Search

ObjectiveTo evaluate the efficacy of corneal neurotisation using sural nerve graft coaptation of the contralateral supratrochlear nerve in unilateral neurotrophic keratopathy and corneal anesthesia. Corneal neuralization has emerged as a potential option in the treatment of neurotropic keratopathy, however not free from the predicament. We evaluated the long-term outcome of corneal neurotisation in the treatment of unresponsive unilateral neurotropic keratopathy using surgical variations to mimic and expedient the surgical procedure.MethodsA Prospective interventional study involving patients with unilateral neurotrophic keratopathy (NK) who did not respond to medical measures was conducted. The study parameters evaluated were best-corrected visual acuity improvement, ocular surface evaluation parameters [tear break-up time (TBUT), Schirmer's 1, and ocular surface staining scores (corneal and conjunctival staining)], central corneal sensation (Cochet Bonnet esthesiometer), sub-basal nerve fiber length (SBNFL), and sub-basal nerve fiber density (SBNFD) determined by central confocal microscopy at recruitment and during follow-up at 1-month, 3-month, 6-month, 9-month and 12-month respectively, following corneal neurotization.ResultsEleven eyes of 11 patients with unilateral neurotrophic keratopathy (NK) who underwent corneal neurotisation were studied. The mean follow-up was 10.09±2.31months (range, 6-12). Mean best corrected visual acuity in log MAR at baseline, 1.35±0.52 improved significantly to 1.06±0.76 (P = 0.012) at 3 months and continued to 0.55±0.60 (P = 0.027) at 12 months. There was a significant reduction in NK grade severity and improvement in the ocular surface as early as 1 month, and central corneal sensations (P = 0.024) as soon as 3 months. Mean corneal SBNF improved from 3.12±1.84 mm/mm2 to 4.49±1.88 at 1 month (P = 0.008), 13.31±3.61 mm/mm2 (P = 0.028) at 12 months. Mean central corneal SBNFD evident at 6 months was 1.83±2.54no/mm2 (P = 0.018) and 4.90±3.12no/mm2 (P = 0.028) at 12 months.ConclusionThis study substantiates the routine practice of corneal neurotisation by simplifying the intricacies observed during the procedure.

Background Registering a donation decision is fundamental to increasing the number of people who donate the organs and tissues essential for transplantation, but the number of registered organ donors is insufficient to meet this demand. Most people in Australia support organ donation, but only a third have registered their decision on the Australian Organ Donor Register (AODR). We addressed this paradox by investigating how feelings of community, engendered through an ethic of hospitality and care and a non-proselytizing dialogue about organ donation, facilitated the decision to register. Methods An Immediate Registration Opportunity was set up in a large public hospital in NSW, Australia. The public was approached and invited to engage in an open, respectful dialogic interaction that met people where their beliefs were and allowed their concerns and fears about donation to be discussed. This included a survey that measured positive and negative beliefs about organ donation, mood, atmosphere, and feelings of community coupled with an on-the-spot opportunity to register their donation decision. Results Over four days, we interacted with 357 participants; 75.5% (210) of eligible-to-register participants registered on the AODR. Generalized Structural Equation Modelling highlighted that as connection to community increased, so did the salience of positive beliefs about organ donation. Positive beliefs, in turn, were negatively correlated with negative beliefs about donation and, as the strength of negative beliefs decreased, the probability of registration on the AODR increased. Participants who registered on the AODR reported stronger connection to the broader community than participants who did not register. Conclusion A respectful non-judgmental interaction that allows beliefs and concerns about organ donation to be discussed, coupled with an immediate opportunity to register, encouraged registration. Within this framework, feelings of belonging to a community were a key determinant that enabled many to make the decision to register.

BackgroundRegistering a donation decision is fundamental to increasing the number of people who donate the organs and tissues essential for transplantation, but the number of registered organ donors is insufficient to meet this demand. Most people in Australia support organ donation, but only a third have registered their decision on the Australian Organ Donor Register (AODR). We addressed this paradox by investigating how feelings of community, engendered through an ethic of hospitality and care and a non-proselytizing dialogue about organ donation, facilitated the decision to register.MethodsAn Immediate Registration Opportunity was set up in a large public hospital in NSW, Australia. The public was approached and invited to engage in an open, respectful dialogic interaction that met people where their beliefs were and allowed their concerns and fears about donation to be discussed. This included a survey that measured positive and negative beliefs about organ donation, mood, atmosphere, and feelings of community coupled with an on-the-spot opportunity to register their donation decision.ResultsOver four days, we interacted with 357 participants; 75.5% (210) of eligible-to-register participants registered on the AODR. Generalized Structural Equation Modelling highlighted that as connection to community increased, so did the salience of positive beliefs about organ donation. Positive beliefs, in turn, were negatively correlated with negative beliefs about donation and, as the strength of negative beliefs decreased, the probability of registration on the AODR increased. Participants who registered on the AODR reported stronger connection to the broader community than participants who did not register.ConclusionA respectful non-judgmental interaction that allows beliefs and concerns about organ donation to be discussed, coupled with an immediate opportunity to register, encouraged registration. Within this framework, feelings of belonging to a community were a key determinant that enabled many to make the decision to register.

The Arab population encompasses over 420 million people characterized by genetic admixture and a consequent rich genetic diversity. A number of genetic diseases have been reported for the first time from the population. Additionally a high prevalence of some genetic diseases including autosomal recessive disorders such as hemoglobinopathies and familial mediterranean fever have been found in the population and across the region. There is a paucity of databases cataloguing genetic variants of clinical relevance from the population. The availability of such a catalog could have implications in precise diagnosis, genetic epidemiology and prevention of disease. To fill in the gap, we have compiled DALIA, a comprehensive compendium of genetic variants reported in literature and implicated in genetic diseases reported from the Arab population. The database aims to act as an effective resource for population-scale and sub-population specific variant analyses, enabling a ready reference aiding clinical interpretation of genetic variants, genetic epidemiology, as well as facilitating rapid screening and a quick reference for evaluating evidence on genetic diseases.

Rapid adaptive responses were evident from reciprocal host-plant switches on performance, digestive physiology and relative gene expression of gut serine proteases in larvae of crucifer pest P. brassicae transferred from cauliflower (CF, Brassica oleracea var. botrytis, family Brassicaceae) to an alternate host, garden nasturtium, (GN, Tropaeolum majus L., family Tropaeolaceae) and vice-versa under laboratory conditions. Estimation of nutritional indices indicated that larvae of all instars tested consumed the least food and gained less weight on CF-GN diet (significant at p≤0.05) as compared to larvae feeding on CF-CF, GN-GN and GN-CF diets suggesting that the switch to GN was nutritionally less favorable for larval growth. Nevertheless, these larvae, especially fourth instars, were adroit in utilizing and digesting GN as a new host plant type. In vitro protease assays conducted to understand associated physiological responses within twelve hours indicated that levels and properties of gut proteases were significantly influenced by type of natal host-plant consumed, change in diet as well as larval age. Activities of gut trypsins and chymotrypsins in larvae feeding on CF-GN and GN-CF diets were distinct, and represented shifts toward profiles observed in larvae feeding continuously on GN-GN and CF-CF diets respectively. Results with diagnostic protease inhibitors like TLCK, STI and SBBI in these assays and gelatinolytic zymograms indicated complex and contrasting trends in gut serine protease activities in different instars from CF-GN diet versus GN-CF diet, likely due to ingestion of plant protease inhibitors present in the new diet. Cloning and sequencing of serine protease gene fragments expressed in gut tissues of fourth instar P. brassicae revealed diverse transcripts encoding putative trypsins and chymotrypsins belonging to at least ten lineages. Sequences of members of each lineage closely resembled lepidopteran serine protease orthologs including uncharacterized transcripts from Pieris rapae. Differential regulation of serine protease genes (Pbr1-Pbr5) was observed in larval guts of P. brassicae from CF-CF and GN-GN diets while expression of transcripts encoding two putative trypsins (Pbr3 and Pbr5) were significantly different in larvae from CF-GN and GN-CF diets. These results suggested that some gut serine proteases that were differentially expressed in larvae feeding on different species of host plants were also involved in rapid adaptations to dietary switches. A gene encoding nitrile-specifier protein (nsp) likely involved in detoxification of toxic products from interactions of ingested host plant glucosinolates with myrosinases was expressed to similar levels in these larvae. Taken together, these snapshots reflected contrasts in physiological and developmental plasticity of P. brassicae larvae to nutritional challenges from wide dietary switches in the short term and the prominent role of gut serine proteases in rapid dietary adaptations. This study may be useful in designing novel management strategies targeting candidate gut serine proteases of P. brassicae using RNA interference, gene editing or crops with transgenes encoding protease inhibitors from taxonomically-distant host plants.

In the present study we examined the effect of nanogypsum and Pseudomonas taiwanensis strain BCRC 17751on plant and soil health using conventional and metagenomics approaches. Soil physicochemical properties and agronomical parameters of maize plants were reported to be better when applied with nanogypsum and bacterial inoculum together. When compared to control a significant increase in total bacterial counts, nitrogen, phosphorus, potassium (NPK) solubilizing bacterial population and soil enzyme activities (fluorescein diacetate, alkaline phosphatase, dehydrogenase, β-glucosidase, arylesterase and amylase) was reported in treatments. The metagenomics studies revealed dominance of beneficial bacteria such as Proteobacteria, Bacteriodetes, Planctomycetes, Acidobacteria and Nitrospirae in treated soil. On the other hand some novel bacterial diversity was also reported in treated soil which was evident from presence of taxonomically unclassified sequences. Hence, it can be concluded that combined application of nanogypsum and Pseudomonas taiwanensis in maize help in improving the structure and function of soil which affects the plant health without causing any toxic effect. However, in situ validation of the prescribed treatment is required under field conditions on different crops in order to give maximum benefits to the farmers and the environment.

In recent times, injudicious use of paclobutrazol (PBZ) in mango orchards deteriorates the soil quality and fertility by persistence nature and causes a serious ecosystem imbalance. In this study, a new Klebsiella pneumoniae strain M6 (MW228061) was isolated from mango rhizosphere and characterized as a potent plant growth promoter, biocontrol, and PBZ degrading agent. The strain M6 efficiently utilizes PBZ as carbon, energy and nitrogen source and degrades up to 98.28% (50 mgL-1 initial conc.) of PBZ at 15th day of incubation in MS medium. In the soil system first order degradation kinetics and linear model suggested 4.5 days was the theoretical half-life (t1/2 value) of PBZ with strain M6. Box Behnken design (BBD) model of Response surface methodology (RSM) showed pH 7.0, 31°C temperature, and 2.0 ml inoculum size (8 x 109 CFU mL-1) was optimized condition for maximum PBZ degradation with strain M6. Plant growth promoting attributes such as Zn, K, PO4 solubilization IAA, HCN and NH3 production of strain M6 showed positive results and were assessed quantitatively. The relation between plant growth promotion and PBZ degradation was analyzed by heat map, principal component analysis (PCA) and, clustal correlation analysis (CCA). Strain M6 was also showing a significant biocontrol activity against pathogenic fungi such as Fusarium oxysporum (MTCC-284), Colletotrichum gloeosporioides (MTCC- 2190), Pythium aphanidermatum (MTCC- 1024), Tropical race 1 (TR -1), and Tropical race 4 (TR -4). Hence, results of the study suggested that strain M6 can be utilized as an effective bio-agent to restore degraded land affected by persistent use of paclobutrazol.

BackgroundWe conducted a mediation analysis of the provider team's role in changes to chronic condition medication adherence among cancer survivors.MethodsWe used a retrospective, longitudinal cohort design following Medicare beneficiaries from 18-months before through 24-months following cancer diagnosis. We included beneficiaries aged ≥66 years newly diagnosed with breast, colorectal, lung or prostate cancer and using medication for non-insulin anti-diabetics, statins, and/or anti-hypertensives and similar individuals without cancer from Surveillance, Epidemiology, and End Results-Medicare data, 2008-2014. Chronic condition medication adherence was defined as a proportion of days covered ≥ 80%. Provider team structure was measured using two factors capturing the number of providers seen and the historical amount of patient sharing among providers. Linear regressions relying on within-survivor variation were run separately for each cancer site, chronic condition, and follow-up period.ResultsThe number of providers and patient sharing among providers increased after cancer diagnosis relative to the non-cancer control group. Changes in provider team complexity explained only small changes in medication adherence. Provider team effects were statistically insignificant in 13 of 17 analytic samples with significant changes in adherence. Statistically significant provider team effects were small in magnitude (ConclusionsIncreased complexity in the provider team associated with cancer diagnosis did not lead to meaningful reductions in medication adherence. Interventions aimed at improving chronic condition medication adherence should be targeted based on the type of cancer and chronic condition and focus on other provider, systemic, or patient factors.

ObjectivesThe aim of this study was to utilise corneal confocal microscopy to quantify corneal nerve morphology and establish the presence of sub-clinical small fibre damage and peripheral neuropathy in children with celiac disease.MethodsThis is a cross-sectional cohort study of twenty children with celiac disease and 20 healthy controls who underwent clinical and laboratory assessments and corneal confocal microscopy. Corneal nerve fiber density (no.mm2), corneal nerve branch density (no.mm2), corneal nerve fiber length (mm.mm2), corneal nerve fiber tortuosity and inferior whorl length (mm.mm2) were quantified manually.ResultsCorneal nerve fiber density (34.7±8.6 vs. 32.9±8.6; P = 0.5), corneal nerve branch density (47.2±24.5 vs. 47.3±20.0; P = 0.1) and corneal nerve fiber length (20.0±5.1 vs. 19.5±4.5; P = 0.8) did not differ between children with celiac disease and healthy controls. Corneal nerve fiber tortuosity (11.4±1.9 vs 13.5±3.0; P = 0.01) was significantly lower and inferior whorl length (20.0±5.5 vs 23.0±3.8; P = 0.06) showed a non-significant reduction in children with celiac disease compared to healthy controls. Inferior whorl length correlated significantly with corneal nerve fiber density (P = 0.005), corneal nerve branch density (P = 0.04), and corneal nerve fiber length (P = 0.002).ConclusionCorneal confocal microscopy demonstrates minimal evidence of neuropathy in children with celiac disease.

ObjectivesTo assess the effects of different types of cataract surgeries and intraocular lenses on generic as well as vision related quality of life of cataract patients, using EQ-5D and IND-VFQ 33 instruments respectively.MethodsAn observational, longitudinal study of patients undergoing cataract surgery was carried out at three ophthalmology centres. Patients were prospectively admitted for surgery for age-related cataract. Generic quality of life was assessed by using Euroqol's EQ5D-5L questionnaire and vision related quality of life was assessed by the IND-VFQ-33 questionnaire. Data pertaining to vision function and quality of life were collected pre surgery and 4 weeks after the surgery.ResultsOut of total patients (n = 814) recruited for the study, 517 patients were interviewed for both pre-surgery and post-surgery for EQ5D and 519 patients were interviewed for both pre-surgery and post-surgery for IND VFQ 33 tool. The combined data from all three centres showed that Quality Adjusted Life Year (QALY) gains observed in patients undergoing phacoemulsification with foldable lens implantation (2.25 QALY) were significantly higher (0.57 QALY) as compared to Small Incision Cataract Surgery (SICS) with PMMA lens implantation (1.68 QALY). Highest improvement however, in all three subscales of IND-VFQ-33 tool were clearly observed for SICS with PMMA lens implantation.ConclusionsThe study has elicited the Health related and vision related Quality of Life scores for cataract surgeries and subsequent lens implantation. This study also offers Health State Utility Values along with visual outcomes for different surgical procedures, lenses and for the combination of surgery with lens implantation for cataract procedures providing a useful resource for future economic evaluation studies.

Objectives To assess the effects of different types of cataract surgeries and intraocular lenses on generic as well as vision related quality of life of cataract patients, using EQ-5D and IND-VFQ 33 instruments respectively. Methods An observational, longitudinal study of patients undergoing cataract surgery was carried out at three ophthalmology centres. Patients were prospectively admitted for surgery for age-related cataract. Generic quality of life was assessed by using Euroqol’s EQ5D-5L questionnaire and vision related quality of life was assessed by the IND-VFQ-33 questionnaire. Data pertaining to vision function and quality of life were collected pre surgery and 4 weeks after the surgery. Results Out of total patients (n = 814) recruited for the study, 517 patients were interviewed for both pre-surgery and post-surgery for EQ5D and 519 patients were interviewed for both pre-surgery and post-surgery for IND VFQ 33 tool. The combined data from all three centres showed that Quality Adjusted Life Year (QALY) gains observed in patients undergoing phacoemulsification with foldable lens implantation (2.25 QALY) were significantly higher (0.57 QALY) as compared to Small Incision Cataract Surgery (SICS) with PMMA lens implantation (1.68 QALY). Highest improvement however, in all three subscales of IND-VFQ-33 tool were clearly observed for SICS with PMMA lens implantation. Conclusions The study has elicited the Health related and vision related Quality of Life scores for cataract surgeries and subsequent lens implantation. This study also offers Health State Utility Values along with visual outcomes for different surgical procedures, lenses and for the combination of surgery with lens implantation for cataract procedures providing a useful resource for future economic evaluation studies.

OBJECTIVE:We examined the incremental cost-effectiveness between two mHealth programs, implemented from 2011 to 2015 in rural Bangladesh: (1) Comprehensive mCARE package as an intervention group and (2) Basic mCARE package as a control group. METHODS:Both programs included a core package of census enumeration and pregnancy surveillance provided by an established cadre of digitally enabled community health workers (CHWs). In the comprehensive mCARE package, short message service (SMS) and home visit reminders were additionally sent to pregnant women (n = 610) and CHWs (n = 70) to promote the pregnant women's care-seeking of essential maternal and newborn care services. Economic costs were assessed from a program perspective inclusive of development, start-up, and implementation phases. Effects were calculated as disability adjusted life years (DALYs) and the number of newborn deaths averted. For comparative purposes, we normalized our evaluation to estimate total costs and total newborn deaths averted per 1 million people in a community for both groups. Uncertainty was assessed using probabilistic sensitivity analyses with Monte Carlo simulation. RESULTS:The addition of SMS and home visit reminders based on a mobile phone-facilitated pregnancy surveillance system was highly cost effective at a cost per DALY averted of $31 (95% uncertainty range: $19-81). The comprehensive mCARE program had at least 88% probability of being highly cost-effective as compared to the basic mCARE program based on the threshold of Bangladesh's GDP per capita. CONCLUSION:mHealth strategies such as SMS and home visit reminders on a well-established pregnancy surveillance system may improve service utilization and program cost-effectiveness in low-resource settings.

Recent study shows that 617 million children and adolescents-or six out of 10 globally- are not acquiring minimum levels in literacy and mathematics, indicating the magnitude of the learning acquisition problem. For children with disabilities in context of conflict, the situation is arguably even worse: the literature shows that they face difficulties to access the education system due to multiple barriers, and when they do access, they are not learning. Our paper examines if an active education policy promoting inclusion since 2005 in Afghanistan, a protracted crisis context, has been effective. Using two cross sectional household surveys carried out eight years apart (2005-2013), our study shows that access to school and literacy did not improve between 2005 and 2013 for children and youth with disabilities. Both access and literacy outcomes were worse for girls with disabilities, those with a mental, learning or associated disability and those living in household where the head was uneducated. Finally, odds of being mentally distressed significantly declined between 2005 and 2013 indicating that schools might play a protective role for children with disabilities in Afghanistan. Our findings suggest that a multilevel multi-pronged adaptation of the existing system to improve the learning experience and promote children's resilience, particularly for children with disabilities, in conflict context such as Afghanistan, is required.

Modified nucleosides in tRNAs are critical for protein translation. N1-methylguanosine-37 and N1-methylinosine-37 in tRNAs, both located at the 3'-adjacent to the anticodon, are formed by Trm5. Here we describe Arabidopsis thaliana AtTRM5 (At3g56120) as a Trm5 ortholog. Attrm5 mutant plants have overall slower growth as observed by slower leaf initiation rate, delayed flowering and reduced primary root length. In Attrm5 mutants, mRNAs of flowering time genes are less abundant and correlated with delayed flowering. We show that AtTRM5 complements the yeast trm5 mutant, and in vitro methylates tRNA guanosine-37 to produce N1-methylguanosine (m1G). We also show in vitro that AtTRM5 methylates tRNA inosine-37 to produce N1-methylinosine (m1I) and in Attrm5 mutant plants, we show a reduction of both N1-methylguanosine and N1-methylinosine. We also show that AtTRM5 is localized to the nucleus in plant cells. Proteomics data showed that photosynthetic protein abundance is affected in Attrm5 mutant plants. Finally, we show tRNA-Ala aminoacylation is not affected in Attrm5 mutants. However the abundance of tRNA-Ala and tRNA-Asp 5' half cleavage products are deduced. Our findings highlight the bifunctionality of AtTRM5 and the importance of the post-transcriptional tRNA modifications m1G and m1I at tRNA position 37 in general plant growth and development.

ObjectiveTo study the role of miRNA-181a and augmenter of liver regeneration in TGF-β-induced fibrosis in hepatic stellate cells.MethodsLX2 cells were treated with 20 ng/ml TGF-β for 24 h. miRNA-181a, ALR plasmid and empty vectors were transfected using siPORT NeoFx reagent. Cells were harvested after 48 h or 72 h of transfection for protein or RNA analysis. Western blotting was performed for ALR, TGF-β receptor II (TGFβ-RII), collagen 1A1 (COLL1A1), alpha-smooth muscle cell actin (α-SMA), rac1, E-cadherin and β-actin. Quantitative RT-PCR was performed for ALR, GAPDH, miRNA-181a or 5S rRNA.ResultsTGF-β induced the expression of miRNA-181a, which in turn down-regulated ALR thereby induced the fibrosis markers, such as COLL1A1, α-SMA and rac1 in hepatic stellate cells. Over-expression of miRNA-181a down-regulated expression of ALR and up-regulated expression of fibrosis markers. On the other hand, ALR over-expression resulted in a decrease in miRNA-181a expression and fibrosis markers. Over-expression of ALR also inhibited the expression of TGFβ-RII and increased expression E-cadherin.ConclusionTGF-β induced miRNA-181a, which in turn induced fibrosis, at least in part, by inhibiting ALR. ALR inhibited TGF-β action by decreasing the expression of TGFβ-RII, thereby inhibiting miRNA-181a expression and fibrosis markers. ALR could serve as a potential molecule to inhibit liver fibrosis.

Ancient DNA (aDNA) analysis of extinct ratite species is of considerable interest as it provides important insights into their origin, evolution, paleogeographical distribution and vicariant speciation in congruence with continental drift theory. In this study, DNA hotspots were detected in fossilized eggshell fragments of ratites (dated ≥25000 years B.P. by radiocarbon dating) using confocal laser scanning microscopy (CLSM). DNA was isolated from five eggshell fragments and a 43 base pair (bp) sequence of a 16S rRNA mitochondrial-conserved region was successfully amplified and sequenced from one of the samples. Phylogenetic analysis of the DNA sequence revealed a 92% identity of the fossil eggshells to Struthio camelus and their position basal to other palaeognaths, consistent with the vicariant speciation model. Our study provides the first molecular evidence for the presence of ostriches in India, complementing the continental drift theory of biogeographical movement of ostriches in India, and opening up a new window into the evolutionary history of ratites.

Birth weight, length and circumferences of the head, chest and arm are key measures of newborn size and health in developing countries. We assessed maternal socio-demographic factors associated with multiple measures of newborn size in a large rural population in Bangladesh using partial least squares (PLS) regression method. PLS regression, combining features from principal component analysis and multiple linear regression, is a multivariate technique with an ability to handle multicollinearity while simultaneously handling multiple dependent variables. We analyzed maternal and infant data from singletons (n = 14,506) born during a double-masked, cluster-randomized, placebo-controlled maternal vitamin A or β-carotene supplementation trial in rural northwest Bangladesh. PLS regression results identified numerous maternal factors (parity, age, early pregnancy MUAC, living standard index, years of education, number of antenatal care visits, preterm delivery and infant sex) significantly (p

Sophorolipids (SLs) are glycolipid biosurfactants that have been shown to display anticancer activity. In the present study, we report anti-proliferative studies on purified forms of novel SLs synthesized using cetyl alcohol as the substrate (referred as SLCA) and their anticancer mechanism in human cervical cancer cells. Antiproliferative effect of column purified SLCA fractions (A, B, C, D, E and F) was examined in panel of human cancer cell lines as well as primary cells. Among these fractions, SLCA B and C significantly inhibited the survival of HeLa and HCT 116 cells without affecting the viability of normal human umbilical vein endothelial cells (HUVEC). The two fractions were identified as cetyl alcohol sophorolipids with non-hydroxylated tail differing in the degree of acetylation on sophorose head group. At an IC50 concentration SLCA B (16.32 μg ml-1) and SLCA C (14.14 μg ml-1) blocked the cell cycle progression of HeLa cells at G1/S phase in time-dependent manner. Moreover, SLCA B and SLCA C induced apoptosis in HeLa cells through an increase in intracellular Ca2+ leading to depolarization of mitochondrial membrane potential and increase in the caspase-3, -8 and -9 activity. All these findings suggest that these SLCAs could be explored for their chemopreventive potential in cervical cancer.

BACKGROUND:In the new Sustainable Development Goal 4, quality of education defined as equity and inclusion alongside traditional learning outcomes, has replaced the narrow goal of access to primary education stipulated in the Millennium Development Goal 2. Since 2000, considerable progress has been made towards improving access to school for children in India, yet questions remain regarding not just children with disabilities' access and acquisition of basic learning skills, but also completion of learning cycles. METHODS AND FINDINGS:Between November, 2, 2011 and June 20th 2012, we interviewed 1294 households about activity limitations and functioning difficulties associated with a health problem among all family members using a validated screening instruments, as well as questions about access, retention and barriers to education. We found that vulnerable children, particularly children with disabilities are less likely to start school and more likely to drop out of school earlier and before completing their high school education than non-disabled children, showing that the learning process is not inclusive in practice. The gap is wider for girls, economically deprived children, or children from households where the head is uneducated. CONCLUSIONS:Firstly, in order to fill the existing knowledge gap on education of children with disabilities in line with SDG4, not only is there a necessity for relevant data with regards to learning outcomes, but also an urgent requirement for more innovative information pertaining to relational aspects of learning that reflect inclusion. Secondly, a stronger understanding of the implications of early assessment would further promote equity in education. Finally, research should tackle learning as a complex and dynamic phenomenon. Education needs to fulfil its instrumental value, but must also re-claim its intrinsic value that often gets watered down in the journey from policies to implementation.

In plants, several cellular and metabolic pathways interact with each other to regulate processes that are vital for their growth and development. Carbon (C) and Nitrogen (N) are two main nutrients for plants and coordination of C and N pathways is an important factor for maintaining plant growth and development. In the present work, influence of nitrogen and sucrose (C source) on growth parameters and expression of genes involved in nitrogen transport and assimilatory pathways was studied in B. juncea seedlings. For this, B. juncea seedlings were treated with four combinations of C and N source viz., N source alone (-Suc+N), C source alone (+Suc-N), with N and C source (+Suc+N) or without N and C source (-Suc-N). Cotyledon size and shoot length were found to be increased in seedlings, when nitrogen alone was present in the medium. Distinct expression pattern of genes in both, root and shoot tissues was observed in response to exogenously supplied N and C. The presence or depletion of nitrogen alone in the medium leads to severe up- or down-regulation of key genes involved in N-uptake and transport (BjNRT1.1, BjNRT1.8) in root tissue and genes involved in nitrate reduction (BjNR1 and BjNR2) in shoot tissue. Moreover, expression of several genes, like BjAMT1.2, BjAMT2 and BjPK in root and two genes BjAMT2 and BjGS1.1 in shoot were found to be regulated only when C source was present in the medium. Majority of genes were found to respond in root and shoot tissues, when both C and N source were present in the medium, thus reflecting their importance as a signal in regulating expression of genes involved in N-uptake and assimilation. The present work provides insight into the regulation of genes of N-uptake and assimilatory pathway in B. juncea by interaction of both carbon and nitrogen.

Abiotic stresses such as salinity, drought and extreme temperatures affect nitrogen (N) uptake and assimilation in plants. However, little is known about the regulation of N pathway genes at transcriptional level under abiotic stress conditions in Brassica juncea. In the present work, genes encoding nitrate transporters (NRT), ammonium transporters (AMT), nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), glutamate synthase (GOGAT), glutamate dehydrogenase (GDH), asparagines synthetase (ASN) were cloned from Brassica juncea L. var. Varuna. The deduced protein sequences were analyzed to predict their subcellular localization, which confirmed localization of all the proteins in their respective cellular organelles. The protein sequences were also subjected to conserved domain identification, which confirmed presence of characteristic domains in all the proteins, indicating their putative functions. Moreover, expression of these genes was studied after 1h and 24h of salt (150 mM NaCl), osmotic (250 mM Mannitol), cold (4°C) and heat (42°C) stresses. Most of the genes encoding nitrate transporters and enzymes responsible for N assimilation and remobilization were found to be downregulated under abiotic stresses. The expression of BjAMT1.2, BjAMT2, BjGS1.1, BjGDH1 and BjASN2 was downregulated after 1hr, while expression of BjNRT1.1, BjNRT2.1, BjNiR1, BjAMT2, BjGDH1 and BjASN2 was downregulated after 24h of all the stress treatments. However, expression of BjNRT1.1, BjNRT1.5 and BjGDH2 was upregulated after 1h of all stress treatments, while no gene was found to be upregulated after 24h of stress treatments, commonly. These observations indicate that expression of most of the genes is adversely affected under abiotic stress conditions, particularly under prolonged stress exposure (24h), which may be one of the reasons of reduction in plant growth and development under abiotic stresses.

BACKGROUND:Although 80% of persons with disabilities live in low and middle-income countries, there is still a lack of comprehensive, cross-culturally validated tools to identify persons facing activity limitations and functioning difficulties in these settings. In absence of such a tool, disability estimates vary considerably according to the methodology used, and policies are based on unreliable estimates. METHODS AND FINDINGS:The Disability Screening Questionnaire composed of 27 items (DSQ-27) was initially designed by a group of international experts in survey development and disability in Afghanistan for a national survey. Items were selected based on major domains of activity limitations and functioning difficulties linked to an impairment as defined by the International Classification of Functioning, Disability and Health. Face, content and construct validity, as well as sensitivity and specificity were examined. Based on the results obtained, the tool was subsequently refined and expanded to 34 items, tested and validated in Darfur, Sudan. Internal consistency for the total DSQ-34 using a raw and standardized Cronbach's Alpha and within each domain using a standardized Cronbach's Alpha was examined in the Asian context (India and Nepal). Exploratory factor analysis (EFA) using principal axis factoring (PAF) evaluated the lowest number of factors to account for the common variance among the questions in the screen. Test-retest reliability was determined by calculating intraclass correlation (ICC) and inter-rater reliability by calculating the kappa statistic; results were checked using Bland-Altman plots. The DSQ-34 was further tested for standard error of measurement (SEM) and for the minimum detectable change (MDC). Good internal consistency was indicated by Cronbach's Alpha of 0.83/0.82 for India and 0.76/0.78 for Nepal. We confirmed our assumption for EFA using the Kaiser-Meyer-Olkin measure of sampling well above the accepted cutoff of 0.40 for India (0.82) and Nepal (0.82). The criteria for Bartlett's test of sphericity were also met for both India (< .001) and Nepal (< .001). Estimates of reliability from the two countries reached acceptable levels of ICC of 0.75 (p

The tubercle complex consists of closely related mycobacterium species which appear to be variants of a single species. Comparative genome analysis of different strains could provide useful clues and insights into the genetic diversity of the species. We integrated genome assemblies of 96 strains from Mycobacterium tuberculosis complex (MTBC), which included 8 Indian clinical isolates sequenced and assembled in this study, to understand its pangenome architecture. We predicted genes for all the 96 strains and clustered their respective CDSs into homologous gene clusters (HGCs) to reveal a hard-core, soft-core and accessory genome component of MTBC. The hard-core (HGCs shared amongst 100% of the strains) was comprised of 2,066 gene clusters whereas the soft-core (HGCs shared amongst at least 95% of the strains) comprised of 3,374 gene clusters. The change in the core and accessory genome components when observed as a function of their size revealed that MTBC has an open pangenome. We identified 74 HGCs that were absent from reference strains H37Rv and H37Ra but were present in most of clinical isolates. We report PCR validation on 9 candidate genes depicting 7 genes completely absent from H37Rv and H37Ra whereas 2 genes shared partial homology with them accounting to probable insertion and deletion events. The pangenome approach is a promising tool for studying strain specific genetic differences occurring within species. We also suggest that since selecting appropriate target genes for typing purposes requires the expected target gene be present in all isolates being typed, therefore estimating the core-component of the species becomes a subject of prime importance.

Inflammation is a condition stemming from complex host defense and tissue repair mechanisms, often simply characterized by plasma levels of a single acute reactant. We attempted to identify candidate biomarkers of systemic inflammation within the plasma proteome. We applied quantitative proteomics using isobaric mass tags (iTRAQ) tandem mass spectrometry to quantify proteins in plasma of 500 Nepalese children 6-8 years of age. We evaluated those that co-vary with inflammation, indexed by α-1-acid glycoprotein (AGP), a conventional biomarker of inflammation in population studies. Among 982 proteins quantified in >10% of samples, 99 were strongly associated with AGP at a family-wise error rate of 0.1%. Magnitude and significance of association varied more among proteins positively (n = 41) than negatively associated (n = 58) with AGP. The former included known positive acute phase proteins including C-reactive protein, serum amyloid A, complement components, protease inhibitors, transport proteins with anti-oxidative activity, and numerous unexpected intracellular signaling molecules. Negatively associated proteins exhibited distinct differences in abundance between secretory hepatic proteins involved in transporting or binding lipids, micronutrients (vitamin A and calcium), growth factors and sex hormones, and proteins of largely extra-hepatic origin involved in the formation and metabolic regulation of extracellular matrix. With the same analytical approach and the significance threshold, seventy-two out of the 99 proteins were commonly associated with CRP, an established biomarker of inflammation, suggesting the validity of the identified proteins. Our findings have revealed a vast plasma proteome within a free-living population of children that comprise functional biomarkers of homeostatic and induced host defense, nutrient metabolism and tissue repair, representing a set of plasma proteins that may be used to assess dynamics and extent of inflammation for future clinical and public health application.

Prenatal multiple micronutrient (MM) supplementation improves birth weight through increased fetal growth and gestational age, but whether maternal or fetal growth factors are involved is unclear. Our objective was to examine the effect of prenatal MM supplementation on intrauterine growth factors and the associations between growth factors and birth outcomes in a rural setting in Bangladesh. In a double-blind, cluster-randomized, controlled trial of MM vs. iron and folic acid (IFA) supplementation, we measured placental growth hormone (PGH) at 10 weeks and PGH and human placental lactogen (hPL) at 32 weeks gestation in maternal plasma (n = 396) and insulin, insulin-like growth factor-1 (IGF-1), and IGF binding protein-1 (IGFBP-1) in cord plasma (n = 325). Birth size and gestational age were also assessed. Early pregnancy mean (SD) BMI was 19.5 (2.4) kg/m2 and birth weight was 2.68 (0.41) kg. There was no effect of MM on concentrations of maternal hPL or PGH, or cord insulin, IGF-1, or IGFBP-1. However, among pregnancies of female offspring, hPL concentration was higher by 1.1 mg/L in the third trimester (95% CI: 0.2, 2.0 mg/L; p = 0.09 for interaction); and among women with height

The integrity of the epithelium is maintained by a complex but regulated interplay of processes that allow conversion of a proliferative state into a stably differentiated state. In this study, using human embryonic stem cell (hESC) derived Retinal Pigment Epithelium (RPE) cells as a model; we have investigated the molecular mechanisms that affect attainment of the epithelial phenotype. We demonstrate that RPE undergo a Mesenchymal-Epithelial Transition in culture before acquiring an epithelial phenotype in a FOXM1 dependent manner. We show that FOXM1 directly regulates proliferation of RPE through transcriptional control of cell cycle associated genes. Additionally, FOXM1 modulates expression of the signaling ligands BMP7 and Wnt5B which act reciprocally to enable epithelialization. This data uncovers a novel effect of FOXM1 dependent activities in contributing towards epithelial fate acquisition and furthers our understanding of the molecular regulators of a cell type that is currently being evaluated as a cell therapy.

Although economic development is generally accompanied by improvements in the overall nutritional status of the country's population the 'nutritional transition' often involves a shift to high energy diets and less exercise with negative consequences. This pilot study was done to examine if education of parents operates at the household level to influence dietary choices and the nutritional status of children in a small community of hospital workers.3 groups of persons with varying skill and education levels participated. Weighed food logs were used in all households to calculate 'adult equivalent' per-capita-consumption. Nutrients were calculated using nutrients calculator software. BMI was used to classify children as underweight, normal weight and overweight.128 individuals participated from 30 families included 47 children. 10 children (21%) were underweight, 29 (62%) were normal and 8 (17%) were overweight. Energy consumption was highest in families with overweight children 2692 +/-502 compared to 2259 +/-359 in families with normal weight and 2031+/-354 in the family of underweight children. These differences were statistically significant. 42% underweight children belonged to Class 1 at the lowest skill level and there were no overweight children in this group. Most of the overweight children belonged to Class 2. In Class 3 there were no underweight children and the majority was normal weight children.Underweight children came from the poorer households. Per capita intake of the family as a whole correlated well with BMI in the children. There was increased obesity in middle income families belonging to Class 2-probably in families who move up the scale from deprivation. Nutritional status in children correlated mostly with maternal education status.

The Gcn5-related N-acetyltransferases (GNATs) are ubiquitously expressed in nature and perform a diverse range of cellular functions through the acetylation of small molecules and protein substrates. Using activated acetyl coenzyme A as a common acetyl donor, GNATs catalyse the transfer of an acetyl group to acceptor molecules including aminoglycoside antibiotics, glucosamine-6-phosphate, histones, serotonin and spermidine. There is often only very limited sequence conservation between members of the GNAT superfamily, in part, reflecting their capacity to bind a diverse array of substrates. In contrast, the secondary and tertiary structures are highly conserved, but then at the quaternary level there is further diversity, with GNATs shown to exist in monomeric, dimeric, or tetrameric states. Here we describe the X-ray crystallographic structure of a GNAT enzyme from Staphylococcus aureus with only low sequence identity to previously solved GNAT proteins. It contains many of the classical GNAT motifs, but lacks other hallmarks of the GNAT fold including the classic β-bulge splayed at the β-sheet interface. The protein is likely to be a dimer in solution based on analysis of the asymmetric unit within the crystal structure, homology with related GNAT family members, and size exclusion chromatography. The study provides the first high resolution structure of this enzyme, providing a strong platform for substrate and cofactor modelling, and structural/functional comparisons within this diverse enzyme superfamily.

BackgroundThe DEAD-box helicases are required mostly in all aspects of RNA and DNA metabolism and they play a significant role in various abiotic stresses, including salinity. The p68 is an important member of the DEAD-box proteins family and, in animal system, it is involved in RNA metabolism including pre-RNA processing and splicing. In plant system, it has not been well characterized. Here we report the cloning and characterization of p68 from pea (Pisum sativum) and its novel function in salinity stress tolerance in plant.ResultsThe pea p68 protein self-interacts and is localized in the cytosol as well as the surrounding of cell nucleus. The transcript of pea p68 is upregulated in response to high salinity stress in pea. Overexpression of p68 driven by constitutive cauliflower mosaic virus-35S promoter in tobacco transgenic plants confers enhanced tolerances to salinity stress by improving the growth, photosynthesis and antioxidant machinery. Under stress treatment, pea p68 overexpressing tobacco accumulated higher K+ and lower Na+ level than the wild-type plants. Reactive oxygen species (ROS) accumulation was remarkably regulated by the overexpression of pea p68 under salinity stress conditions, as shown from TBARS content, electrolyte leakage, hydrogen peroxide accumulation and 8-OHdG content and antioxidant enzyme activities.ConclusionsTo the best of our knowledge this is the first direct report, which provides the novel function of pea p68 helicase in salinity stress tolerance. The results suggest that p68 can also be exploited for engineering abiotic stress tolerance in crop plants of economic importance.

1α-25(OH)2 vitamin D3 (1-25D), an active hormonal form of Vitamin D3, is a well-known chemopreventive and pro-differentiating agent. It has been shown to inhibit the growth of several prostate cancer cell lines. Gap junctions, formed of proteins called connexins (Cx), are ensembles of cell-cell channels, which permit the exchange of small growth regulatory molecules between adjoining cells. Cell-cell communication mediated by gap junctional channels is an important homeostatic control mechanism for regulating cell growth and differentiation. We have investigated the effect of 1-25D on the formation and degradation of gap junctions in an androgen-responsive prostate cancer cell line, LNCaP, which expresses retrovirally-introduced Cx32. Connexin32 is expressed by the luminal and well-differentiated cells of normal prostate and prostate tumors. Our results document that 1-25D enhances the expression of Cx32 and its subsequent assembly into gap junctions. Our results further show that 1-25D prevents androgen-regulated degradation of Cx32, post-translationally, independent of androgen receptor (AR)-mediated signaling. Finally, our findings document that formation of gap junctions sensitizes Cx32-expressing LNCaP cells to the growth inhibitory effects of 1-25D and alters their morphology. These findings suggest that the growth-inhibitory effects of 1-25D in LNCaP cells may be related to its ability to modulate the assembly of Cx32 into gap junctions.

This analysis was conducted to explore the association between 5 birth size measurements (weight, length and head, chest and mid-upper arm [MUAC] circumferences) as dependent variables and 10 maternal factors as independent variables using canonical correlation analysis (CCA). CCA considers simultaneously sets of dependent and independent variables and, thus, generates a substantially reduced type 1 error. Data were from women delivering a singleton live birth (n = 14,506) while participating in a double-masked, cluster-randomized, placebo-controlled maternal vitamin A or β-carotene supplementation trial in rural Bangladesh. The first canonical correlation was 0.42 (P

BACKGROUND:Today's uncertain HIV funding landscape threatens to slow progress towards treatment goals. Understanding the costs of antiretroviral therapy (ART) will be essential for governments to make informed policy decisions about the pace of scale-up under the 2013 WHO HIV Treatment Guidelines, which increase the number of people eligible for treatment from 17.6 million to 28.6 million. The study presented here is one of the largest of its kind and the first to describe the facility-level cost of ART in a random sample of facilities in Ethiopia, Malawi, Rwanda, South Africa and Zambia. METHODS & FINDINGS:In 2010-2011, comprehensive data on one year of facility-level ART costs and patient outcomes were collected from 161 facilities, selected using stratified random sampling. Overall, facility-level ART costs were significantly lower than expected in four of the five countries, with a simple average of $208 per patient-year (ppy) across Ethiopia, Malawi, Rwanda and Zambia. Costs were higher in South Africa, at $682 ppy. This included medications, laboratory services, direct and indirect personnel, patient support, equipment and administrative services. Facilities demonstrated the ability to retain patients alive and on treatment at these costs, although outcomes for established patients (2-8% annual loss to follow-up or death) were better than outcomes for new patients in their first year of ART (77-95% alive and on treatment). CONCLUSIONS:This study illustrated that the facility-level costs of ART are lower than previously understood in these five countries. While limitations must be considered, and costs will vary across countries, this suggests that expanded treatment coverage may be affordable. Further research is needed to understand investment costs of treatment scale-up, non-facility costs and opportunities for more efficient resource allocation.

Yeast sporulation efficiency is a quantitative trait and is known to vary among experimental populations and natural isolates. Some studies have uncovered the genetic basis of this variation and have identified the role of sporulation genes (IME1, RME1) and sporulation-associated genes (FKH2, PMS1, RAS2, RSF1, SWS2), as well as non-sporulation pathway genes (MKT1, TAO3) in maintaining this variation. However, these studies have been done mostly in experimental populations. Sporulation is a response to nutrient deprivation. Unlike laboratory strains, natural isolates have likely undergone multiple selections for quick adaptation to varying nutrient conditions. As a result, sporulation efficiency in natural isolates may have different genetic factors contributing to phenotypic variation. Using Saccharomyces cerevisiae strains in the genetically and environmentally diverse SGRP collection, we have identified genetic loci associated with sporulation efficiency variation in a set of sporulation and sporulation-associated genes. Using two independent methods for association mapping and correcting for population structure biases, our analysis identified two linked clusters containing 4 non-synonymous mutations in genes - HOS4, MCK1, SET3, and SPO74. Five regulatory polymorphisms in five genes such as MLS1 and CDC10 were also identified as putative candidates. Our results provide candidate genes contributing to phenotypic variation in the sporulation efficiency of natural isolates of yeast.

BACKGROUND:Multinucleated giant cells (MGC) are the histologic hallmark of granuloma which is known to limit tuberculosis infection. Both Th1 and Th2 type of cytokines regulate the immune response occurring within the granulomas. The objective of the study was to determine whether tuberculosis patient monocytes differed in their MGC forming ability as compared to healthy controls. METHODS:In vitro MGC formation was carried out by treatment of monocytes with cytokine containing culture supernatant of ConA or PPD stimulated peripheral mononuclear cells. IL-2, TNF-α, IL-4, IL-10 and TGF-β cytokine levels were analysed in culture supernatants using ELISA. IL-4 and IL-10 were added to culture supernatant separately and simultaneously along with their respective neutralizing antibodies and their consequent effect on MGC formation was evaluated. RESULTS:MGC formation was significantly low in patient monocytes incubated with autologous culture supernatant as compared to control culture supernatant. Cytokine analysis of the culture supernatants revealed that while IL-4 levels were similar in patients and controls, increased IL-10 levels were found in patients. Exogenous addition of IL-10 resulted in reduced MGC formation. Contrastingly, when IL-4 was added exogenously, it led to increased MGC formation. The effects of both IL-10 and IL-4 were reversed upon addition of their respective antibodies. CONCLUSION:The findings suggest that one of the factors contributing to the disease could be the effect of cytokines on the functionality of monocytes, which are crucial in the fight against the organism. Significantly reduced MGC formation was observed on addition of IL-10. The findings imply an overriding role of IL-10 in MGC formation. The suppressive effect of IL-10 on MGC formation was further confirmed by addition of IL-10 neutralizing antibody.

Breast tumor metastasis is a leading cause of cancer-related deaths worldwide. Breast tumor cells frequently metastasize to brain and initiate severe therapeutic complications. The chances of brain metastasis are further elevated in patients with HER2 overexpression. In the current study, we evaluated the anti-metastatic effects of phenethyl isothiocyanate (PEITC) in a novel murine model of breast tumor metastasis. The MDA-MB-231-BR (BR-brain seeking) breast tumor cells stably transfected with luciferase were injected into the left ventricle of mouse heart and the migration of cells to brain was monitored using a non-invasive IVIS bio-luminescent imaging system. In order to study the efficacy of PEITC in preventing the number of tumor cells migrating to brain, mice were given 10 µmol PEITC by oral gavage for ten days prior to intra-cardiac injection of tumor cells labeled with quantum dots. To evaluate the tumor growth suppressive effects, 10 µmol PEITC was given to mice every day starting 14(th) day after intra-cardiac cell injection. Based on the presence of quantum dots in the brain section of control and treated mice, our results reveal that PEITC significantly prevented the metastasis of breast cancer cells to brain. Our results demonstrate that the growth of metastatic brain tumors in PEITC treated mice was about 50% less than that of control. According to Kaplan Meir's curve, median survival of tumor bearing mice treated with PEITC was prolonged by 20.5%. Furthermore as compared to controls, we observed reduced HER2, EGFR and VEGF expression in the brain sections of PEITC treated mice. To the best of our knowledge, our study for the first time demonstrates the anti-metastatic effects of PEITC in vivo in a novel breast tumor metastasis model and provides the rationale for further clinical investigation.

Stress has long been known to increase susceptibility to health disorders. In 2009, American Psychological Association further established association of stress to serious health problems. However, a quantitative and accurate way to evaluate and estimate stress status of individuals is still a big challenge. It has been shown, in large animal models using cattle, that psychological stress can be quantified as well as disease susceptibility could be predicted through biomarker discovery. Taking cue from those studies, we have evaluated and estimated psychological stress level of individuals theoretically and validated experimentally. Various biomarkers have also been identified which can be associated to psychological stress to predict stress status of unknown individuals.

The benzylisoquinoline alkaloid papaverine, synthesized in low amount in most of the opium poppy varieties of Papaver somniferum, is used as a vasodilator muscle relaxant and antispasmodic. Papaverine biosynthesis remains controversial as two different routes utilizing either (S)-coclaurine or (S)-reticuline have been proposed with uncharacterized intermediate steps. In an attempt to elucidate papaverine biosynthesis and identify putative genes involved in uncharacterized steps, we carried out comparative transcriptome analysis of high papaverine mutant (pap1) and normal cultivar (BR086) of P. somniferum. This natural mutant synthesizes more than 12-fold papaverine in comparison to BR086. We established more than 238 Mb transcriptome data separately for pap1 and BR086. Assembly of reads generated 127,342 and 106,128 unigenes in pap1 and BR086, respectively. Digital gene expression analysis of transcriptomes revealed 3,336 differentially expressing unigenes. Enhanced expression of (S)-norcoclaurine-6-O-methyltransferase (6OMT), (S)-3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase (4'OMT), norreticuline 7-O-methyltransferase (N7OMT) and down-regulation of reticuline 7-O-methyltransferase (7OMT) in pap1 in comparison to BR086 suggest (S)-coclaurine as the route for papaverine biosynthesis. We also identified several methyltransferases and dehydrogenases with enhanced expression in pap1 in comparison to BR086. Our analysis using natural mutant, pap1, concludes that (S)-coclaurine is the branch-point intermediate and preferred route for papaverine biosynthesis. Differentially expressing methyltransferases and dehydrogenases identified in this study will help in elucidating complete biosynthetic pathway of papaverine. The information generated will be helpful in developing strategies for enhanced biosynthesis of papaverine through biotechnological approaches.

Withania somnifera is one of the most valuable medicinal plants used in Ayurvedic and other indigenous medicine systems due to bioactive molecules known as withanolides. As genomic information regarding this plant is very limited, little information is available about biosynthesis of withanolides. To facilitate the basic understanding about the withanolide biosynthesis pathways, we performed transcriptome sequencing for Withania leaf (101L) and root (101R) which specifically synthesize withaferin A and withanolide A, respectively. Pyrosequencing yielded 8,34,068 and 7,21,755 reads which got assembled into 89,548 and 1,14,814 unique sequences from 101L and 101R, respectively. A total of 47,885 (101L) and 54,123 (101R) could be annotated using TAIR10, NR, tomato and potato databases. Gene Ontology and KEGG analyses provided a detailed view of all the enzymes involved in withanolide backbone synthesis. Our analysis identified members of cytochrome P450, glycosyltransferase and methyltransferase gene families with unique presence or differential expression in leaf and root and might be involved in synthesis of tissue-specific withanolides. We also detected simple sequence repeats (SSRs) in transcriptome data for use in future genetic studies. Comprehensive sequence resource developed for Withania, in this study, will help to elucidate biosynthetic pathway for tissue-specific synthesis of secondary plant products in non-model plant organisms as well as will be helpful in developing strategies for enhanced biosynthesis of withanolides through biotechnological approaches.

The retinoids, the natural or synthetic derivatives of Vitamin A (retinol), are essential for the normal development of prostate and have been shown to modulate prostate cancer progression in vivo as well as to modulate growth of several prostate cancer cell lines. 9-cis-retinoic acid and all-trans-retinoic acid are the two most important metabolites of retinol. Gap junctions, formed of proteins called connexins, are ensembles of intercellular channels that permit the exchange of small growth regulatory molecules between adjoining cells. Gap junctional communication is instrumental in the control of cell growth. We examined the effect of 9-cis-retinoic acid and all-trans retinoic acid on the formation and degradation of gap junctions as well as on junctional communication in an androgen-responsive prostate cancer cell line, LNCaP, which expressed retrovirally introduced connexin32, a connexin expressed by the luminal cells and well-differentiated cells of prostate tumors. Our results showed that 9-cis-retinoic acid and all-trans retinoic acid enhanced the assembly of connexin32 into gap junctions. Our results further showed that 9-cis-retinoic acid and all-trans-retinoic acid prevented androgen-regulated degradation of gap junctions, post-translationally, independent of androgen receptor mediated signaling. Finally, our findings showed that formation of gap junctions sensitized connexin32-expressing LNCaP cells to the growth modifying effects of 9-cis-retinoic acid, all-trans-retinoic acid and androgens. Thus, the effects of retinoids and androgens on growth and the formation and degradation of gap junctions and their function might be related to their ability to modulate prostate growth and cancer.

Ovarian cancer is the leading cause of cancer related deaths in women. Genetic alterations including overexpression of EGFR play a crucial role in ovarian carcinogenesis. Here we evaluated the effect of phenethyl isothiocyanate (PEITC) in ovarian tumor cells in vitro and in vivo. Oral administration of 12 µmol PEITC resulted in drastically suppressing ovarian tumor growth in a preclinical mouse model. Our in vitro studies demonstrated that PEITC suppress the growth of SKOV-3, OVCAR-3 and TOV-21G human ovarian cancer cells by inducing apoptosis in a concentration-dependent manner. Growth inhibitory effects of PEITC were mediated by inhibition of EGFR and AKT, which are known to be overexpressed in ovarian tumors. PEITC treatment caused significant down regulation of constitutive protein levels as well as phosphorylation of EGFR at Tyr1068 in various ovarian cancer cells. In addition, PEITC treatment drastically reduced the phosphorylation of AKT which is downstream to EGFR and disrupted mTOR signaling. PEITC treatment also inhibited the kinase activity of AKT as observed by the down regulation of p-GSK in OVCAR-3 and TOV-21G cells. AKT overexpression or TGF treatment blocked PEITC induced apoptosis in ovarian cancer cells. These results suggest that PEITC targets EGFR/AKT pathway in our model. In conclusion, our study suggests that PEITC could be used alone or in combination with other therapeutic agents to treat ovarian cancer.

A majority of the studies examining the molecular regulation of human labor have been conducted using single gene approaches. While the technology to produce multi-dimensional datasets is readily available, the means for facile analysis of such data are limited. The objective of this study was to develop a systems approach to infer regulatory mechanisms governing global gene expression in cytokine-challenged cells in vitro, and to apply these methods to predict gene regulatory networks (GRNs) in intrauterine tissues during term parturition. To this end, microarray analysis was applied to human amnion mesenchymal cells (AMCs) stimulated with interleukin-1β, and differentially expressed transcripts were subjected to hierarchical clustering, temporal expression profiling, and motif enrichment analysis, from which a GRN was constructed. These methods were then applied to fetal membrane specimens collected in the absence or presence of spontaneous term labor. Analysis of cytokine-responsive genes in AMCs revealed a sterile immune response signature, with promoters enriched in response elements for several inflammation-associated transcription factors. In comparison to the fetal membrane dataset, there were 34 genes commonly upregulated, many of which were part of an acute inflammation gene expression signature. Binding motifs for nuclear factor-κB were prominent in the gene interaction and regulatory networks for both datasets; however, we found little evidence to support the utilization of pathogen-associated molecular pattern (PAMP) signaling. The tissue specimens were also enriched for transcripts governed by hypoxia-inducible factor. The approach presented here provides an uncomplicated means to infer global relationships among gene clusters involved in cellular responses to labor-associated signals.

Saliva of blood sucking arthropods contains compounds that antagonize their hosts' hemostasis, which include platelet aggregation, vasoconstriction and blood clotting; saliva of these organisms also has anti-inflammatory and immunomodullatory properties. Perhaps because hosts mount an active immune response against these compounds, the diversity of these compounds is large even among related blood sucking species. Because of these properties, saliva helps blood feeding as well as help the establishment of pathogens that can be transmitted during blood feeding.We have obtained 1,626,969 reads by pyrosequencing a salivary gland cDNA library from adult females Amblyomma maculatum ticks at different times of feeding. Assembly of this data produced 72,441 sequences larger than 149 nucleotides from which 15,914 coding sequences were extracted. Of these, 5,353 had >75% coverage to their best match in the non-redundant database from the National Center for Biotechnology information, allowing for the deposition of 4,850 sequences to GenBank. The annotated data sets are available as hyperlinked spreadsheets. Putative secreted proteins were classified in 133 families, most of which have no known function.This data set of proteins constitutes a mining platform for novel pharmacologically active proteins and for uncovering vaccine targets against A. maculatum and the diseases they carry.

BACKGROUND: The Mycobacterium tuberculosis genome encodes two peptide transporters encoded by Rv3665c-Rv3662c and Rv1280c-Rv1283c. Both belong to the family of ABC transporters containing two nucleotide-binding subunits, two integral membrane proteins and one substrate-binding polypeptide. However, little is known about their functions in M. tuberculosis. Here we report functional characterization of the Rv1280c-Rv1283c-encoded transporter and its substrate-binding polypeptide OppA(MTB). METHODOLOGY/PRINCIPAL FINDINGS: OppA(MTB) was capable of binding the tripeptide glutathione and the nonapeptide bradykinin, indicative of a somewhat broad substrate specificity. Amino acid residues G109, N110, N230, D494 and F496, situated at the interface between domains I and III of OppA, were required for optimal peptide binding. Complementaton of an oppA knockout mutant of M. smegmatis with OppA(MTB) confirmed the role of this transporter in importing glutathione and the importance of the aforesaid amino acid residues in peptide transport. Interestingly, this transporter regulated the ability of M. tuberculosis to lower glutathione levels in infected compared to uninfected macrophages. This ability was partly offset by inactivation of oppD. Concomitantly, inactivation of oppD was associated with lowered levels of methyl glyoxal in infected macrophages and reduced apoptosis-inducing ability of the mutant. The ability to induce the production of the cytokines IL-1beta, IL-6 and TNF-alpha was also compromised after inactivation of oppD. CONCLUSIONS: Taken together, these studies uncover the novel observations that this peptide transporter modulates the innate immune response of macrophages infected with M. tuberculosis.

Angiogenesis is a complex process orchestrated by both growth factors and cell adhesion and is initiated by focal degradation of the vascular basement membrane with subsequent migration and proliferation of endothelial cells. The Ras/Raf/MEK/ERK pathway is required for EC function during angiogenesis. Although in vitro studies implicate ERK1 and ERK2 in endothelial cell survival, their precise role in angiogenesis in vivo remains poorly defined. Cre/loxP technology was used to inactivate Erk1 and Erk2 in endothelial cells during murine development, resulting in embryonic lethality due to severely reduced angiogenesis. Deletion of Erk1 and Erk2 in primary endothelial cells resulted in decreased cell proliferation and migration, but not in increased apoptosis. Expression of key cell cycle regulators was diminished in the double knockout cells, and decreased DNA synthesis could be observed in endothelial cells during embryogenesis. Interestingly, both Paxillin and Focal Adhesion Kinase were expressed at lower levels in endothelial cells lacking Erk1 and Erk2 both in vivo and in vitro, leading to defects in the organization of the cytoskeleton and in cell motility. The regulation of Paxillin and Focal Adhesion Kinase expression occurred post-transcriptionally. These results demonstrate that ERK1 and ERK2 coordinate endothelial cell proliferation and migration during angiogenesis.

Chronic pain is a major clinical problem and opiates are often the only treatment, but they cause significant problems ranging from sedation to deadly respiratory depression. Resiniferatoxin (RTX), a potent agonist of Transient Receptor Potential Vanilloid 1 (TRPV1), causes a slow, sustained and irreversible activation of TRPV1 and increases the frequency of spontaneous excitatory postsynaptic currents, but causes significant depression of evoked EPSCs due to nerve terminal depolarization block. Intrathecal administration of RTX to rats in the short-term inhibits nociceptive synaptic transmission, and in the long-term causes a localized, selective ablation of TRPV1-expressing central sensory nerve terminals leading to long lasting analgesia in behavioral models. Since RTX actions are selective for central sensory nerve terminals, other efferent functions of dorsal root ganglion neurons can be preserved. Preventing nociceptive transmission at the level of the spinal cord can be a useful strategy to treat chronic, debilitating and intractable pain.