Question: Individuals with the alcohol aldehyde dehydrogenase deficiency mutation consume less alcohol than those without the mutation.

Alcohol Consumption and Cardiovascular Health: An Exploration through Mendelian Randomization

INTRODUCTION Alcohol has long been recognized as a modifiable risk factor for hypertension, a condition that significantly impacts cardiovascular health. Yet, traditional observational studies can be limited by confounding variables related to lifestyle and socioeconomic factors, while clinical trials often suffer from short follow-up periods and practical implementation challenges. Mendelian randomization, leveraging genetic variants as natural experiments, offers a robust approach to elucidate the relationship between alcohol consumption and hypertension. Specifically, the focus is on a genetic variant in the aldehyde dehydrogenase 2 (ALDH2) enzyme, which plays a crucial role in alcohol metabolism. Individuals with the null variant (ALDH2 *2*2) exhibit adverse reactions to alcohol, leading to reduced consumption compared to those with the wild-type allele (ALDH2 *1*1) or heterozygotes.

METHODS AND RESULTS We conducted a systematic review and meta-analysis of studies examining the ALDH2 genotype in relation to blood pressure and hypertension risk. The analysis included five studies with a total of 7,658 participants for blood pressure measurements and three studies with 4,219 participants for hypertension assessment. Our findings revealed that, in men, the odds ratio for hypertension was 2.42 (95% CI 1.66-3.55, p = 4.8 x 10(-6)) when comparing ALDH2 *1*1 homozygotes with *2*2 homozygotes. Similarly, heterozygotes, who can be considered moderate drinkers, had an odds ratio of 1.72 (95% CI 1.17-2.52, p = 0.006) for hypertension compared to *2*2 homozygotes. In terms of blood pressure, systolic pressure was observed to be 7.44 mmHg (95% CI 5.39-9.49, p = 1.1 x 10(-12)) higher in *1*1 homozygotes and 4.24 mmHg (95% CI 2.18-6.31, p = 0.00005) higher in heterozygotes when contrasted with *2*2 homozygotes.

CONCLUSIONS Our results provide strong evidence supporting the notion that alcohol consumption has a

BACKGROUND Results of the few cohort studies from countries with low incomes or middle incomes suggest a lower incidence of dementia than in high-income countries. We assessed incidence of dementia according to criteria from the 10/66 Dementia Research Group and Diagnostic and Statistical Manual of Mental Disorders (DSM) IV, the effect of dementia at baseline on mortality, and the independent effects of age, sex, socioeconomic position, and indicators of cognitive reserve. METHODS We did a population-based cohort study of all people aged 65 years and older living in urban sites in Cuba, the Dominican Republic, and Venezuela, and rural and urban sites in Peru, Mexico, and China, with ascertainment of incident 10/66 and DSM-IV dementia 3-5 years after cohort inception. We used questionnaires to obtain information about age in years, sex, educational level, literacy, occupational attainment, and number of household assets. We obtained information about mortality from all sites. For participants who had died, we interviewed a friend or relative to ascertain the likelihood that they had dementia before death. FINDINGS 12,887 participants were interviewed at baseline. 11,718 were free of dementia, of whom 8137 (69%) were reinterviewed, contributing 34,718 person-years of follow-up. Incidence for 10/66 dementia varied between 18·2 and 30·4 per 1000 person-years, and were 1·4-2·7 times higher than were those for DSM-IV dementia (9·9-15·7 per 1000 person-years). Mortality hazards were 1·56-5·69 times higher in individuals with dementia at baseline than in those who were dementia-free. Informant reports suggested a high incidence of dementia before death; overall incidence might be 4-19% higher if these data were included. 10/66 dementia incidence was independently associated with increased age (HR 1·67; 95% CI 1·56-1·79), female sex (0·72; 0·61-0·84), and low education (0·89; 0·81-0·97), but not with occupational attainment (1·04; 0·95-1·13). INTERPRETATION Our results provide supportive evidence for the cognitive reserve hypothesis, showing that in middle-income countries as in high-income countries, education, literacy, verbal fluency, and motor sequencing confer substantial protection against the onset of dementia. FUNDING Wellcome Trust Health Consequences of Population Change Programme, WHO, US Alzheimer's Association, FONACIT/ CDCH/ UCV.

The weight lowering potential of glucagon-like peptide 1 (GLP-1) receptor agonists (RAs) is inter-individually different and clinically unpredictable. The potential role of genetic variability of GLP-1R on body weight response to GLP-1 RAs in obese women with polycystic ovary syndrome (PCOS) has not yet been evaluated. Fifty-seven obese women with PCOS (aged 30.7 ± 7.0, BMI 38.6 ± 5.3 kg/m2) were assigned to liraglutide 1.2 mg QD s.c. for 12 weeks and classified as strong responders regarding weight loss if they lost 5 % or more of their initial body weight. They were genotyped for common GLP-1R single nucleotide polymorphisms (SNPs) rs6923761 and rs10305420. Changes of measures of obesity were measured before and at the end of the treatment. Twenty out of 57 subjects were strong responders and lost 7.38 ± 1.74 compared to 2.11 ± 2.17 kg lost in poor responders. Carriers of at least one polymorphic rs10305420 allele had poor treatment response compared to carriers of two wild type alleles (OR = 0.27, 95 % CI = 0.09–0.85, P = 0.025). Carriers of at least one polymorphic rs6923761 allele tended to have stronger treatment response compared to carriers of two wild type alleles (OR = 3.06, 95 % CI = 0.96–9.74, P = 0.058). Fasting glucose and glucose after oral glucose tolerance test (OGTT) comparably decreased in both groups when compared to baseline, whereas no within treatment differences were found in androgen profile. Gastrointestinal adverse events were transit and balanced between strong and poor responders. GLP-1R rs10305420 polymorphism explained some of the inter-individual differences in response to liraglutide regarding weight loss in obese PCOS women.

Calorie restriction extends life-span in a wide variety of organisms. Although it has been suggested that calorie restriction may work by reducing the levels of reactive oxygen species produced during respiration, the mechanism by which this regimen slows aging is uncertain. Here, we mimicked calorie restriction in yeast by physiological or genetic means and showed a substantial extension in life-span. This extension was not observed in strains mutant for SIR2 (which encodes the silencing protein Sir2p) or NPT1 (a gene in a pathway in the synthesis of NAD, the oxidized form of nicotinamide adenine dinucleotide). These findings suggest that the increased longevity induced by calorie restriction requires the activation of Sir2p by NAD.

We performed genome-wide analyses to identify genomic loci that interact with sodium to influence blood pressure (BP) using single-marker-based (1 and 2 df joint tests) and gene-based tests among 1876 Chinese participants of the Genetic Epidemiology Network of Salt-Sensitivity (GenSalt) study. Among GenSalt participants, the average of 3 urine samples was used to estimate sodium excretion. Nine BP measurements were taken using a random zero sphygmomanometer. A total of 2.05 million single-nucleotide polymorphisms were imputed using Affymetrix 6.0 genotype data and the Chinese Han of Beijing and Japanese of Tokyo HapMap reference panel. Promising findings (P<1.00×10(-4)) from GenSalt were evaluated for replication among 775 Chinese participants of the Multi-Ethnic Study of Atherosclerosis (MESA). Single-nucleotide polymorphism and gene-based results were meta-analyzed across the GenSalt and MESA studies to determine genome-wide significance. The 1 df tests identified interactions for UST rs13211840 on diastolic BP (P=3.13×10(-9)). The 2 df tests additionally identified associations for CLGN rs2567241 (P=3.90×10(-12)) and LOC105369882 rs11104632 (P=4.51×10(-8)) with systolic BP. The CLGN variant rs2567241 was also associated with diastolic BP (P=3.11×10(-22)) and mean arterial pressure (P=2.86×10(-15)). Genome-wide gene-based analysis identified MKNK1 (P=6.70×10(-7)), C2orf80 (P<1.00×10(-12)), EPHA6 (P=2.88×10(-7)), SCOC-AS1 (P=4.35×10(-14)), SCOC (P=6.46×10(-11)), CLGN (P=3.68×10(-13)), MGAT4D (P=4.73×10(-11)), ARHGAP42 (P≤1.00×10(-12)), CASP4 (P=1.31×10(-8)), and LINC01478 (P=6.75×10(-10)) that were associated with at least 1 BP phenotype. In summary, we identified 8 novel and 1 previously reported BP loci through the examination of single-nucleotide polymorphism and gene-based interactions with sodium.

Objective:A number of case reports describe multiple family members with gastroesophageal reflux disease and Barrett's esophagus. The wider importance of familial factors in gastroesophageal reflux disease has not been established. Therefore, we have studied the prevalence of reflux symptoms and medication use among relatives of patients with documented gastroesophageal reflux disease. Methods:A postal questionnaire study of the first degree relatives of six groups of matched patients. The groups comprised patients with 1) no dyspeptic symptoms; 2) reflux symptoms and a normal pH study; 3) reflux symptoms, an abnormal pH study, and a lower esophageal sphincter (LOS) pressure more than 10 mm Hg; 4) reflux symptoms, an abnormal pH study, and a LOS pressure less than 10 mm Hg; 5) Barrett's esophagus; and 6) peptic stricture. Results:Four hundred eighteen subjects replied (78% response). Infrequent reflux symptoms were equally common in all groups of relatives. Frequent reflux symptoms, however, were more common among relatives of patients with an abnormal pH study and normal (26%, p= 0.007) or low LOS pressure (27%, p= 0.01) or Barrett's esophagus (30%, p= 0.003), compared with relatives of nondyspeptic patients (9%). Frequent reflux symptoms were no more common among relatives of patients with a normal pH study (16%) or peptic stricture (18%). Reflux medication use showed a similar pattern. Conclusions:Familial clustering of reflux symptoms is seen in relatives of patients with reflux symptoms and increased esophageal acid exposure and in relatives of patients with Barrett's esophagus.

OBJECTIVES We estimated the relationship between soft drink consumption and obesity and diabetes worldwide. METHODS We used multivariate linear regression to estimate the association between soft drink consumption and overweight, obesity, and diabetes prevalence in 75 countries, controlling for other foods (cereals, meats, fruits and vegetables, oils, and total calories), income, urbanization, and aging. Data were obtained from the Euromonitor Global Market Information Database, the World Health Organization, and the International Diabetes Federation. Bottled water consumption, which increased with per-capita income in parallel to soft drink consumption, served as a natural control group. RESULTS Soft drink consumption increased globally from 9.5 gallons per person per year in 1997 to 11.4 gallons in 2010. A 1% rise in soft drink consumption was associated with an additional 4.8 overweight adults per 100 (adjusted B; 95% confidence interval [CI] = 3.1, 6.5), 2.3 obese adults per 100 (95% CI = 1.1, 3.5), and 0.3 adults with diabetes per 100 (95% CI = 0.1, 0.8). These findings remained robust in low- and middle-income countries. CONCLUSIONS Soft drink consumption is significantly linked to overweight, obesity, and diabetes worldwide, including in low- and middle-income countries.

CONTEXT Sugar-sweetened beverages like soft drinks and fruit punches contain large amounts of readily absorbable sugars and may contribute to weight gain and an increased risk of type 2 diabetes, but these relationships have been minimally addressed in adults. OBJECTIVE To examine the association between consumption of sugar-sweetened beverages and weight change and risk of type 2 diabetes in women. DESIGN, SETTING, AND PARTICIPANTS Prospective cohort analyses conducted from 1991 to 1999 among women in the Nurses' Health Study II. The diabetes analysis included 91,249 women free of diabetes and other major chronic diseases at baseline in 1991. The weight change analysis included 51,603 women for whom complete dietary information and body weight were ascertained in 1991, 1995, and 1999. We identified 741 incident cases of confirmed type 2 diabetes during 716,300 person-years of follow-up. MAIN OUTCOME MEASURES Weight gain and incidence of type 2 diabetes. RESULTS Those with stable consumption patterns had no difference in weight gain, but weight gain over a 4-year period was highest among women who increased their sugar-sweetened soft drink consumption from 1 or fewer drinks per week to 1 or more drinks per day (multivariate-adjusted means, 4.69 kg for 1991 to 1995 and 4.20 kg for 1995 to 1999) and was smallest among women who decreased their intake (1.34 and 0.15 kg for the 2 periods, respectively) after adjusting for lifestyle and dietary confounders. Increased consumption of fruit punch was also associated with greater weight gain compared with decreased consumption. After adjustment for potential confounders, women consuming 1 or more sugar-sweetened soft drinks per day had a relative risk [RR] of type 2 diabetes of 1.83 (95% confidence interval [CI], 1.42-2.36; P<.001 for trend) compared with those who consumed less than 1 of these beverages per month. Similarly, consumption of fruit punch was associated with increased diabetes risk (RR for > or =1 drink per day compared with <1 drink per month, 2.00; 95% CI, 1.33-3.03; P =.001). CONCLUSION Higher consumption of sugar-sweetened beverages is associated with a greater magnitude of weight gain and an increased risk for development of type 2 diabetes in women, possibly by providing excessive calories and large amounts of rapidly absorbable sugars.

Serotonin signaling suppresses generation of amyloid-β (Aβ) in vitro and in animal models of Alzheimer’s disease (AD). We show that in an aged transgenic AD mouse model (APP/PS1 plaque-bearing mice), the antidepressant citalopram, a selective serotonin reuptake inhibitor, decreased Aβ in brain interstitial fluid in a dose-dependent manner. Growth of individual amyloid plaques was assessed in plaque-bearing mice that were chronically administered citalopram. Citalopram arrested the growth of preexisting plaques and reduced the appearance of new plaques by 78%. In healthy human volunteers, citalopram’s effects on Aβ production and Aβ concentrations in cerebrospinal fluid (CSF) were measured prospectively using stable isotope labeling kinetics, with CSF sampling during acute dosing of citalopram. Aβ production in CSF was slowed by 37% in the citalopram group compared to placebo. This change was associated with a 38% decrease in total CSF Aβ concentrations in the drug-treated group. The ability to safely decrease Aβ concentrations is potentially important as a preventive strategy for AD. This study demonstrates key target engagement for future AD prevention trials.

BACKGROUND The prevalence of Alzheimer disease (AD) is increasing in the elderly, and vascular risk factors may increase its risk. OBJECTIVE To explore the association of the aggregation of vascular risk factors with AD. METHODS The authors followed 1,138 individuals without dementia at baseline (mean age 76.2) for a mean of 5.5 years. The presence of vascular risk factors was related to incident possible and probable AD. RESULTS Four risk factors (diabetes, hypertension, heart disease, and current smoking) were associated with a higher risk of AD (p < 0.10) when analyzed individually. The risk of AD increased with the number of risk factors (diabetes + hypertension + heart disease + current smoking). The adjusted hazards ratio of probable AD for the presence of three or more risk factors was 3.4 (95% CI: 1.8, 6.3; p for trend < 0.0001) compared with no risk factors. Diabetes and current smoking were the strongest risk factors in isolation or in clusters, but hypertension and heart disease were also related to a higher risk of AD when clustered with diabetes, smoking, or each other. CONCLUSIONS The risk of Alzheimer disease (AD) increased with the number of vascular risk factors. Diabetes and current smoking were the strongest risk factors, but clusters including hypertension and heart disease also increased the risk of AD. These associations are unlikely to be explained by misclassification of the outcome, given strong associations when only probable AD is considered.

Background: The relationship of LPA single nucleotide polymorphisms (SNPs), apolipoprotein(a) isoforms, and lipoprotein(a) [Lp(a)] levels with major adverse cardiovascular events (MACE) in different ethnic groups is not well known. Methods: LPA SNPs, apolipoprotein(a) isoforms, Lp(a), and oxidized phospholipids on apolipoprotein B-100 (OxPL-apoB) levels were measured in 1792 black, 1030 white, and 597 Hispanic subjects enrolled in the Dallas Heart Study. Their interdependent relationships and prospective association with MACE after median 9.5-year follow-up were determined. Results: LPA SNP rs3798220 was most prevalent in Hispanics (42.38%), rs10455872 in whites (14.27%), and rs9457951 in blacks (32.92%). The correlation of each of these SNPs with the major apolipoprotein(a) isoform size was highly variable and in different directions among ethnic groups. In the entire cohort, Cox regression analysis with multivariable adjustment revealed that quartiles 4 of Lp(a) and OxPL-apoB were associated with hazard ratios (95% confidence interval) for time to MACE of 2.35 (1.50–3.69, P<0.001) and 1.89 (1.26–2.84, P=0.003), respectively, versus quartile 1. Addition of the major apolipoprotein(a) isoform and the 3 LPA SNPs to these models attenuated the risk, but significance was maintained for both Lp(a) and OxPL-apoB. Evaluating time to MACE in specific ethnic groups, Lp(a) was a positive predictor and the size of the major apolipoprotein(a) isoform was an inverse predictor in blacks, the size of the major apolipoprotein(a) isoform was an inverse predictor in whites, and OxPL-apoB was a positive predictor in Hispanics. Conclusions: The prevalence and association of LPA SNPs with size of apolipoprotein(a) isoforms, Lp(a), and OxPL-apoB levels are highly variable and ethnicity-specific. The relationship to MACE is best explained by elevated plasma Lp(a) or OxPL-apoB levels, despite significant ethnic differences in LPA genetic markers.


Question: Cataract and trachoma are the leading reasons for blindness in Southern Sudan.

Incidence and Etiology of Visual Impairment in Southern Sudan
Background: It is widely believed that blindness and low vision are prevalent in the southern region of Sudan, yet the exact extent and regional dispersion of these conditions remain largely undocumented. This study was undertaken to assess the prevalence of blindness and low vision, pinpoint the primary causes of these visual impairments, and establish benchmarks for blindness prevention initiatives in the Mankien payam (district) of southern Sudan.

The investigation reveals that cataracts and trachoma are indeed the leading causes of blindness in the area, with environmental factors and lack of access to healthcare playing significant roles. The data collected not only confirms the high prevalence of blindness and low vision but also underscores the urgent need for targeted interventions aimed at reducing these conditions. The findings provide a crucial foundation for developing effective blindness prevention programs tailored to the specific needs of the southern Sudanese population.

Prior studies have found only a modest relationship between objective and subjective crowding, defying logic and commonsensical notions of why people feel crowded. Using data from a representative sample of Bangkok, Thailand, where the level of household crowding is four times that in western societies, we explore several possibilities of why this is the case. Examining seven different indicators of objective crowding, our analyses suggest that the modest relationship is not an artifact of measurement. Contrary to the assumption of prior investigations, the findings indicate that the objective-subjective crowding relationship is nonlinear and that there is a ceiling effect muting the impact of increased objective crowding. The analyses further suggest that the strength of the relationship is mitigated somewhat, with part of the feeling of being crowded accounted for by household circumstances, such as the degree of control an individual has over the use of household space.

Detailed information on drowning in children is not routinely collected by offices of national statistics. Few studies have been carried out in the United Kingdom, and none has been done on British children abroad. In 1988-9, two of the authors (AMK and JRS) combined information from national statistical offices, police forces (Royal Life Saving Society), and from a press cutting service (Royal Society for Prevention of Accidents) for a detailed analysis of deaths by drowning in children.1, 2, 3 This analysis found that 149 children had drowned in the United Kingdom during 1998-9. It also identified a safety agenda, which focused on young children in garden ponds and pools and on older children swimming without supervision. Over the past 10 years there have been initiatives on children's safety in water, particularly swimming. We obtained similar information for 1998-9 to identify changes that have …

Metabolic syndrome is diagnosed when three or more of the following criteria are met: abdominal obesity (waist circumference more than 102 cm in men and 88 cm in women); hypertriglyceridemia of 150 mg/dl or above; a high-density lipoprotein (HDL) cholesterol level less than 40 mg/dl in men or 50 mg/dl in women; blood pressure of 130/85 mm Hg or higher; or fasting glucose of at least 110 mg/dl. Individuals with metabolic syndrome are likelier than others to develop diabetes and cardiovascular disease and have increased mortality from all causes (and from cardiovascular disease in particular). The investigators attempted to determine the prevalence of the syndrome in the United States by analyzing data on 8814 men and women 20 years of age or older who took part in the Third National Health and Nutrition Examination Survey in the years 1988 to 1994. This is a cross-sectional health survey of a sample of the noninstitutionalized civilian American population. The overall age-adjusted prevalence of metabolic syndrome was 23.7%. The prevalence rose from 6.7% in persons 20 to 29 years of age to 42% in those aged 70 years and more. There was virtually no gender-related difference in prevalence rates for the combined racial groups. Metabolic syndrome was most prevalent in Mexican Americans and least prevalent in whites, African Americans, and "others. " Among both African Americans and Mexican Americans, women had higher prevalence rates than men. Extrapolating from age-specific prevalence rates and US census counts from the year 2000, 47 million US residents have metabolic syndrome. Considering its prevalence, it seems important to estimate the direct medical costs of metabolic syndrome. In the great majority of cases the critical causes are improper nutrition and insufficient physical activity, emphasizing the importance of controlling obesity and encouraging physical activity in the United States.

Correction for:    Kurreeman FAS, Padyukov L, Marques RB, Schrodi SJ, Seddighzadeh M, et al. (2007) A Candidate Gene Approach Identifies the TRAF1/C5 Region as a Risk Factor for Rheumatoid Arthritis. PLoS Med 4(9): e278. doi:10.1371/journal.pmed.0040278    In Table 1, the allele ratio in column eight (Allele Ratiosb: Cases, Controls) refers to allele A: allele B and not allele1:allele2 as described in footnote b, with Allele A being the Susceptibility Allele as denoted in column seven. The footnote should read:    bNumber of alleles were compared in cases versus controls: allele A: allele B cases, allele A: allele B controls. Allele A refers to the susceptibility alleles as given in column seven.

OBJECTIVES This paper examines policy implementation gaps of user fees plus exemptions and health insurance in providing financial access to primary clinical care for children under five in Ghana. METHODS Methods included analysis of routine data, focus group discussions, in-depth interviews, and administration of a structured questionnaire. RESULTS Providers modified exemptions policy implementation arrangements, sometimes giving partial or no exemptions. Clients who knew or suspected exemption entitlements failed to request them because of fear of negative reactions from providers. Providers attributed their modification of implementation arrangements and negative reactions to the threat posed to the financial viability of their institutions by reimbursement uncertainty and delays. At the time of the study insurance coverage was low and frontline workers were not noticeably modifying implementation arrangements. However, the underlying goal conflicts, resource scarcity, conditions of work and relationships between frontline workers and clients that fueled the exemptions policy implementation gaps were unchanged. The potential for the health insurance policy to stumble over implementation gaps as happened with the exemptions policy therefore remained. CONCLUSIONS Policies that do not take into account the incentives for frontline worker adherence and align them better with policy objectives may experience implementation gaps.

Homeless youth suffer from high rates of health problems, yet little is known about their perceptions of or context for their own health issues. In this study, a combination of qualitative techniques from participatory rural appraisal and rapid assessment procedures was used to investigate the perceptions of health needs of shelter-based youth in Baltimore, MD in the U.S.A. The most common youth-identified health problems included STDs, HIV/AIDS, pregnancy, depression, drug use and injuries. These correlate well with more objective health status data for the same youth. The youth spoke of environmental safety threats of violence and victimization by adults, as well as racism and sexism in their lives. Youth reported that trusted adult figures such as grandmothers are important sources of health advice. Many homeless youth from less than ideal family situations remain in contact with and continue to seek advice from parents and other family members. Health interventions with urban street youth need to acknowledge the primacy of the social context for these youth, as well as the reality of violence as a daily health threat.

This paper describes the Estimation and Projection Package (EPP) for estimating and projecting HIV prevalence levels in countries with generalised epidemics. The paper gives an overall summary of the software and interface. It describes the process of defining and modelling a national epidemic in terms of locally relevant sub-epidemics and the four epidemiological parameters used to fit a curve to produce the prevalence trends in the epidemic. It also provides an example of using the EPP in a country with a generalised epidemic. The paper discusses the strengths and weaknesses of the software and its envisaged future developments.

OBJECTIVE To determine the incidence of surgically managed pelvic organ prolapse and urinary incontinence in a population-based cohort, and to describe their clinical characteristics. METHODS Our retrospective cohort study included all patients undergoing surgical treatment for prolapse and incontinence during 1995; all were members of Kaiser Permanente Northwest, which included 149,554 women age 20 or older. A standardized data-collection form was used to review all inpatient and outpatient charts of the 395 women identified. Variables examined included age, ethnicity, height, weight, vaginal parity, smoking history, medical history, and surgical history, including the preoperative evaluation, procedure performed, and details of all prior procedures. Analysis included calculation of age-specific and cumulative incidences and determination of the number of primary operations compared with repeat operations performed for prolapse or incontinence. RESULTS The age-specific incidence increased with advancing age. The lifetime risk of undergoing a single operation for prolapse or incontinence by age 80 was 11.1%. Most patients were older, postmenopausal, parous, and overweight. Nearly half were current or former smokers and one-fifth had chronic lung disease. Reoperation was common (29.2% of cases), and the time intervals between repeat procedures decreased with each successive repair. CONCLUSION Pelvic floor dysfunction is a major health issue for older women, as shown by the 11.1% lifetime risk of undergoing a single operation for pelvic organ prolapse and urinary incontinence, as well as the large proportion of reoperations. Our results warrant further epidemiologic research in order to determine the etiology, natural history, and long-term treatment outcomes of these conditions.

BACKGROUND Despite recent achievements to reduce child mortality, neonatal deaths continue to remain high, accounting for 41% of all deaths in children under five years of age worldwide, of which over 90% occur in low- and middle-income countries (LMICs). Infections are a leading cause of death and limitations in care seeking for ill neonates contribute to high mortality rates. As estimates for care-seeking behaviors in LMICs have not been studied, this review describes care seeking for neonatal illnesses in LMICs, with particular attention to type of care sought. METHODS AND FINDINGS We conducted a systematic literature review of studies that reported the proportion of caregivers that sought care for ill or suspected ill neonates in LMICs. The initial search yielded 784 studies, of which 22 studies described relevant data from community household surveys, facility-based surveys, and intervention trials. The majority of studies were from South Asia (n = 17/22), set in rural areas (n = 17/22), and published within the last 4 years (n = 18/22). Of the 9,098 neonates who were ill or suspected to be ill, 4,320 caregivers sought some type of care, including care from a health facility (n = 370) or provider (n = 1,813). Care seeking ranged between 10% and 100% among caregivers with a median of 59%. Care seeking from a health care provider yielded a similar range and median, while care seeking at a health care facility ranged between 1% and 100%, with a median of 20%. Care-seeking estimates were limited by the few studies conducted in urban settings and regions other than South Asia. There was a lack of consistency regarding illness, care-seeking, and care provider definitions. CONCLUSIONS There is a paucity of data regarding newborn care-seeking behaviors; in South Asia, care seeking is low for newborn illness, especially in terms of care sought from health care facilities and medically trained providers. There is a need for representative data to describe care-seeking patterns in different geographic regions and better understand mechanisms to enhance care seeking during this vulnerable time period.

Despite its key role in determining the stability and intensity of malaria transmission, the infectiousness of human populations to mosquitoes has rarely been estimated. Field-based analyses of malaria transmission have frequently relied on the prevalence of asexual parasites or gametocytes as proxies for infectiousness. We now summarize empirical data on human infectiousness from Africa and Papua New Guinea. Over a wide range of transmission intensities there is little relationship between the infectiousness of human populations to vector mosquitoes and mosquito-to-human transmission intensity. We compare these data with the predictions of a stochastic simulation model of Plasmodium falciparum epidemiology. This model predicted little variation in the infectiousness of the human population for entomologic inoculation rates (EIRs) greater than approximately 10 infectious bites per year, demonstrating that the lack of relationship between the EIR and the infectious reservoir can be explained without invoking any effects of acquired transmission-blocking immunity. The near absence of field data from areas with an EIR < 10 per year precluded validation of the model predictions for low EIR values. These results suggest that interventions reducing mosquito-to-human transmission will have little or no effect on human infectiousness at the levels of transmission found in most rural areas of sub-Saharan Africa. Unless very large reductions in transmission can be achieved, measures to prevent mosquito-to-human transmission need to be complemented with interventions that reduce the density or infectiousness of blood stage parasites.


Question: Cell autonomous sex determination within somatic cells doesn't happen in Galliformes.

Cell Autonomous Sex Determination in Avian Somatic Cells: Evidence from Chicken Gynandromorphs. The prevailing model of sex determination in vertebrates, exemplified by mammals, posits that embryos are initially sexually indifferent until a sex-determining gene triggers gonadal differentiation. While this model is presumed to be universal, it has not been conclusively validated across all vertebrates. To explore the sex-determining mechanism in birds, we investigated three naturally occurring chicken gynandromorphs (organisms displaying both male and female characteristics simultaneously on different parts of their body). Our findings confirmed that these birds are true male:female chimaeras, suggesting that avian somatic cells may inherently possess a sex identity. To further test this hypothesis, we conducted reciprocal sex transplantation experiments, moving presumptive mesodermal cells between embryos of different sexes. In contrast to mammalian chimaeras, where donor cells can contribute to the gonadal structures, chicken chimaeras exhibited exclusion of donor cells from the functional components of the host gonad. A notable case was observed when female tissue was transplanted into a male host, where the donor cells maintained their female identity and expressed markers indicative of female function within the developing testis. This research substantiates that avian somatic cells have an intrinsic sex identity, and thus, sexual differentiation in birds is significantly cell autonomous.

The objective of the present study was to determine if chicken melanoma-differentiation-associated gene 5 (MDA5) senses infectious bursal disease virus infection to induce innate immunity that bridges to adaptive immunity. During IBDV infection in HD11 cells, IBDV titers and RNA loads increased up to 3.4 × 107 plaque-forming units (PFU)/mL and 1114 ng/µL, respectively, at 24 hours postinfection (hpi). IBDV infection in HD11 cells induced significantly upregulated (p < 0.05) expression levels of chicken MDA5 (59-fold), interferon-β (IFN-β) (693-fold), dsRNA-dependent protein kinase (PKR) (4-fold), 2’, 5’-oligoadenylate synthetase (OAS) (286-fold), myxovirus resistance gene (Mx) (22-fold), interleukin-1β (IL-1β) (5-fold), IL-6 (146-fold), IL-8 (4-fold), IL-10 (4-fold), inducible nitric oxide synthase (iNOS) (15-fold), and major histocompatibility complex class I (MHC class I) (4-fold). Nitric oxide production in the culture supernatants increased significantly (p < 0.05) up to 6.5 μM at 24 hpi. The expressed chMDA5 and IBDV-derived dsRNA were localized in the cytoplasm of HD11 cells during IBDV infection. ChMDA5-knockdown HD11 cells had significantly higher (p < 0.05) IBDV RNA loads at 24 hpi and significantly lower (p < 0.05) nitric oxide production and expression levels of chicken MDA5, IFN-β, PKR, OAS, Mx, IL-1β, IL-6, IL-8, IL-12(p40), IL-18, IL-10, iNOS, MHC class I and CD86 at 24 hpi. In addition, chMDA5 overexpression in HD11 cells resulted in significantly reduced (p < 0.05) IBDV titers and RNA loads and significantly increased (p < 0.05) nitric oxide production at 16 and 24 hpi. It also resulted in significantly higher (p < 0.05) expression levels of chicken MDA5, IFN-β, PKR, OAS, Mx, IL-1β, IL-6, IL-8, IL-12(p40), IL-10 and iNOS at 2 hpi. In conclusion, the results indicate that chMDA5 senses IBDV infection in chicken macrophages, and this is associated with IBDV-induced expression of IFN-β and initiation of an innate immune response that in turn activates the adaptive immune response and limits IBDV replication.

The earliest aspects of human embryogenesis remain mysterious. To model patterning events in the human embryo, we used colonies of human embryonic stem cells (hESCs) grown on micropatterned substrate and differentiated with BMP4. These gastruloids recapitulate the embryonic arrangement of the mammalian germ layers and provide an assay to assess the structural and signaling mechanisms patterning the human gastrula. Structurally, high-density hESCs localize their receptors to transforming growth factor β at their lateral side in the center of the colony while maintaining apical localization of receptors at the edge. This relocalization insulates cells at the center from apically applied ligands while maintaining response to basally presented ones. In addition, BMP4 directly induces the expression of its own inhibitor, NOGGIN, generating a reaction-diffusion mechanism that underlies patterning. We develop a quantitative model that integrates edge sensing and inhibitors to predict human fate positioning in gastruloids and, potentially, the human embryo.

Regulatory T (Treg) cells, whose differentiation and function are controlled by X chromosome-encoded transcription factor Foxp3, are generated in the thymus (tTreg) and extrathymically (peripheral, pTreg), and their deficiency results in fatal autoimmunity. Here, we demonstrate that a Foxp3 enhancer, conserved noncoding sequence 1 (CNS1), essential for pTreg but dispensable for tTreg cell generation, is present only in placental mammals. CNS1 is largely composed of mammalian-wide interspersed repeats (MIR) that have undergone retrotransposition during early mammalian radiation. During pregnancy, pTreg cells specific to a model paternal alloantigen were generated in a CNS1-dependent manner and accumulated in the placenta. Furthermore, when mated with allogeneic, but not syngeneic, males, CNS1-deficient females showed increased fetal resorption accompanied by increased immune cell infiltration and defective remodeling of spiral arteries. Our results suggest that, during evolution, a CNS1-dependent mechanism of extrathymic differentiation of Treg cells emerged in placental animals to enforce maternal-fetal tolerance.

Females live longer than males in many species, including humans. We have traced a possible explanation for this phenomenon to the beneficial action of estrogens, which bind to estrogen receptors and increase the expression of longevity-associated genes, including those encoding the antioxidant enzymes superoxide dismutase and glutathione peroxidase. As a result, mitochondria from females produce fewer reactive oxygen species than those from males. Administering estrogens has serious drawbacks, however--they are feminizing (and thus cannot be administered to males) and may increase the incidence of serious diseases such as uterine cancer in postmenopausal women. Phytoestrogens, which are present in soy or wine, may have some of the favorable effects of estrogens without their undesirable effects. Study of gender differences in longevity may help us to understand the basic processes of aging and to devise practical strategies to increase the longevity of both females and males.

The ontogeny of haematopoietic stem cells (HSCs) during embryonic development is still highly debated, especially their possible lineage relationship to vascular endothelial cells. The first anatomical site from which cells with long-term HSC potential have been isolated is the aorta-gonad-mesonephros (AGM), more specifically the vicinity of the dorsal aortic floor. But although some authors have presented evidence that HSCs may arise directly from the aortic floor into the dorsal aortic lumen, others support the notion that HSCs first emerge within the underlying mesenchyme. Here we show by non-invasive, high-resolution imaging of live zebrafish embryos, that HSCs emerge directly from the aortic floor, through a stereotyped process that does not involve cell division but a strong bending then egress of single endothelial cells from the aortic ventral wall into the sub-aortic space, and their concomitant transformation into haematopoietic cells. The process is polarized not only in the dorso-ventral but also in the rostro-caudal versus medio-lateral direction, and depends on Runx1 expression: in Runx1-deficient embryos, the exit events are initially similar, but much rarer, and abort into violent death of the exiting cell. These results demonstrate that the aortic floor is haemogenic and that HSCs emerge from it into the sub-aortic space, not by asymmetric cell division but through a new type of cell behaviour, which we call an endothelial haematopoietic transition.

Mechanical forces play important roles during tissue organization in developing animals. Many tissues are organized into adjacent, nonmixing groups of cells termed compartments. Boundaries between compartments display a straight morphology and are associated with signaling centers that are important for tissue growth and patterning. Local increases in mechanical tension at cell junctions along compartment boundaries have recently been shown to prevent cell mixing and to maintain straight boundaries. The cellular mechanisms by which local increases in mechanical tension prevent cell mixing at compartment boundaries, however, remain poorly understood. Here, we have used live imaging and quantitative image analysis to determine cellular dynamics at and near the anteroposterior compartment boundaries of the Drosophila pupal abdominal epidermis. We show that cell mixing within compartments involves multiple cell intercalations. Frequency and orientation of cell intercalations are unchanged along the compartment boundaries; rather, an asymmetry in the shrinkage of junctions during intercalation is biased, resulting in cell rearrangements that suppress cell mixing. Simulations of tissue growth show that local increases in mechanical tension can account for this bias in junctional shrinkage. We conclude that local increases in mechanical tension maintain cell populations separate by influencing junctional rearrangements during cell intercalation.

Haematopoietic stem cells (HSCs) and their subsequent progenitors produce blood cells, but the precise nature and kinetics of this production is a contentious issue. In one model, lymphoid and myeloid production branch after the lymphoid-primed multipotent progenitor (LMPP), with both branches subsequently producing dendritic cells. However, this model is based mainly on in vitro clonal assays and population-based tracking in vivo, which could miss in vivo single-cell complexity. Here we avoid these issues by using a new quantitative version of ‘cellular barcoding’ to trace the in vivo fate of hundreds of LMPPs and HSCs at the single-cell level. These data demonstrate that LMPPs are highly heterogeneous in the cell types that they produce, separating into combinations of lymphoid-, myeloid- and dendritic-cell-biased producers. Conversely, although we observe a known lineage bias of some HSCs, most cellular output is derived from a small number of HSCs that each generates all cell types. Crucially, in vivo analysis of the output of sibling cells derived from single LMPPs shows that they often share a similar fate, suggesting that the fate of these progenitors was imprinted. Furthermore, as this imprinting is also observed for dendritic-cell-biased LMPPs, dendritic cells may be considered a distinct lineage on the basis of separate ancestry. These data suggest a ‘graded commitment’ model of haematopoiesis, in which heritable and diverse lineage imprinting occurs earlier than previously thought.

The invertebrate's innate immune system was reported to show some form of adaptive features, termed trained immunity. However, the memory characteristics of innate immune system and the mechanisms behind such phenomena remain unclear. Using the invertebrate model Artemia, we verified the possibility or impossibility of trained immunity, examining the presence or absence of enduring memory against homologous and heterologous antigens (Vibrio spp.) during a transgenerational study. We also determined the mechanisms behind such phenomenon. Our results showed the occurrence of memory and partial discrimination in Artemia's immune system, as manifested by increased resistance, for three successive generations, of the progenies of Vibrio-exposed ancestors towards a homologous bacterial strain, rather than to a heterologous strain. This increased resistance phenotype was associated with elevated levels of hsp70 and hmgb1 signaling molecules and alteration in the expression of key innate immunity-related genes. Our results also showed stochastic pattern in the acetylation and methylation levels of H4 and H3K4me3 histones, respectively, in the progenies whose ancestors were challenged. Overall results suggest that innate immune responses in invertebrates have the capacity to be trained, and epigenetic reprogramming of (selected) innate immune effectors is likely to have central place in the mechanisms leading to trained immunity.

Differentiating somatic cells are progressively restricted to specialized functions during ontogeny, but they can be experimentally directed to form other cell types, including those with complete embryonic potential. Early nuclear reprogramming methods, such as somatic cell nuclear transfer (SCNT) and cell fusion, posed significant technical hurdles to precise dissection of the regulatory programmes governing cell identity. However, the discovery of reprogramming by ectopic expression of a defined set of transcription factors, known as direct reprogramming, provided a tractable platform to uncover molecular characteristics of cellular specification and differentiation, cell type stability and pluripotency. We discuss the control and maintenance of cellular identity during developmental transitions as they have been studied using direct reprogramming, with an emphasis on transcriptional and epigenetic regulation.

All organisms must trade off resource allocation between different life processes that determine their survival and reproduction. Malaria parasites replicate asexually in the host but must produce sexual stages to transmit between hosts. Because different specialized stages are required for these functions, the division of resources between these life-history components is a key problem for natural selection to solve. Despite the medical and economic importance of these parasites, their reproductive strategies remain poorly understood and often seem counterintuitive. Here, we tested recent theory predicting that in-host competition shapes how parasites trade off investment in in-host replication relative to between-host transmission. We demonstrate, across several genotypes, that Plasmodium chabaudi parasites detect the presence of competing genotypes and facultatively respond by reducing their investment in sexual stages in the manner predicted to maximize their competitive ability. Furthermore, we show that genotypes adjust their allocation to sexual stages in line with the availability of exploitable red blood cell resources. Our findings are predicted by evolutionary theory developed to explain life-history trade-offs in more traditionally studied multicellular taxa and suggest that the answer to the long-standing question of why so few transmission stages are produced is that in most natural infections heavy investment in reproduction may compromise in-host survival.


Question: Cell autonomous sex determination within somatic cells happens in Passeriformes.

Cell Autonomous Sex Determination in Avian Somatic Cells: Evidence from Chicken Models. The prevailing mammalian model of sex determination, characterized by initial sexual indifference followed by gonadal differentiation triggered by a transient sex-determining gene, is often assumed to be universal across vertebrates. However, this assumption has not been universally validated. In this study, we investigated the sex-determining mechanism in birds by examining three rare, naturally occurring lateral gynandromorph chickens, where one side of the animal exhibits male characteristics while the other side is female. Our findings confirmed that gynandromorph chickens are true male:female chimaeras, suggesting that avian somatic cells may possess an inherent sex identity. To further explore this hypothesis, we conducted reciprocal sex transplantation experiments using presumptive mesoderm between embryos of different sexes, resulting in chimaeric embryos with mixed-sex gonads. Unlike in mammalian chimaeras, where donor cells can contribute to the host gonad, our chicken chimaeras showed that donor cells were excluded from the functional structures of the host gonad. In a specific case where female tissue was transplanted into a male host, the donor cells maintained their female identity and expressed a marker indicative of female function even within a developing testis. These results strongly support the notion that avian somatic cells have a cell-autonomous sex identity, indicating that sexual differentiation in birds is substantially cell autonomous.

All organisms must trade off resource allocation between different life processes that determine their survival and reproduction. Malaria parasites replicate asexually in the host but must produce sexual stages to transmit between hosts. Because different specialized stages are required for these functions, the division of resources between these life-history components is a key problem for natural selection to solve. Despite the medical and economic importance of these parasites, their reproductive strategies remain poorly understood and often seem counterintuitive. Here, we tested recent theory predicting that in-host competition shapes how parasites trade off investment in in-host replication relative to between-host transmission. We demonstrate, across several genotypes, that Plasmodium chabaudi parasites detect the presence of competing genotypes and facultatively respond by reducing their investment in sexual stages in the manner predicted to maximize their competitive ability. Furthermore, we show that genotypes adjust their allocation to sexual stages in line with the availability of exploitable red blood cell resources. Our findings are predicted by evolutionary theory developed to explain life-history trade-offs in more traditionally studied multicellular taxa and suggest that the answer to the long-standing question of why so few transmission stages are produced is that in most natural infections heavy investment in reproduction may compromise in-host survival.

Nuclear holoploid genome sizes (C-values) have been estimated to vary about 800-fold in angiosperms, with the smallest established 1C-value of 157 Mbp recorded in Arabidopsis thaliana. In the highly specialized carnivorous family Lentibulariaceae now three taxa have been found that exhibit significantly lower values: Genlisea margaretae with 63 Mbp, G. aurea with 64 Mbp, and Utricularia gibba with 88 Mbp. The smallest mitotic anaphase chromatids in G. aurea have 2.1 Mbp and are thus of bacterial size (NB: E. coli has ca. 4 Mbp). Several Utricularia species range somewhat lower than A. thaliana or are similar in genome size. The highest 1C-value known from species of Lentibulariaceae was found in Genlisea hispidula with 1510 Mbp, and results in about 24-fold variation for Genlisea and the Lentibulariaceae. Taking into account these new measurements, genome size variation in angiosperms is now almost 2000-fold. Genlisea and Utricularia are plants with terminal positions in the phylogeny of the eudicots, so that the findings are relevant for the understanding of genome miniaturization. Moreover, the Genlisea-Utricularia clade exhibits one of the highest mutational rates in several genomic regions in angiosperms, what may be linked to specialized patterns of genome evolution. Ultrasmall genomes have not been found in Pinguicula, which is the sister group of the Genlisea-Utricularia clade, and which does not show accelerated mutational rates. C-values in Pinguicula varied only 1.7-fold from 487 to 829 Mbp.

Synchronous oscillations of thousands of cellular clocks in the suprachiasmatic nucleus (SCN), the circadian centre, are coordinated by precisely timed cell-cell communication, the principle of which is largely unknown. Here we show that the amount of RGS16 (regulator of G protein signalling 16), a protein known to inactivate Gαi, increases at a selective circadian time to allow time-dependent activation of intracellular cyclic AMP signalling in the SCN. Gene ablation of Rgs16 leads to the loss of circadian production of cAMP and as a result lengthens circadian period of behavioural rhythm. The temporally precise regulation of the cAMP signal by clock-controlled RGS16 is needed for the dorsomedial SCN to maintain a normal phase-relationship to the ventrolateral SCN. Thus, RGS16-dependent temporal regulation of intracellular G protein signalling coordinates the intercellular synchrony of SCN pacemaker neurons and thereby defines the 24 h rhythm in behaviour.

The adult Drosophila hindgut was recently reported to contain active, tissue-replenishing stem cells, like those of the midgut, but located within an anterior ring so as to comprise a single giant crypt. In contrast to this view, we observed no active stem cells and little cell turnover in adult hindgut tissue based on clonal marking and BrdU incorporation studies. Again contradicting the previous proposal, we showed that the adult hindgut is not generated by anterior stem cells during larval/pupal development. However, severe tissue damage within the hindgut elicits cell proliferation within a ring of putative quiescent stem cells at the anterior of the pylorus. Thus, the hindgut does not provide a model of tissue maintenance by constitutively active stem cells, but has great potential to illuminate mechanisms of stress-induced tissue repair.

The Heliconius butterflies are a diverse recent radiation comprising multiple levels of divergence with ongoing gene flow between species. The recently sequenced genome of Heliconius melpomene allowed us to investigate the genomic evolution of this group using dense RAD marker sequencing. Phylogenetic analysis of 54 individuals robustly supported reciprocal monophyly of H. melpomene and Heliconius cydno and refuted previous phylogenetic hypotheses that H. melpomene may be paraphylectic with respect to H. cydno. Heliconius timareta also formed a monophyletic clade closely related but distinct from H. cydno with Heliconius heurippa falling within this clade. We find evidence for genetic admixture between sympatric populations of the sister clades H. melpomene and H. cydno/timareta, particularly between H. cydno and H. melpomene from Central America and between H. timareta and H. melpomene from the eastern slopes of the Andes. Between races, divergence is primarily explained by isolation by distance and there is no detectable genetic population structure between parapatric races, suggesting that hybrid zones between races are not zones of secondary contact. Our results also support previous findings that colour pattern loci are shared between populations and species with similar colour pattern elements. Furthermore, this pattern is almost unique to these genomic regions, with only a very small number of other loci showing significant similarity between populations and species with similar colour patterns.

The change in avian clutch size with latitude is a celebrated example of geographic variation in a vertebrate life-history trait. Alternative hypotheses for this pattern invoke nest predation, limited food for nestlings, or post-fledging juvenile mortality as selection pressures leading to small clutch size of tropical birds. We manipulated the clutch size of Spotted Antbirds (Hylophylax naevioides) in central Panama to test these hypotheses. We observed that rates of nest predation were not influenced by parental activity at nests and parents could successfully feed nestlings in enlarged broods. Although larger broods produced the most fledged juveniles, these individuals were less likely to survive to dispersal than were juveniles that fledged from smaller broods. Consequently, nest productivity did not vary with clutch size. Post-fledging mortality was not related to nestling mass two to three days prior to fledging. Rather, differences in the allocation of parental investment per juvenile among differently sized broods appeared to influence juvenile survival probability. These results identify parental care during the post-fledging period as a potential key factor influencing the evolution of small clutch size in tropical birds.

Partitioning tissues into compartments that do not intermix is essential for the correct morphogenesis of animal embryos and organs. Several hypotheses have been proposed to explain compartmental cell sorting, mainly differential adhesion, but also regulation of the cytoskeleton or of cell proliferation. Nevertheless, the molecular and cellular mechanisms that keep cells apart at boundaries remain unclear. Here we demonstrate, in early Drosophila melanogaster embryos, that actomyosin-based barriers stop cells from invading neighbouring compartments. Our analysis shows that cells can transiently invade neighbouring compartments, especially when they divide, but are then pushed back into their compartment of origin. Actomyosin cytoskeletal components are enriched at compartmental boundaries, forming cable-like structures when the epidermis is mitotically active. When MyoII (non-muscle myosin II) function is inhibited, including locally at the cable by chromophore-assisted laser inactivation (CALI), in live embryos, dividing cells are no longer pushed back, leading to compartmental cell mixing. We propose that local regulation of actomyosin contractibility, rather than differential adhesion, is the primary mechanism sorting cells at compartmental boundaries.

The aim of this study was to confirm the presence of stem cells in the ovarian surface epithelium of patients with premature ovarian failure and no mature follicles and oocytes. In these patients, small round cells of unknown origin expressing SOX-2 marker of pluripotency were observed among the epithelial cells just after the ovarian surface epithelium scraping. These cells were an integral part of the ovarian surface epithelium. When the scraped cells were cultured in a medium with added follicular fluid to provide some ovarian niche, primitive oocyte-like cells and typical round-shaped cell clusters positively stained on alkaline phosphatase, and markers of pluripotency, such as SOX-2 and SSEA-4, were developed. These markers were expressed early and also later in the culture. Single oocyte-like cells expressed genes OCT4A, SOX-2, NANOG, NANOS, STELLA, CD9, LIN28, KLF4, GDF3, and MYC, characteristic for pluripotent stem cells. The results of this study confirmed the presence of putative stem cells in the ovarian surface epithelium of these patients and provided some basis to create a stem cell line in the future.

The expression of clock genes in vertebrates is widespread and not restricted to classical clock structures. The expression of the Clock gene in zebrafish shows a strong circadian oscillation in many tissues in vivo and in culture, showing that endogenous oscillators exist in peripheral organs. A defining feature of circadian clocks is that they can be set or entrained to local time, usually by the environmental light-dark cycle. An important question is whether peripheral oscillators are entrained to local time by signals from central pacemakers such as the eyes or are themselves directly light-responsive. Here we show that the peripheral organ clocks of zebrafish are set by light-dark cycles in culture. We also show that a zebrafish-derived cell line contains a circadian oscillator, which is also directly light entrained.

The Tasmanian devil (Sarcophilus harrisii), the largest marsupial carnivore, is endangered due to a transmissible facial cancer spread by direct transfer of living cancer cells through biting. Here we describe the sequencing, assembly, and annotation of the Tasmanian devil genome and whole-genome sequences for two geographically distant subclones of the cancer. Genomic analysis suggests that the cancer first arose from a female Tasmanian devil and that the clone has subsequently genetically diverged during its spread across Tasmania. The devil cancer genome contains more than 17,000 somatic base substitution mutations and bears the imprint of a distinct mutational process. Genotyping of somatic mutations in 104 geographically and temporally distributed Tasmanian devil tumors reveals the pattern of evolution and spread of this parasitic clonal lineage, with evidence of a selective sweep in one geographical area and persistence of parallel lineages in other populations.


Question: Cells deficient in clpC display a flaw in sporulation efficacy in Bacillus subtilis.

Advanced Genetic Screening Techniques Uncover Novel Sporulation Genes in Bacillus subtilis

The developmental pathway of Bacillus subtilis leading to spore formation is a well-studied model in biology. Extensive genetic screens over several decades have identified numerous factors critical for each phase of this complex morphological transformation. More recently, advancements in transcriptional profiling have shed light on additional sporulation-induced genes that are essential for successful spore development. In this study, we employed transposon sequencing (Tn-seq) to explore whether there were any undiscovered sporulation genes remaining. Our comprehensive screen successfully identified 133 out of 148 genes known to have sporulation defects. Remarkably, we also discovered 24 new genes that had not been previously associated with spore formation.

To elucidate the functions of these genes, we utilized fluorescence microscopy to monitor the progression of sporulation in null mutants derived from the B. subtilis ordered knockout collection. This approach revealed mutants that exhibit delays in sporulation initiation, defects in membrane remodeling, and impairments in spore maturation. Some mutants displayed unique sporulation phenotypes not previously observed. We conducted detailed analyses of two newly identified factors that play roles in cell-cell signaling during sporulation. One factor (SpoIIT) is involved in the activation of σE in the mother cell, while the other (SpoIIIL) is necessary for σG activity in the forespore.

Our investigation also uncovered that as many as 36 sporulation-induced genes, previously lacking mutant phenotypes, are crucial for timely spore maturation. Additionally, we identified a substantial number of transposon insertions that unexpectedly trigger premature sporulation initiation. These findings underscore the efficacy of Tn-seq for the discovery of new genes and pathways in sporulation. Coupled with the recently completed null mutant collection, this opens up new opportunities for similar high-throughput screens in less well-understood biological processes, further expanding our knowledge of bacterial development.

Chaperone-mediated autophagy (CMA), a selective mechanism for degradation of cytosolic proteins in lysosomes, contributes to the removal of altered proteins as part of the cellular quality-control systems. We have previously found that CMA activity declines in aged organisms and have proposed that this failure in cellular clearance could contribute to the accumulation of altered proteins, the abnormal cellular homeostasis and, eventually, the functional loss characteristic of aged organisms. To determine whether these negative features of aging can be prevented by maintaining efficient autophagic activity until late in life, in this work we have corrected the CMA defect in aged rodents. We have generated a double transgenic mouse model in which the amount of the lysosomal receptor for CMA, previously shown to decrease in abundance with age, can be modulated. We have analyzed in this model the consequences of preventing the age-dependent decrease in receptor abundance in aged rodents at the cellular and organ levels. We show here that CMA activity is maintained until advanced ages if the decrease in the receptor abundance is prevented and that preservation of autophagic activity is associated with lower intracellular accumulation of damaged proteins, better ability to handle protein damage and improved organ function.

Cooperation among individuals is necessary for evolutionary transitions to higher levels of biological organization. In such transitions, groups of individuals at one level (such as single cells) cooperate to form selective units at a higher level (such as multicellular organisms). Though the evolution of cooperation is difficult to observe directly in higher eukaryotes, microorganisms do offer such an opportunity. Here we report the evolution of novel cooperative behaviour in experimental lineages of the bacterium Myxococcus xanthus. Wild-type strains of M. xanthus exhibit socially dependent swarming across soft surfaces by a mechanism known as ‘S-motility’ that requires the presence of extracellular type IV pili. In lineages of M. xanthus unable to make pili, a new mechanistic basis for cooperative swarming evolved. Evolved swarming is mediated, at least in part, by enhanced production of an extracellular fibril matrix that binds cells—and their evolutionary interests—together. Though costly to individuals, fibril production greatly enhanced population expansion in groups of interconnected cells. These results show that fundamental transitions to primitive cooperation can readily occur in bacteria.

Bactericidal antibiotics kill by modulating their respective targets. This traditional view has been challenged by studies that propose an alternative, unified mechanism of killing, whereby toxic reactive oxygen species (ROS) are produced in the presence of antibiotics. We found no correlation between an individual cell's probability of survival in the presence of antibiotic and its level of ROS. An ROS quencher, thiourea, protected cells from antibiotics present at low concentrations, but the effect was observed under anaerobic conditions as well. There was essentially no difference in survival of bacteria treated with various antibiotics under aerobic or anaerobic conditions. This suggests that ROS do not play a role in killing of bacterial pathogens by antibiotics.

The ability to maintain a functional proteome, or proteostasis, declines during the ageing process. Damaged and misfolded proteins accumulate with age, impairing cell function and tissue homeostasis. The accumulation of damaged proteins contributes to multiple age-related diseases such as Alzheimer's, Parkinson's or Huntington's disease. Damaged proteins are degraded by the ubiquitin-proteasome system or through autophagy-lysosome, key components of the proteostasis network. Modulation of either proteasome activity or autophagic-lysosomal potential extends lifespan and protects organisms from symptoms associated with proteostasis disorders, suggesting that protein clearance mechanisms are directly linked to ageing and age-associated diseases.

In animal cells, most microtubules are nucleated at centrosomes. At the onset of mitosis, centrosomes undergo a structural reorganization, termed maturation, which leads to increased microtubule nucleation activity. Centrosome maturation is regulated by several kinases, including Polo-like kinase 1 (Plk1). Here, we identify a centrosomal Plk1 substrate, termed Nlp (ninein-like protein), whose properties suggest an important role in microtubule organization. Nlp interacts with two components of the gamma-tubulin ring complex and stimulates microtubule nucleation. Plk1 phosphorylates Nlp and disrupts both its centrosome association and its gamma-tubulin interaction. Overexpression of an Nlp mutant lacking Plk1 phosphorylation sites severely disturbs mitotic spindle formation. We propose that Nlp plays an important role in microtubule organization during interphase, and that the activation of Plk1 at the onset of mitosis triggers the displacement of Nlp from the centrosome, allowing the establishment of a mitotic scaffold with enhanced microtubule nucleation activity.

Recently many exciting advances have been achieved in our understanding of Drosophila meiosis due to combined cytological and genetic approaches. New techniques have permitted the characterization of chromosome position and spindle formation in female meiosis I. The proteins encoded by the nod and ncd genes, two genes known to be needed for the proper partitioning of chromosomes lacking exchange events, have been identified and found to be kinesin-like motors. The effects of mutations in these genes on the spindle and chromosomes, together with the localization of the proteins, have yielded a model for the mechanism of female meiosis I. In male meiosis I, the chromosomal regions responsible for homolog pairing have been resolved to the level of specific DNA sequences. This provides a foundation for elucidating the molecular basis of meiotic pairing. The cytological techniques available in Drosophila also have permitted inroads into the regulation of sister-chromatid segregation. The products of two genes (mei-S332 and ord) essential for sister-chromatid cohesion have been identified recently. Additional advances in understanding Drosophila meiosis are the delineation of a functional centromere by using minichromosome derivatives and the identification of several regulatory genes for the meiotic cell cycle.

Staphylococcus aureus is a human commensal that can also cause systemic infections. This transition requires evasion of the immune response and the ability to exploit different niches within the host. However, the disease mechanisms and the dominant immune mediators against infection are poorly understood. Previously it has been shown that the infecting S. aureus population goes through a population bottleneck, from which very few bacteria escape to establish the abscesses that are characteristic of many infections. Here we examine the host factors underlying the population bottleneck and subsequent clonal expansion in S. aureus infection models, to identify underpinning principles of infection. The bottleneck is a common feature between models and is independent of S. aureus strain. Interestingly, the high doses of S. aureus required for the widely used "survival" model results in a reduced population bottleneck, suggesting that host defences have been simply overloaded. This brings into question the applicability of the survival model. Depletion of immune mediators revealed key breakpoints and the dynamics of systemic infection. Loss of macrophages, including the liver Kupffer cells, led to increased sensitivity to infection as expected but also loss of the population bottleneck and the spread to other organs still occurred. Conversely, neutrophil depletion led to greater susceptibility to disease but with a concomitant maintenance of the bottleneck and lack of systemic spread. We also used a novel microscopy approach to examine abscess architecture and distribution within organs. From these observations we developed a conceptual model for S. aureus disease from initial infection to mature abscess. This work highlights the need to understand the complexities of the infectious process to be able to assign functions for host and bacterial components, and why S. aureus disease requires a seemingly high infectious dose and how interventions such as a vaccine may be more rationally developed.

Many cellular proteins perform their roles within macromolecular assemblies. Hence, an understanding of how these multiprotein complexes form is a fundamental question in cell biology. We developed a translation-controlled pulse-chase system that allows time-resolved isolation of newly forming multiprotein complexes in chemical quantities suitable for biochemical and cell biological analysis. The "pulse" is triggered by an unnatural amino acid, which induces immediate translation of an amber stop codon repressed mRNA encoding the protein of interest with a built-in tag for detection and purification. The "chase" is elicited by stopping translation of this bait via a riboswitch in the respective mRNA. Over the course of validating our method, we discovered a distinct time-resolved assembly step during NPC biogenesis and could directly monitor the spatiotemporal maturation of preribosomes via immunofluorescence detection and purification of a pulse-labeled ribosomal protein. Thus, we provide an innovative strategy to study dynamic protein assembly within cellular networks.

Oncogene-induced DNA replication stress has been implicated as a driver of tumorigenesis. Many chromosomal rearrangements characteristic of human cancers originate from specific regions of the genome called common fragile sites (CFSs). CFSs are difficult-to-replicate loci that manifest as gaps or breaks on metaphase chromosomes (termed CFS ‘expression’), particularly when cells have been exposed to replicative stress. The MUS81–EME1 structure-specific endonuclease promotes the appearance of chromosome gaps or breaks at CFSs following replicative stress. Here we show that entry of cells into mitotic prophase triggers the recruitment of MUS81 to CFSs. The nuclease activity of MUS81 then promotes POLD3-dependent DNA synthesis at CFSs, which serves to minimize chromosome mis-segregation and non-disjunction. We propose that the attempted condensation of incompletely duplicated loci in early mitosis serves as the trigger for completion of DNA replication at CFS loci in human cells. Given that this POLD3-dependent mitotic DNA synthesis is enhanced in aneuploid cancer cells that exhibit intrinsically high levels of chromosomal instability (CIN+) and replicative stress, we suggest that targeting this pathway could represent a new therapeutic approach.

The bacterial type VI secretion system (T6SS) is an organelle that is structurally and mechanistically analogous to an intracellular membrane-attached contractile phage tail. Recent studies determined that a rapid conformational change in the structure of a sheath protein complex propels T6SS spike and tube components along with antibacterial and antieukaryotic effectors out of predatory T6SS(+) cells and into prey cells. The contracted organelle is then recycled in an ATP-dependent process. T6SS is regulated at transcriptional and posttranslational levels, the latter involving detection of membrane perturbation in some species. In addition to directly targeting eukaryotic cells, the T6SS can also target other bacteria coinfecting a mammalian host, highlighting the importance of the T6SS not only for bacterial survival in environmental ecosystems, but also in the context of infection and disease. This review highlights these and other advances in our understanding of the structure, mechanical function, assembly, and regulation of the T6SS.


Question: Cells experiencing methionine restriction might trigger miRNAs.

microRNAs: A Protective Mechanism in Stress Adaptation?
Recent evidence indicates that microRNAs (miRNAs) play a crucial role in stress response mechanisms, in addition to their established function in developmental processes. Intriguingly, these small regulatory RNAs, known for their capacity to repress target gene expression, can switch to become activators of gene expression under stressful conditions. This phenomenon could be attributed to the altered interactions between miRNA/Argonaute complexes and RNA-binding proteins, which are redistributed from various cellular compartments as a response to stress. This dynamic interplay between miRNAs and other cellular components might be a key factor in the adaptive responses of cells to environmental challenges.

Transcription factor-based cellular reprogramming has opened the way to converting somatic cells to a pluripotent state, but has faced limitations resulting from the requirement for transcription factors and the relative inefficiency of the process. We show here that expression of the miR302/367 cluster rapidly and efficiently reprograms mouse and human somatic cells to an iPSC state without a requirement for exogenous transcription factors. This miRNA-based reprogramming approach is two orders of magnitude more efficient than standard Oct4/Sox2/Klf4/Myc-mediated methods. Mouse and human miR302/367 iPSCs display similar characteristics to Oct4/Sox2/Klf4/Myc-iPSCs, including pluripotency marker expression, teratoma formation, and, for mouse cells, chimera contribution and germline contribution. We found that miR367 expression is required for miR302/367-mediated reprogramming and activates Oct4 gene expression, and that suppression of Hdac2 is also required. Thus, our data show that miRNA and Hdac-mediated pathways can cooperate in a powerful way to reprogram somatic cells to pluripotency.

BACKGROUND MicroRNA-196 (miR-196), which is highly up-regulated in oral cancer cells, has been reported to be aberrantly expressed in several cancers; however, the significance of miR-196 in oral cancer has not yet been addressed. METHODS Cellular functions in response to miR-196 modulation were examined, including cell growth, migration, invasion and radio/chemosensitivity. Algorithm-based studies were used to identify the regulatory target of miR-196. The miR-196 target gene and downstream molecular mechanisms were confirmed by RT-qPCR, western blot, luciferase reporter and confocal microscopy analyses. miR-196 expression was determined in paired cancer and adjacent normal tissues from oral cancer patients. RESULTS Both miR-196a and miR-196b were highly over-expressed in the cancer tissue and correlated with lymph node metastasis (P = 0.001 and P = 0.006, respectively). Functionally, miR-196 actively promoted cell migration and invasion without affecting cell growth. Mechanistically, miR-196 performed it's their function by inhibiting NME4 expression and further activating p-JNK, suppressing TIMP1, and augmenting MMP1/9. CONCLUSION miR-196 contributes to oral cancer by promoting cell migration and invasion. Clinically, miR-196a/b was significantly over-expressed in the cancer tissues and correlated with lymph node metastasis. Thus, our findings provide new knowledge of the underlying mechanism of cancer metastasis. miR-196 may serve as a promising marker for better oral cancer management.

Mouse microRNAs (miRNAs) miR-290-miR295 are encoded by a cluster of partially homologous pre-miRNA hairpins and are likely to be functionally important in embryonic stem (ES) cells and preimplantation embryos. We present evidence that a spliced, capped, and polyadenylated primary transcript spans this entire Early Embryonic microRNA Cluster (EEmiRC). Partial Drosha processing yields additional large nuclear RNA intermediates. A conserved promoter element containing a TATA-box directs EEmiRC transcription. Sequence analysis shows that the EEmiRC transcription unit is remarkably variable and can only be identified bioinformatically in placental (eutherian) mammals. Consistent with eutherian-specific function, EEmiRC is expressed in trophoblastic stem (TS) cells. When analyzing evolutionary and functional relationships, the organization of the entire miRNA loci should be considered in addition to the mature miRNA sequences. Application of this concept suggests that EEmiRC is a recently acquired rapidly evolving gene important for eutherian development.

Despite being surrounded by diverse nutrients, mammalian cells preferentially metabolize glucose and free amino acids. Recently, Ras-induced macropinocytosis of extracellular proteins was shown to reduce a transformed cell's dependence on extracellular glutamine. Here, we demonstrate that protein macropinocytosis can also serve as an essential amino acid source. Lysosomal degradation of extracellular proteins can sustain cell survival and induce activation of mTORC1 but fails to elicit significant cell accumulation. Unlike its growth-promoting activity under amino-acid-replete conditions, we discovered that mTORC1 activation suppresses proliferation when cells rely on extracellular proteins as an amino acid source. Inhibiting mTORC1 results in increased catabolism of endocytosed proteins and enhances cell proliferation during nutrient-depleted conditions in vitro and within vascularly compromised tumors in vivo. Thus, by preventing nutritional consumption of extracellular proteins, mTORC1 couples growth to availability of free amino acids. These results may have important implications for the use of mTOR inhibitors as therapeutics.

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) resets the epigenome to an embryonic-like state. Vitamin C enhances the reprogramming process, but the underlying mechanisms are unclear. Here we show that the histone demethylases Jhdm1a/1b are key effectors of somatic cell reprogramming downstream of vitamin C. We first observed that vitamin C induces H3K36me2/3 demethylation in mouse embryonic fibroblasts in culture and during reprogramming. We then identified Jhdm1a/1b, two known vitamin-C-dependent H3K36 demethylases, as potent regulators of reprogramming through gain- and loss-of-function approaches. Furthermore, we found that Jhdm1b accelerates cell cycle progression and suppresses cell senescence during reprogramming by repressing the Ink4/Arf locus. Jhdm1b also cooperates with Oct4 to activate the microRNA cluster 302/367, an integral component of the pluripotency machinery. Our results therefore reveal a role for H3K36me2/3 in cell fate determination and establish a link between histone demethylases and vitamin-C-induced reprogramming.

The microRNA (miRNA) "sponge" method was introduced three years ago as a means to create continuous miRNA loss of function in cell lines and transgenic organisms. Sponge RNAs contain complementary binding sites to a miRNA of interest, and are produced from transgenes within cells. As with most miRNA target genes, a sponge's binding sites are specific to the miRNA seed region, which allows them to block a whole family of related miRNAs. This transgenic approach has proven to be a useful tool to probe miRNA functions in a variety of experimental systems. Here we will discuss the ways sponge and related constructs can be optimized and review recent applications of this method with particular emphasis on stable expression in cancer studies and in transgenic animals.

Epstein-Barr virus (EBV), an oncogenic human herpesvirus, induces cell proliferation after infection of resting B lymphocytes, its reservoir in vivo. The viral latent proteins are necessary for permanent B cell growth, but it is unknown whether they are sufficient. EBV was recently found to encode microRNAs (miRNAs) that are expressed in infected B cells and in some EBV-associated lymphomas. EBV miRNAs are grouped into two clusters located either adjacent to the BHRF1 gene or in introns contained within the viral BART transcripts. To understand the role of the BHRF1 miRNA cluster, we have constructed a virus mutant that lacks all its three members (Δ123) and a revertant virus. Here we show that the B cell transforming capacity of the Δ123 EBV mutant is reduced by more than 20-fold, relative to wild type or revertant viruses. B cells exposed to the knock-out virus displayed slower growth, and exhibited a two-fold reduction in the percentage of cells entering the cell cycle S phase. Furthermore, they displayed higher latent gene expression levels and latent protein production than their wild type counterparts. Therefore, the BHRF1 miRNAs accelerate B cell expansion at lower latent gene expression levels. Thus, this miRNA cluster simultaneously enhances expansion of the virus reservoir and reduces the viral antigenic load, two features that have the potential to facilitate persistence of the virus in the infected host. Thus, the EBV BHRF1 miRNAs may represent new therapeutic targets for the treatment of some EBV-associated lymphomas.

CaMKII is activated by oxidation of methionine residues residing in the regulatory domain. Oxidized CaMKII (ox-CaMKII) is now thought to participate in cardiovascular and pulmonary diseases and cancer. This invited review summarizes current evidence for the role of ox-CaMKII in disease, considers critical knowledge gaps and suggests new areas for inquiry.

Maternally expressed gene 3 (MEG3) is a maternally expressed imprinted gene representing a large noncoding RNA in which microRNAs (miRNAs) and small nucleolar RNAs are also hosted. It is capable of interacting with cyclic AMP, p53, murine double minute 2 (MDM2) and growth differentiation factor 15 (GDF15) playing a role in cell proliferation control. MEG3 expression is under epigenetic control, and aberrant CpG methylation has been observed in several types of cancer. Moreover, gene copy number loss has been reported as additional mechanism associated with tumorigenesis. MEG3 deletion seems to upregulate the paternally expressed genes and on the other hand downregulate the expression of downstream maternally expressed genes and tumor suppressor miRNAs, although there are conflicting data on the topic. MEG3 could represent a tumor suppressor gene located in chromosome 14q32 and its association with tumorigenesis is growing every day.

The regulation and coordination of mitochondrial metabolism with hematopoietic stem cell (HSC) self-renewal and differentiation is not fully understood. Here we report that depletion of PTPMT1, a PTEN-like mitochondrial phosphatase, in inducible or hematopoietic-cell-specific knockout mice resulted in hematopoietic failure due to changes in the cell cycle and a block in the differentiation of HSCs. Surprisingly, the HSC pool was increased by ∼40-fold in PTPMT1 knockout mice. Reintroduction of wild-type PTPMT1, but not catalytically deficient PTPMT1 or truncated PTPMT1 lacking mitochondrial localization, restored differentiation capabilities of PTPMT1 knockout HSCs. Further analyses demonstrated that PTPMT1 deficiency altered mitochondrial metabolism and that phosphatidylinositol phosphate substrates of PTPMT1 directly enhanced fatty-acid-induced activation of mitochondrial uncoupling protein 2. Intriguingly, depletion of PTPMT1 from myeloid, T lymphoid, or B lymphoid progenitors did not cause any defects in lineage-specific knockout mice. This study establishes a crucial role of PTPMT1 in the metabolic regulation of HSC function.