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[This corrects the article DOI: 10.1371/journal.pone.0278550.].

BackgroundNonpharmacological interventions, such as personal protective equipment for example, surgical masks and respirators, and maintenance of hand hygiene along with COVID-19 vaccines have been recommended to reduce viral transmission in the community and health care settings. There is evidence from the literature that surgical and N95 masks may reduce the initial degree of exposure to the virus. A limited research that has studied the cost-effective analysis of surgical masks and N95 masks among health care workers in the prevention of COVID-19 in India. The objective of this study was to estimate the cost-effectiveness of N95 and surgical mask compared to wearing no mask in public hospital settings for preventing COVID-19 infection among Health care workers (HCWs) from the health care provider's perspective.MethodsA deterministic baseline model, without any mask use, based on Eikenberry et al was used to form the foundation for parameter estimation and to estimate transmission rates among HCWs. Information on mask efficacy, including the overall filtering efficiency of a mask and clinical efficiency, in terms of either inward efficiency(ei) or outward efficiency(e0), was obtained from published literature. Hospitalized HCWs were assumed to be in one of the disease states i.e., mild, moderate, severe, or critical. A total of 10,000 HCWs was considered as representative of the size of a tertiary care institution HCW population. The utility values for the mild, moderate and severe model health states were sourced from the primary data collection on quality-of-life of HCWs COVID-19 survivors. The utility scores for mild, moderate, and severe COVID-19 conditions were 0.88, 0.738 and 0.58, respectively. The cost of treatment for mild sickness (6,500 INR per day), moderate sickness (10,000 INR per day), severe (require ICU facility without ventilation, 15,000 INR per day), and critical (require ICU facility with ventilation per day, 18,000 INR) per day as per government and private COVID-19 treatment costs and capping were considered. One way sensitivity analyses were performed to identify the model inputs which had the largest impact on model results.ResultsThe use of N95 masks compared to using no mask is cost-saving of $1,454,632 (INR 0.106 billion) per 10,000 HCWs in a year. The use of N95 masks compared to using surgical masks is cost-saving of $63,919 (INR 0.005 billion) per 10,000 HCWs in a year. the use of surgical masks compared to using no mask is cost-saving of $1,390,713 (INR 0.102 billion) per 10,000 HCWs in a year. The uncertainty analysis showed that considering fixed transmission rate (1.7), adoption of mask efficiency as 20%, 50% and 80% reduces the cumulative relative mortality to 41%, 79% and 94% respectively. On considering ei = e0 (99%) for N95 and surgical mask with ei = e0 (90%) the cumulative relative mortality was reduced by 97% and the use of N95 masks compared to using surgical masks is cost-saving of $24,361 (INR 0.002 billion) per 10,000 HCWs in a year.DiscussionBoth considered interventions were dominant compared to no mask based on the model estimates. N95 masks were also dominant compared to surgical masks.

BackgroundAntihypertensive drug supply is sometimes inadequate in public sector health facilities in India. One of the core strategies of the India Hypertension Control Initiative (IHCI) is to improve the availability of antihypertensive drugs in primary and secondary care facilities. We quantified the availability of antihypertensive drugs in 2019-20 and described the practices in supply chain management in 22 districts across four states of India.MethodsTwenty-two districts from 4 states (Punjab, Madhya Pradesh, Telangana, and Maharashtra) were studied. We described the practices and challenges in supply chain management. We collected data on drug procurement from 2018 to 2020 and drug availability from April 2019 to March 2020. Quantity procured, the proportion of facilities with stockout at the end of each quarter, and availability of drugs in patient days were tabulated.ResultsAll states selected drug- and dose-specific protocols with Amlodipine as the initial drug and shifted to morbidity-based forecasting. The total number of antihypertensive tablets procured for the 22 districts increased from 16 million in 2017-2018 to 160 million in 2019-2020. The proportion of facilities with Amlodipine stock-out was below 5% during the study period. Amlodipine stock was available for at least 60 patient days from the third quarter of 2019 onward in all districts.ConclusionsThis study demonstrates that including best practices can gradually strengthen the procurement and supply chain for antihypertensives in a low-resource setting. As the program was rapidly growing, there were still gaps in the procurement and distribution system which needed to be addressed to ensure the adequacy of drugs. We recommend that best practices, including choosing a single protocol, basing supply on projected patient load rather than an increment from historical levels, and using simple stock management tools, be replicated in other districts in India to increase and sustain coverage of hypertension treatment.

One of the biggest challenges to be addressed in world agriculture is low nitrogen (N) use efficiency (

BackgroundFilter clotting is a major issue in continuous kidney replacement therapy (CKRT) that interrupts treatment, reduces delivered effluent dose, and increases cost of care. While a number of variables are involved in filter life, treatment modality is an understudied factor. We hypothesized that filters in pre-filter continuous venovenous hemofiltration (CVVH) would have shorter lifespans than in continuous venovenous hemodialysis (CVVHD).MethodsThis was a single center, pragmatic, unblinded, quasi-randomized cluster trial conducted in critically ill adult patients with severe acute kidney injury (AKI) at the University of Iowa Hospitals and Clinics (UIHC) between March 2020 and December 2020. Patients were quasi-randomized by time block to receive pre-filter CVVH (convection) or CVVHD (diffusion). The primary outcome was filter life, and secondary outcomes were number of filters used, number of filters reaching 72 hours, and in-hospital mortality.ResultsIn the intention-to-treat analysis, filter life in pre-filter CVVH was 79% of that observed in CVVHD (mean ratio 0.79, 95% CI 0.65-0.97, p = 0.02). Median filter life (with interquartile range) in pre-filter CVVH was 21.8 (11.4-45.3) and was 26.6 (13.0-63.5) for CVVHD. In addition, 11.8% of filters in pre-filter CVVH were active for >72 hours, versus 21.2% in the CVVHD group. Finally, filter clotting accounted for the loss of 26.7% of filters in the CVVH group compared to 17.5% in the CVVHD group. There were no differences in overall numbers of filters used or mortality between groups.ConclusionsAmong critically patients with severe AKI requiring CKRT, use of pre-filter CVVH resulted in significantly shorter filter life compared to CVVHD.Trial registrationClinicalTrials.gov, NCT04762524. Registered 02/21/21-Retroactively registered, https://clinicaltrials.gov/ct2/show/NCT04762524?cond=The+Impact+of+CRRT+Modality+on+Filter+Life&draw=2&rank=1.

Antibiotic resistance threatens provision of healthcare and livestock production worldwide with predicted negative socioeconomic impact. Antibiotic stewardship can be considered of importance to people living in rural communities, many of which depend on agriculture as a source of food and income and rely on antibiotics to control infectious diseases in livestock. Consequently, there is a need for clarity of the structure of antibiotic value chains to understand the complexity of antibiotic production and distribution in community settings as this will facilitate the development of effective policies and interventions. We used a value chain approach to investigate how relationships, behaviours, and influences are established during antibiotic distribution. Interviews were conducted with key informants (n = 17), value chain stakeholders (n = 22), and livestock keeping households (n = 36) in Kolkata, and two rural sites in West Bengal, India. Value chain mapping and an assessment of power dynamics, using manifest content analysis, were conducted to investigate antibiotic distribution and identify entry points for antibiotic stewardship. The flow of antibiotics from manufacturer to stockists is described and mapped and two local level maps showing distribution to final consumers presented. The maps illustrate that antibiotic distribution occurred through numerous formal and informal routes, many of which circumvent antibiotic use legislation. This was partly due to limited institutional power of the public sector to govern value chain activities. A 'veterinary service lacuna' existed resulting in livestock keepers having higher reliance on private and informal providers, who often lacked legal mandates to prescribe and dispense antibiotics. The illegitimacy of many antibiotic prescribers blocked access to formal training who instead relied on mimicking the behaviour of more experienced prescribers-who also lacked access to stewardship guidelines. We argue that limited institutional power to enforce existing antibiotic legislation and guide antibiotic usage and major gaps in livestock healthcare services make attempts to curb informal prescribing unsustainable. Alternative options could include addressing public sector deficits, with respect to both healthcare services and antibiotic provision, and by providing resources such as locally relevant antibiotic guidelines to all antibiotic prescribers. In addition, legitimacy of informal prescribers could be revised, which may allow formation of associations or groups to incentivise good antibiotic practices.

Glycogen storage disorders occur due to enzyme deficiencies in the glycogenolysis and gluconeogenesis pathway, encoded by 26 genes. GSD's present with overlapping phenotypes with variable severity. In this series, 57 individuals were molecularly confirmed for 7 GSD subtypes and their demographic data, clinical profiles and genotype-phenotype co-relations are studied. Genomic DNA from venous blood samples was isolated from clinically affected individuals. Targeted gene panel sequencing covering 23 genes and Sanger sequencing were employed. Various bioinformatic tools were used to predict pathogenicity for new variations. Close parental consanguinity was seen in 76%. Forty-nine pathogenic variations were detected of which 27 were novel. Variations were spread across GSDIa, Ib, III, VI, IXa, b and c. The largest subgroup was GSDIII in 28 individuals with 24 variations (12 novel) in AGL. The 1620+1G>C intronic variation was observed in 5 with GSDVI (PYGL). A total of eleven GSDIX are described with the first Indian report of type IXb. This is the largest study of GSDs from India. High levels of consanguinity in the local population and employment of targeted sequencing panels accounted for the range of GSDs reported here.

IntroductionAvoidable blindness is a significant public health problem in India. Nationally representative RAAB surveys (Rapid Assessment of Avoidable Blindness) are being conducted periodically in the country to know the current status of blindness in the country. The current study describes the findings from the RAAB survey conducted during 2015-19 in India.MethodologyA cross-sectional, population-based survey was conducted across the entire country among persons aged 50 years and above using RAAB version 6 methodology. Presenting and pinhole visual acuity was recorded followed by lens examination using a torchlight. In order to estimate the prevalence of blindness and visual impairment in overall population in India, district weights were assigned to each of the 31 surveyed districts and the prevalence was standardized using the RAAB software.ResultsThe overall weighted, age-gender standardized, prevalence of blindness (presenting visual acuity ConclusionThe results of the survey demonstrate that currently more than one fourth of persons aged 50 years and above are visually impaired (PVA

ObjectivesTo assess and classify all private and government schools located in a northern city of India for accreditation as health promoting schools and comparative health profile assessment of selected higher accredited schools with lower accredited and non-accredited schools.DesignQuasi experimental study with pre and post assessment with comparison of higher with lower accredited schools.SettingsThe current study was conducted in 206 schools of Chandigarh City of Northern India. Comparative health profile assessment was undertaken in 8 schools with 754 children from higher accredited (platinum, gold, silver) and 8 schools with 700 children from lower accredited (bronze) and non-accredited (below bronze) schools.InterventionsMulticomponent and multilevel intervention was undertaken with self-quality improvement by schools with help of a manual of accreditation of school as health promoting schools. Key intervention included capacity building, technical visits, supportive supervision, sensitization of policymakers and key stakeholders, implementation of policy initiatives, use of social media, technical support and monitoring of activities.OutcomesAccreditation levels (bronze, silver, gold and platinum levels) as health promoting schools after pre and post intervention.ResultsOut of 206 schools, 203 participated in the baseline assessment and 204 in the endline assessment. The response rate was 99%. Two schools which refused participation were excluded and not assessed. Schools (N = 17) which participated in the 2011-2013 study were excluded from analysis. There was a statistically difference (p = 0.01) in the improvement of accreditation level of the baseline and endline assessment after intervention(pConclusionsThe health promoting school programme was found to be feasible and effective and lead to significant improvement in accreditation level as compared to baseline assessment after continuous self-quality improvement by schools(p

ObjectiveTo examine the relationship between current cigarette smoking patterns and three established risk factors for suicide using nationally representative data of high school students in the United States.MethodsWe analyzed cross-sectional data from the national Youth Risk Behavior Surveillance Survey (YRBSS)-United States, 2017. Multivariable, logistic regressions examined the association between 3 cigarette smoking behaviors [i.e., past 30-day cigarette (n = 13,731), frequent (n = 1,093) and heavy (n = 880) smoking] and 3 risk factors for suicidal outcomes [feeling sad or hopeless, suicidal ideation, suicide plan] assessed over the previous year.ResultsAmong high school cigarette smokers, smoking 11 or more cigarettes per day (i.e., heavy smoking) was associated with 3.43 (95% CI: 1.69, 6.94) greater odds of reporting feeling sad or hopeless, 2.97 (95% CI: 1.60, 5.51) greater odds of reporting suicidal ideations, and 2.11 (95% CI: 1.34, 3.32) greater odds of reporting having ever planned a suicide attempt, controlling for covariates.ConclusionsOur study shows that it is not simply cigarette smoking, but heavy cigarette smoking that is a risk factor for suicidal outcomes among adolescents.Public health implicationsA comprehensive plan is needed to accommodate heavy adolescent smokers who are at increased suicidal risk.

PURPOSE:To determine the prevalence of cataract and its association with sun exposure and other environmental risk factors in three different geographically diverse populations of India. DESIGN:Population based cross sectional study during 2010-2016. PARTICIPANTS:People aged ≥ 40 years residing in randomly sampled villages were enumerated (12021) and 9735 (81%) underwent ophthalmic evaluation from plains, hilly and coastal regions (3595, 3231, 2909 respectively). METHODS:A detailed questionnaire-based interview about outdoor activity in present, past and remote past, usage of sun protective measures, exposure to smoke, and detailed ophthalmic examination including assessment of uncorrected and best corrected visual acuity, measurement of intraocular pressure, slit lamp examination, lens opacities categorization using LOCS III and posterior segment evaluation was done. Lifetime effective sun exposure was calculated using Melbourne formula and expressed as quintiles. These were supplemented with physical environmental measurements. MAIN OUTCOME MEASURES:Lifetime sun exposure hours, smoking, indoor kitchen smoke exposure and their association with cataract and subtypes. Prevalence of cataract calculated based on lens opacities or evidence of cataract surgery. RESULTS:Cataract was identified in 3231 (33.3%) participants. Prevalence of cataract in males (32.3%) and females (34.1%) was similar. Nuclear cataract was the commonest sub-type identified in 94.7% of affected eyes. Sun exposure had a significant association with cataract with odds ratio (OR) increasing from 1.6 (95% Confidence Intervals [CI]: 1.4, 1.9) in 3rd quintile, to 2.6 (CI: 2.2, 3.1) in 4th quintile and 9.4 (CI: 7.9, 11.2) in 5th quintile (p

AIM:To assess prevalence, clinical presentation and multimodal imaging characteristics of polypoidal choroidal vasculopathy (PCV) in a hospital-based setting in South India. METHODS:Electronic medical records (EMR) of new patients presenting with suspected clinical signs of wet age-related macular degeneration (AMD) in a tertiary hospital from January to December 2016 were retrospectively analyzed using keywords and filtered for patient who underwent multimodal imaging. Clinical presentations were categorized into predominantly hemorrhagic, exudative or mixed pattern. The imaging features were compared in these clinical groups. The multimodal images were graded by two masked graders and discrepancies between them were settled by a senior arbitrator. RESULTS:Of the 147 clinically suspicious cases of PCV out of 785 patients with clinical presentation of AMD as recorded in the EMR, 73 (49.7%) patients had a multimodal imaging diagnosis of PCV. There was no difference in the demography, distribution of polyps, ICGA and OCT characteristics in eyes presenting with hemorrhagic, exudative or mixed clinical features. CONCLUSION:Approximately half of South Asian patients presenting with clinical features of neovascular AMD harbor PCV irrespective of their clinical presentation and so we recommend that multimodal imaging is done in all cases of suspicious neovascular AMD in Indian population.

Safety critical events in robotic applications can often be characterized by forces between the robot end-effector and the environment. One application in which safe interaction between the robot and environment is critical is in the area of medical robots. In this paper, we propose a novel Compact Form Dynamic Linearization Model-Free Prediction (CFDL-MFP) technique to predict future values of any time-series sensor data, such as interaction forces. Existing time series forecasting methods have high computational times which motivates the development of a novel technique. Using Autoregressive Integrated Moving Average (ARIMA) forecasting as benchmark, the performance of the proposed model was evaluated in terms of accuracy, computation efficiency, and stability on various force profiles. The proposed algorithm was 11% more accurate than ARIMA and maximum computation time of CFDL-MFP was 4ms, compared to ARIMA (7390ms). Furthermore, we evaluate the model in the special case of predicting needle buckling events, before they occur, by using only axial force and needle-tip position data. The model was evaluated experimentally for robustness with steerable needle insertions into different tissues including gelatin and biological tissue. For a needle insertion velocity of 2.5mm/s, the proposed algorithm was able to predict needle buckling 2.03s sooner than human detections. In biological tissue, no false positive or false negative buckling detections occurred and the rates were low in artificial tissue. The proposed forecasting model can be used to ensure safe robot interactions with delicate environments by predicting adverse force-based events before they occur.

The extensively employed limited-gene coverage NGS panels lead to clinically inadequate molecular profiling of myeloid neoplasms. The aim of the present investigation was to assess performance and clinical utility of a comprehensive DNA panel for myeloid neoplasms. Sixty-one previously well characterized samples were sequenced using TSO500 library preparation kit on NextSeq550 platform. Variants with a VAF ≥ 5% and a total read depth of >50X were filtered for analysis. The following results were recorded-for clinical samples: clinical sensitivity (97%), specificity (100%), precision (100%) and accuracy (99%) whereas reference control results were 100% for analytical sensitivity, specificity, precision and accuracy, with high intra- and inter-run reproducibility. The panel identified 880 variants across 292 genes, of which, 749 variants were in genes not covered in the 54 gene panel. The investigation revealed 14 variants in ten genes, and at least one was present in 96.2% patient samples that were pathogenic/ likely pathogenic in myeloid neoplasms. Also, 15 variants in five genes were found to be pathogenic/ likely pathogenic in other tumor types. Further, the TMB and MSI scores ranged from 0-7 and 0-9, respectively. The high analytical performance and clinical utility of this comprehensive NGS panel makes it practical and clinically relevant for adoption in clinical laboratories for routine molecular profiling of myeloid neoplasms.

This study assesses the dollar benefit of a neighborhood approach intervention on disaster risk reduction in small-sized, densely populated, and hazard-prone informal settlements across Latin American and Caribbean countries. We use a life satisfaction approach that assigns a dollar value to gains in wellbeing associated with the neighborhood approach intervention. Our primary data was a survey to a sample of 349 beneficiaries from small towns in Haiti, Guatemala, and Jamaica, and in major cities' surrounded areas of Peru, Colombia, and Honduras. Out of 14 interventions, we found that community empowerment, physical works in public spaces and urban gardens/food approaches produced a gain of USD1,038 to USD1,241 to individual beneficiaries. Our study suggests a large benefit associated with the neighborhood approach intervention. It also shows that the life satisfaction approach is a promising method for the valuation of non-market and public goods, especially for countries where data on hazards and risks is not available to help monetize risk reductions.

A field experiment was conducted at the experimental farm of CSIR-Institute of Himalayan Bioresource Technology, Palampur, India for two consecutive years (2015-16 and 2016-17). The aim of the study was to test the hypothesis whether different shade level and mulch type would influence the growth, flower yield and essential oil profile of R. damascena. Yield attributes viz., numbers of flowers plant-1, fresh flower weight plant-1, flower yield, and essential oil yield were significantly higher under open sunny conditions as compared to 25% and 50% shade levels. However, plants grown under 50% shade level recorded significantly higher plant height (cm), plant spread (cm) and the lowest numbers of branches as compared to control. Among mulches, black polyethylene mulch recorded significantly higher growth, and yield attributes of damask rose as compared to other mulches. Black polyethylene mulch recorded 74.5 and 39.2% higher fresh flower yield as compared to without mulch, during 2015-16 and 2016-17, respectively. Correlation studies showed a positively significant correlation between quality and quantity traits. A total of twenty-six essential oil compounds were identified which accounted for a total of 88.8 to 95.3%. Plants grown under open sunny conditions along with the applications of black polyethylene mulch produced a higher concentration of citronellol and trans-geraniol. Damask rose planted in open sunny conditions and mulched with black polyethylene sheet recorded significantly higher flower yield.

Francisella tularensis is a Gram-negative, facultative intracellular pathogen and the causative agent of a lethal human disease known as tularemia. Due to its extremely high virulence and potential to be used as a bioterror agent, F. tularensis is classified by the CDC as a Category A Select Agent. As an intracellular pathogen, F. tularensis during its intracellular residence encounters a number of oxidative and nitrosative stresses. The roles of the primary antioxidant enzymes SodB, SodC and KatG in oxidative stress resistance and virulence of F. tularensis live vaccine strain (LVS) have been characterized in previous studies. However, very fragmentary information is available regarding the role of peroxiredoxin of the AhpC/TSA family (annotated as AhpC) of F. tularensis SchuS4; whereas the role of AhpC of F. tularensis LVS in tularemia pathogenesis is not known. This study was undertaken to exhaustively investigate the role of AhpC in oxidative stress resistance of F. tularensis LVS and SchuS4. We report that AhpC of F. tularensis LVS confers resistance against a wide range of reactive oxygen and nitrogen species, and serves as a virulence factor. In highly virulent F. tularensis SchuS4 strain, AhpC serves as a key antioxidant enzyme and contributes to its robust oxidative and nitrosative stress resistance, and intramacrophage survival. We also demonstrate that there is functional redundancy among primary antioxidant enzymes AhpC, SodC, and KatG of F. tularensis SchuS4. Collectively, this study highlights the differences in antioxidant defense mechanisms of F. tularensis LVS and SchuS4.

Background and objectiveAs of 2016, 43 US states have policies regarding alcohol use during pregnancy. A recent study found that out of eight state-level alcohol/pregnancy policies, six are significantly associated with poorer birth outcomes, and two are not associated with any outcomes. Here we estimate the excess numbers of low birthweight (LBW) and preterm births (PTB) related to these policies and their associated additional costs in the first year of life.MethodsCost study using birth certificate data for 155,446,714 singleton live births in the United States between 1972-2015. Exposures were state- and month/year-specific indicators of having each of eight alcohol/pregnancy policies in place. Outcomes were excess numbers of LBW and PTB and associated costs in the first year of life. Fixed effects regressions with state-specific time trends were used for statistical analyses in 2018.ResultsIn 2015, policies mandating warning signs were associated with an excess of 7,375 LBW; policies defining alcohol use during pregnancy as child abuse/neglect were associated with an excess of 12,372 PTB; these excess adverse outcomes are associated with additional costs of $151,928,002 and $582,698,853 in the first year of life, respectively.ConclusionsMultiple state-level alcohol pregnancy policies lead to increased prevalence of LBW and PTB, which cost hundreds of millions of dollars annually. Policymakers should consider adverse public health impacts of alcohol/pregnancy policies before expanding extant policies to new substances or adopting existing policies in new states.

Francisella tularensis is a Gram-negative bacterium responsible for causing tularemia in the northern hemisphere. F. tularensis has long been developed as a biological weapon due to its ability to cause severe illness upon inhalation of as few as ten organisms and, based on its potential to be used as a bioterror agent is now classified as a Tier 1 Category A select agent by the CDC. The stringent response facilitates bacterial survival under nutritionally challenging starvation conditions. The hallmark of stringent response is the accumulation of the effector molecules ppGpp and (p)ppGpp known as stress alarmones. The relA and spoT gene products generate alarmones in several Gram-negative bacterial pathogens. RelA is a ribosome-associated ppGpp synthetase that gets activated under amino acid starvation conditions whereas, SpoT is a bifunctional enzyme with both ppGpp synthetase and ppGpp hydrolase activities. Francisella encodes a monofunctional RelA and a bifunctional SpoT enzyme. Previous studies have demonstrated that stringent response under nutritional stresses increases expression of virulence-associated genes encoded on Francisella Pathogenicity Island. This study investigated how stringent response governs the oxidative stress response of F. tularensis. We demonstrate that RelA/SpoT-mediated ppGpp production alters global gene transcriptional profile of F. tularensis in the presence of oxidative stress. The lack of stringent response in relA/spoT gene deletion mutants of F. tularensis makes bacteria more susceptible to oxidants, attenuates survival in macrophages, and virulence in mice. This work is an important step forward towards understanding the complex regulatory network underlying the oxidative stress response of F. tularensis.

Methylsulfonylmethane (MSM) is a naturally occurring, sulfate-containing, organic compound. It has been shown to stimulate the differentiation of mesenchymal stem cells into osteoblast-like cells and bone formation. In this study, we investigated whether MSM influences the differentiation of stem cells from human exfoliated deciduous teeth (SHED) into osteoblast-like cells and their osteogenic potential. Here, we report that MSM induced osteogenic differentiation through the expression of osteogenic markers such as osterix, osteopontin, and RUNX2, at both mRNA and protein levels in SHED cells. An increase in the activity of alkaline phosphatase and mineralization confirmed the osteogenic potential of MSM. These MSM-induced effects were observed in cells grown in basal medium but not osteogenic medium. MSM induced transglutaminase-2 (TG2), which may be responsible for the cross-linking of extracellular matrix proteins (collagen or osteopontin), and the mineralization process. Inhibition of TG2 ensued a significant decrease in the differentiation of SHED cells and cross-linking of matrix proteins. A comparison of mineralization with the use of mineralized and demineralized bone particles in the presence of MSM revealed that mineralization is higher with mineralized bone particles than with demineralized bone particles. In conclusion, these results indicated that MSM could promote differentiation and osteogenic potential of SHED cells. This osteogenic property is more in the presence of mineralized bone particles. TG2 is a likely cue in the regulation of differentiation and mineral deposition of SHED cells in response to MSM.

The extensive use of daptomycin (DAP) for treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections in the last decade has led to the emergence of DAP non-susceptible (DNS) Staphylococcus aureus strains. A better understanding of the molecular changes underlying DAP-non-susceptibility is required for early diagnosis and intervention with alternate combination therapies. The phenotypic changes associated with DNS strains have been well established. However, the genotypic changes-especially the kinetics of expression of the genes responsible for DAP-non-susceptibility are not well understood. In this study, we used three clinically derived isogenic pairs of DAP-susceptible (DAP-S) and DNS S. aureus strains to study gene expression profiles with the objective of identifying the potential genotypic changes associated with DAP-nonsusceptibility. We determined the expression profiles of genes involved in cell membrane (CM) charge, autolysis, cell wall (CW) synthesis, and penicillin binding proteins in DAP-S and DNS isogenic pairs. Our results demonstrate characteristic expression profiles for mprF, dltABCD, vraS, femB, and pbp2a genes, which are common to all the DNS S. aureus strains tested. Whole genome sequencing of DAP-S and DNS clinical isolates of S. aureus showed non-synonymous mutations in all DNS strains in genes involved in CM charge, CM composition, CW thickness and CW composition. To conclude, this study unravels some of the complex molecular changes involved in the development of DAP-nonsusceptibility by demonstrating distinct differences in gene expression profiles and mutations in the DNS S. aureus strains. This knowledge will aid in rapid identification of DNS S. aureus in clinical settings.

Mutations in gyrA are the primary cause of quinolone resistance encountered in gram-negative clinical isolates. The prospect of this work was to analyze the role of gyrA mutations in eliciting high quinolone resistance in uropathogenic E.coli (UPEC) through molecular docking studies. Quinolone susceptibility testing of 18 E.coli strains isolated from UTI patients revealed unusually high resistance level to all the quinolones used; especially norfloxacin and ciprofloxacin. The QRDR of gyrA was amplified and sequenced. Mutations identified in gyrA of E.coli included Ser83Leu, Asp87Asn and Ala93Gly/Glu. Contrasting previous reports, we found Ser83Leu substitution in sensitive strains. Strains with S83L, D87N and A93E (A15 and A26) demonstrated norfloxacin MICs ≥1024mg/L which could be proof that Asp87Asn is necessary for resistance phenotype. Resistance to levofloxacin was comparatively lower in all the isolates. Docking of 4 quinolones (ciprofloxacin, ofloxacin, levofloxacin and norfloxacin) to normal and mutated E.coli gyrase A protein demonstrated lower binding energies for the latter, with significant displacement of norfloxacin in the mutated GyrA complex and least displacement in case of levofloxacin.

The objectives of the study were to develop a framework for automatic outer and inner breast tissue segmentation using multi-parametric MRI images of the breast tumor patients; and to perform breast density and tumor tissue analysis. MRI of the breast was performed on 30 patients at 3T-MRI. T1, T2 and PD-weighted(W) images, with and without fat saturation(WWFS), and dynamic-contrast-enhanced(DCE)-MRI data were acquired. The proposed automatic segmentation approach was performed in two steps. In step-1, outer segmentation of breast tissue from rest of body parts was performed on structural images (T2-W/T1-W/PD-W without fat saturation images) using automatic landmarks detection technique based on operations like profile screening, Otsu thresholding, morphological operations and empirical observation. In step-2, inner segmentation of breast tissue into fibro-glandular(FG), fatty and tumor tissue was performed. For validation of breast tissue segmentation, manual segmentation was carried out by two radiologists and similarity coefficients(Dice and Jaccard) were computed for outer as well as inner tissues. FG density and tumor volume were also computed and analyzed. The proposed outer and inner segmentation approach worked well for all the subjects and was validated by two radiologists. The average Dice and Jaccard coefficients value for outer segmentation using T2-W images, obtained by two radiologists, were 0.977 and 0.951 respectively. These coefficient values for FG tissue were 0.915 and 0.875 respectively whereas for tumor tissue, values were 0.968 and 0.95 respectively. The volume of segmented tumor ranged over 2.1 cm3-7.08 cm3. The proposed approach provided automatic outer and inner breast tissue segmentation, which enables automatic calculations of breast tissue density and tumor volume. This is a complete framework for outer and inner breast segmentation method for all structural images.

Coxsackie B Virus (CBV) infection has been linked to the aetiology of type 1 diabetes (T1D) and vaccination has been proposed as prophylaxis for disease prevention. Serum neutralising antibodies and the presence of viral protein and RNA in tissues have been common tools to examine this potential disease relationship, whilst the role of anti-CBV cytotoxic T cell responses and their targets have not been studied. To address this knowledge gap, we augmented conventional HLA-binding predictive algorithm-based epitope discovery by cross-referencing epitopes with sites of positive natural selection within the CBV3 viral genome, identified using mixed effects models of evolution. Eight epitopes for the common MHC class I allele HLA-A*0201 occur at sites that appear to be positively selected. Furthermore, such epitopes span the viral genome, indicating that effective anti-viral responses may not be restricted to the capsid region. To assess the spectrum of IFNy responses in non-diabetic subjects and recently diagnosed type 1 diabetes (T1D) patients, we stimulated PBMC ex vivo with pools of synthetic peptides based on component-restricted sequences identified in silico. We found responders were more likely to recognize multiple rather than a single CBV peptide pool, indicating that the natural course of infection results in multiple targets for effector memory responses, rather than immunodominant epitopes or viral components. The finding that anti-CBV CD8 T cell immunity is broadly targeted has implications for vaccination strategies and studies on the pathogenesis of CBV-linked diseases.

Sealing ring formation is a requirement for osteoclast function. We have recently identified the role of an actin-bundling protein L-plastin in the assembly of nascent sealing zones (NSZs) at the early phase of sealing ring formation in osteoclasts. TNF-α signaling regulates this actin assembly by the phosphorylation of L-plastin on serine -5 and -7 residues at the amino-terminal end. These NSZs function as a core for integrin localization and coordinating integrin signaling required for maturation into fully functional sealing rings. Our goal is to elucidate the essential function of L-plastin phosphorylation in actin bundling, a process required for NSZs formation. The present study was undertaken to determine whether targeting serine phosphorylation of cellular L-plastin would be the appropriate approach to attenuate the formation of NSZs. Our approach is to use TAT-fused small molecular weight amino-terminal L-plastin peptides (10 amino acids) containing phospho- Ser-5 and Ser-7. We used peptides unsubstituted (P1) and substituted (P2- P4) at serine-to-alanine residues. Immunoblotting, actin staining, and dentine resorption analyses were done to determine cellular L-plastin phosphorylation, NSZ or sealing ring formation, and osteoclast function, respectively. Immunoblotting for bone formation markers, Alizarin red staining and alkaline phosphatase activity assay have been done to determine the effect of peptides on the mineralization process mediated by osteoblasts. Transduction of unsubstituted (P1) and substituted peptides at either Serine 5 or Serine 7 with Alanine (P3 and P4) demonstrated variable inhibitory effects on the phosphorylation of cellular L-plastin protein. Peptide P1 reduces the following processes substantially: 1) cellular L-plastin phosphorylation; 2) formation of nascent sealing zones and sealing rings; 3) bone resorption. Substitution of both Serine-5 and -7 with Alanine (P2) had no effects on the inhibitory activities described above. Furthermore, either the L-plastin (P1-P5) or (P6) control peptides had a little or no impact on the a) assembly/disassembly of podosomes and migration of osteoclasts; b) mineralization process mediated by osteoblasts in vitro. Small molecular weight peptidomimetics of L-plastin inhibits bone resorption by osteoclasts via attenuation of NSZ and sealing ring formation but not bone formation by osteoblasts in vitro. The L-plastin may be a valuable therapeutic target to treat and prevent diseases associated with bone loss without affecting bone formation.

BACKGROUND AND AIM:Due to a dysregulated immune response, patients with acute-on-chronic liver failure (ACLF) have increased risk of infection and multi organ failure in comparison to compensated cirrhosis. The comparative data on the presence of 'immune paresis' in patients with ACLF and decompensated cirrhosis without ACLF is not available. Aim of the present study was to compare the immunological parameters in patients with decompensated cirrhosis with and without ACLF. METHODOLOGY:In a prospective study, 76 patients with decompensated cirrhosis with (n = 38) and without (n = 38) ACLF and 10 healthy controls (HC) were evaluated for monocytic human leukocyte antigen-antigen D Related (HLA-DR) expression, mean density of HLA-DR expressed on the surface of these cells, neutrophil oxidative burst (NOB) capacity and serum levels of cytokines (IL-1, IL-6, IL-8, IL10, IL-12, and TNF-α). RESULTS:Patients of decompensated cirrhosis with and without ACLF demonstrated significantly lower mean percentage of monocytes expressing HLA-DR and quantitative increase in the NOB after stimulation with PMA when compared to HC. However there was no difference in mean percentage of monocytes with HLA-DR expression (43.61±26.56% vs. 43.10±20.98%) (p = 0.91), mean density of HLA-DR expression on the surface (30.34±29.32 vs. 41.71±52.13) (p = 0.42) and quantitative increase in NOB after stimulation with PMA (16.55±11.91 vs. 17.24±16.18) (p = 0.47) amongst patients with decompensated cirrhosis with and without ACLF. Patients with ACLF had significantly higher pro-inflammatory and anti-inflammatory cytokines in comparison to patients with decompensated cirrhosis without ACLF. CONCLUSION:Patients with decompensated cirrhosis demonstrate a component of immune-paresis, however there is similar impairment in HLA-DR expression and NOB capacity in patients with and without ACLF. Both inflammatory and anti-inflammatory cytokines are increased in patients with ACLF in comparison to patients with decompensated cirrhosis without ACLF.

Clinical studies have revealed a higher risk of breast tumor recurrence in African-American (AA) patients compared to European-American (EA) patients, contributing to the alarming inequality in clinical outcomes among the ethnic groups. However, distinctions in recurrence patterns upon receiving hormone, radiation, and/or chemotherapy between the races remain poorly characterized.We compared patterns and rates (per 1000 cancer patients per 1 year) of recurrence following each form of treatment between AA (n = 1850) and EA breast cancer patients (n = 7931) from a cohort of patients (n = 10504) treated between 2005-2015 at Northside Hospital in Atlanta, GA.Among patients who received any combination of adjuvant therapy, AA displayed higher overall rates of recurrence than EA (p = 0.015; HR: 1.699; CI: 1.108-2.606). Furthermore, recurrence rates were higher in AA than EA among stage I (p = 0.031; HR: 1.736; CI: 1.052-2.864) and T1 classified patients (p = 0.003; HR: 2.009; CI: 1.263-3.197). Interestingly, among patients who received neoadjuvant chemotherapy, AA displayed higher rates of local recurrence than EA (p = 0.024; HR: 7.134; CI: 1.295-39.313).Our analysis revealed higher incidence rates of recurrence in AA compared to EA among patients that received any combination of adjuvant therapy. Moreover, our data demonstrates an increased risk of tumor recurrence in AA than EA among patients diagnosed with minimally invasive disease. This is the first clinical study to suggest that neoadjuvant chemotherapy improves breast cancer recurrence rates and patterns in AA.

BACKGROUND:Burn-induced inflammation leads to impaired immune responses resulting in increased morbidity and mortality. T-cells are central in the immune response and circulating CD4 and CD8 T-cells have been used to evaluate immune status; however, the role of these T-cell subsets in the burn wound is unknown. METHODS:Male C57BL/6 mice were subjected to a major 3rd degree scald burn or sham treatment. Twenty-four hours later, full thickness skin samples from sham mice and the burn wounds were collected and single cells were isolated and analyzed for αβ TCR, γδ TCR, CD3, CD4, CD8 and CD69 expressions by flow cytometry. RESULTS:The burn wound contained significantly greater numbers of T-cells than skin from sham mice, due to a profound infiltration of αβ T-cells. These infiltrating αβ T-cells were primarily suppressor T-cells with a CD8+ or CD8-CD4- phenotype. The 15-fold increase in CD8+ αβ T-cells caused a decrease in the CD4:CD8 ratio from 0.7 in sham skin to 0.3 in the burn wound. In contrast, the majority of the γδ T-cells in sham skin were CD4-CD8-, which decreased 9-fold in the burn wound. CD69 expression was suppressed on burn wound αβ T-cells, but increased on γδ T-cells in the burn wound. CONCLUSIONS:The infiltrating burn wound αβ T-cells likely act to quell inflammation. In contrast wound γδ T-cells were activated with elevated CD4 and CD69 expression. Thus, these two distinct T-cell subsets likely differentially regulate the burn wound inflammatory response.

Next generation sequencing based tumor tissue genotyping involves complex workflow and a relatively longer turnaround time. Semiconductor based next generation platforms varied from low throughput Ion PGM to high throughput Ion Proton and Ion S5XL sequencer. In this study, we compared Ion PGM and Ion Proton, with a new Ion S5XL NGS system for workflow scalability, analytical sensitivity and specificity, turnaround time and sequencing performance in a clinical laboratory.Eighteen solid tumor samples positive for various mutations as detected previously by Ion PGM and Ion Proton were selected for study. Libraries were prepared using DNA (range10-40ng) from micro-dissected formalin-fixed, paraffin-embedded (FFPE) specimens using the Ion Ampliseq Library Kit 2.0 for comprehensive cancer (CCP), oncomine comprehensive cancer (OCP) and cancer hotspot panel v2 (CHPv2) panel as per manufacturer's instructions. The CHPv2 were sequenced using Ion PGM whereas CCP and OCP were sequenced using Ion Proton respectively. All the three libraries were further sequenced individually (S540) or multiplexed (S530) using Ion S5XL. For S5XL, Ion chef was used to automate template preparation, enrichment of ion spheres and chip loading. Data analysis was performed using Torrent Suite 4.6 software on board S5XL and Ion Reporter. A limit of detection and reproducibility studies was performed using serially diluted DLD1 cell line.A total of 241 variant calls (235 single nucleotide variants and 6 indels) expected in the studied cohort were successfully detected by S5XL with 100% and 97% concordance with Ion PGM and Proton, respectively. Sequencing run time was reduced from 4.5 to 2.5 hours with output range of 3-5 GB (S530) and 8-9.3Gb (S540). Data analysis time for the Ion S5XL is faster 1 h (S520), 2.5 h (S530) and 5 h (S540) chip, respectively as compared to the Ion PGM (3.5-5 h) and Ion Proton (8h). A limit detection of 5% allelic frequency was established along with high inter-run reproducibility.Ion S5XL system simplified workflow in a clinical laboratory, was feasible for running smaller and larger panels on the same instrument, had a shorter turnaround time, and showed good concordance for variant calls with similar sensitivity and reproducibility as the Ion PGM and Proton.

Single nucleotide polymorphisms (SNPs) in the human angiotensinogen (hAGT) gene may modulate its transcription and affect the regulation of blood pressure via activation of the renin-angiotensin aldosterone system (RAAS). In this regard, we have identified polymorphisms in the 2.5 Kb promoter of the hAGT gene that form two haplotype (Hap) blocks: -6A/G (-1670A/G, -1562C/T, -1561T/C) and -217A/G (-532T/C, -793A/G, -1074T/C & -1178G/A). hAGT gene with Hap -6A/-217A (Hap I) is associated with increased blood pressure whereas, Hap -6G/-217G (Hap II) is associated with normal blood pressure in human subjects. Since RAAS over activity contributes to hypertension in obesity, we have made transgenic mice (TG) containing either Hap I or Hap II of the hAGT gene to understand the role of obesity on its transcriptional regulation. Although, a high-fat diet (60% Kcal from fat, 12 weeks) elevates hAGT and mAGT regardless of haplotype, this effect is significantly (p

Wheat cultivars with wide introgression have strongly impacted global wheat production. Aegilops geniculata (MgUg) is an important wild relative with several useful traits that can be exploited for wheat improvement. Screening of Ae. geniculata addition lines indicated a negative effect of 1Ug and the positive effect of 1Mg chromosome on wheat dough strength. Negative effect of 1Ug is probably associated with variation in number and position of the tripeptide repeat motif in the high molecular weight glutenin (HMW-G) gene. To utilize the positive potential of 1Mg chromosome, three disomic substitution lines (DSLs) 1Mg(1A), 1Mg(1B) and 1Mg(1D) were created. These lines were characterized for morphological, cytogenetic properties and biochemical signatures using FISH, 1D-, 2D-PAGE and RP-HPLC. Contribution of wheat 1A, 1B and 1D chromosomes towards dough mixing and baking parameters, chapatti quality, Fe/Zn content and glume color were identified. Observed order of variation in the dough mixing and baking parameters {1Mg(1D) ≤wheat ≤1Mg(1B) ≤1Mg(1A)} indicated that chromosome specific introgression is desirable for best utilization of wild species' potential.

Microtubule structure and functions have been widely studied in vitro and in cells. Research has shown that cysteines on tubulin play a crucial role in the polymerization of microtubules. Here, we show that blocking sulfhydryl groups of cysteines in taxol-stabilized polymerized microtubules with a commonly used chemical crosslinker prevents temporal end-to-end annealing of microtubules in vitro. This can dramatically affect the length distribution of the microtubules. The crosslinker sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, sulfo-SMCC, consists of a maleimide and an N-hydroxysuccinimide ester group to bind to sulfhydryl groups and primary amines, respectively. Interestingly, addition of a maleimide dye alone does not show the same interference with annealing in stabilized microtubules. This study shows that the sulfhydryl groups of cysteines of tubulin that are vital for the polymerization are also important for the subsequent annealing of microtubules.

Starch and proteins are major components in the wheat endosperm that affect its end product quality. Between the two textural classes of wheat i.e. hard and soft, starch granules are loosely bound with the lipids and proteins in soft wheat due to higher expression of interfering grain softness proteins. It might have impact on starch granules properties. In this work for the first time the physiochemical and structural properties of different sized starch granules (A-, B- and C-granules) were studied to understand the differences in starches with respect to soft and hard wheat. A-, B- and C-type granules were separated with >95% purity. Average number and proportion of A-, B-, and C-type granules was 18%, 56%, 26% and 76%, 19%, 5% respectively. All had symmetrical birefringence pattern with varied intensity. All displayed typical A-type crystallites. A-type granules also showed V-type crystallinity that is indicative of starch complexes with lipids and proteins. Granules differing in gelatinization temperature (ΔH) and transition temperature (ΔT), showed different enthalpy changes during heating. Substitution analysis indicated differences in relative substitution pattern of different starch granules. Birefringence, percentage crystallinity, transmittance, gelatinization enthalpy and substitution decreased in order of A>B>C being higher in hard wheat than soft wheat. Amylose content decreased in order of A>B>C being higher in soft wheat than hard wheat. Reconstitution experiment showed that starch properties could be manipulated by changing the composition of starch granules. Addition of A-granules to total starch significantly affected its thermal properties. Effect of A-granule addition was higher than B- and C-granules. Transmittance of the starch granules paste showed that starch granules of hard wheat formed clear paste. These results suggested that in addition to differences in protein concentration, hard and soft wheat lines have differences in starch composition also.

The UK Functional Assessment Measure (UKFIM+FAM) is the principal outcome measure for the UK Rehabilitation Outcomes Collaborative (UKROC) national database for specialist rehabilitation. Previously validated in a mixed neurorehabilitation cohort, this study is the first to explore its psychometric properties in a stroke population, and compare left and right hemispheric strokes (LHS vs RHS). We analysed in-patient episode data from 62 specialist rehabilitation units collated through the UKROC database 2010-2013. Complete data were analysed for 1,539 stroke patients (LHS: 588, RHS: 566 with clear localisation). For factor analysis, admission and discharge data were pooled and randomised into two equivalent samples; the first for exploratory factor analysis (EFA) using principal components analysis, and the second for confirmatory factor analysis (CFA). Responsiveness for each subject (change from admission to discharge) was examined using paired t-tests and differences between LHS and RHS for the entire group were examined using non-paired t-tests. EFA showed a strong general factor accounting for >48% of the total variance. A three-factor solution comprising motor, communication and psychosocial subscales, accounting for >69% total variance, provided acceptable fit statistics on CFA (Root Mean Square Error of Approximation was 0.08 and Comparative Fit Index/ Tucker Lewis Index 0.922/0.907). All three subscales showed significant improvement between admission and discharge (p0.5). Total scores between LHS and RHS were not significantly different. However, LHS showed significantly higher motor scores (Mean 5.7, 95%CI 2.7, 8.6 p

Humans are exposed to numerous xenobiotics, a majority of which are in the form of pharmaceuticals. Apart from human enzymes, recent studies have indicated the role of the gut bacterial community (microbiome) in metabolizing xenobiotics. However, little is known about the contribution of the plethora of gut microbiome in xenobiotic metabolism. The present study reports the results of analyses on xenobiotic metabolizing enzymes in various human gut microbiomes. A total of 397 available gut metagenomes from individuals of varying age groups from 8 nationalities were analyzed. Based on the diversities and abundances of the xenobiotic metabolizing enzymes, various bacterial taxa were classified into three groups, namely, least versatile, intermediately versatile and highly versatile xenobiotic metabolizers. Most interestingly, specific relationships were observed between the overall drug consumption profile and the abundance and diversity of the xenobiotic metabolizing repertoire in various geographies. The obtained differential abundance patterns of xenobiotic metabolizing enzymes and bacterial genera harboring them, suggest their links to pharmacokinetic variations among individuals. Additional analyses of a few well studied classes of drug modifying enzymes (DMEs) also indicate geographic as well as age specific trends.

Gamma delta T-cells have been shown to be important to the early immunoinflammatory response to injury, independent of infection. This unique T-cell population acts to regulate cell trafficking and the release of cytokines and growth factors. We propose this sterile inflammatory response is in part associated with damage associated molecular patterns (DAMPs) generated by major injury, such as burn, and mediated via toll-like receptors (TLRs). It is unknown whether DAMPs can activate resident γδ T-cells that reside in skin.Gamma delta T-cells were isolated from the skin of male C57BL/6 mice by enzymatic digestion. Mitochondrial DAMPs (MTDs) were generated from mitochondria isolated from mouse livers by sonication and centrifugation. Dermal γδ T-cells were incubated with MTDs (0-500 μg/ml) for 24 hr and cells and supernatants were collected for analysis.MTDs activated dermal γδ T-cells, as evidenced by increased TLR2 and TLR4 expression following in vitro exposure. MTDs also induced the production of inflammatory cytokines (IL-1β, IL-6), and growth factors (PDGF and VEGF) by γδ T-cells.These findings herein support the concept that MTDs released after tissue/cellular injury are capable of activating dermal γδ T-cells. We propose that the activation of this unique T-cell population is central in the initiation of sterile inflammation and also contributes to the subsequent healing processes.

Schmallenberg virus (SBV) is an emerging Orthobunyavirus, first described in 2011 in cattle in Germany and subsequently spread throughout Europe, affecting mainly ruminant livestock through the induction of foetal malformations. To gain a better understanding of the spectrum of susceptible species and to assess the value of current SBV serological assays, screening of serum samples from exotic artiodactyls and perissodactyls collected at the Living Collections from the Zoological Society of London (Whipsnade and London Zoos) and Chester Zoo was carried out. There was compelling evidence of SBV infection in both zoological collections. The competitive ELISA has proved to be applicable for the detection of SBV in exotic Bovidae, Cervidae, Suidae, Giraffidae and most notably in endangered Asian elephants (Elephas maximus), but unreliable for the screening of Camelidae, for which the plaque reduction neutralisation test was considered the assay of choice.

BackgroundHousehold contacts of diagnostically established tuberculosis (TB) patients are highly susceptible to disease development. It is surmised that cytokines perhaps play a synergistic and a prognostic role in the activation of the otherwise latent infection in these house hold contacts. Evaluation of the cytokines and any of their inherent polymorphisms might provide a useful diagnostic tool in evaluating the immune regulation and the progression of the disease. The cytokines thus released in a paracrine manner in serum may also provide an indirect measure of the cytokine function.ObjectiveThe present study was aimed to evaluate the levels of TNF-α, IL-10 & IL-6 cytokines and their correlation with genotype variants amongst tuberculosis patients and their household contacts.MethodsThe cytokine levels were estimated in serum by enzyme-linked immunosorbent assay (ELISA) and their polymorphisms were studied by amplification refractory mutation system polymerase chain reaction (ARMs PCR) in active pulmonary tuberculosis patients (APTB = 150), household contacts (HHC = 190), and healthy controls (HC = 150).ResultsThe median values of TNF-α cytokine were significantly high among APTB and HHC compared to HCs (P< 0.0001 and 0.0001). IL-6 levels also were elevated among APTB compared to HHC and HC, and a significant difference was observed between APTB and HHC at PConclusionLarge sample size with follow-up at different time points may further illuminate the role of IL-10 and IL-6 cytokines as a prognostic marker in house hold contacts.

BackgroundFamilies in high mortality settings need regular contact with high quality services, but existing population-based measurements of contacts do not reflect quality. To address this, in 2012, we designed linked household and frontline worker surveys for Gombe State, Nigeria, Ethiopia, and Uttar Pradesh, India. Using reported frequency and content of contacts, we present a method for estimating the population level coverage of high quality contacts.Methods and findingsLinked cluster-based household and frontline health worker surveys were performed. Interviews were conducted in 40, 80 and 80 clusters in Gombe, Ethiopia, and Uttar Pradesh, respectively, including 348, 533, and 604 eligible women and 20, 76, and 55 skilled birth attendants. High quality contacts were defined as contacts during which recommended set of processes for routine health care were met. In Gombe, 61% (95% confidence interval 50-72) of women had at least one antenatal contact, 22% (14-29) delivered with a skilled birth attendant, 7% (4-9) had a post-partum check and 4% (2-8) of newborns had a post-natal check. Coverage of high quality contacts was reduced to 11% (6-16), 8% (5-11), 0%, and 0% respectively. In Ethiopia, 56% (49-63) had at least one antenatal contact, 15% (11-22) delivered with a skilled birth attendant, 3% (2-6) had a post-partum check and 4% (2-6) of newborns had a post-natal check. Coverage of high quality contacts was 4% (2-6), 4% (2-6), 0%, and 0%, respectively. In Uttar Pradesh 74% (69-79) had at least one antenatal contact, 76% (71-80) delivered with a skilled birth attendant, 54% (48-59) had a post-partum check and 19% (15-23) of newborns had a post-natal check. Coverage of high quality contacts was 6% (4-8), 4% (2-6), 0%, and 0% respectively.ConclusionsMeasuring content of care to reflect the quality of contacts can reveal missed opportunities to deliver best possible health care.

Francisella tularensis is a facultative intracellular pathogen, and is the causative agent of a fatal human disease known as tularemia. F. tularensis is classified as a Category A Biothreat agent by the CDC based on its use in bioweapon programs by several countries in the past and its potential to be used as an agent of bioterrorism. No licensed vaccine is currently available for prevention of tularemia. In this study, we used a novel approach for development of a multivalent subunit vaccine against tularemia by using an efficient tobacco mosaic virus (TMV) based delivery platform. The multivalent subunit vaccine was formulated to contain a combination of F. tularensis protective antigens: OmpA-like protein (OmpA), chaperone protein DnaK and lipoprotein Tul4 from the highly virulent F. tularensis SchuS4 strain. Two different vaccine formulations and immunization schedules were used. The immunized mice were challenged with lethal (10xLD100) doses of F. tularensis LVS on day 28 of the primary immunization and observed daily for morbidity and mortality. Results from this study demonstrate that TMV can be used as a carrier for effective delivery of multiple F. tularensis antigens. TMV-conjugate vaccine formulations are safe and multiple doses can be administered without causing any adverse reactions in immunized mice. Immunization with TMV-conjugated F. tularensis proteins induced a strong humoral immune response and protected mice against respiratory challenges with very high doses of F. tularensis LVS. This study provides a proof-of-concept that TMV can serve as a suitable platform for simultaneous delivery of multiple protective antigens of F. tularensis. Refinement of vaccine formulations coupled with TMV-targeting strategies developed in this study will provide a platform for development of an effective tularemia subunit vaccine as well as a vaccination approach that may broadly be applicable to many other bacterial pathogens.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a rare hereditary stroke caused by mutations in NOTCH3 gene. We report the first case of CADASIL in an indigenous Rungus (Kadazan-Dusun) family in Kudat, Sabah, Malaysia confirmed by a R54C (c.160C>T, p.Arg54Cys) mutation in the NOTCH3. This mutation was previously reported in a Caucasian and two Korean cases of CADASIL. We recruited two generations of the affected Rungus family (n = 9) and found a missense mutation (c.160C>T) in exon 2 of NOTCH3 in three siblings. Two of the three siblings had severe white matter abnormalities in their brain MRI (Scheltens score 33 and 50 respectively), one of whom had a young stroke at the age of 38. The remaining sibling, however, did not show any clinical features of CADASIL and had only minimal changes in her brain MRI (Scheltens score 17). This further emphasized the phenotype variability among family members with the same mutation in CADASIL. This is the first reported family with CADASIL in Rungus subtribe of Kadazan-Dusun ethnicity with a known mutation at exon 2 of NOTCH3. The penetrance of this mutation was not complete during the course of this study.

Severely immunocompromised state during advanced stage of HIV-1 infection has been linked to functionally defective antigen presentation by dendritic cells (DCs). The molecular mechanisms behind DC impairment are still obscure. We investigated changes in DC function and association of key regulators of cytokine signaling during different stages of HIV-1 infection and following antiretroviral therapy (ART).Phenotypic and functional characteristics of circulating myeloid DCs (mDCs) in 56 ART-naive patients (23 in early and 33 in advanced stage of disease), 36 on ART and 24 healthy controls were evaluated. Sixteen patients were studied longitudinally prior-to and 6 months after the start of ART. For functional studies, monocyte-derived DCs (Mo-DCs) were evaluated for endocytosis, allo-stimulation and cytokine secretion. The expression of suppressor of cytokine signaling (SOCS)-1 and other regulators of cytokine signaling was evaluated by real-time RT-PCR.The ability to respond to an antigenic stimulation was severely impaired in patients in advanced HIV-1 disease which showed partial recovery in the treated group. Mo-DCs from patients with advanced HIV-disease remained immature with low allo-stimulation and reduced cytokine secretion even after TLR-4 mediated stimulation ex-vivo. The cells had an increased expression of negative regulatory factors like SOCS-1, SOCS-3, SH2-containing phosphatase (SHP)-1 and a reduced expression of positive regulators like Janus kinase (JAK)2 and Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)1. A functional recovery after siRNA mediated silencing of SOCS-1 in these mo-DCs confirms the role of negative regulatory factors in functional impairment of these cells.Functionally defective DCs in advanced stage of HIV-1 infection seems to be due to imbalanced state of negative and positive regulatory gene expression. Whether this is a cause or effect of increased viral replication at this stage of disease, needs further investigation. The information may be useful in design of novel therapeutic targets for better management of disease.

We previously reported the generation of a reporter line of human embryonic stem cells (hESCs) with enhanced green fluorescent protein (eGFP) expression driven by the α-myosin heavy chain (αMHC) promoter. The GFP+/αMHC+ cells derived from this cell line behave as multipotent, human myocardial precursors (hMPs) in vitro. In this study, we evaluated the therapeutic effects of GFP+/αMHC+ cells isolated from the reporter line in a mouse model of myocardial infarction (MI).MI was generated in immunodeficient mice. hMPs were injected into murine infarcted hearts under ultrasound guidance at 3 days post-MI. Human fetal skin fibroblasts (hFFs) were injected as control. Cardiac function was evaluated by echocardiography. Infarct size, angiogenesis, apoptosis, cell fate, and teratoma formation were analyzed by immunohistochemical staining.Compared with control, hMPs resulted in improvement of cardiac function post-MI with smaller infarct size, induced endogenous angiogenesis, and reduced apoptosis of host cardiomyocytes at the peri-infarct zone at 28 days post-MI.Intramyocardial injection of hMPs improved cardiac function post-MI. The engraftment rate of these cells in the myocardium post-MI was low, suggesting that the majority of effect occurs via paracrine mechanisms.

Tularemia is caused by a gram-negative, intracellular bacterial pathogen, Francisella tularensis (Ft). The history weaponization of Ft in the past has elevated concerns that it could be used as a bioweapon or an agent of bioterrorism. Since the discovery of Ft, three broad approaches adopted for tularemia vaccine development have included inactivated, live attenuated, or subunit vaccines. Shortcomings in each of these approaches have hampered the development of a suitable vaccine for prevention of tularemia. Recently, we reported an oxidant sensitive mutant of Ft LVS in putative EmrA1 (FTL_0687) secretion protein. The emrA1 mutant is highly sensitive to oxidants, attenuated for intramacrophage growth and virulence in mice. We reported that EmrA1 contributes to oxidant resistance by affecting the secretion of antioxidant enzymes SodB and KatG. This study investigated the vaccine potential of the emrA1 mutant in prevention of respiratory tularemia caused by Ft LVS and the virulent SchuS4 strain in C57BL/6 mice. We report that emrA1 mutant is safe and can be used at an intranasal (i. n.) immunization dose as high as 1x106 CFU without causing any adverse effects in immunized mice. The emrA1 mutant is cleared by vaccinated mice by day 14-21 post-immunization, induces minimal histopathological lesions in lungs, liver and spleen and a strong humoral immune response. The emrA1 mutant vaccinated mice are protected against 1000-10,000LD100 doses of i.n. Ft LVS challenge. Such a high degree of protection has not been reported earlier against respiratory challenge with Ft LVS using a single immunization dose with an attenuated mutant generated on Ft LVS background. The emrA1 mutant also provides partial protection against i.n. challenge with virulent Ft SchuS4 strain in vaccinated C57BL/6 mice. Collectively, our results further support the notion that antioxidants of Ft may serve as potential targets for development of effective vaccines for prevention of tularemia.

Equine influenza viruses (EIV)-H3N8 continue to circulate in equine population throughout the world. They evolve by the process of antigenic drift that leads to substantial change in the antigenicity of the virus, thereby necessitating substitution of virus strain in the vaccines. This requires frequent testing of the new vaccines in the in vivo system; however, lack of an appropriate laboratory animal challenge model for testing protective efficacy of equine influenza vaccine candidates hinders the screening of new vaccines and other therapeutic approaches. In the present investigation, BALB/c mouse were explored for suitability for conducting pathogenecity studies for EIV. The BALB/c mice were inoculated intranasally @ 2×106.24 EID50 with EIV (H3N8) belonging to Clade 2 of Florida sublineage and monitored for setting up of infection and associated parameters. All mice inoculated with EIV exhibited clinical signs viz. loss in body weights, lethargy, dyspnea, etc, between 3 and 5 days which commensurate with lesions observed in the respiratory tract including rhinitis, tracheitis, bronchitis, bronchiolitis, alveolitis and diffuse interstitial pneumonia. Transmission electron microscopy, immunohistochemistry, virus quantification through titration and qRT-PCR demonstrated active viral infection in the upper and lower respiratory tract. Serology revealed rise in serum lactate dehydrogenase levels along with sero-conversion. The pattern of disease progression, pathological lesions and virus recovery from nasal washings and lungs in the present investigations in mice were comparable to natural and experimental EIV infection in equines. The findings establish BALB/c mice as small animal model for studying EIV (H3N8) infection and will have immense potential for dissecting viral pathogenesis, vaccine efficacy studies, preliminary screening of vaccine candidates and antiviral therapeutics against EIV.

G beta 5 (Gbeta5, Gβ5) is a unique G protein β subunit that is thought to be expressed as an obligate heterodimer with R7 regulator of G protein signaling (RGS) proteins instead of with G gamma (Gγ) subunits. We found that D2-dopamine receptor (D2R) coexpression enhances the expression of Gβ5, but not that of the G beta 1 (Gβ1) subunit, in HEK293 cells, and that the enhancement of expression occurs through a stabilization of Gβ5 protein. We had previously demonstrated that the vast majority of D2R either expressed endogenously in the brain or exogenously in cell lines segregates into detergent-resistant biochemical fractions. We report that when expressed alone in HEK293 cells, Gβ5 is highly soluble, but is retargeted to the detergent-resistant fraction after D2R coexpression. Furthermore, an in-cell biotin transfer proximity assay indicated that D2R and Gβ5 segregating into the detergent-resistant fraction specifically interacted in intact living cell membranes. Dopamine-induced D2R internalization was blocked by coexpression of Gβ5, but not Gβ1. However, the same Gβ5 coexpression levels had no effect on agonist-induced internalization of the mu opioid receptor (MOR), cell surface D2R levels, dopamine-mediated recruitment of β-arrestin to D2R, the amplitude of D2R-G protein coupling, or the deactivation kinetics of D2R-activated G protein signals. The latter data suggest that the interactions between D2R and Gβ5 are not mediated by endogenously expressed R7 RGS proteins.

Mutations in the autosomal genes TMPRSS3, TMC1, USHIC, CDH23 and TMIE are known to cause hereditary hearing loss. To study the contribution of these genes to autosomal recessive, non-syndromic hearing loss (ARNSHL) in India, we examined 374 families with the disorder to identify potential mutations. We found four mutations in TMPRSS3, eight in TMC1, ten in USHIC, eight in CDH23 and three in TMIE. Of the 33 potentially pathogenic variants identified in these genes, 23 were new and the remaining have been previously reported. Collectively, mutations in these five genes contribute to about one-tenth of ARNSHL among the families examined. New mutations detected in this study extend the allelic heterogeneity of the genes and provide several additional variants for structure-function correlation studies. These findings have implications for early DNA-based detection of deafness and genetic counseling of affected families in the Indian subcontinent.

Wolbachia, an endosymbiont of filarial nematode, is considered a promising target for treatment of lymphatic filariasis. Although functional characterization of the Wolbachia peptidoglycan assembly has not been fully explored, the Wolbachia genome provides evidence for coding all of the genes involved in lipid II biosynthesis, a part of peptidoglycan biosynthesis pathway. UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is one of the lipid II biosynthesis pathway enzymes and it has inevitably been recognized as an antibiotic target. In view of the vital role of MurA in bacterial viability and survival, MurA ortholog from Wolbachia endosymbiont of Brugia malayi (wBm-MurA) was cloned, expressed and purified for further molecular characterization. The enzyme kinetics and inhibition studies were undertaken using fosfomycin. wBm-MurA was found to be expressed in all the major life stages of B. malayi and was immunolocalized in Wolbachia within the microfilariae and female adults by the confocal microscopy. Sequence analysis suggests that the amino acids crucial for enzymatic activity are conserved. The purified wBm-MurA was shown to possess the EPSP synthase (3-phosphoshikimate 1-carboxyvinyltransferase) like activity at a broad pH range with optimal activity at pH 7.5 and 37°C temperature. The apparent affinity constant (Km) for the substrate UDP-N-acetylglucosamine was found to be 0.03149 mM and for phosphoenolpyruvate 0.009198 mM. The relative enzymatic activity was inhibited ∼2 fold in presence of fosfomycin. Superimposition of the wBm-MurA homology model with the structural model of Haemophilus influenzae (Hi-MurA) suggests binding of fosfomycin at the same active site. The findings suggest wBm-MurA to be a putative antifilarial drug target for screening of novel compounds.

BackgroundAlthough the importance of the Pap smear in reducing cancer incidence and mortality is known, many countries in Africa have not initiated yet widespread national cervical cancer screening programs. The World Health Organization (WHO) has published Clinical Practice Guidelines (CPGs) on cervical cancer screening in developing countries; however, there is a gap between expectations and clinical performance. Thus, the aim of this study was to conduct a systematic review and meta-summary to identify factors affecting compliance with CPGs for Pap screening among healthcare providers in Africa.MethodsAnd Findings: MEDLINE, Scirus, Opengate and EMBASE databases were searched in January 2012. Studies involving medical personnel practicing in Africa, whose outcome measured any factors that affect medical personnel from using a Pap smear to screen for cervical cancer, were included. Two reviewers independently evaluated titles and abstracts, then full-texts, extracted data and assessed quality of the included studies. A descriptive analysis of the included studies was conducted. We calculated Frequency effect sizes (FES) for each finding and Intensity effect sizes (IES) for each article to represent their magnitudes in the analyses. Of 1011 studies retrieved, 11 studies were included (2045 individuals). Six different themes related to the factors affecting compliance with CPGs were identified: Insufficient Knowledge/Lack of awareness (FES = 82%), Negligence/Misbeliefs (FES = 82%), Psychological Reasons (FES = 73%), Time/Cost Constraint (FES = 36%), Insufficient infrastructure/training (FES = 45%) and also no reason given (FES = 36%). IES for articles ranged between 33 and 83%.ConclusionsThese results suggest that prevention initiatives should be comprehensive to include education and resources needs assessments and improvement, Pap smear test training, strategies on costing, and practitioner time studies.