Analysis of serotonin receptor 2A gene (HTR2A): Association study with autism spectrum disorder in the Indian population and investigation of the gene expression in peripheral blood leukocytes

Several studies suggest involvement of serotoninergic system in the pathophysiology of Autism Spectrum Disorder (ASD). The 5-HT receptor binding studies using ³H-lysergic acid diethylamide (³H-LSD) and linkage analysis provided evidences to consider HTR2A as a potential candidate gene for ASD. The three SNPs, −1438A/G (rs6311), 102T/C (rs6313) and 1354C/T (rs6314) of HTR2A have been well studied in the etiology of various neuropsychiatric disorders. But studies on association of this gene with ASD are limited to two reports from American and Korean populations. Additionally there are reports, which demonstrated paternal imprinting of HTR2A with expression from only one allele. So far no reports are available on HTR2A and its association with any neuropsychiatric disorders from Indian population. Therefore, the present study investigates association of the above mentioned three markers of HTR2A with ASD in Indian population using population and family-based approaches. The study also deals with allelic expression pattern of HTR2A in Peripheral Blood Leukocytes (PBLs) to understand the parental imprinting status. The genotyping analyses were carried out for probands, parents and controls. The subsequent association analyses did not show association of these markers with ASD. So, HTR2A is unlikely to be a genetic marker for ASD in Indian population. The expression analyses showed absence of monoallelic expression, suggesting lack of parental imprinting of HTR2A gene. However, we noticed methylation of the CpG sites at −1438A/G and 102T/C loci of HTR2A gene. Further bioinformatics analysis revealed absence of CpG islands in the promoter of the gene supporting biallelic expression pattern of HTR2A in PBLs.     

Interaction of rs316019 variants of SLC22A2 with metformin and other drugs- an in silico analysis

Metformin is one of the first-line and most widely prescribed drugs to treat type 2 diabetes (T2D). Its clearance from circulation is mostly facilitated by SLC22A2 (OCT2) in the renal cells. SLC22A2 is a polyspecific organic cation transporter and mediate transport of structurally unrelated endogenous and exogenous compounds including many drugs. rs316019 (p.270A?>?S) is the most common variant of SLC22A2 with a frequency as high as 15% or more in many populations. The 270S form of SLC22A2 clears metformin from circulation at much reduced level compared to the 270A form. If accumulated, metformin increases plasma lactate level in a concentration-dependent manner which can lead to a condition known as metformin-associated lactic acidosis (MALA). MALA is a potentially life-threatening complication with a mortality rate of 30–50%. Pre-existing clinical conditions, such as renal impairment, sepsis, anoxia, etc may make individuals more prone to MALA. In this study, we used computational approaches to investigate the effect of 270A?>?S change in SLC22A2 on interaction with metformin and other drugs. Based on the structural models, all substrates bind to the same pocket of SLC22A2. The substrates fit better to the binding site of 270A form of SLC22A2. The binding site has a few core interacting residues, among which SER358 appears to be the most important. It is an in silico prediction that the T2D patients, who are under metformin regimen, should be cautious in taking ranitidine (an over-the-counter sold drug) on a regular basis as it may lead to metformin associated lactate accumulation in blood.     

Cdc14 Phosphatase Promotes TORC1-Regulated Autophagy in Yeast

Cdc14 protein phosphatase is critical for late mitosis progression in budding yeast, although its orthologs in other organisms, including mammalian cells, function as stress-responsive phosphatases. We found herein unexpected roles of Cdc14 in autophagy induction after nutrient starvation and target of rapamycin complex 1 (TORC1) kinase inactivation. TORC1 kinase phosphorylates Atg13 to repress autophagy under nutrient-rich conditions, but if TORC1 becomes inactive upon nutrient starvation or rapamycin treatment, Atg13 is rapidly dephosphorylated and autophagy is induced. Cdc14 phosphatase was required for optimal Atg13 dephosphorylation, pre-autophagosomal structure formation, and autophagy induction after TORC1 inactivation. In addition, Cdc14 was required for sufficient induction of ATG8 and ATG13 expression. Moreover, Cdc14 activation provoked autophagy even under normal conditions. This study identified a novel role of Cdc14 as the stress-responsive phosphatase for autophagy induction in budding yeast.     

Salicylic acid-mediated changes in photosynthesis,nutrients content and antioxidant metabolism in two mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis> L.) cultivars differing in salt tolerance

Mustard (Brassica juncea L.) cultivars Alankar (salt-tolerant) and PBM16 (salt-sensitive) plants were grown with 50 mM NaCl and were sprayed with 0.1, 0.5, and 1.0 mM salicylic acid (SA) to study the physiological processes determining salt tolerance and to observe the influence of SA application on the alleviation of NaCl-induced adverse effects. The content of leaf Na⁺, Cl⁻, H₂O₂, TBARS, and electrolyte leakage and the activity of SOD were higher in PBM16 than Alankar. In contrast, nutrients content, activity of APX and GR, glutathione content, photosynthetic and growth characteristics were higher in Alankar. Treatment of 50 mM NaCl resulted in increase of Na⁺ and Cl⁻, oxidative stress, activity of antioxidant enzymes and glutathione content, while nutrients content, photosynthetic, and growth characteristics decreased in both the cultivars. Application of 0.5 mM SA alleviated the negative effects of 50 mM NaCl maximally, but 1.0 mM SA proved inhibitory. The effect of SA was more conspicuous in Alankar than PBM16. It is concluded that the higher tolerance of Alankar was due to its lower leaf Na⁺ and Cl⁻ content, higher nutrients content, and efficient antioxidant metabolism. The application of 0.5 mM SA substantially alleviated salt-induced adverse effects in Alankar.     

HIV-1 promotes renal tubular epithelial cell protein synthesis: role of mTOR pathway

Tubular cell HIV-infection has been reported to manifest in the form of cellular hypertrophy and apoptosis. In the present study, we evaluated the role of mammalian target of rapamycin (mTOR) pathway in the HIV induction of tubular cell protein synthesis. Mouse proximal tubular epithelial cells (MPTECs) were transduced with either gag/pol-deleted NL4-3 (HIV/MPTEC) or empty vector (Vector/MPTEC). HIV/MPTEC showed enhanced DNA synthesis when compared with Vector/MPTECs by BRDU labeling studies. HIV/MPTECs also showed enhanced production of β-laminin and fibronection in addition to increased protein content per cell. In in vivo studies, renal cortical sections from HIV transgenic mice and HIVAN patients showed enhanced tubular cell phosphorylation of mTOR. Analysis of mTOR revealed increased expression of phospho (p)-mTOR in HIV/MPTECs when compared to vector/MPTECs. Further downstream analysis of mTOR pathway revealed enhanced phosphorylation of p70S6 kinase and associated diminished phosphorylation of eEF2 (eukaryotic translation elongation factor 2) in HIV/MPTECs; moreover, HIV/MPTECs displayed enhanced phosphorylation of eIF4B (eukaryotic translation initiation factor 4B) and 4EBP-1 (eukaryotic 4E binding protein). To confirm our hypothesis, we evaluated the effect of rapamycin on HIV-induced tubular cell downstream signaling. Rapamycin not only attenuated phosphorylation of p70S6 kinase and associated down stream signaling in HIV/MPTECs but also inhibited HIV-1 induced tubular cell protein synthesis. These findings suggest that mTOR pathway is activated in HIV-induced enhanced tubular cell protein synthesis and contributes to tubular cell hypertrophy.     

Salicylic acid alleviates decreases in photosynthesis under salt stress by enhancing nitrogen and sulfur assimilation and antioxidant metabolism differentially in two mungbean cultivars

Salicylic acid (SA) is known to affect photosynthesis under normal conditions and induces tolerance in plants to biotic and abiotic stresses through influencing physiological processes. In this study, physiological processes were compared in salt-tolerant (Pusa Vishal) and salt-sensitive (T44) cultivars of mungbean and examined how much these processes were induced by SA treatment to alleviate decrease in photosynthesis under salt stress. Cultivar T44 accumulated higher leaf Na⁺ and Cl⁻ content and exhibited greater oxidative stress than Pusa Vishal. Activity of antioxidant enzymes, ascorbate peroxidase (APX) and glutathione reductase (GR) was greater in Pusa Vishal than T44. Contrarily, activity of superoxide dismutase (SOD) was greater in T44. The greater accumulation of leaf nitrogen and sulfur through higher activity of their assimilating enzymes, nitrate reductase (NR) and ATP-sulfurylase (ATPS) increased reduced glutathione (GSH) content more conspicuously in Pusa Vishal than T44. Application of 0.5 mM SA increased nitrogen and sulfur assimilation, GSH content and activity of APX and GR. This resulted in the increase in photosynthesis under non-saline condition and alleviated the decrease in photosynthesis under salt stress. It also helped in restricting Na⁺ and Cl⁻ content in leaf, and maintaining higher efficiency of PSII, photosynthetic N-use efficiency (NUE) and water relations in Pusa Vishal. However, application of 1.0 mM SA resulted in inhibitory effects. The effect of SA was more pronounced in Pusa Vishal than T44. These results indicate that SA application alleviates the salt-induced decrease in photosynthesis mainly through inducing the activity of NR and ATPS, and increasing antioxidant metabolism to a greater extent in Pusa Vishal than T44.     

Crowdsourcing as a Novel Technique for Retinal Fundus Photography Classification: Analysis of Images in the EPIC Norfolk Cohort on Behalf of the UKBiobank Eye and Vision Consortium

Aim

Crowdsourcing is the process of outsourcing numerous tasks to many untrained individuals. Our aim was to assess the performance and repeatability of crowdsourcing for the classification of retinal fundus photography.

Methods

One hundred retinal fundus photograph images with pre-determined disease criteria were selected by experts from a large cohort study. After reading brief instructions and an example classification, we requested that knowledge workers (KWs) from a crowdsourcing platform classified each image as normal or abnormal with grades of severity. Each image was classified 20 times by different KWs. Four study designs were examined to assess the effect of varying incentive and KW experience in classification accuracy. All study designs were conducted twice to examine repeatability. Performance was assessed by comparing the sensitivity, specificity and area under the receiver operating characteristic curve (AUC).

Results

Without restriction on eligible participants, two thousand classifications of 100 images were received in under 24 hours at minimal cost. In trial 1 all study designs had an AUC (95%CI) of 0.701(0.680–0.721) or greater for classification of normal/abnormal. In trial 1, the highest AUC (95%CI) for normal/abnormal classification was 0.757 (0.738–0.776) for KWs with moderate experience. Comparable results were observed in trial 2. In trial 1, between 64–86% of any abnormal image was correctly classified by over half of all KWs. In trial 2, this ranged between 74–97%. Sensitivity was ≥96% for normal versus severely abnormal detections across all trials. Sensitivity for normal versus mildly abnormal varied between 61–79% across trials.

Conclusions

With minimal training, crowdsourcing represents an accurate, rapid and cost-effective method of retinal image analysis which demonstrates good repeatability. Larger studies with more comprehensive participant training are needed to explore the utility of this compelling technique in large scale medical image analysis.     

Exogenously-sourced ethylene increases stomatal conductance, photosynthesis, and growth under optimal and deficient nitrogen fertilization in mustard

In order to ascertain the stomatal and photosynthetic responses of mustard to ethylene under varying N availability, photosynthetic characteristics of mustard grown with optimal (80 mg N kg(-1) soil) or low (40 mg N kg(-1) soil) N were studied after the application of an ethylene-releasing compound, ethephon (2-chloroethyl phosphonic acid) at 40 days after sowing (DAS). The availability of N influenced ethylene evolution and affected stomatal conductance and photosynthesis. The effect of ethylene was smaller under deficient N where plants contained higher glucose (Glc) sensitivity, despite high ethylene evolution even in the absence of ethephon, potentially because the plants were less sensitive to ethylene per se. Ethephon application at each level of N increased ethylene and decreased Glc sensitivity, which increased photosynthesis via its effect on the photosynthetic machinery and effects on stomatal conductance. Plants grown with sufficient-N and treated with 200 μl l(-1) ethephon exhibited optimal ethylene, the greatest stomatal conductance and photosynthesis, and growth. These plants made maximum use of available N and exhibited the highest nitrogen-use efficiency (NUE).