 97.4% of students, a 1.3% of workers, and a 1.3% who were both workers and students. Among the students, 46.1% and 47.4%, respectively, were high school and primary school students, and only 1.3% and 5.1% were university and professional career students.The group of participants who were at probable risk of gambling activity (122 participants) comprised a 96.5% of students, a 2.9% of workers, and a 0.9% of both students and workers. Among the students, 50.5% were high school students, 37.4% were primary school students, 9.6% were professional career students, 0.9% were university students, and 1.7% were drop-outs.Those with a total score of zero in the SOGS-RA were excluded from this study\u00b4s analyses.The South Oaks Gambling Screen-Revised for Adolescents to 5 (Strongly agree). The original scale is composed of three factors. Based on previous literature, in this study, we only used the DIF factor, which refers to problems identifying emotions and confusing them with physical symptoms. The global scale has shown good internal consistency , and the DIF subscale had a Cronbach's alpha of 0.78. In the current study, Cronbach's alpha was 0.87 for the global scale, and 0.86 for the DIF subscale.The Cognitive Emotion Regulation Questionnaire  to 5 . The nine strategies are grouped into two different factors. The first factor refers to emotional well-being and adaptive behaviors . The second factor includes maladaptive strategies associated with distress and psychopathological disorders . Self-blame\u00a0refers to thoughts putting the blame for what you have experienced on yourself; (2)\u00a0other-blame\u00a0refers to thoughts of putting the blame for what you have experienced on the environment or another person; (3)\u00a0rumination\u00a0consists of obsessively focusing on the feelings and thoughts associated with a negative event; (4)\u00a0catastrophizing\u00a0refers to emphasizing and overestimating the negative experience or its consequences; (5)\u00a0putting into perspective\u00a0consists of thoughts brushing aside the seriousness of the event or considering its relativity when compared to other events; (6)\u00a0positive refocusing\u00a0refers to redirecting attention to joyful or pleasant themes; (7)\u00a0positive reappraisal\u00a0refers to reinterpreting the event in positive terms of personal growth; (8)\u00a0acceptance\u00a0includes thoughts of nonjudgmental resignation; and (9)\u00a0refocusing on planning\u00a0refers to thinking about the steps that should be taken to handle the situation resulting from the event. The original instrument presents adequate psychometric properties . In this study, Cronbach's alpha of the global scale was 0.84, while the respective subscales presented Cronbach's alphas of 0.73 and 0.81.The Positive and Negative Affect Schedule . Scores on each subscale range from 10 to 50 points, with higher scores indicating more affect. Both subscales show high internal consistency . In the current study, the Positive and Negative Affect subscales showed alphas of 0.84 and 0.85, respectively.Clinical and non-clinical samples were collected. The non-clinical sample was obtained through educational institutions of the Basque Country (northern Spain) and other nearby educational institutions, using convenience sampling.. An email was sent to the educational institutions that were likely to have adolescent and/or young adult students. In total, 545 school directors were contacted, of whom 18 directors responded and 10 agreed to participate. On the other hand, the clinical sample was recruited from gambling disorder treatment centers. The institutions participating in the study received feedback on the results of the research. This sample was divided into two groups (with and without at-risk gambling profiles) as a function of their total score on the SOGS-RA. The group of gamblers without at-risk gambling profile included those who had engaged in at least one gambling activity in the last year (a SOGS-RA score of 1). The second included those who were at probable risk of gambling activity (SOGS-RA score of 2 or more).All participants were informed about the study and gave informed consent. Parental consent was requested for minors. Participation was voluntary, and confidentiality and anonymity were guaranteed. Participants did not receive any reward for collaborating in the study. Participants completed the questionnaire in online or offline versions. The questionnaire included general information about the main goals of the study, informing that there were no right or wrong answers and that participants could email the research team if they required further information about the study. The Institutional Review Board of the first author\u2019s university approved the study.1 and a2, respectively); whether negative/positive affect predicted MERS ; and whether DIF predicted MERS (path c). In parallel mediation, two variables (M1 and M2) were proposed to mediate the association between X and Y; thus, two indirect effects (a1b1 and a2b2) will be explored. Next, this model tested the direct effects of DIF on MERS (path c\u2019). Age and sex were included as covariates of no interest in the mediation analyses performed for both groups. A 95% bias-corrected confidence interval based on 5000 samples was calculated. In the ARG, we also controlled for the SOGS total score of the at-risk participants.To perform the parallel mediation analysis among our variables of interest, we first carried out Spearman's two-tailed correlations with SPSS. To complement the above analyses, we tested whether the relationship between higher difficulties to identify feelings  and higher use of MERS (Y) was mediated by negative (M1) or/and positive (M2) affect, using parallel mediation analysis (PROCESS macro v3.5 by Hayes) implemented in SPSS . This mediation analysis tested whether DIF predicted the negative/positive affect  and higher Positive Affect . Secondly, higher Negative Affect was related to more use of MERS , whereas higher Positive Affect was not significantly related to MERS . A 95% bias-corrected confidence interval based on 5000 samples indicated that the indirect effect through Negative Affect , holding Positive Affect constant, was significant  as the confidence intervals did not include zero. Conversely, the indirect effect through Positive Affect  was not significant . In other words, individuals with higher difficulties identifying feelings employ more MERS when they feel negative affect, but not when they feel positive affect. Furthermore, regarding the direct effect, higher DIF was associated with MERS when taking into account DIF\u00b4s indirect effect through the positive and negative dimensions of affect .Results from the parallel mediation analysis in the ARG group showed that greater difficulties to identify feelings (DIF) were indirectly related to higher use of MERS through its relationship with the Negative Affect subscale of the PANAS, but not with the Positive Affect subscale. First, as seen in Fig.\u00a0This study\u2019s first aim was to analyze the differences in MERS, alexithymia, and negative and positive affect among adolescents balanced for gender and sex, with and without risky gambling behavior. According to the results, the differences were significant in negative affect and gambling severity. These results follow the line of those obtained by K\u00e4llm\u00e9n et al. , who fouThe second aim of the study was to analyze the relationship between the studied variables separately, as a function of the presence of problematic gambling behavior. Results showed that MERS were related to negative and positive affect in the ARG group, whereas positive affect was not related in the group without gambling problems. This agrees with previous studies in which gambling disorder was related to difficulties in emotion regulation, sensation-seeking, and negative emotional states such as depression and anxiety (Tarrega et al., Finally, the third aim of this study included the analysis of the mediating role of positive and negative affect in the relationship between alexithymia and MERS in the ARG group, finding that negative affect mediated the relationship between difficulties to identify emotions and the use of MERS in this group but not in the non-problematic gambling group. These results agree with previous studies that show that negative affect mediated between alexithymia and the increase of the severity of gambling disorder (N\u00f6el et al., This study presents some limitations. First, it is a cross-sectional study, so causal relationships cannot be established. Additionally, SOGS-RA (Winters et al., As a conclusion, this study highlights the importance of understanding alexithymia and emotion regulation processes in individuals with at-risk gambling, in which a relationship between difficulties to identify emotions, negative affect, and MERS was found. According to these results, the acquisition of adequate emotion identification skills, especially negative emotions, may be relevant to avoid the use of MERS that may promote problematic gambling behaviors. These results are of interest for clinical interventions, as they increase our comprehension of the processes underlying gambling behavior and specify which variables should be addressed. These results may also be of interest for developing prevention programs that focus on better emotion identification and management."}
+{"text": "The purpose of the present paper is to predict the grain size of steel during the hot-rolling process. The basis represents a macroscopic simulation system that can cope with temperatures, stresses and strains of steel in a complete continuous rolling mill, including reversible pre-rolling and finishing rolling with several tenths of rolling passes. The grain size models, newly introduced in the present paper, are one-way coupled to the macro-scale calculations performed with the slice model assumption. Macroscale solution is based on a novel radial basis function collocation method. This numerical method is truly meshless by involving the space discretization in arbitrarily distributed nodes without meshing. A new efficient node generation algorithm is implemented in the present paper and demonstrated for irregular domains of the slice as they appear in different rolling passes. Multiple grain size prediction models are considered. Grain size prediction models are based on empirical relations. Austenite grain size at each rolling pass as well as the ferrite grain size at the end of rolling are predicted in this simulation. It is also shown that based on the rolling schedule, it is highly likely that recrystallization takes place at each pass throughout a continuous rolling mill. The simulation system is coded as a user-friendly computer application for industrial use based on programming language C# and an open source developer platform NET and runs on regular personal computers the computational time for a typical rolling simulation is usually less than one hour and can thus be straightforwardly used to optimize the rolling mill design in a reasonable time. In the present paper, the macroscopic simulation system, coping with a complete continuous rolling mill, including reversible pre-rolling and finishing rolling with several tenths of rolling passes ,2,3,4,5,Shaping any metal, such as in the hot rolling process, requires consideration of large plastic deformation. An overview of related multiscale physics is given in the following paragraphs. The start of this plastic deformation process is triggered when the dislocations can move inside a grain. This threshold is the yield stress, and its value depends on the dislocation density. By applying deformation and heat treatment, dislocation density and the grain sizes face a significant variation.If the strain rate and temperature are high enough, a different phenomenon, dynamic recrystallization, occurs. The dynamic recrystallization depends on the critical strain. As a result, new grain sizes appear, either partially or fully. However, not all recrystallization takes place immediately after exceeding the critical strain. After a particular time, meta-dynamic crystallization may take place.When the deformation is small, or there is no deformation, a softening process may occur due to sufficient time at high temperatures. The softening can again lead to partial or complete static recrystallization. Static recrystallization usually leads to larger grains with higher temperatures . The stoThe numerical prediction of grain size constitutes an essential aspect of microstructure studies . SmallerIn this paper, multiple grain size prediction models ,15,16,17Over the years, a comprehensive rolling simulation system has been developed for industrial use. The supporting steel factory provides rolling schedules in use, and macro-scale simulations have been up to now run by a successful macroscopic hot rolling simulation system . The mesIn the present paper, the possibility of complete or partial recrystallization is modelled. This makes more realistic the assumption of complete recrystallization at each roll pass, as assumed in some publications . FurtherIt is pointed out by Sellars  that theThermo-mechanical simulation of rolling is based on 2D slices aligned towards the rolling direction. The slices were simulated one after another, and both thermal and mechanical models were solved in a coupled wayThe temperature in the thermal model and the mechanical model\u2019s displacement field are interpolated with the multi-quadric (MQ) radial basis functions (RBF). The details of the solution of these two models have been previously published in .The boundary condition for the thermal model is of the Robin type,Interpolating an unknown field with regular node distribution over a rectangular domain works perfectly with meshless Local Radial basis Function Collocation Method (LRBFCM) and is also easy to implement numerically. However, severe deformation simulations, such as rolling or forging, require redistribution of the computational nodes. In other words, a solution with the regular node distribution becomes impossible in large deformations. An essential advantage of the LRBFCM is that it gives stable results also with scattered node distribution. A comparison of the results is shown later in this paper where a rectangular domain is interpolated with regular and scattered node distribution.LRBFCM interpolates the temperature field in the thermal model, and the governing equation is solved through explicit time stepping. The time step is chosen as 1 ms. The nodes of the mechanical and thermal models coincide. The solution is obtained through local interpolation for each influence domain without creating a global matrix. In the mechanical model, interpolation of displacement filed over each local influence domain can be assembled into a global solution matrix  occurs when the critical strain is reached, but time is not long enough for meta-dynamic recrystallization. The grain size is predicted from the following equationThe dynamic recrystallization (The coefficients of Equation (10) are defined in MDRX) occurs. If this is the case, the grain sizes must be recalculated from the following equation,The meta-dynamic crystallization is also called post dynamic recrystallization. Its effect on the grain size is similar to the dynamic recrystallization; however, there is a time-based criterion that includes strain rate. In this paper, the following definition is used ,(11)tMDThe grain size prediction model for the meta-dynamic and dynamic recrystallization is the same, but the coefficients are different. The constants of Equation (12) are defined in There is a time criterion for static recrystallization (SRX) to occur, and in this paper, a model defined by Zhang et al.  for highThe coefficients for Equation (14) are defined in After complete recrystallization, the grains will grow until the next partial or complete recrystallization. In the present paper, the following grain growth model to calculate the new grain size is considered for three types of steel.The coefficients for Equation (15) are defined in It is known from the experiments that recrystallization may not occur every time, even though the conditions are met. This limitation comes from the temperature value. If it is below a certain value (given in (16)Tnr=After the steel leaves the last rolling stand, it is cooled in a controlled way at the cooling bed. During the cooling process the austenite phase transforms into ferrite and perlite, especially for C-Mn steels. In this paper, only the ferrite transformation is considered, and the temperature at which the transformation occurs is called Ferrite Transformation Temperature (FTT). (FTT) for any type of steel in \u00b0C is calculated based on the following equation given in :(17)TFTTThe steel has to cool down until the maximum temperature goes below the FTT, and with that specific cooling rate at that temperature, the ferrite grain size can be predicted based on the following equation.The necessary coefficients are defined in d Beynon  is consiMacro-scale simulation is followed by micro-scale simulation for each slice at a time except for the final ferrite grain size, which is calculated at the end. The major steps of the macro and micro-simulation steps and the coupling between them can be seen in After the continuous casting in a typical steel production line, the strand proceeds to a reheating furnace to remove the cast dendritic structures and homogenize most alloying elements . The strMost of the rolling schedules used in the simulations may be categorized into two groups when rolling the billets. In the first group, oval grooves were used to achieve more oval or round-shaped forms. In the second group, primarily flat rolls were used to get more slab-like shapes. Regular node distribution was adequate for most of the slab simulations; however, regular nodes faced stability issues for a schedule that turns a rectangular billet into a round bar. Previously we used an Elliptic Node Generation (ENG) algorithm ,28 for pA fast, irregular node generation algorithm  is numerDue to the different texture of internal points near the boundaries and the rest of the internal points, an iterative node adjustment algorithm was used. In this way, the neighbouring nodes have a more uniform distance to the central nodes. The adjustment is based on a node repelling algorithm. The vector component of displacement In this section, multiple numerical results from the represented multiscale rolling simulation system  are illustrated. Macro-scale results are compared with regular and irregular collocation nodes and also compared with Finite Element Method (FEM) results. Later, an actual rolling schedule which consists of eight rolling stands, is applied. Micro-scale results are demonstrated in terms of grain size and recrystallization throughout the rolling schedule. The necessary values and definitions are given in 2 is flat-rolled into 34.5 mm height. The material is elastic with Young\u2019s modulus 106 Pa and Poisson\u2019s ratio 0.35. A regular node distribution with 546 nodes and irregular with 571 nodes are used, as shown in An initial slab with 217 \u00d7 45 mmThe results in terms of effective strain can be seen in More distinct differences occur when starting from an initial shape with curved corners, as obtained from the reversing rolling mill. The node arrangement is kept regular with ENG  and irreThe instability issue at the edge of the first contact is still carried out until the end of the simulation in the regular node arrangement. In The same flat rolling example as in FEM code , and a g2 end shape within a half millimetre error range.In this section, a rolling schedule from the industry with specific grooves are shown in Effective strain and temperature fields are the most critical inputs of the micro-scale models. The corresponding position and time of each slice have to be known throughout the simulation. In the simulations, the deformation occurred so rapidly that the time for meta-dynamic or static recrystallization was never reached. However, at each pass, soon after the first contact, a complete dynamic recrystallization was always achieved except for the model developed by Yada . In thatThe results of partial dynamic recrystallization obtained by the rolling simulation system are shown in A rolling schedule is used as described in The model by Yada  is shownFor the calculation of the final grain size in the ferrite phase, 16MnCr5 steel is considered. This steel grade has 0.16% carbon, 1.15% manganese and 0.95% chromium. The ferrite transformation temperature is based on the model given by Hodgson and Gibbs, calculated as 754.15 \u00b0C, and the user interface is displayed in Based on the model expressed by Yang, et al. , no-recrThe positions of roll passes which are used in Micro-scale calculations are based on a multitude of empirical correlations obtained from the experiments in the last four decades. The simulation system is capable of checking for meta-dynamic, dynamic or static recrystallization. When recrystallization occurs, a new grain size is calculated. If there is a full recrystallization, grain growth will start. At the end of rolling, cooling is simulated with the thermal model until it reaches the ferrite transformation temperature. The simulation ends with ferrite grain size prediction. A comparison of different models based on maximum and minimum grain sizes is shown. The accuracy of those results are limited to the accuracy of the input data which is simulated by the macro-scale model and the accuracy of the empirical micro-scale models. It is known from the industrial user experience that the accuracy of the macro-scale results is highly satisfactory. Therefore, if the empirical models are based on enough experiments, the expected grain-related results should be between the maximum and minimum simulated values.Another outcome of this research is to be able to come up with new grain size prediction models in the future. This is why all the definitions are implemented as user-defined functions. Numerous additional experimental results are needed to obtain this goal.The simulation system is currently limited to austenite and ferrite grain size predictions and assumes only one phase at a time. This will be improved in the future with multiple initial phase definitions and phase transformation criteria. The micro-scale models are also limited to the adequacy of those coefficients used. All the necessary coefficients for each steel type may not be found. It is a long and complicated experimental process to define all those parameters by analyzing numerous grain size data obtained by either using electron microscope  or ultraIn the present paper, a rolling simulation system capable of predicting the grain structure is described. First, a macroscopic thermo-mechanical simulation is performed, based on a 2D solution over each cross-sectional slice. Displacement, strain, stress and temperature fields are calculated considering the defined process parameters (rolling schedule), heat transfer and material properties. The solution is achieved through a truly meshless radial basis function collocation method. A new irregular node generation algorithm is used, which is shown, by FEM comparison, to perform better on irregular domains. This method requires only the distribution of collocation nodes over a domain, without classical meshing. It is possible to represent the complex domain shapes more accurately with the irregular node distribution as with the regular node distribution. Due to the high possibility of recrystallization at each pass, strain filed during the last rolling stand leads to relatively smaller ferrite grain size. It was shown in the simulations that dynamic recrystallization always took place at each roll pass. Meta-dynamic and static recrystallizations did not occur due to time restrictions. It was also expressed that no-recrystallization temperatures most probably does not stop recrystallization during hot rolling of steel above 1100 \u00b0C. A list of all the correlations used in this paper may be found in"}
+{"text": "Heroin-induced pancreatitis (HIP) is\u00a0rare\u00a0with only a few\u00a0cases reported previously in the literature and the\u00a0pathophysiology mechanism is yet to be investigated. We present two cases of acute pancreatitis (AP) in the setting of acute heroin (diacetylmorphine) intoxication. Both patients presented with nausea, vomiting and severe abdominal pain after intranasal heroin use. On laboratory analysis were found to have elevated serum lipase, positive urine toxicology for opioids, without any other obvious causes for AP. Both patients had a full recovery with supportive treatment. As a general approach, drug-induced pancreatitis is a diagnosis of exclusion and a high index of suspicion is required when the most common etiologies are ruled out. Drug-induced pancreatitis is relatively rare and often poses a diagnostic challenge for clinicians as more than 30 drugs are implicated in the definitive causality according to the World Health Organization, and opiates being one of them . To dateOpiates such as codeine, morphine are known to cause elevated serum amylase levels, pancreatic and biliary duct dilatation with spasm of the sphincter of Oddi -9; howevCase 1A 50-year-old male with past medical\u00a0history of hypertension, heroin use disorder presented to the emergency department (ED) with nausea, vomiting, and abdominal pain started after using 2-3 grams of intranasal heroin, daily,\u00a0for the past two weeks. Vital signs on presentation were: temperature 38\u2103, heart rate 100 beats per minute, blood pressure 157/102 mmHg, respiratory rate 20 breaths/min, and oxygen saturation 95% on room air. Physical examination was pertinent for moderate diffuse abdominal tenderness to palpation. Laboratory data revealed leukocytosis with left shift, acute kidney injury with serum creatinine 1.3 mg/dL, elevated liver enzymes with alanine transaminase/aspartate transaminase (ALT/AST) 45/45 IU/L, high serum lipase 389 U/L , negative blood alcohol,\u00a0calcium 10.4 mg/dL  is metabolized to morphine via cytochrome P450 2D6 (CYP2D6), a polymorphic gene that has three variations when it comes to O-demethylation, and depending on the gene activity, will generate ultra-rapid metabolizers, extensive metabolizers, and poor metabolizers ,16. UltrMorphine is broadly used for pain management in patients with acute pancreatitis, although on literature review, it is also associated with dysfunction of sphincter Oddi, including elevation in bile duct pressure, a significant increase in contractile amplitude and frequency of the sphincter of Oddi ,17,18.\u00a0ITreatment of drug-induced pancreatitis involves the withdrawal of the offending agent and supportive therapy; thus, an early diagnosis would help in reducing morbidity and length of hospital stay as seen in our patients. Within 24 hours of hospitalization, we were able to rule out the most common causes of AP and in the setting of known exposure to heroin, this was the most appropriate explanation of AP, and our patients did not receive any opioids for pain management.\u00a0Our patients can be categorized as mild AP as per Ranson\u2019s criteria in the setting of intranasal heroin use with negative CT findings, whereas previously reported cases ,10 had sGenerally, the subjects with drug-induced pancreatitis have a good prognosis with an uneventful clinical course ,7 as notAP is a serious condition with potential high morbidity and mortality if it is not diagnosed and treated in a timely manner. Drug-induced pancreatitis is a diagnosis of exclusion. A strong understanding of the drugs associated with its causality is needed, as knowledge of HIP may help clinicians to have a high index of suspicion and establish an early diagnosis. Further studies are necessary to evaluate the underlying mechanism, actual incidence, and the risk of HIP."}
+{"text": "The association between perinatal depression and infant cognitive development has been well documented in research based in high-income contexts, but the literature regarding the same relationship in low and middle-income countries (LMICs) is less developed. The aim of this study is to systematically review what is known in this area in order to inform priorities for early intervention and future research in LMICs. The review protocol was pre-registered on Prospero (CRD42018108589) and relevant electronic databases were searched using a consistent set of keywords and 1473 articles were screened against the eligibility criteria. Sixteen articles were included in the review, seven focusing on the antenatal period, eight on the postnatal period, and one which included both. Five out of eight studies found a significant association between antenatal depression (d = .21-.93) and infant cognitive development, while four out of nine studies found a significant association with postnatal depression (d = .17-.47). Although the evidence suggests that LMICs should prioritise antenatal mental health care, many of the studies did not adequately isolate the effects of depression in each period. Furthermore, very few studies explored more complex interactions that may exist between perinatal depression and other relevant factors. More high-quality studies are needed in LMIC settings, driven by current theory, that test main effects and examine moderating or mediating pathways to cognitive development. The global prevalence of perinatal depression has been estimated at 11.9%, but a recent meta-regression has shown a significant difference between high income countries (HICs) and low- and middle-income countries (LMICs) . Both stCognitive development refers to the whole range of mental activities and skills, including memory, language, learning, problem solving, perception and social cognition . During The perinatal period, and beyond into infancy, has received particular attention in HIC research because it represents an important and sensitive stage where children are at their most receptive stage of development \u20137. From The possible detrimental effects of perinatal depression on infant health and development have been well documented in research from HICs. Significant deleterious impacts have been observed in brain development, regulatory behaviours, acquisition of developmental milestones and various other behavioural, emotional, and physical outcomes \u201313. In aIn LMICs, where infants are generally exposed to a larger number and range of adversities, the impact of factors such as perinatal depression are likely to be magnified . AlthougFinally, although there now have been a number of individual studies that have explored the relationship between perinatal depression and infant cognitive development in LMIC settings, the overall literature in this context is not yet well developed and has not been synthesised. As such, it is expected that the majority of research identified in this review will be focused on investigating the presence of main effects of perinatal depression on infant outcomes, rather than on testing the more complex interplay between factors that has more recently emerged in the HIC literature.An important distinction demonstrated in research from HICs is that depression during the antenatal and postnatal periods exert independent effects on development and act on development through different mechanisms . AccordiThe primary purpose of this review was to synthesise the evidence for the independent and joint effects of antenatal and postnatal depression on the cognitive development of infants aged 0\u20133 years in LMICs. The strength of the evidence for the independent effects will be discussed in terms of whether studies have adequately controlled for concurrent depressive symptoms and depressive episodes at other time-points in the perinatal period. These findings can then be used to inform recommendations for early intervention and the progression of the literature in this area in terms of how studies in LMICs can be designed to more effectively test current theory.This research adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines for systematic review . A protoObservational studies  were eligible for inclusion if they quantitatively assessed the association between maternal depression during pregnancy and/or the first 12 months post-birth and cognitive development in infants aged 0\u20133 years, and if a) maternal depression was diagnosed through a standardised diagnostic interview, or a validated symptom questionnaire or screening tool, b) cognitive development was assessed using a direct, validated measure of cognitive or language ability, and c) the research was carried out in a LMIC as designated by the World Bank.Due to the emerging nature of the literature, this review has leaned toward overinclusion of studies, and therefore RCTs were also eligible for inclusion if they a) presented findings on the control arm of the study, b) present findings using baseline data, or c) they appropriately adjusted for intervention effects in the full sample. No historical time-limits were imposed on publication dates. Final searches included studies published in May 2020.Studies were excluded if a) depressive symptoms were assessed as part of a general mental health assessment from which it was not possible to isolate the effects of depression (e.g. SRQ-20), b) they examined specific patient populations (mother or infant) with any physical disease or disorder that may impact cognitive development, other than maternal depression, c) development was assessed using implied measures of cognition, or d) they were case-series\u2019 or case-studies.Defined as a non-psychotic depressive episode of mild to major severity that occurs during pregnancy or up to 12 months postnatal , diagnosThis review utilises an inclusive conceptualisation of global infant cognitive development that includes early mental, language and psychomotor milestones.A country designated as a LMIC by the World Bank at the point of data collection.Eligible studies were identified using electronic and manual searches in October 2018. Follow-up searches were completed in May 2020 to ensure that all relevant studies were included. PubMed, PsychInfo and CINAHL were identified as the most relevant databases and searched for relevant articles. Keywords relating to the research questions were identified and an overall search strategy was devised using Boolean operators to combine free text and MeSH terms . Free teStudy selection was completed in two phases . First, \u201cDemonstration that outcome of interest was not present at the start of the study\u201d) was not appropriate to the study design in question since many aspects of cognitive function cannot be assessed early in the perinatal period. However, rather than remove this item and adjust the standardised scoring system, it was decided to simply give all the studies a score of zero for this item. Thus, it may be that the quality assessments slightly underestimate the quality of each study. Following independent review, any disagreements regarding quality were solved by consensus.Study quality was evaluated independently by two reviewers (MBD and DP) using the Newcastle-Ottawa Scale (NOS) adapted for cohort studies . StudiesData extraction was completed independently by two reviewers (MBD and DP) using a standardised data extraction form that was created specifically for this review. Data extracted included country/setting, study design, aims/hypotheses, sample size, sample characteristics, measurement of exposure, measurement of outcome, perinatal stage at exposure, infant age at outcome, statistical approach, confounding variables, mediating or moderating variables, and main results . Following an initial piloting phase, data were extracted and compared. Any disagreements were resolved by consensus, and any issues that still lacked clarity were referred to HS. Where relevant data were missing study authors were contacted to request specific information.Extraction included the recording of variables that were defined a priori: socioeconomic status, maternal age, maternal education, infant gender, birthweight, intervention (if RCT). If meta-analysis had been possible this set would have represented a minimum set of adjustment variables to compare the effect size of \u201cadequately\u201d controlled studies and \u201cinadequately\u201d controlled studies. In practice, meta-analysis was not possible due to the variation between studies in terms of their context and the measurement tools used. However, this set of variables were considered when examining patterns in the data through narrative synthesis. Articles presenting findings regarding exposure to antenatal and postnatal depression were synthesised and discussed separately.The adjusted standardised mean difference (d) between depressed and non-depressed group was calculated wherever possible. If this was not possible, unadjusted effect sizes were calculated using group means and standard deviations. The strength of the effect size can be interpreted as follows: 0.2 = small; 0.5 = medium; 0.8 = large .16 studies were selected for inclusion in this review , 7 with Findings are grouped by perinatal period according to the main focus of each paper. Significant results are presented first, followed by non-significant findings. Findings are then briefly synthesised in terms of the adherence of each study to the requirements for assessing the independent effects of maternal depression within each period and the inclusion by each study of the a priori key adjustment variables.Unless otherwise stated, beta coefficients given for main effects are unstandardized and refer to the change in the number of points scored on a particular measure when a mother is depressed, compared to when a mother is non-depressed. Means and standard deviations are reported where available. All depression assessments are maternal self-report unless otherwise stated.rd trimester, one during the 2nd trimester and one simply stated that the assessment was conducted during the antenatal period. The Bayley Scales of Infant Development (BSID) were most commonly utilised for assessing infant outcome, with four studies using the Bayley-III [yley-III  and two yley-III . Anotheryley-III  and the yley-III  and an eyley-III . Age at Five out of eight studies found a significant association between antenatal depression and infant cognitive development . Four ofd = 0.15) on the Bayley-III. Importantly, the authors demonstrated that a significant association between depression and cognitive development at 6 months remained after controlling for both postpartum and concurrent depressive symptoms . Lin et al. [Arguably the most methodologically robust study in this review is Tran et al. . Althougn et al.  also conThe three other studies that found a significant association between antenatal depression and cognitive outcomes did not account for postnatal or concurrent depression in their analyses and so their results need to be interpreted with caution. Outside of this consideration, however, all are robust studies and are discussed next.d = 0.93) and they also scored significantly lower on the language subscale  than those of the non-depressed group . In the full Drakenstein Health Study sample, Donald et al. [d = 0.21). Final results for the language subscale were not reported.Two studies utilised data from the same prospective sample, the Drakenstein Health Study in South Africa. Breen et al.  used a nd et al.  reportedrd trimester blood manganese as a covariate, depression during the 3rd trimester was associated with significant deficits in both cognitive  and language skills  assessed by the Bayley-III. However, in the second model, which excluded antenatal levels of blood manganese but included levels of cord blood manganese during birth, the coefficient was of similar magnitude but was only marginally significant for both cognitive  and language skills .Similarly, Munoz-Rocha et al.  found evThe three studies that did not find a significant association between antenatal depression and cognitive outcomes received good  and fairp < 0.03) and 6 month (p < 0.05) postnatal depression. However, the effect size was attenuated and became marginal once intervention, risk and the intervention by risk interaction term were included in the final model . Risk was a composite measure that included items relating to maternal age, maternal education and SES. Although non-significant, these findings are indicative of a role for antenatal depression in infant cognitive development.The key difference between Murray et al.  and manyRotheram-Fuller et al.  utilisedFinally, Bandoli et al.  found noOut of nine studies reviewed, four found an association between postnatal depression and cognitive outcomes . One wasd = 0.07), current  and postpartum/current . Adjusted analyses showed that infants of mothers who reported depression had significantly lower language scores  and post-hoc tests revealed that this effect was only significant for the combined postpartum and current depression group.Quevedo et al.  was the d = 0.44) groups. When treated as dichotomous, with scores <85 considered to be at risk for developmental delay, infants of postnatally depressed mothers were significantly more likely to be delayed than infants of non-depressed mothers . Galler et al. [All other studies with significant findings did not account for either antenatal or concurrent depressive symptoms. Patel et al.  used a cr et al.  assessedth month of follow-up ) and the 12th month of follow-up ). There was no significant association between depression and language.Finally, Ali et al.  utilisedFive out of nine postnatal studies did not find a significant association between postnatal depression and cognitive outcomes. Of these five studies, four were fair quality , 66, 67 d = 0.32).Tran et al.  was the d = 0.08) in a sample from Bangladesh. Familiar et al. [p = 0.15).Hamadani et al.  found nor et al.  assessedd = 0.08) on Bayley-II mental development index scores at 18 months (\u03b2 = 0.08 p = 0.905). There were also no significant associations between early postpartum depression and performance on any of the tasks that formed the EF battery at 36 months . Similarly, there were no significant effects for any of the other depression groups. It is unclear whether beta coefficients reported are standardised or not.Garman et al.  utilisedp < 0.05), but the adjusted model, including earlier cognitive development at 6 months, revealed a non-significant association . Although an unadjusted association is reported, this may reflect the effects of the micronutrients given within the RCT.Finally, Black et al.  used a cThis systematic review of 16 studies investigating the association between maternal perinatal depression and infant cognitive outcomes in LMICs provides relatively strong evidence for an effect of antenatal depression but more inconsistent findings amongst postnatal studies. Five out of eight antenatal studies found a prospective negative association between depression and cognitive outcomes. However, only four out of nine postnatal studies found evidence of a prospective or cross-sectional negative association with infant cognitive development. While limited in number and mixed in design, most studies were of fair to good quality, with only three studies being rated as poor. It is important to consider and interpret this pattern of findings in the context of key study design features, as well as the size and quality of the evidence base, and the potential moderated effects that were not tested. This will be discussed in the following sections.d = 0.21\u20130.93) between antenatal depression and infant cognitive outcomes [There is fairly consistent evidence of a significant association between antenatal depression and infant cognitive development. All five antenatal studies that used a prospective, observational design report a significant association  between postnatal depression and cognitive outcomes [Evidence for an independent postnatal effect is far less consistent. Three studies, all rated as fair quality, found a significant and negative prospective association , this systematic review gives valuable insight into what is known about the relationship between perinatal depression and infant cognitive development in LMIC settings. A key strength of this study is the focus on, and in-depth exploration of, the association between cognitive development and perinatal depression alone. This contrasts with previous systematic reviews which have focused more broadly on a range of mental health disorders and neurodevelopmental outcomes, thereby retaining a high degree of novelty and adding considerable value to the existing literature. The findings provide a platform for the progression of research in this area by highlighting methodological and theoretical improvements that can be made to increase its relevance and impact.Evidence for a direct association between antenatal depression and cognitive development was quite consistent, especially when restricted to prospective studies. There was some evidence that this association is independent of postnatal depression and endures into the child\u2019s second and third year. This provides a strong case for the inclusion of screening for maternal depression as part of routine antenatal care and for early intervention to be implemented in LMIC settings. Evidence for the direct influence of postnatal depression is more equivocal at present.Importantly, findings from HICs suggest that the inconsistent findings regarding postnatal depression may be the result of its effect being evident in interaction with other factors. While there is some evidence of possible moderated effects of postnatal depression on cognitive development in LMICs, the majority of studies reviewed did not investigate these underlying pathways. The paucity of studies exploring anything more than a direct relationship between both postnatal and antenatal depression and cognitive development precludes drawing firm conclusions about this. More high-quality studies are needed in LMIC settings, driven by current theory, that test direct associations and examine moderating or mediating pathways to child cognitive development.S1 File(PDF)Click here for additional data file.S2 File(DOC)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file. 19 Mar 2021PONE-D-21-04110A systematic review of the association between perinatal depression and cognitive development in infancy in low and middle-income countriesPLOS ONEDear Dr. Bluett-Duncan,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE\u2019s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.plosone@plos.org. 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The PLOS ONE style templates can be found athttps://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf andhttps://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf2. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article\u2019s retracted status in the References list and also include a citation and full reference for the retraction notice.3. Please ensure that you refer to Figure 2 in your text as, if accepted, production will need this reference to link the reader to the figure.Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1:\u00a0YesReviewer #2:\u00a0Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1:\u00a0YesReviewer #2:\u00a0N/A**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data\u2014e.g. participant privacy or use of data from a third party\u2014those must be specified.The Reviewer #1:\u00a0YesReviewer #2:\u00a0No**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1:\u00a0YesReviewer #2:\u00a0Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1:\u00a0The question of whether or not maternal depression during the antenatal or postnatal periods influences cognitive development from birth to 3-years old in low- and middle-income countries is an important research question. This meta-analyses included 8 antenatal studies, 4 from South Africa, one from the Ukraine, one from China, one from Mexico and one from Vietnam. Postnatal studies were included from 8 countries, Pakistan, Bangladesh, Uganda, Barbados, South Africa, India, Brazil and Vietnam.The study from Vietnam found that antenatal depression was associated with lower cognitive scores at 6 months after controlling for postpartum and concurrent depression symptoms. In China, maternal depression was associated with lower language scores on the Gesell Scale at 24 - 30 months.Three other studies found a relationship between antenatal depression on cognitive outcomes but did not control for postnatal or concurrent maternal depression.Authors reviewed 9 studies of the relationship of postnatal depression on infant cognition at different time points between birth and 36 months, depending on the study. Studies took place in Pakistan, Bangladesh, Uganda, Barbados, South Africa, India, Brazil and Vietnam. Of these studies, 2 were assessed as poor quality, 6 as fair quality and one as good quality. Four of the studies found a main effect of postnatal depression resulting in lower scores on infant language development.The study also reviewed evidence for an effect of postnatal depression on infant development. In the settings of LMIC, these studies did not find an effect of postnatal depression on infant language development.What does this study add?Maternal depression during pregnancy adversely influences infant cognitive development. However, postnatal depression did not influence infant cognitive development in these settings. This finding raises questions about the role of environment in low and middle income countries on infant development and on the mother.The reference list is comprehensive.The focus on low and middle-income countries is important.Reviewer #2:\u00a0Review of --\u201c A systematic review of the association between perinatal depression and cognitive development in infancy in low and middle-income countries\u201d.By Matthew Bluett-Duncan, M. Thomas Kishore, Divya M. Patil, Veena A. Satyanarayana and Helen SharpThe impact of perinatal depression on cognitive outcome of offspring is an important area that still blurred and requires more insights for the future-direction of child-focused interventions. The authors made a good attempt to review the existing assorted literatures on this complex topic and summarize the take-home messages.The authors very clearly and elaborately described all the steps of the systematic review with references. They also detailed the working definitions of both exposure and outcome variables.However, they are encouraged to address the following concerns:\u2022For simplifying the findings, the authors made lots of sub-headings in result and discussion section that caused repetition of similar information This repetition is distorted the flow of reading due to lack of customization. So, some sections could be merged, e.g. \u201ckey adjustment sections\u201d does not need to be separately reported. Rather \u201cadjustment information\u201d can be blend with specific studies--- but the tables( 3 & 4) can be presented for better understanding.\u2022 Table 1 & 2 on antenatal & postnatal findings, need to be more informative. At least those cab include positive/negative findings. Otherwise it is difficult to relate results and discussion on antenatal or postnatal findings with several tables in main paper and in supplementary document.\u2022Sample size of study population and their follow-up percentages need careful checking. For example, sample size and follow up % of the study done by Garman et al in Table-2 is not matching with the journal information. In the journal, at follow-up, 58% children were assessed for developmental measures. Please clarify if the sample size in the tables refers to the numbers of mother-child dyads.\u2022Reference numbers are wrong in many places e.g. page 17, section 3.3.1, 2nd line, reference 56 will be 58; page 19, ref of Rotheram-Fuller will be 55 etc.\u2022 Throughout the write up please correct \u201cBSID-III\u201d-- It should be Bayley-III.\u2022 Finally based on the nature of majority of the studies , heterogeneous nature of the exposure/outcome assessments and huge drop-outs at follow up-- it will be better to say \u201csignificant association\u201d of antenatal or postnatal depression with cognition, rather than \u201csignificant effect\u201d, which is a more robust terminology.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,\u00a0AttachmentReview Comments_Perinatal Depression and ECD_PONE-D-21-04110.docSubmitted filename: Click here for additional data file. 8 Jun 2021Reviewer 1 comments to the author: no queries raised.Reviewer 2 Comment: For simplifying the findings, the authors made lots of sub-headings in result and discussion section that caused repetition of similar information This repetition is distorted the flow of reading due to lack of customization. So, some sections could be merged, e.g. \u201ckey adjustment sections\u201d does not need to be separately reported. Rather \u201cadjustment information\u201d can be blend with specific studies--- but the tables( 3 & 4) can be presented for better understanding.Author Response: The \u201ckey adjustment\u201d section was originally included as a distinct section as one of the key questions being asked by the review pertains to the ability of each study to isolate the variance in cognitive development that could be attributed to perinatal depression. It was felt that including this as a separate section was therefore justified. However, it is a valid point that its inclusion as a distinct section may not be necessary due to the inclusion of the information elsewhere, and that it serves to break up the flow of the narrative. Following the advice of the reviewer, this section has now been removed but the tables have been retained. The tables are now referenced in the initial descriptions of the antenatal and postnatal studies and the relevance of the information regarding the \u201ckey adjustment variables\u201d is referred to and discussed in the discussion section. We believe this retains the emphasis on the importance of these factors but removes some of the repetition.  We also considered whether any of the other sub-sections could be reduced or merged together, particularly in the \u201cResults\u201d section. However, we believe that the way they are currently set is optimal as it allows the reader to clearly distinguish between antenatal (section 3.2) and postnatal studies (section 3.3), and within them, those studies which found significant associations and those that did not. Similarly the layout of the discussion section allows the reader to clearly distinguish between the synthesis of the antenatal studies and the synthesis of the postnatal studies. We also believe that a separate section for methodological considerations is justified due to the emphasis in the review on assessing the robustness of the studies carried out in LMIC setting and the importance of clearly evaluating how different methodological decisions may have impacted results. While it may be possible to merge the discussion regarding methodological issues with the discussion of overall findings, the result would be a loss of clarity for both areas.Reviewer 2 Comment: Table 1 & 2 on antenatal & postnatal findings, need to be more informative. At least those cab include positive/negative findings. Otherwise it is difficult to relate results and discussion on antenatal or postnatal findings with several tables in main paper and in supplementary document.Author Response: We acknowledge that it would be beneficial to the reader to be able to easily see from the tables whether a study found a significant association or not. Therefore, we have now added a column to tables 1 & 2 to indicate whether or not a significant association has been found. We have not added any further information to these tables as the results section already describes the key findings from each study and we did not want to add in overly repetitive information. The two supplementary tables show a detailed breakdown of the study results for the antenatal and postnatal periods. Reviewer 2 Comment: Sample size of study population and their follow-up percentages need careful checking. For example, sample size and follow up % of the study done by Garman et al in Table-2 is not matching with the journal information. In the journal, at follow-up, 58% children were assessed for developmental measures. Please clarify if the sample size in the tables refers to the numbers of mother-child dyads.Author response: The reviewer is correct that the follow-up rate for Garman et al was incorrect on the table due to a miscalculation by the lead author. This has now been rectified and all the other follow-up rates checked carefully, with a couple of further minor revisions being made. Follow-up rates were calculated from the information provided by each study regarding the number of mother-infant dyads who completed developmental assessments and/or were included in the final analyses. This has now been clarified in the text as well.Reviewer 2 Comments: Reference numbers are wrong in many places e.g. page 17, section 3.3.1, 2nd line, reference 56 will be 58; page 19, ref of Rotheram-Fuller will be 55 etc.Author Response: The reviewer was correct in noticing that references were incorrectly labelled in a few instances. References have now been checked and corrected where necessary.Reviewer 2 Comments: Throughout the write up please correct \u201cBSID-III\u201d-- It should be Bayley-IIIAuthor Response: This has now been changed to Bayley-III. BSID-II has also been changed to Bayley II for consistency.Reviewer 2 Comments: Finally based on the nature of majority of the studies , heterogeneous nature of the exposure/outcome assessments and huge drop-outs at follow up-- it will be better to say \u201csignificant association\u201d of antenatal or postnatal depression with cognition, rather than \u201csignificant effect\u201d, which is a more robust terminology.Author Response: This is a very good point and we have now changed the majority of references to \u201csignificant effects\u201d to \u201csignificant associations\u201d. There are a few instances where the use of the word \u201ceffect\u201d remains more suitable and so it has been retained. Importantly, key sentences that serve to summarise findings in the abstract, discussion and conclusion sections have now been changed to reflect the nature of the studies that were synthesized.AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 14 Jun 2021A systematic review of the association between perinatal depression and cognitive development in infancy in low and middle-income countriesPONE-D-21-04110R1Dear Dr. Bluett-Duncan,We\u2019re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you\u2019ll receive an e-mail detailing the required amendments. When these have been addressed, you\u2019ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they\u2019ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Angela Lupattelli, PhDAcademic EditorPLOS ONE 18 Jun 2021PONE-D-21-04110R1 A systematic review of the association between perinatal depression and cognitive development in infancy in low and middle-income countries Dear Dr. Bluett-Duncan:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofDr. Angela Lupattelli Academic EditorPLOS ONE"}
+{"text": "Lactobacillus brevis BGZLS10-17 and Lb. plantarum BGPKM22, effectively attenuated LPS-induced nuclear factor-\u03baB (NF-\u03baB) nuclear translocation. Moreover, BGZLS10-17 and BGPKM22 reduced the activation of p38, extracellular signal-related kinase (ERK), and c-Jun amino-terminal kinase (JNK) signaling cascade resulting in a reduction of pro-inflammatory mediator expressions in BEAS-2B cells. Collectively, the LAB strains BGZLS10-17 and BGPKM22 exhibited anti-inflammatory effects in BEAS-2B cells and could be employed to balance immune response in lungs and replenish diminished lung microbiota in chronic lung diseases.Bronchial epithelial cells are exposed to environmental influences, microbiota, and pathogens and also serve as a powerful effector that initiate and propagate inflammation by the release of pro-inflammatory mediators. Recent studies suggested that lung microbiota differ between inflammatory lung diseases and healthy lungs implicating their contribution in the modulation of lung immunity. Lactic acid bacteria (LAB) are natural inhabitants of healthy human lungs and also possess immunomodulatory effects, but so far, there are no studies investigating their anti-inflammatory potential in respiratory cells. In this study, we investigated immunomodulatory features of 21 natural LAB strains in lipopolysaccharide (LPS)-stimulated human bronchial epithelial cells (BEAS-2B). Our results show that several LAB strains reduced the expression of pro-inflammatory cytokine and chemokine genes. We also demonstrated that two LAB strains,  The interior of the airways is lined with epithelial cells acting as a defensive barrier . Bronchiinterleukin (IL)-1\u03b2, IL-6, and IL-8 (CXCL-8) and the tumor necrosis factor (TNF) [The epithelial barrier also mediates the clearance of microbial pathogens and the adaptation to the commensal bacteria by immune responses that minimize inflammation and maintain tissue homeostasis . Bronchior (TNF) ,8. InflaChronic inflammatory lung diseases such as chronic obstructive pulmonary disease (COPD), neutrophilic asthma, cystic fibrosis (CF), bronchiectasis, and pulmonary fibrosis are characterized by activated immune and structural lung cells producing a variety of pro-inflammatory mediators, leading to excessive and self-perpetuating airway inflammation and tissue destruction ,11,12,13Hence, it has been presumed that the alteration of lung microbiota contributes to the impairment in lung immunity and pulmonary inflammation and promotes infections, susceptibility, and exacerbation of chronic lung diseases leading to the destruction of the lungs ,18,19,20Lactic acid bacteria (LAB) are well known for their immunomodulatory effects, and particular strains belonging to the genus Lactobacillus may have health-promoting effects and are denoted as probiotics . A vast IL-1\u03b2, IL-6, IL-8, TNF, and monocyte chemoattractant protein-1 (MCP-1). Additionally, we demonstrated that two LAB strains are able to attenuate the LPS-induced activity of NF-\u03baB and MAPK signaling cascade resulting in the reduction of pro-inflammatory cytokine and chemokine expressions in bronchial epithelial cells.For this study, we selected 21 LAB strains from WBC artisanal dairy products together with human intestinal isolates from the collection at the Institute of Molecular Genetics and Genetic Engineering (IMGGE). In particular, the strains selected for this study were chosen based on preliminary results pointing to their antioxidant and anti-inflammatory activity, with the aim to select the best probiotic candidates that could be used in the prevention or treatment of inflammatory lung diseases. To the best of our knowledge, the present study, for the first time, investigates the immunomodulatory abilities of 21 natural LAB strains in LPS-stimulated human bronchial epithelial cells. We showed that LAB strains have an immunostimulatory effect, as well as an anti-inflammatory effect reducing the expression of pro-inflammatory mediators such as Lactobacillus plantarum (6), Lb. paraplantarum (1), Lb. paracasei subsp. paracasei (3), Lb. helveticus (1), Lb. brevis (2), Lb. rhamnosus (2), Lactococcus lactis subsp. lactis (4), Lc. lactis subsp. cremoris (1), and Streptococcus thermophilus (1) species. LAB strains and their sources are listed in In this study, 21 LAB strains were chosen from the IMGGE collection in order to evaluate their immunomodulatory properties in LPS-induced BEAS-2B cells. The strains belong to the: A safety evaluation of the LAB strains was performed by minimal inhibitory concentration (MIC) determination of bacterial antimicrobial susceptibility and an analysis of hemolytic and gelatinase activities. The results reveal that none of the strains exhibited hemolytic and/or gelatinase activity (data not shown). Moreover, in line with the European Food Safety Authority (EFSA) recommendations, the susceptibility of the strains to nine antibiotic groups was evaluated . All of The cytotoxicity was tested by measuring the level of released lactate dehydrogenase (LDH) from the BEAS-2B cells exposed to the LAB strains at a 1:10 ratio or LPS (100 ng/mL). Our results show that there was no cytotoxicity (\u22645%) of the chosen LAB strains or LPS used in the treatments of BEAS-2B cells, as shown in IL-1\u03b2, IL-6, IL-8, MCP-1, and TNF genes  and MCP-1 ; Lb. plantarum BGPKM22 for IL-1\u03b2 , IL-8 , and TNF ; Lb. plantarum BGGO7-29 for IL-6 ; Lc. lactis BGAR8 for TNF ; Lb. rhamnosus BGHI22 for IL-6  , IL-8 , MCP-1 , and TNF ; Lc. cremoris BGTRM1-22 for IL-8  and MCP-1 ; and St. thermophilus BGKMJ1-36 for IL-6 , IL-8 , and TNF  gene expression in comparison to treatment with LPS ; Lc. lactis subsp. lactis BGTRK4-21 for MCP-1 ; and Lb. helveticus BGRA43 for IL-1\u03b2  and IL-6  gene expression compared to the LPS treatment . Treatment with LPS significantly induced the expression of NF genes B,C. A to < 0.05) B; Lb. pawith LPS B,C. On treatment B. AccordThe ability of the five selected LAB strains to adhere to the BEAS-2B cells was tested by the adhesion assay. According to our results, all selected LAB strains were able to adhere to the BEAS-2B cells though the levels of adhesion varied among the strains, as shown in In order to scrutinize the molecular mechanisms implicated in the anti-inflammatory effects of selected LAB, the ability of selected strains to affect the NF-\u03baB nuclear translocation was tested. The activation of TLR4 by LPS leads to the translocation of the phosphorylated NF-\u03baB transcription factor from the cytosol to the nucleus, consequently inducing the expression of pro-inflammatory genes. The BEAS-2B cells treated with LPS showed a significant increase in nuclear NF-\u03baB compared to the control cells. The effects of the LAB strains on the LPS-induced nuclear translocation of NF-\u03baB are shown in Lb. brevis BGZLS10-17 and Lb. plantarum BGPKM22 showed the ability to attenuate the LPS-induced nuclear translocation of NF-\u03baB. The level of nuclear NF-\u03baB was significantly lower in cells treated with BGZLS10-17  or BGPKM22  and LPS compared to cells treated with LPS only  or BGPKM22  and LPS in comparison to LPS  or BGPKM22  and LPS , suggesting immunostimulatory effects of both strains. However, the levels of phosphorylated ERK1/2 were decreased if the cells were treated with BGZLS10-17  or BGPKM22  and LPS, as shown in Our results show a decrease in activated JNK when the cells were treated with BGZLS10-17 or BGPKM22 strains for one or both isoforms of JNK, p46, and p54 C. The leThe obtained results show that both LAB strains, BGZLS10-17 and BGPKM22, are able to attenuate LPS-induced MAPK signaling that is responsible for pro-inflammatory gene expression via suppressing the activation of p38, ERK1/2, and JNK kinases.Chronic inflammatory lung diseases, such as COPD and asthma, are among the leading causes of severe illness and death, affecting more than 540 million people worldwide . Smoking3 per cm2, due to the generally inhospitable lung environment for bacterial growth, and are constantly renewed and replaced by immigration, microaspiration, and elimination [Parabacteroides goldsteinii has the ability to block NF-\u03baB activation and inhibit lung inflammation by acting through the gut\u2013lung axis [Although long considered as a sterile organ, lungs are characterized by a relatively low density of a complex microbial community of commensal bacteria. In contrast to the gastrointestinal tract, as an organ with the most studied bacterial community until now, the role of lung microbiota is relatively poorly investigated, and a comprehensive meta-analysis is still missing . Recent mination ,18. Gut mination ,49. It hmination ,50. For ung axis . Moreoveung axis . It has ung axis ,53. MoreLactobacillus spp. strains with probiotic properties could maintain physiological homeostasis of the lung and may provide a novel perspective for treating pulmonary diseases [As natural inhabitants of healthy human lungs, LAB are also well-known for a variety of health-promoting effects including anti-inflammatory abilities, which are of particular interest in this study as they may express alleviating effects related to lung inflammation. Moreover, it has been reported that decreased bacterial diversity related to Firmicutes phyla is associated with the pathogenesis of emphysema and airway remodeling ,50. Due diseases . Probiotdiseases ,51,54,55Lb. brevis BGZLS10-17 and Lb. plantarum BGPKM22, possess anti-inflammatory properties by attenuating the activity of the NF-\u03baB transcription factor and MAPK signaling cascade, consequently leading to lower pro-inflammatory gene expression. However, so far, immunomodulatory effects of LAB (Firmicutes) have not been investigated in the respiratory epithelial cells. Hence, the main objective of this study was to investigate the ability of natural LAB strains to modulate the inflammatory response in human bronchial epithelial cells used as an in vitro model system. Here, we showed that two LAB strains, In this study, the effect of 21 natural LAB strains on the expression of LPS-induced pro-inflammatory genes in normal human bronchial epithelial cells was tested. The obtained results reveal that the strains exhibited a strain-specific effect, which is in line with previously published data .Lb. plantarum BGAN8, Lc. lactis subsp. lactis BGTRK4-21, and Lb. helveticus BGRA43 showed an immunostimulatory response increasing the expression of the IL-1\u03b2, IL-6, and MCP-1 genes. On the contrary, several LAB strains exhibited an anti-inflammatory effect by reducing the expression of pro-inflammatory genes. In particular, the strains Lb. brevis BGZLS10-17, Lb. plantarum BGPKM22, BGGO7-29, Lb. paracasei BGAR88-2, and St. thermophilus BGKMJ1-36 showed significant anti-inflammatory abilities in LPS-stimulated BEAS-2B cells. Since LAB are lung commensals, the immune response of the lung may vary depending on the strains that come into the composition of lung microbiota. This opens up the possibility for the manipulation of lung microbiota, and LAB strains capable of reducing the inflammatory response, may be employed in the prevention or treatment of lung diseases characterized by diminished lung microbiota.In particular, the strains Lactobacillus spp. and Bifidobacterium spp., can suppress inflammation by interfering with NF-\u03baB and MAPK signaling checkpoints, which was mostly studied in intestinal epithelial or immune cells [Lb. brevis BGZLS10-17 and Lb. plantarum BGPKM22, possess the capability to attenuate the LPS-induced nuclear translocation of NF-\u03baB in bronchial epithelial cells resulting in a decrease in inflammatory gene expression. Additionally, we showed that these strains, BGZLS10-17 and BGPKM22, inhibit the activity of MAPK signaling components including p38, ERK1/2, and JNK, contributing to anti-inflammatory effects, too. The pro-inflammatory transcription factor NF-\u03baB and MAPK kinases are activated in chronic lung diseases and implicated in the amplification of the inflammatory response in lungs [Lb. brevis BGZLS10-17 and Lb. plantarum BGPKM22, isolated from semi-hard cheese and artisanal sour milk that attenuate NF-\u03baB and MAPK signaling may be employed for the alleviation or prevention of signs of chronic lung diseases.It is well-known that probiotics, such as ne cells ,51,54,55in lungs ,15. TheyThe ability of the strains BGZLS10-17 and BGPKM22 to colonize human bronchial epithelial cells and to persist in the lungs, which is the prerequisite for their potential application as a probiotic for lungs, was tested in BEAS-2B cells . The obtLb. brevis BGZLS10-17 and Lb. plantarum BGPKM22, capable to inhibit prominent mediators of lung inflammation NF-\u03baB and MAPK, could balance immune response and replenish diminished lung microbiota. These strains fulfil the demands for preclinical testing in an animal model in order to advance their application for the treatment of chronic lung diseases. Furthermore, a precise characterization of the LAB components responsible for anti-inflammatory effects will enable us to gain deeper insight into potential health benefit effects of Lactobacillus spp. strains.In conclusion, this is the first study providing insight into the immunomodulatory effects of autochthonous natural LAB strains from Western Balkan countries in LPS-stimulated human bronchial epithelial cells. Lactobacillus strains were grown in De Man-Rogosa-Sharpe (MRS) medium , while Lactococcus and Streptococcus strains were grown in M17 medium  supplemented with 0.5% (w/v) glucose (GM17). Bacteria were grown at 30 or 37 \u00b0C under aerobic or anaerobic conditions depending on the strain. Solid medium was prepared by adding agar   to the broth medium. The strains were stored at \u221280 \u00b0C in an appropriate medium (GM17 or MRS) supplemented with 15% (v/v) glycerol. For the in vitro immunomodulatory assays, overnight cultures were harvested by centrifugation, washed with sterile phosphate-buffered saline (PBS), counted in a Petroff\u2013Hausser counting chamber, and resuspended in Roswell Park Memorial Institute (RMPI) medium until use.For the purpose of this study, 21 natural LAB strains were chosen from the IMGGE collection of microorganisms. The 6. Cell density was monitored by OD600 measurements after 24 h of incubation at appropriate temperature in a microtiter plate reader . The lowest concentration of antibiotic at which no growth of bacteria was detected was taken as MIC. Experiments were performed in triplicate.Minimal inhibitory concentrations (MICs) were determined by microdilution testing according to the European Food Safety Authority  guidance for bacterial antimicrobial susceptibility. Microbiological cut-off values (mg/L) of 9 antibiotics  for each bacterial species were determined in accordance to the EFSA . Determiv/v) defibrinated horse blood after 48 hours of incubation at 37 \u00b0C [w/v) gelatin  [Hemolytic activity was determined on Columbia Blood Agar  containing 5%  .Gibco), 100 \u00b5g/mL streptomycin, and 100 U/mL penicillin  at 37 \u00b0C and 5% CO2. For immunomodulatory assay, BEAS-2B cells with 80% confluence were pre-treated with natural LAB strains  was cultured in RPMI medium  supplemented with 10% fetal bovine serum (FBS) , 100 ng/mL for 4 h. The activity of LDH was determined by measuring the absorbance at 490 nm and 680 nm, using a microtiter plate reader . The cytotoxicity was calculated as the percentage relative to unstimulated cells.The cellular cytotoxicity was determined by measuring the activity of LDH from the culture medium. The activity of LDH was measured spectrophotometrically by CyQUANT LDH Cytotoxicity Assay Kit , as per the manufacturer\u2019s instructions. Briefly, 1.7 \u00d7 105 BEAS-2B cells per well were seeded, and the next day the cells were pre-treated with LAB in a 1:10 ratio for two hours. Afterwards, the medium was removed and replaced with fresh medium with LPS 100 ng/mL for 4 h. Total RNA was isolated from BEAS-2B cells using the RNeasy Plus Mini Kit  according to the manufacturer\u2019s instructions. RNA purity and concentration were determined by BioSpec-nano .A total of 2.5 \u00d7 10IL-1\u03b2, IL-6, IL-8, MCP-1, TNF, and endogenous control, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene, were used for RT-PCR on 7500 Real-Time PCR system  following manufacturer\u2019s protocols. Relative quantification by \u0394\u0394CT method was used to calculate gene expression relative to positive control (LPS), which was used as a calibrator. Results were analyzed using Applied Biosystems 7500 System Software.cDNA was synthesized from 1\u2009\u03bcg of total RNA using the High Capacity cDNA Reverse Transcription Kit  as per the manufacturer\u2019s instructions. TaqMan Gene Expression Assays  for the 5 BEAS-2B cells, seeded the day before, were washed twice with PBS and treated with bacterial suspensions using a ratio of 1:10 (BEAS-2B cells: LAB). Following co-incubation for two hours at 37 \u00b0C and 5% CO2, the cells were gently washed with PBS and lysed with 0.25% Trypsin\u2013EDTA solution . Serial dilutions of samples, before and after adhesion, were plated on appropriate agar plates. The adhesion was calculated as (%) = (CFU/mL adhered bacteria/CFU/mL added bacteria) \u00d7 100.The ability of selected LAB to adhere to BEAS-2B cells was analyzed by adhesion assay according to Mushtaq et al. with minor modifications . Overnig\u00ae 594, Abcam) was diluted 1:400 in 1% BSA in PBS and applied for one hour at RT. Coverslips were washed 3 times for 10 min in 0.2% Tween 20 in PBS. Nuclei were stained with 0.1 mg/mL diamino phenylindole . Coverslips were mounted on to the glass slides using Fluorescent Mounting Medium . BEAS-2B cells were seeded on coverslips and pre-treated the next day with LAB in 1:10 ratio for one hour and treated with LPS 100 ng/mL for 30 min. The cells were fixed using 3% paraformaldehyde and 2% sucrose for 20 min at room temperature (RT) and permeabilized in 0.2% Triton X-100 for 10 min. Non-specific binding was blocked in 10% normal goat serum  and 1% bovine serum albumin (BSA)  in PBS for one hour at RT. An anti-NF-kB p65 antibody . The AF594 labelled antibody was excited with an OPSL 552 laser, while DAPI was excited with 405 diode laser. DAPI  and AF594  fluorescence were captured using sequential acquisition to give separate image files for each. A pin hole of 1 Airy (95.5 \u03bcm), scan speed of 200 Hz and 4 frame averaging, was used. Photomultiplier tube gain and offset were adjusted to give sub-saturating fluorescence intensity with optimal signal to noise ratio. Image acquisition settings were the same for all examined samples in order to compare fluorescence intensities. Six Z stacks in the range of 1.47 \u00b5m were acquired. Quantification of NF-\u03baB nuclear translocation was performed by image analysis using LAS X software . Mean fl5 BEAS-2B cells were seeded, and the next day the medium was changed to serum-free RPMI for 24 hours. The cells were pre-treated with LAB in a 1:10 ratio for one hour and treated with LPS 100 ng/mL for 20 min. The cells were washed with ice-cold PBS and lysed with Radio Immuno Precipitation Assay buffer  containing protease inhibitor cocktail tablets  and 1 mM phenylmethylsulphonyl fluoride , for 30 min on ice. The total protein concentrations were determined using the BCA protein assay kit . Equal amounts of proteins (15 \u03bcg) were separated by 12.5% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoresed proteins were transferred from the gel to a 0.2 \u03bcm nitrocellulose membrane  using a Bio-Rad Mini trans-blot system . Immunoblots were blocked in a 5% non-fat dry milk in PBS-Tween  overnight at 4 \u00b0C. After that, the immunoblots were incubated with primary antibodies anti-phospho-p38 MAPK (Thr180/Tyr182), anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), and anti-phospho-JNK (Thr183/Tyr185)  and anti-\u03b1-tubulin and anti-\u03b2-actin as a loading controls  for two hours at RT. The membranes were subsequently washed and incubated with appropriate horseradish peroxidase (HRP) conjugated secondary antibody  for one hour at RT. Proteins were detected by enhanced chemiluminescence Immobilon Western .A total of 10p-value of less than 0.05 was considered significant. Statistical analysis was carried out, and graphs were prepared by using the GraphPad Prism 6 software.The data are expressed as mean \u00b1 standard effort of mean (SEM). The statistical significance of the differences between treatment and control was tested by Student\u2019s t-test. A"}
+{"text": "Die zystische Fibrose (CF) ist eine multisystemische progrediente Stoffwechselerkrankung mit vorwiegend abdomineller und pulmonaler Beteiligung. Schmerzen sind f\u00fcr Betroffene ein weiteres komplexes und von den Behandlern untersch\u00e4tztes Problem.Eine Literaturrecherche deutschsprachiger Leitlinien und englischsprachiger Studien zum Thema CF und Schmerzen wurde durchgef\u00fchrt, zus\u00e4tzlich die Beobachtungen zur Diagnostik und Therapie eines CF-Patienten mit progredienten thorakalen Schmerzen ausgewertet.Die Recherche ergab, dass zur Diagnostik und Therapie thorakaler Schmerzen bei CF keine deutschsprachigen Leitlinien oder Konsenspapiere existieren. Die europ\u00e4ischen und amerikanischen Erhebungen zeigen aber die gro\u00dfe Relevanz des Themas und postulieren einen Zusammenhang von Schmerzintensit\u00e4t mit einer erh\u00f6hten Mortalit\u00e4t. Sie enthalten jedoch keine Daten zur Effektivit\u00e4t der Schmerztherapie. Anhand dieser Daten und des Fallberichts eines jungen CF-Patienten mit st\u00e4rksten Thoraxschmerzen bei pulmonalen Exazerbationen lassen sich die CF-spezifischen Herausforderungen der Schmerztherapie illustrieren. Neben den Schmerzen an sich sind auch Analgetika angesichts der multiplen Organdysfunktionen mit besonderen Risiken wie gastrointestinalen Blutungen, opioidinduzierter Atemdepression oder opioidinduzierter Obstipation verbunden.Schmerztherapie bei Patienten mit zystischer Fibrose und Multiorganbeteiligung erfordert ein sorgf\u00e4ltiges Monitoring und interdisziplin\u00e4res Handeln. Empfehlungen zum Schmerzmanagement sollten in die deutschsprachigen CF-Leitlinien aufgenommen werden. Die zystische Fibrose (CF) ist eine seltene genetische multisystemische Stoffwechselerkrankung. Aufgrund z\u00e4her Sekrete kommt es zu progredienten inflammatorischen Ver\u00e4nderungen an Pankreas und Lunge. Vor allem die pulmonalen Exazerbationen sind lebensbegrenzend und gehen oft mit ausgepr\u00e4gten, mit Progredienz der Erkrankung zunehmenden thorakalen Schmerzen einher. Anhand eines exemplarischen Fallberichts sowie der aktuellen Literatur illustrieren wir die Komplexit\u00e4t der Multiorganerkrankung mit intermittierenden Schmerzexazerbationen gefolgt von Phasen der Schmerzfreiheit. Dies erfordert nicht nur eine individualisierte Schmerztherapie, sondern auch Kenntnis und Beachtung diverser krankheitsspezifischer Risiken.Die CF ist die h\u00e4ufigste autosomal-rezessiv vererbte multisystemische Stoffwechselkrankheit in Deutschland . MutatiDie Therapie der CF war jahrzehntelang symptomatisch . Seit Kurzem k\u00f6nnen f\u00fcr bestimmte Mutationen durch CFTR-Modulatoren pulmonale Exazerbationen reduziert und die Lungenfunktion stabilisiert werden . PatientEs wurde eine umfassende Literaturrecherche in der PubMed-Datenbank zu den Begriffen \u201ecystic fibrosis\u201c AND \u201epain\u201c AND \u201eadults\u201c OR \u201echildren\u201c AND \u201etreatment\u201c OR \u201eadverse events\u201c durchgef\u00fchrt. Dar\u00fcber hinaus wurden die deutschsprachigen Zystische Fibrose (CF)-Leitlinien (AWMF) nach Empfehlungen zu Schmerztherapie bei CF durchsucht. Und es wurden klinische Beobachtungen der erw\u00fcnschten und unerw\u00fcnschten Wirkungen der analgetischen Therapie eines jungen CF-Patienten mit thorakalen Schmerzen ausgewertet.Die Recherche ergab, dass in Deutschland zur Schmerztherapie bei CF weder Studien noch spezielle Leitlinien existieren. Diverse europ\u00e4ische und amerikanische Erhebungen der letzten zwanzig Jahren zeigen aber, dass Schmerzen ein h\u00e4ufiges und gravierendes Problem f\u00fcr die Betroffenen darstellen und oft inad\u00e4quat behandelt werden , 28. UnsSchmerzen bei der CF sind durch die erh\u00f6hte Inflammation mit vermehrter Aussch\u00fcttung proinflammatorischer Zytokine bedingt . Zudem iW\u00e4hrend zun\u00e4chst Abdominalschmerzen dominieren, treten bei Jugendlichen die Thoraxschmerzen mit zunehmender H\u00e4ufigkeit von Exazerbationen in den Vordergrund. Zwei Studien zeigten einen Zusammenhang zwischen dem vermehrten Auftreten von Thoraxschmerzen und einem erh\u00f6hten Mortalit\u00e4tsrisiko , 28. Je Die Schmerzen sind meist intermittierend und in ihrer Frequenz bei Kindern und Erwachsenen vergleichbar, bei Letzteren chronifizieren sie h\u00e4ufiger. Auch unter der meist lebenslang notwendigen symptomatischen Therapie (insbesondere autogene Drainage und Atemphysiotherapie) sowie bei der Diagnostik (z.\u202fB. Spirometrie) treten starke Schmerzen auf . LangfriH\u00e4ufig beschrieben wird das geringe Problembewusstsein der Behandler f\u00fcr die Schmerzen und umgekehrt auch die selten aktive Thematisierung der Schmerzen durch die Patienten. Viele Patienten behandeln sich mit frei verk\u00e4uflichen Analgetika selbst. Neben Paracetamol (59\u202f%) nehmen die Patienten nichtsteroidale Antiphlogistika NSAR; 10\u202f%), Acetylsalicyls\u00e4ure (5\u202f%) und Spasmolytika ein . Opioide\u202f%, AcetyWir berichten von einem jungen Erwachsenen (ca.\u00a050\u202fkg) mit genetisch gesicherter Zystischer Fibrose (CF) und zunehmenden Schmerzen im Rahmen der Progression seiner Grunderkrankung.Im Kindesalter standen bei ihm abdominelle Probleme im Vordergrund: Neben einem einmalig aufgetretenen distalen intestinalen Obstruktionssyndrom (DIOS) litt er an rezidivierenden Gastritiden mit zweimalig nachweisbarem Ulcus duodeni. Damals traten wellenartige g\u00fcrtelf\u00f6rmig in den R\u00fccken ausstrahlende Oberbauchschmerzen (NRS 7\u20138/10) auf, die mit Metamizol und Butylscopolamin sowie unregelm\u00e4\u00dfig mit NSAR behandelt wurden. Ab diesem Alter fiel eine milde Hepatopathie auf.Seit dem 12.\u00a0Lebensjahr stieg die Zahl krankenhauspflichtiger pulmonaler Exazerbationen an, seit dem 18.\u00a0Lebensjahr auf bis zu sechsmal j\u00e4hrlich. Neben vermehrtem Husten und verminderter Belastbarkeit klagte der Patient \u00fcber zunehmende Dyspnoe. Die Lungenfunktion verschlechterte sich jetzt kontinuierlich, teilweise lag die Einsekundenkapazit\u00e4t (FEV1) unter 20\u202f% (aktuell 45\u202f%).Es erfolgte stets eine resistogrammgerechte antibiotische Therapie, Atemphysio- und Inhalationstherapien. Bei zus\u00e4tzlichem Asthma bronchiale erhielt er inhalative und im Intervall systemische Kortikosteroide.Seit 2\u00a0Jahren klagte der Patient im Rahmen seiner Exazerbationen vermehrt nachts sowie beim Husten und bei Inspiration \u00fcber linksseitige parasternale und paravertebrale als \u201ebrennend\u201c beschriebene, vermutlich \u00fcberwiegend pleuritisch bedingte Thoraxschmerzen von hoher Intensit\u00e4t NRS 7\u20138/10). Die Schmerzintensit\u00e4t war wechselhaft und trat auch bei der Atemphysiotherapie bzw. bei Durchf\u00fchrung einer Spirometrie auf. Ein Pneumothorax wurde stets ausgeschlossen. Im letzten Halbjahr traten pulmonale Exazerbationen mit st\u00e4rksten Thoraxschmerzen (NRS 7\u20139/10) alle 2\u20134\u00a0Wochen auf. Die Schmerzintensit\u00e4t war wechselhaft, verst\u00e4rkt bei Stress und durch \u00c4ngste und wurde gemildert bei Ablenkung. In der konkreten Akutsituation ergab sich unseres Ermessens keine Option f\u00fcr eine psychotherapeutische Intervention. Wie zuvor war er in den Wochen und Monaten zwischen den Episoden schmerzfrei. Zuletzt fanden sich in einer CT neben Bronchiektasen und narbigen Residuen zudem bei nachgewiesener Osteopenie unterschiedlich alte Rippenfrakturen (Abb.\u00a00. Die ScSeit ca.\u00a02\u00a0Jahren mussten in Akutphasen intraven\u00f6s appliziertes Metamizol und Paracetamol sowie Ibuprofen immer h\u00e4ufiger durch Piritramid erg\u00e4nzt werden. Hierunter waren zwar die Schmerzen geringer (NRS 3\u20135/10), aber der Patient atmete flacher und konnte weniger Sputum mobilisieren. 2020 erfolgte ein Behandlungsversuch mit Gabapentin neben einer festen Analgesie mit Metamizol. Dieses wurde von dem Patienten als wirksam empfunden und daher mehrfach wiederholt. Zus\u00e4tzlich erfolgten mehrfach manualmedizinische Behandlungen, die nur eine leichte Beschwerdelinderung erbrachten.Im Herbst 2020 war die beschriebene, bislang in der Klinik \u00fcbliche Kombinationstherapie nicht mehr ausreichend. Es wurde daraufhin, da in der Vergangenheit mehrfach bei CF-Patienten erfolgreich durchgef\u00fchrt (s.\u00a0unten), in H\u00f6he Th7/8 ein Epiduralkatheter angelegt. Dar\u00fcber wurden Ropivacain und Sufentanil appliziert. Dennoch klagte der Patient weiter \u00fcber starke Schmerzen, sodass am ersten Tag parallel Piritramid injiziert wurde. Hierunter kam es zu n\u00e4chtlichen Hypoxien (min. Sauerstoffs\u00e4ttigung 85\u202f%) mit intermittierendem Sauerstoffbedarf von max. 3\u202fl/min \u00fcber einige Stunden. Am 2.\u00a0Tag erfolgte eine Umstellung von Piritramid auf Buprenorphin sublingual. Danach traten keine Hypoxien mehr auf, obwohl er bis zum 4.\u00a0Tag parallel Sufentanil epidural erhielt, danach 3\u00a0Tage nur Ropivacain (Liegedauer: 7\u00a0Tage).Zus\u00e4tzlich entwickelte er rasch eine zunehmende opioidinduzierte Obstipation (OIC) mit beginnendem Subileus und Gefahr eines distalen intestinalen Obstruktionssyndrom (DIOS). Sie konnte durch Absetzen von Piritramid und mehrt\u00e4gige Naloxegol- sowie Macrogolgabe gebessert werden.Bei zwei weiteren station\u00e4ren Aufenthalten gelang mit einer Infusion von Parecoxib sowie einer Epiduralanalgesie mit Sufentanil und Ropivacain eine ausreichende Schmerzkontrolle ohne parallele Gabe systemischer Opioide. Dennoch traten erneut n\u00e4chtliche Hypoxien auf, die erst nach Absetzen des Sufentanils verschwanden. Nach den Entlassungen beendete er bei Schmerzfreiheit die analgetische Therapie jeweils vollst\u00e4ndig.Unsere selbstkritisch intendierte Fallstudie und die Literatur\u00fcbersicht unterstreichen die Notwendigkeit eines schmerztherapeutischen Konzepts. Sie illustrieren aber auch die Schwierigkeiten bei der Umsetzung. Das \u00dcbersehen bestimmter Neben- und Wechselwirkungen der Analgetika kann den Teufelskreis von Schmerz und Hypoxie noch verst\u00e4rken Abb.\u00a0. Das F\u00fcrParacetamol (PCM) sowie Nicht steroidale Antirheumatika (NSAR) und in Deutschland\u00a0(D) auch Metamizol werden von den meisten Patienten mit Thoraxschmerzen, wie auch im Fallbericht beschrieben, als Basismedikation verwendet .Aufgrund ihrer antiinflammatorischen Wirkung ist der Einsatz von NSAR rational begr\u00fcndbar. Allerdings sind NSAR bei Dauergebrauch und in Kombination mit anderen in der CF-Therapie erforderlichen Medikamenten (z.\u202fB. Aminoglykosiden) potenziell nephrotoxisch . Das UlkAber auch PCM, ein nur schwach wirksames Analgetikum , ist keiDas ebenfalls h\u00e4ufig auch bei Kindern in\u00a0D eingesetzte Metamizol hat im Vergleich ein geringeres gastrointestinales, renales und vermutlich auch hepatisches Risiko . Im vorlGabapentin, das auch unser Patient als wirksam beschrieb, wird in den USA durch fast 20\u202f% der CF-Behandler im Erwachsenenbereich eingesetzt . GabapenBei zunehmender Progression der Erkrankung sind, wie auch aus dem Fallbericht ersichtlich, Opioide meist unvermeidlich, in der Palliativphase fast immer geboten , 28, 32.Bei unstrittiger analgetischer Effektivit\u00e4t sind vor allem die Dosisfindung und die Wahl des Opioids bei der CF aus zwei Gr\u00fcnden von herausragender Bedeutung.Das erste Problem ist eine opioidinduzierte Obstipation (OIC), welche bei der CF, wie oben beschrieben, leicht zu einem DIOS bzw. Subileus f\u00fchren kann  aller Altersgruppen. Schmerzen erschweren die Therapie und gef\u00e4hrden so die Patienten. Eine falsch gew\u00e4hlte oder unzureichend \u00fcberwachte Schmerztherapie ist mit erheblichen Risiken verbunden. Zur Integration der Schmerztherapie in die CF-Leitlinien ist ein Stufenkonzept erforderlich. Als n\u00e4chster Schritt muss die Versorgungssituation auch in Deutschland erfasst werden, um dann gezielt Versorgungsl\u00fccken zu erkennen, mittels geeigneter Studien zu beseitigen und ein Therapiekonzept zu erarbeiten. Hierzu ist eine bessere Zusammenarbeit von P\u00e4diatern, Pneumologen und Schmerzmedizinern erforderlich."}
+{"text": "Docking studies indicated that ribociclib interacted with the drug-substrate binding site of P-gp. The short-term and long-term intracellular accumulation of doxorubicin greatly increased in the KB-C2 cells co-cultured with ribociclib, indicating ribociclib inhibited the drug efflux activity of P-gp. The results of our study indicate that LEE011 may be a potential agent for combined therapy of the cancers with P-gp mediated MDR.The efficacy of cancer chemotherapy can be attenuated or abrogated by multidrug resistance (MDR) in cancer cells. In this study, we determined the effect of the CDK4/6 inhibitor, ribociclib (or LEE011), on P-glycoprotein (P-gp)-mediated MDR in the human epidermoid carcinoma MDR cell line, KB-C2, which is widely used for studying P-gp-mediated MDR in cancers. The incubation of KB-C2 cells with ribociclib (3\u20139\u00a0\u00b5M) increased the efficacy of colchicine, a substrate for P-gp. The cell expression of P-gp was down-regulated at both translation and transcription levels. Furthermore, ribociclib produced a 3.5-fold increase in the basal activity of P-gp ATPase, and the concentration required to increase basal activity by 50% (EC They bictively) . Ribocice arrest . In addie arrest  and 2) ie arrest . Currente arrest .P-glycoprotein (P-gp/ABCB1) is an ATP-binding cassette (ABC) transporter that extrudes various anticancer drugs  from cancer cells, as well as various endogenous molecules . The strIt has been reported that P-gp can significantly decrease the levels of ribociclib in the brain, suggesting that its efficacy may be limited by cancer cells that overexpress P-gp . Recentl2 at 37\u00b0C. CRISPR/Cas9 all-in-one plasmids, encoding single guide RNA (SgRNA) and Cas9, were purchased from GeneCopoeia Inc . Because KB-C2 cells are widely used for studying P-gp-mediated MDR in cancers, KB-C2 cells were used to determine the reversal of P-gp-mediated MDR.The human epidermoid carcinoma cell line, KB-3-1, was used as the parental cell line. KB-C2, an MDR cell line that is resistant to colchicine due to the overexpression of P-gp, was created by exposing KB-3-1 cells to increasing concentrations of colchicine over a period of at least 2\u00a0months . Both ceRibociclib was kindly provided by ChemieTek . Paclitaxel, colchicine, and doxorubicin were purchased form Sigma Chemical Co. . Mouse anti-P-gp, HRP ligated or fluorescent secondary rabbit or goat-anti mouse antibodies, were purchased from Invitrogen, Thermo Fisher . Mouse-anti-CDK4 and CDK6 antibodies were purchased from R&D Systems . All other reagents were purchased from VWR International .3 cells/well. These experiments were conducted in triplicate. After 72\u00a0h of incubation, 20\u00a0\u03bcl of MTT (5\u00a0mg/ml) was added to each well. After incubation for an additional 4\u00a0h, the medium containing MTT was discarded and replaced with 150\u00a0\u03bcl of DMSO. The plates were gently shaken until the dark blue-purple crystal were completely dissolved in DMSO. The absorbance was measured at a wavelength of 490\u00a0nm, using an ELx 800 Universal Microplate Reader . The relative survival rate (%) for the cells was analyzed using the SPSS 20 program  and the survival rate - drug concentration curves were generated using Origin 9.0 software . The concentration of drug required to inhibit cell viability by 50%  was determined using Origin 9.0 software.Exponentially growing cells were seeded into 96 well plates at 5 \u00d7 103 cells per well) and cultured overnight. The cells were incubated with ribociclib  for 1 or 2\u00a0h, followed by incubation with gradient concentrations of colchicine and paclitaxel, which are substrates for the P-gp transporter. The IC50 values of the anti-cancer drugs were determined using the MTT assay as described above.Cells were seeded into 96-well plates  assays were conducted to determine the effect of ribociclib on the expression of the P-gp protein. For the Western blot assay, the cells were lysed with SDS lysate reagent and separated on a gradient polyacrylamide gel . The proteins on the SDS-PAGE gel were transferred to a PVDF membrane. After blocking with 5% milk, the membrane was washed with TBST buffer  three times, incubated with mouse anti-P-gp antibody at 4\u00b0C for 2\u00a0h, adequately washed with TBST, and incubated with goat anti-mouse IgG-HRP (horseradish peroxidase) at RT for 2\u00a0h. The membrane was then washed with TBST four times and exposed to the SignalFire\u2122 ECL Reagent developing reagent , and the results were quantified using an AI600 RGB GEL Imaging System  set for the chemiluminescence mode.ABCB1 mRNA level was performed to investigate the influence of ribociclib on the ABCB1 expression on the transcription level. Ribociclib (9\u00a0\u00b5M) showing the effect of reversing ABCB1 mediated MDR in cancer cells was added to the cell culture medium. The cells cultured without ribociclib were set as control. After 48\u00a0h of cell culture, the cells were sampled for mRNA extraction and RT-q-PCR . ABCB1 and GAPDH were the objective and internal reference genes, respectively. Primers for amplification of ABCB1 gene were: GAAAGTGAAAAGGTTG (Forward), and CTGGCGCTTTGTTCCA (Reverse). Primers for amplification of GAPDH gene were ATTGACCTCAACTACA (Forward), and AGAGATGATGACCCTT (Reverse). Expression deviation was calculated according to the formular: Ratio Sample/Control = 2\u2212\u0394\u0394CT, where CT value was automatically calculated by QuantStudio\u2122 Design and Analysis Software v1.3.1 according to the \u0394Rn-Cycle curve.RT-PCR of 2). The membrane vesicles from Sf9 cells were provided by the manufacturer, expressing high levels of human ABCB1, were incubated with ribociclib  for 3\u00a0min. ATP hydrolysis was initiated by adding 5\u00a0mM of Mg-ATP and the reaction was terminated using a 5% SDS solution. Subsequently, the light absorption was measured at 800\u00a0nm using a Bio-Rad SmartSpec 30,000 UV/Vis spectrophotometer .The ATPase activity of P-gp was determined using the PREDEASY ATPase Kits , according to the manufacturer\u2019s protocol . Briefly\u03b2-glycerophosphate, 1\u00a0mM of EDTA, 1\u00a0mM of Na3VO4 and 0.5\u00a0\u03bcg/ml of leupeptin). The fluorescence intensity, an indicator of doxorubicin accumulation, was determined using a SynergyTM 4 Multi-Mode Microplate Reader .Because doxorubicin is a fluorescent substrate that can be extruded from cancer cells by the P-gp transporter , its int3 cells per well, cultured for 8\u00a0h, and incubated with 9\u00a0\u03bcM of ribociclib and doxorubicin (1\u00a0\u00b5M for the MDR KB-C2 cells and 0.1\u00a0\u00b5M for drug-sensitive KB-3-1 cells to maintain MDR and cell viability above 60%) for 72\u00a0h. Because the membrane of the dead or severely apoptotic cells was damaged, which could lead to the inaccurate quantification of the intercellular doxorubicin levels, the distribution of doxorubicin was determined in triplicate, using fluorescent microscopy.For the long-term evaluation of the efflux of doxorubicin, cells were seeded at 3 \u00d7 10https://swissmodel.expasy.org/). The interactions between ribociclib and P-gp, CDK4 or CDK6 were calculated using HEX 8.0 software , based on the most stable calculated structures of the complexes by molecular docking (We first determined the transcripts for the P-gp and CDK4/6 proteins in the KB-C2 cell line. According to the open reading frame (ORF) sequences detected in the major transcripts of P-gp in KB-C2 cell line, the structure was achieved by structure modelling using a Swiss model platform ( docking . PyMOL (50 = 13.15\u00a0nM) the viability of the colchicine-sensitive KB-3-1 cancer cells  in MDR KB-C2 cancer cells. As previously reported, colchicine potently decreased  of ribociclib for 72\u00a0h produced a significant decrease in the expression of P-gp protein levels compared to cells incubated with vehicle . The remABCB1 transcription in the KB-C2 cells (ABCB1 mRNA amounts was detected in the cells treated with ribociclib (9\u00a0\u00b5M), as compared with that detected in the cells without ribociclib treatment. This conclusion was coherent with the that supported by Western blot  on the intracellular accumulation on doxorubicin, a substrate for the P-gp transporter, in KB-C2 cancer cells. As previously reported , the accThese results indicated that ribociclib increases the ATPase activity of P-gp and inhibits the drug-efflux function of P-gp, suggesting that ribociclib can interact with P-gp directly, which may contribute to the reversal of P-gp-mediated MDR in KB-C-2 cells.These studies were conducted to ascertain if the prolonged incubation (72\u00a0h) of KB-3-1 and KB-C2 cancer cells with ribociclib would inhibit the efflux of doxorubicin. The incubation of the parental KB-3-1 cells with 9\u00a0\u00b5M of ribociclib for 72\u00a0h did not significantly alter the intracellular accumulation of doxorubicin compared to cells incubated with vehicle . We conc50 value of colchicine  in the KB-C2 cells, whereas it had no significant effect on the efficacy of colchicine in the parental KB-3-1 cells , reversal effects on MDR in KB-C2 cells increased. But the cell proliferation was also inhibited when using only ribiciclib (instead of colchicine) higher concentrations. This could be caused by the enhanced inhibition effect on CDK4/6 by a greater number of ribociclib molecules that had entered the cells in addition to their interaction with the membrane P-gp transporters. Thus, the functions of inhibiting cancer cell proliferation and reversing P-gp mediated MDR could synergize each other during combined chemotherapy based on ribociclib and P-gp substrate drugs.\u03b1 and 110\u03b2, and PI3K 110\u03b1/110\u03b2 deficiency in-return downregulated CDK6. CDK6-PI3K axis synergizes in regulating ABCB1 expression, which further strengthened the regulation of ABCB1 over single regulation by either CDK6 or PI3K 110\u03b1/110\u03b2.By single gene knockout using CRISPR/Cas9 technique in human epidermoid carcinoma MDR cell line KB-C2, we recently revealed that CDK6-PI3K signaling axis is an efficient target for attenuating ABCB1/P-gp mediated MDR in cancer cells . CDK6 knIt will be instructive if we know the mechanisms of the interaction between ribociclib and P-gp, which will benefit modification of the structure of ribociclib and improving its affinity to P-gp. Till now, no study about this aspect has been reported, however, we are still making efforts on its exploration, which is an undergoing project in our laboratories.In our laboratories, we are currently performing modification and optimization of a serial of combined drug-systems that can reverse MDR in cancers and inhibit cancer cell growth as well. Based on these studies, our tasks in the next stage will contain animal study to testify the biocompatibility and tumor killing efficacy of these drug systems.In conclusion, the results of this study indicated that in KB-C2 cells, ribociclib inhibited the efflux of the P-gp transporter substrates, doxorubicin and colchicine and decreased the expression of the P-gp transporter, resulting in the reversal of MDR. P-gp expression was downregulated by ribociclib. Furthermore, protein docking data reveals that ribociclib binds near the P-gp transporter drug-substrate binding site and it stimulates the basal activity of the P-gp ATPase. ATPase analysis and drug accumulation experiments further demonstrated that the activity of P-gp was inhibited by ribociclib. Thus, ribiciclib may be a promising inhibiter for the application in combination of anticancer therapies against solid tumor cells with P-gp mediated MDR."}
+{"text": "Background: Inappropriate cycling positions may affect muscle usage strategy and raise the level of fatigue or risk of sport injury. Dynamic bike fitting is a growing trend meant to help cyclists select proper bikes and adjust them to fit their ergometry. The purpose of this study is to investigate how the \u201cknee forward of foot\u201d (KFOF) distance, an important dynamic bike fitting variable, influences the muscle activation, muscle usage strategy, and rate of energy expenditure during cycling. Methods: Six amateur cyclists were recruited to perform the short-distance ride test (SRT) and the graded exercise tests (GXT) with pedaling positions at four different KFOF distances . The surface electromyographic (EMG) and portable energy metabolism systems were used to monitor the muscle activation and energy expenditure. The outcome measures included the EMG root-mean-square (RMS) amplitudes of eight muscles in the lower extremity during the SRT, the regression line of the changes in the EMG RMS amplitude and median frequency (MF), and the heart rate and oxygen consumption during the GXT. Results: Our results revealed significant differences in the muscle activation of vastus lateralis, vastus medialis, and semitendinosus among four different pedaling positions during the SRT. During GXT, no statistically significant differences in muscle usage strategy and energy expenditure were found among different KFOF. However, most cyclists had the highest rate of energy expenditure with either KFOF at \u221240 mm or 20 mm. Conclusions: The KFOF distance altered muscle activation in the SRT; however, no significant influence on the muscle usage strategy was found in the GXT. A higher rate of energy expenditure in the extreme pedaling positions of KFOF was observed in most amateur cyclists, so professional assistance for proper bike fitting was recommended. Cycling exercise has become popular in recent years. Although originally considered as a means of transportation, biking has now become a recreational activity and a type of competitive athletics. Many people start riding and become professional or amateur cyclists. Due to the long distance of biking, many cyclists suffer from various sport injuries, such as hip pain, anterior knee pain, patellar tendinitis, iliotibial band friction syndrome, quadriceps tendinitis, Achilles tendon tendinitis, etc. ,2. TheseEfficiency, as a key factor in cycling performance, is defined as the ratio of power output to energy expenditure . A crankBike fitting, either static or dynamic, is a good way to assess cycling posture. It is defined as \u2018the detailed process of evaluating the physical and performance requirements of the cyclist, and systematically adjusting the bicycle to meet the goals and needs\u2019 . Bike fiMost previous studies changed the dimensions of the bike in static fitting condition to investigate their effects on muscle activation, muscle fatigue level, and oxygen consumption. However, these changes would alter the positions of multiple body segments simultaneously. The special bike of a dynamic fitting system makes it possible to change the cyclist\u2019s KFOF distance only while maintaining the same positions in other joints. The purposes of this study were (1) to investigate the effects of different KFOF distances on muscle activation of eight muscles in the lower limb in a short-distance ride; and (2) to investigate the effects of different KFOF distances on the muscle usage strategy and energy expenditure rate during a long period of cycling. Our hypotheses were that cycling in extreme KFOF distances that were away from the vertical line would result in less efficient pedaling, showing in higher muscle activation, easier muscle fatigue, and more energy expenditure.This is an exploratory study. Male amateur cyclists were screened and recruited in this study. The inclusion criteria were: (i) age from 20 to 50, (ii) capable of completing the Taiwan Wu-Ling highest point (3275 m) challenge  in 4 h, (iii) completion of the ACSM Health/Fitness Facility Pre-participation Screening Questionnaire with fewer than three items checked. This study received ethical approval from the Institutional Review Board of China Medical University Hospital (IRB number CMUH105-REC3-053). Subjects fully understood the testing procedure and signed an informed consent before the study began.2) and heart rate during the cycling exercise testing.We used a special bike (Ret\u00fcl M\u00fcve SL Dynamic Fit Bike) and a dynamic bike fitting system  to control the standard cycling position while allowing us to change the KFOF parameter only. The Delsys Trigno Wireless System  was used to record muscle activation in the lower limb. The surface electrodes had four silver bar contacts (5 \u00d7 1 mm) spaced 10 mm apart in a rectangular configuration. Before the riding tests, the electrodes were placed at the vastus lateralis (VL), vastus medialis (VM), semimembranosus (SEMI), biceps femoris (BF), gluteus maximus (GM), tensor fascia lata (TFL), medial gastrocnemius (MG), and tibialis anterior (TA) of the right leg according to the method described in the previous study . A portaFour different pedaling positions were investigated in our study. The KFOF distance was measured at the forward pedaling position of 90\u00b0 during dynamic cycling, and set at +20, 0, \u221220, and \u221240 mm as the pedaling positions in our study  .The experiment was divided into a short-distance ride test (SRT) and a graded exercise test (GXT). The SRT was designed to examine muscle activations in four different KFOF positions under the same riding powers. Four riding powers  were tested on four non-consecutive days, and one power level was assigned randomly for each day. After the EMG electrodes were properly placed, the maximal voluntary isometric test (MVIC) was conducted first, using standard manual muscle testing methods, and the EMG data were recorded and analyzed for normalization. Then, the cyclist started with a warm-up phase for 5 min at 100 W in each short-distance ride. The sequence of the four pedaling positions during the SRT was randomly assigned. During the test, the cyclist was asked to maintain cadence at 90 rpm. When they reached the selected power level at each pedaling position, the myoelectric data would be recorded for 1 min. When adjusting the pedal position, the cyclist would keep pedaling at 100 W power level to prevent cooling down.After a 5-min rest, the metabolic system (COSMED K4b2) and the POLAR heart rate monitor were equipped. Then, the GXT was conducted to examine the muscle activation level (%MVC), muscle fatigue (MF change), and energy expenditure during a long-distance ride. One randomly assigned pedaling position was tested on each day. After proper dynamic fitting, the riding power for the GXT was set starting at 120 W and increased incrementally by 10 W every minute until the cyclist could no longer continue. During the test, each cyclist was asked to maintain a ride cadence at around 90 rpm. If the cadence was less than 80 rpm, the researchers would remind and encourage the cyclist. However, if the cadence dropped to less than 70 rpm, the test would be ended. In the cool down phase, the cyclist would continue riding 120 W until the heart rate returned to the baseline at the start of the GXT. The main reason to stop the test\u2014i.e., due to leg soreness or breathlessness\u2014would be documented.2) was calculated and recorded breath-by-breath by the metabolic system, and the heart rate measured by the POLAR strap was recorded every 30 s during the whole GXT.The recorded myoelectric signals in the middle 30 s of every testing minute in the SRT and GXT were used for further analysis. The oxygen consumption , and then the root-mean-square (RMS) values were calculated by using a moving window of 0.02 s with 0.01 s overlapped. These data were then normalized with the MVIC value of each muscle after the same signal processing to represent the amplitude of the muscle activation. For every 30 s data of every testing minute, the peaks of the normalized RMS extracted from the pedaling cycles were then averaged.2, and heart rate of each testing minute during the GXT was further analyzed using linear regression, and the coefficient of the variable in the regression equation represented their changing trend as the riding power and time increased  of each muscle\u2014reflecting the shift in recruitment of muscle fiber types\u2014in each pedaling cycle and then averaged in every minute. Since we set the increment of the cycling power constant, the changes in these variables were expected to have a linear trend. Therefore, the mean RMS, MF, VOncreased . SPSS soSix male cyclists (age 32.5 \u00b1 2.7 years) participated voluntarily and completed the tests in this study. Their mean height and weight were 173.7 \u00b1 1.5 cm and 72.0 \u00b1 11.0 kg, respectively. During the SRT, we found significant differences in VL and VM among four different pedaling positions at 180 W workload and SM at 210 W workload. For VL and VM muscles, the activation during cycling with KFOF at 20 mm was the greatest, while for the SEMI muscle, the activation with KFOF at 0 mm was the least. Comparing among the four increasing workloads, significantly increasing trends in VM muscle activation at KFOF at +20, 0, and \u221240 mm were found. The muscle activation of the VM would increase significantly as the workload increased from 120 to 210 W at KFOF at +20 and 0 mm and in comparison of 210 W with 150 W at KFOF at \u221240 mm .2 among the four pedaling positions  distance could alter the muscle activation of VL, VM, and SEMI in the SRT. Only the activation level in VM was found to be increased with the increasing workloads in the SRT. With various responses in the amateur cyclists in the GXT, no significant alteration or consistent pattern in the muscle usage strategy was found. However, a higher rate of energy expenditure in extreme pedaling positions of KFOF at +20 mm or \u221240 mm was observed in the GXT for most cyclists. It is recommended that cyclists find professional assistance for the proper bike fitting."}
+{"text": "Cancer vaccine, which can promote tumor-specific immunostimulation, is one of the most important immunotherapeutic strategies and holds tremendous potential for cancer treatment/prevention. Here, we prepare a series of nanoparticles composed of doxorubicin- and tyrosine kinase inhibitor-loaded and hyaluronic acid-coated dendritic polymers (termed HDDT\u00a0nanoparticles) and find that the HDDT nanoparticles can convert various cancer cells to micrometer-sized vesicles  with ~100% cell-to-HMV conversion efficiency. We confirm in two tumor-bearing mouse models that the nanoparticles can restrain tumor growth, induce robust immunogenic cell death, and convert the primary tumor into an antigen depot by producing HMVs in situ to serve as personalized vaccines for cancer immunotherapy. Furthermore, the HDDT-healed mice show a\u00a0strong immune memory effect and the HDDT treatment can realize long-term protection against tumor rechallenge. Collectively, the present work provides a general strategy for the preparation of tumor-associated antigen-containing vesicles and the development of personalized cancer vaccines. Autologous tumor cell vaccines can elicit anti-tumor immune responses. Here, the authors report the design of dendritic polymer-based nanoparticles loaded with doxorubicin and the tyrosine kinase inhibitor apatinib that can induce immunogenic cell death and the in situ production of immuno-stimulatory micrometer-sized vesicles, promoting anti-tumor immune responses in preclinical models. Various kinds of cancer immunotherapeutic approaches, such as immune checkpoint blockade (ICB) therapy12, cancer vaccines16, and chimeric antigen receptor T-cell therapy18, have been extensively investigated with desirable outcomes. Among them, cancer vaccine, which can arouse tumor-specific immunostimulation, is one of the most important immunotherapeutic strategies and holds tremendous potential for cancer treatment23. Besides, the neoantigen- and messenger RNA (mRNA)-based vaccines have achieved encouraging results in cancer patients25. However, despite great efforts made to develop cancer vaccines, they are still in the stage of cancer prophylaxis and evoking substantial immune responses in cancerous patients remains a huge challenge, mainly due to the weak immunogenicity of these vaccines, the immunosuppressive tumor microenvironment (TME), and the low relevance between the antigens in these vaccines and the tumors of specific patients27.Cancer immunotherapy has shown great promise in cancer treatment during the past decade33. Benefiting from their tumor-specific tumor-associated antigens (TAAs), the autologous tumor-cell vaccines exhibit a stronger immune response-inducing ability than traditional cancer vaccines36. However, in vitro preparation of autologous vaccines faces the problems of complex procedure, low yield, and unsatisfactory efficacy, limiting the clinical applications of autologous cancer\u00a0vaccines. On the other hand, the massive in situ production of autologous tumor-cell vaccines in vivo, which avoids the complicated vaccine preparation procedures in vitro, is a highly desirable choice for producing cancer vaccines.To improve the therapeutic outcomes of cancer vaccines, autologous tumors have been used to produce cancer vaccines  in vitro or in vivo48, most of them require complicated and rigorous physical stimulation conditions , making it difficult or even impossible to mildly produce cancer cell-derived vesicles in situ. In contrast, our vesicle-producing strategy based on the HDDT NBs, which induces cancer cell death and realizes the in situ massive production of cancer cell-derived vesicles, requires no external stimuli and avoids the strict storage conditions required for the non-in-situ vaccines. Further studies reveal that after systemic administration, the HDDT NBs (taking the HA\u2212dendrimer (Den)\u2212DOX\u2212apatinib (Apa) (HDDA) NBs as an example) can actively accumulate in the tumor regions, restrain the tumor growth in different tumor models, massively induce immunogenic cell death (ICD) and produce HMVs in situ, translate the tumor tissues into the antigen depots, evoke intratumoral and systemic immune responses alone or in combination with ICB therapy, and establish strong immunological memory effects to efficiently protect the cured mice from tumor rechallenge.In this work, we fabricate a series of nanobombs (NBs) comprising doxorubicin (DOX)- and tyrosine kinase inhibitor (TKI)-loaded and hyaluronic acid -coated dendritic polymers (termed HDDT NBs) Fig.\u00a0. We findFour representative TKIs , sorafinib (Sor), and dasatinib (Das)) were used in our study. Considering that Apa is one of the most widely used TKIs and numerous studies have shown its good biosafety and superb anticancer activity, we chose Apa as the representative TKI in our study. As a representative example, the HDDA NBs were prepared according to the procedure shown in Fig.\u00a0To study the anticancer effect of HDDA NBs, we first examined the internalization of HDDA by murine mammary carcinoma 4T1) cells. The time-dependent confocal microscopic images , 5-amiloride hydrochloride (amiloride), and genistein could also partially affect the uptake of HDDA NBs by the cells, while chlorpromazine hydrochloride (CPZ) had little influence on the endocytosis process , B16F10 cells (a murine melanoma cell line), MCF-7 cells (a human breast cancer cell line), and MCF-7/ADR cells . Not surprisingly, these cells were also changed to HMVs with different diameters , which usually have diameters of 1\u20135\u2009\u03bcm and high expression levels of annexin V and cleaved caspase-3Then, to figure out the mechanism of HMV formation, the transcriptomic analysis of the 4T1 cells after HDDA treatment was carried out. The 4T1 cells with culture medium treatment were set as the control group. To be specific, DEG  analysis, GO (Gene Ontology) enrichment analysis, and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis were carried out to compare the transcriptomes of the culture medium (control)- and HDDA-treated 4T1 cells. After HDDA treatment, 3425 DEGs were detected, among which 1523 DEGs were upregulated and 1902 DEGs were downregulated. The distribution of these DEGs was shown by the volcano plot Fig.\u00a0, and theThen to further study the protein profile of HMVs, the proteomic analysis of HMVs was carried out. As one of the most widely used cell debris, the sonication-induced cell debris (termed \u201cSoni\u201d sample) was also subjected to proteomic analysis for comparison purpose. The 4T1 cells were also used as another control group. First, the SDS-PAGE assay was carried out to study the cytoproteins (control), \u201cSoni\u201d sample proteins, and HMV proteins. The results shown in Supplementary Fig.\u00a0Next, to further study the protein profile of HMVs, the proteomic analyses of 4T1 cells (control), 4T1 cell-derived \u201cSoni\u201d sample, and HMVs were carried out. The statistical results of the total detected proteins and the Venn diagram revealed that the 4T1 cells (control) and the \u201cSoni\u201d sample contained more types of proteins than the HMVs  . Thus, three vital DAMPs, i.e., calreticulin , high-mobility group box 1 protein , and adenosine triphosphate , were analyzed to study the HDDA-induced ICD effect Fig.\u00a0 and flow60  and present it to immature DCs for DC maturation60 Fig.\u00a0, which i60 Fig.\u00a0, the HMV+CD86+MHC II+)  and M1-like repolarization of macrophages induced by the \u201csecretome\u201d of HDDA-treated cells were investigated. The DC maturation was studied first by using bone marrow-derived dendritic cells (BMDCs). 4T1 cells were seeded into the upper chambers of the transwells  and pretreated with culture medium (control), DOX, Apa, DOX\u2009+\u2009Apa, HDD, HDA, and HDDA, respectively. Next, the culture medium was replaced by fresh culture medium, and the immature BMDCs were seeded into the lower chambers of the above transwells  Fig.\u00a0. Not sur62. M1-like TAMs exhibit anticancer activity and immunostimulatory effect, while M2-like TAMs promote tumor growth and exhibit immunosuppressive effect. Thus, the repolarization of the M2-like TAMs to the M1 phenotype is a promising strategy to reverse the immunosuppressive TME and improve antitumor immunity. It has been reported that ROS have the ability to repolarize the M2-phenotype TAMs to the M1-phenotype ones63. Inspired by the ROS-producing ability of the HDDA NBs, we next investigated whether the HDDA NBs could repolarize the M2-phenotype TAMs by using the M2-like RAW 264.7 mouse macrophages, and the procedure was similar to that shown in Fig.\u00a0+CD11c+) (94.13%) was observed in the HDDA group  (64.78%) and M1-like macrophages (F4/80+CD11c+) (31.25%) were remarkably increased after HMV treatment  is another important marker of immunostimulation leveloup Fig.\u00a0, indicatTo assess whether the HDDA NBs could also induce ICD in vivo and make tumor cells produce HMVs for cancer immunotherapy, 4T1 breast tumor-bearing BALB/c mouse models were established. First, the hemolytic effect of the HDDA NBs was studied. The results indicated that the incubation of red blood cells (RBCs) with HDDA NBs only caused little hemolysis even at a DOX concentration of 14.5\u2009\u03bcg/mL  injected with different drugs for 3 times Fig.\u00a0. NotablyBased on the in vitro experiments, we deduced that after the HDDA-induced tumor ablation, the HMVs would be produced and acted as the autologous tumor-cell vaccines for intratumoral and systemic immunostimulation. Therefore, the in situ HMV production in the tumor tissues from\u00a0the\u00a0HDDA-treated mice was studied. First, the membrane marker CD44 was used to track the HMVs in tumor tissues. The Western blot analysis and immunofluorescence staining results of the CD44 in HMVs in vitro revealed the high CD44 expression level in the HMVs  which overexpress CD11c , MDSCs (CD11b+Gr-1+), and Tregs (CD3+CD4+FoxP3+) in the tumor regions were also analyzed by flow cytometry , which can present antigens to T cells, was also elevated in the HDDA group  and cytotoxic T cells (CD3+CD8+)  signal was detected in the tumor tissues from HDDA-treated mice ice Fig.\u00a0, furtherNext, to further understand the underlying mechanisms of the antitumor and immunostimulatory effects of HDDA NBs and the HMV production in the tumor tissues of the HDDA-treated mice, we then studied the transcriptomic features of the 4T1 tumor tissues after HDDA NB treatment. The tumor tissues of the PBS-treated 4T1 tumor-bearing BALB/c mice were set as the control group. As shown by the volcano plot and heat map, 1396 DEGs were detected, among which 830 DEGs were upregulated and 566 DEGs were downregulated after the HDDA NB treatment  in TDLNs and spleens among all the groups. Besides, the HDDA NB treatment could also markedly elevate the proportion of the M1-like TAMs (F4/80+CD11c+) in TDLNs and spleens  and spleens were retrieved from 4T1-bearing BALB/c mice 7 d after various treatments. As illustrated by the flow cytometric results Fig.\u00a0, the micens Fig.\u00a0, which aens Fig.\u00a0 and 42.FBesides, the concentrations of various proinflammatory and antiinflammatory cytokines in serum were measured. The levels of proinflammatory cytokines  in serum were significantly elevated and the antiinflammatory IL-10 was close to the baseline level after HDDA treatment Fig.\u00a0. In addiCollectively, the HDDA treatment could induce strong antitumor systemic immune responses.CM, CD3+CD8+CD44+CD62L+) and effector memory T cells  in the spleens of the 4T1-bearing BALB/c mice 30 d after different treatments. The flow cytometric results indicated that the percentages of TEM and TCM in the CD3+CD8+ T cells in the HDDA group showed a significant increase compared with those in the control mice  injected with tumor cells was almost completely suppressed, all the mice in the HDDA group survived within the 40 d observation period, and no tumor nodules were observed in the lungs of the mice that were i.v. injected with tumor cells Fig.\u00a0, indicatFurther, the in vivo biocompatibility of the HDDA NBs was analyzed. The PBS-treated mice were set as the control group. The results showed that neither abnormal changes in the routine blood analysis and biochemical analysis results nor tissue damage in the histopathologic images of the major organs in the HDDA-treated mice were observed  in lymph nodes  and helper T cells (CD3+CD4+) in the HMVs group were also markedly increased  (forming DiD@HMVs). The BALB/c mice were s.c. injected with DiD@HMV suspension   and cytotoxic T cells (CD3+CD8+) in lymph nodes after DiD@HMV treatment were also elevated  , HMVs, or the \u201cSoni\u201d sample was evaluated. Compared with the other two groups, the HMVs showed the most significant tumor suppression during the 30 d observation period with negligible body weight loss  and i.v. injected with various samples  for 3 times, respectively  blockade therapy) was also introduced to enhance the immunostimulation effect of the HDDA NBs. The experiments were performed using the B16F10 tumor-bearing C57BL/6 mice, which were randomly divided into five groups  in the TME after \u201cHDDA\u2009+\u2009anti-PD-1\u201d treatment was remarkably elevated  and cytotoxic T cells (CD3+CD8+) in the \u201cHDDA\u2009+\u2009anti-PD-1\u201d group were further increased  were significantly decreased  in TDLNs and spleens was observed in the \u201cHDDA\u2009+\u2009anti-PD-1\u201d group   Fig.\u00a0. As for oup Fig.\u00a0, and simoup Fig.\u00a0. Additiooup Fig.\u00a0. The aboFurthermore, the B16F10 tumor-bearing C57BL/6 mice that survived after the first \u201cHDDA\u2009+\u2009anti-PD-1\u201d treatment were rechallenged with B16F10 tumor cells by intravenous injection. Notably, no tumor nodules in the lungs were observed after intravenous rechallenge  to induce robust cancer cell death and realize the in situ massive production of HMVs without the need of external stimuli like light irradiation, sonication, and electric field. Importantly, we found that the cancer cells could be turned into HMVs (average size: 1.6\u20133.2\u2009\u03bcm) with ~100% cell-to-HMV conversion efficiency. We also found that the HDDT NBs (taking the HDDA NBs as an example) could accumulate in the tumor region, induce robust ICD, reverse the immunosuppressive TME, and massively produce HMVs. As a result, the tumor tissue was transformed into a depot of autologous vaccines that triggered the systemic tumor-specific immune responses . Moreover, we found that the HDDA NBs-triggered systemic tumor-specific immune responses could be further enhanced by ICB therapy. As shown in two tumor models, the HDDA NBs could restrain the tumor growth and evoke intratumoral and systemic immune responses alone or in combination with ICB therapy. In addition, the HDDA NBs could establish strong immunological memory in the cured mice, which could efficiently protect the cured mice from tumor rechallenge.On the other hand, traditional cancer vaccines containing specific antigens have poor therapeutic outcomes owing to the low relevance between the antigens in these vaccines and the tumors of specific patients. In our strategy, the primary tumor itself acts as the depot of the in situ autologous tumor-cell vaccines containing all the tumor-specific DAMPs and TAAs for immunostimulation. In addition, the massive and highly efficient in situ production of HMVs avoids the complicated vaccine manufacture in vitro and the strict storage conditions required for these non-in-situ vaccines. Further, the two drugs (DOX and TKIs) we used in the HDDT NBs have already been widely applied in clinic for many years and the Den and HA have been proved to have satisfactory biocompatibility, which facilitate the potential clinical applications of the HDDT NBs. Collectively, the HMV production strategy provides a simple and promising nanotechnology-based strategy for in situ generation of autologous tumor-cell vaccines and may shed light on the development of personalized cancer vaccines.\u03b2CD were obtained from Aladdin . Lap was ordered from Energy Chemical . The ethylenediamine core amine-terminated G4 polyamidoamine (PAMAM) dendrimer (Den) (polydispersity index\u2009<\u20091.08) was bought from Dendritech . Collagenase IV was obtained from BioFroxx . Deoxyribonuclease I was ordered from Leagene Biotechnology Co., Ltd. . Methylthiazolyldiphenyl-tetrazolium bromide (MTT) was bought from Shanghai Yuanye Bio-Technology Co., Ltd. Dimethyl sulfoxide (DMSO) and methanol were obtained from Shanghai Lingfeng Chemical Reagent Co., Ltd. IL-4 and GM-CSF were bought from Pepro Tech Inc. . DiD and DiR were obtained from KeyGEN BioTECH Co., Ltd. . Hoechst\u00a033342 was purchased from Beyotime Institute Biotechnology . FITC-labeled goat anti-rabbit immunoglobulin G (IgG) antibody  bs-0296G-FITC), anti-mouse CD44-FITC antibody (cat. no. bs-0521R-FITC), and enhanced chemiluminescence (ECL) reagent were obtained from Bioss Antibodies . The horseradish peroxidase (HRP)-labeled goat antirabbit IgG (H\u2009+\u2009L) antibody (cat. no. E-AB-1003) was bought from Elabscience Biotechnology Co., Ltd. . Anti-mouse CD44 (cat. no. mAb #3570) and anti-mouse caspase-3 (cat. no. mAb #9668) were bought from Cell Signaling Technology . Anti-mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (cat. no. ab8245) was ordered from Abcam . Anti-mouse cleaved caspase-3 (cat. no. Asp175) was obtained from Affinity Biosciences . PD-1 monoclonal antibody (cat. no. BP1046) was obtained from BioXcell . Anti-mouse CRT (cat. no. 27298-1-ap) was purchased from Proteintech . Anti-mouse CD4 (cat. no. GB13064-2), anti-mouse CD8 (cat. no. GB13429), anti-mouse granzyme B (cat. no. GB14092), anti-mouse Gr-1 (cat. no. GB11229), anti-mouse FoxP3 (cat. no. GB11093), anti-mouse CD206 (cat. no. GB13438), and cyanine3 (Cy3)-labeled goat anti-rabbit IgG antibody (cat. no. GB21303) were obtained from Wuhan Servicebio Technology Co., Ltd. (China). Anti-mouse CD3-PE (cat. no. 12-0032-82), anti-mouse CD4-FITC (cat. no. 11-0041-82), anti-mouse CD8a (CD8)-APC (cat. no. 17-0081-81), anti-mouse CD8-PE-Cy7 (cat. no. 25-0081-81), anti-mouse MCH II-PE (cat. no. 12-5321-82), anti-mouse CD11c-FITC (cat. no. 11-0114-82), anti-mouse CD80-PE (cat. no. 12-0801-82), anti-mouse CD86-PE-Cy7 (cat. no. 25-0862-82), and TRIzol reagent were bought from Invitrogen . Anti-mouse CD11c-PE (cat. no. 117308) and anti-mouse F4/80-FITC (cat. no.124611) were purchased from BioLegend . The HMGB1 ELISA kit was bought from Cusabio Biotech Co., Ltd. . Mouse cytokine ELISA kits for IL-1\u03b2 (cat. no. 88-7013-22), IL-6 (cat. no. 88-7064-22), and TNF-\u03b1 (cat. no. 88-7324-22) were purchased from Invitrogen , and IL-10 (cat. no. EK210/4-96) and IFN-\u03b3 (cat. no. EK280/3-48) were bought from MultiSciences (Lianke) Biotech, Co., Ltd. . Deionized water (18.2 M\u2126\u2022cm) was obtained from a Milli-Q system . Dialysis membranes with a molecular weight cut-off (MWCO) of 250\u2009kDa were purchased from Spectrum Labs . Ultrafiltration tubes\u00a0(MWCO = 100 kDa) were obtained from Merck Millipore Ltd. .\u00a0Unless otherwise mentioned, all the antibodies are diluted 200 times before use.DOX\u2022HCl was ordered from Beijing Huafeng United Technology Co., Ltd. (China). Sodium dodecyl sulfate (SDS), LPS, Triton X-100, and glutaraldehyde  were ordered from Sigma-Aldrich . HA (110\u2009kDa) was purchased from Shanghai Macklin Biochemical Co., Ltd. . Apa, Das, Sor, trimethylamine, CPZ, bovine serum albumin (BSA), genistein, amiloride, and MDen was dissolved in methanol with a concentration of 1\u2009mM. DOX\u2022HCl was dissolved in methanol and was neutralized with triethylamine to obtain the HCl-free DOX solution (10\u2009mM). Apa was dissolved in methanol with a concentration of 5.8\u2009mg/mL. Then 50\u2009\u03bcL of DOX solution, 25\u2009\u03bcL of Apa solution, and 150\u2009\u03bcL of Den solution were mixed and the suspension was vortexed for 20\u2009s, followed by evaporation under nitrogen gas. The obtained powder was resuspended with 1\u2009mL of deionized water under bath sonication for 2\u2009min, and the resultant suspension was centrifuged  to remove the precipitates. The DDA NPs were in the supernatant and the concentrations of DOX and Apa in the NPs were quantified by UV\u2212vis spectroscopy.For HA coating, 1\u2009mL of the above-obtained DDA suspension (with the DOX concentration of 0.5\u2009mM) was added to 1\u2009mL of HA solution (1\u2009mg/mL) dropwise under agitation and the mixture was further vortexed for 20\u2009s to form the HDDA NBs. The resultant HDDA suspension was dialyzed (MWCO\u2009=\u2009250\u2009kDa) to remove free HA molecules. For comparison purpose, the HDD and HDA NPs were prepared in a similar way.2O with a concentration of 1\u2009mM. DOX\u2022HCl was dissolved in DMSO and was neutralized with triethylamine to obtain the HCl-free DOX solution (20\u2009mM). Lap was dissolved in DMSO with a concentration of 5.8\u2009mg/mL. Then, 150\u2009\u03bcL of Den solution was added into 800\u2009\u03bcL H2O. Next, 25\u2009\u03bcL of DOX solution and 25\u2009\u03bcL of Lap solution were mixed and vortexed for 20\u2009s, and the resultant mixture was added to the above Den solution dropwise under stirring to obtain the Den\u2212DOX\u2212Lap NPs. The resultant Den\u2212DOX\u2212Lap suspension was centrifuged (6700\u2009g for 10\u2009min) to remove the precipitates. For HA coating, 1\u2009mL of the above obtained Den\u2212DOX\u2212Lap suspension (with the DOX concentration of 0.5\u2009mM) was added to 1\u2009mL of HA solution (1\u2009mg/mL) dropwise under agitation and the mixture was further vortexed for 20\u2009s to form the HDDL NBs. The resultant HDDL suspension was dialyzed (MWCO\u2009=\u2009250\u2009kDa) to remove the free HA molecules. The HDDS and HDDD NBs were obtained in a similar way.For the fabrication of HDDL, Den was dissolved in H7 4T1 cells were collected via centrifugation , and were resuspended in PBS to obtain the 4T1 cell suspension. Then, the cell suspension was treated with bath sonication for 10\u2009min and washed with PBS via ultrafiltration (MW\u2009=\u2009100\u2009kDa) for at least 3 times to obtain the PBS suspension of the \u201cSoni\u201d sample. The protein contents of HMVs and the \u201cSoni\u201d sample were determined by a BCA protein assay kit .To prepare and isolate HMVs, 4T1 cells were seeded into 6-well plates and incubated for 24\u2009h. Next, the cells were treated with HDDA NBs (DOX: 14.5\u2009\u03bcg/mL) for 24\u2009h. The suspensions were collected, washed with PBS, and purified via ultrafiltration (MWCO\u2009=\u2009100\u2009kDa) for at least 3 times to obtain the PBS suspension of HMVs. To prepare the \u201cSoni\u201d sample, 1\u2009\u00d7\u200910The encapsulation ratio (ER) and loading coefficient (DL) of Apa or DOX in HDDA NBs were calculated via the equations shown below (1 and 2):The morphology and size of HDDA and DDA were studied using a transmission electron microscope . The zeta potentials and hydrodynamic sizes of HDDA and DDA were characterized by a zetasizer instrument . The UV\u2013vis absorption spectra of DOX, Apa, HA, Den, and HDDA solutions/suspensions were measured by a Shimadzu UV-2600 spectrophotometer (Japan).oC in 5% CO2. B16F10, HPAEpiC,\u00a0HMEC-1, MDA-MB-231, and NIH 3T3 cells were incubated in Dulbecco\u2019s modified Eagle\u2019s medium (DMEM)  containing 10% FBS, 0.08\u2009mg/mL streptomycin, and 80\u2009U/mL penicillin at 37\u2009oC in 5% CO2.4T1 (cat. no. KG338), B16F10 (cat. no. KG078), MCF-7 (cat. no. KG031), MCF-7/ADR (cat. no. KG0311), NIH 3T3 (cat. no. KG085), and RAW 264.7 (cat. no. KG240) cells were obtained from KeyGEN BioTECH, China. HMEC-1 (cat. no. bio-74149), HPAEpiC (cat. no. bio-73467), and MDA-MB-231 (cat. no. bio-69126) cells were purchased from American Type Culture Collection (ATCC), USA. MCF-7, 4T1, RAW 264.7, and MCF-7/ADR cells were cultured in Roswell Park Memorial Institute (RPMI) 1640  containing 10% fetal bovine serum (FBS), 0.08\u2009mg/mL streptomycin, and 80\u2009U/mL penicillin at 37\u2009oC) with 12\u2009h dark\u2212light cycles and a relative humidity of 40\u201370%. All mice had access to food and water ad libitum. The 4T1 xenograft tumor model was built by inoculating 8\u2009\u00d7\u2009106 4T1 cells into the back of BALB/c mice. The B16F10 xenograft tumor model was built by inoculating 8\u2009\u00d7\u2009106 B16F10 cells into the back of C57BL/6 mice. For the maximal tumor size/burden, we adhered to the guideline of the maximal tumor diameter of 20\u2009mm according to the national standard ), and the maximal tumor size/burden in this study was not exceeded.BALB/c and C57BL/6 mice  were ordered from Yangzhou University Medical Center . Mice were housed in groups of 5 mice per individually ventilated cage with constant room temperature  were separately added to 4T1 cells and incubated for 2\u2009h at 37\u2009oC in serum-free culture medium. After the pretreatment, the culture medium was carefully replaced with HDDA (DOX: 3.6\u2009\u03bcg/mL) in complete RPMI 1640 containing different inhibitors (except M\u03b2CD) with further incubation for 2\u2009h. To study the energy-dependent endocytosis, 4T1 cells were also incubated at 4\u2009oC for 2\u2009h. After that, the culture medium was carefully replaced with HDDA (DOX: 3.6\u2009\u03bcg/mL)-containing culture medium and the cells were further incubated at 4\u2009oC for 2\u2009h. Then, the treated cells were collected and analyzed by a flow cytometer  or a confocal microscope .To investigate the endocytic pathways, different endocytosis inhibitors such as amiloride , CPZ , genistein , and MTo study the composition of the 4T1 cell-produced HMV proteins, the cytoproteins, cytoplasmic proteins, cell membrane proteins, and HMV proteins of 4T1 cells were collected using a membrane and cytosol protein extraction kit  and the SDS-PAGE assay was performed following a standard protocol.3 cells/well. 24\u2009h later, the cell culture medium was replaced by fresh culture medium containing various concentrations of DOX, Apa, DOX\u2009+\u2009Apa, HDD, HDA, or HDDA. After another 24\u2009h, MTT assay was performed to determine the relative cell viabilities. The viability of untreated cells (control) was set as 100%.4T1 cells were seeded in 96-well plates at a density of 5\u2009\u00d7\u2009104 cells/well) with 450\u2009\u03bcL culture medium\u00a0in each well. Next, different drugs were added separately by replacing the initial culture media with the drug-containing media when the cell index (CI) value reached 2.8\u22124.5. The real-time proliferation processes of the cells were recorded based on an impedance-based system and were presented as cell growth curves by the software of the instrument.The real-time cytotoxicities of culture medium (control), DOX, Apa, DOX\u2009+\u2009Apa, HDA, HDD, and HDDA  were studied by a real-time cell analyzer\u2014the iCELLigence system . Briefly, 4T1 cells were seeded into 8-well L8 E-plates . Then all the cells were measured by the flow cytometer.The production of ROS was determined by the ROS probe DCFH-DA . Specifically, 4T1 cells after different treatments were collected and treated with DCFH-DA (10\u2009\u03bcM) for 30\u2009min (37\u2009To study the cellular apoptosis/necrosis induced by culture medium (control), DOX, Apa, DOX\u2009+\u2009Apa, HDA, HDD, and HDDA , we carried out the annexin V-FITC/propidium iodide (PI) apoptosis detection assay. In brief, the 4T1 cells after respective treatments were collected, washed three times with PBS, treated with an apoptosis detection kit , and then measured by the flow cytometer.4 cells/well) and incubated for 1 d. Then, the cells were incubated with various drugs. Confocal images were taken by the confocal microscope.For confocal imaging, 4T1 cells were seeded in Lab-Tek 8-well chamber slides  for various time periods. Next, the media were replaced by the Hoechst working solution (10\u2009\u03bcg/mL) and further incubated for 10\u2009min. Then, the cells were washed with PBS and observed by the confocal microscope at the excitation wavelengths of 405 (for Hoechst) and 552 (for DOX) nm, respectively.To study the endo-lysosomal escape of HDDA NBs, 4T1 cells were incubated with HDDA NBs (DOX: 3.6\u2009\u03bcg/mL) for different time periods. Next, the culture media were replaced by the media containing Hoechst (10\u2009\u03bcg/mL) and LysoTracker Green  and further incubated for 15\u2009min. Finally, the 4T1 cells were washed with PBS and observed by the confocal microscope at the excitation wavelengths of 405 (for Hoechst), 488 (for LysoTracker Green), and 552 (for DOX) nm, respectively.To investigate the cellular internalization of HDDA NBs by 4T1 and MDA-MB-231 cells, the two types of cells were treated with HDDA (DOX: 3.6\u2009\u03bcg/mL) for 3\u2009h. Then, the treated cells were collected and analyzed by the flow cytometer.To perform Western blot analysis, the cytoproteins of 4T1 cells, cell membranes (4T1), cytoplasm (4T1), and HDDA-induced HMVs (obtained from 4T1 cells) were collected as mentioned before. The Western blot analyses of CD44, caspase-3, and cleaved caspase-3 were performed following standard protocols. GAPDH was used as the loading control.4 cells per well and cultured for 1 d. After that, culture medium (control), DOX, Apa, DOX\u2009+\u2009Apa, HDA, HDD, and HDDA  were added to the cells separately and incubated for 4\u2009h. After being washed with PBS, the cells were fixed in 4% glutaraldehyde solution for 10\u2009min. Next, the cells were blocked by 5% BSA for 3\u2009h (25\u2009oC), treated with the anti-CRT antibody (1/400) overnight (4\u2009oC), washed with PBS, and then treated with the FITC-labeled goat anti-rabbit IgG antibody (1/300) for 3\u2009h (25\u2009oC). After being stained with Hoechst, the cells were observed under the confocal microscope. The sample treatment procedures of the flow cytometry analysis were similar to those of confocal microscopic imaging.The CRT expression on the cell membrane was studied via immunofluorescence assay and flow cytometry. For immunofluorescence assay, 4T1 cells were seeded into Lab-Tek 8-well chamber slides at a density of 1\u2009\u00d7\u200910The contents of the HMGB1 and ATP released by the 4T1 cells after different treatments were analyzed by the HMGB1 ELISA kit and enhanced ATP assay kit  following the manufacturers\u2019 protocols.BMDCs were collected from femurs and tibias of BALB/c mice . Briefly, the mouse bone marrow cells were collected and incubated in the X-VIVO 15 culture medium  with IL-4 (10\u2009ng/mL) and GM-CSF (20\u2009ng/mL) for 5 d to obtain the immature BMDCs. Besides, 4T1 cells were seeded into the upper chambers of the transwells  and incubated for 1 d. Next, the cells were incubated separately with culture medium, DOX, Apa, DOX\u2009+\u2009Apa, HDD, HDA, and HDDA  for 4\u2009h.\u00a0 Next, the culture medium was replaced by fresh culture medium. After that, the immature BMDCs were seeded into the lower chambers of the above transwells. After 1 d, BMDCs were collected and stained with anti-mouse CD11c-FITC, anti-mouse CD86-PE-Cy7, and anti-mouse MHC II-PE, and analyzed by flow cytometry. The immature BMDCs treated with LPS (25\u2009ng/mL) and GM-CSF (20\u2009ng/mL) for 1 d were set as positive control and negative control, respectively.For macrophage polarization experiment, the procedures were similar to those in the above experiment, and M2-like RAW 264.7 cells (pretreated with IL-4) were used to replace the above BMDCs. The RAW 264.7 cells treated with different samples were stained with anti-mouse F4/80-FITC and anti-mouse CD11c-PE antibodies, and then analyzed by flow cytometry. The RAW 264.7 cells treated with culture medium, IL-4 (20\u2009ng/mL), and IFN-\u03b3 (100\u2009ng/mL) + LPS (25\u2009ng/mL) for 1 d were set as the M0, M2, and M1 types of macrophages, respectively.The blood sample (1\u2009mL) was collected from healthy BALC/c mice. RBCs were obtained by the centrifugation  of the above blood sample and resuspended by PBS (50\u2009mL) to obtain an RBC suspension. Next, various concentrations of HDDA NB suspensions  were mixed with the RBC suspensions (0.5\u2009mL), respectively. After treatment for 2\u2009h, the supernatants were collected via centrifugation . RBCs in water and PBS were used as positive control and negative control, respectively.n\u2009=\u20099/group) for different treatments as follows for three times : (1) i.v. injected with PBS\u00a0(control), (2) i.v. injected with DOX solution, (3) i.v. injected with HDA suspension, (4) i.v. injected with HDD suspension, and (5) i.v. injected with HDDA suspension. Note that the groups 2\u20135 had the same DOX dose of 5\u2009mg/kg and the same injection volume (100\u2009\u03bcL). The tumor size and body weight were recorded for 38 and\u00a030 d, respectively. Tumor volume (V) was calculated as width2\u2009\u00d7\u2009length/2.The 4T1 tumor-bearing BALB/c mice were divided into five groups (n\u2009=\u200910/group) and i.v. injected with PBS\u00a0(control), DOX, HDA, HDD, and HDDA for 3 times (DOX: 5\u2009mg/kg), respectively. On the next day of each injection, half of the mice in each group were i.p. injected with the anti-PD-1 antibody (10\u2009mg/kg). The tumor size and body weight were recorded for 30 and 22 d, respectively.The B16F10 tumor-bearing C57BL/6 mice were divided into 5 groups  was used following the instructions from the manufacturer.oC overnight, followed by immobilization in OsO4 solution (1%) at room temperature for 1\u2009h. Next, the tumor tissues were dehydrated using ethanol with graded concentrations and then embedded in epoxy resin. An ultramicrotome  was used to obtain ultrathin tissue sections. Then, the obtained sections were stained with uranyl acetate/lead citrate and imaged under a transmission electron microscope (H-600(4), Hitachi, Japan).Briefly, the 4T1 tumor-bearing BALB/c mice were sacrificed after HDDA or PBS (control) treatment, and their tumors were excised for Bio-TEM measurements. First, the tumor tissues were fixed with 4% glutaraldehyde at 4\u2009For total cellular transcriptomic analysis, 4T1 cells were incubated with culture medium (control) or HDDA NBs (DOX: 3.6\u2009\u03bcg/mL) for 1 d, and the RNA samples were extracted following the TRIzol-based procedure. For tumor transcriptomic analysis, the 4T1 tumor-bearing BALB/c mice were sacrificed 7 d after PBS (control) or HDDA NB treatment (DOX: 5\u2009mg/kg), and the RNA samples of the tumors were collected following the TRIzol-based procedure. Total cellular RNA and tumor RNA were analyzed by Novogene .Proteomic analysis was performed using mass spectrometry in data-dependent acquisition mode. The proteins of 4T1 cells, \u201cSoni\u201d sample, and HMVs were analyzed by Shanghai Applied Protein Technology Co., Ltd. .The ELISA analyses of the extracellular levels of TNF-\u03b1, IL-1\u03b2, and TGF-\u03b2 produced by RAW 264.7 cells that were treated with the culture media of differently treated 4T1 cells, and the intratumoral and serum levels of IL-6, IL-1\u03b2, IFN-\u03b3, TNF-\u03b1, and IL-10 were performed following the instructions of the ELISA kits.oC for 30\u2009min. The spleens and TDLNs were soaked in PBS and passed through a cell strainer (70 \u03bcm) to obtain the single-cell suspensions. The single-cell suspensions were washed with 1\u2009wt% BSA buffer (solvent: PBS), and then stained with anti-CD11c-FITC, anti-CD80-PE (or anti-MHC II-PE), anti-CD86-PE-Cy7, anti-CD11c-PE, anti-F4/80-FITC, anti-CD3-PE, anti-CD4-FITC, and/or anti-CD8-PE-Cy7 following the instructions of the manufacturers to detect the maturation of DCs (CD11c+CD86+MHC II+ or CD11c+CD86+CD80+), the M1-like TAMs (F4/80+CD11c+), the helper T cells (CD3+CD4+), and the cytotoxic T cells (CD3+CD8+), respectively. All the antibodies were used at a dilution of 1:200. The antibodies-stained single-cell suspensions were analyzed by flow cytometry.The tumor tissues, spleens, and TDLNs of 4T1 tumor-bearing BALB/c mice and B16F10 tumor-bearing C57BL/6 mice after different treatments were retrieved for flow cytometry analyses. The tumor tissues were cultured in the dissociation buffer (100\u2009\u03bcg/mL deoxyribonuclease I and 1\u2009mg/mL collagenase IV) at 37\u2009The BALB/c mice were i.v. injected with PBS (control), DOX, HDA, HDD, or HDDA. The C57BL/6 mice were i.v. injected with PBS (control), DOX, HDA, HDD, or HDDA and i.p. injected with the anti-PD-1 antibody (10\u2009mg/kg). 7 d after administration, the major organs of the treated mice were collected for H&E assay following a standard protocol. Besides, the routine blood analyses were carried out by an automatic hematology analyzer , and the biochemical tests were performed on an automated biochemical analyzer . For the PBS- and HDDA-treated BALB/c mice, their major organs were also collected for Masson, Van Gieson (VG), hexamine silver, and TUNEL staining assays following standard protocols, respectively.n\u2009=\u20094/group) for different treatments as follows for three times : (1) subcutaneously (s.c.) injected with PBS; (2) s.c. injected with the \"Soni\" sample\u00a0(protein content: 1\u2009mg per mouse); (3)\u00a0s.c. injected with the HMV suspension (protein content: 1\u2009mg per mouse). Next, the mice were s.c. injected with 4T1 cells (8\u2009\u00d7\u2009106 cells per mouse) to build the subcutaneous 4T1 tumor models. The tumor size and body weight were recorded for 30 and 28\u00a0d, respectively.\u00a0To study the immune memory effect of HDDA, the\u00a0HDDA-healed BALB/c\u00a0mice were s.c. or i.v. rechallenged by 4T1\u00a0tumor cells\u00a0(s.c. injected with\u00a08\u2009\u00d7\u2009105\u00a0cells per mouse or\u00a0i.v. injected with\u00a08\u2009\u00d7\u2009104\u00a0cells per mouse), and the tumor size or the number of\u00a0tumor nodules were recorded. The untreated mice were set as the control group. To study the immune memory effect of\u00a0\"HDDA + anti-PD-1\", the\u00a0\"HDDA + anti-PD-1\"-healed C57BL6 mice were i.v.\u00a0rechallenged by B16F10 tumor cells (8\u2009\u00d7\u2009104\u00a0cells per mouse), and\u00a0the number of\u00a0tumor nodules was\u00a0recorded. The\u00a0untreated mice were set as the control group.To study the tumor prevention\u00a0effect of HMVs, BALB/c mice were divided into 3 groups  with Tukey\u2019s post-hoc test was adopted. P values of less than 0.05 were considered significant. *P\u2009<\u20090.05, **P\u2009<\u20090.01, ***P\u2009<\u20090.001, ****P\u2009<\u20090.0001. All statistical analyses were performed by GraphPad Prism 9, Excel 2016, or Excel 2019. The flow cytometry data were processed using NovoExpress .Most of the numeric data are expressed as mean\u2009\u00b1\u2009SD unless otherwise indicated. The significance between two groups was analyzed by two-tailed Student\u2019s Further information on research design is available in the\u00a0Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary data 2Supplementary Data 3Reporting Summary"}
+{"text": "Models of networks play a major role in explaining and reproducing empirically observed patterns. Suitable models can be used to randomize an observed network while preserving some of its features, or to generate synthetic graphs whose properties may be tuned upon the characteristics of a given population. In the present paper, we introduce the Fitness-Corrected Block Model, an adjustable-density variation of the well-known Degree-Corrected Block Model, and we show that the proposed construction yields a maximum entropy model. When the network is sparse, we derive an analytical expression for the degree distribution of the model that depends on just the constraints and the chosen fitness-distribution. Our model is perfectly suited to define maximum-entropy data-driven spatial social networks, where each block identifies vertices having similar position  and age, and where the expected block-to-block adjacency matrix can be inferred from the available data. In this case, the sparse-regime approximation coincides with a phenomenological model where the probability of a link binding two individuals is directly proportional to their sociability and to the typical cohesion of their age-groups, whereas it decays as an inverse-power of their geographic distance. We support our analytical findings through simulations of a stylized urban area. The definition of a suitable data-driven spatial social network model is a widely studied problem in computational social sciences. Various dynamical processes  can be represented on such networks, and the topology of the network has a direct impact on the evolution of the process. Having general models for social interactions, based on available data and capable of reconstructing stylized facts known from the literature, is of utmost importance to prevent from reaching conclusions biased by incorrect or ill-defined assumptions. With widely available survey and census data, it is now possible to generate synthetic geo-localized populations, stratified by age and organized into households. However, there is no equally direct way to accurately model interpersonal relationships.1. This property can be reproduced using the so-called Stochastic Block Model (SBM), risen to prominence as a way to generate networks with a known community structure3. In the SBM, the vertex set is partitioned into disjoint blocks and the probability of an edge between two nodes depends on the blocks to which the two nodes belong. In the original formulation of the SBM, all vertices belonging to the same block are indistinguishable, so that the degree distribution within each block tends to be Poisson-like for large graphs2. To produce a more realistic network, a few extensions to the model have been proposed in the literature, including the Degree-Corrected Block-Model (DCBM)2 and its maximum-entropy version4. These models are, in some sense, the SBM-equivalent of the well-known configuration model. They are based on enforcing both the desired group mixing and a target degree-sequence, either exactly or in expectation. If I and J \u2013 or twice that number, if I, and the density of the network is fixed equal to N is the size of the network.Many real social networks exhibit some form of group mixing, driven by the tendency of individuals to socialize with their peersp is a configuration parameter. In the FCBM the block-level mixing is specified in terms of a matrix of edge-densities 6 measures the propensity of each vertex to establish links and can be used to enforce the desired intra-block heterogeneity. In the DCBM the constants p, In this paper, we define and analyze the Fitness-Corrected Block Model (FCBM), a variation of the DCBM where the network density 4 for the DCBM, and later clarified and generalized in Ref.7. In a nutshell, the approach consists in: (1) the definition of an ensemble of networks, each with the same number of nodes, but with all possible configurations of links; (2) the constrained maximization of the entropy associated to the network ensemble, via the method of Lagrangian multipliers. By imposing the conditions I, J, i, the probability per graph in the ensemble factorises in terms of probabilities per link. The numerical value of the Lagrangian multipliers has to be calculated solving the system of nonlinear equations given by the model constraints. The resolution of this system might become expensive when the network is large and, to the best of our knowledge, it is not implemented in any publicly available software library. For the present paper, we implemented a parallel version of the solver for the maximum-entropy DCBM, written in C making use of the Intel MKL scientific library and the OpenMP API, which can also be used to solve our FCBM. The solver is released as open-source software at https://gitlab.com/cranic-group/dcbm_solver.To the purpose of having a model that is maximally random, the FCBM is defined following the approach first presented in Ref.i and j is directly proportional to their sociability and to the cohesion of their blocks, i.e., A known sparse-regime approximation for the edge probability of the DCBM implies that, in the sparse FCBM, the probability of a link binding two individuals 9.Among the many possible applications, the FCBM is especially well suited for generating a data-driven social network of geo-referenced and age-stratified individuals. The partition of the population into blocks can be obtained by grouping the individuals having similar position  and age, whereas the expected block-to-block edge-density matrix 11 tool to extract age-based social mixing patterns, and we embrace the widely-accepted assumption that an inverse-power-law relation binds the distance between two individuals and the frequency of their social interactions13. We generate instances of our FCBM using either the exact model or its sparse-regime approximation, varying both the spatial density of the individuals in the territory and the fitness distribution. We show that the empirical degree distribution obtained with all considered configurations is in sharp agreement with the analytical estimates. The implementation of the FCBM is publicly available as part of the Urban Social Networks (USN) framework at https://gitlab.com/cranic-group/usn.To show how the FCBM can be used in practice, and to provide empirical support to our analytical findings, we generate a set of synthetic social networks for a stylized urban population distributed on a disk of radius 2.5\u00a0Km. We use the SOCRATESIn the following, we summarize the main contributions and present the main analytical and experimental results of this paper. For all methodological details, we refer the reader to the \u201cMethods\u201d section.We propose the Fitness-Corrected Block Model (FCBM), a new maximum-entropy model for modular networks, parameterized by the network density V be a vertex set of size N partitioned into n blocks P, over all networks having vertex set V, fulfilling the following two conditions:P.Formally, let The FCBM can be seen as a generalization of the Degree-Corrected Block Model (DCBM). However, contrarily to the DCBM, we show that the FCBM is consistent for any choice of the configuration parameters, making it suitable for generating random graphs with tunable topological properties.P(G) of generating a specific graph G with the FCBM can be factorized as the product of independent edge probabilities, namely Leveraging on the framework of entropy-based null-models, we prove that the probability mentclass2pt{minim14, using Newton\u2019s method for the Hessian approximation. To the best of our knowledge, this is the first publicly available solver for the DCBM. The source code is publicly released as open-source software at https://gitlab.com/cranic-group/dcbm_solver.The system of nonlinear equations, needed to explicitly calculate edge probabilities, becomes computationally expensive as the size of the network increases. Already with a few thousand nodes, a straightforward implementation may be too slow to be used in practical applications. We implemented a parallel C-program that efficiently solves this system, as well as the analogous system arising from the DCBM. The solver follows the Sequential Quadratic Programming approach presented in Ref.The fitness sequence entclass1pt{minimaWhen the network is sparse, we show that the system from which all 9 and to study the spread of an epidemic process17, on it.We propose an application for our FCBM as a maximum-entropy model for data-driven spatial social networks. In particular, we envision its application to generate geographic networks informed by census data, contact surveys, and geospatial data. Indeed, the phenomenological model described at the end of this section has already been employed to develop a realistic social network at the urban scaleV describe an age-stratified population of N individuals living in a territory tessellated into square tiles of side l. Each Let the vertex set 20, can be used to reconstruct a data-driven age-based mixing matrix S9, whose Social mixing patterns, derived from heterogeneous data sources such as surveys, cell phones or wearable sensors13.The frequency of social relations between individuals living in To define a data-driven block-wise mixing matrix This leads to:i,\u00a0j, compute the edge probability P guarantees that: (1) the expected number of links between Finally, we extract the fitness vector In this case, the sparse-regime approximation yields9, where the probability of two individuals being connected is proportional to their sociability and to the cohesion of their age-groups, while decaying as a power of their distance. Clearly, the estimates obtained in (G is controlled by the available data and by the chosen fitness distribution Expression  defines ained in  and 4)  9, wherehttps://gitlab.com/cranic-group/usn.We implemented both the exact FCBM and its sparse-regime approximation. The code is released, as open-source software, as part of the Urban Social Networks (USN) framework at p so that the average degree of the network is S from data released by the POLYMOD project21. We considered three possible spatial densities\u2014uniform, and increasing or decreasing with the distance from the disk\u2019s center\u2014and three possible fitness distributions\u2014pareto, lognormal and exponential. For all nine combinations, we generated 10 independent graph instances.We used our data-driven FCBM to generate instances of a spatial social network for a stylized city of 10K inhabitants living in disk of radius 2.5\u00a0Km. We set In Fig.\u00a0Developing realistic network models for social interactions is of paramount importance to understand the underlying mechanisms that lead to the observed features of such networks and to study all dynamical processes, such as disease spreading, that are strongly influenced by the network topology. Extreme care must be taken to avoid that any bias is unintentionally injected in the model. For this reason, maximum-entropy models are extensively used in network analysis, either as null models, or to generate synthetic networks that have specific characteristics, but are otherwise maximally random.In this paper, we introduced the maximum-entropy Fitness-Corrected Block Model (FCBM), an adjustable-density model for modular and heterogeneous networks, whose block-wise mixing pattern is known in expectation. The model has a general and flexible formulation, but it was designed with a key application in mind: providing a working tool for building synthetic social networks informed with census, survey and geospatial data. Publicly available spatial density and demographic data are in fact necessarily discrete, thus inducing a partition of the population into blocks of agents who belong to the same age-group and live in the same area. The expected block-to-block edge-density can be estimated based on empirical findings that quantify the dependence of contact frequencies upon geographic and demographic features. However, both the density and the degree of heterogeneity of real-world social networks depend on the considered type of interpersonal relations and are rarely explicitly known beforehand. Contrarily to other block-models in the literature, the FCBM makes both these network features adjustable. In particular, the desired intra-block heterogeneity can be enforced through a vertex-intrinsic social fitness, possibly modelled upon observable population-level variables, such as wealth, employment or mobility.We implemented the FCBM and made it publicly available as open-source software. The released software includes a parallel code that speeds up the computation of the most expensive part of the required maximization procedure, a step of the algorithm that is also needed in the well-known Degree-Corrected Block Model. We tested our implementation of the FCBM by reconstructing instances of a social network connecting the individuals of a stylized city of 10K inhabitants. The experiments allowed to verify that the exact model and its efficient sparse-regime approximation yield networks with almost identical degree distributions. We also showed and experimentally verified that, in the sparse-regime, the expected degree distribution of the output network can be estimated by two closed-form expressions. Thanks to these two estimates, the shape of the degree distribution can be predicted based on the chosen fitness distribution.In the next future, we plan to use the FCBM\u2014and, possibly, a temporal extension of the model\u2014to simulate dynamical processes in real-world territories and understand how socio-demographic features and social habits affect the outcomes of these processes. To this end, we will work towards gaining a better understanding of how the topological properties of the FCBM depend on the configuration parameters, with special attention paid to a set of network properties, such as the clustering coefficient and the excess degree distribution, that can be used to study percolation and diffusion dynamics on the network.29. World-scale meta-population models and agent-based systems describing small and large areas can be informed by a variety of data sources. Census data and/or grid based population counts can be integrated to reconstruct populations that are statistically indistinguishable from real ones, including age, geographic distribution, education and wealth. The number and intensity of contacts in specific settings, such as workplaces, schools, or households, can be collected through surveys, questionnaires, diaries, and, if possible, supplemented with data obtained from digital technologies such as cell phones or wearable sensors20. These data are then used to reconstruct contact matrices, individual or group schedules, and are widely used to reproduce synthetic interactions31.Under the pressure of the COVID-19 pandemic, several simulation frameworks have been developed to provide realistic descriptions of the disease spread process on different spatial and temporal scales, from a single building to complex urban areas up to a global scale32. These networks show a heavy-tailed  degree distribution, often with a finite cutoff in agreement with Dunbar\u2019s number. The transitivity of the networks is high, compared to a random graph model, as a consequence of the well-established principle that \u201cfriends of my friends are my friends\u201d. Moreover, they show positive assortativity by degree and type. By quantitatively looking at ego networks, mobile phone networks, and online social networks we now have a better understanding of some of their peculiar features and underline mechanisms. Education, wellness, age and spatial proximity are regarded as critical elements in the formation of friendship bonds34. Among these, age is probably the most studied, possibly due to the fact that age-related data is actually available at different spatial scales35. While there is wide evidence that geographical factors alone cannot explain the structure of real-world spatial social networks37, the dependence of friendship on distance is widely assumed to follow an inverse power-law with exponent 41\u2014and this surprisingly holds even for online relationships42. In particular, 13 and for urban networks40, in line with sociological studies43. However, contrary to other real-world networks45, such networks do not present very large hubs46. Their degree distribution is right skewed and relatively long-tailed37, and it has been, at times, approximated by a power-law with a large (5\u20138) exponent46 or by a lognormal distribution13. Within cities, population density impacts on the frequency of close-range contacts, but usually not on the overall size of each person\u2019s network41. While geographical proximity and community structure appear to be related41, some authors argue that only small clusters 3, on the other hand, have been specifically developed to reproduce networks with a community structure, a typical characteristic of real-world social networks, which follows from the presence of some kind of group-level homophily. In this type of network models, the nodes are partitioned into disjoint sets named blocks and the probability of an edge existing between two nodes depends on the blocks to which the two nodes belong. The SBM and its generalization have gained their success in the last decades as they can be used to discover and understand the structure of a network, as well as for clustering purposes52.Defining simple models that capture all of these features is not an easy task. Models designed to mimic the scale-free degree distribution emerging in many real networks, for instance, may fail to yield the expected clustering structure53. Introducing a penalty on \u201clong\u201d edges, which mimics a penalty in maintaining long-distance relationships, has an impact on clusters, path lengths, degree distributions, and more54. Recently, network instances having suitable features have been generated by means of the so-called random geometric models57, where the popularity and similarity of the nodes depend on their position in some latent metric space58. Embedding the vertices into a hyperbolic disk55 has proved a way to obtain both high clustering and heavy-tailed degree distribution.Spatial network models are often obtained by incorporating vertices into a metric space and induced constraints can determine some of the network properties7. Among the very first fundamental papers, the work of Park and Newman has a particular relevance59: based on Jaynes\u2019 derivation of Statistical Physics from Information Theory60, they proposed a general maximum entropy approach for the randomization of complex networks. Among the extension to a different context, the main innovations of Ref.59 are the introduction of local constraints, as the degree sequence, and the interpretation of the general framework of Exponential Random Graphs (ERGs) in terms of maximum entropy models. The present construction was later extended to the analysis of real networks62, tailoring the entropy-based model on the observed network. As a matter of fact, the various Lagrangian multipliers, introduced for the entropy maximisation in Ref.59, can be numerically calculated by maximising the (Log-)Likelihood associated with the real network. Such a construction represents a perfect benchmark for the analysis of real systems, since it is maximally random (due to the entropy maximisation) and tailored on the observed system (due to the Likelihood maximisation). It is not surprising that it has been extensively applied to the study of non trivial structural patterns of different systems, as financial and trade networks, biological systems and online social networks63. Moreover, the general framework can be easily extended to tackle different kinds of networks, as undirected, directed, weighted, directed and weighted66, bipartite67, bipartite weighted68 and degree corrected block models4. Another relevant branch of research focuses on the reconstruction of networks from limited information69. This application is of particular interest for risk assessment of financial networks, and limited information is available due to privacy concerns70. Reconstruction approaches based on entropy-based null models have proven particularly effective in this context, and the maximally random nature of the framework described here is critical to avoid the introduction of bias into the predictions73.In the present paper, we leverage on the framework of entropy-based null-models for real complex networks, revised in Ref.N vertices. If P is a probability distribution over P(G) is the probability of graph P. The vertex set n blocks I is I,\u00a0J, i,\u00a0j) with Let G is I is I,\u00a0J, G, or, if G will be often omitted in the following.Each P over I. If H(P) denotes the Shannon entropy of P, the sought P can be obtained by finding the stationary points ofP(G). Since the functional derivatives with respect to P(G) are The maximum entropy Degree-Corrected Block Model (DCBM) is defined as the maximum entropy probability distribution i and all This results inWhen the average degree  rewrite  and 12)\\documentK and Since the constants =\u2211i\u2208Iki,  and 14)K and \\doGiven a scalar p is a configuration parameter. As in the original stochastic block model, the block-level mixing is specified in terms of a set of edge-densities 6. p. By design, p is a coFor fixed given by  and the ogous of  and 12)\\documentIn the sparse-regime, i.e, when ution to  and 18)\\documentIn this regime, the probability per edge can thus be rewritten asG with respect to both P. If 5, we haveI. Equation  the expected number of links between Finally, we extract the fitness vector G is controlled by the available data and by the chosen fitness distribution In this case, the sparse-regime approximation yieldspression  and 24)25\\docume"}
+{"text": "Desmoid tumors (DTs), also known as desmoid fibromatosis or aggressive fibromatosis, are rare, locally invasive, non-metastatic soft tissue tumors. Although histological results represent the gold standard diagnosis, imaging represents the fundamental tool for the diagnosis of these tumors. Although histological analysis represents the gold standard for diagnosis, imaging represents the fundamental tool for the diagnosis of these tumors. DTs represent a challenge for the radiologist, being able to mimic different pathological conditions. A proper diagnosis is required to establish an adequate therapeutic approach. Multimodality imaging, including ultrasound (US), computed tomography (CT) and Magnetic Resonance Imaging (MRI), should be preferred. Different imaging techniques can also guide minimally invasive treatments and monitor their effectiveness. The purpose of this review is to describe the state-of-the-art multidisciplinary imaging of DTs; and its role in patient management. Desmoid tumors (DTs), also known as desmoid fibromatosis or aggressive fibromatosis, is an unusual and locally aggressive monoclonal, fibroblastic proliferation characterized by a variable and often unpredictable clinical course. According to the World Health Organization (WHO), DT is a \u201cclonal fibroblastic proliferation that arises in the deep soft tissues and is characterized by infiltrative growth and a tendency toward local recurrence but an inability to metastasize\u201d, even though it may be multifocal in the same limb or body part . AlthougThe purpose of this review is to describe state-of-the-art nature of multimodality imaging evaluation, highlighting the advantages and disadvantages of the different techniques in abdominal and extra-abdominal DTs. We assessed their role during the different phases of patient management, including the possibility of guiding interventional radiology treatment.Desmoid tumors commonly affect individuals between the age of 15 and 60 years, with a peak incidence at 35\u201340 years and a reported incidence of 2\u20134 per million population, mainly in women of reproductive age ,31,32,33To date, two different clinical\u2013pathological entities have been identified: sporadic DT and DT associated with adenomatous polyposis coli (APC) gene mutation . Most ofIt was demonstrated that a third variant known as \u201cwild-type\u201d DT (without CTNNB1 or APC mutations) does not exist and is the result of a diagnostic error (other proliferations of spindle cells that mimic DT) or DT with unrecognized CTNNB1 or APC mutations ,3.The definitive diagnosis is histopathological with evidence of the proliferation of uniform spindle cells resembling myofibroblasts in the background of abundant collagenous stroma and the vascular network and characterized immunohistochemistry stains positive for nuclear B-catenin, vimentin, cyclooxygenase 2, tyrosine kinase PDGFRb, androgen receptor and estrogen receptor beta but negative for desmin, S-100, h-caldesmon, CD34 and c-KIT . Ne. Ne80]. Shi et al. evaluated the imaging features in 13 patients with desmoid fibroma of the extremities, finding that the tumors showed a lower density (69.23%), a higher degree of enhancement (61.54%) and unclear boundaries (84.62%) ; a CT vaTo the best of our knowledge, no one study described the role of DECT in DTs.As in US, CT also plays a pivotal role in guiding ablative treatment. In particular, CT-guided cryoablation is safe, effective and offers some important advantages. First, it presents the possibility of treating even large and multiple lesions in one session, creating a large area of ablation with low risks of spreading the pathological cells. Second, the technique is minimally invasive and requires low hospitalization times and mild anesthesia. Third, the functional recovery is satisfyingly immediate. Fourth, the procedure can be repeated with no risks for the patient in case of partial treatment ,151,152.Thanks to the multiparametric approach, the exceptional contrast resolution and the possibility to exploit several advanced sequences, magnetic resonance imaging is the diagnostic gold standard for the study, characterization and follow-up of extra-abdominal DTs, with a pivotal role for intra-abdominal ones ,142,143.Some characteristic but not specific findings of DTs on MRI have been identified. Low-signal-intensity non-enhancing linear bands in all sequences, known as the band sign, are present in 60% to 90% of DTs and can be seen in other benign (giant cell tumor of the tendon sheath) and malignant , likely corresponding to the dense collagenous stroma often found at histologic examination . Extra-aBeyond information on morphology, several MRI sequences can be used to obtain functional, ultrastructural information on tissue and deepen the diagnosis.Using DWI imaging, the DWI signal and the ADC values reflect the cellularity of the tissues, so even if there are no normal cutoff values, DWI can characterize the biological activity of the tissues ,159,160.Dynamic perfusion MRI is another functional imaging technique often used to evaluate tumors, mainly to depict the early intravascular and interstitial distribution of gadolinium ,171,172.MRI can guide ablative treatment as US and CT. High-intensity focused ultrasound (HIFU) ablation is a noninvasive treatment that has been successfully used for the treatment of various solid tumors . In the The critical weakness of MR assessment is related to the long examination and interpretation time, as well as higher costs, which still represent barriers to MRI use . AbbreviRegarding the abdominal wall, several pathological processes can cause wall lesions to comprise infection, hematoma, endometriosis and neoplasm ,182. RegDesmoid tumors in FAP patients are characterized considering clinical data, surgical history and imaging studies. CT and MRI allow us to characterize the lesion and to determine the relationship between lesions and surrounding organs for proper treatment planning. During CT study, the lesion density is uniform with uniform enhancement during the contrast study ,137. TheRegarding extra-abdominal DTs, several soft tissue lesions , occurring in extremities, head and neck and trunk, may mimic these entities. During CT studies, DTs of the extremities show a low density, mild enhancement and unclear boundary . In MRI So, CT or MRI scans can not only help for diagnosis but also in determining the relationship between tumors and the surrounding organs to obtain proper pre-treatment planning. Otherwise, although US assessment is safe since it does not use ionizing radiation so that the examination can be repeated several times ,115,116,Although imaging assessment could help characterization, a definitive diagnosis requires histopathological confirmation . PatholoDesmoid tumors represent a challenge for the radiologist, being able to mimic different pathological conditions. A proper diagnosis is required to establish the proper therapeutic approach in relation to the location, clinic and evolution of the disease. Imaging plays a pivotal role in the detection and assessment of these lesions. In the correct radiological disease management, multimodality imaging, including US, CT and MRI, should be preferred. Furthermore, in relation to the different phases of DTs approaches, detection and characterization, adjacent structures involvement assessment, treatment response evaluation and surveillance should also be considered. These different imaging techniques can also guide minimally invasive treatments and monitor their effectiveness.Regarding differential diagnoses, although imaging assessment could aid characterization, a definitive diagnosis requires histopathological confirmation."}
+{"text": "Results showed that the extension services significantly affected irrigated wheat yield with an average increase of 0.66\u00a0t/ha. Based on the results, out of the Marginal product value ($69 USD) of the extension services provided to the main farmers, 13.3% ($9 USD) had been spent as the cost of extension services and 86.7% ($60 USD) had been net profit gained by farmers. Farmers\u2019 behaviors changed as a result of the AEMs with reduced input costs for growing the crop and increased production and profit from the crop.Agricultural extension is a key policy to accomplish sustainable agricultural development by improving farmers\u2019 knowledge. Agricultural Extension Model Sites (AEMSs) is a new agricultural extension approach for empowering farmers in Iran. Therefore, the purpose of this research is the economic effect assessment of extension services of AEMSs for irrigated wheat production in Iran. Surveys were conducted with 180 people of the main farmers from irrigated wheat AEMSs throughout Iran. The research tool was a questionnaire, the validity of which was examined using the opinions of a group of experts from the Agricultural Education and Extension Institute in Iran. Data analysis was done using econometric analysis and One-way ANOVA analysis through SHAZAM And are subject to uncertain weather and a changing climate, which jeopardizes food security even more8. Consulting, education, and extension services to smallholder farmers have been found effective in assisting them to adapt their farm practices and reduce the economic costs of climatic impacts9. This is important in Iran where smallholding is a substantial proportion of farming. The improvement of agricultural productivity is a policymaking concern in developing countries. In 2017 while 60% of the population of less-developed economies were employed in agriculture, but they only produced 24% of the gross domestic product10. In addition, global population growth, economic development, and climate change have meant that farmers are now increasingly overusing their land, leading to serious environmental pollution, land degradation, and ecosystem imbalance11. Agriculture is increasingly influenced by global forces, such as new scientific discoveries, demographic variations, changes in social and economic characteristics, changes in society\u2019s consumption pattern, and mutual dependence on global markets. These changes influence the agricultural production system and affect the future of the agricultural sector12. To increase agricultural productivity a link is needed between farmers and modern technology. Agricultural extension is an important area of the investigation of effects to enhance the performance of agricultural extension agents and thereby improve the delivery of extension services in developing regions like Iran15. The consulting services of agricultural and rural extension are a key instrument for promoting technical changes, escalating the growth of agricultural productivity, and finally improving farmers\u2019 livelihoods16. Access to agricultural information and knowledge is essential for sustainable production, and the main roles played by the extension system are to facilitate information sharing among scientists, extension agents, and farmers and to guide farmers with the sound use of technology17. It seems that to effectively increase the adoption of improved technologies, it is necessary to pay more attention to designing and implementing public extension programs20. Agricultural advisory services facilitate a switch to more efficient production methods, higher yields, and higher farm incomes. Besides influencing farmer behavior towards new technologies, farm expansion can also disseminate knowledge about the optimal use of inputs, cultivation techniques, prices, pest and risk management, access to credit, etc.21. The effect of agricultural extension is positively associated with technical efficiency, and it is shown that the agricultural extension service can enhance farmers\u2019 productivity and livelihood by educating them about the correct use of resources23.Developing countries are faced with low productivity in the agricultural sector and food insecurity24.Given the increased global demand for food when considering the prediction that the global population will reach 9.7 billion by 2050, i.e., 2 billion more than the current population. So, based on the information provided by the Food and Agriculture Organization of the United Nations (FAO), crop production needs to be increased by 60% in order to be able to supply the future food security of the world25. 17% of the world's arable land space and 50% of it is allocated to wheat production. Now, the cultivated area of irrigated and rainfed wheat in Iran is approximately 2 and 4 million hectares, respectively. Iranian people consume 12.5 million tons of wheat annually and the per capita consumption of wheat is c 141\u00a0kg26. It is acceptable that agricultural extension by providing educational services can be effective in nature conservation. The belief of agricultural extension services and their role in the conservation of agriculture can be effective in improving farmers' reactions to climate change and developing sustainable agriculture27. Agricultural extension approach can be effective, by improving farmers\u2019 knowledge to improve agricultural management through the improvement of agricultural productivity28. Achieving this goal will be possible only through the provision of effective agricultural knowledge transfer services29.Today, wheat cultivation is considered as a key food source in the world. For many years, bread and durum wheat have been cultivated in European countries to provide the energy and nutrients needed by humans. Nowadays, China and India are the major producers of wheat, mostly because wheat requires less water, and at the same time, it is a central component of a variety of processed products in modern and more urban life. For over 100\u00a0years, farmers have continuously developed wheat by focusing on components influencing crop yield and, more recently, quality technology by the Agricultural Extension Services30.As shown in Fig.\u00a0As shown in Fig.\u00a034. Since AEMSs is a relatively new concept in Iran, limited studies have focused on them. In a study on the effect of agricultural extension programs on rural wheat growers\u2019 knowledge and productivity in Kermanshah County, Iran, Ali-baygi and Ghanbar-ali35 concluded that the implementation of the farmers field school program in the study site influenced farmers\u2019 knowledge and revenues significantly.A literature review shows that many studies have been published on the effect of agricultural extension services over the years, with mixed results36 found that the farmers participating in the AEMSs had gained significantly higher quantity and quality of field crops, horticulture, and vegetables production. Degrees of reduction in water consumption, chemical fertilizers, and pesticides application were observed in MU sites and subordinate units. Moreover, in each site, some new technologies were transferred to subordinate farmers.Shahpasand37 found that the farmers of Shoushtar County in Khuzestan province who attended the educational and extension courses, extension exhibitions, multi-day educational courses, scientific seminars, and model farms significantly differed in the extent of their chemical use from those who didn\u2019t.Noorivandi38.In other countries researchers have found that for every $1000 increase in extension costs, the farm yield increases by $2173 over a two-year period 39 in his research on A meta-analysis of the impact of agricultural extension services It has been concluded that the empirical results show that, on average, agricultural extension services have statistically significant and positive impacts on the potential outcomes identified in the primary studies. However, the magnitude of the impact is considered medium-sized. Other results show that the effect size estimates of agricultural extension services\u2019 impact significantly vary with the data type , research design  and econometric methods employed in the primary studies.Ogundari40 in their research on Impact of Agricultural Extension Services on Fertilizer Use and Farmers\u2019 Welfare: Evidence from Bangladesh found that farmers who access extension regularly use significantly less urea than farmers who only access it once. Farmers who used extension more often were also statistically significantly more productive and profitable. Access to private agricultural extension appears to lead to statistically significantly higher incomes but does not reduce the rate of urea use. Our results suggest that a more nuanced understanding can be gained from modeling the therapeutic effects of the source and frequency of extension than in the presence or absence of the binary variable formulation. Most common extension in the literature.Rahman and Connor41addressed the role of public agricultural extension services on fertilizer use for rice production in China and found that about 22% of paddy farmers adopted public extension services. Also, the effects of the adoption of public extension services on fertilizer consumption were heterogeneous across different provinces. These results imply that movements should be toward changing the productivity-based agricultural policy regime, reinforcing the socialization agricultural service system, and changing the content and approaches of agricultural extension services. Gebresilasse1 studied the concurrent but independent expansions of rural roads and extension in Ethiopia, and find that, while ineffective in isolation, access to both a road and extension increases productivity by 6%. The key mechanisms include increased take up of advice, adoption of modern inputs, and advantageous shifts in crop choice and labor allocation.Lin et al.42 evaluated wheat production in Uzbekistan empirically. The results showed that there were still significant gaps in wheat production. Irrespective of farmers\u2019 characteristics, the frequency of extension visits and the cooperative extension approaches were specifically important in accounting for the differences in the technical efficiencies of wheat growers, so they can bridge the gaps in productivity.In another study on the effect of agricultural extension type and form on technical efficiency under transition, Djuraeva et al.According to the literature review in this research, we came to the conclusion that there is a gap in the knowledge of agricultural extension and that gap is the lack of systematic studies on the economic effect of extension services. Although few and scattered studies have been done in this field. But none of them have evaluated the agricultural extension as a purposeful economic activity. Rather, they have only been satisfied with a survey of the studied people. The contribution of this research is the identification of variables related to the economic assessment of extension services in different levels of agricultural production, which can be used as a pattern to assess extension services in other countries. Therefore, this research aims to provide an economic effect assessment of extension services of AEMSs for the irrigated wheat production in Iran for providing solutions for the improvement of these sites' performance. Also, at the end of this section the conceptual framework of the study is presented in Fig.\u00a0This research is an applied study in terms of goal, a field study in terms of the extent of supervision and control over the variables, and a descriptive-correlational study in terms of data collection methodology and generalizability, which was conducted by the survey method as one of the descriptive research methodologies.To determine the sample size, the standard deviation of the dependent variable (wheat yield) was first calculated. Using Cochran\u2019s formula Eq.\u00a0 the requTemperate and humid climate (southern coasts of the Caspian Sea),Cold climate (western mountainous areas),Hot and arid climate ,Hot and humid climate (southern coast).The measurement accuracy (d) was equal to 0.003 of the range of the highest and lowest wheat yield (0.03 * 10.83\u20133.70)\u2009=\u20090.21) across the country (3.70\u201310.83 t/ha). We divided into four zones whose information is provided in Table 0.83\u20133.70The sample was taken for the research by the multi-step technique for which first some provinces were purposefully selected from each climate, because agricultural operations are affected by climatic conditions. The remaining samples were taken by systematic randomization with proportional allocation.The research instrument was a questionnaire composed of a section for farmers\u2019 demographical and professional characteristics in terms of age, education, and revenue. AEMS characteristics were identified in terms of area, number of farmers, experts of Agricultural institute of education and extension in Iran involved. The features of the extension services provided to the main farmers about wheat cultivation in terms of the type and extension service and its extent were also identified. An economic assessment of the agricultural extension services conducted in the AEMS was also made. According to the multiple stages of agricultural production, the research questionnaire included several sections. The first part is the personal-professional information of the farmer, the second part is the information about the site, including the activity history of the site, the number of researchers/experts/extension agents on the site, the area of the site, the number Farmers of the site, the third part was the status of the extension services provided to the wheat farmers regarding the cultivation of irrigated wheat. The fourth part was related to the quantitative and qualitative comparison of extension services provided on the site. And the fifth part was related to the economic assessment of the extension services provided on the site in the period before receiving the extension services and after receiving the extension services.In the next step, the estimation of an extended quadratic regression model was used to estimate the effectiveness of the agricultural extension services on increasing production. It should be noted that agricultural extension services include a wide range of services, that the most important of which include educational course, Extension agent\u2019s visit to the farmer\u2019s farm, Practical training at the site, Organized field visit to the model farms, Distribution of extension journals and brochures, sending extension SMS, Attending the programs of finding transfer and farm day, Educational documentaries, Extension apps and Radio and TV programs.43. The Durbin-Watson (DW) test showed that the extended quadratic function did not encounter the problem of collinearity between the independent variables, autocorrelation between the noise components, and heteroscedasticity in the variance of the error term. So, this research also selected the suitable function based on the criteria of selecting the best function out of different functions.There are various econometric criteria for selecting a suitable form among flexible functions. Consistency of the signs and values of the function parameters and elasticities with economic theories is an important criterion for identifying the best pattern according to ThompsonAll interviewees were informed about data protection issues by the enumerators and gave their consent orally at the beginning of each interview. Informed consent was obtained from all individual participants included in the study. All materials and methods are performed in accordance with the instructions and regulations and this research has been approved by an ethics committee at Tarbiat Modares University, Tehran, Iran. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. It should also be mentioned that in this research, no plant sample or materials was taken during the study.11 software suite. Indeed, to calculate the final productivity of the extension course, we needed to calculate the marginal product (MP) for the extension courses. If the extended quadratic function is in the form of Eq.\u00a0(x represents production inputs (including extension courses) and MP represents the marginal product of the ith input (including extension courses). As such, the production elasticity of the input is calculated by Eq.\u00a0. Finally, the economic value of the extension courses can be estimated if the marginal product of the input (extension courses) is multiplied by the crop price as represented by Eq.\u00a0 and Py represents the selling price of the crop.The model was estimated by the SHAZAMm of Eq.\u00a0, Marginam of Eq.\u00a02\\documend by Eq.\u00a0 as follod by Eq.\u00a0.5\\documeIn order to evaluate the indicators, the draft framework and questionnaire were piloted, reviewed and confirmed by an academic panel consisting of agricultural extension expertise, before deployment of the questionnaire in the field. In addition, we used the Split-half coefficient and composite reliability coefficient to evaluate the reliability of the research questions. Split-half reliability is determined by dividing the total set of items  relating to a construct of interest into halves  and comparing the results obtained from the two subsets of items thus created. Finally, the reliability coefficient of this questionnaire was 0.82. The face validity of the questionnaire was checked by a panel of extension experts including 5 peoples of progressive AEMS farmers, 3 peoples of AEMS experts, 10 peoples of extension agents that were responsible for the zone, and 5 peoples of experts of the Agricultural Knowledge and Technology Extension Office of the Agricultural Education and Extension Institute in Iran. Pearson\u2019s correlation coefficient was used to provide a measure of importance of the relationship between the variable of crop performance and the variable of extension investment. A Paired-Samples t-test to check the difference in the production variables in the AEMS before and after receiving the extension services.27 software. In fact, the Paired-Samples T Test procedure compares the means of two variables for a single group. The procedure computes the differences between values of the two variables for each case and tests whether the average differs from 0. The procedure also automates the t-test effect size computation. Also, to calculate of the comparison of means of variables between the four climates was performed using One-way ANOVA in SPSS27. On the other hand, the estimation of the effect of extension services on improving the production yield of irrigated wheat was performed using calculate of production elasticity of the variables in SHAZAM11. Production elasticity is a criterion to measure the amount of production reaction in exchange for a change in the consumption of production inputs. The larger the elasticity of production, it means that the reaction of production to the increase in the use of production inputs is more intense.Data analysis of the comparison of means for the variables related to irrigated wheat production in the studied AEMSs was performed using Paired-Samples t-test between before and after receiving the extension services in SPSSInformed consent was obtained from all individual participants included in the study.The participants\u2019 mean age was 50\u00a0years. Nearly all (96%) of the studied farmers gained their livelihood from crop farming, and the rest by horticulture, ranching, or other sources. Most respondents (72%) stated that they had no other job and that farming was their main source of income. The average number of irrigated wheat land parcels cultivated by each farmer was two, and the mean area of the farmers\u2019 irrigated wheat farms was 15.9\u00a0ha. The AEMSs in this study had on average, been active for 2\u00a0years, and had one main farmer, 20 secondary farmers, one researcher, one extension agent, and three experts. The sites had, on average, 18 sub-units.The status of the extension activity for irrigated wheat farming in these sites Table  revealedThe classification of AEMSs based on the agricultural land class in Table Distribution of the main farmers based on the climatic classification in the studied irrigated wheat AEMSs revealed that 47% of them belonged to cold climate, 34% to hot and dry climate, 16% to hot and humid climate and 4% to moderate and humid climate.The results about the type of extension services for the community of main farmers Table  revealedBased on the results for the status of extension services for irrigated wheat AEMSs in terms of the cultivation stage Table , most exTable Table Table 27 before receiving the extension services and after receiving them revealed significant differences between the four climates, except for the hot and dry climate and the hot and humid climate.The comparison of means by the Paired-Samples t-test between before and after receiving the extension services in Table On the other hand, the comparison of the mean values of the economic variables by the Paired-Samples t-test between before and after receiving the extension services in Table 44. So, although the principles AEMSs in the four climates were the same, but a significant difference was observed in terms of wheat crop performance and productivity. In other research in Iran, the results showed that the highest response coefficient of irrigated wheat to rain is in hot semi-arid climates. In addition, the highest reaction coefficient of irrigated wheat to temperature is found in moderate semi-arid climates16. After checking different functions, the quadratic model was selected as the suitable function form for estimating the production function. The results showed that the variable of extension influenced irrigated wheat yield significantly with a coefficient of 0.32 at the P\u2009<\u20090.01 level and a t-statistic of 6.1 (T\u2009>\u20092)  Table . The squ11 software suite  were related to the growing stage. Extension services do not completely cover the full crop supply chain. Most extension services are related to the methods of tillage and crop growth and, to a lesser extent, to the methods of crop harvest which has helped them increase their production. To help growers to take advantage of the increased production and gain more profit farmers need to be familiar with crop marketing. We suggest that comprehensive extension and educational programs and policies should be developed and implemented that focus on the whole crop supply chain. Data on the rate of seed consumption in the irrigated wheat AEMSs revealed an almost 36% decrease in this variable. So, it can be concluded that the extension agents that were responsible for the zone were able to prevent seed overuse by recommending seeders instead of seed spreaders. It was found about water consumption in the AEMSs that water consumption had been reduced by 6% after presenting the extension services. This saving was related to the adoption of approaches proposed by the extension agents. So, it can generally be claimed that AEMSs had been effective in increasing irrigated wheat production in Iran, so the new agricultural achievements can be introduced and presented through these sites to the stakeholders. Results showed that presenting each extension service to the community of AEMS main farmers enhanced irrigated wheat yield by 0.66\u00a0t/ha these results were supported by Lin et al.A limitation of this research is that there were no previous surveys of extension services we cannot make comparisons with previous years or conduct the statistical analysis of time series. We suggest that further research need to be done to further expand on the findings of this study including the different extension outcomes across different climate zones. Further research could be undertaken to explore the role that extension has in helping farmers adapt to a changing climate. At the end this research, the topic that suggest for future research is Designing a pattern for AEMSs to adapt to different climatic conditions in Iran."}
+{"text": "Zinc biofortified wheat may be a sustainable strategy to increase zinc intake in areas where fortification and dietary diversification are not feasible or are limited by household purchasing power. This convergent mixed methods study aimed to explore the farmers\u2019 and millers\u2019 experiences and attitudes towards the production and processing of zinc biofortified wheat in Pakistan.n\u2009=\u2009418) who were provided with Zincol-2016 biofortified wheat seed for the 2019\u20132020 growing season, as part of a wheat grain micronutrient mapping study across Punjab Province. The survey explored the farmers\u2019 experiences of growing Zincol-2016 and whether they opted to grow it again in the subsequent season. Semi-structured focus group discussions were undertaken in a separate group of farmers in Khyber Pakhtunkhwa (KP) province (n\u2009=\u200912) who grew Zincol-2016 for the BiZiFED2 RCT. Millers were also interviewed in KP, both those who had processed Zincol-2016 for the trial (n\u2009=\u200912) and those who had no experience of processing biofortified wheat (n\u2009=\u200912). Survey data were analyzed using descriptive statistics and transcripts of focus groups were analyzed using thematic analysis.A telephone survey was conducted with farmers  re-cultivated Zincol-2016 in the following season. The drivers for Zincol-2016 re-cultivation were seed availability (100%), grain yield and growth resistance (98%), quality of the flour from the previous harvest (97%) and nutritional benefit (94.5%). Discussions with farmers suggested that the main motivators for potential scale-up of biofortified wheat were the perceived quality of the grain, wheat, and flour. Millers saw it as an opportunity to expand their business. Farmers and millers valued the health benefits of the wheat. Challenges for scale-up include the need of additional support to produce it, unfamiliarity with the biofortification process, production costs, and external threats to the supply chain.Farmers and millers showed a strong implicit preference for Zincol-2016 over alternative varieties. Crop performance and product yield were the most cited motivators for growing Zincol-2016. Farmers and millers are willing to produce and process biofortified wheat if financial and educational support is provided. Mild-to-moderate zinc deficiency may lead to growth faltering in children, impaired immune function, and altered integrity and function of the gastrointestinal tract . Zinc deLeading public health strategies to address zinc deficiency include supplementation, fortification and increasing dietary diversity. However, the sustainability of these strategies can be challenging in remote impoverished rural areas where access to affordable diverse diets, supplementation interventions and centrally fortified food products are limited . BiofortThere is increasing evidence that consumption of biofortified foods improve micronutrient status , 8. BiofThe success of biofortification strategies to improve micronutrient status on a population scale depends not only on the evidence of a positive impact on relevant health outcomes, but also on high rates of adoption and consumption by the producers and intended beneficiaries , 12. SysIn 2016 a new variety of zinc biofortified wheat (Zincol-2016) was released by HarvestPlus in partnership with the Research & Development Institutions of Pakistan. As wheat is the main staple food and cultivated on the largest acreages in Pakistan , this biThis study is part of the BiZiFED2 project (Biofortification with Zinc and Iron for Eliminating Deficiency) . One of The study used a mixed methods convergent parallel design in which two independent strands of complementary quantitative and qualitative data were collected and analyzed independently and merged in the integration phase . This apIn 2019, 686 farmers across the Punjab region were recruited to participate in the BiZiFED trial. They were provided (free of charge) with 25\u2009kg Zincol-2016 wheat seed sufficient for 0.5 acre (~0.2 hectare) using standard broadcast sowing practices. Farmers who had granted their permission to be followed up during the initial recruitment period were contacted to take part in a survey. Up to three attempts were made to reach each farmer by telephone. Upon contact, the nature of the study was explained to each potential participant and consent was reconfirmed. Farmers were provided with the contact details of the researchers and encouraged to ask any questions and seek clarification from the research time if required. The survey was conducted by an experienced agronomy extension worker (SA) who was fluent in the local language and trained in the skills required to collect data for this project by telephone, using KoboCollect software . The surThe survey was designed to capture the extent of biofortified wheat cultivation, the farmers\u2019 experiences of growing Zincol-2016 in the growing season 2019\u20132020, and whether they had continued to grow Zincol-2016 in the 2020\u20132021 growing season. The full suite of questions is provided in Four FGDs were planned of which two were with farmers and two with millers. The farmers were recruited from a total pool of 59 tenant farmers cultivating land belonging to two landlords in the Peshawar area. Farmers were small scale farmers, relying mostly on manual techniques for sowing and harvesting and who were given resources (Zincol-2016 seed and zinc fertilizer) to grow Zincol-2016 which was then purchased for use in the BiZiFED2 effectiveness trial. The millers were recruited from two mills that were initially chosen to process the wheat for the trial flour. These mills were selected according to the affordability, accessibility, and condition of the machinery. Of these two mills only one (Mill 1) was chosen to grind the Zincol-2016 wheat grain for the trial. The second mill (Mill 2) acted as a potential substitute in case of any mechanical fault or power shortages in the area. Only non-biofortified wheat was processed in Mill 2, which was used at the start of the trial as a source of control flour. For the FGDs, potential participants were eligible if: they were over the age of 18, were employed at the selected farms or mills, and could willingly give informed consent. The selection of the participants for the FGDs was conducted by the BiZiFED2 RCT trial management team who identified individuals who were willing to speak openly.A total of four FGDs with a duration of between 30 and 60\u2009min were conducted between November and December 2020. The location of the FGDs was selected based on ease of access for the participants and where COVID-19 safety measures could be ensured. Topic guides were used to lead the FGDs See  and wereSimple descriptive statistics, such as frequencies and percentages, were used to analyze the survey data, using Microsoft Excel for Microsoft 365 MSO .\u00ae 12  for analysis. An inductive thematic analysis was conducted to generate themes following the approach by Braun and Clarke  reported that they chose to grow Zincol-2016 again in the following season (2020\u201321). Nearly two thirds  of all farmers were aware of Akbar-19, a recently released zinc biofortified wheat variety.Of all farmers surveyed, 278 (67%) kept some or all their harvested Zincol-2016 grain separated from their usual variety post-harvest, while the remaining 33% mixed it with any other variety. Of the farmers who kept some separate, some 227 (82%) reported storing this seed for growing in the 2020\u20132021 season. Almost half of the farmers .When asked what factors would influence their decision to apply basal or foliar zinc fertilizer, which would typically incur additional expense for the farmer, the most frequent responses were cost of zinc fertilizer (n\u2009=\u2009138), whether there was sufficient market demand/buyers interest in the biofortified product (n\u2009=\u2009130), and lack of knowledge on how to apply foliar zinc fertilizer (n\u2009=\u2009102), while discoloration of leaves (n\u2009=\u200925), preference of organic matter (n\u2009=\u200926) and access to credit (n\u2009=\u200932) were less commonly identified as influencing factors. Most farmers  indicated they would be more likely to use foliar zinc on wheat intended for their own consumption. Most participants (84% n\u2009=\u2009341/407) indicated that they would appreciate some training on this approach.The majority of farmers did not use foliar zinc fertilization on their crops to increase grain zinc , growth and disease resistance (n\u2009=\u2009117/190), grain yield (120/188) and the quality of flour from the previous harvest (107/180) were very important motivators to growing Zincol-2016 again. Nutritional benefit was identified as \u201cimportant\u201d (n\u2009=\u200986/166) or \u201cvery important\u201d (n\u2009=\u200971/166) by most participants. The cost of seed was rated as \u201cnot important\u201d by 28% of those who responded to this option (n\u2009=\u200950/177) The 197 participants who reported growing Zincol-2016 again in the following season were asked to rate how important certain factors were in their decision to grow this variety again. The farmers indicated that the availability of Zincol-2016 seed (n\u2009=\u2009221), gifted or shared with neighbors (n\u2009=\u2009144), or was stored for multiplication sowing in the following season (n\u2009=\u2009227). Of those who consumed the Zincol-2016 grain within their own household, 29% had consumed it for less than 3\u2009months (n\u2009=\u200964), 57% consumed it for 3\u20136\u2009months (n\u2009=\u2009127), and 14% consumed it for more than 6\u2009months (n\u2009=\u200930). Of those who had consumed bread made with Zincol-2016 grain, most felt it had a better taste , better texture  and lighter color  than bread made with their usual variety (n\u2009=\u20096) or as payment to laborers (n\u2009=\u200931) and 19% (n\u2009=\u200952) reported that they had sold the Zincol-2016 grain.Farmers who had indicated that they had kept some Zincol-2016 grain separate from their other variety post-harvest were asked how they used the retained grain. Of the 278 farmers who responded, the majority (79%) reported having more than one use for the grain. As shown in  variety . Only a Farmers were asked if the COVID-19 pandemic impacted on the amount of grain they sold in 2020 to which 49% responded that they had sold the same amount as usual, 23% had sold less than usual, and 28% had sold more than usual.A total of 12 farmers and 12 millers agreed to take part on the FGDs. Information derived from the thematic analysis enabled the identification of two main themes: (1) Enablers for scaling up of biofortified wheat and (2) Challenges and considerations for scaling up.Among the enablers for scaling up we identified four subthemes: (1.1) Perceived health benefits; (1.2) Improved grain quality and production; (1.3) Willingness to produce and process biofortified wheat if provided with support; and (1.4) An opportunity for millers to expand their business.The farmers and the millers who had produced and processed the biofortified wheat, recognized that adding minerals to the wheat or flour through biofortification or fortification was beneficial to alleviate mineral deficiencies among the population. The health benefits were seen as an incentive to produce and/or process biofortified wheat.\u201cSir, the reason we consented (to grow biofortified wheat) was that there was deficiency in our country, children weakness, their mothers\u2019 weakness, vitamin deficiency, when we read your description we found good things in it, and that wheat was beneficial for us. We consented for the purpose of having a healthy society\u201d \u2013 A farmer.The farmers acknowledged that the process of growing biofortified wheat differed from their usual ways of farming  and required additional care. However, farmers did not verbalize any concerns about the process of growing biofortified wheat or about the wheat grain provided to them during the trial. On the contrary, farmers expressed satisfaction with the quality of seeds and believed that the zinc sprays enabled the grain to grow stronger and be of better quality. Farmers also believed that the sprays were beneficial to the soil and felt that the chemicals they received increased their yield.\u201cIt came up very good and its production was beautiful, we were very much happy with it. Every grain of wheat was very big and beautiful, every grain of it was like a red berry, it was the mercy of Allah and it happened due to the grace and mercy of Allah\u201d \u2013 A farmer.\u201cIt was very good because when we grew it the production was more than it used to be and additionally zinc and everything were given to it, so it brought a very good result\u201d \u2013 A farmer.The farmers expressed that they did not have any issues while growing Zincol-2016 for the BiZiFED trial, other than those that they would normally face while growing a non-biofortified variety. These common challenges included disease and weather issues.\u201cIn the name of Allah, the most merciful, the most compassionate Mr. Rashid, when we would do farming previously, it would be on our (inaudible) but when you came, the method you did the wheat farming it was in a very good way, and we were to get its products but unfortunately some storms and rain occurred which caused the wheat\u2019s grain to remain incomplete\u201d \u2013 A farmer.The farmers and millers who produced and processed the Zincol-2016 wheat grain expressed that, given the benefits of biofortified wheat for the population, their production and business, they would be willing to produce and process it if given support  from the government or other organizations.\u201cAbsolutely. We will be growing yeah (multiple voices). This is beneficial for us, this thing is good for our coming future, we like it very much, yes. We want the government to help us like that then God willing we will keep growing this. We request the government to help us the way you came last year and helped us in terms of chemicals, soils, wheat seeds\u201d \u2013 A farmer.\u201cIf the government provides us with biofortified wheat, then we are ready by all means to grind it. If we get this sort of wheat we will grind and sell in the bazaar\u201d \u2013 A miller, Mill 1.Some participants indicated the need for government initiatives to promote the scale up of biofortified wheat. A miller expressed his belief that improved community benefit would arise from a government supported program of biofortification.\u201cIf the government supports this program as this work is done for the welfare and good of our people. Therefore, we should participate in it, and we should become part of it. If this done by the government then it would be carried out with enforcing people and it would develop easily. Instead of doing it privately if it is carried out through the government then it would bring up better results\u201d \u2013 A miller, Mill 1.The farmers expressed that receiving support during the BiZiFED2 trial in the form of soil, seeds, fertilizers and knowledge enabled farmers to increase their production and allowed unused lands to be re-used. Farmers expressed their interest for continuing to receive the support provided during the trial and expressed their concerns of a decline in production when the trial came to an end.\u201cThe chemicals and the seeds increased our products and as Mr. (Name) told you that before when we would plough a field then we would plant a mound (37.324\u2009kg) or two in it, but this time, Praise be to Allah, they showed us a proper limit and we used Urea as well in its proper limit, God willing. We were expecting after that as well, but it did not happen. God willing, we will fully cooperate with you if we receive all these things next time again\u201d \u2013 A farmer.Discussions with millers also revealed that milling biofortified wheat was beneficial, not only for the potential health benefits that this could confer to the population, but as an opportunity to expand their business and possibly gain interest from the consumers, as had occurred when millers fortified wheat with support of the World Food Program (WFP).\u201cIf we get it (biofortified wheat) then we would accept it with happiness, because we try to expand our business so it would help us to expand our business. And we would be able to provide people with such standard flour which would be good for their health, and it would meet their nutritional requirements as well\u201d \u2013 A miller, Mill 1.Participants from both mills believed that improving the nutritional value of the wheat through the WFP fortification initiative had boosted consumer demand, and one participant suggested that an identifier label or stamp may help consumers differentiate fortified products from standard varieties.\u201cOur production has increased with it (fortification) because the item that is put into it, which is iron, that is in fact for some requirement such as some illness and so. Therefore, people consume it with good interest\u201d \u2013 A miller, Mill 2.\u201cWhichever market this flour goes, we have told all of our customers regularly that you should first look at its own fortified monogram and then buy the flour so that you make sure this thing is available in it\u201d \u2013 A miller, Mill 1.Four sub-themes were identified as possible challenges and considerations for scaling up. These were: (1) Unfamiliarity with the biofortification process and crop; (2) Production costs; (3) Need of support from the landlord; (4) Millers\u2019 beliefs about local wheat; and (5) External threats to supply chain of wheat and resources .Prior to the BiZiFED2 trial, it appeared that neither farmers nor millers had previously produced or processed biofortified wheat, nor did they know what biofortification was. The farmers that produced biofortified wheat for the first time during the BiZiFED2 trial suggested to the research team that they use a simple local name to refer to biofortified wheat as they would find easier to understand.\u201cMy suggestion is that you give us some advice, for this wheat which you call it biofortified, choose a name which a farmer finds that name sweet and dignified, yeah. Just call it \u2018Sona\u2019  wheat as a lot of hard work is done on it. As \u2018Sona\u2019 has a high quality in the soil so is this wheat\u201d \u2013 A farmer.Some millers described not being aware of what biofortified wheat was despite previously having ground it for the BiZiFED2 trial, but they were open to receiving more information about it. A miller believed that the farmers should be given training on the process since they would be the ones that grew the wheat.\u201cThose who need to be told first are the farmers as they are the one who grow the wheat and that wheat comes to us after that. We have not been briefed about this, so when they come then we will receive briefing about it. When they teach us, then good results will be achieved\u201d \u2013 A miller, Mill 1.The millers that had not ground Zincol-2016 before, expressed their beliefs that the quality of the local wheat was poor, hence they would not be interested in milling biofortified wheat if it was grown locally unless they had evidence that the quality of the flour produced by the biofortified grain was good.\u201cWe will not be paying attention to this wheat firstly because its bread is weak, and secondly, we will see next year, if the wheat is good then we will be buying it. Although we do not buy the local wheat because even if we use it then it gets returned to us from every side, the wheat that particularly the wheat that is native to Peshawar\u201d \u2013 A miller, Mill 2.For the farmers, the costs of the chemicals required to support biofortified wheat crops was the main barrier to its adoption. Farmers expressed not being able to afford the chemicals as they needed to prioritize other expenditures due to severe resource limitations.\u201cYeah, white fertilizer black fertilizer, chemicals, if these things are expensive then we will not be able to afford it, because we would be wondering whether to spend what we earn through labor work on the field or on ourselves. So, if we cannot do the crops, it would be due to the poverty and helplessness\u201d \u2013 A farmer.\u201cYes, it does require expenditure and we cannot afford it; it needs fertilizers, garbage , and we cannot afford all of these. We can only put soil and so into it\u201d \u2013 A farmer.The price of the biofortified wheat was not directly mentioned by millers as a barrier to processing it, but they did state that when wheat prices increased, for example as they did during the COVID-19 pandemic due to supply chain issues, the price of their product also increased, and consumers may choose not to buy their flour. This suggests that millers may not be willing to mill biofortified wheat if the cost to consumers exceeded the cost of government-produced wheat.\u201cNo, no, there is shortage of wheat. It is not available at all. When it is available then its price is 5,000 Rupees, yeah, when we flour it then people do not buy it because there is a big difference between the 5,000 and the price of the government wheat, the difference is almost 1,200 Rupees at this time\u201d \u2013 A farmer.Farmers and millers both described a requirement for third party support if they were to produce or process biofortified wheat. Decisions concerning the type of crops cultivated by the farmers was not only dependent on the farmers themselves but also on their landlords, which would ultimately impact on whether they would be able to grow biofortified wheat crops.\u201cWe have (Name of landlord) land, we cultivate it. Whatever he orders us we cultivate the land in accordance to that\u201d \u2013 A farmer.Typically, farmers gave a proportion of their crop to their landlord and the rest was retained by the famers for self-consumption or for selling, either to millers or in the markets. Some of the money earned from the farmers\u2019 sales was used to buy farming resources, but they also expressed a need for more support from their landlords for such expenses.\u201cThere are a lot of expenses, the farmers spend money on tractor. The landlord just halves it [the harvest] and they do not care about anything else. There are expenses, at least half of those expenses should be accepted by the landlords\u201d \u2013 A farmer.Millers also believed that if there was any special equipment or training to process biofortified wheat they would need support from a third party.\u201cWhatever machinery is necessary for this, the government should provide us with, and we will go along with it\u201d \u2013 A miller, Mill 1.Market and road closures and transportation disruption were a threat to the wheat and resource supply chain  and economy of the farmers and millers during the COVID-19 pandemic. Both groups described how the disruption in transportation would cause delays or an inability to deliver their products to the market or to their customers, making it difficult to sell their products.\u201cThe disease that Corona [COVID-19] has brought has had a big impact. As my friend said when we take a crop to the bazaar, it is a challenge. Firstly, vehicles are not available and so going to the bazaar is difficult but when we manage to take it to the bazaar then there is the issue of selling; when we sell it, it goes for cheaper but when we buy something it cost expensively for us\u201d \u2013 A farmer.\u201cDue to the Corona [COVID-19] the transportation has been affected very much. A work that would have been done for ten rupees before, has turned to be done for 100 rupees. Therefore, for a poor man wheat in the open market became very expensive. The wheat that the government would subsidize, and we would grind, that flour was available to the public with subsidy. But the private wheat\u2019s transport cost doubled for the flour mills because of the closing of vehicles stations, and lack of transportation. So, this was a loss for the mills\u201d \u2013 A Miller, Mill 1.Farmers and millers also noted the impact of reduced opening times at the markets where they usually sold their products. To ensure that products were sold, farmers would directly sell their products to the mills or sell them at a lower price which represented losses for them. Both farmers and millers expressed that the market closures would cause shortages and therefore an increase in the price of the resources  required for producing their products.\u201cIt would certainly have been affected because it is about the bazaar, when the bazaar is closed then it definitely gets affected. When a product does not reach the bazaar on time then the product that was supposed to be sold for ten, now it gets sold for eight. This issue is there yeah\u201d \u2013 A Farmer.However, these adverse circumstances may have been short-lived as participants in subsequent focus groups (conducted in February 2021) did not describe market closures and attributed this to support from the government. The millers acknowledged the work of the government in reducing the shortage of wheat during the pandemic and reducing its price.\u201cThe price was high at that time because the wheat was short and wasn\u2019t available. But at the present time the wheat is gradually increasing from every side and the government provides a lot of facilities, so a month ago our wheat was 1,250 Rupees and now it is 1,100 Rupees, and due to the government wheat, the flour is getting cheaper\u201d \u2013 A miller, Mill 2.The farmers expressed that the COVID-19 pandemic led to an unstable labor force as they were not able to leave their homes to go to work. This would affect their farming activities as those who had other jobs in addition to farming would not be able to afford the resources required to grow the wheat. The farmers explained that this instability in the labor force would cause economic difficulties among their consumers and hence their products would not get sold.\u201cIt affects the work of a laborer as he cannot find work, so it impacts it in a big way. The farmers\u2019 products do not get sold\u201d \u2013 A farmer.Millers, however, believed that people would continue to consume the same amount of flour during the pandemic therefore they would still be able to sell their products.\u201cFlour is something which is for daily consumption, therefore there has been no impact on it\u201d \u2013 A miller, Mill 2.Zinc biofortified wheat may be a sustainable strategy to increase the dietary zinc intake among populations with low access to nutritious diets . Given tThe survey and focus group data provided evidence that the farmers were satisfied with the Zincol-2016 wheat variety. Almost half of farmers (47%) who received Zincol-2016 seed to grow in the 2019\u20132020 season, stored a portion of the grain harvest and expanded its production in the following season (2020\u20132021), which suggests a preference of Zincol-2016 over their existing varieties. In addition, in the focus groups the farmers expressed their willingness to grow the biofortified crop given the perceived health benefits of the grain. Our findings are similar to previous studies in Uganda and Nigeria that investigated stakeholders\u2019 perceptions towards the adoption of biofortified crops, where farmers expressed a positive response to agronomic biofortification , 17, parIn our study we were able to identify some potential motivators that could increase the likelihood of biofortified wheat adoption among producers and processors. These included perceptions related to the superiority of the grain yield and flour quality, perceived health benefits of the biofortified flour, acceptability of the flour among consumers, and potential for increased marketing opportunities. It was also noted that adoption is likely to be improved if support, in the form of training and resources, is provided alongside the biofortified seed.Crop performance was the most cited motivators for growing Zincol-2016 among farmers, who observed that the quality of the Zincol-2016 seeds, together with the fertilizers, increased their yield. Two thirds of farmers who cultivated the Zincol-2016 grain in the following season (2020\u20132021) cited good growth, disease resistance and yield as reasons for why they chose to do so. Farmers who took part in the focus groups had no concerns about the health of their Zincol-2016 crops above and beyond the common challenges they regularly faced . These findings provide promising indicators for the success of scaling up of biofortified wheat, since maximizing yield and reducing production threats due to disease positively impact the farm income potential . Crop reProduction costs are a key factor that influence producers\u2019 intention to produce biofortified crops, particularly in relation to the cost of foliar zinc fertilizer application, which may be in addition to soil enrichment with zinc and organic fertilizers. Foliar zinc fertilizers increase the zinc content and quality of the wheat crops and have been used with Zincol-2016 to enhance the grain\u2019s performance , 27. HowA particularly striking finding of the farmer\u2019s survey is that, with a starting provision of sufficient grain for half an acre of cultivation, seed saving, and multiplication saw this extend to nearly one third (31%) of the cultivated wheat area just 1\u2009year later, for the 47% of initial farmers who continued to grow Zincol-2016. It also showed a strong preference of home consumption and gifting of seed to neighbors, rather than more commercial transfers to landowners, workers, or sale. Data from both focus groups and survey shows that seeds were retained to sow for the next season, providing evidence that an initial investment for the provision of seed could be a sustainable strategy to support the reduction of micronutrient deficiencies if the crop maintains its high zinc trait over successive growing season.Compared to control flour, the additional zinc content of the Zincol-2016 flour produced in this study was 3.69\u2009mg Zinc/kg . These fThe results of our study suggest that decisions to grow biofortified wheat does not only rely on the farmers, but also on the landowners who may have ultimate control of what the farmers grow. Moreover, it is possible that farmers may not want to invest in zinc fertilizers or improvements in the soil if their tenure is uncertain. A study in Pakistan  suggesteThe present study was conducted during a critical period of the COVID-19 pandemic, when intermittent lockdowns were in place across Pakistan. In the FGDs farmers described their experiences during lockdown, including difficulties with access to markets affecting the price of fertilizers and pesticides and their ability to sell their products, and increased prices of basic food items. This concurs with similar reports from Pakistan  and IndiEvidence suggests that a key driver to the adoption of Zincol-2016 and other biofortified wheat varieties in rural areas in Pakistan is the provision of adequate support for both producers and processors of the grain. Our survey and focus group discussions suggested that most farmers were willing to grow biofortified wheat if given affordable access to foliar zinc fertilizers and training on their application. A previous study in Pakistan exploring the factors influencing the adoption of improved wheat varieties in rural areas revealed that access to extension services and micro credit schemes was found to be a key factor . In thisFinally, our study revealed that the millers considered consumer acceptability and the quality of the food staple when deciding whether to adopt new technologies. The millers who did not process Zincol-2016 grain in our study expressed that they would not adopt it until they had evidence that the flour and bread it produced were of good quality. Our survey data indicated that the majority of farmers who had consumed the Zincol-2016 flour believed that the bread it produced had a better taste and texture compared to their usual varieties. Similarly, our earlier study of consumers\u2019 perceptions of Zincol-2016  revealedTo our knowledge, this is the first study that has been conducted exploring producers\u2019 experiences of growing biofortified wheat in Pakistan. The quantitative survey 418 of farmers, together with the qualitative exploration of experiences and attitudes allowed the triangulation of data and added richness and depth to the findings. This study also has some limitations. The farmers in the focus group discussions received support from the BiZiFED2 project, which included the provision of the seed, fertilizers, and the purchase of their crop at a favorable rate. The farmers in the survey received free Zincol-2016 seed the previous growing season and soil fertility data at the time of their selection, which could have introduced bias to their responses: they do not represent an unbiased sample of Punjab Province wheat growers in relation to biofortified wheat cultivation. Moreover, our survey did not collect the reasons for why some farmers did not grow Zincol-2016 in the following season. Additionally, the views of farmers not involved in the BiZiFED2 trial were not sought, and it is possible that their views may differ from those of farmers involved in the study.In conclusion, our study suggests that biofortified wheat was well received among the producers in KP and Punjab provinces. Although both farmers and millers valued the nutritional qualities of the crop, farmers felt that crop performance and yield were among the most valued characteristics of the grain, while millers saw it as a marketing strategy and would be willing to adopt it due to the high quality of the product. Further awareness and training about the benefits of biofortification is required among both farmers and millers. Farmers require support to acquire and utilize zinc foliar fertilizers to optimize the zinc content of the Zincol-2016 grain. The results of this study strongly suggest that farmers and millers are willing to produce biofortified wheat providing support is given in the form of resources and training. These finding can inform the scaling up of biofortification for the provision of more nutritious foods to populations, particularly in areas outside the reach of centralized fortification interventions.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving humans were approved by Ethical approval granted by the University of Central Lancashire (REFTEMH1014), Khyber Medical College Peshawar (reference BZ/000628) and University of Nottingham (BIO-1819-001A) ethics committees. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.NML, VHM, ELA, EJMJ, HO, MZa, MZi, and SA contributed to the design and development of the study. UM, HO, SA, ELA, MZi, and MZa managed data collection. MC-R and UM completed initial coding and analysis of the qualitative data, which were double checked by VHM and EJMJ. SA and ELA cleaned and analyzed the survey data. M-CR integrated the qualitative and quantitative data and wrote the manuscript. VHM, NML, and EJMJ revised the paper and finalized the manuscript. All authors contributed to the article and approved the submitted version."}
+{"text": "Furthermore, the depolymerization product BHET was used as the raw material and Ti/Si\u2013EG was used as the catalyst for the polycondensation reaction to synthesize PET, and the amount of the catalyst added was 0.015% by mass (in terms of BHET). The performance of the synthesized PET was tested to be the same as that of PET synthesized by the PTA method under the same conditions, thus achieving the resourceful treatment and high-value recycling of waste PET. The introduction of Si in the catalyst reduced the catalytic activity of Ti and avoided the problem of yellowing of polyester products, and the use of the glycol salt as a catalyst in the depolymerization and polycondensation reaction process did not introduce impurity groups.In this study, a Ti\u2013Si\u2013ethylene glycol salt (Ti/Si\u2013EG) was synthesized and used as a catalyst for the depolymerization of PET\u2013ethylene glycol to produce bis(hydroxyethyl)terephthalate (BHET), and the optimum conditions for the depolymerization were determined by response surface analysis:  In this study, a Ti\u2013Si\u2013ethylene glycol salt (Ti/Si\u2013EG) was synthesized and used as a catalyst for the depolymerization of PET\u2013ethylene glycol to form bis(hydroxyethyl)terephthalate (BHET), and catalysts for the resynthesis of PET by BHET. Currently, the global production of PET continues to rise, but the recycling rate is less than 10%.4\u20139 which is highly efficient in terms of the reagents used to depolymerize PET and is an effective way to realize the sustainable use of plastic recycling; physical recycling, which uses high-temperature melt extrusion to recycle PET for a second time, but the quality index of the recycled PET will be reduced, limiting the number of recycling times and compromising the quality of the recycled product; and biological recycling,10\u201313 which is similar to chemical recycling and depolymerizes PET, but it is more costly. At present, the use of ethylene glycol as a solvent has been reported to depolymerize PET, and the yield of the depolymerization product BHET is mostly in the range of 50\u201380% and seldom exceeds 90%.14 In this regard, it is necessary to develop a suitable depolymerization catalyst.Currently, waste PET recycling and reuse methods are divided into three categories: chemical recycling15,16 which have a high catalytic activity and are environment friendly and inexpensive. However, studies have shown that titanium catalysts have certain drawbacks, such as a very high catalytic activity and too many catalytic side reactions in the process, which limits their large-scale application.Re-polymerization of the depolymerization product BHET can undoubtedly achieve the closed-loop utilization of PTE. At present, more than 90% of the catalysts used in the industrial synthesis of PET are antimony-based, but they are potentially toxic to the environment and organisms; therefore, more and more studies are being focused on titanium-based catalysts,17\u201321In order to recycle PET, it is required that the catalyst meets the following conditions: (1) the catalyst is safe and has no direct or potential toxicity toward human beings and organisms. (2) The catalytic activity should not be too high, so as to avoid problems such as deepening of the color of the product and side reactions caused by the catalytic activity during the depolymerization and polycondensation processes. (3) No impurity groups should be introduced into the depolymerization and polycondensation system to avoid the introduction of impurity groups causing the quality of BHET to be lowered, as well as the introduction of the impurities in the process of polycondensation, resulting in the PET product being prematurely capped, the molecular weight of the product difficult to increase and other problems.22\u201327 The ethylene glycol salt of Ti\u2013Si (Ti/Si\u2013EG) was prepared for use as a catalyst in this study. Because the arrangement of the extra-nuclear electron cloud of the element silicon (Si) makes it weakly catalytic when used alone, its use in combination with the element titanium (Ti) can reduce the activity of the titanium catalyst.28 As a glycol salt, the catalyst will not introduce impurity groups during depolymerization and polycondensation, thus affecting the quality of the product. Moreover, after Ti/Si\u2013EG is catalyzed, the deactivated catalyst is uniformly dispersed in PET and can be used as a matting agent.29In this study, Si, which has a huge stock in nature, is non-toxic, non-hazardous, cheap, and easily available, was chosen to be used in conjunction with Ti.2.2.12.1.1Waste PET, tetrabutyl titanate, tetraethyl orthosilicate, xylene, anhydrous ethanol, terephthalic acid (PTA), ethylene glycol (EG), and the raw materials used in the experiments were all analytically pure reagents.2.1.2A balance (FA2204B), China Shanghai Precision Scientific Instruments, viscometer, China, field emission scanning electron microscope  Thermo Fisher Scientific, U.S.A., infrared spectrometer (TENROR-II) Bruker, Germany, thermogravimetric analyzer , Bruker, Germany, X-ray diffraction spectrometer  Bruker, Germany, differential scanning calorimeter  METTLER TOLEDO, Switzerland, colorimeter (ADCI-60-C) China, circulating water vacuum pump (SHZ-D), China, laser particle size distribution meter (BT-800), China, gel permeation chromatography  Agilent, U.S.A.2.22.2.1Tetrabutyl titanate, tetraethyl orthosilicate, and ethylene glycol were mixed in a 1\u2009:\u20091\u2009:\u20094 (mol) ratio and added to the reactor. The mixture was then heated to 115 \u00b0C and refluxed for 3 h. Xylene was added as a solvent. The reaction mechanism was changed from reflux to distillation. Cooling was performed once the desired amount of the product was obtained. The solid product was washed with anhydrous ethanol, dried, and pulverized to produce the Ti\u2013Si\u2013EG catalyst .2.2.2Waste PET was washed, dried, and added to the reactor. Ethylene glycol was added, and the temperature was raised to approximately 100 \u00b0C. The Ti/Si\u2013EG catalyst was added, and the mixture was refluxed at around 200 \u00b0C for 3\u20134 hours. After the reaction was complete, the temperature was reduced, and distilled water was added to the reaction system. When the temperature reached 60 \u00b0C, the mixture was filtered, to separate the incompletely depolymerized polymers, and the filtrate was cooled at 5 \u00b0C for 5 hours. The resulting crystallized BHET product was filtered, vacuum-dried, and obtained as the final product.2.2.3The synthesis steps for PET are shown in x = 1).(1) The synthesis of PET by the PTA method begins with an esterification stage, wherein terephthalic acid and ethylene glycol undergo an esterification reaction to produce ethylene glycol terephthalate (2CH2OH) from each end in the beginning stage, and the two are combined to regenerate EG. Subsequently, the monomers with the groups are removed from both ends and are connected to each other to form polyethylene terephthalate with a low degree of polymerization (x = 1\u20134 or so), which is the pre-condensation stage.(2) Glycol terephthalate removes the hydroxyl group (\u2013OH) and the hydroxyethyl group (\u2013CH(3) The polycondensation stage repeats the chain-enhancing reaction on the basis of (2) at reduced pressure and elevated temperature to generate large molecular weight PET.The synthesis of PET using the BHET method omits the esterification stage and proceeds directly to the pre-condensation and condensation steps of (2) and (3), as described above. The specific method is as follows the raw material terephthalic acid (PTA) and ethylene glycol were added to the reactor, and the total amount of the catalyst added was 0.015% of the raw material amount, and the catalyst was uniformly dispersed in ethylene glycol before use. The esterification reaction was carried out under the conditions of atmospheric pressure esterification, and temperature between 185\u2013195 \u00b0C, and a stirring speed of about 100 rpm. The polycondensation test was carried out between 260\u2013280 \u00b0C, stirring speed of 100 rpm, and pressure of \u22120.1 MPa.2.2.4The molecular weight parameters presented herein were obtained from a solvent mixture of phenol\u2009:\u2009tetrachloroethane of 1\u2009:\u20091 (w%), tested in a constant temperature water tank at 25 \u00b0C using a Ubbelohde viscometer.\u03b7r is the relative viscosity, t1 is the solution flow time through the capillary (s), t0 is the solvent flow time through the capillary (s), \u03b7sp is the incremental viscosity, [\u03b7] is the characteristic viscosity (dL g\u22121), and c is the solution concentration (g dL\u22121). The average molecular weight of PET was deduced from the measured characteristic viscosity.According to 3.3.13.1.1The scanning electron microscope test results of the Ti/Si\u2013EG catalyst are shown in 3.1.22\u2013SiO2 (PDF #43-0055) and Cu6(Si6O18) (PDF #79-0988).3.1.3\u22121 correspond to the stretching vibration of \u2013CH2, the strong absorption peak at 1045.9 cm\u22121 corresponds to the stretching vibration of the Ti\u2013O\u2013C bond, and the absorption peak at 587.9 cm\u22121 corresponds to the stretching vibration of Ti\u2013O. The absorption peak at 1150.1 cm\u22121 corresponds to the vibration of the Si\u2013O bond.3.1.4TG curves of Ti/Si\u2013EG are shown in 3.23.2.12 atmosphere at a rate of 15\u00b0C min\u22121. It can be seen that the melting point of the BHET monomer is approximately 112.7 \u00b0C, and therefore DSC testing of the depolymerization product can be used to initially determine the presence of BHET.3.2.2\u22121 corresponds to the stretching vibration of the BHET terminal hydroxy \u2013O\u2013H, the strong absorption peak at 1709.5 cm\u22121 corresponds to the stretching vibration of the ester C PBM data was replaced with SVG by xgml2pxml:<glyph-data id=\"z.dbd\" format=\"PBM\" resolution=\"300\" x-size=\"8\" y-size=\"10\" xml:space=\"preserve\">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns=\"http://www.w3.org/2000/svg\" version=\"1.0\" width=\"13.200000pt\" height=\"16.000000pt\" viewBox=\"0 0 13.200000 16.000000\" preserveAspectRatio=\"xMidYMid meet\"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform=\"translate scale\" fill=\"currentColor\" stroke=\"none\"><path d=\"M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z\"/></g></svg>O, vibration peak of the aromatic \u2013C\u2013H at 1405.3 cm\u22121, C\u2013O vibration peaks at 1261.4 cm\u22121 and 1125.8 cm\u22121, and the bending vibration in the aromatic ring at 896.1 cm\u22121. Vibration peaks at 2952.2 cm\u22121 and 2868.2 cm\u22121 are due to \u2013C\u2013H of the methylene in the EG chain. The infrared spectrum of the product is consistent with the infrared spectrum of BHET, which confirmed that the copolymer product was BHET.From 3.2.31H NMR spectrum of the depolymerization product is shown in 1H NMR  \u03b4 8.12 , 4.52\u20134.43 , 4.07\u20133.98 , 2.09 . Therefore, it can be determined that the product is BHET.3.33.3.1\u22121 correspond to the telescopic vibration of ester CO, aromatic \u2013C\u2013H vibration peaks at 1390\u20131450 cm\u22121, C\u2013O vibration peaks at 1160\u20131300 cm\u22121 and 1050\u20131150 cm\u22121, and \u2013C\u2013O vibration peaks at 850\u2013890 cm\u22121. The peaks at 1160\u20131300 cm\u22121, 1050\u20131150 cm\u22121, and 850\u2013890 cm\u22121 correspond to aromatic ring bending vibrations. The C\u2013H vibration of the methylene group in the EG chain was observed at 2850\u20132980 cm\u22121. The infrared spectra of the catalyst-synthesized products were consistent with those of PET, which confirmed that the catalytic products were all PET.The FT-IR spectrum of the synthesized PET is shown in 3.3.2\u22121, and the program temperature was 10\u00b0C min\u22121 from \u221210 \u00b0C to 280 \u00b0C, holding for 10 min, and then 10\u00b0C min\u22121 to \u221210 \u00b0C, holding for 10 min for the DSC analysis. As shown in The nitrogen flow rate was 40 mL min3.3.3The GPC effluent profile of the synthesized PET is shown in 3.430 experiments were designed. Response surface modeling was performed using software for the analysis. The experimental results of the corresponding results of the response surface curves are as follows.Based on the Box\u2013Behnken model,The 3D and 2D response surface plots created to show the effect of the interaction between the catalyst use and reaction time on the yield of BHET are shown in As shown in As shown in Ultimately, the variance of the regression equation showed that the yield of BHET increased upon increasing the reaction temperature, reaction time, and catalyst dosage. The interactions were significant between catalyst dosage and reaction time, less significant between the catalyst dosage and reaction temperature, and more significant between the reaction time and reaction temperature. The optimal depolymerization conditions were determined to be a reaction temperature of 203 \u00b0C, reaction time of 3.8 h, and maximum yield of 90.1% for BHET when the catalyst mass dosage was 0.56% (in terms of the PET mass).b* of 1.2 and an L* of 97.1, and the BHET monomer was depolymerized using Ti\u2013EG catalyst under the same conditions was significantly darker than the BHET depolymerized using Ti/Si\u2013EG alone. This phenomenon is related to the excessive catalytic activity and poor catalytic selectivity of Ti\u2013EG.As shown in 3.5L*, b*) and molecular weight indexes of PET under the above PET synthesis conditions were examined under different cases: (1) PET synthesized using PTA as the raw material and Ti/Si\u2013EG as the catalyst had the lowest b*, the highest L*, and the lightest overall color. (2) PET synthesized using PTA as the raw material and Ti\u2013EG as the catalyst had the highest b*, lowest L*, and darkest color. (3) PET synthesized using BHET as the feedstock and Ti/Si\u2013EG as the catalyst had in between b* and L* values.As can be seen in The reason for this phenomenon is the high catalytic activity and poor selectivity of the single Ti\u2013EG catalyst at high temperatures. The catalyst catalyzes the polymerization of ethylene glycol resulting in the darkening of the PET color. Under the joint action of Ti and Si elements, it can be seen from the above data that the activity of Ti was suppressed, and the color of the product was reduced.PET synthesized from the depolymerization product BHET using Ti/Si\u2013EG as the catalyst showed a small but insignificant increase in chromaticity compared with that synthesized by the PTA method. The reasons may include polymerization of the glycol during depolymerization, excessive depolymerization of PET, and oxidation of some additives in commercial PET. However, the color deepening of BHET is not obvious and meets the index of reuse.3.6Based on the experimental results of this study, the experimental mechanism of ethylene glycol depolymerization of waste PET using Ti/Si\u2013EG as a catalyst is shown in O bond on the PET ester group is broken, and the electrons on the other oxygen of the PET ester group are transferred to form a new \u2013C\u2013O bond. As \u2013OCH2CH2\u2013 is detached from PET, the PET chain is broken. The above process is repeated until the monomer is formed.The depolymerization process was carried out in two steps. The first step was the activation process of the catalyst, in which Ti, Si, and O in Ti/Si\u2013EG formed a coordination structure with the hydroxyl groups of ethylene glycol, which increased the electronegativity of the hydroxyl oxygen in EG. Ti and Si cations interacted with the oxygen in the PET ester group, which makes it easier for the hydroxyl oxygen on EG to attack the positively charged carbon ions in the PET ester group and nucleophilic substitution occurs to form tetrahedral structural intermediates. Subsequently, the H on EG is detached, the C2CH2\u2013 in the other molecule attacks the carbonyl carbon atom, and the nucleophilic attack of the ester carbonyl oxygen on the central titanium atom increases the positive charge of the carbonyl carbon, thereby increasing the rate of reaction.During the polycondensation reaction of PET, the catalyst ligand first undergoes an exchange reaction between the end hydroxyl groups of the monomer or oligomer to form a metal alcohol salt. The oxygen in \u2013OCH4.In this study, Ti\u2013Si\u2013ethylene glycol salt (Ti\u2013Si\u2013EG) was synthesized by transesterification of titanate and silicate with ethylene glycol, combining Si, of low polyester catalytic activity, with Ti, of high catalytic activity. This solved the problem of high activity when using a single Ti catalyst in PET catalysis. Also, the catalyst as a glycol salt avoids the problem of introducing impurity groups during PET synthesis and depolymerization.w(EG)\u2009:\u2009w(PET) ratio of 4\u2009:\u20091, a catalyst dosage of 0.56% (based on the mass of PET), a reaction temperature of 203 \u00b0C, and a depolymerization time of 3.8 hours. As a result, the yield of the product (BHET) could reach 90.10%.Glycol depolymerization of PET was carried out using the synthesized catalysts. Response surface methodology was utilized to determine the optimum process conditions, which included a b* of the depolymerization product and the condensation product. This study provides a reference for the resource recovery of waste PET through the synthesis of catalysts.The same catalyst was used again for the polycondensation of the depolymerized product BHET, and the obtained product PET was tested for color, molecular weight, and thermal properties. It can be concluded that PET synthesized by this method is the same as the PET synthesized by the PTA method in terms of various indexes. Compared with a single titanium catalyst, the titanium\u2013silicon composite catalyst can significantly reduce the chromaticity Prof. Guoliang Shen and Prof. Tie Jun Xu provided the idea for the experiment, Yang Yu led the experimental work, Wen Ruiyang completed the data organization, Yue Huo provided the data analysis and other aspects of the work, Yun Chang Qiao and Ru Chao Cheng assisted to complete the work for sample testing.The authors declare that they have no conflicts of interest."}
+{"text": "Populus abla \u00d7 Populus glandulosa) was used as a template to clone the full length of the PagMYB205 gene fragment, and transgenic poplar lines with PagMYB205 overexpression (OX) or inhibited expression  were cultivated. The role of PagMYB205 in poplar growth and development and salt tolerance was detected using morphological and physiological methods. The full-length CDS sequence of PagMYB205 was 906 bp, encoding 301 amino acids, and the upstream promoter sequence contained abiotic stress-related cis-acting elements. The results of subcellular localization and transactivation assays showed that the protein had no self-activating activity and was localized in the nucleus. Under salt stress, the rooting rate and root vitality of RNAi were higher than OX and wild type (WT). However, the malondialdehyde (MDA) content of the RNAi lines was significantly lower than that of the wild-type (WT) and OX lines, but the reactive oxygen species (ROS) scavenging ability, such as the peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) enzyme activities, was dramatically more powerful. Most significantly of all, the RNAi3 line with the lowest expression level of PagMYB205 had the lowest MDA content, the best enzyme activity and root vitality, and the best salt stress tolerance compared to the other lines. The above results suggest that the transcription factor PagMYB205 could negatively regulate salt stress tolerance by regulating antioxidant enzyme activity and root vitality.Salt stress is one of the major abiotic stresses that limits plant growth and development. The MYB transcription factor family plays essential roles in plant growth and development, as well as stress tolerance processes. In this study, the cDNA of the 84K poplar ( Soil salinization is one of the main environmental stresses limiting plant growth and production . ResearcGmDREB1 improved salt tolerance and leaf protein in response to high salt concentration in transgenic wheat [TabZIP14-B was involved in the regulation of salt tolerance and frost resistance in transgenic plants, and overexpressing lines showed dramatical salt tolerance ability compared to the wild type [PeNAC1, was successfully isolated from poplar and played an important role in salt stress endurance by regulating Na+/K+ homeostasis [ATAF1 of the Arabidopsis NAC gene was significantly induced by high salt levels and abscisic acid (ABA) [RmMYB108 gene was induced by low temperature, salt, and drought stress [Transcription factors (TFs) are often key regulators of plant responses to biotic and abiotic stresses . The peric wheat . The wheild type . NAC (NAild type . A streseostasis . The expid (ABA) . The RmMMYB7 played a role in regulating carotenoid and chlorophyll accumulation in tissues by transcriptionally activating metabolic pathway genes [MdMYB308L positively regulated cold tolerance and anthocyanin accumulation in apples by interacting with MdbHLH33 [RmMYB108 responded to low-temperature stress in Rosa multiflora and cold hardiness in Arabidopsis thaliana [MYB12 and MYB75 in transgenic Arabidopsis significantly increased the accumulation of flavonoids with potent antioxidant activity, resulting in enhanced tolerance to drought and oxidative stress [MYB94 and MYB96 together activated epidermal wax biosynthesis to promote drought tolerance in Arabidopsis [MYB60 was specifically expressed in guard cells and enhanced plant drought tolerance by reducing water dissipation. The expression of MYB60 was induced by signals that promote stomatal opening, such as white and blue light, but was inhibited by darkness, desiccation, and ABA treatment [PYL8 directly interacted with transcription factors MYB77, MYB44, and MYB73 to increase the binding ability of MYB77 to the target motif MBSI in the promoters of multiple growth factor responsive genes and promoted lateral root growth [MYB49 positively regulated salt tolerance by modulating stratum corneum formation and antioxidant defenses [MYB12 overexpression induced the expression of ABA and proline biosynthesis-related genes, as well as improved flavonoid biosynthesis and reactive oxygen species (ROS) scavenging to confer salt tolerance in plants [MYB42 positively regulated salt tolerance by regulating the expression of SOS2  [MYB30 regulated salt tolerance in plants by regulating mitochondrial alternative oxidase AOX1a  [The MYB transcription factor family is one of the largest families of transcription factors in plants and plays an important role in the regulation of plant growth, development, and metabolism . MYB TFsay genes . MdMYB30MdbHLH33 . RmMYB10e stress . MYB94 abidopsis . MYB60 wreatment . PYL8 dit growth . MYB49 pdefenses . Many MYn plants . MYB42 pitive 2) . MYB30 rdase 1a) .Potri.019G081500.1) in plant abiotic stress endurance and the breeding of salt-tolerant, superior tree species.Poplar is an important raw material for biofuels and is widely used as green forest, timber forest, and protection forest for production, road greening, and desertification control . CurrentPagMYB205 was 906 bp and encoded 301 amino acids. The molecular weight of the protein was 34.16 kDa, the molecular formula was C1473H2348N436O472S14, and the number of atoms was 4743  and MeJA hormone response element, as well as some light response elements and seed-specific regulation elements  by using the Agrobacterium rhizogenes-mediated leaf disk method to cultivate PagMYB205 overexpression (OX) and inhibited-expression (RNAi) transgenic lines, respectively. Transgenic seedlings that could root normally in 1/2 Murashige and Skoog (MS) medium containing 50 mg/mL Kanamycin and 200 mg/mL Timentin were selected as positive lines (PagMYB205 in the 13 transgenic lines was then detected with qRT-PCR. As shown in PagMYB205 was significantly up-regulated in nine OX lines compared to the wild-type (WT) line and significantly down-regulated in four RNAi lines. Three RNAi  and three OX  lines with high, medium, and low expression levels were selected for further physiological experiments, respectively , peroxidase (POD), and catalase (CAT) play an important role in ROS scavenging, we further determined the activities of SOD, POD, and CAT in each poplar line under both treatments. After salt treatment, the SOD, POD, and CAT activities of the RNAi were significantly higher than those of the other two lines, with RNAi3 having the highest enzyme activity A\u2013C. The Plant growth and development are severely affected by abiotic and biotic stresses from the environment . The secPagMYB205 through bioinformatics analysis. The PagMYB205 protein was found to be an unstable hydrophilic soluble protein localized in the nucleus, without transmembrane structural domains and self-activation. The homology sequence alignment and phylogenetic tree indicated that PagMYB205 belonged to the R2R3MYB subfamily that is involved in plant growth and development, primary and secondary plant metabolism, hormone regulation, and stress response stages [Arabidopsis, the R2R3MYB member AtMYB44 responds to drought stress by participating in stomatal regulation and ROS accumulation [GmMYB118 maintains cellular homeostasis by regulating osmotic and oxidative substances to enhance tolerance to drought and salt stress in soybean [PtsrMYB enhances tolerance to salt, dehydration, and cold in transgenic tobacco [MYB15, MYB37, and MYB96 were involved in the ABA-mediated inhibition of seed germination [GhODO1 positively regulates resistance to yellow wilt in cotton through the lignin biosynthesis and jasmonic acid signaling pathways [PagMYB205 may be involved in the stress-related response of poplar.In this study, abiotic stress-related cis-acting elements were detected in the promoter region of e stages . In Arabmulation ,34. GmMY soybean . The ove tobacco . MYB15, mination ,39. GhODpathways . These sPagMYB205, we cloned PagMYB205 from 84K poplar and cultivated transgenic poplar OX and RNAi. By subjecting transgenic plants to non-salt and salt stress treatment for 30d, it was found that there were mostly no significant differences between the morphology of transgenic and non-transgenic lines, but the rooting rate and root vitality of RNAi was higher than that of other lines under salt treatment conditions. These were consistent with other transcription factors in responding to abiotic stresses. For example, LbTRY was involved in root hair development and specifically reduced salt tolerance by increasing ion accumulation and reducing osmoregulatory substances when heterologously expressed in Arabidopsis [SiMYB75 promoted Arabidopsis root growth and regulated ABA-mediated drought and salt stress tolerance [To reveal the function of bidopsis . The ectolerance .AcoMYB4 overexpression decreased POD, SOD, and CAT antioxidant enzyme activities, but significantly increased MDA levels [OsMYBR1 significantly increased soluble sugar and proline levels in transgenic rice [SiMYB56 decreased the MDA content [FvMYB82 resulted in higher proline and chlorophyll content, as well as higher SOD, POD, and CAT activities in Arabidopsis under salt and cold treatments, which improved salt and cold tolerance [PagMYB205, were the highest. However, the enzyme activities of OX were lower or similar to those of WT. MDA is one of the membrane lipid peroxidation products and can be used as a sign to measure the degree of damage of cell membranes caused by abiotic stress [OsMYBR1 and SiMYB56 improved the tolerance of rice to drought stress by increasing soluble sugar and proline content but decreasing MDA in transgenic rice plants [PagMYB205 made a contribution to the salt stress tolerance of poplar; that is to say, PagMYB205 may negatively regulate the salt stress tolerance of poplar through interfering with ROS scavenging and root vitality . The roots, stems, and leaves used in this experiment were excised from the plants, immediately frozen in liquid nitrogen, and then stored at \u221280 \u00b0C [The 84K poplars used in this study were grown on a 1/2 MS plant medium in a greenhouse . cDNA was synthesized using FastKing gDNA Dispelling RT SuperMix .  primers  and liga primers  and inse05-F1/R1  and liga05-F2/R2  and simi5-BD-F/R  and ligaPagMYB205. ProtParam from the ExPASy database  was used for the basic physicochemical properties analysis. The protein signal peptide, transmembrane structure, phosphorylation site, and secondary structure of proteins were analyzed with SignalP 5.0 , TMHMM-2.0 , NetPhos 3.1 , and SOP 2021 , respectively. The protein domain was identified with NCBI\u2019s CD-search . The 2000 bp upstream sequence of PagMYB205 was obtained from PlantCare  and used for promoter cis-element analysis. The amino acid sequence of homologous genes of PagMYB205 was blasted and downloaded from NCBI  to construct a neighbor-joining phylogenetic tree and amino acid sequence comparison using BioEdit 7.0.9.1 [The bioinformatics analysis was performed based on the sequences of CDs and amino acids of  7.0.9.1 .Agrobacterium containing subcellular localization vectors 35S:PagMYB205-GFP or no-load control 35S:GFP were injected into tobacco leaves using 5 mL sterile needleless syringes. After being grown in the dark for 2\u20133 day, the fluorescence signals under different fields of view were observed and photographed under a confocal laser scanning microscope .PagMYB205 gene into the pGBKT7 vector to obtain the pGBKT7-PagMYB205 plasmid. Yeast cells transfected with the pGBKT7 empty vector and the pGBKT7-53/pGBKT7-T were designed as the negative and positive control, respectively. Yeast cells were incubated on SD/-Trp and SD/-Trp/-His/-Ade/X-\u03b1-Gal solid media to observe the yeast growth status and to test the self-activation ability.To verify whether PagMYB205 has self-activating activity, we fused the Agrobacterium-mediated transformation method [PagMYB205 in each line using SuperReal PreMix Plus  with actin as the internal reference gene. The fold change of gene expression was calculated by using the 2\u2212\u0394\u0394CT method.Transgenic poplars were obtained by using the n method . The traSalt tolerance tests were performed using transgenic poplar and WT. For each line, 30-day-old plants of uniform height and vigorous growth were selected, and the terminal buds were cut and subcultured in 1/2 MS medium containing 0 or 50 mmol/L NaCl. After 30 d, fresh weight, plant height, and number of leaves were statistically determined. The roots were scanned and photographed using an EPSON EXPRESSION 10,000 XL root scanning system (resolution set at 400 dpi) to detect the primary root length, root diameter, root area, root volume, etc., and WinRHIZO 2016p software  was used for statistical analysis . The 3rdLeaves of 30 day seedlings were selected for the determination of relevant physiological indices. SOD activity was measured using the nitrogen blue tetrazolium (NBT) photoreduction method. CAT activity was determined using UV spectrophotometry and POD activity was determined using the guaiacol method. MDA content was determined using the colorimetric method using barbituric acid. Root vitality was determined using the triphenyltetrazolium chloride (TTC) method ,57. At lp < 0.05 was used to test the significance of differences between the experimental and control groups. Plots were made using Origin 2019b  and Adobe Photoshop 2022. Each experiment was performed with three technical repeats.The data were processed using SPSS 22.0 software . PagMYB205 reduced the MDA content of the RNAi, but increased SOD and CAT enzyme activities and root vitality under salt stress. Above all, this study preliminarily revealed the negative function of PagMYB205 in poplar salt stress responses.PagMYB205 protein was an unstable hydrophilic soluble protein, localized in the nucleus without a transmembrane structural domain and self-activating activity. PagMYB205 had cis-acting elements related to abiotic stresses in the promoter region. Inhibiting the expression of"}
+{"text": "Creativity is a primary factor in driving transformational change. This study explored the impact of leader humor on employee creativity  from the perspective of employee voice. Data were collected from 812 Chinese employees through multipoint surveys. Through the surveys, we found that (1) leader humor has a significant positive effect on employee incremental and radical creativity; (2) promotive/prohibitive voice mediates the relationship between leader humor and employee incremental/radical creativity separately; (3) contradictory thinking moderates the effect of leader humor on prohibitive voice and further moderates the indirect effect of leader humor on radical creativity; and (4) the moderated mediation model mainly applies to state-owned enterprises rather than private-owned enterprises. The theoretical and practical implications of these findings are discussed. In recent years, industries have struggled in economically uncertain times, especially due to the COVID pandemic and trade war between China and the United States, and some enterprises are on the verge of bankruptcy. However, some enterprises driven by creativity have shown strong adaptability and even achieved tremendous development during this period. This is because creativity is the key to the survival and development of enterprises . Leader humor has a stronger indirect effect on the incremental creativity of employees with high contradictory thinking than on the incremental creativity of employees with low contradictory thinking.H4b: Contradictory thinking has a moderating effect on the mediating role of a prohibitive voice in the relationship between leader humor and radical creativity (H2b). Leader humor has a stronger indirect effect on the radical creativity of employees with high levels of contradictory thinking than the radical creativity of employees with low levels of contradictory thinking.Based on H4a and H4b, the present study further proposes that the moderated mediation effect of contradictory thinking may also be influenced by corporate ownership. Ownership is a common characteristic of Chinese enterprises , 812 valid questionnaires were obtained.Among the participants, 427 (52.6%) of them were from SOEs, including 150 men (35.1%) and 277 women (64.9%), with an average age of 31.37 years (SD = 8.319); 164 (38.4%) of them were unmarried, and 263 (61.6%) were married; 103 (24.1%) of participants had a senior high school or lower level of education, 124 (29.0%) had a junior college education, and 200 (46.9%) had an undergraduate or higher level of education. The average number of years they had worked in the current firm was 7.03 years (SD = 7.923). Moreover, 385 (47.4%) participants were from POEs, including 201 men (52.2%) and 184 women (47.8%), with an average age of 32.58 years (SD = 7.722); 144 (37.4%) of these participants were unmarried and 241 (62.6%) of them were married; 45 (11.7%) of them had a senior high school or lower level of education, 59 (15.3%) had a junior college education, 281 (73%) had an undergraduate or higher level of education; and the average years they worked in their current firm was 4.79 years (SD = 5.413).All items in the questionnaire were rated on a 7-point Likert scale, ranging from 1 (completely disagree) to 7 (completely agree).The Leader Humor Scale developed by Cooper et al.  was usedThe scale proposed by Liang et al.  was adopThe instrument proposed by Gilson et al.  includesThe subscale of contradictory thinking in the Dialectical Thinking Scale for Chinese People developed by Hou  was usedAccording to the recommendations of Chow , this st2 = 489.046, \u03c72/df = 4.328, TLI = 0.954, CFI = 0.945, SRMR = 0.040, RMSEA = 0.064); among the \u201cleader humor + promotive voice + incremental creativity + contradictory thinking\u201d models, the four-factor model fit the data better than alternative models .The present study used Mplus Version 7.4 to conduct confirmatory factor analysis. The results showed that, among the \u201cleader humor + prohibitive voice + radical creativity + contradictory thinking\u201d models, the four-factor model fit the data better than alternative models  and incremental creativity . Leader humor was also positively correlated with a promotive voice , a prohibitive voice , and contradictory thinking . Moreover, a prohibitive voice was positively correlated with radical creativity , while a promotive voice was positively correlated with incremental creativity . These findings provide a basis for further hypothesis testing.adjusted R2 = 0.117, B = 0.213, p < 0.01) and incremental creativity  (collected at time point 3). Hence, H1a and H1b were supported. In addition, leader humor was positively correlated with a promotive voice  and a prohibitive voice  (collected at time point 2). Next, Model 4 was constructed in PROCESS . Promotive voice mediated the relationship between leader humor and incremental creativity . Therefore, H2a and H2b were supported.An ordinary least squares (OLS) regression was used to test H1 and H2, and the results are shown in S Hayes,  to test B = 0.073, p < 0.01) and contradictory thinking . Then, the interaction effect was significant , and the model explained more variance . Model 1 was then used in PROCESS , and when contradictory thinking was low, the effect was not significant . Thus, H3b was supported. Model 7 was used in PROCESS , the indirect effect was not significant when contradictory thinking was low , and that the indirect effect was significant when contradictory thinking was medium . The moderated mediating effect of the overall model was significant . Hence, H4b was supported. The same methods were used to test H3a and H4a. Leader humor was found to be significantly correlated with promotive voice . However, there was no significant correlation between contradictory thinking and promotive voice . In addition, the effect of the interaction term was also not significant . Therefore, H3a was rejected  . The resS Hayes,  to furthS Hayes,  to test rejected , and furindex = 0.037, SE = 0.010, 95% CI = \u22120.014 to 0.023). However, in SOEs, the moderated mediation model was significant . In POEs, the moderated mediation model was not significant ; thus, H5b was partially supported.Finally, Model 11 was applied in PROCESS Hayes,  to conduFurthermore, the present study used the simple slope method to draw the chart. Low/high levels of contradictory thinking were calculated using \u2013/+ one standard deviation from the mean of the variable. This study makes the following four theoretical contributions. First, the results showed that leader humor had a significant positive effect on employee incremental and radical creativity. Previous studies have conceptualized and operationalized creativity as a single concept, which overlooks its inherent complexity. This study further divided creativity into radical and incremental creativity according to the recommendations of Madjar et al. . In thisThird, this study found that contradictory thinking moderates the effect of leader humor on employee voice. Compared to employees with low levels of contradictory thinking, employees with high levels of contradictory thinking are more likely to perceive leader humor and thus express their voice. However, it was also found that contradictory thinking only moderates the relationship between leader humor and prohibitive voice but not between leader humor and promotive voice. This may be because Chinese culture emphasizes maintaining and prioritizing harmony. Thus, compared to the prohibitive voice that may put employees' relationships with leaders at risk, Chinese employees are more inclined to propose suggestions that maintain internal unity and stability, such as a promotive voice. Consequently, such social desirability for a promotive voice may make the participants exhibit bias in the process of completing the questionnaire.Fourth, the moderated mediation effect was supported in SOEs but not in POEs. This conclusion was contrary to our hypothesis. The result further found that the indirect effect of leader humor on the radical creativity of employees with high levels of contradictory thinking through prohibitive voice is greater in SOEs than in POEs (\u201cSOE-high contradiction\u201d > \u201cPOE-high contradiction\u201d). We attributed this phenomenon to the following reasons: There has been a growing demand for employment in China over recent years, which may result in increased difficulty in finding a new job. As a result, employees may be less willing to risk losing their current job to exhibit a prohibitive voice. Compared to employees in POEs, employees in SOEs are better protected  and attempting to replicate the outcomes. Third, we used questionnaires to explore how leader humor influences two types of creativity, but this method may not provide evidence for causal relations between variables. Future studies should investigate causality through an experimental design.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Peking University. The patients/participants provided their written informed consent to participate in this study.YC and KZ developed the study concept, drafted the manuscript, and were performed the testing and data collection. YH and RM contributed to the study design and provided critical revisions. YC and YW performed the data analysis and interpretation under the supervision of YH and RM. All authors approved the final version of the manuscript for submission."}
+{"text": "This suggests that the RBDdiffuses between the open and closed positions without significantenergetic hindrance. This interpretation appears consistent with experimentsbut is at odds with some previous simulations. Modeling the RBD motionas diffusive dynamics along the computed free energy profile, we findthat the overall time required for the transition is only about 2 \u03bcs,which is 5 orders of magnitude shorter than experimentally measuredtransition times. We speculate that the most likely reason for thetransition time mismatch is our use of very short glycans, which wasrequired to make the simulations performed here feasible. Despitethe long simulation times, the final free energy profile is not fullyconverged with statistical errors of \u22431.16 kcal/mol, whichwere found to be consistent with the slow time decay in the autocorrelationof the conformational motions of the protein. The simulation lengthsthat would be required to obtain fully converged results remain unknown,but the present calculations would benefit from at least an order-of-magnitudeextension.Infection by sarbecovirusesbegins with the attachment of the homotrimericviral \u201cspike\u201d protein to the angiotensin-convertingenzyme 2 receptor on the surface of mammalian cells. This requiresone or more receptor-binding domains (RBDs) to be in the open (up)position. Here, we present the results of long molecular dynamicssimulations with umbrella sampling (US) to compute a one-dimensionalfree energy profile of RBD opening/closing and the associated transitiontimes. After \u22433.58 To address this challenge in the frameworkof rational antigen design, a detailed molecular-level understandingof the determinants of coronavirus infectivity is necessary. Becauseinfection with sarbecoviruses begins with the attachment of the trimericviral spike to the angiotensin-converting enzyme 2 (ACE2) receptorfound in many cells throughout the body, the spike is the target ofmost antibodies that neutralize COVID-19. The binding occurs via anexposed receptor-binding domain (RBD), which is relatively looselyattached to the rest of the spike (stem), as different positions andorientations of the RBD have been inferred from solution studies.10 The dynamics and energetics of RBD motion between the two positionalextremes, \u201cclosed\u201d (RBD down) and \u201copen\u201d(RDB up), appear to have a significant effect on the virulence ofthe virus. For example, the mutation D614G that is near the base ofthe attachment of the RBD to the spike increases the prevalence ofopen configurations,10 which are believedto be responsible for the higher infectivity of this strain.11 However, open positions also expose the RBDto the immune system, which elicits the maturation of anti-RBD antibodies,increasing the evolutionary pressure on the virus to mutate to avoidimmune detection.As the descendantsof the original Wuhan strain of COVID-19 arelikely to become endemic,12 is oftenused to increase vaccine efficacy. Here, we present results from longMD simulations that use umbrella sampling to compute the free energy(FE) profile and the associated rates via diffusion theory along a1D reaction coordinate of RBD opening in the proline-stabilized SARS2spike mutant.9 The main purpose of thestudy is 2-fold: (i) to establish the amount of sampling requiredto obtain converged FE profiles in such a large and partially disorderedbiomolecular system and (ii) to explain the origin, at the atom level,of the rather high  free energy barriers to openingthat have been observed in some earlier studies. To further motivatethis study and to place it into proper context, we summarize in thefollowing the computational studies that are most relevant to ourwork. The informed reader may wish to skip to the next section.While the majority of the information neededfor rational coronavirusvaccine design comes from experiments in structural biology and biochemistry,molecular dynamics (MD) simulations are increasingly able to aid inthe interpretation of such experiments by providing dynamic informationon temporal and spatial scales that are finer than the resolutionof the experiments. In the context of rational vaccine design, itis useful to be able to predict computationally how amino acid mutationsaffect the distribution of antigenic epitopes that are exposed tothe immune system. For example, stabilization of antigenic spikesin the prefusion conformation13 investigatedthe closed-to-open(C\u2194O) transition in a fully glycosylated spike model14 using the weighted ensemble (WE) method. Briefly,multiple unbiased simulations were launched in parallel from the closedstate and run for 100 ps; at the end of this iteration, the trajectorywhich had progressed the farthest toward the open state, on the basisof a preselected 2D progress coordinate, was used to reinitializethe next trajectory ensemble. The process was repeated until the RBDof the simulated spike sampled the open conformations. Several openconformations were observed that were similar to previously determinedexperimental cryo-EM structures.15 Theauthors also reported that typical transition lengths were hundredsof nanoseconds. However, because of the basing inherent in WE, theycould not be related to the transition rates or relative populationsof different states. Consequently, these results were not reported.An important prediction from the simulations was that a glycan atposition N343 promotes RBD-open conformations. This appears to beconsistent with an experimental measurement that showed reduced ACE2binding by the N343A mutant.13Sztain et al.16 proposed a model of theC\u2194O transition on the basis of steered MD (SMD)17 simulations. Starting from intermediate structuresobtained from SMD, the authors generated conformational ensemblesusing unbiased MD. They were projected onto principal components havingthe highest variance, which were computed from MD simulations of theend points, to visualize a 2D transition landscape. The authors didnot describe whether they removed the bias arising from the use ofthe steering forces in generating the intermediate trajectories. However,they reported a metastable state, which may be consistent with experiments.8 Another approach using SMD simulations was reportedby Ray et al.,18 but their nearly monotonicFE profiles suggest that the transition conformations sampled arefar from equilibrium.Guret al.19 computed an initialpathway for the C\u2194O transition making use of the equilibratedstructures of Casalino et al.14 The pathwaywas represented by 32 backbone structures of the spike, computed usingthe nudged elastic band (NEB) method,20 followed by SMD in the full coordinate space to steer an all-atomstructure to each of the NEB structures. The resulting pathway wasinput as initial conditions for 2D umbrella sampling (US) simulations,which were then used to construct 2D free energy landscapes spanningthe C\u2194O transition for the wild-type (WT) spike as well asfor three mutants. The NEB method for path finding has the advantagethat it does not introduce directional bias, unlike steered or targeteddynamics,21 and potentially requires lessMD equilibration to converge to paths of high equilibrium probability.With around 300 US windows, each simulated for 16 ns for each mutant,the authors could only interpret the FE landscapes qualitatively becauseof the high uncertainties in modeling and statistics. Generally, thesimulations revealed landscapes with relatively shallow basins, theopen and closed states being separated by barriers of between 2.5and 5 kcal/mol and differing in free energy by \u22123 to 3 kcal/mol,depending on the mutant simulated.Fallon et al.22 used parallel cascade MD,23 avariant of the WE method, to generate a C\u2194Otransition pathway, which was subsequently used to initialize US simulations.The reaction coordinate was the root-mean-square difference (RMSD)between the simulation and the open structure. Unfortunately, someimportant simulation details were not clearly stated, such as whetherthe simulation structures included glycosylation, the duration ofsampling within the US windows, and the type of statistical or erroranalysis used. The FE profile computed for the transition showed thatthe closed structure is more stable by \u22433 kcal/mol than theopen structure. However, the RMSD used as a reaction coordinate canbe associated with a large Jacobian correction factor due to the coordinatetransformations from Cartesian coordinates , which 25 performed US simulationsto characterize the C\u2194O transition for three SARS-CoV-2 spikevariants, the fully glycosylated wild-type (WT), the deglycosylatedWT spike, and the deglycosylated double proline mutant.15 For each system, a partial 2D landscape containing the minimum-free-energypath was computed. It was composed of 1000\u20131200 windows, withtotal simulation lengths of 12\u201391 \u03bcs, depending on thespike variant. To improve convergence of the US simulations, the authorsused Monte Carlo (MC) replica exchange, in which adjacent windowscan swap their bias values, according to the Metropolis MC criterion.26 With this method, the authors report impressivelylow statistical errors, most of which are in the range 0.1\u20130.15kcal/mol. However, for the fully glycosylated spike, they predictedthe open conformation to be unfavorable by 5.2 \u00b1 0.1 kcal/mol,which appears to be at odds with the experimental observations ofequal populations of the spike in the two positions, inferred fromfluorescence resonance transfer (FRET) data8 and analysis of cryo-EM images.10 It is noteworthythat the glycan-free simulations correctly predict the two conformationsto be nearly isoenergetic,25 but in viewof the importance of glycans noted in other studies, this findingmay not be biologically significant. The authors also computed themean first passage time (MFPT) for the C\u2194O transition in thesystems and noted that their high value of \u223c1.5s in the forwarddirection, compared to \u223c0.3s from the experiment of Lu et al.,8 could be caused by insufficient sampling of glycanconformations. However, the most likely cause of the long MFPT inthe calculations of Pang et al.25 couldbe the high FE barrier, which is 11 \u00b1 0.1 kcal/mol. By contrast,the distribution of fluorescence intensities that were used to identifythe degree of spike opening was essentially flat,8 suggesting the absence of significant barriers. However,the \u201creaction coordinates\u201d, i.e., the variables chosento measure the extent of opening, were different between the calculationand the experiment, so that the two profiles may not be directly relatable.Pang et al.\u03bcs simulations of Pang et al.25 appear similarly converged as the 91\u03bcsones. They have also not explained the molecular origins of the  free energy barriers to RBD opening or closing that havebeen observed. In this study, we provide more detail on the energeticsand dynamics of the RBD transition using very long free energy simulationsin one dimension.The above listing of studies illustrates the challenge of reconcilingcomputational with experimental studies and also of different computationalstudies with each other. Further, the existing simulations have notestablished the extent of sampling required to obtain converged FEprofiles in this large and partly disordered biomolecular system;e.g., the 1227 as implemented in CHARMM.29 Specifically,we refined by minimization of an all-atom linearly interpolated pathbetween the C (PDB: 6VXX)9 and O (PDB: 6VSB)15 end points, with the missing loops modeled by Casalinoet al.14 This initialization approach ischosen because it does not introduce directional bias into the path21 and is similar to the NEB method used by Fallonet al.19 The ZTS was represented by 64discrete structures (replicas). To generate the interpolant whileavoiding steric clashes, we used the method of No\u00e9 et al.,30 in which the side chains are shrunk before interpolation, and the equilibrium bondlengths are restored automatically during subsequent minimization.To prepare initial structuresfor the umbrella sampling (US) simulations,we used the zero-temperature string (ZTS) method,25 which included only 63K protein and glycan atoms,and the remaining were solvent atoms. To reduce the calculation costand memory demand, we performed the US simulations using a quasi-equilibriumsolvation shell model,31 in which the spikewas surrounded by a thin layer of solvent, whose evaporation was preventedusing restraint potentials implemented as a plugin for the MD OpenMMlibrary.32 The solvation model has beenvalidated in unbiased MD, as well as free energy simulations, andfound to introduce only minor artifacts, in the form of a slight butcontrollable departure from equilibrium dynamics, and increased solventpressure on the biomolecule.31 Comparisonsof potential of mean force (PMF) profiles of antigen\u2013antibodyseparation produced with the shell model vs the periodic solvent boxmodel showed that the differences between them were within statisticalerror31 . Sodium and chlorideions were added to the solvent shell to achieve a total ion concentrationof 150 mM.Previous simulations of the fully glycosylated spike protein immersedin a periodic box of solvent required more than 750 K atoms,25 To avoid this problem, we truncated all glycans to theN-acetylglucosamine moieties present in the PDB coordinates; however,the use of the short glycans also implies that our system better approximatesthe deglycosylated spike. In addition, we replaced the sequence containingthe furin site (677\u2013688) that was unresolved in the PDB  with thelinker GSA at residue position 677. The use of the linker is unlikelyto impact the properties of the spike; Gobeil et al.33 found that the removal of the furin site resulted in aspike that was similar to the wild-type in terms of thermal stabilityand binding to ACE2.As mentioned in the 32 library with the CHARMM36energy function35 in TIP3 water.36The use of shorter glycans, the linker,and the solvent shell layerof 10 \u00c5 resulted in relatively small solvated systems, rangingfrom \u2243125 to \u2243130 K atoms in total, depending on theZTS replica (1\u201364) and permitting over two hundred microsecondsof aggregate MD simulation time. The solvated systems were simulatedusing the OpenMM\u20131 for plain USsimulations and to 5 ps\u20131 for computing diffusionconstants. The reason for the higher value used in computing the diffusionconstants was to reduce the enhancement of self-diffusion by the TIP3water model.37The MD simulation setup included particle-mesh Ewald(PME) electrostatics,a 9 \u00c5 nonbonded cutoff for Lennard-Jones interactions, and hydrogen-massrepartitioning, which transfers 3 amu to every hydrogen from the parentheavy atom, reducing corresponding angle vibration frequencies andallowing a 4 fs simulation time step. Covalent bonds to hydrogensand water molecules were kept rigid, and the equations of motion wereintegrated using a Langevin dynamics integrator at 300 K with theatomic friction set equal to 1 ps25 is illustrated in Supporting Information. The chosen distance is convenient because it increases graduallyfrom \u223c49 to \u223c69 \u00c5 during the transition, separatingthe end points and possible intermediates (see 8The reaction coordinate(RC) for the opening motion was chosento be the distance between the centers-of-mass (COMs) of the RBD ofone monomer in the spike protein (RBD1), and a part of the spike formedby another monomer that excludes the RBD STEM2\u0394RBD2). ThisRC, which is similar to one of the coordinates used by Pang et al.,\u0394RBD2. Thates see 2. The fr2. The statistics from the flat-bottom (FB)part of the windows were used to compute the derivative of the FEprofile under the assumption that it is linear over the FB window.Specifically,38 for a window with FB width\u0394 centered on x0, the FE derivative F\u2032 is computed from the average of the RC value (x\u0305MD) in the FB portion of the window bynumerically solving F\u2032f is the monotonicfunctionkBT)\u22121, where kB is Boltzmann\u2019s constantand T = 300 K isthe temperature. The FE profile was obtained by integrating F\u2032 using the trapezoid rule. Statistical uncertaintyin F\u2032 was estimated using the block averagemethod for correlated time series.39Umbrella sampling simulationsused the harmonic flat-bottom restraintswith a flat-bottom width of 0.5 \u00c5 and a force constant of 25kcal/mol/\u00c531 to maintain the solventdensity in the outer solvation shell at \u22431g/mL by dynamicallyadjusting the solvent layer thickness.The first 4 nanoseconds of each simulation were performed withdensity controlO end pointscentered on d = 49 and 69\u00c5, respectively, which gives a window spacing of \u22430.3175\u00c5. Simulations corresponding to different windows were run inparallel, but asynchronously, on multiple computing nodes inside NERSCPerlmutter supercomputer, with each simulation using one NVIDIA A100GPU and 16 AMD EPYC 7763 CPU cores, running at a speed of 90\u2013115ns/day, with the exact speed depending on the particular simulationwindow and overall system load. Using asynchronous, rather than synchronousparallel execution, as would be required with more sophisticated samplingstrategies, e.g., replica exchange,26 allowedfor up to an order-of-magnitude reduction in queue wait times.Sixty-four equispacedUS windows were used, with the C and 39 Simulations were stoppedafter 3583.4 ns (per window), at which point the block averages goingfrom 3 to 2 blocks showed evidence of a nascent plateau, indicatinga correlation length for FE derivatives of \u223c1.5 \u03bcs.Simulations were run in 20\u201325 ns increments while monitoringthe FE profile for convergence using the blocking method for correlatedtime series.40 we also computed the diffusion D coefficient in each RC window. Briefly, the simulationtimes at which the RC value crosses the boundaries of the flat-bottom(FB) regions are recorded, and the portions of the trajectories thatare outside of the FB region are discarded and the simulation clockis stopped until the trajectory re-enters the FB region. In accordancewith the diffusion model and the assumption of constant F\u2032 over the FB region, the diffusion constant is related tothe average roundtrip time between the FB boundaries38F\u2032/(kBT), and F\u2032 is the FE derivative in the simulationwindow. The above relation is accurate provided that the FE profileis well approximated by a line over the FB window. To investigatethe effect of \u0394, we performed 20\u201340 ns of MD simulationsin each window with \u0394 = {0.5, 1, 1.5, 2}.To characterize the motion of the RC using the Smoluchowski diffusionmodel,Supporting Information.Principal componentanalysis (PCA) was performed essentially asin ref  but usinkBT). Although thestatistical error in the profile remains rather high, reaching \u223c1.16kcal/mol at the O end point, it is sufficiently small that the O stateis predicted to be lower in the FE than the C state.This value is consistent with the result of Pang et al.,25 who show a profile with \u0394FEC\u2192O \u2243 \u22121.25 \u00b1 0.2 kcal/mol. While experiments onthe WT spike show a slightly favored C state , the K987P substitutionin the proline mutant simulated here cannot form transient salt bridgeswith D427 and D428, both of which would stabilize the C state, asnoted before.25 Furthermore, the FRET dataof Lu et al.8 actually showed a higherenergy state near the C state , for which the \u0394FEC\u2192O is \u2243 \u22120.27 kcal/mol. Thus, our computed\u0394FEC\u2192O, though not very precise, appears tobe consistent with the existing data. However, the fact that we usedshort glycans may limit its biological significance.The freeenergy profile, block average analysis, and the runningerror estimate of the FE change of the C\u2194O transition are shownin 8 do not show significant transitionbarriers .Qualitatively similar behavior was observed in simulations of theA522L/V mutants by Fallon et al.,19 whereasthe more recent work of Pang et al.25 showedan \u223c9 kcal/mol barrier, approximately 33% into the transition. Although we also observed a small barrier of \u22431 kcal/mol,relative to the C state, in the first few hundrednanoseconds of simulation , the barrier progressively disappeared as the duration of the simulationwas increased.It is noteworthythat the present FE profile and the FRET histogramsof Lu et al.43 or away from44 prescribed states usingapplied forces or selection. Our previous work on a much simpler proteinsuggests that the two types of methods can produce significantly differentpaths, with respect to the order of conformational events, as wellas the energetics.45 Sampling differencescould also arise from the use of different RCs. While we use a singledistance, Pang et al.25 use an additionalangle to describe the orientation of the RBD relative to the spike.However, their 2D FE landscapes suggest that integrating over theangle dimension would not substantially reduce the observed barrier.Fallon et al.19 also compute a 2D landscape,using a linear and a dihedral angle as coordinates; however, the barriersobserved in their landscapes vary in the range 2\u20135 kcal/mol,depending on the mutation studied. Unfortunately, investigating theabove possibilities in the current context would require systematicallyrepeating simulations starting from different initial conditions,possibly with different restraints, which would be computationallyprohibitive, as the present FE profile alone required \u223c230\u03bcs of simulation on a supercomputer.Speculations about the reasons for the discrepanciesin the FEbarriers are difficult. One possible explanation is that differentregions of conformational space are sampled by different methods.The differences begin with the choice of initial configurations forumbrella sampling. While the end point states are specified by PDBstructures , intermediate structures arenot known and thus must be created entirely by computation. Here,and in ref , thesta40 to compute a position-dependent diffusion coefficient D, in addition to the FE profile.The FE profile byitself is related only to the probabilities ofobserving a particular extent of RBD \u201copenness\u201d, asmeasured by the distance defined above. To obtain a kinetic description,we used the formalism of 1D diffusion in the overdamped regimeD is variable across the RC, with thevariability generally increasing with the size of flat-bottom window\u0394. The source of the variability is mainly statistical uncertainty,which increases with \u0394 because longer times are needed to crosslarger FB widths (the Taylor expansion of Trt \u2243 (\u03942 + O(\u03944))/D). Consequently, fewer crossing eventsare observed for larger \u0394 values in a fixed simulation time.For this reason, the simulations with \u0394 = 2 \u00c5 were twiceas long as the others (40 ns per window). Further, there were windowsfor which we did not observe any crossing events; for those windows,we used linear interpolation to approximate the D from neighboring windows.The results of thediffusion calculations are summarized in D along theRC, the average diffusion constant \u27e8D\u27e9across all windows was similar for all values of \u0394, varyingin the range 6 \u00d7 108 to 7.5 \u00d7 108 \u00c52/s  \u2243 10.8 \u00d7 109 \u00c52/s, where \u03b7 is the dynamic viscosity for water at 20\u00b0C (\u22431 mPa\u00b7s), and we took r tobe the radius of gyration of the RBD, computed from PDB 6M0J from the end point states to any other stateby evaluating the following equations40 using linear interpolation and the trapezoid rule integrationd(C) and d(O) are thedistances that correspond to the C and O states, respectively, and d* is any intermediate distance. The results of the integrationare shown in With the FE and D does not lead to significant differences between the MFPT profiles.This result is to be expected from D appearsthrough its reciprocal, whereas the free energy F enters exponentially. Consequently, variations in D have a smaller effect than those in F. This observationalso suggests that there is no compelling reason to improve the precisionof the computed D profile using longer simulationsbefore improving the precision of F.First, from O\u2194C transition, corresponding to2.2 \u00b1 0.6 \u03bcs or the rate of 0.45 \u00b10.12 \u03bcs\u20131 = 450 \u00d7 103 s\u20131. The computed rate is more than 5 orders of magnitudelarger than the experimental rate for the slowest substep in the transition,8 which was estimated at 1.56 \u00b1 0.01 or 1.99\u00b1 0.02 s\u20131, depending on the type of nanoparticleused. A possible explanation for the large discrepancy is that D is significantly overestimated in the calculations becauseof the very short glycans used. Longer glycans could provide frictionalresistance to RBD motion through many transient interactions witheach other and with protein residues. Computing the diffusion constantfor the RBD in a fully glycosylated spike using the present methodwould be prohibitively expensive. For example, the 40 ns long simulationsused here to compute D for the deglycosylated spikewould need to be orders of magnitude longer and would probably requiresubstantially more solvent to maintain hydration of the glycans, regardlessof their length or conformation. Such simulations might need to beperformed on special computing hardware like the Anton2 computer.47Second,we note that the longest MFPT computed here is for the 37 For example, the friction value of 1 ps\u20131 leads to an overestimate by a factor of \u223c2. For this reason, as mentioned inthe \u20131, which improvesthe calculated self-diffusion coefficient to \u223c3 \u00d7 1011 \u00c52/s.37 Thus,we expected that the D computed for the RC motionwould be sufficiently accurate that it could not explain the discrepanciesin the MFPTs. To check this possibility further, we recomputed D along the RC using the Langevin friction value of 1 ps\u20131 . We obtainedthe average D value of 1.09 \u00d7 109 \u00c52/s, which is less than twice those obtained abovewith the Langevin friction of 5 ps\u20131 and indicatesthat the accelerated diffusion in the water model could not accountfor the MFPT underestimate.We note that the standard TIP3 water modelis known to overestimatethe rates of self-diffusion at the typical values used for Langevinfriction in MD simulations with a Langevin thermostat.25 computed the MFPTs forthe C\u2194O transition in the deglycosylated spike equal to 143and 497 \u03bcs in the forward and reverse directions, respectively.Using the FE profile from their publication and D value consistent with their results to be D \u22431.5 \u00d7 108 \u00c52/s, which is roughly consistentwith our values; it is similar to the smallest value of D \u2243 1.4 \u00d7 108 \u00c52/s computedby us in the \u0394 = 2 \u00c5 case , which results in a modestincrease of the O\u2194C MFPT to 5.6 \u00b1 1.2 \u03bcs.Pang et al.case see 4B. Thus,C\u2194O FE profilesare the longest reported for this transition. Specifically, each ofthe 64 windows was simulated for 3.583 \u03bcs, for a total of 229\u03bcs. By comparison, Fallon et al.19 performed 16 ns of simulation in each of 328 or fewer windows (dependingon the mutant) inside a 2D reaction coordinate landscape for a totalof 5.25 \u03bcs. Pang et al.25 performeda total of 91 \u03bcs of simulation over 1211 windows in a 2D reactioncoordinate landscape. We note, however, that they used Hamiltonianreplica exchange26 to accelerate samplingby swapping RC values between adjacent windows, which could significantlyimprove conformational sampling. In spite of our long simulation times,we were surprised that the statistical error in the \u0394F estimate remained rather high at 1.16 kcal/mol  of the mass-weightedspike coordinates41 in each simulationwindow. To accelerate the matrix diagonalization required to computethe PCs, each residue was coarse-grained to a single particle locatedat the residue COM with a mass equal to the residue mass. Time scalesof conformational motions can be visualized from the autocorrelationfunctions (ACFs) of displacements along PCs, as shown in To the best of our knowledge, the simulations performed here tocompute the /mol see 3. Furthe28 crossesthe vertical axis around 700\u2013800 ns but does not decay to zero,instead showing anticorrelation. Higher PCs (#3\u2013#9) show morerapidly decaying ACFs, with most of them fluctuating around zero inthe neighborhood of 1 \u03bcs. Qualitatively, the results appearto be insensitive to the choice of domain considered. For example,the ACFs computed along PCs using a single RBD only  for thetwo solvation treatments and statistically the same value for thePMF of separation.To check that the long decay times inthe ACFs were not introducedby the shell solvation model used here, we reanalyzed microsecond-longtrajectories of the smaller proteins dihydrofolate reductase (DHFR)and myosin 6 (M6) studied before.F in Figure S5A,B) suggests that this featuremay be common in conformational free energy simulations of proteins.A noteworthy feature of the very slow convergencebehavior of \u03948 and some of the calculations of Fallon et al.19 In contrast, Pang et al.25 foundbarriers of \u223c5 and \u223c8.5 kcal/mol, respectively, forthe wild-type spike and the double proline mutant (the latter wassimulated here). Despite the long total simulation times, which, toour knowledge, are the longest yet reported for this transition, theFE profile shows rather poor convergence, with statistical uncertaintyfor the FE difference equal to \u22431.16 kcal/mol. The large uncertaintyis attributed to long trajectory decorrelation times, which suggeststhat strong memory effects are present in the conformational dynamicsof the system. Consistent with this interpretation are the slowlydecaying autocorrelation functions of displacements along the principalcomponents (PCs) of the motions, which take hundreds of nanosecondsor longer to cross the horizontal axis and often remain negative forhundreds of nanoseconds longer. Modeling the transition as an overdampeddiffusion on the FE profile, we computed the average diffusion constantassociated with the motion along the RC to be around 6 \u00b1 2 \u03bcm2/s, which is about four times higher than the value inferredfrom the simulation data of Pang et al.25 However, combined with the FE profile, this value yields predictionsfor the transition rate of about 450 \u00d7 103 s\u20131, which is over 5 orders of magnitude faster than the experimentallymeasured result of \u22432 s\u20131 of its viral spike protein transitions to an \u201copen\u201d(or \u201cup\u201d) conformation, which is able to bind the angiotensin-convertingenzyme 2 receptor, initiating viral cell entry. We have describedthe results of very long molecular dynamics simulations that use umbrellasampling (US) to compute the free energy (FE) profile and rates oftransition between the two conformations along a 1D reaction coordinate(RC), chosen to be the distance between one of the RBDs and part ofthe stem of another spike protomer. The MD calculations were performedfor \u22433.58 \u03bcs per US window, totaling 229 \u03bcs over64 windows spanning the RC. The FE computed profile is relativelyflat, with energy barriers of the order of 1 kcal/mol, which is broadlyconsistent with the experiment of Lu et al.48 For example,for the dimeric 416-residue protein phosphoglycerate kinase (PGK),ACFs of selected inter-residue distances decreased below 0.01 onlyafter several microseconds; the authors interpreted the protein dynamicsto be subdiffusive on a fractal energy landscape, which did not appearto be converged even after microseconds of MD simulations. Whetherthe simulations performed here could yield a converged and precisefree energy profile using currently available computer resources remainsunclear. However, at least an order-of-magnitude extension of thesimulations presented here would almost certainly be required. Inaddition, the use of enhanced sampling methods for molecular simulation,especially those based on Hamiltonian replica exchange (HREX), couldaccelerate the decay in the ACFs, facilitating more rapid convergence.However, the exact degree of acceleration that would be achieved withHREX is not clear, as it is dependent on the simulation system, aswell as on the simulation parameters, e.g., the number of replicassimulated concurrently, or the frequency of exchanges.Slow decay of ACFs in protein simulations is notunexpected andhas been observed before in MD simulations of smaller proteins, rangingin size from 21 to 101 kDa."
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