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People living with HIV/AIDS (PLH) experience high rates of depression and related psychosocial risk factors that vary by gender. This study examines gender differences in depression severity among antiretroviral therapy (ART) patients (n = 362) from a large government ART clinic in Kolkata, India. Hypotheses for multiple linear regression models were guided by an integrated gendered stress process model focusing on variables reflecting social status (age, partner status), stressors (stigma), and resources (income, social support). Depressive symptoms were assessed with the Hospital Anxiety and Depression Scale (HADS); 22% of the sample reached the cutoff for severe depression, 56% moderate, and 13% mild depression. Compared to men, women reported lower income, education (50% no formal education vs. 20% men), availability of emotional and instrumental support, and were less likely to be married or cohabiting (53% women vs. 72% of men). However, more women had partners who were HIV-positive (78% women vs. 46% men). Overall, depression severity was negatively associated with availability of emotional support and self-distraction coping, and positively associated with internalized HIV/AIDS stigma, availability of instrumental support, and behavioral disengagement coping. Interactions for instrumental support by income and partner status by age varied significantly by gender. Analyses stratified by gender indicated that: 1) Frequently seeking instrumental support from others was protective for men at all income levels, but only for high-income women; and 2) having a partner was protective for men as they aged, but not for women. These results suggest that gender disparities in depression severity are created and maintained by women's lower social status and limited access to resources. The effect of stigma on depression severity did not vary by gender. These findings may inform the tailoring of future interventions to address mental health needs of PLH in India, particularly gender disparities in access to material and social resources for coping with HIV. Trial Registration: ClinicalTrials.gov registration #NCT02118454, registered April 2014.

Amphibian populations are vanishing worldwide. Declines and extinctions of many populations have been attributed to chytridiomycosis, a disease induced by the pathogenic fungus Batrachochytrium dendrobatidis (Bd). In Africa, however, changes in amphibian assemblages were typically attributed to habitat change. We conducted a retrospective study utilizing field surveys from 2004-2012 of the anuran faunas on two mountains in western Cameroon, a hotspot of African amphibian diversity. The number of species detected was negatively influenced by year, habitat degradation, and elevation, and we detected a decline of certain species. Because another study in this region revealed an emergence of Bd in 2008, we screened additional recent field-collected samples and also pre-decline preserved museum specimens for the presence of Bd supporting emergence before 2008. When comparing the years before and after Bd detection, we found significantly diminished frog species richness and abundance on both mountains after Bd emergence. Our analyses suggest that this may be the first disease-driven community-level decline in anuran biodiversity in Central Africa. The disappearance of several species known to tolerate habitat degradation, and a trend of stronger declines at higher elevations, are consistent with Bd-induced declines in other regions. Not all species decreased; populations of some species remained constant, and others increased after the emergence of Bd. This variation might be explained by species-specific differences in infection probability. Increased habitat protection and Bd-mitigation strategies are needed for sustaining diverse amphibian communities such as those on Mt. Manengouba, which contains nearly half of Cameroon's frog diversity.

Introduction Cervix, breast and oral cancers account for about one-third of all cancers in India which as a group is a major contributor to all non-communicable disease-related morbidity and mortality among women. Existing evidence suggests that early diagnosis plays a pivotal role in the prevention and intervention of these cancers, and many community-based early screening and awareness programs have been in place in developed countries. Currently, there is not enough research evidence regarding the sociodemographic correlates of cervix, breast and oral cancer screening among Indian women. In the present study, we aimed to assess the self-reported percentage and sociodemographic factors associated with the use of these three types of cancer screening services among Indian women aged 15–49 years. Methods Data were collected from National Family Health Survey conducted during 2015–16. Sample population was 699,686 women aged 15–49 years. Associations between self-reported cervical, breast and oral cancer screening status and the associated sociodemographic factors were analyzed using multivariable logistic regression methods. Results The percentage of screening for cervical (21%), breast (8.95%), and oral cancers (13.45%) varied significantly across the population sub-groups. Higher age, urban residence, higher education, having employment, health insurance, use of electronic media, higher household wealth quintile, having healthcare autonomy, showed a positive effect on taking screening services. Further analyses revealed that the strength of the associations varied considerably between urban and rural residents, denoting the need for region-specific intervention strategies. Sex of household head, age, watching TV, using radio, and having health insurance were the most significant contributors to the outcome effects. Conclusions The present study provides important insights regarding the current scenario of seeking cancer screening services among women in India. These findings could inform policy analysis and make an avenue for further in-depth analysis for future studies. Our findings conclude that cancer prevention policies should focus on leveraging the positive effects of better socioeconomic status, employment, health insurance ownership, exposure to electronic media, and better healthcare autonomy to improve the cancer screening service uptake among Indian women.

It is hypothesized that same driver gene mutations should be present in both oral leukoplakia and cancer tissues. So, we attempted to find out mutations at one of the driver genes, CASP8, in cancer and adjacent leukoplakia tissues. Patients (n = 27), affected by both of cancer and adjacent leukoplakia, were recruited for the study. Blood and tissue DNA samples were used to identify somatic mutations at CASP8 by next generation sequencing method. In total, 56% (15 out of 27) cancer and 30% (8 out of 27) leukoplakia tissues had CASP8 somatic mutations. In 8 patients, both cancer and adjacent leukoplakia tissues, located within 2-5 cm of tumor sites, had identical somatic mutations. But, in 7 patients, cancer samples had somatic mutations but none of the leukoplakia tissues, located beyond 5cm of tumor sites, had somatic mutations. Mutated allele frequencies at CASP8 were found to be more in cancer compared to adjacent leukoplakia tissues. This study provides mutational evidence that oral cancer might have progressed from previously grown leukoplakia lesion. Leukoplakia tissues, located beyond 5cm of cancer sites, were free from mutation. The study implies that CASP8 mutation could be one of the signatures for some of the leukoplakia to progress to oral cancer.

Common scab disease in potato has become a widespread issue in major potato production areas, leading to increasing economic losses. Varietal resistance is seen as a viable and long-term scab management strategy. However, the genes and mechanisms of varietal resistance are unknown. In the current study, a comparative RNA transcriptome sequencing and differential gene signaling and priming sensitization studies were conducted in two potato cultivars that differ by their response to common scab (Streptomyces scabies), for unraveling the genes and pathways potentially involved in resistance within this pathosystem. We report on a consistent and contrasted gene expression pattern from 1,064 annotated genes differentiating a resistant (Hindenburg) and a susceptible (Green Mountain) cultivars, and identified a set of 273 co-regulated differentially expressed genes in 34 pathways that more likely reflect the genetic differences of the cultivars and metabolic mechanisms involved in the scab pathogenesis and resistance. The data suggest that comparative transcriptomic phenotyping can be used to predict scab lesion phenotype in breeding lines using mature potato tuber. The study also showed that the resistant cultivar, Hindenburg, has developed and maintained a capacity to sense and prime itself for persistent response to scab disease over time, and suggests an immune priming reaction as a mechanism for induced-resistance in scab resistant potato cultivars. The set of genes identified, described, and discussed in the study paves the foundation for detailed characterizations towards tailoring and designing procedures for targeted gene knockout through gene editing and phenotypic evaluation.

BackgroundThe general benefit of physical activity (PA) to one's mental health has been widely acknowledged. Nevertheless, the specific type and amount of PA that associates with lower risk of depression in China awaits further investigation. The present study was conducted on middle- and older-aged Chinese population with two objectives: 1) to understand the patterns of PA; 2) to measure the associations between depression and PA at different levels from various aspects.MethodsUsing data from the China Health and Retirement Longitudinal Study (CHARLS, 2015), we selected 9118 community residents aged 45 years and older. Depressive symptoms were measured by 10-item Center for Epidemiologic Studies (CES-D 10). Multivariate logistic regression model was performed to examine the association between risk of depression and PA from four aspects including intensity, frequency, duration, and volume.ResultsSpending 1-2 days/week (OR = 0.58, 95% CI: 0.36, 0.91), less than 30 minutes each time (OR = 0.66, 95% CI: 0.42, 1.03) or 150-299 min/week (OR = 0.49, 95% CI: 0.28, 0.87) on Moderate Physical Activity (MPA) was associated with lower odds of depression in women. Spending 3-5 days/week (OR = 1.98, 95% CI: 1.29, 3.05) or 6-7 days/week (OR = 1.50, 95% CI: 1.07, 2.11), 4 hours and longer each time (OR = 1.65, 95% CI: 1.18, 2.32), 300 min/week or longer (OR = 1.65, 95% CI: 1.22, 2.24) on Vigorous Physical Activity (VPA) in total, or 2250 Metabolic Equivalent of Task (OR = 1.73, 95% CI: 1.26, 2.38) on Moderate-to-Vigorous PA was associated with higher risk of depression in men.ConclusionsThe association between depression and PA depended largely on intensity and gender. Lower frequency, shorter duration, and moderate amount of MPA was associated with lower risk of depression in women. Risk of depression was higher in men who spent higher frequency, longer duration, and overlong time on VPA.

ObjectivesPrevious studies have attempted to assess the role of health insurance on health care utilization in African settings. However, there is limited evidence on the effects of health insurance on use of maternal health care. In the present study our objective was to measure the prevalence of insurance ownership, types of services covered by the insurance and the association of insurance ownership with the utilization of respective maternal health services in Ghana.MethodsThis study was based on nationally representative Demographic and Health Survey in Ghana (GDHS 2014) encompassing 4,293 mothers aged 15-49 years. Outcome variables were use of early antenatal care (ANC), facility delivery, and postnatal care (PNC) for mothers and children, and the explanatory variables were insurance coverage for these services. Associations were analysed using logistic regression models whilst controlling for potentially confounding variables.ResultsPrevalence of health insurance ownership was 66.8% (95%CI = 64.5-68.9) with significant socioeconomic disparities. The prevalence was higher particularly among women who were urban residents, had higher educational and wealth status. In general, insurance coverage for services such as ANC, childbirth and postnatal care was higher in rural areas, but that of cash benefit was higher in urban areas. Findings of multivariate analysis indicated that women who had their ANC services covered had significantly higher odds of attending at least one and four ANC visits, as well as receiving PNC for child. Insurance coverage for childbirth services showed significant association with the PNC for child, not with choice of health facility delivery. Women who had cash benefit were twice as likely to use early ANC visit (OR = 2.046, pConclusionOverall prevalence of health insurance coverage has increased since 2008, with significant disparities across demographic and socioeconomic groups. Insurance ownership for different types of maternal health services showed positive association with service uptake, with exceptions for place of delivery, indicating that insurance coverage alone may not be able to promote facility delivery. More studies are required to measure the progress in maternal healthcare utilisation through the insurance programmes.

[This corrects the article DOI: 10.1371/journal.pone.0207055.].

BackgroundUnmet need for contraception and unwanted pregnancy are recognised as significant barriers to promoting women's reproductive health and well-being. Currently there is no research evidence on these two crucial indicators of reproductive care in Angola. Therefore, we conducted this study with the objectives of exploring the current prevalence of unmet need for contraception and unintended pregnancy as well as their relationship among married women in Angola.MethodsThis study was based on cross-sectional data from Angola Demographic and Health Survey (DHS) conducted in 2015-16. Participants were 7,808 married women aged 15-49 years. Unwanted pregnancy was measured in terms of the mistimed and unintended conception for the last-born child. Unmet need for contraception included those who reported unmet need for spacing and limiting. Data were analysed using bivariate and multivariable techniques.ResultsThe combined prevalence of mistimed and unwanted pregnancy was 38.3% (95%CI = 35.9-40.7), and that of unmet need for contraception for spacing and limiting was 51.7% (49.9-53.5). Among the 18 regions, Luanda had the highest prevalence of unmet need for contraception and of unwanted pregnancy with the prevalence being higher than more than one-third of the women. Multivariable analysis significantly revealed a significantly positive association between unmet need and unwanted pregnancy. In all the models, the odds of unwanted pregnancy were found to be as high as four times among women with unmet need compared with those had no unmet need. Compared to women who had no unmet need, those who had unmet need had respectively four (OR = 4.380; 95%CI = 3.690-5.198) and seven (OR = 6.951; 95%CI = 4.642-10.410) times higher odds of experiencing unwanted pregnancy.ConclusionThis study concludes that the prevalence of unmet need for contraception and unwanted pregnancy are high with significant disparities across the regions. Women in the capital city had the highest prevalence of both unmet need for contraception and unwanted pregnancy. Although the data were cross-sectional and do not indicate causal relationships, the findings showed a strong positive association between unmet need for contraception and unwanted pregnancy. However, it is recommended to conduct further studies to replicate the findings and to explore the influence behavioural and cultural practices on unwanted pregnancy.

IntroductionPatients with severe malaria or sepsis are at risk of developing life-threatening acute respiratory distress syndrome (ARDS). The objective of this study was to evaluate point-of-care lung ultrasound as a novel tool to determine the prevalence and early signs of ARDS in a resource-limited setting among patients with severe malaria or sepsis.Materials and methodsSerial point-of-care lung ultrasound studies were performed on four consecutive days in a planned sub study of an observational cohort of patients with malaria or sepsis in Bangladesh. We quantified aeration patterns across 12 lung regions. ARDS was defined according to the Kigali Modification of the Berlin Definition.ResultsOf 102 patients enrolled, 71 had sepsis and 31 had malaria. Normal lung ultrasound findings were observed in 44 patients on enrolment and associated with 7% case fatality. ARDS was detected in 10 patients on enrolment and associated with 90% case fatality. All patients with ARDS had sepsis, 4 had underlying pneumonia. Two patients developing ARDS during hospitalisation already had reduced aeration patterns on enrolment. The SpO2/FiO2 ratio combined with the number of regions with reduced aeration was a strong prognosticator for mortality in patients with sepsis (AUROC 91.5% (95% Confidence Interval: 84.6%-98.4%)).ConclusionsThis study demonstrates the potential usefulness of point-of-care lung ultrasound to detect lung abnormalities in patients with malaria or sepsis in a resource-constrained hospital setting. LUS was highly feasible and allowed to accurately identify patients at risk of death in a resource limited setting.

BACKGROUND:Under-5 mortality rate in the sub-Saharan region has remained unabated. Worse still, information on the regional trend and associated determinants are not readily available. Knowledge of the trend and determinants of under-5 mortality are essential for effective design of intervention programmes that will enhance their survival. We aimed to examine the mortality patterns in under-5 children and maternal factors associated with under-5 deaths. METHODS:Demographic and Health Survey (DHS) data from five sub-Sahara Africa countries; Chad, Democratic Republic of Congo, Mali, Niger and Zimbabwe were used in this study. The sample size consisted of 68,085 women aged 15-49 years with at least one history of childbirth. The outcome variable was under-five mortality rate. Relevant information on maternal factors were extracted for analysis. Multivariable Cox proportional hazards regression was used to model maternal factors associated with under-five mortality. RESULTS:The current under-5 mortality rate (per 1,000 live births) was; 133 in Republic of Chad, 104 in Democratic Republic of Congo, 95 in Mali, 127 in Niger, and 69 in Zimbabwe. Several maternal and child level factors were found to be significantly associated with under-five mortality. Lack of spousal support (not currently married) resulted to increase in under-five mortality (Chad- Hazard Ratio [HR] = 1.11, 95%CI = 0.97-1.25; DR Congo- HR = 1.24, 95%CI = 1.11-1.40; Mali- HR = 2.43, 95%CI = 1.63-3.64; Niger- HR = 1.59, 95%CI = 1.24-2.03; Zimbabwe- HR = 1.33, 95%CI = 1.06-1.67). Delivery by caesarean section was significantly associated with under-five mortality (Chad- HR = 1.32, 95%CI = 1.00-1.77; DR Congo- HR = 1.20, 95%CI = 1.01-1.43; Mali- HR = 1.42, 95%CI = 1.08-1.85; Niger- HR = 1.43, 95%CI = 1.06-1.92; Zimbabwe- HR = 1.49, 95%CI = 1.03-2.15). CONCLUSION:Despite concerted effort by government and several stakeholders in health to improve childhood survival, the rate of under-5 mortality is still high. Our findings provided evidence on the contribution of maternal age, place of residence, household wealth index, level of education, employment, marital status, religious background, birth type, birth order and interval, sex and size of child, place and mode of delivery, to Under-5 mortality rate in SSA. The position of prominent risk factors for under-five mortality should be addressed through effective design of timely and efficient intervention aimed at reducing childhood mortality.

The nick translation property of DNA polymerase I (Pol I) ensures the maturation of Okazaki fragments by removing primer RNAs and facilitating ligation. However, prolonged nick translation traversing downstream DNA is an energy wasting futile process, as Pol I simultaneously polymerizes and depolymerizes at the nick sites utilizing energy-rich dNTPs. Using an in vitro assay system, we demonstrate that the β-clamp of the Escherichia coli replisome strongly inhibits nick translation on the DNA substrate. To do so, β-clamp inhibits the strand displacement activity of Pol I by interfering with the interaction between the finger subdomain of Pol I and the downstream primer-template junction. Conversely, β-clamp stimulates the 5' exonuclease property of Pol I to cleave single nucleotides or shorter oligonucleotide flaps. This single nucleotide flap removal at high frequency increases the probability of ligation between the upstream and downstream DNA strands at an early phase, terminating nick translation. Besides β-clamp-mediated ligation helps DNA ligase to seal the nick promptly during the maturation of Okazaki fragments.

BACKGROUND:Healthcare systems in Sub-Saharan Africa (SSA) are fraught with numerous governance and infrastructural issues including lack of access to quality care and health worker shortage. Policy makers are becoming increasingly interested in adopting novel technologies such as web-based interventions within the scope of e-Health to bridge the gaps in care delivery in a cost-effective and sustainable manner. Successful implementation of these policies is reliant on evidences regarding people's access to these technologies, which are scarce for countries in SSA. OBJECTIVES:To 1) investigate the variation in the prevalence of accessing computer and internet across regional and socioeconomic groups, and 2) assess association between ever accessing computer and internet and knowledge of routes and risk factors of HIV transmission in selected SSA countries. METHODS:We analyzed cross-sectional datasets from UNICEF Multiple Indicator Cluster Surveys. Participants were16,194 men and 39,121 women from Ghana, Guinea Bissau, Malawi and Zimbabwe. Main outcome variable was ever-accessing computer and Internet for any purpose. Associations were assessed by multivariable regression methods. RESULTS:Lifetime computer usage in Ghana, Guinea Bissau, Malawi and Zimbabwe was respectively 21.5%, 13.4%, 12.3%, 28.4% among men, and 12.5%, 8.3%, 4.8%, 20.5% among women, and that of internet was 14.9%, 11.7%, 10.8%, 34% among men, and 6.4%, 6.9%, 4.2%, 21.6% among women in the aforementioned order. Participants who reported ever using computer and Internet were more likely to have higher knowledge regarding the transmission of HIV compared to those who did not. CONCLUSIONS:Prevalence of lifetime access to computer and Internet was considerably low in all four countries. Several socioeconomic factors appeared to be associated with the access to computer and Internet, addressing which might prove beneficial for the successful expansion e-Health in these countries.

With less than 3200 wild tigers in 2010, the heads of 13 tiger-range countries committed to doubling the global population of wild tigers by 2022. This goal represents the highest level of ambition and commitment required to turn the tide for tigers in the wild. Yet, ensuring efficient and targeted implementation of conservation actions alongside systematic monitoring of progress towards this goal requires that we set site-specific recovery targets and timelines that are ecologically realistic. In this study, we assess the recovery potential of 18 sites identified under WWF's Tigers Alive Initiative. We delineated recovery systems comprising a source, recovery site, and support region, which need to be managed synergistically to meet these targets. By using the best available data on tiger and prey numbers, and adapting existing species recovery frameworks, we show that these sites, which currently support 165 (118-277) tigers, have the potential to harbour 585 (454-739) individuals. This would constitute a 15% increase in the global population and represent over a three-fold increase within these specific sites, on an average. However, it may not be realistic to achieve this target by 2022, since tiger recovery in 15 of these 18 sites is contingent on the initial recovery of prey populations, which is a slow process. We conclude that while sustained conservation efforts can yield significant recoveries, it is critical that we commit our resources to achieving the biologically realistic targets for these sites even if the timelines are extended.

People living with HIV/AIDS (PLH) experience high rates of depression and related psychosocial risk factors that vary by gender. This study examines gender differences in depression severity among antiretroviral therapy (ART) patients (n = 362) from a large government ART clinic in Kolkata, India. Hypotheses for multiple linear regression models were guided by an integrated gendered stress process model focusing on variables reflecting social status (age, partner status), stressors (stigma), and resources (income, social support). Depressive symptoms were assessed with the Hospital Anxiety and Depression Scale (HADS); 22% of the sample reached the cutoff for severe depression, 56% moderate, and 13% mild depression. Compared to men, women reported lower income, education (50% no formal education vs. 20% men), availability of emotional and instrumental support, and were less likely to be married or cohabiting (53% women vs. 72% of men). However, more women had partners who were HIV-positive (78% women vs. 46% men). Overall, depression severity was negatively associated with availability of emotional support and self-distraction coping, and positively associated with internalized HIV/AIDS stigma, availability of instrumental support, and behavioral disengagement coping. Interactions for instrumental support by income and partner status by age varied significantly by gender. Analyses stratified by gender indicated that: 1) Frequently seeking instrumental support from others was protective for men at all income levels, but only for high-income women; and 2) having a partner was protective for men as they aged, but not for women. These results suggest that gender disparities in depression severity are created and maintained by women's lower social status and limited access to resources. The effect of stigma on depression severity did not vary by gender. These findings may inform the tailoring of future interventions to address mental health needs of PLH in India, particularly gender disparities in access to material and social resources for coping with HIV. Trial Registration: ClinicalTrials.gov registration #NCT02118454, registered April 2014.

Obstetric complications and maternal deaths can be prevented through safe delivery process. Facility based delivery significantly reduces maternal mortality by increasing women's access to skilled personnel attendance. However, in sub-Saharan Africa, most deliveries take place without skilled attendants and outside health facilities. Utilization of facility-based delivery is affected by socio-cultural norms and several other factors including cost, long distance, accessibility and availability of quality services. This study examined country-level variations of the self-reported causes of not choosing to deliver at a health facility.Cross-sectional data on 37,086 community dwelling women aged between 15-49 years were collected from DHS surveys in Ethiopia (n = 13,053) and Nigeria (n = 24,033). Outcome variables were the self-reported causes of not delivering at health facilities which were regressed against selected sociodemographic and community level determinants. In total eight items complaints were identified for non-use of facility delivery: 1) Cost too much 2) Facility not open, 3) Too far/no transport, 4) don't trust facility/poor service, 5) No female provider, 6) Husband/family didn't allow, 7) Not necessary, 8) Not customary. Multivariable regression methods were used for measuring the associations.In both countries a large proportion of the women mentioned facility delivery as not necessary, 54.9% (52.3-57.9) in Nigeria and 45.4% (42.0-47.5) in Ethiopia. Significant urban-rural variations were observed in the prevalence of the self-reported causes of non-utilisation. Women in the rural areas are more likely to report delivering at health facility as not customary/not necessary and healthy facility too far/no transport. However, urban women were more likely to complain that husband/family did not allow and that the costs were too high.Women in the rural were more likely to regard facility delivery as unnecessary and complain about transportation and financial difficulties. In order to achieving the maternal mortality related targets, addressing regional disparities in accessing maternal healthcare services should be regarded as a priority of health promotion programs in Nigeria and Ethiopia.

The source populations of tigers are mostly confined to protected areas, which are now becoming isolated. A landscape scale conservation strategy should strive to facilitate dispersal and survival of dispersing tigers by managing habitat corridors that enable tigers to traverse the matrix with minimal conflict. We present evidence for tiger dispersal along transboundary protected areas complexes in the Terai Arc Landscape, a priority tiger landscape in Nepal and India, by comparing camera trap data, and through population models applied to the long term camera trap data sets. The former showed that 11 individual tigers used the corridors that connected the transboundary protected areas. The estimated population growth rates using the minimum observed population size in two protected areas in Nepal, Bardia National Park and Suklaphanta National Park showed that the increases were higher than expected from growth rates due to in situ reproduction alone. These lines of evidence suggests that tigers are recolonizing Nepal's protected areas from India, after a period of population decline, and that the tiger populations in the transboundary protected areas complexes may be maintained as meta-population. Our results demonstrate the importance of adopting a landscape-scale approach to tiger conservation, especially to improve population recovery and long term population persistence.

INTRODUCTION:Bangladesh has made remarkable progress towards reducing its maternal mortality rate (MMR) over the last two decades and is one of the few countries on track to achieving the MMR-related Millennium Development Goals (MDG-5A). However, the provision of universal access to reproductive healthcare (MDG-5B) and the utilization of maternal healthcare services (MHS) such as institutional delivery, which are crucial to the reduction of maternal mortality, are far behind the internationally agreed-upon target. Effective policymaking to promote the utilization of MHS can be greatly facilitated by the identification of the factors that hinder service uptake. In this study, we therefore aim to measure the prevalence of institutional delivery services and explore the factors associated with their utilization in Bangladesh. METHODS:Data for this study were extracted from the 2011 Bangladesh Demographic and Health Survey (BDHS, 2011); participants were 7,313 women between the ages of 15 and 49 years, selected from both urban and rural households. Data were analyzed using Chi-square analysis, and conditional logistic regression. RESULTS:According to the findings, fewer than one in three women reported delivering at a health facility. The multivariable regression analysis showed that participants from rural areas were 46.9% less likely to have institutional deliveries compared to urban dwellers (OR = 0.531; p

Clinical trials in severe falciparum malaria require a large sample size to detect clinically meaningful differences in mortality. This means few interventions can be evaluated at any time. Using a validated surrogate endpoint for mortality would provide a useful alternative allowing a smaller sample size. Here we evaluate changes in coma score and plasma lactate as surrogate endpoints for mortality in severe falciparum malaria.Three datasets of clinical studies in severe malaria were re-evaluated: studies from Chittagong, Bangladesh (adults), the African 'AQUAMAT' trial comparing artesunate and quinine (children), and the Vietnamese 'AQ' study (adults) comparing artemether with quinine. The absolute change, relative change, slope of the normalization over time, and time to normalization were derived from sequential measurements of plasma lactate and coma score, and validated for their use as surrogate endpoint, including the proportion of treatment effect on mortality explained (PTE) by these surrogate measures.Improvements in lactate concentration or coma scores over the first 24 hours of admission, were strongly prognostic for survival in all datasets. In hyperlactataemic patients in the AQ study (n = 173), lower mortality with artemether compared to quinine closely correlated with faster reduction in plasma lactate concentration, with a high PTE of the relative change in plasma lactate at 8 and 12 hours of 0.81 and 0.75, respectively. In paediatric patients enrolled in the 'AQUAMAT' study with cerebral malaria (n = 785), mortality was lower with artesunate compared to quinine, but this was not associated with faster coma recovery.The relative changes in plasma lactate concentration assessed at 8 or 12 hours after admission are valid surrogate endpoints for severe malaria studies on antimalarial drugs or adjuvant treatments aiming at improving the microcirculation. Measures of coma recovery are not valid surrogate endpoints for mortality.

In recent years, Bangladesh government has accomplished the ambitious project of establishing hospitals 18,000 Community Health Clinics in sub-districts across the country. Operating under the affiliation of the government hospitals, these community health clinics aim to provide free healthcare services and to increase health-awareness among the extreme poor communities in the rural areas. However, a great proportion of the people are still not well aware of the services offered by the community health clinics. Thus, it is imperative to identify the factors of awareness regarding the community clinics. Research-based evidence is necessary to improve the efficacy and service coverage of community clinics among key population.Cross-sectional data of size 11,673 women aged 15 to 49 years living in rural settings across seven divisions were extracted from the latest Bangladesh Demographic and Health Survey 2014. The main outcome measures of our study were awareness and utilization of Community Clinic Services (CCs). Descriptive statistics were used to present the baseline socio-demographic and economic characteristics; Chi-square test and logistic regression were performed to identify the factors associated with awareness of community clinics.About one-third (36.7%) of the women were aware of community clinics. Geographical location, level of education, household wealth status and frequency of reading newspaper were found to be significantly associated with awareness about community clinic services. Services reported to be obtained in the community clinics include family planning, immunization, tetanus, antenatal care, vitamin A, and health care for children and child growth monitoring. In the multivariate logistic regression, the odds of awareness among participants with primary education [p

Malaria remains a major public health issue in most southern African countries as the disease remains hyper endemic. Burkina Faso continues to face challenges in the treatment of malaria, as the utilization of preventive measures remains low on a national scale. While it has been acknowledged that understanding women's health-seeking behaviours, perception of malaria and its preventive measures will aid in the control of malaria, there is paucity of information on Knowledge, Attitudes and Practices among women in the reproductive age of 15-49 years in Burkina Faso. This study investigated women's knowledge of malaria, attitudes towards malaria, and practices of malaria control in order to create a synergy between community efforts and governmental/non-governmental malaria control interventions in Burkina Faso.The analysis used data from the 2014 Burkina Faso Malaria Indicator Survey (MIS). In total 8111 women aged between 15-49 years were included in the present study. We assessed women's knowledge about 1) preventive measures, 2) causes and 3) symptoms of malaria, as well as malaria prevention practices for their children and during pregnancy. The socio-demographic characteristics were considered for Age, Religion, Education, Wealth index, Number of household members, Sex of household head, Household possession of radio, TV and Received antenatal care. Data were analyzed using STATA, version 14. Associations between variables were tested using a Chi-square and logistic regression, with the level of statistical significance set at 95%.A preponderant proportion of respondents were aged 15-29 years (mean age was 28.63±9.41). About three-quarters of the respondents had no formal education. An estimated two-third of the participants were of Islamic faith, while access to media and behavioural communication were generally poor. The level of knowledge was 53% for rural women and 68.2% for urban dwellers. In sum, there was 56.1% level of accurate knowledge of malaria among women in Burkina Faso. In the multivariable logistic regression, women in rural location had 40% reduction in the odds of having accurate knowledge of malaria when compared to urban women (aOR = 0.60; 95%CI: 0.52-0.68). The educational level was a key factor in the knowledge of malaria. The odds of having accurate knowledge of malaria increased as the educational level increased, hence, women with secondary and higher education had 29% and 93% increase in the odds of having accurate knowledge of malaria when compared to the women without formal education. Results indicate that antenatal care (ANC) services were major sources of information on malaria. Women who reportedly received ANC were 3.9 times more likely to have accurate knowledge of malaria when compared to those who did not utilize skilled ANC services (aOR = 3.90; 95%CI = 3.34-4.56).The overall knowledge of malaria prevention practices among a large proportion of women was found to be low, which implies that the knowledge about the prevention of malaria should be improved upon by both urban and rural dwellers. There is need for concerted behavioural communication intervention to improve the knowledge of malaria especially for rural dwellers regarding malaria prevention measures, causes and symptoms. Consistent efforts at providing relevant information by health organizations are needed to reduce and control incidences of malaria in the general public.

Although ANC services are increasingly available to women in low and middle-income countries, their inadequate use persists. This suggests a misalignment between aims of the services and maternal beliefs and circumstances. Owing to the dearth of studies examining the timing and adequacy of content of care, this current study aims to investigate the timing and frequency of ANC visits in Ethiopia.Data was obtained from the nationally representative 2011 Ethiopian Demographic and Health Survey (EDHS) which used a two-stage cluster sampling design to provide estimates for the health and demographic variables of interest for the country. Our study focused on a sample of 10,896 women with history of at least one childbirth event. Percentages of timing and adequacy of ANC visits were conducted across the levels of selected factors. Variables which were associated at 5% significance level were examined in the multivariable logistic regression model for association between timing and frequency of ANC visits and the explanatory variables while controlling for covariates. Furthermore, we presented the approach to estimate marginal effects involving covariate-adjusted logistic regression with corresponding 95%CI of delayed initiation of ANC visits and inadequate ANC attendance. The method used involved predicted probabilities added up to a weighted average showing the covariate distribution in the population.Results indicate that 66.3% of women did not use ANC at first trimester and 22.3% had ANC less than 4 visits. The results of this study were unique in that the association between delayed ANC visits and adequacy of ANC visits were examined using multivariable logistic model and the marginal effects using predicted probabilities. Results revealed that older age interval has higher odds of inadequate ANC visits. More so, type of place of residence was associated with delayed initiation of ANC visits, with rural women having the higher odds of delayed initiation of ANC visits (OR = 1.65; 95%CI: 1.26-2.18). However, rural women had 44% reduction in the odds of having inadequate ANC visits. In addition, multi-parity showed higher odds of delayed initiation of ANC visit when compared to the primigravida (OR = 2.20; 95%CI: 1.07-2.69). On the contrary, there was 36% reduction in the odds of multigravida having inadequate ANC visits when compared to the women who were primigravida. There were higher odds of inadequacy in ANC visits among women who engaged in sales/business, agriculture, skilled manual and other jobs when compared to women who currently do not work, after adjusting for covariates. From the predictive margins, assuming the distribution of all covariates remained the same among respondents, but everyone was aged 15-19 years, we would expect 71.8% delayed initiation of ANC visit. If everyone was aged 20-24years, 73.4%; 25-29years, 66.5%; 30-34years, 64.8%; 35-39years, 65.6%; 40-44years, 59.6% and 45-49years, we would expect 70.1% delayed initiation of ANC visit. If instead the distribution of age was as observed and for other covariates remained the same among respondents, but no respondent lived in the rural, we would expect about 61.4% delayed initiation of ANC visit; if however, everyone lived in the rural, and we would expect 71.6% delayed initiation in ANC visit. Model III revealed the predictive margins of all factors examined for delayed initiation for ANC visits, while Model IV presented the predictive marginal effects of the determinants of adequacy of ANC visits.The precise mechanism by which these factors affect ANC visits remain blurred at best. There may be factors on the demand side like the women's empowerment, financial support of the husband, knowledge of ANC visits in the context of timing, frequency and the expectations of ANC visits might be mediating the effects through the factors found associated in this study. Supply side factors like the quality of ANC services, skilled staff, and geographic location of the health centers also mediate their effects through the highlighted factors. Irrespective of the knowledge about the precise mechanism of action, policy makers could focus on improving women's empowerment, improving women's education, reducing wealth inequity and facilitating improved utilization of ANC through modifications on the supply side factors such as geographic location and focus on hard to reach women.

Asian pangolins are a highly-threatened species group, mainly due to the perceived medicinal value of their scales. Increased demand from China has resulted in pangolins being the most trafficked mammal in the world. Three pangolin species are reported to occur in Bangladesh: Manis pentadactyla, M. crassicaudata, and M. javanica. No peer-reviewed studies exist detailing these species' current distribution or status within Bangladesh. A literature review was conducted resulting in the clarification of conflicting reports and misidentified observations and specimen records. In this paper, we also report the current status of pangolins (Manis spp.) in Bangladesh based on semi-structured interviews, camera trapping, media queries, and field surveys employing traditional ecological knowledge and non-randomized transect surveys. Ethnozoological knowledge pertaining to the natural history of M. pentadactyla is also reported from experienced Mro tribal hunters. The critically endangered M. pentadactyla was verified to occur in northwest, northeast, and southeast Bangladesh in natural and degraded habitats. Interviews with the Mro tribe in the southeast indicate that pangolin populations there were likely extirpated in 2014 due to skilled commercial collection beginning in 2010. Evidence of extant M. crassicaudata and M. javanica populations remain unverified and questionable, and historical records of M. crassicaudata and M. javanica are likely a result of misidentification.

Amphibian populations are vanishing worldwide. Declines and extinctions of many populations have been attributed to chytridiomycosis, a disease induced by the pathogenic fungus Batrachochytrium dendrobatidis (Bd). In Africa, however, changes in amphibian assemblages were typically attributed to habitat change. We conducted a retrospective study utilizing field surveys from 2004-2012 of the anuran faunas on two mountains in western Cameroon, a hotspot of African amphibian diversity. The number of species detected was negatively influenced by year, habitat degradation, and elevation, and we detected a decline of certain species. Because another study in this region revealed an emergence of Bd in 2008, we screened additional recent field-collected samples and also pre-decline preserved museum specimens for the presence of Bd supporting emergence before 2008. When comparing the years before and after Bd detection, we found significantly diminished frog species richness and abundance on both mountains after Bd emergence. Our analyses suggest that this may be the first disease-driven community-level decline in anuran biodiversity in Central Africa. The disappearance of several species known to tolerate habitat degradation, and a trend of stronger declines at higher elevations, are consistent with Bd-induced declines in other regions. Not all species decreased; populations of some species remained constant, and others increased after the emergence of Bd. This variation might be explained by species-specific differences in infection probability. Increased habitat protection and Bd-mitigation strategies are needed for sustaining diverse amphibian communities such as those on Mt. Manengouba, which contains nearly half of Cameroon's frog diversity.

The role of nuclear receptor PXR in detoxification and clearance of xenobiotics and endobiotics is well-established. However, its projected role in hepatic cancer is rather illusive where its expression is reported altered in different cancers depending on the tissue-type and microenvironment. The expression of PXR, its target genes and their biological or clinical significance have not been examined in hepatic cancer. In the present study, by generating DEN-induced hepatic cancer in mice, we report that the expression of PXR and its target genes CYP3A11 and GSTa2 are down-regulated implying impairment of hepatic detoxification capacity. A higher state of inflammation was observed in liver cancer tissues as evident from upregulation of inflammatory cytokines IL-6 and TNF-α along with NF-κB and STAT3. Our data in mouse model suggested a negative correlation between down-regulation of PXR and its target genes with that of higher expression of inflammatory proteins (like IL-6, TNF-α, NF-κB). In conjunction, our findings with relevant cell culture based assays showed that higher expression of PXR is involved in reduction of tumorigenic potential in hepatic cancer. Overall, the findings suggest that inflammation influences the expression of hepatic proteins important in drug metabolism while higher PXR level reduces tumorigenic potential in hepatic cancer.

OBJECTIVES:The present study was undertaken to investigate the mutations that are present in mexR gene of multidrug resistant (MDR) isolates of Pseudomonas aeruginosa collected from a tertiary referral hospital of north east India. METHODS:76 MDR clinical isolates of P. aeruginosa were obtained from the patients who were admitted to or attended the clinics of Silchar medical college and hospital. They were screened phenotypically for the presence of efflux pump activity by an inhibitor based method. Acquired resistance mechanisms were detected by multiplex PCR. Real time PCR was performed to study the expression of mexA gene of MexAB-OprM efflux pump in isolates with increase efflux pump activity. mexR gene of the isolates with overexpressed MexAB-OprM efflux pump was amplified, sequenced and analysed. RESULTS:Out of 76 MDR isolates, 24 were found to exhibit efflux pump activity phenotypically against ciprofloxacin and meropenem. Acquired resistance mechanisms were absent in 11 of them and among those isolates, 8 of them overexpressed MexAB-OprM. All the 8 isolates possessed mutation in mexR gene. 11 transversions, 4 transitions, 2 deletion mutations and 2 insertion mutations were found in all the isolates. However, the most significant observation was the formation of a termination codon at 35th position which resulted in the termination of the polypeptide and leads to overexpression of the MexAB-OprM efflux pump. CONCLUSIONS:This study highlighted emergence of a novel mutation which is probably associated with multi drug resistance. Therefore, further investigations and actions are needed to prevent or at least hold back the expansion and emergence of newer mutations in nosocomial pathogens which may compromise future treatment options.

Flax secoisolariciresinol diglucoside (SDG) lignan is a natural phytoestrogen for which a positive role in metabolic diseases is emerging. Until recently however, much less was known about SDG and its monoglucoside (SMG) biosynthesis. Lately, flax UGT74S1 was identified and characterized as an enzyme sequentially glucosylating secoisolariciresinol (SECO) into SMG and SDG when expressed in yeast. However, the amino acids critical for UGT74S1 glucosyltransferase activity were unknown. A 3D structural modeling and docking, site-directed mutagenesis of five amino acids in the plant secondary product glycosyltransferase (PSPG) motif, and enzyme assays were conducted. UGT74S1 appeared to be structurally similar to the Arabidopsis thaliana UGT72B1 model. The ligand docking predicted Ser357 and Trp355 as binding to the phosphate and hydroxyl groups of UDP-glucose, whereas Cys335, Gln337 and Trp355 were predicted to bind the 7-OH, 2-OCH3 and 17-OCH3 of SECO. Site-directed mutagenesis of Cys335, Gln337, His352, Trp355 and Ser357, and enzyme assays revealed an alteration of these binding sites and a significant reduction of UGT74S1 glucosyltransferase catalytic activity towards SECO and UDP-glucose in all mutants. A complete abolition of UGT74S1 activity was observed when Trp355 was substituted to Ala355 and Gly355 or when changing His352 to Asp352, and an altered metabolite profile was observed in Cys335Ala, Gln337Ala, and Ser357Ala mutants. This study provided for the first time evidence that Trp355 and His352 are critical for UGT74S1's glucosylation activity toward SECO and suggested the possibility for SMG production in vitro.

δ-Catenin, an adherens junctions protein, is not only involved in early development, cell-cell adhesion and cell motility in neuronal cells, but it also plays an important role in vascular endothelial cell motility and pathological angiogenesis. In this study, we report a new function of δ-catenin in lymphangiogenesis. Consistent with expression of δ-catenin in vascular endothelial cells, we detected expression of the gene in lymphatic endothelial cells (LECs). Ectopic expression of δ-catenin in LECs increased cell motility and lymphatic vascular network formation in vitro and lymphangiogenesis in vivo in a Matrigel plug assay. Conversely, knockdown of δ-catenin in LECs impaired lymphangiogenesis in vitro and in vivo. Biochemical analysis shows that δ-catenin regulates activation of Rho family small GTPases, key mediators in cell motility. δ-catenin activates Rac1 and Cdc42 but inhibits RhoA in LECs. Notably, blocking of Rac1 activation impaired δ-catenin mediated lymphangiogenesis in a Matrigel assay. Consistently, loss of δ-catenin in mice inhibited the growth of tumor metastases. Taken together, these findings identify a new function of δ-catenin in lymphangiogenesis and tumor growth/metastasis, likely through modulation of small Rho GTPase activation. Targeting δ-catenin may offer a new way to control tumor metastasis.

Latent fingerprint examination is a complex task that, despite advances in image processing, still fundamentally depends on the visual judgments of highly trained human examiners. Fingerprints collected from crime scenes typically contain less information than fingerprints collected under controlled conditions. Specifically, they are often noisy and distorted and may contain only a portion of the total fingerprint area. Expertise in fingerprint comparison, like other forms of perceptual expertise, such as face recognition or aircraft identification, depends on perceptual learning processes that lead to the discovery of features and relations that matter in comparing prints. Relatively little is known about the perceptual processes involved in making comparisons, and even less is known about what characteristics of fingerprint pairs make particular comparisons easy or difficult. We measured expert examiner performance and judgments of difficulty and confidence on a new fingerprint database. We developed a number of quantitative measures of image characteristics and used multiple regression techniques to discover objective predictors of error as well as perceived difficulty and confidence. A number of useful predictors emerged, and these included variables related to image quality metrics, such as intensity and contrast information, as well as measures of information quantity, such as the total fingerprint area. Also included were configural features that fingerprint experts have noted, such as the presence and clarity of global features and fingerprint ridges. Within the constraints of the overall low error rates of experts, a regression model incorporating the derived predictors demonstrated reasonable success in predicting objective difficulty for print pairs, as shown both in goodness of fit measures to the original data set and in a cross validation test. The results indicate the plausibility of using objective image metrics to predict expert performance and subjective assessment of difficulty in fingerprint comparisons.

Current, commercial, electrode micro-drives that allow independent positioning of multiple electrodes are expensive. Custom designed solutions developed by individual laboratories require fabrication by experienced machinists working in well equipped machine shops and are therefore difficult to disseminate into widespread use. Here, we present an easy to assemble modular micro-drive system for acute primate neurophysiology (PriED) that utilizes rapid prototyping (3-d printing) and readily available off the shelf-parts. The use of 3-d printed parts drastically reduces the cost of the device, making it available to labs without the resources of sophisticated machine shops. The direct transfer of designs from electronic files to physical parts also gives researchers opportunities to easily modify and implement custom solutions to specific recording needs. We also demonstrate a novel model of data sharing for the scientific community: a publicly available repository of drive designs. Researchers can download the drive part designs from the repository, print, assemble and then use the drives. Importantly, users can upload their modified designs with annotations making them easily available for others to use.

Deregulation of miRNA expression may contribute to tumorigenesis and other patho-physiology associated with cancer. Using TLDA, expression of 762 miRNAs was checked in 18 pairs of gingivo buccal cancer-adjacent control tissues. Expression of significantly deregulated miRNAs was further validated in cancer and examined in two types of precancer (leukoplakia and lichen planus) tissues by primer-specific TaqMan assays. Biological implications of these miRNAs were assessed bioinformatically. Expression of hsa-miR-1293, hsa-miR-31, hsa-miR-31* and hsa-miR-7 were significantly up-regulated and those of hsa-miR-206, hsa-miR-204 and hsa-miR-133a were significantly down-regulated in all cancer samples. Expression of only hsa-miR-31 was significantly up-regulated in leukoplakia but none in lichen planus samples. Analysis of expression heterogeneity divided 18 cancer samples into clusters of 13 and 5 samples and revealed that expression of 30 miRNAs (including the above-mentioned 7 miRNAs), was significantly deregulated in the cluster of 13 samples. From database mining and pathway analysis it was observed that these miRNAs can significantly target many of the genes present in different cancer related pathways such as "proteoglycans in cancer", PI3K-AKT etc. which play important roles in expression of different molecular features of cancer. Expression of hsa-miR-31 was significantly up-regulated in both cancer and leukoplakia tissues and, thus, may be one of the molecular markers of leukoplakia which may progress to gingivo-buccal cancer.

Polycystic ovary syndrome (PCOS) represents the most common cause of anovulatory infertility and affects 6-15% of women of reproductive age. However, the underlying etiology is still poorly understood. In this study, we attempted to examine the association between circulating heat shock protein 70 (Hsp70) concentrations and PCOS in a non-obese Chinese population.Human peripheral blood from 52 patients with PCOS and 57 healthy controls, matched for age and BMI, were analyzed. Women with PCOS were found to have significantly higher fasting insulin (FI) levels, as well as Insulin resistance index (HOMA-IR) (P < 0.05). Identically, markers of oxidative stress (malondialdehyde (MDA), 8-Hydroxy-desoxyguanosine (8-OHdG), Nitric oxide (NO)) and inflammation (tumor necrosis factor-alpha (TNF-α), C-reactive protein (CRP)) were markedly increased when compared to controls (P < 0.05). Elevated serum Hsp70 was positively correlated with IR, oxidative stress and inflammation in PCOS, even after adjustment for age, BMI and gynecologic inflammation (GI). The receiver-operating characteristic curve (ROC) analysis yielded notably different discriminative value for PCOS, with or without an addition of Hsp70 (areas under the curves were 0.884 (95% CI 0.822-0.946) vs. 0.822 (95% CI 0.744-0.900); P for difference = 0.015).Increased serum Hsp70 levels are associated with the combination of IR, oxidative stress and low-grade chronic inflammation in PCOS individuals, which provides supportive evidence that Hsp70 plays a key role in the pathogenesis of PCOS. More consequent studies were warranted to confirm the clinical utility of circulating Hsp70, especially in diagnosis and prognosis of PCOS and its long-term health cost.

Glycine N-methyltransferase (GNMT), an abundant cytosolic enzyme, catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to glycine generating S-adenosylhomocysteine and sarcosine (N-methylglycine). This reaction is regulated by 5-methyltetrahydrofolate, which inhibits the enzyme catalysis. In the present study, we observed that GNMT is strongly down regulated in human cancers and is undetectable in cancer cell lines while the transient expression of the protein in cancer cells induces apoptosis and results in the activation of ERK1/2 as an early pro-survival response. The antiproliferative effect of GNMT can be partially reversed by treatment with the pan-caspase inhibitor zVAD-fmk but not by supplementation with high folate or SAM. GNMT exerts the suppressor effect primarily in cells originated from malignant tumors: transformed cell line of non-cancer origin, HEK293, was insensitive to GNMT. Of note, high levels of GNMT, detected in regenerating liver and in NIH3T3 mouse fibroblasts, do not produce cytotoxic effects. Importantly, GNMT, a predominantly cytoplasmic protein, was translocated into nuclei upon transfection of cancer cells. The presence of GNMT in the nuclei was also observed in normal human tissues by immunohistochemical staining. We further demonstrated that the induction of apoptosis is associated with the GNMT nuclear localization but is independent of its catalytic activity or folate binding. GNMT targeted to nuclei, through the fusion with nuclear localization signal, still exerts strong antiproliferative effects while its restriction to cytoplasm, through the fusion with nuclear export signal, prevents these effects (in each case the protein was excluded from cytosol or nuclei, respectively). Overall, our study indicates that GNMT has a secondary function, as a regulator of cellular proliferation, which is independent of its catalytic role.

Protein kinases play key roles in oncogenic signaling and are a major focus in the development of targeted cancer therapies. Imatinib, a BCR-Abl tyrosine kinase inhibitor, is a successful front-line treatment for chronic myelogenous leukemia (CML). However, resistance to imatinib may be acquired by BCR-Abl mutations or hyperactivation of Src family kinases such as Lyn. We have used multiplexed kinase inhibitor beads (MIBs) and quantitative mass spectrometry (MS) to compare kinase expression and activity in an imatinib-resistant (MYL-R) and -sensitive (MYL) cell model of CML. Using MIB/MS, expression and activity changes of over 150 kinases were quantitatively measured from various protein kinase families. Statistical analysis of experimental replicates assigned significance to 35 of these kinases, referred to as the MYL-R kinome profile. MIB/MS and immunoblotting confirmed the over-expression and activation of Lyn in MYL-R cells and identified additional kinases with increased (MEK, ERK, IKKα, PKCβ, NEK9) or decreased (Abl, Kit, JNK, ATM, Yes) abundance or activity. Inhibiting Lyn with dasatinib or by shRNA-mediated knockdown reduced the phosphorylation of MEK and IKKα. Because MYL-R cells showed elevated NF-κB signaling relative to MYL cells, as demonstrated by increased IκBα and IL-6 mRNA expression, we tested the effects of an IKK inhibitor (BAY 65-1942). MIB/MS and immunoblotting revealed that BAY 65-1942 increased MEK/ERK signaling and that this increase was prevented by co-treatment with a MEK inhibitor (AZD6244). Furthermore, the combined inhibition of MEK and IKKα resulted in reduced IL-6 mRNA expression, synergistic loss of cell viability and increased apoptosis. Thus, MIB/MS analysis identified MEK and IKKα as important downstream targets of Lyn, suggesting that co-targeting these kinases may provide a unique strategy to inhibit Lyn-dependent imatinib-resistant CML. These results demonstrate the utility of MIB/MS as a tool to identify dysregulated kinases and to interrogate kinome dynamics as cells respond to targeted kinase inhibition.

The mechanisms by which MAP kinases recognize and phosphorylate substrates are not completely understood. Efforts to understand the mechanisms have been compromised by the lack of MAPK-substrate structures. While MAPK-substrate docking is well established as a viable mechanism for bringing MAPKs and substrates into close proximity the molecular details of how such docking promotes phosphorylation is an unresolved issue. In the present study computer modeling approaches, with restraints derived from experimentally known interactions, were used to predict how the N-terminus of Ets-1 associates with ERK2. Interestingly, the N-terminus does not contain a consensus-docking site ((R/K)(2-3)-X(2-6)-Φ(A)-X-Φ(B), where Φ is aliphatic hydrophobic) for ERK2. The modeling predicts that the N-terminus of Ets-1 makes important contributions to the stabilization of the complex, but remains largely disordered. The computer-generated model was used to guide mutagenesis experiments, which support the notion that Leu-11 and possibly Ile-13 and Ile-14 of Ets-1 1-138 (Ets) make contributions through binding to the hydrophobic groove of the ERK2 D-recruiting site (DRS). Based on the modeling, a consensus-docking site was introduced through the introduction of an arginine at residue 7, to give the consensus (7)RK-X(2)-Φ(A)-X-Φ(B) (13). This results in a 2-fold increase in k(cat)/K(m) for the phosphorylation of Ets by ERK2. Similarly, the substitution of the N-terminus for two different consensus docking sites derived from Elk-1 and MKK1 also improves k(cat)/K(m) by two-fold compared to Ets. Disruption of the N-terminal docking through deletion of residues 1-23 of Ets results in a 14-fold decrease in k(cat)/K(m), with little apparent change in k(cat). A peptide that binds to the DRS of ERK2 affects K(m), but not k(cat). Our kinetic analysis suggests that the unstructured N-terminus provides 10-fold uniform stabilization of the ground state ERK2•Ets•MgATP complex and intermediates of the enzymatic reaction.

Huntington's disease (HD) is caused by the expansion of N-terminal polymorphic poly Q stretch of the protein huntingtin (HTT). Deregulated microRNAs and loss of function of transcription factors recruited to mutant HTT aggregates could cause characteristic transcriptional deregulation associated with HD. We observed earlier that expressions of miR-125b, miR-146a and miR-150 are decreased in STHdh(Q111)/Hdh(Q111) cells, a model for HD in comparison to those of wild type STHdh(Q7)/Hdh(Q7) cells. In the present manuscript, we show by luciferase reporter assays and real time PCR that decreased miR-146a expression in STHdh(Q111)/Hdh(Q111) cells is due to decreased expression and activity of p65 subunit of NFkB (RelA/NFkB). By reporter luciferase assay, RT-PCR and western blot analysis, we also show that both miR-150 and miR-125b target p53. This partially explains the up regulation of p53 observed in HD. Elevated p53 interacts with RelA/NFkB, reduces its expression and activity and decreases the expression of miR-146a, while knocking down p53 increases RelA/NFkB and miR-146a expressions. We also demonstrate that expression of p53 is increased and levels of RelA/NFkB, miR-146a, miR-150 and miR-125b are decreased in striatum of R6/2 mice, a mouse model of HD and in cell models of HD. In a cell model, this effect could be reversed by exogenous expression of chaperone like proteins HYPK and Hsp70. We conclude that (i) miR-125b and miR-150 target p53, which in turn regulates RelA/NFkB and miR-146a expressions; (ii) reduced miR-125b and miR-150 expressions, increased p53 level and decreased RelA/NFkB and miR-146a expressions originate from mutant HTT (iii) p53 directly or indirectly regulates the expression of miR-146a. Our observation of interplay between transcription factors and miRNAs using HD cell model provides an important platform upon which further work is to be done to establish if such regulation plays any role in HD pathogenesis.