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IntroductionUncorrected refractive errors pose a significant challenge globally, particularly in remote regions of low-middle income countries where access to optometric care is often limited. Telerefraction, which involves refraction by a trained technician followed by real-time consultation with remote optometrist, is a promising approach for such remote settings. This study aimed to evaluate the accuracy of this model.MethodsThis prospective study, conducted in New Delhi, compared tele-refraction to in-person examinations. Trained technicians used a simple device, Click-check, to perform objective refraction and a tele-refraction platform to enter the findings of objective refraction. Final prescription was made after consulting a remote optometrist on that platform. Masked face-to-face optometrists served as the gold standard. The study involved refraction in 222 patients and 428 eyes.ResultsTele-refraction demonstrated a strong agreement with in-person optometry, achieving 84.6% in spherical correction and 81% conformity in spherical equivalent. The mean difference of spherical equivalent between the two arms was only 0.11 D. The consultation with a remote optometrist improved conformity of spherical equivalent by 14.8% over objective refraction. 82 percent eyes matched in best corrected visual acuity and 92 percent were within 0.1 logMAR difference. For cylindrical axis, 74% eye were within acceptable 10 degrees of difference. The mismatch amongst the individual trained technicians, in terms of difference between the tele-refraction arm and the face-to face optometrist arm was found to be significant for cylindrical axis and not for spherical power and spherical equivalent.ConclusionOur study found tele-refraction by a trained technician comparable to refraction done by face-to-face optometrist. Tele-refraction, coupled with remote optometrist guidance can address the optometry resource gap in underserved areas. Thus, this model offers a transformative approach to enhancing the accessibility and quality of eye care services, which can significantly contribute to our efforts in achieving the global targets set by the World Health Organization for effective refractive error coverage. More standardized training for these technicians on ClickCheckTM for detecting the cylindrical axis with better accuracy, can improve this model further.

Today, supply chain (SC) networks are facing more disruptions compared to the past. While disruptions are rare, they can have catastrophic long-term economic or societal repercussions, and the recovery processes can be lengthy. These can tremendously affect the SC and make it vulnerable, as observed during the COVID-19 pandemic. The identification of these concerns has prompted the demand for improved disruption management by developing resilient, agile, and adaptive SC. The aim of this study is to introduce an assessment framework for prioritizing and evaluating the determinants to supply chain resilience (SCR). To analyze the empirical data, fuzzy criteria importance through intercriteria correlation (fuzzy CRITIC) and fuzzy technique for order of preference by similarity to ideal solution (fuzzy TOPSIS) have been incorporated. Fuzzy CRITIC method was used to identify the critical determinants and fuzzy TOPSIS method was applied for determining relative ranking of some real-world companies. Finally, by developing propositions an interpretive triple helix framework was proposed to achieve SCR. This research stands out for its originality in both methodology and implications. By introducing the novel combination of Fuzzy CRITIC and Fuzzy TOPSIS in the assessment of determinants to SCR and applying these determinants with the help of interpretive triple helix framework to establish a resilient SC, this study offers a unique and valuable contribution to the field of SCR. The key findings suggest that 'Responsiveness' followed by 'Managerial coordination and information integration' are the most significant determinant to achieve SCR. The outcome of this work can assist the managers to achieve SCR with improved agility and adaptivity.

DNA from formalin-preserved tissue could unlock a vast repository of genetic information stored in museums worldwide. However, formaldehyde crosslinks proteins and DNA, and prevents ready amplification and DNA sequencing. Formaldehyde acylation also fragments the DNA. Treatment with proteinase K proteolyzes crosslinked proteins to rescue the DNA, though the process is quite slow. To reduce processing time and improve rescue efficiency, we applied the mechanical energy of a vortex fluidic device (VFD) to drive the catalytic activity of proteinase K and recover DNA from American lobster tissue (Homarus americanus) fixed in 3.7% formalin for >1-year. A scan of VFD rotational speeds identified the optimal rotational speed for recovery of PCR-amplifiable DNA and while 500+ base pairs were sequenced, shorter read lengths were more consistently obtained. This VFD-based method also effectively recovered DNA from formalin-preserved samples. The results provide a roadmap for exploring DNA from millions of historical and even extinct species.

Globally, traditional knowledge systems are a powerhouse of information which can revolutionise the world, if decoded accurately and logically. Plant-based ethno-traditional and folklore curatives/medicines has a firm basis in the psyche of the common masses of West Bengal and Holarrhena pubescens is a representative example of it. This article communication on depicting the anthelmintic efficacy of ethanolic extract and Ethyl acetate fraction of the stem bark of Holarrhena pubescens against the cestode Raillietina spp. through efficacy studies, ultra-structural observations, histochemical and biochemical analysis on some tegumental enzymes i.e., Acid Phosphatase (AcPase), Alkaline Phosphatase (AlkPase), Adenosine Triphosphatase (ATPase) and 5'-Nucleotidase (5'-Nu) along with Gray Level Co-occurrence Matrix (GLCM) analysis of histochemical study. Praziquantel was used as the reference drug. Investigations revealed 10mg/ml dosage of crude extract was the most efficacious dose and amongst the fractions the ethyl acetate fraction showed the most anthelmintic property. Ultrastructural studies through Scanning Electron Microscope (SEM) and Transmission Electron Microscope (TEM) clearly depicted the damage in head, sucker, proglottids, proximal and distal cytoplasm (DC), microtriches (MT), basal lamina (BL), nuclear membrane (NM), and, nucleolus (NL) in the treated worms. Histochemical studies revealed decrease in staining intensity for all the tegumental enzymes in the treated worms compared to control. The GLCM analysis strongly supported the result of histochemical studies. Biochemical studies revealed marked reduction in enzyme activity in the treated worms with maximum reduction in the activity of 5'- Nu (77.8%) followed by ATPase (63.17%).

BackgroundSexually transmitted infections (STIs) are one of the leading causes of health, and economic burdens in the developing world, leading to considerable morbidity, mortality, and stigma. The incidence and prevalence of the four curable STIs viz. syphilis, gonorrhoea, chlamydia, and trichomoniasis vary remarkably across different geographical locations. In India, the prevalence of four curable STI among general populations is in between 0 to 3.9 percent. However, it is assumed that STI prevalence is much higher among subpopulations practicing high-risk behaviour. Like men who have sex with men (MSM), transgender (TG), injecting drug users (IDU), and female sex workers (FSW).ObjectivesIn the present circumstances, a systematic review is necessary to integrate the available data from previously published peer-reviewed articles and published reports from several competent authorities to provide the prevalence and geographical distribution of the four curable STIs among the key population of India.MethodsAll available articles will be retrieved from PubMed, Google Scholar, Cochrane database, Scopus, Science Direct, and the Global Health network using the appropriate search terms. The data will be extracted through data extraction form as per PICOS (population, intervention, comparison, outcome, study design) framework. Risk of bias and quality assessment will be performed according to the situation with the help of available conventional protocol.DiscussionThe future systematic review, generated from the present protocol, may provide evidence of the prevalence and geographical distribution of the four curable STIs among the key population of India. We hope that the findings of the future systematic review will strengthen the existing surveillance system in India, to determine the above-mention STIs prevalence among key populations in India. Protocol registration number: International Prospective Register for Systematic Reviews (PROSPERO) number CRD42022346470.

BackgroundBuilding on a distinguished history of community medicine training, public health programs have been expanding in India in recent years. The COVID-19 pandemic has brought additional attention to the importance of public health programs and the need for a strong workforce. This paper aims to assess the current capacity for public health education and training in India and provide recommendations for improved approaches to meet current and future public health needs.MethodsWe conducted a desk review of public health training programs via extensive internet searches, literature reviews, and expert faculty consultations. Among those programs, we purposively selected faculty members to participate in in-depth interviews. We developed summary statistics based on the desk review. For qualitative analysis, we utilized a combination of deductive and inductive coding to identify key themes and systematically reviewed the strengths and weaknesses of each theme.ResultsThe desk review captured 59 institutions offering public health training across India. The majority of training programs were graduate level degrees including Master of Public Health and Master of Science degrees. Key factors impacting these programs included collaborations, mentorship, curriculum standardization, tuition and funding, and student demand for public health education and careers. Collaborations and mentorship were highly valued but varied in quality across institutions. Curricula lacked standardization but also contained substantial flexibility and innovation as a result. Public sector programs were perceived to be affordable though fees and stipends varied across institutions. Further development of career opportunities in public health is needed.ConclusionPublic health education and training in India have a strong foothold. There are numerous opportunities for continued expansion and strengthening of this field, to support a robust multi-disciplinary public health workforce that will contribute towards achieving the sustainable development goals.

Introduction: Uncorrected refractive errors pose a significant challenge globally, particularly in remote regions of low-middle income countries where access to optometric care is often limited. Telerefraction, which involves refraction by a trained technician followed by real-time consultation with remote optometrist, is a promising approach for such remote settings. This study aimed to evaluate the accuracy of this model. Methods: This prospective study, conducted in New Delhi, compared tele-refraction to in-person examinations. Trained technicians used a simple device, Click-check, to perform objective refraction and a tele-refraction platform to enter the findings of objective refraction. Final prescription was made after consulting a remote optometrist on that platform. Masked face-to-face optometrists served as the gold standard. The study involved refraction in 222 patients and 428 eyes. Results: Tele-refraction demonstrated a strong agreement with in-person optometry, achieving 84.6% in spherical correction and 81% conformity in spherical equivalent. The mean difference of spherical equivalent between the two arms was only 0.11 D. The consultation with a remote optometrist improved conformity of spherical equivalent by 14.8% over objective refraction. 82 percent eyes matched in best corrected visual acuity and 92 percent were within 0.1 logMAR difference. For cylindrical axis, 74% eye were within acceptable 10 degrees of difference. The mismatch amongst the individual trained technicians, in terms of difference between the tele-refraction arm and the face-to face optometrist arm was found to be significant for cylindrical axis and not for spherical power and spherical equivalent. Conclusion: Our study found tele-refraction by a trained technician comparable to refraction done by face-to-face optometrist. Tele-refraction, coupled with remote optometrist guidance can address the optometry resource gap in underserved areas. Thus, this model offers a transformative approach to enhancing the accessibility and quality of eye care services, which can significantly contribute to our efforts in achieving the global targets set by the World Health Organization for effective refractive error coverage. More standardized training for these technicians on ClickCheckTM for detecting the cylindrical axis with better accuracy, can improve this model further. [ABSTRACT FROM AUTHOR]

Consistent clinical observations of characteristic findings of COVID-19 pneumonia on chest X-rays have attracted the research community to strive to provide a fast and reliable method for screening suspected patients. Several machine learning algorithms have been proposed to find the abnormalities in the lungs using chest X-rays specific to COVID-19 pneumonia and distinguish them from other etiologies of pneumonia. However, despite the enormous magnitude of the pandemic, there are very few instances of public databases of COVID-19 pneumonia, and to the best of our knowledge, there is no database with annotation of abnormalities on the chest X-rays of COVID-19 affected patients. Annotated databases of X-rays can be of significant value in the design and development of algorithms for disease prediction. Further, explainability analysis for the performance of existing or new deep learning algorithms will be enhanced significantly with access to ground-truth abnormality annotations. The proposed COVID Abnormality Annotation for X-Rays (CAAXR) database is built upon the BIMCV-COVID19+ database which is a large-scale dataset containing COVID-19+ chest X-rays. The primary contribution of this study is the annotation of the abnormalities in over 1700 frontal chest X-rays. Further, we define protocols for semantic segmentation as well as classification for robust evaluation of algorithms. We provide benchmark results on the defined protocols using popular deep learning models such as DenseNet, ResNet, MobileNet, and VGG for classification, and UNet, SegNet, and Mask-RCNN for semantic segmentation. The classwise accuracy, sensitivity, and AUC-ROC scores are reported for the classification models, and the IoU and DICE scores are reported for the segmentation models.

Aim To develop an accurate lab score based on in-hospital patients’ potent clinical and biological parameters for predicting COVID-19 patient severity during hospital admission. Methods To conduct this retrospective analysis, a derivation cohort was constructed by including all the available biological and clinical parameters of 355 COVID positive patients (recovered = 285, deceased = 70), collected in November 2020-September 2021. For identifying potent biomarkers and clinical parameters to determine hospital admitted patient severity or mortality, the receiver operating characteristics (ROC) curve and Fischer’s test analysis was performed. Relative risk regression was estimated to develop laboratory scores for each clinical and routine biological parameter. Lab score was further validated by ROC curve analysis of the validation cohort which was built with 50 COVID positive hospital patients, admitted during October 2021-January 2022. Results Sensitivity vs. 1-specificity ROC curve (>0.7 Area Under the Curve, 95% CI) and univariate analysis (pConclusion The development of proper lab scores, based on patients’ clinical parameters and routine biomarkers, would help physicians to predict patient risk at the time of their hospital admission and may improve hospital-admitted COVID-19 patients’ survivability.

Inhaled Corticosteroids (ICS) are commonly prescribed to patients with severe COPD and recurrent exacerbations. It is not known what impact ICS cause in terms of COVID-19 positivity or disease severity in COPD. This study examined 27,810 patients with COPD from the Cleveland Clinic COVID-19 registry between March 8th and September 16th, 2020. Electronic health records were used to determine diagnosis of COPD, ICS use, and clinical outcomes. Multivariate logistic regression was used to adjust for demographics, month of COVID-19 testing, and comorbidities known to be associated with increased risk for severe COVID-19 disease. Amongst the COPD patients who were tested for COVID-19, 44.1% of those taking an ICS-containing inhaler tested positive for COVID-19 versus 47.2% who tested negative for COVID-19 (p = 0.033). Of those who tested positive for COVID-19 (n = 1288), 371 (28.8%) required hospitalization. In-hospital outcomes were not significantly different when comparing ICS versus no ICS in terms of ICU admission (36.8% [74/201] vs 31.2% [53/170], p = 0.30), endotracheal intubation (21.9% [44/201] vs 16.5% [28/170], p = 0.24), or mortality (18.4% [37/201] vs 20.0% [34/170], p = 0.80). Multivariate logistic regression demonstrated no significant differences in hospitalization (adj OR 1.12, CI: 0.90-1.38), ICU admission (adj OR: 1.31, CI: 0.82-2.10), need for mechanical ventilation (adj OR 1.65, CI: 0.69-4.02), or mortality (OR: 0.80, CI: 0.43-1.49). In conclusion, ICS therapy did not increase COVID-19 related healthcare utilization or mortality outcome in patients with COPD followed at the Cleveland Clinic health system. These findings should encourage clinicians to continue ICS therapy for COPD patients during the COVID-19 pandemic.

We provide an assessment of the impact of government closure and containment measures on deaths from COVID-19 across sequential waves of the COVID-19 pandemic globally. Daily data was collected on a range of containment and closure policies for 186 countries from January 1, 2020 until March 11th, 2021. These data were combined into an aggregate stringency index (SI) score for each country on each day (range: 0-100). Countries were divided into successive waves via a mathematical algorithm to identify peaks and troughs of disease. Within our period of analysis, 63 countries experienced at least one wave, 40 countries experienced two waves, and 10 countries saw three waves, as defined by our approach. Within each wave, regression was used to assess the relationship between the strength of government stringency and subsequent deaths related to COVID-19 with a number of controls for time and country-specific demographic, health system, and economic characteristics. Across the full period of our analysis and 113 countries, an increase of 10 points on the SI was linked to 6 percentage points (P < 0.001, 95% CI = [5%, 7%]) lower average daily deaths. In the first wave, in countries that ultimately experiences 3 waves of the pandemic to date, ten additional points on the SI resulted in lower average daily deaths by 21 percentage points (P < .001, 95% CI = [8%, 16%]). This effect was sustained in the third wave with reductions in deaths of 28 percentage points (P < .001, 95% CI = [13%, 21%]). Moreover, interaction effects show that government policies were effective in reducing deaths in all waves in all groups of countries. These findings highlight the enduring importance of non-pharmaceutical responses to COVID-19 over time.

Exposure to ultrafine combustion aerosols such as particulate matter (PM) from residential woodburning, forest fires, cigarette smoke, and traffic emission have been linked to adverse health outcomes. Excitation-emission matrix (EEM) spectroscopy presents a sensitive and cost-effective alternative for analysis of PM organic fraction. However, as with other analytical chemistry methods, the miniaturization is hindered by a solvent extraction step and a need for benchtop instrumentation. We present a methodology for collecting and in-situ analysis of airborne nanoparticles that eliminates labor-intensive sample preparation and miniaturizes the detection platform. Nanoparticles are electrostatically collected onto a transparent substrate coated with solid-phase (SP) solvent-polydimethylsiloxane (PDMS). The PM organic fraction is extracted into PDMS and analyzed in-situ, thus avoiding liquid-phase extraction. In the SP-EEM analysis, we evaluated external and internal excitation schemes. Internal excitation shows the lowest scattering interference but leads to signal masking from PDMS fluorescence for λ

Melanopsin-expressing retinal ganglion cells (mRGCs) in the eye play an important role in many light-activated non-image-forming functions including neonatal photoaversion and the adult pupillary light reflex (PLR). MRGCs rely on glutamate and possibly PACAP (pituitary adenylate cyclase-activating polypeptide) to relay visual signals to the brain. However, the role of these neurotransmitters for individual non-image-forming responses remains poorly understood. To clarify the role of glutamatergic signaling from mRGCs in neonatal aversion to light and in adult PLR, we conditionally deleted vesicular glutamate transporter (VGLUT2) selectively from mRGCs in mice. We found that deletion of VGLUT2 in mRGCs abolished negative phototaxis and light-induced distress vocalizations in neonatal mice, underscoring a necessary role for glutamatergic signaling. In adult mice, loss of VGLUT2 in mRGCs resulted in a slow and an incomplete PLR. We conclude that glutamatergic neurotransmission from mRGCs is required for neonatal photoaversion but is complemented by another non-glutamatergic signaling mechanism for the pupillary light reflex in adult mice. We speculate that this complementary signaling might be due to PACAP neurotransmission from mRGCs.

BackgroundSeveral agents are available to treat osteoporosis while addressing patient-specific medical needs. Individuals' residual risk to severe fracture may require changes in treatment strategy. Data at osseous cellular and microstructural levels due to a therapy switch between agents with different modes of action are rare. Our study on a series of five consecutively taken bone biopsies from an osteoporotic individual over a six-year period analyzes changes in cellular characteristics, bone microstructure and mineralization caused by a therapy switch from an antiresorptive (bisphosphonate) to a dual action bone agent (strontium ranelate).Methodology/principal findingsBiopsies were progressively taken from the iliac crest of a female patient. Four biopsies were taken during bisphosphonate therapy and one biopsy was taken after one year of strontium ranelate (SR) treatment. Furthermore, serum bone markers and dual x-ray absorptiometry measurements were acquired. Undecalcified histology was used to assess osteoid parameters and bone turnover. Structural indices and degree of mineralization were determined using microcomputed tomography, quantitative backscattered electron imaging, and combined energy dispersive x-ray/µ-x-ray-fluorescence microanalysis.Conclusions/significanceMicrostructural data revealed a notable increase in bone volume fraction after one year of SR treatment compared to the bisphosphonate treatment period. Indices of connectivity density, structure model index and trabecular bone pattern factor were predominantly enhanced indicating that the architectural transformation from trabecular rods to plates was responsible for the bone volume increase and less due to changes in trabecular thickness and number. Administration of SR following bisphosphonates led to a maintained mineralization profile with an uptake of strontium on the bone surface level. Reactivated osteoclasts designed tunneling, hook-like intratrabecular resorption sites. The appearance of tunneling resorption lacunae and the formation of both mini-modeling units and osteon-like structures within increased plate-like cancellous bone mass provides additional information on the mechanisms of strontium ranelate following bisphosphonate treatment, which may deserve special attention when monitoring a treatment switch.

The identification of potential interactions between drugs and target proteins is crucial in pharmaceutical sciences. The experimental validation of interactions in genomic drug discovery is laborious and expensive; hence, there is a need for efficient and accurate in-silico techniques which can predict potential drug-target interactions to narrow down the search space for experimental verification. In this work, we propose a new framework, namely, Multi-Graph Regularized Nuclear Norm Minimization, which predicts the interactions between drugs and target proteins from three inputs: known drug-target interaction network, similarities over drugs and those over targets. The proposed method focuses on finding a low-rank interaction matrix that is structured by the proximities of drugs and targets encoded by graphs. Previous works on Drug Target Interaction (DTI) prediction have shown that incorporating drug and target similarities helps in learning the data manifold better by preserving the local geometries of the original data. But, there is no clear consensus on which kind and what combination of similarities would best assist the prediction task. Hence, we propose to use various multiple drug-drug similarities and target-target similarities as multiple graph Laplacian (over drugs/targets) regularization terms to capture the proximities exhaustively. Extensive cross-validation experiments on four benchmark datasets using standard evaluation metrics (AUPR and AUC) show that the proposed algorithm improves the predictive performance and outperforms recent state-of-the-art computational methods by a large margin. Software is publicly available at https://github.com/aanchalMongia/MGRNNMforDTI.

Yield gaps of maize (Zea mays L.) in the smallholder farms of eastern India are outcomes of a complex interplay of climatic variations, soil fertility gradients, socio-economic factors, and differential management intensities. Several machine learning approaches were used in this study to investigate the relative influences of multiple biophysical, socio-economic, and crop management features in determining maize yield variability using several machine learning approaches. Soil fertility status was assessed in 180 farms and paired with the surveyed data on maize yield, socio-economic conditions, and agronomic management. The C&RT relative variable importance plot identified farm size, total labor, soil factors, seed rate, fertilizer, and organic manure as influential factors. Among the three approaches compared for classifying maize yield, the artificial neural network (ANN) yielded the least (25%) misclassification on validation samples. The random forest partial dependence plots revealed a positive association between farm size and maize productivity. Nonlinear support vector machine boundary analysis for the eight top important variables revealed complex interactions underpinning maize yield response. Notably, farm size and total labor synergistically increased maize yield. Future research integrating these algorithms with empirical crop growth models and crop simulation models for ex-ante yield estimations could result in further improvement.

The current study was undertaken to investigate the effect of differentially formulated polyphenolic compound Essential Turmeric Oil-Curcumin (ETO-Cur), and Tocotrienol-rich fraction (TRF) of vitamin E isomers on colorectal cancer (CRC) cells that produce aggressive tumors. Combinations of ETO-Cur and TRF were used to determine the combinatorial effects of ETO-Cur and TRF-mediated inhibition of growth of CRC cells in vitro and HCT-116 cells xenograft in SCID mice. 16S rRNA gene sequence profiling was performed to determine the outcome of gut microbial communities in mice feces between control and ETO-Cur-TRF groups. Bacterial identifications were validated by performing SYBR-based Real Time (RT) PCR. For metagenomics analysis to characterize the microbial communities, multiple software/tools were used, including Quantitative Insights into Microbial Ecology (QIIME) processing tool. We found ETO-Cur and TRF to synergize and that the combination of ETO-Cur-TRF significantly inhibited growth of HCT-116 xenografts in SCID mice. This was associated with a marked alteration in microbial communities and increased microbial OTU (operation taxonomic unit) number. The relative abundance of taxa was increased and the level of microbial diversity after 34 days of combinatorial treatment was found to be 44% higher over the control. Shifting of microbial family composition was observed in ETO-Cur-TRF treated mice as evidenced by marked reductions in Bacteroidaceae, Ruminococcaceae, Clostridiales, Firmicutes and Parabacteroids families, compared to controls. Interestingly, during the inhibition of tumor growth in ETO-Cur treated mice, probiotic Lactobacillaceae and Bifidobacteriaceae were increased by 20-fold and 6-fold, respectively. The relative abundance of anti-inflammatory Clostridium XIVa was also increased in ETO-Cur-TRF treated mice when compared with the control. Our data suggest that ETO-Cur-TRF show synergistic effects in inhibiting colorectal cancer cell proliferation in vitro and in mouse xenografts in vivo, and might induce changes in microbial diversity in mice.

DNA from formalin-preserved tissue could unlock a vast repository of genetic information stored in museums worldwide. However, formaldehyde crosslinks proteins and DNA, and prevents ready amplification and DNA sequencing. Formaldehyde acylation also fragments the DNA. Treatment with proteinase K proteolyzes crosslinked proteins to rescue the DNA, though the process is quite slow. To reduce processing time and improve rescue efficiency, we applied the mechanical energy of a vortex fluidic device (VFD) to drive the catalytic activity of proteinase K and recover DNA from American lobster tissue (Homarus americanus) fixed in 3.7% formalin for >1-year. A scan of VFD rotational speeds identified the optimal rotational speed for recovery of PCR-amplifiable DNA and while 500+ base pairs were sequenced, shorter read lengths were more consistently obtained. This VFD-based method also effectively recovered DNA from formalin-preserved samples. The results provide a roadmap for exploring DNA from millions of historical and even extinct species.

[This corrects the article DOI: 10.1371/journal.pone.0220956.].

In the present two-year study, an attempt was made to estimate the grain yield, grain nutrient uptake, and oil quality of three commonly grown maize (Zea mays L.) hybrids fertilized with varied levels of nitrogen (N), phosphorus (P) and potassium (K). Results obtained from both the experimental years indicated that application of 125% of recommended dose of fertilizer (RDF) recorded maximum grain yield (10.37 t ha-1; 124% higher than control). When compared with 100% RDF, grain yield reduction with nutrient omission was 44% for N omission, 17% for P omission, and 27% for K omission. Nitrogen uptake was increased with increasing NPK levels up to 150% RDF that was statistically at par (p ≥ 0.01) with 125% RDF. Increasing trend in P and K uptake was observed with successive increase in NPK levels up to 125% RDF, above which it declined. The protein content was significantly higher in grains of var. P 3396 with 125% RDF. Nutrient management has significant (p ≤ 0.01) role in the grain oil content. Saturated fatty acids (palmitic, stearic and arachidic acid) content decreased, and unsaturated fatty acid (oleic, linoleic and linolenic acid) increased with increasing NPK levels. The average oleic acid desaturation and linoleic acid desaturation ratios were increased with increasing NPK levels up to 100 and 125% RDF, respectively. However, average monounsaturated fatty acids (MUFA): poly-unsaturated fatty acids (PUFA), saturated: unsaturated as well as linoleic: linolenic acid ratios were increased on receiving 75% RDF, and beyond that it showed decreasing trend. The omission of K had the highest inhibitory effect on corn oil quality followed by N and P omission.

ObjectiveSevere hypoglycemia (blood glucose < 50 mg/dl) in hospitalized patients with diabetes mellitus is associated with poor outcomes such as increased mortality and readmission rates. We study the effects of system based interventions in managing severe hypoglycemia and its impact on outcomes.Research design and methodsWe performed retrospective review of pre- and post- intervention study to quantify severe hypoglycemia in patients admitted in the general internal medicine wards with primary or secondary diagnosis of diabetes mellitus based on ICD-9 and ICD-10 codes. We implemented multidisciplinary interventions including standardization of treatment, education of in-patient medical teams and physician notification and feedback immediately after severe hypoglycemia. The endpoints were the comparative analysis of incidence of severe hypoglycemia, in-patient mortality rate, 30-day mortality rate, 30-day readmission rate, recovery time from hypoglycemia, time to next glucose measurements, use of standardized treatment and physician notification rate pre-and post-intervention.ResultsThe incidence of severe hypoglycemia per patient with diabetes was reduced from 9.6% (233/2416) to 5.6% (202/3607) (pConclusionsOur study shows that multidisciplinary strategies improves the process of early detection and management of severe hypoglycemia and reduce incidence and in-patient mortality rate.

Ti-6Al-4V is commonly used in orthopaedic implants, and fabrication techniques such as Powder Bed Fusion (PBF) are becoming increasingly popular for the net-shape production of such implants, as PBF allows for complex customisation and minimal material wastage. Present research into PBF fabricated Ti-6Al-4V focuses on new design strategies (e.g. designing pores, struts or lattices) or mechanical property optimisation through process parameter control-however, it is pertinent to examine the effects of altering PBF process parameters on properties relating to bioactivity. Herein, changes in Ti-6Al-4V microstructure, mechanical properties and surface characteristics were examined as a result of varying PBF process parameters, with a view to understanding how to tune Ti-6Al-4V bio-activity during the fabrication stage itself. The interplay between various PBF laser scan speeds and laser powers influenced Ti-6Al-4V hardness, porosity, roughness and corrosion resistance, in a manner not clearly described by the commonly used volumetric energy density (VED) design variable. Key findings indicate that the relationships between PBF process parameters and ultimate Ti-6Al-4V properties are not straightforward as expected, and that wide ranges of porosity (0.03 ± 0.01% to 32.59 ± 2.72%) and corrosion resistance can be achieved through relatively minor changes in process parameters used-indicating volumetric energy density is a poor predictor of PBF Ti-6Al-4V properties. While variations in electrochemical behaviour with respect to the process parameters used in the PBF fabrication of Ti-6Al-4V have previously been reported, this study presents data regarding important surface characteristics over a large process window, reflecting the full capabilities of current PBF machinery.

The histone demethylase LSD1 plays a pivotal role in cellular differentiation, particularly in silencing lineage-specific genes. However, little is known about how LSD1 regulates neurosensory differentiation in the inner ear. Here we show that LSD1 interacts directly with the transcription factor Pax2 to form the NuRD co-repressor complex at the Pax2 target gene loci in a mouse otic neuronal progenitor cell line (VOT-N33). VOT-N33 cells expressing a Pax2-response element reporter were GFP-negative when untreated, but became GFP positive after forced differentiation or treatment with a potent LSD inhibitor. Pharmacological inhibition of LSD1 activity resulted in the enrichment of mono- and di-methylation of H3K4, upregulation of sensory neuronal genes and an increase in the number of sensory neurons in mouse inner ear organoids. Together, these results identify the LSD1/NuRD complex as a previously unrecognized modulator for Pax2-mediated neuronal differentiation in the inner ear.

Sertoli cells (Sc) are unique somatic cells of testis that are the target of both FSH and testosterone (T) and regulate spermatogenesis. Although Sc of neonatal rat testes are exposed to high levels of FSH and T, robust differentiation of spermatogonial cells becomes conspicuous only after 11-days of postnatal age. We have demonstrated earlier that a developmental switch in terms of hormonal responsiveness occurs in rat Sc at around 12 days of postnatal age during the rapid transition of spermatogonia A to B. Therefore, such "functional maturation" of Sc, during pubertal development becomes prerequisite for the onset of spermatogenesis. However, a conspicuous difference in robust hormone (both T and FSH) induced gene expression during the different phases of Sc maturation restricts our understanding about molecular events necessary for the spermatogenic onset and maintenance. Here, using microarray technology, we for the first time have compared the differential transcriptional profile of Sc isolated and cultured from immature (5 days old), maturing (12 days old) and mature (60 days old) rat testes. Our data revealed that immature Sc express genes involved in cellular growth, metabolism, chemokines, cell division, MAPK and Wnt pathways, while mature Sc are more specialized expressing genes involved in glucose metabolism, phagocytosis, insulin signaling and cytoskeleton structuring. Taken together, this differential transcriptome data provide an important resource to reveal the molecular network of Sc maturation which is necessary to govern male germ cell differentiation, hence, will improve our current understanding of the etiology of some forms of idiopathic male infertility.

SETTING:Myanmar National AIDS Program has had significant scale-up of services and changes in CD4 eligibility criterion for ART initiation from 2013 to 2016. This study assessed early death within 6 months and attrition (death and loss to follow-up, LTFU) after ART initiation and their associated factors. DESIGN:A retrospective cohort study on people living with HIV (PLHIV >15 year of age) enrolled at three specialist hospitals in Yangon from 1st June 2013 to 30th June 2016. Cox regression was used to calculate hazard ratios (HRs) of early death and attrition. RESULTS:Of 11,727 adults enrolled, 11,186 (95%) were initiated on ART, providing 15,964 person-years of follow-up. At baseline, median age was 36 years [IQR: 30-43], 58% were men and median CD4 count was 151 cells/mm3 (IQR: 54-310). There were 733(6%) early deaths, 961(9%) total deaths and 1371 (12%) LTFU during the study period. Independent risk factors for early death were older age (41-50 and ≥51 years) [aHR 1.38, 1.07-1.78 and 1.68, 1.21-2.34], male (1.84, 1.44-2.35), low weight (2.06, 1.64-2.59), bedridden, (3.81, 2.57-5.66) and CD4 count ≤ 50 cells/mm3 (6.83, 2.52-18.57). In addition to above factors, high attrition was associated with an abacavir-based regimen. CONCLUSION:Although there was a low rate of early deaths, patients were being diagnosed late and there was a high attrition rate from specialist hospitals. Concerted effort is required to increase early diagnosis and ART initiation, and strengthen community systems for HIV care to achieve ambitious goal of ending AIDS epidemic by 2030.

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.

Statins, the 3-hydroxy-3-methyl-glutaryl (HMG)-CoA reductase inhibitors, are widely prescribed for treatment of hypercholesterolemia. Although statins are generally well tolerated, up to ten percent of statin-treated patients experience myalgia symptoms, defined as muscle pain without elevated creatinine phosphokinase (CPK) levels. Myalgia is the most frequent reason for discontinuation of statin therapy. The mechanisms underlying statin myalgia are not clearly understood. To elucidate changes in gene expression associated with statin myalgia, we compared profiles of gene expression in skeletal muscle biopsies from patients with statin myalgia who were undergoing statin re-challenge (cases) versus those of statin-tolerant controls. A robust separation of case and control cohorts was revealed by Principal Component Analysis of differentially expressed genes (DEGs). To identify putative gene expression and metabolic pathways that may be perturbed in skeletal muscles of patients with statin myalgia, we subjected DEGs to Ingenuity Pathways (IPA) and DAVID (Database for Annotation, Visualization and Integrated Discovery) analyses. The most prominent pathways altered by statins included cellular stress, apoptosis, cell senescence and DNA repair (TP53, BARD1, Mre11 and RAD51); activation of pro-inflammatory immune response (CXCL12, CST5, POU2F1); protein catabolism, cholesterol biosynthesis, protein prenylation and RAS-GTPase activation (FDFT1, LSS, TP53, UBD, ATF2, H-ras). Based on these data we tentatively conclude that persistent myalgia in response to statins may emanate from cellular stress underpinned by mechanisms of post-inflammatory repair and regeneration. We also posit that this subset of individuals is genetically predisposed to eliciting altered statin metabolism and/or increased end-organ susceptibility that lead to a range of statin-induced myopathies. This mechanistic scenario is further bolstered by the discovery that a number of single nucleotide polymorphisms (e.g., SLCO1B1, SLCO2B1 and RYR2) associated with statin myalgia and myositis were observed with increased frequency among patients with statin myalgia.

Some patients experience a delayed discharge from the intensive care unit (ICU) where the intended and actual discharge times do not coincide. The clinical implications of this remain unclear.To determine the incidence and duration of delayed ICU discharge, identify the reasons for delay and evaluate the clinical consequences.Prospective multi-centre observational study involving five ICUs over a 3-month period. Delay in discharge was defined as >6 hours from the planned discharge time. The primary outcome measure was hospital length stay after ICU discharge decision. Secondary outcome measures included ICU discharge after-hours, incidence of delirium, survival to hospital discharge, discharge destination, the incidence of ICU acquired infections, revocation of ICU discharge decision, unplanned readmissions to ICU within 72 hours, review of patients admitting team after ICU discharge decision.A total of 955 out of 1118 patients discharged were included in analysis. 49.9% of the patients discharge was delayed. The most common reason (74%) for delay in discharge was non-availability of ward bed. The median duration of the delay was 24 hours. On univariable analysis, the duration of hospital stay from the time of ICU discharge decision was significantly higher in patients who had ICU discharge delay (Median days-5 vs 6; p = 0.003). After-hours discharge was higher in patients whose discharge was delayed (34% Vs 10%; p

Dramatic increase of diabetes over the globe is in tandem with the increase in insulin requirement. This is because destruction and dysfunction of pancreatic β-cells are of common occurrence in both Type1 diabetes and Type2 diabetes, and insulin injection becomes a compulsion. Because of several problems associated with insulin injection, orally active insulin mimetic compounds would be ideal substitute. Here we report a small molecule, a peroxyvanadate compound i.e. DmpzH[VO(O2)2(dmpz)], henceforth referred as dmp, which specifically binds to insulin receptor with considerable affinity (KD-1.17μM) thus activating insulin receptor tyrosine kinase and its downstream signaling molecules resulting increased uptake of [14C] 2 Deoxy-glucose. Oral administration of dmp to streptozotocin treated BALB/c mice lowers blood glucose level and markedly stimulates glucose and fatty acid uptake by skeletal muscle and adipose tissue respectively. In db/db mice, it greatly improves insulin sensitivity through excess expression of PPARγ and its target genes i.e. adiponectin, CD36 and aP2. Study on the underlying mechanism demonstrated that excess expression of Wnt3a decreased PPARγ whereas dmp suppression of Wnt3a gene increased PPARγ expression which subsequently augmented adiponectin. Increased production of adiponectin in db/db mice due to dmp effected lowering of circulatory TG and FFA levels, activates AMPK in skeletal muscle and this stimulates mitochondrial biogenesis and bioenergetics. Decrease of lipid load along with increased mitochondrial activity greatly improves energy homeostasis which has been found to be correlated with the increased insulin sensitivity. The results obtained with dmp, therefore, strongly indicate that dmp could be a potential candidate for insulin replacement therapy.

Visceral leishmaniasis (VL), which is endemic as a major infectious disease in the tropical and subtropical countries, is caused by a protozoan parasite Leishmania donovani. At present, restricted treatment options and lack of vaccines intensify the problem of controlling VL. Therefore, finding a novel immunoprophylactic or therapeutic principle is a pressing need. Here, we report that arabinosylated lipoarabinomannan (Ara-LAM), a TLR2-ligand isolated from Mycobacterium smegmatis, exhibits a strong immunomodulatory property that conferred protection against L. donovani infection. Although, Ara-LAM modulates TLR2 and MAPK signaling, it is not known whether Ara-LAM involves IFN-γ signaling for effective parasite clearance. Because, it is reported that IFN-γ signaling, a principle mediator of NO generation and macrophage and Tcell activation, is hampered during leishmanial pathogenesis. Ara-LAM increases IFN-γ receptor expression and potentiates IFN-γ receptor signaling through JAK-STAT pathway. Moreover, Ara-LAM reciprocally modulates IRF4 and IRF8 expression and reinstates anti-leishmanial Th1 response that eventuates in significantly reduced parasite load in spleen and liver of L. donovani-infected BALB/c mice. IFN-γRα silencing resulted in the suppression of these host-protective mechanisms affected by Ara-LAM. Thus, Ara-LAM-mediated restoration of IFN-γ responsiveness is a novel immuno-modulatory principle for protection against L. donovani susceptible host.

Leishmania donovani, a protozoan parasite, causes the disease visceral leishmanisis (VL), characterized by inappropriate CD8+ T-cell activation. Therefore, we examined whether the Toll-like Receptor 2 (TLR2) ligand Ara-LAM, a cell wall glycolipid from non-pathogenic Mycobacterium smegmatis, would restore CD8+ T-cell function during VL. We observed that by efficient upregulation of TLR2 signaling-mediated NF-κB translocation and MAPK signaling in CD8+ T-cells (CD25+CD28+IL-12R+IFN-γR+), Ara-LAM triggered signaling resulted in the activation of T-bet, which in turn, induced transcription favourable histone modification at the IFN-γ, perforin, granzyme-B promoter regions in CD8+ T-cells. Thus, we conclude that Ara-LAM induced efficient activation of effector CD8+ T-cells by upregulating the expression of IFN-γ, perforin and granzyme-B in an NF-κB and MAPK induced T-bet dependent manner in VL.

The rhesus macaque (Macaca mulatta) is widely used in molecular evolutionary analyses, particularly to identify genes under adaptive or unique evolution in the human lineage. For such studies, it is necessary to align nucleotide sequences of homologous protein-coding genes among multiple species. The validity of these analyses is dependent on high quality genomic data. However, for most mammalian species (other than humans and mice), only draft genomes are available. There has been concern that some results obtained from evolutionary analyses using draft genomes may not be correct. The rhesus macaque provides a unique opportunity to determine whether an improved genome (MacaM) yields better results than a draft genome (rheMac2) for evolutionary studies. We compared protein-coding genes annotated in the rheMac2 and MacaM genomes with their human orthologs. We found many genes annotated in rheMac2 had apparently spurious sequences not present in genes derived from MacaM. The rheMac2 annotations also appeared to inflate a frequently used evolutionary index, ω (the ratio of nonsynonymous to synonymous substitution rates). Genes with these spurious sequences must be filtered out from evolutionary analyses to obtain correct results. With the MacaM genome, improved sequence information means many more genes can be examined for indications of selection. These results indicate how upgrading genomes from draft status to a higher level of quality can improve interpretation of evolutionary patterns.

Currently, only 62% of incident tuberculosis (TB) cases are reported to the national programme in Pakistan. Several innovative interventions are being recommended to detect the remaining 'missed' TB cases. One such intervention involved expanding contact investigation to the community using the Xpert MTB/RIF test.This was a before and after intervention study involving retrospective record review. Passive case finding and household contact investigation was routinely done in the pre-intervention period July 2011-June 2013. Four districts with a high concentration of slums were selected as intervention areas; Lahore, Rawalpindi, Faisalabad and Islamabad. Here, in the intervention period, July 2013-June 2015, contact investigation beyond household was conducted: all people staying within a radius of 50 metres (using Geographical Information System) from the household of smear positive TB patients were screened for tuberculosis. Those with presumptive TB were investigated using smear microscopy and the Xpert MTB/RIF test was performed on smear negative patients. All the diagnosed TB patients were linked to TB treatment and care.A total of 783043 contacts were screened for tuberculosis: 23741(3.0%) presumptive TB patients were identified of whom, 4710 (19.8%) all forms and 4084(17.2%) bacteriologically confirmed TB patients were detected. The contribution of Xpert MTB/RIF to bacteriologically confirmed TB patients was 7.6%. The yield among investigated presumptive child TB patients was 5.1%. The overall yield of all forms TB patients among investigated was 22.3% among household and 19.1% in close community. The intervention contributed an increase of case detection of bacteriologically confirmed tuberculosis by 6.8% and all forms TB patients by 7.9%.Community contact investigation beyond household not only detected additional TB patients but also increased TB case detection. However, further long term assessments and cost-effectiveness studies are required before national scale-up.

Mutational processes and signatures that drive early tumorigenesis are centrally important for early cancer prevention. Yet, to date, biomarkers and risk factors for polyps (adenomas) that inordinately and rapidly develop into colon cancer remain poorly defined. Here, we describe surprisingly high mutational profiles through whole-genome sequence (WGS) analysis in 2 of 4 pairs of benign colorectal adenoma tissue samples. Unsupervised hierarchical clustered transcriptomic analysis of a further 7 pairs of adenomas reveals distinct mutational signatures regardless of adenoma size. Transitional single nucleotide substitutions of C:G>T:A predominate in the adenoma mutational spectrum. Strikingly, we observe mutations in the TGF-β pathway and CEA-associated genes in 4 out of 11 adenomas, overlapping with the Wnt pathway. Immunohistochemical labeling reveals a nearly 5-fold increase in CEA levels in 23% of adenoma samples with a concomitant loss of TGF-β signaling. We also define a functional role by which the CEA B3 domain interacts with TGFBR1, potentially inactivating the tumor suppressor function of TGF-β signaling. Our study uncovers diverse mutational processes underlying the transition from early adenoma to cancer. This has broad implications for biomarker-driven targeting of CEA/TGF-β in high-risk adenomas and may lead to early detection of aggressive adenoma to CRC progression.

Testis of Hemidactylus flaviviridis, commonly known as Indian wall lizard, displays a lack of cellular and metabolic activity in regressed phase of testis during non-breeding season of the year. Retracted Sertoli cells (Sc), fibroid myoid cells and pre-meiotic resting spermatogonia are observed in such testis. This situation is akin to certain forms of infertility in men where hormone supplementation fails to generate sperm despite the presence of Sc and germ cells (Gc) in testis. In testis of lizard, spermatogenesis is reinitiated upon increased level of hormones during appropriate season (phase of recrudescence). Study of genes associated with generation of sperm, from regressed adult testis in lizard, may provide valuable information for understanding certain forms of male idiopathic infertility. Subtractive hybridization using testicular RNA obtained from the regressed and active phases of lizard reproductive cycle led to identify eight partial mRNA sequences that showed sequence homology with mice genes. We further evaluated the gene expression prolife by real-time PCR in three different reproductive phases of H. flaviviridis: regressed (pre-meiotic), recrudescent (meiotic) and active (post meiotic), for comparison with the corresponding testicular phases found in testis of 5 days (pre-meiotic), 20 days (meiotic) and 60 days (post-meiotic) old mouse. This is the first report where genes associated with progression of spermatogenesis during active phase, which follows a regressed state of adult testis, were identified in lizard and found to be conserved in mouse. Six important genes, Hk1, Nme5, Akap4, Arih1, Rassf7 and Tubb4b were found to be strictly associated with active spermatogenesis in both mouse and lizard. Factors interfering with the expression of any of these genes may potentially abrogate the process of spermatogenesis leading to infertility. Such information may shed light on unknown causes of idiopathic male infertility.

INTRODUCTION:This research article reports on factors influencing initial voluntary uptake of community-based health insurance (CBHI) schemes in low- and middle-income countries (LMIC), and renewal decisions. METHODS:Following PRISMA protocol, we conducted a comprehensive search of academic and gray literature, including academic databases in social science, economics and medical sciences (e.g., Econlit, Global health, Medline, Proquest) and other electronic resources (e.g., Eldis and Google scholar). Search strategies were developed using the thesaurus or index terms (e.g., MeSH) specific to the databases, combined with free text terms related to CBHI or health insurance. Searches were conducted from May 2013 to November 2013 in English, French, German, and Spanish. From the initial search yield of 15,770 hits, 54 relevant studies were retained for analysis of factors influencing enrolment and renewal decisions. The quantitative synthesis (informed by meta-analysis) and the qualitative analysis (informed by thematic synthesis) were compared to gain insight for an overall synthesis of findings/statements. RESULTS:Meta-analysis suggests that enrolments in CBHI were positively associated with household income, education and age of the household head (HHH), household size, female-headed household, married HHH and chronic illness episodes in the household. The thematic synthesis suggests the following factors as enablers for enrolment: (a) knowledge and understanding of insurance and CBHI, (b) quality of healthcare, (c) trust in scheme management. Factors found to be barriers to enrolment include: (a) inappropriate benefits package, (b) cultural beliefs, (c) affordability, (d) distance to healthcare facility, (e) lack of adequate legal and policy frameworks to support CBHI, and (f) stringent rules of some CBHI schemes. HHH education, household size and trust in the scheme management were positively associated with member renewal decisions. Other motivators were: (a) knowledge and understanding of insurance and CBHI, (b) healthcare quality, (c) trust in scheme management, and (d) receipt of an insurance payout the previous year. The barriers to renewal decisions were: (a) stringent rules of some CBHI schemes, (b) inadequate legal and policy frameworks to support CBHI and (c) inappropriate benefits package. CONCLUSION AND POLICY IMPLICATIONS:The demand-side factors positively affecting enrolment in CBHI include education, age, female household heads, and the socioeconomic status of households. Moreover, when individuals understand how their CBHI functions they are more likely to enroll and when people have a positive claims experience, they are more likely to renew. A higher prevalence of chronic conditions or the perception that healthcare is of good quality and nearby act as factors enhancing enrolment. The perception that services are distant or deficient leads to lower enrolments. The second insight is that trust in the scheme enables enrolment. Thirdly, clarity about the legal or policy framework acts as a factor influencing enrolments. This is significant, as it points to hitherto unpublished evidence that governments can effectively broaden their outreach to grassroots groups that are excluded from social protection by formulating supportive regulatory and policy provisions even if they cannot fund such schemes in full, by leveraging people's willingness to exercise voluntary and contributory enrolment in a community-based health insurance.

Interferon-gamma (Ifnγ), a key macrophage activating cytokine, plays pleiotropic roles in host immunity. In this study, the ability of Ifnγ to induce the aggregation of resident mouse adherent peritoneal exudate cells (APECs), consisting primarily of macrophages, was investigated. Cell-cell interactions involve adhesion molecules and, upon addition of Ifnγ, CD11b re-localizes preferentially to the sites of interaction on APECs. A functional role of CD11b in enhancing aggregation is demonstrated using Reopro, a blocking reagent, and siRNA to Cd11b. Studies with NG-methyl-L-arginine (LNMA), an inhibitor of Nitric oxide synthase (Nos), NO donors, e.g., S-nitroso-N-acetyl-DL-penicillamine (SNAP) or Diethylenetriamine/nitric oxide adduct (DETA/NO), and Nos2-/- mice identified Nitric oxide (NO) induced by Ifnγ as a key regulator of aggregation of APECs. Further studies with Nos2-/- APECs revealed that some Ifnγ responses are independent of NO: induction of MHC class II and CD80. On the other hand, Nos2 derived NO is important for other functions: motility, phagocytosis, morphology and aggregation. Studies with cytoskeleton depolymerizing agents revealed that Ifnγ and NO mediate the cortical stabilization of Actin and Tubulin which contribute to aggregation of APECs. The biological relevance of aggregation of APECs was delineated using infection experiments with Salmonella Typhimurium (S. Typhimurium). APECs from orally infected, but not uninfected, mice produce high amounts of NO and aggregate upon ex vivo culture in a Nos2-dependent manner. Importantly, aggregated APECs induced by Ifnγ contain fewer intracellular S. Typhimurium compared to their single counterparts post infection. Further experiments with LNMA or Reopro revealed that both NO and CD11b are important for aggregation; in addition, NO is bactericidal. Overall, this study elucidates novel roles for Ifnγ and Nos2 in regulating Actin, Tubulin, CD11b, motility and morphology during the aggregation response of APECs. The implications of aggregation or "group behavior" of APECs are discussed in the context of host resistance to infectious organisms.

Collagen crosslinking is a relatively new treatment for structural disorders of corneal ectasia, such as keratoconus. However, there is a lack of animal models of keratoconus, which has been an obstacle for carefully analyzing the mechanisms of crosslinking and evaluating new therapies. In this study, we treated rabbit eyes with collagenase and chondroitinase enzymes to generate ex vivo corneal ectatic models that simulate the structural disorder of keratoconus. The models were then used to evaluate the protective effect of soluble collagen in the UVA crosslinking system. After enzyme treatment, the eyes were exposed to riboflavin/UVA crosslinking with and without soluble type I collagen. Corneal morphology, collagen ultrastructure, and thermal stability were evaluated before and after crosslinking. Enzyme treatments resulted in corneal curvature changes, collagen ultrastructural damage, decreased swelling resistance and thermal stability, which are similar to what is observed in keratoconus eyes. UVA crosslinking restored swelling resistance and thermal stability, but ultrastructural damage were found in the crosslinked ectatic corneas. Adding soluble collagen during crosslinking provided ultrastructural protection and further enhanced the swelling resistance. Therefore, UVA crosslinking on the ectatic model mimicked typical clinical treatment for keratoconus, suggesting that this model replicates aspects of human keratoconus and could be used for investigating experimental therapies and treatments prior to translation.

The great promise of digital PCR is the potential for unparalleled precision enabling accurate measurements for genetic quantification. A challenge associated with digital PCR experiments, when testing unknown samples, is to perform experiments at dilutions allowing the detection of one or more targets of interest at a desired level of precision. While theory states that optimal precision (Po) is achieved by targeting ~1.59 mean copies per partition (λ), and that dynamic range (R) includes the space spanning one positive (λL) to one negative (λU) result from the total number of partitions (n), these results are tempered for the practitioner seeking to construct digital PCR experiments in the laboratory. A mathematical framework is presented elucidating the relationships between precision, dynamic range, number of partitions, interrogated volume, and sensitivity in digital PCR. The impact that false reaction calls and volumetric variation have on sensitivity and precision is next considered. The resultant effects on sensitivity and precision are established via Monte Carlo simulations reflecting the real-world likelihood of encountering such scenarios in the laboratory. The simulations provide insight to the practitioner on how to adapt experimental loading concentrations to counteract any one of these conditions. The framework is augmented with a method of extending the dynamic range of digital PCR, with and without increasing n, via the use of dilutions. An example experiment demonstrating the capabilities of the framework is presented enabling detection across 3.33 logs of starting copy concentration.

Leishmania donovani, a protozoan parasite, causes a strong immunosuppression in a susceptible host and inflicts the fatal disease visceral leishmaniasis. Relatively high toxicity, low therapeutic index, and failure in reinstating host-protective anti-leishmanial immune responses have made anti-leishmanial drugs patient non-compliant and an immuno-modulatory treatment a necessity. Therefore, we have tested the anti-leishmanial efficacy of a combination of a novel immunomodulator, Mycobacterium indicus pranii (Mw), and an anti-leishmanial drug, Amphotericin B (AmpB). We observe that Mw alone or with a suboptimal dose of AmpB offers significant protection against L. donovani infection by activating the macrophages. Our experiments examining the anti-leishmanial activity of Mw alone or with AmpB also indicate a p38MAPK and ERK-1/2 regulated pro-inflammatory responses. The Mw-AmpB combination induced nitric oxide production, restored Th1 response, and significantly reduced parasite burden in wild type macrophages but not in IL-12-deficient macrophages indicating a pivotal role for IL-12 in the induction of host-protection by Mw and AmpB treatments. In addition, we observed that Mw alone or in combination with suboptimal dose of AmpB render protection against L. donovani infection in susceptible BALB/c mice. However, these treatments failed to render protection in IL-12-deficient mice in vivo which added further support that IL-12 played a central role in this chemo immunotherapeutic approach. Thus, we demonstrate a novel chemo-immunotherapeutic approach- Mw and AmpB crosstalk eliminating the parasite-induced immunosuppression and inducing collateral host-protective effects.

The parasitic protozoan Leishmania donovani is the causative organism for visceral leishmaniasis (VL) which persists in the host macrophages by deactivating its signaling machinery resulting in a critical shift from proinflammatory (Th1) to an anti-inflammatory (Th2) response. The severity of this disease is mainly determined by the production of IL-12 and IL-10 which could be reversed by use of effective immunoprophylactics. In this study we have evaluated the potential of Arabinosylated Lipoarabinomannan (Ara-LAM), a cell wall glycolipid isolated from non pathogenic Mycobacterium smegmatis, in regulating the host effector response via effective regulation of mitogen-activated protein kinases (MAPK) signaling cascades in Leishmania donovani infected macrophages isolated from BALB/C mice. Ara-LAM, a Toll-like receptor 2 (TLR2) specific ligand, was found to activate p38 MAPK signaling along with subsequent abrogation of extracellular signal-regulated kinase (ERKs) signaling. The use of pharmacological inhibitors of p38MAPK and ERK signaling showed the importance of these signaling pathways in the regulation of IL-10 and IL-12 in Ara-LAM pretreated parasitized macrophages. Molecular characterization of this regulation of IL-10 and IL-12 was revealed by chromatin immunoprecipitation assay (CHIP) which showed that in Ara-LAM pretreated parasitized murine macrophages there was a significant induction of IL-12 by selective phosphorylation and acetylation of histone H3 residues at its promoter region. While, IL-10 production was attenuated by Ara-LAM pretreatment via abrogation of histone H3 phosphorylation and acetylation at its promoter region. This Ara-LAM mediated antagonistic regulations in the induction of IL-10 and IL-12 genes were further correlated to changes in the transcriptional regulators Signal transducer and activator of transcription 3 (STAT3) and Suppressor of cytokine signaling 3 (SOCS3). These results demonstrate the crucial role played by Ara-LAM in regulating the MAPK signaling pathway along with subsequent changes in host effector response during VL which might provide crucial clues in understanding the Ara-LAM mediated protection during Leishmania induced pathogenesis.

In the present study, we observed a marked variation in the expression of PKCα and PKCδ isotypes in B16F10 melanoma tumor cells compared to the normal melanocytes. Interestingly, the tumor instructed expression or genetically manipulated overexpression of PKCα isotype resulted in enhanced G1 to S transition. This in turn promoted cellular proliferation by activating PLD1 expression and subsequent AKT phosphorylation, which eventually resulted in suppressed ceramide generation and apoptosis. On the other hand, B16F10 melanoma tumors preferentially blocked the expression of PKCδ isotype, which otherwise could exhibit antagonistic effects on PKCα-PLD1-AKT signaling and rendered B16F10 cells more sensitive to apoptosis via generating ceramide and subsequently triggering caspase pathway. Hence our data suggested a reciprocal PKC signaling operational in B16F10 melanoma cells, which regulates ceramide generation and provide important clues to target melanoma cancer by manipulating the PKCδ-ceramide axis.