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BackgroundIn order for low and middle income countries (LMIC) to transition to Human Papilloma Virus (HPV) test based cervical cancer screening, a greater understanding of how to implement these evidence based interventions (EBI) among vulnerable populations is needed. This paper documents outcomes of an implementation research on HPV screening among women from tribal, rural, urban slum settings in India.MethodsA mixed-method, pragmatic, quasi-experimental trial design was used. HPV screening on self-collected cervical samples was offered to women aged 30-60 years. Implementation strategies were 1) Assessment of contextual factors using both qualitative and quantitative methods like key informant interviews (KII), focus group discussions (FGDs), pre-post population sample surveys, capacity assessment of participating departments 2) enhancing provider capacity through training workshops, access to HPV testing facility, colposcopy, thermal ablation/cryotherapy at the primary health care centers 3) community engagement, counselling for self-sampling and triage process by frontline health care workers (HCWs). Outcomes were assessed using the RE-AIM (Reach, Effectiveness, adoption, implementation, maintenance) framework.ResultsScreening rate in 8 months' of study was 31.0%, 26.7%, 32.9%, prevalence of oncogenic HPV was 12.1%, 3.1%, 5.5%, compliance to triage was 53.6%, 45.5%, 84.6% in tribal, urban slum, rural sites respectively. Pre-cancer among triage compliant HPV positive women was 13.6% in tribal, 4% in rural and 0% among urban slum women. Unique challenges faced in the tribal setting led to programme adaptations like increasing honoraria of community health workers for late-evening work and recalling HPV positive women for colposcopy by nurses, thermal ablation by gynaecologist at the outreach camp site.ConclusionsSelf-collection of samples combined with HCW led community engagement activities, flexible triage processes and strengthening of health system showed an acceptable screening rate and better compliance to triage, highlighting the importance of identifying the barriers and developing strategies suitable for the setting.Trial registrationCTRI/2021/09/036130.

We determined weights for a multi-criteria tool for assessing the relative merits of clinical-trial research proposals, and investigated whether the weights vary across relevant stakeholder groups. A cross-sectional, adaptive discrete choice experiment using 1000minds online software was administered to consumers, researchers and funders affiliated with the Australian Clinical Trials Alliance (ACTA). We identified weights for four criteria-Appropriateness, Significance, Relevance, Feasibility-and their levels, representing their relative importance, so that research proposals can be scored between 0% (nil or very low merit) and 100% (very high merit). From 220 complete survey responses, the most important criterion was Appropriateness (adjusted for differences between stakeholder groups, mean weight 28.9%) and the least important was Feasibility (adjusted mean weight 19.5%). Consumers tended to weight Relevance more highly (2.7% points difference) and Feasibility less highly (3.1% points difference) than researchers. The research or grant writing experience of researchers or consumers was not associated with the weights. A multi-criteria tool for evaluating research proposals that reflects stakeholders' preferences was created. The tool can be used to assess the relative merits of clinical trial research proposals and rank them, to help identify the best proposals for funding.

A healthy lifestyle is defined as ‘a way of living that lowers the risk of being seriously ill or dying early.’ Although barriers and facilitators of healthy lifestyles have been well-studied among populations like those with chronic non-communicable diseases, adolescents, and older adults in Asia, less information is available on barriers and facilitators perceived by the general adult population. Using a qualitative methodology and leveraging the socio-ecological model, the current study aimed to understand the barriers and facilitators of a healthy lifestyle in a sample of Singapore residents. Overall, 30 semi-structured interviews were conducted in English and other local languages from August 2020 to March 2021. Transcripts were analysed using framework analysis. Five main themes pertaining to personal, interpersonal, environmental, socio-cultural, and policy-level factors were classified under the two overarching categories of barriers and facilitators of healthy lifestyles. The results of this study offer important insights into understanding the barriers and facilitators to the adoption of a healthy lifestyle among people in Singapore. Furthermore, our findings illustrate the complex interplay between individuals, social relationships, environment, and policy that can act as either a barrier or a facilitator to adopting a healthy lifestyle.

BackgroundGlobally, complications due to preterm birth are the leading contributor to neonatal mortality, resulting in an estimated one million deaths annually. Kangaroo Mother Care (KMC) has been endorsed by the World Health Organisation as a low cost, safe, and effective intervention in reducing morbidity and mortality among preterm infants. The objective of this study was to describe the implementation of a KMC model among preterm infants and its impact on neonatal outcomes at a tertiary level hospital in Lusaka, Zambia.MethodsWe conducted a prospective descriptive study using data collected from the KMC room at the University Teaching Hospital between January 2016 and September 2017. Mothers and government nurses were trained in KMC. We monitored skin-to-skin and breastfeeding practices, weight at admission, discharge, and length of admission.ResultsWe enrolled 573 neonates into the study. Thirteen extremely low weight infants admitted to the KMC room had graduated to Group A (1,000g-1,499g) at discharge, with a median weight gain of 500g. Of the 419 very low weight neonates at admission, 290 remained in Group A while 129 improved to Group B (1,500g-2,499g), with a median weight gain of 280g. Among the 89 low weight neonates, 1 regressed to Group A, 77 remained in Group B, and 11 improved to Group C (≥2,500g), individually gaining a median of 100g. Of the seven normal weight neonates, 6 remained in Group C individually gaining a median of 100g, and 1 regressed to Group B. Among all infants enrolled, two (0.35%) died in the KMC room.ConclusionsBased on the RE-AIM metrics, our results show that KMC is a feasible intervention that can improve neonatal outcomes among preterm infants in Zambia. The study findings show a promising, practical approach to scaling up KMC in Zambia.Trial registrationThe trial is registered under ClinicalTrials.gov under the following ID number: NCT03923023.

Stereotypical locomotor movements can be made without input from the brain after a complete spinal transection. However, the restoration of functional gait requires descending modulation of spinal circuits to independently control the movement of each limb. To evaluate whether a brain-machine interface (BMI) could be used to regain conscious control over the hindlimb, rats were trained to press a pedal and the encoding of hindlimb movement was assessed using a BMI paradigm. Off-line, information encoded by neurons in the hindlimb sensorimotor cortex was assessed. Next neural population functions, or weighted representations of the neuronal activity, were used to replace the hindlimb movement as a trigger for reward in real-time (on-line decoding) in three conditions: while the animal could still press the pedal, after the pedal was removed and after a complete spinal transection. A novel representation of the motor program was learned when the animals used neural control to achieve water reward (e.g. more information was conveyed faster). After complete spinal transection, the ability of these neurons to convey information was reduced by more than 40%. However, this BMI representation was relearned over time despite a persistent reduction in the neuronal firing rate during the task. Therefore, neural control is a general feature of the motor cortex, not restricted to forelimb movements, and can be regained after spinal injury.

Threatened and endangered green turtles (Chelonia mydas) are unique because as juveniles they recruit from pelagic to near-shore waters and shift from an omnivorous to primarily herbivorous diet (i.e. seagrass and algae). Nevertheless, when injured and ill animals are admitted to rehabilitation, animal protein (e.g. seafood) is often offered to combat poor appetite and emaciation. We examined how the fecal microbiome of juvenile green turtles changed in response to a dietary shift during rehabilitation. We collected fecal samples from January 2014 -January 2016 from turtles (N = 17) in rehabilitation at the Georgia Sea Turtle Center and used next generation sequencing to analyze bacterial community composition. Samples were collected at admission, mid-rehabilitation, and recovery, which entailed a shift from a mixed seafood-vegetable diet at admission to a primarily herbivorous diet at recovery. The dominant phyla changed over time, from primarily Firmicutes (55.0%) with less Bacteroidetes (11.4%) at admission, to primarily Bacteroidetes (38.4%) and less Firmicutes (31.8%) at recovery. While the microbiome likely shifts with the changing health status of individuals, this consistent inversion of Bacteroidetes and Firmicutes among individuals likely reflects the increased need for protein digestion, for which Bacteroidetes are important. Firmicutes are significant in metabolizing plant polysaccharides; thus, fewer Firmicutes may result in underutilization of wild diet items in released individuals. This study demonstrates the importance of transitioning rehabilitating green turtles to an herbivorous diet as soon as possible to afford them the best probability of survival.

Antibiotic administration can be a cause of gastrointestinal disease in horses, creating a disruption in the normal population and function of bacteria found in the hindgut. The objective of this study was to describe the changes in the cecal and fecal microbiomes and metabolomes of clinically healthy horses before and after metronidazole administration. Metronidazole (15 mg/kg BID PO) was given to five horses with cecal cannulas. The study was suspended on Day 3 due to adverse gastrointestinal effects. Cecal and fecal samples were obtained before (Days minus52, m28, m14, and 0) and after (Days 7, 14, 28, and 52) metronidazole administration. DNA was extracted from the cecal and fecal samples, and 16S rRNA genes were sequenced. Richness and evenness indices were significantly decreased by metronidazole administration in both cecal and fecal samples, but the overall composition was only significantly changed in fecal samples on Day 3 (ANOSIM, p = 0.008). The most dominant phyla were Bacteroidetes and Firmicutes in all groups examined. In fecal samples, significant changes of the phyla Actinobacteria, Spirochaetes, Lentisphaerae, and Verrucomicrobia occurred on Day 3, which correlated with clinical signs of gastrointestinal disease. The metabolome was characterized by mass spectrometry-based methods and only named metabolites were included in the analysis. Fecal, but not cecal, metabolites were significantly affected by metronidazole. The fecal metabolites affected represent diverse metabolic pathways, such as the metabolism of amino acids, carbohydrates, lipids, nucleic acids and cofactors and vitamins. Metronidazole administration has potential to cause adverse effects in horses, alters the bacterial composition of the horse's cecal and fecal content, and the metabolome of fecal samples.

We previously reported that feeding Se-biofortified alfalfa hay to weaned beef calves in a preconditioning program decreases morbidity and mortality during the feedlot period. To understand the mode of action by which supranutritional Se supplementation supports calf health, we examined the effect of agronomic Se-biofortification on nasal microbiome and fecal parasites. Recently weaned Angus-cross beef calves (n = 30) were randomly assigned to two groups and fed an alfalfa hay-based diet for 9 weeks in a preconditioning program. Alfalfa hay was harvested from fields fertilized with sodium selenate at a rate of 0 or 90 g Se/ha. Calculated Se intake from dietary sources was 1.09 and 27.45 mg Se/calf per day for calves consuming alfalfa hay with Se concentrations of 0.06 and 3.47 mg Se/kg dry matter, respectively. Feeding Se-biofortified alfalfa hay for 9 weeks was effective at increasing whole-blood Se concentrations (556 ± 11 vs 140 ± 11 ng/mL; P < 0.001) and increasing body weight (PTreatment, = 0.03) in weaned beef calves. Slaughter yield grades were higher for calves that had been fed Se-enriched alfalfa hay during the preconditioning period (PTreatment = 0.008). No significant differences were observed in fecal parasite load, which remained low. The nasal microbiome and microbiota diversity within calves and across calves expanded from weaning (week 0) to the feedlot period (week 12), which was promoted by feeding Se-biofortified alfalfa hay. Especially concerning was the expansion of nasal Mycoplasmataceae in the feedlot, which reached over 50% of the total microbiota in some calves. In conclusion, we identified dietary Se-biofortified alfalfa hay as a potential promoter of nasal microbiome genome and microbiota diversity, which may explain in part high-Se benefits for prevention of bovine respiratory disease complex in beef calves.

Wireless Body Area Network (WBAN) has become the emerging technology due to its ability to provide intelligent and cost-effective healthcare monitoring solution. The biological sensors used in WBAN are energy-constrained and required to be functional for a longer duration. Also, the sensed data should be communicated in reasonable time. Therefore, network lifetime and delay have become the primary concerns in the design of WBAN. In this paper, Receive Diversity based Transmission Data Rate Optimization (RDTDRO) scheme is proposed to improve the network lifetime and delay efficiency of Multi level-Quadrature Amplitude Modulation (M-QAM) based WBAN. In the proposed RDTDRO scheme, minimum energy consumption is ensured by optimizing the transmission data rate with respect to a given transmission distance and number of receive antennas while satisfying the Bit Error Rate (BER) requirements. The performance of proposed RDTDRO is analyzed in terms of network lifetime and delay difference and is compared with conventional Baseline and Rate optimized schemes. The results show that at a transmission distance of 0.3 m, the proposed RDTDRO scheme with a receive diversity order of 4 achieves 1.30 times and 1.27 times improvement in network lifetime over conventional Baseline and Rate optimized schemes respectively. From the results, it is also evident that at a transmission distance of 0.3 m, the proposed RDTDRO scheme with a receive diversity order of 4 is delay efficient as it achieves delay difference of 0.75 μs and 0.29 μs over conventional Baseline and Rate optimized schemes respectively.

The marker-trait association for complex traits using genotyping by sequencing (GBS) method is being widely spread in plants. The study aimed to identify significant single nucleotide polymorphism (SNP) associations for rachis length (RL), leaf area (LA) and total dry weight (TrDW) in oil palm among diverse African germplasm. The Illumina NextSeq platform has been used for SNP genotyping and retained 4031 fully informative SNPs after applying the filter criterion. These 4031 SNPs were used for genome wide association study for the above three traits. The LD decay rates of the African germplasm using GBS data of SNP is observed to be 25 Kb at 0.45 of average pair wise correlation coefficient (r2). Association mapping led to the identification of seven significant associations for three traits using MLM approach at a P value of ≤ 0.001. Three associations were identified for total dry weight, two each for leaf area index and rachis length. The qtlLA1 was found to be highly significant at a P value of 7.39E-05 (18.4% phenotypic variance) which is located on chromosome 4. Two QTLs (qtlLA2 and qtlRL1) were located on chromosome 1, which explained 11.9% and 12.4% of phenotypic variance respectively. Three QTLs for total dry weight were located on chromosome 2, 14 and 16, all-together explained 40% phenotypic variance. The results showed that the SNP-trait associations identified in the present study could be used in selection of elite oil palm germplasm for higher yields.

ObjectivesOcular hypertension is a primary risk factor for glaucoma and results in retinal ganglion cell (RGC) degeneration. Current animal models of glaucoma lack severe RGC cell death as seen in glaucoma, making assessment of physiological mediators of cell death difficult. We developed a modified mouse model of ocular hypertension whereby long-lasting elevation of intraocular pressure (IOP) is achieved, resulting in significant reproducible damage to RGCs.ResultsIn this model, microbeads are mixed with hyaluronic acid and injected into the anterior chamber of C57BL/6J mice. The hyaluronic acid allows for a gradual release of microbeads, resulting in sustained blockage of Schlemm's canal. IOP elevation was bimodal during the course of the model's progression. The first peak occurred 1 hours after beads injection, with an IOP value of 44.69 ± 6.00 mmHg, and the second peak occurred 6-12 days post-induction, with an IOP value of 34.91 ± 5.21 mmHg. RGC damage was most severe in the peripheral retina, with a loss of 64.1% compared to that of untreated eyes, while the midperiphery exhibited a 32.4% loss, 4 weeks following disease induction.ConclusionsThese results suggest that sustained IOP elevation causes more RGC damage in the periphery than in the midperiphery of the retina. This model yields significant and reproducible RGC degeneration.

It was estimated that 1.2 million people live with HIV/AIDS in Zambia by 2015. Zambia has developed and implemented diverse programs to reduce the prevalence in the country. HIV-testing is a critical step in HIV treatment and prevention, especially among all the key populations. However, there is no systematic review so far to demonstrate the trend of HIV-testing studies in Zambia since 1990s or synthesis the key factors that associated with HIV-testing practices in the country. Therefore, this study conducted a systematic review to search all English literature published prior to November 2016 in six electronic databases and retrieved 32 articles that meet our inclusion criteria. The results indicated that higher education was a common facilitator of HIV testing, while misconception of HIV testing and the fear of negative consequences were the major barriers for using the testing services. Other factors, such as demographic characteristics, marital dynamics, partner relationship, and relationship with the health care services, also greatly affects the participants' decision making. The findings indicated that 1) individualized strategies and comprehensive services are needed for diverse key population; 2) capacity building for healthcare providers is critical for effectively implementing the task-shifting strategy; 3) HIV testing services need to adapt to the social context of Zambia where HIV-related stigma and discrimination is still persistent and overwhelming; and 4) family-based education and intervention should involving improving gender equity.

[This corrects the article DOI: 10.1371/journal.pone.0171345.].

Candida albicans is one of the most common causes of hospital-acquired urinary tract infections (UTIs). However, azoles are poorly active against biofilms, echinocandins do not achieve clinically useful urinary concentrations, and amphotericin B exhibits severe toxicities. Thus, novel strategies are needed to prevent Candida UTIs, which are often associated with urinary catheter biofilms. We previously demonstrated that cranberry-derived proanthocyanidins (PACs) prevent C. albicans biofilm formation in an in vitro urinary model. To elucidate functional pathways unique to urinary biofilm development and PAC inhibition, we investigated the transcriptome of C. albicans in artificial urine (AU), with and without PACs. C. albicans biofilm and planktonic cells were cultivated with or without PACs. Genome-wide expression analysis was performed by RNA sequencing. Differentially expressed genes were determined using DESeq2 software; pathway analysis was performed using Cytoscape. Approximately 2,341 of 6,444 total genes were significantly expressed in biofilm relative to planktonic cells. Functional pathway analysis revealed that genes involved in filamentation, adhesion, drug response and transport were up-regulated in urinary biofilms. Genes involved in carbon and nitrogen metabolism and nutrient response were down-regulated. In PAC-treated urinary biofilms compared to untreated control biofilms, 557 of 6,444 genes had significant changes in gene expression. Genes downregulated in PAC-treated biofilms were implicated in iron starvation and adhesion pathways. Although urinary biofilms share key features with biofilms formed in other environments, many genes are uniquely expressed in urinary biofilms. Cranberry-derived PACs interfere with the expression of iron acquisition and adhesion genes within urinary biofilms.

Multiple Sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) where pathology is thought to be regulated by autoreactive T cells of the Th1 and Th17 phenotype. In this study we sought to understand the functions of Presenilin 1 (PSEN1) in regulating T cell effector responses in the experimental autoimmune encephalomyelitis (EAE) murine model of MS. PSEN1 is the catalytic subunit of γ-secretase a multimolecular protease that mediates intramembranous proteolysis. γ-secretase is known to regulate several pathways of immune importance. Here we examine the effects of disrupting PSEN1 functions on EAE and T effector differentiation using small molecule inhibitors of γ-secretase (GSI) and T cell-specific conditional knockout mice (PSEN1 cKO). Surprisingly, blocking PSEN1 function by GSI treatment or PSEN1 cKO had little effect on the development or course of MOG35-55-induced EAE. In vivo GSI administration reduced the number of myelin antigen-specific T cells and suppressed Th1 and Th17 differentiation following immunization. In vitro, GSI treatment inhibited Th1 differentiation in neutral but not IL-12 polarizing conditions. Th17 differentiation was also suppressed by the presence of GSI in all conditions and GSI-treated Th17 T cells failed to induce EAE following adoptive transfer. PSEN cKO T cells showed reduced Th1 and Th17 differentiation. We conclude that γ-secretase and PSEN1-dependent signals are involved in T effector responses in vivo and potently regulate T effector differentiation in vitro, however, they are dispensable for EAE.

Little is known about physiological factors that affect the sense of olfaction in dogs. The objectives of this study were to describe the canine nasal and oral microbiota in detection dogs. We sought to determine the bacterial composition of the nasal and oral microbiota of a diverse population of detection canines. Nasal and oral swabs were collected from healthy dogs (n = 81) from four locations-Alabama, Georgia, California, and Texas. Nasal and oral swabs were also collected from a second cohort of detection canines belonging to three different detection job categories: explosive detection dogs (SP-E; n = 22), patrol and narcotics detection dogs (P-NDD; n = 15), and vapor wake dogs (VWD-E; n = 9). To understand if the nasal and oral microbiota of detection canines were variable, sample collection was repeated after 7 weeks in a subset of dogs. DNA was extracted from the swabs and used for 454-pyrosequencing of the16S rRNA genes. Nasal samples had a significantly lower diversity than oral samples (P

BACKGROUND:The Asian tiger mosquito, Aedes albopictus is currently an important vector for dengue, chikungunya and Zika virus, and its role in transmission of arthropod-borne viruses (arboviruses) may increase in the future due to its ability to colonize temperate regions. In contrast to Aedes aegypti, the dominant vector of dengue, chikungunya and Zika virus, genetic responses of Ae. albopictus upon infection with an arbovirus are not well characterized. Here we present a study of the changes in transcript expression in Ae. albopictus exposed to dengue virus serotype 2 via feeding on an artificial bloodmeal. METHODOLOGY/PRINCIPAL FINDINGS:We isolated midguts and midgut-free carcasses of Ae. albopictus fed on bloodmeals containing dengue virus as well as controls fed on virus-free control meals at day 1 and day 5 post-feeding. We confirmed infection of midguts from mosquitoes sampled on day 5 post-feeding via RT-PCR. RNAseq analysis revealed dynamic modulation of the expression of several putative immunity and dengue virus-responsive genes, some of whose expression was verified by qRT-PCR. For example, a serine protease gene was up-regulated in the midgut at 1 day post infection, which may potentially enhance mosquito susceptibility to dengue infection, while 14 leucine-rich repeat genes, previously shown to be involved in mosquito antiviral defenses, were down-regulated in the carcass at 5 days post infection. The number of significantly modulated genes decreased over time in midguts and increased in carcasses. CONCLUSION/SIGNIFICANCE:Dengue virus exposure results in the modulation of genes in a time- and site-specific manner. Previous literature on the interaction between mosquitoes and mosquito-borne pathogens suggests that most of the changes that occurred in Ae. albopictus exposed to DENV would favor virus infection. Many genes identified in this study warrant further characterization to understand their role in viral manipulation of and antiviral response of Ae. albopictus.

Selenium (Se) is an essential trace mineral important for immune function and overall health of cattle. The nasopharyngeal microbiota in cattle plays an important role in overall respiratory health, especially when stresses associated with weaning, transport, and adaptation to a feedlot affect the normal respiratory defenses. Recent evidence suggests that cattle diagnosed with bovine respiratory disease complex have significantly less bacterial diversity. The objective of this study was to determine whether feeding weaned beef calves Se-enriched alfalfa (Medicago sativa) hay for 9 weeks in a preconditioning program prior to entering the feedlot alters nasal microbiota. Recently weaned beef calves (n = 45) were blocked by sex and body weight, randomly assigned to 3 treatment groups with 3 pens of 5 calves per treatment group, and fed an alfalfa hay based diet for 9 weeks. Alfalfa hay was harvested from fields fertilized with sodium selenate at a rate of 0, 45.0 or 89.9 g Se/ha. Blood samples were collected biweekly and analyzed for whole-blood Se concentrations. Nasal swabs were collected during week 9 from one or two calves from each pen (total n = 16). Calculated Se intake from dietary sources was 3.0, 15.6, and 32.2 mg Se/head/day for calves consuming alfalfa hay with Se concentrations of 0.34 to 2.42 and 5.17 mg Se/kg dry matter, respectively. Whole-blood Se concentrations after 8 weeks of feeding Se-fertilized alfalfa hay were dependent upon Se-application rates (0, 45.0, or 89.9 g Se/ha) and were 155, 345, and 504 ng/mL (PLinear < 0.0001). Microbial DNA was extracted from nasal swabs and amplified and sequenced. Alpha rarefaction curves comparing the species richness (observed OTUs) and overall diversity (Chao1, Observed OTU, and Shannon index) between calves fed selenium-biofortified alfalfa hay compared with control calves showed that Se-supplementation tended to be associated with an enriched nasal microbiota. ANOSIM of unweighted UniFrac distances showed that calves fed high Se-biofortified alfalfa hay clustered separately when compared with control calves in the PCoA plot (R = 0.216, P = 0.04). The bacterial orders Lactobacillales and Flavobacteriales were increased in healthy control calves compared with Clostridiales and Bacteroidales being increased in calves fed Se-biofortified alfalfa hay. Although there were strong trends, no significant differences were noted for any of the bacterial taxa. Based upon these findings, we suggest that weaned beef calves fed Se-biofortified hay tend to have an enriched nasal microbiota. Feeding Se-biofortified alfalfa hay to weaned beef calves prior to entering the feedlot is a strategy for increasing nasopharyngeal microbial diversity.

Public attitudes to mental illness could influence how the public interact with, provide opportunities for, and help people with mental illness.This study aims to explore the underlying factors of the Attitudes to Mental Illness questionnaire among the general population in Singapore and the socio-demographic correlates of each factor.From March 2014 to April 2015, a nation-wide cross-sectional survey on mental health literacy with 3,006 participants was conducted in Singapore.Factor analysis revealed a 4-factor structure for the Attitudes to Mental Illness questionnaire among the Singapore general population, namely social distancing, tolerance/support for community care, social restrictiveness, and prejudice and misconception. Older age, male gender, lower education and socio-economic status were associated with more negative attitudes towards the mentally ill. Chinese showed more negative attitudes than Indians and Malays (except for prejudice and misconception).There is a need for culture-specific interventions, and the associated factors identified in this study should be considered for future attitude campaigns.

Evidence suggests neuroprotective effects of fluoxetine, a selective serotonin reuptake inhibitor (SSRI), on the developed neurons in the adult brain. In contrast, the drug may be deleterious to immature or undifferentiated neural cells, although the mechanism is unclear. Recent investigations have suggested that microRNAs (miRNA) may be critical for effectiveness of psychotropic drugs including SSRI. We investigated whether fluoxetine could modulate expressions of neurologically relevant miRNAs in two neuroblastoma SK-N-SH and SH-SY5Y cell lines. Initial screening results revealed that three (miR-489, miR-572 and miR-663a) and four (miR-320a, miR-489, miR-572 and miR-663a) miRNAs were up-regulated in SK-N-SH cells and SH-SY5Y cells, respectively, after 24 hours treatment of fluoxetine (1-25 μM). Cell viability was reduced according to the dose of fluoxetine. The upregulation of miR-572 and miR-663a was consistent in both the SH-SY5Y and SK-N-SH cells, confirmed by a larger scale culture condition. Our data is the first in vitro evidence that fluoxetine could increase the expression of miRNAs in undifferentiated neural cells, and that putative target genes of those miRNAs have been shown to be involved in fundamental neurodevelopmental processes.

Transient transfection promoter reporter assays are commonly used in the study of transcriptional regulation, and can be used to define and characterize both cis-acting regulatory sequences and trans-acting factors. In the process of using a variety of reporter assays designed to study regulation of the rhodopsin (rho) promoter, we discovered that rhodopsin promoter-driven reporter expression could be activated by certain species of shRNA in a gene-target-independent but shRNA sequence-specific manner, suggesting involvement of a specific shRNA associated pathway. Interestingly, the shRNA-mediated increase of rhodopsin promoter activity was synergistically enhanced by the rhodopsin transcriptional regulators CRX and NRL. Additionally, the effect was cell line-dependent, suggesting that this pathway requires the expression of cell-type specific factors. Since microRNA (miRNA) and interferon response-mediated processes have been implicated in RNAi off-target phenomena, we performed miRNA and gene expression profiling on cells transfected with shRNAs that do target a specific gene but have varied effects on rho reporter expression in order to identify transcripts whose expression levels are associated with shRNA induced rhodopsin promoter reporter activity. We identified a total of 50 miRNA species, and by microarray analysis, 320 protein-coding genes, some of which were predicted targets of the identified differentially expressed miRNAs, whose expression was altered in the presence of shRNAs that stimulated rhodopsin-promoter activity in a non-gene-targeting manner. Consistent with earlier studies on shRNA off-target effects, a number of interferon response genes were among those identified to be upregulated. Taken together, our results confirm the importance of considering off-target effects when interpreting data from RNAi experiments and extend prior results by focusing on the importance of including multiple and carefully designed controls in the design and analysis of the effects of shRNA on transient transfection-based transcriptional assays.

Natural killer (NK) cells belong to the innate arm of the immune system and though activated NK cells can modulate immune responses through the secretion of cytokines, their primary effector function is through target cell lysis. Accordingly, cytotoxicity assays are central to studying NK cell function. The 51Chromium release assay, is the "gold standard" for cytotoxicity assay, however, due to concerns over toxicity associated with the use and disposal of radioactive compounds there is a significant interest in non-radioactive methods. We have previously used the calcein release assay as a non-radioactive alternative for studying NK cell cytotoxicity. In this study, we show that the calcein release assay varies in its dynamic range for different tumor targets, and that the entrapped calcein could remain unreleased within apoptotic bodies of lysed tumor targets or incompletely released resulting in underestimation of percent specific lysis. To overcome these limitations, we developed a novel cytotoxicity assay using the Cellometer Vision Image Cytometer and compared this method to standard calcein release assay for measuring NK cell cytotoxicity. Using tumor lines K562, 721.221, and Jurkat, we demonstrate here that image cytometry shows significantly higher percent specific lysis of the target cells compared to the standard calcein release assay within the same experimental setup. Image cytometry is able to accurately analyze live target cells by excluding dimmer cells and smaller apoptotic bodies from viable target cell counts. The image cytometry-based cytotoxicity assay is a simple, direct and sensitive method and is an appealing option for routine cytotoxicity assay.

Acute kidney injury (AKI) is a highly morbid condition in critically ill patients that is associated with high mortality. Previous clinical studies have demonstrated the safety and efficacy of the Selective Cytopheretic Device (SCD) in the treatment of AKI requiring continuous renal replacement therapy in the intensive care unit (ICU).A randomized, controlled trial of 134 ICU patients with AKI, 69 received continuous renal replacement therapy (CRRT) alone and 65 received SCD therapy.No significant difference in 60-day mortality was observed between the treated (27/69; 39%) and control patients (21/59; 36%, with six patients lost to follow up) in the intention to treat (ITT) analysis. Of the 19 SCD subjects (CRRT+SCD) and 31 control subjects (CRRT alone) who maintained a post-filter ionized calcium (iCa) level in the protocol's recommended range (≤ 0.4 mmol/L) for greater or equal to 90% of the therapy time, 60-day mortality was 16% (3/19) in the SCD group compared to 41% (11/27) in the CRRT alone group (p = 0.11). Dialysis dependency showed a borderline statistically significant difference between the SCD treated versus control CRRT alone patients maintained for ≥ 90% of the treatment in the protocol's recommended (r) iCa target range of ≤ 0.4 mmol/L with values of, 0% (0/16) and 25% (4/16), respectively (P = 0.10). When the riCa treated and control subgroups were compared for a composite index of 60 day mortality and dialysis dependency, the percentage of SCD treated subjects was 16% versus 58% in the control subjects (p

Microorganisms within the gastrointestinal tract significantly influence metabolic processes within their mammalian host, and recently several groups have sought to characterise the gastrointestinal microbiota of individuals affected by metabolic disease. Differences in the composition of the gastrointestinal microbiota have been reported in mouse models of type 2 diabetes mellitus, as well as in human patients. Diabetes mellitus in cats has many similarities to type 2 diabetes in humans. No studies of the gastrointestinal microbiota of diabetic cats have been previously published. The objectives of this study were to compare the composition of the faecal microbiota of diabetic and non-diabetic cats, and secondarily to determine if host signalment and dietary factors influence the composition of the faecal microbiota in cats. Faecal samples were collected from insulin-treated diabetic and non-diabetic cats, and Illumina sequencing of the 16S rRNA gene and quantitative PCR were performed on each sample. ANOSIM based on the unweighted UniFrac distance metric identified no difference in the composition of the faecal microbiota between diabetic and non-diabetic cats, and no significant differences in the proportions of dominant bacteria by phylum, class, order, family or genus as determined by 16S rRNA gene sequencing were identified between diabetic and non-diabetic cats. qPCR identified a decrease in Faecalibacterium spp. in cats aged over ten years. Cat breed or gender, dietary carbohydrate, protein or fat content, and dietary formulation (wet versus dry food) did not affect the composition of the faecal microbiota. In conclusion, the composition of the faecal microbiota was not altered by the presence of diabetes mellitus in cats. Additional studies that compare the functional products of the microbiota in diabetic and non-diabetic cats are warranted to further investigate the potential impact of the gastrointestinal microbiota on metabolic diseases such as diabetes mellitus in cats.

Curcumin and capsaicin are dietary xenobiotics with well-documented anti-inflammatory properties. Previously, the beneficial effect of these spice principles in lowering chronic inflammation was demonstrated using a rat experimental model for arthritis. The extent of lowering of arthritic index by the spice principles was associated with a significant shift in macrophage function favoring the reduction of pro-inflammatory molecules such as reactive oxygen species and production and release of anti-inflammatory metabolites of arachidonic acid. Beyond the cellular effects on macrophage function, oral administration of curcumin and capsaicin caused alterations in serum protein profiles of rats injected with adjuvant to develop arthritis. Specifically, a 72 kDa acidic glycoprotein, GpA72, which was elevated in pre-arthritic rats, was significantly lowered by feeding either curcumin or capsaicin to the rats. Employing the tandem mass spectrometric approach for direct sequencing of peptides, here we report the identification of GpA72 as T-kininogen I also known as Thiostatin. Since T-kininogen I is an early acute-phase protein, we additionally tested the efficiency of curcumin and capsaicin to mediate the inflammatory response in an acute phase model. The results demonstrate that curcumin and capsaicin lower the acute-phase inflammatory response, the molecular mechanism for which is, in part, mediated by pathways associated with the lowering of T-kininogen I.

Activation of the PI3K/Akt pathway, a critical step for survival in cancer cells is often associated with decreased sensitivity to several chemotherapeutic drugs. PIK3CA gene amplification is observed in 16-24% of epithelial ovarian cancer (EOC) patients in conjunction with p53 mutations. A 900 bp long PIK3CA promoter is shown to be negatively regulated by p53 in ovarian surface epithelial cells but the consequence of chemotherapeutic drug treatments on this promoter in ovarian cancer cells is largely unknown. We aim to study the modulation of this promoter by cisplatin using an improved fusion reporter in ovarian cancer cells and tumor xenografts by non-invasive imaging approach. A PIK3CA sensor was developed using a bi-fusion reporter from a newly constructed library of bi- and tri-fusion vectors comprising of two mutant far red fluorescent proteins (mcherry/mch and tdTomato/tdt), a mutant firefly luciferase (fluc2), and a PET reporter protein (ttk). In vivo imaging of mice implanted with 293T cells transiently expressing these bi- and tri-fusion reporters along with respective controls revealed comparable activity of each reporter in the fusion background and fluc2-tdt as the most sensitive one. Repression of the PIK3CA sensor by drugs was inversely proportional to cellular p53 level in a germline (PA1) and in an EOC (A2780) cell line but not in a p53 deficient EOC (SKOV3) cell line. Bioluminescence imaging of tumor xenografts stably expressing the PIK3CA sensor in PA1 and A2780 cells exhibited attenuating activity without any change in SKOV3 tumors expressing the PIK3CA sensor after cisplatin treatment. Sequential mutation at p53 binding sites showed gradual increase in promoter activity and decreased effects of the drugs. These newly developed PIK3CA-fluc2-tdt and the mutant reporter sensors thus would be extremely useful for screening new drugs and for functional assessment of PIK3CA expression from intact cells to living subjects.

The present study aimed at investigating whether neuromotor development, from birth to 14 months of age, shows seasonal, cyclic patterns in association with months of birth. Study participants were 742 infants enrolled in the Hamamatsu Birth Cohort (HBC) Study and followed-up from birth to the 14th month of age. Gross motor skills were assessed at the ages of 6, 10, and 14 months, using Mullen Scales of Early Learning. The score at each assessment was regressed onto a trigonometric function of months of birth, with an adjustment for potential confounders. Gross motor scores at the 6th and 10th months showed significant 1-year-cycle variations, peaking among March- and April-born infants, and among February-born infants, respectively. Changes in gross motor scores between the 10th and 14th months also showed a cyclic variation, peaking among July- and August-born infants. Due to this complementary effect, gross motor scores at the 14th month did not show seasonality. Neuromotor development showed cyclic seasonality during the first year of life. The effects brought about by month of birth disappeared around 1 year of age, and warmer months seemed to accelerate the neuromotor development.

Stereotypical locomotor movements can be made without input from the brain after a complete spinal transection. However, the restoration of functional gait requires descending modulation of spinal circuits to independently control the movement of each limb. To evaluate whether a brain-machine interface (BMI) could be used to regain conscious control over the hindlimb, rats were trained to press a pedal and the encoding of hindlimb movement was assessed using a BMI paradigm. Off-line, information encoded by neurons in the hindlimb sensorimotor cortex was assessed. Next neural population functions, or weighted representations of the neuronal activity, were used to replace the hindlimb movement as a trigger for reward in real-time (on-line decoding) in three conditions: while the animal could still press the pedal, after the pedal was removed and after a complete spinal transection. A novel representation of the motor program was learned when the animals used neural control to achieve water reward (e.g. more information was conveyed faster). After complete spinal transection, the ability of these neurons to convey information was reduced by more than 40%. However, this BMI representation was relearned over time despite a persistent reduction in the neuronal firing rate during the task. Therefore, neural control is a general feature of the motor cortex, not restricted to forelimb movements, and can be regained after spinal injury.

Rab monomeric GTPases regulate specific aspects of vesicle transport in eukaryotes including coat recruitment, uncoating, fission, motility, target selection and fusion. Moreover, individual Rab proteins function at specific sites within the cell, for example the ER, golgi and early endosome. Importantly, the localization and function of individual Rab subfamily members are often conserved underscoring the significant contributions that model organisms such as Caenorhabditis elegans can make towards a better understanding of human disease caused by Rab and vesicle trafficking malfunction. With this in mind, a bioinformatics approach was first taken to identify and classify the complete C. elegans Rab family placing individual Rabs into specific subfamilies based on molecular phylogenetics. For genes that were difficult to classify by sequence similarity alone, we did a comparative analysis of intron position among specific subfamilies from yeast to humans. This two-pronged approach allowed the classification of 30 out of 31 C. elegans Rab proteins identified here including Rab31/Rab50, a likely member of the last eukaryotic common ancestor (LECA). Second, a molecular toolset was created to facilitate research on biological processes that involve Rab proteins. Specifically, we used Gateway-compatible C. elegans ORFeome clones as starting material to create 44 full-length, sequence-verified, dominant-negative (DN) and constitutive active (CA) rab open reading frames (ORFs). Development of this toolset provided independent research projects for students enrolled in a research-based molecular techniques course at California State University, East Bay (CSUEB).

Redox enzyme maturation proteins (REMPs) bind pre-proteins destined for translocation across the bacterial cytoplasmic membrane via the twin-arginine translocation system and enable the enzymatic incorporation of complex cofactors. Most REMPs recognize one specific pre-protein. The recognition site usually resides in the N-terminal signal sequence. REMP binding protects signal peptides against degradation by proteases. REMPs are also believed to prevent binding of immature pre-proteins to the translocon. The main aim of this work was to better understand the interaction between REMPs and substrate signal sequences. Two REMPs were investigated: DmsD (specific for dimethylsulfoxide reductase, DmsA) and TorD (specific for trimethylamine N-oxide reductase, TorA). Green fluorescent protein (GFP) was genetically fused behind the signal sequences of TorA and DmsA. This ensures native behavior of the respective signal sequence and excludes any effects mediated by the mature domain of the pre-protein. Surface plasmon resonance analysis revealed that these chimeric pre-proteins specifically bind to the cognate REMP. Furthermore, the region of the signal sequence that is responsible for specific binding to the corresponding REMP was identified by creating region-swapped chimeric signal sequences, containing parts of both the TorA and DmsA signal sequences. Surprisingly, specificity is not encoded in the highly variable positively charged N-terminal region of the signal sequence, but in the more similar hydrophobic C-terminal parts. Interestingly, binding of DmsD to its model substrate reduced membrane binding of the pre-protein. This property could link REMP-signal peptide binding to its reported proofreading function.

F18 2-Fluoro 2-deoxyglucose (FDG) has been the gold standard in positron emission tomography (PET) oncologic imaging since its introduction into the clinics several years ago. Seeking to complement FDG in the diagnosis of breast cancer using radio labeled fructose based analogs, we investigated the expression of the chief fructose transporter-GLUT 5 in breast cancer cells and human tissues. Our results indicate that GLUT 5 is not over-expressed in breast cancer tissues as assessed by an extensive immunohistochemistry study. RT-PCR studies showed that the GLUT 5 mRNA was present at minimal amounts in breast cancer cell lines. Further knocking down the expression of GLUT 5 in breast cancer cells using RNA interference did not affect the fructose uptake in these cell lines. Taken together these results are consistent with GLUT 5 not being essential for fructose uptake in breast cancer cells and tissues.

Multiple lines of evidence suggest a serotoninergic dysfunction in autism. The role of LMX1B in the development and maintenance of serotoninergic neurons is well known. In order to examine the role, if any, of LMX1B with autism pathophysiology, a trio-based SNP association study using 252 family samples from the AGRE was performed. Using pair-wise tagging method, 24 SNPs were selected from the HapMap data, based on their location and minor allele frequency. Two SNPs (rs10732392 and rs12336217) showed moderate association with autism with p values 0.018 and 0.022 respectively in transmission disequilibrium test. The haplotype AGCGTG also showed significant association (p = 0.008). Further, LMX1B mRNA expressions were studied in the postmortem brain tissues of autism subjects and healthy controls samples. LMX1B transcripts was found to be significantly lower in the anterior cingulate gyrus region of autism patients compared with controls (p = 0.049). Our study suggests a possible role of LMX1B in the pathophysiology of autism. Based on previous reports, it is likely to be mediated through a seretoninergic mechanism. This is the first report on the association of LMX1B with autism, though it should be viewed with some caution considering the modest associations we report.

Daily nevirapine (NVP) prophylaxis to HIV-exposed infants significantly reduces breast-milk HIV transmission. We assessed NVP-resistance in Indian infants enrolled in the "six-week extended-dose nevirapine" (SWEN) trial who received single-dose NVP (SD-NVP) or SWEN for prevention of breast-milk HIV transmission but who also acquired subtype C HIV infection during the first year of life.Standard population sequencing and cloning for viral subpopulations present at > or =5% frequency were used to determine HIV genotypes from 94% of the 79 infected Indian infants studied. Timing of infection was defined based on when an infant's blood sample first tested positive for HIV DNA. SWEN-exposed infants diagnosed with HIV by six weeks of age had a significantly higher prevalence of NVP-resistance than those who received SD-NVP, by both standard population sequencing (92% of 12 vs. 38% of 29; p = 0.002) and low frequency clonal analysis (92% of 12 vs. 59% of 29; p = 0.06). Likelihood of infection with NVP-resistant HIV through breast-milk among infants infected after age six weeks was substantial, but prevalence of NVP-resistance did not differ among SWEN or SD-NVP exposed infants by standard population sequencing (15% of 13 vs. 15% of 20; p = 1.00) and clonal analysis (31% of 13 vs. 40% of 20; p = 0.72). Types of NVP-resistance mutations and patterns of persistence at one year of age were similar between the two groups. NVP-resistance mutations did differ by timing of HIV infection; the Y181C variant was predominant among infants diagnosed in the first six weeks of life, compared to Y188C/H during late breast-milk transmission.Use of SWEN to prevent breast-milk HIV transmission carries a high likelihood of resistance if infection occurs in the first six weeks of life. Moreover, there was a continued risk of transmission of NVP-resistant HIV through breastfeeding during the first year of life, but did not differ between SD-NVP and SWEN groups. As with SD-NVP, the value of preventing HIV infection in a large number of infants should be considered alongside the high risk of resistance associated with extended NVP prophylaxis.ClinicalTrials.gov NCT00061321.