Variability in the Responses of Hepatitis B Virus D-Subgenotypes to Antiviral Therapy: Designing Pan-D-Subgenotypic Reverse Transcriptase Inhibitors.

Nucleos(t)ide analogues entecavir (ETV) and tenofovir disoproxil fumarate (TDF) are recommended as first-line monotherapies for chronic hepatitis B (CHB). Multiple HBV genotypes/subgenotypes have been described, but their impact on treatment response remains largely elusive. We investigated the effectiveness of ETV/TDF on HBV/D-subgenotypes, D1/D2/D3/D5, studied the structural/functional differences in subgenotype-specific reverse transcriptase (RT) domains of viral polymerase, and identified novel molecules with robust inhibitory activity on various D-subgenotypes. Transfection of Huh7 cells with full-length D1/D2/D3/D5 and in vitro TDF/ETV susceptibility assays demonstrated that D1/D2 had greater susceptibility to TDF/ETV while D3/D5 exhibited poorer response. Additionally, HBV load was substantially reduced in TDF-treated CHB patients carrying D1/D2 but minimally reduced in D3/D5-infected patients. Comparison of RT sequences of D-subgenotypes led to identification of unique subgenotype-specific residues, and molecular modeling/docking/simulation studies depicted differential bindings of TDF/ETV to the active site of their respective RTs. Replacement of signature residues in D3/D5 HBV clones with corresponding amino acids seen in D1/D2 improved their susceptibility to TDF/ETV. Using high throughput virtual screening, we identified N(9)-[3-fluoro-2-(phosphonomethoxy)propyl] (FPMP) derivatives of purine bases, including N6-substituted (S)-FPMP derivative of 2,6-diaminopurine (DAP) (OB-123-VK), as potential binders of RT of different D-subgenotypes. We synthesized (S)-FPMPG prodrugs (FK-381-FEE/FK-381-SEE/FK-382) and tested their effectiveness along with OB-123-VK. Both OB-123-VK and FK-381-FEE exerted similar antiviral activities against all D-subgenotypes, although FK-381-FEE was more potent. Our study highlighted the natural variation in therapeutic response of D1/D2/D3/D5 and emphasized the need for HBV subgenotype determination before treatment. Novel molecules described here could benefit future design/discovery of pan-D-subgenotypic inhibitors. IMPORTANCE Current treatment of chronic hepatitis B relies heavily on nucleotide/nucleoside analogs in particular, tenofovir disoproxil fumarate (TDF) and entecavir (ETV) to keep HBV replication under control and prevent end-stage liver diseases. However, it was unclear whether the therapeutic effects of TDF/ETV differ among patients infected with different HBV genotypes and subgenotypes. HBV genotype D is the most widespread of all HBV genotypes and multiple D-subgenotypes have been described. We here report that different subgenotypes of HBV genotype-D exhibit variable response toward TDF and ETV and this could be attributed to naturally occurring amino acid changes in the reverse transcriptase domain of the subgenotype-specific polymerase. Further, we identified novel molecules and also synthesized prodrugs that are equally effective on different D-subgenotypes and could facilitate management of HBV/D-infected patients irrespective of D-subgenotype.

Crp/fnr family protein binds to promoters of atxA and sodmn genes that regulate the expression of exotoxins in Bacillus anthracis.

Bacillus anthracis produces a tripartite exotoxin, which is regulated by AtxA. Sodmn is constitutively expressed during invasion. Crp/Fnr family transcriptional regulators are known to bind promoters of toxin regulators as well as constitutively expressed genes during pathogenesis. B. anthracis fnr gene was cloned, over-expressed in E. coli and recombinant protein was purified. Oligomeric nature of recombinant rFnr protein was studied by diamide treatment and DTT reduction. DNA binding of rFnr protein was studied by EMSA. We observed that rFnr exists in both monomeric and oligomeric forms. It was found that rFnr was able to oligomerize after diamide treatment which was reversible through DTT reduction. Promoter regions of atxA and sodmn show binding to monomeric form of rFnr, however, dimeric form was unable to bind. Fnr might be playing a role in regulation of toxin gene expression via regulation of atxA gene. It can also be involved in regulation of pathogenesis by regulating the sodmn expression. Oligomerization can act as an ON/OFF switch for the Fnr mediated regulation.

Altered chromatin landscape in circulating T follicular helper and regulatory cells following grass pollen subcutaneous and sublingual immunotherapy.

BACKGROUND: Allergen-specific immunotherapy is a disease-modifying treatment that induces long-term T-cell tolerance. OBJECTIVE: We sought to evaluate the role of circulating CXCR5+PD-1+ T follicular helper (cTFH) and T follicular regulatory (TFR) cells following grass pollen subcutaneous immunotherapy (SCIT) and sublingual immunotherapy (SLIT) and the accompanying changes in their chromatin landscape. METHODS: Phenotype and function of cTFH cells were initially evaluated in the grass pollen-allergic (GPA) group (n = 28) and nonatopic healthy controls (NAC, n = 13) by mathematical algorithms developed to manage high-dimensional data and cell culture, respectively. cTFH and TFR cells were further enumerated in NAC (n = 12), GPA (n = 14), SCIT- (n = 10), and SLIT- (n = 8) treated groups. Chromatin accessibility in cTFH and TFR cells was assessed by assay for transposase-accessible chromatin sequencing (ATAC-seq) to investigate epigenetic mechanisms underlying the differences between NAC, GPA, SCIT, and SLIT groups. RESULTS: cTFH cells were shown to be distinct from TH2- and TH2A-cell subsets, capable of secreting IL-4 and IL-21. Both cytokines synergistically promoted B-cell class switching to IgE and plasma cell differentiation. Grass pollen allergen induced cTFH-cell proliferation in the GPA group but not in the NAC group (P < .05). cTFH cells were higher in the GPA group compared with the NAC group and were lower in the SCIT and SLIT groups (P < .01). Time-dependent induction of IL-4, IL-21, and IL-6 was observed in nasal mucosa following intranasal allergen challenge in the GPA group but not in SCIT and SLIT groups. TFR and IL-10+ cTFH cells were induced in SCIT and SLIT groups (all, P < .01). ATAC-seq analyses revealed differentially accessible chromatin regions in all groups. CONCLUSIONS: For the first time, we showed dysregulation of cTFH cells in the GPA group compared to NAC, SCIT, and SLIT groups and induction of TFR and IL-10+ cTFH cells following SCIT and SLIT. Changes in the chromatin landscape were observed following allergen-specific immunotherapy in cTFH and TFR cells.

Outcome of sequential dilatation in achalasia cardia patients: a prospective cohort study.

BACKGROUND: Sequential increment of balloon diameter for endoscopic pneumatic dilatation is a protocol that is used for symptomatic relief in achalasia cardia. However, most of the studies evaluating its effectiveness are retrospective in nature. This study intended to look into the efficacy of the above protocol in a prospective fashion. METHODS: Consecutive patients of achalasia cardia (n = 72) attending gastroenterology department were subjected to graded dilatation with 30, 35, and 40 mm pneumatic balloon and followed up (median 48 weeks; range: 4-96 weeks) with Eckardt score. Efficacy was assessed by proportion of patients achieving and maintaining clinical remission (Eckardt score ≤ 3) without requiring surgery during follow-up. RESULT: Overall 91% of patients (60 out of 66 with follow-up data) remained symptom free without requirement of surgery. Proportion of type 3 achalasia patients was significantly higher in the group requiring surgery compared to those who did not (p = 0.005). Threshold of 12 mm Hg in 4-week post-dilatation integrated relaxation pressure noted to predict future requirement of surgery in type 3 achalasia patients with sensitivity and specificity of 75% and 85%, respectively. Major adverse events requiring in-patient management were 2.9% with perforation noted in 1.9%. CONCLUSION: A sequential increment of balloon diameter for pneumatic dilatation in achalasia is an effective mode of therapy to achieve and maintain clinical remission in achalasia. The incidents of adverse events are low in this approach. Type 3 achalasia patients are more likely to require surgery despite sequential dilatation.

Nailfold capillary changes in newly diagnosed hypertensive patients: An observational analytical study.

INTRODUCTION: Nail Fold capillaroscopy (NFC) is used to evaluate microvascular changes in the horizontally lying capillaries in the proximal nail fold. Arterial hypertension affects the microvascular beds producing structural changes. Our objective was to evaluate qualitative and quantitative NFC changes in newly diagnosed hypertensives as compared to age and sex matched normotensive controls and to determine association, if any, with microvascular changes visualized on fundoscopy. MATERIALS AND METHODS: This observational, analytical study involved 41 newly diagnosed hypertensives (18-60 years) with 41 normotensive age and sex matched normotensive controls. The mean capillary density (MCD) and morphological changes were assessed for all, while fundoscopy was done for study group participants. The collected data was statistically analyzed. RESULTS: The MCD in newly diagnosed hypertensives (5.21 ± 0.90 capillaries/mm) was significantly lower than normotensive controls (6.50 ± 0.65 capillaries/mm) (p < 0.001) in our study. Qualitative morphologic changes were more common in hypertensive patients including meandering capillaries, capillary dilation, avascular areas, bushy capillaries, and microhemorrhages (p value <0.001). Capillary disarray (73.17%) was a unique morphologic change seen significantly more commonly in study group (p < 0.001). Among hypertensives, MCD was lesser in patients with retinopathy (p = 0.125) and with microalbuminuria, while avascular areas and dilated capillaries were significantly more common. CONCLUSION: Our study supports the role of NFC with USB dermatoscope in detecting unique microvascular morphological alterations in hypertensives, which were more frequent as well as distinctive, as compared to healthy controls. A good correlation with fundoscopic features and microalbuminuria suggests that it could be useful in predicting/detecting cardiovascular, or renal complications early, with an advantage of easy accessibility and repeatability.

Reversal of portal gastropathy with partial internal biliary diversion in a case of progressive familial intrahepatic cholestasis.

Progressive intrahepatic cholestasis is a rare, genetic disorder causing bile acid secretion or transport defects. It can result in intrahepatic cholestasis that can progress to end-stage liver disease. Diagnosis is made using a combination of clinical and biochemical approaches. Genetic testing is currently the gold standard for investigation. We report a case of an 18-month-old male child with cholestatic pattern of jaundice from 16 months of life, which was associated with features suggestive of portal gastropathy. Detailed workup led to the diagnosis of progressive intrahepatic cholestasis (type 2). Early diagnosis prevented the need for liver transplant, and the child underwent surgical treatment with partial internal biliary diversion. Portal gastropathy and disease progression dramatically changed with corrective surgery. The patient was symptom-free at 10-week follow-up. Detecting this rare genetic disorder early has very good therapeutic implications from the patient's perspective and their morbidity and mortality profile; if untreated, it has a high propensity to progress to end-stage liver disease. The requirement of surgical interventions and liver transplantation is individualized on a case-to-case basis. An early diagnosis and initiation of treatment can prevent the need for a liver transplant as shown in the present case.

The exosome encapsulated microRNAs as circulating diagnostic marker for hepatocellular carcinoma with low alpha-fetoprotein.

Diagnosis of hepatocellular carcinoma (HCC) remains challenging to clinicians, particularly in a patient with low alpha-fetoprotein. Here, in silico, ex vivo and in vitro data were combined to identify liver-specific exosomal miRNAs as an early diagnostic marker for HCC. Transcriptome profiling for mRNA and small RNA in same HCV-HCC and normal liver tissues followed by cross-validation of 41 deregulated miRNAs (log2 FoldChange > 1.5, Padj < .1) with GEO/TCGA datasets of HCV/HBV related HCC vs normal/adjacent tissue revealed three miRNAs were commonly deregulated (miR-10b/miR-21/miR-182) among all HCC irrespective of viral etiology. Targets of top deregulated miRNAs were identified by TargetScan/miRwalk and validated in mRNA transcriptome data followed by Panther/Gene Ontology enrichment/Cytoscape analysis suggested that targets were mostly from carcinogenesis pathways. Hence, those miRNAs were validated in normal and HCV-HCC tissues by qRT-PCR and subsequently in plasma-derived-exosomes of both HBV/HCV infected non-HCC (chronic hepatitis [CH]/liver cirrhosis [LC]) and HCC samples, and in liver-specific Anti-Asgr2 immuno-enriched exosomes. Exosomes were verified using Nanosight/TEM/immune-blotting with anti-Alix/anti-GRP78/anti-Asgr2. Along with miR-21-5p, miR-10b-5p/miR-221-3p/miR-223-3p was found significantly upregulated in the exosome of HCC patients than CH/non-HCC. The comparable expression pattern was seen in anti-Asgr2 immuno-precipitated exosomes. Interestingly, the AFP level was found below 250 ng/mL in about 94% of HCV-HCC and 62% of HBV-HCC patients. ROC analysis showed that miR-10b-5p + miR-221-3p + miR-223-3p + miR-21-5p could differentiate CH/non-HCC(CH + LC) from HCC with AUROC: 0.86 (97.5% CI: 0.77-0.94)/0.80 (97.5% CI: 0.70-0.89), sensitivity: 74%/58% and specificity: 86%/95% while miR-10b-5p + miR-221-3p + miR-223-3p showed AUROC: 0.84 (97.5% CI: 0.74-0.94)/0.74 (97.5% CI: 0.63-0.84), sensitivity: 86%/86% and specificity:66%/53% for low AFP-HCC vs CH/non-HCC, respectively, having better sensitivity than the combination of four miRNAs. Multivariate analysis further revealed low Albumin and high miR-21-5p as probable independent risk factor for HCC.

Orientational competition in quantum dot growth in Si-Ge heteroepitaxy on pit-patterned Si(001) substrates.

Towards precisely controlled nanostructure growth, patterned substrates are used as templates to direct heteroepitaxial self-assembly. This affects the size, shape and ordering of nanostructures, which are formed as a consequence of the mismatch in strain. In the well-studied case of Si-Ge heteroepitaxy on Si, the lattice mismatch leads to spontaneous formation of quantum dots. On patterned substrates, the competition between the length scale of the pattern and the intrinsic quantum dot size leads to rich behavior, where the localization of dots can be modified with respect to the features of the patterns. We show by continuum modeling that, in cubic elastic materials such as silicon and germanium, there is also a competition between the pattern orientation and the elastically soft directions of the film, which affects the precise location of quantum dots on the surface. When the pattern is between the elastically soft directions, the quantum dots can form purely in the narrow region directly between two neighboring pits, referred to as the saddle region. On the other hand, when the pattern is along the elastically soft directions, the quantum dots prefer to form in the region at the centre of four pits, referred to as the crown region. This resolves a discrepancy between theory and experiments and gives another dimension to control quantum dot formation in strained nanocrystalline systems.

Bacterial community structure analysis of a hot spring soil by next generation sequencing of ribosomal RNA.

In the present study the whole bacterial community structure of Tapovan hot spring soil located in the state of Uttarakhand, India was analysed through next generation sequencing. The hot spring soil is slightly alkaline in nature with abundance of sulphur. The spring soil was rich in various metallic and non-metallic elements required for bacterial survival. The community was found to comprise of 14 bacterial phyla with representation of members belonging to Firmicutes, Proteobacteria, Thermi, Bacteroidetes, Aquificae, Actinobacteria, chloroflexi, TM7, Fusobacteria etc. At the genus level Bacillus, Pseudomonas, Symbiobacterium, Thermus, Geobacillus, Anoxybacillus were found in abundance as compared to other genera like Flavobacterium, Ureibacillus, Clostridium, Meiothermus, Acinetobacter, Desulfotomaculum and Rheinheimera.

Exploiting ensemble learning to improve prediction of phospholipidosis inducing potential.

Phospholipidosis is characterized by the presence of excessive accumulation of phospholipids in different tissue types (lungs, liver, eyes, kidneys etc.) caused by cationic amphiphilic drugs. Electron microscopy analysis has revealed the presence of lamellar inclusion bodies as the hallmark of phospholipidosis. Some phospholipidosis causing compounds can cause tissue specific inflammatory/retrogressive changes. Reliable and accurate in silico methods could facilitate early screening of phospholipidosis inducing compounds which can subsequently speed up the pharmaceutical drug discovery pipelines. In the present work, stacking ensembles are implemented for combining a number of different base learners to develop predictive models (a total of 256 trained machine learning models were tested) for phospholipidosis inducing compounds using a wide range of molecular descriptors (ChemMine, JOELib, Open babel and RDK descriptors) and structural alerts as input features. The best model consisting of stacked ensemble of machine learning algorithms with random forest as the second level learner outperformed other base and ensemble learners. JOELib descriptors along with structural alerts performed better than the other types of descriptor sets. The best ensemble model achieved an overall accuracy of 88.23%, sensitivity of 86.27%, specificity of 90.20%, mcc of 0.765, auc of 0.896 with 88.21 g-means. To assess the robustness and stability of the best ensemble model, it is further evaluated using stratified 10×10 fold cross validation and holdout testing sets (repeated 10 times) achieving 84.83% mean accuracy with 0.708 mean mcc and 88.46% mean accuracy with 0.771 mean mcc respectively. A comparison of different meta classifiers (Generalized linear regression, Gradient boosting machines, Random forest and Deep learning neural networks) in stacking ensemble revealed that random forest is the better choice for combining multiple classification models.

Food allergy and omics.

Food allergy (FA) prevalence has been increasing over the last few decades and is now a global health concern. Current diagnostic methods for FA result in a high number of false-positive results, and the standard of care is either allergen avoidance or use of epinephrine on accidental exposure, although currently with no other approved treatments. The increasing prevalence of FA, lack of robust biomarkers, and inadequate treatments warrants further research into the mechanism underlying food allergies. Recent technological advances have made it possible to move beyond traditional biological techniques to more sophisticated high-throughput approaches. These technologies have created the burgeoning field of omics sciences, which permit a more systematic investigation of biological problems. Omics sciences, such as genomics, epigenomics, transcriptomics, proteomics, metabolomics, microbiomics, and exposomics, have enabled the construction of regulatory networks and biological pathway models. Parallel advances in bioinformatics and computational techniques have enabled the integration, analysis, and interpretation of these exponentially growing data sets and opens the possibility of personalized or precision medicine for FA.

Heat stress-induced alterations in the expression of genes associated with gonadal integrity of the teleost Puntius sophore.

Temperature plays an important role on reproductive physiology of vertebrates including mammals, fish, and birds. It has varying effects on fish reproduction depending on the species; higher temperatures favor the spring-spawning species, while lower temperatures stimulate reproduction in autumn spawners. To evaluate the impact of high temperature on the reproductive physiology of minnow Puntius sophore, we carried out expression analysis of selected genes associated with gamete quality (hsp60, hsp70, hsp90, hsf1, vtg), pleuripotency (sox2, oct4, nanog), and sex determination (dmrt1) in gonads (ovary and testis) of P. sophore, heat stressed for different time periods (36 °C/7 days or 60 days) using real-time quantitative polymerase chain reaction (RT-qPCR). Expression of most of the hsp, vtg, and pleuripotency marker genes sox-2, oct-4, and nanog genes was downregulated in both ovary and testis of heat-stressed fish. The expression of dmrt-1 was upregulated in testis but downregulated in ovary of the heat-stressed fish which could be a male favoring effect of high temperature in P. sophore. This study suggests that the reproductive physiology and health of the nutrient dense P. sophore would be negatively affected by high temperature stress.

Consequences of elastic anisotropy in patterned substrate heteroepitaxy.

The role of elastic anisotropy on quantum dot formation and evolution on a pre-patterned substrate is evaluated within the framework of a continuum model. We first extend the formulation for surface evolution to take elastic anisotropy into account. Using a small slope approximation, we derive the evolution equation and show how it can be numerically implemented up to linear and second order for stripe and egg-carton patterned substrates using an accurate and efficient procedure. The semi-infinite nature of the substrate is used to solve the elasticity problem subject to other boundary conditions at the free surface and at the film-substrate interface. The positioning of the quantum dots with respect to the peaks and valleys of the pattern is explained by a competition between the length scale of the pattern and the wavelength of the Asaro-Tiller-Grinfeld instability, which is also affected by the elastic anisotropy. The alignment of dots is affected by a competition between the elastic anisotropy of the film and the pattern orientation. A domain of pattern inversion, wherein the quantum dots form exclusively in the valleys of the patterns is identified as a function of the average film thickness and the elastic anisotropy, and the time-scale for this inversion as function of height is analyzed.

Perception about use of comics in medical and nursing education among students in health professions' schools in New Delhi.

Background: Graphic narratives can give medical and nursing students a broader concept of health and illness. However, graphic texts are not yet integrated into medical education in India. The present study was undertaken to assess the perception of medical and nursing undergraduate students about the use of comics in health professions' education. Methods: This study was an institution-based cross-sectional study conducted among 130 medical and 108 nursing students of a medical and nursing college, respectively, in Central Delhi. A pretested self-administered questionnaire containing items to assess knowledge and perception about graphic medicine in medical education was used for data collection. Data were analyzed using SPSS 16.0. Results: The mean age of students was 20.82 ± 1.51 years. Out of the 238 students, only 21.8% had heard about graphic medicine. However, 76.9% (n = 183) of students agreed that comics should be used in health professions' education. When asked about the subjects in which comic strips can be useful, the majority of medical students wanted them to be used in teaching community medicine (n = 95; 73.1%), while nursing students more often indicated their use in anatomy (n = 59; 54.6%). Overall large proportion of both nursing and medical students opined that comics aid in better understanding (58.8%) and memorization of concepts and content (57.6%). The majority of respondents felt that comic designing requires expertise (67.6%). Students also stated that comic strips on communicable diseases and reproductive and child health could be developed and used in teaching the subject of community medicine/public health. Discussion: Majority of students felt that comics should be used in medical education in India, preferably for teaching community medicine. Further, maternal and child health are important topics that can be taught using comics in public health. There is a need to explore ways for integrating graphic medicine into health professions' education.

Disinfectant Efficacy of 0.525% Sodium Hypochlorite and Epimax on Alginate Impression Material.

AIM: Species of Streptococcus, Escherichia coli, Staphylococcus, Actinomyces, Pseudomonas, Klebsiella, and Candida are commonly seen in the oral cavity. Impression materials are commonly contaminated with microorganisms. The present study was conducted to assess the disinfection efficacy of Epimax and 0.525% sodium hypochlorite on alginate impression over a period of 10 minutes. MATERIALS AND METHODS: This study was conducted in the Department of Prosthodontics in the year 2015. An alginate impression material was prepared. For each bacteria species, 15 samples were used. Out of 15 samples, 3 were used by 0.525% sodium hypochlorite for disinfection for 5 minutes and 3 others for 10 minutes. Similarly, 3 samples were used by Epimax for 5 minutes and other 3 for 10 minutes. Three samples were used as controls. Each sample was polluted with Candida albicans, Pseudomonas aeruginosa, and Staphylococcus aureus strains. RESULTS: There was no statistical difference in P. aeruginosa and C. albicans after 5 minutes, whereas S. aureus showed significant difference (p < 0.05). Epimax was found to be more effective in removing S. aureus as compared with other disinfectants. Both Epimax and 0.525% sodium hypochlorite did not show significant difference against P. aeruginosa and C. albicans, whereas significant difference was found between both agents against S. aureus (p < 0.05). It was seen that Epimax eliminated S. aureus after 5 minutes and P. aeruginosa after 10 minutes and 99.8% C. albicans after 10 minutes. About 0.525% sodium hypochlorite eliminated 99.1% of C. albicans after 10 minutes, whereas 98.5 and 99% of S. aureus and P. aeruginosa were eliminated after 10 minutes respectively. CONCLUSION: Both Epimax and 0.525% sodium hypochlorite can disinfect the alginate impression material against C. albicans, P. aeruginosa, and S. aureus strains. However, Epimax was found to be more effective against S. aureus as compared with 0.525% sodium hypochlorite. CLINICAL SIGNIFICANCE: Efficacy of disinfection of sodium hypo-chlorite and Epimax on alginate impression.

Suitable reference gene for quantitative real-time PCR analysis of gene expression in gonadal tissues of minnow Puntius sophore under high-temperature stress.

BACKGROUND: High ambient temperature is known to affect fish gonadal development and physiology in a variety of ways depending on the severity and duration of exposure; however, the underlying molecular mechanisms are poorly understood. Gonadal gene expression influence the gonadal development, physiology and the quality of egg/sperm produced in teleosts and the mechanistic understanding of spatio-temporal changes in the gonadal gene expression could be instrumental in controlling the fate of egg/sperm and the quality of seed produced. Real time-quantititative polymerase chain reaction (RT-qCR), is a high throughput, sensitive and reproducible methodology used for understanding gene expression patterns by measuring the relative abundance of mRNA transcripts. However, its accuracy relies upon a suitable reference gene whose expression levels remain stable across various experimental conditions. In the present study, we evaluated the suitability of ten potential reference genes to be used as internal controls in RT-qPCR analysis in gonadal tissues (ovary and testis) of minnow Puntius sophore exposed to high temperature stress for different time periods (7 days, 60 days). Expression analysis of ten different constitutively expressed genes viz. 18S ribosomal RNA (18S rRNA), beta actin (ßactin), ß-2 microglobulin (b2mg), eukaryotic elongation factor-1 (eef1), glyceraldehyde-3phosphate dehydrogenase (gapdh), glucose-6-phosphate dehydrogenase (g6pd), ribosomal binding protein L13 (rpl13), tubulin (tub), tata box binding protein (tbp), ubiquitin (ubi) was carried out by using RT-qPCR and the stability in their expressions were evaluated by using four different algorithms; namely, delta Ct, BestKeeper, geNorm and NormFinder. RESULTS: In ovary, eef1 was found to be the most suitable reference gene in all the algorithms used. In testis, b2mg was found to be the most suitable reference gene in delta Ct, BestKeeper, NormFinder analysis while tbp and eef1 were found to be the most suitable reference genes in geNorm analysis. CONCLUSIONS: In conclusion, eef1 and b2mg were found to be the most suitable reference genes in ovary and testis, respectively, of Puntius sophore exposed to high temperature stress, and could be used as internal controls for gene expression analysis in gonadal tissues of Puntius sophore.

Correlation of macular structure and function in a boy with primary foveomacular retinitis and sequence of changes over 5 years.

PURPOSE: To describe the clinical characteristics, macular structure and function, and to document sequential changes over 5 years in a 10-year-old boy with bilateral primary foveomacular retinitis. METHODS: A 10-year-old boy presented with sudden onset scotoma in both eyes, experienced after getting up from bed on a non-eclipse day. He persistently denied direct sun-gazing. He neither had any significant systemic illness, nor was using any medications. In addition to a detailed examination at presentation that included fundus fluorescein angiogram (FFA), electroretinogram (ERG), pattern ERG and electrooculogram (EOG), he was examined periodically for 5 years with Humphrey visual field (HVF), spectral domain optical coherence tomogram (SDOCT), Amsler grid charting and multifocal ERG. The macular structure and functions were analyzed over the years and correlated with the symptoms. RESULTS: All findings were bilaterally symmetrical at each visit. At presentation, his corrected visual acuity was 20/25 with subfoveal yellow dot similar to solar retinopathy, central scotoma with reduced foveal threshold in HVF 24-2, micropsia in Amsler grid, missing of two plates on Ishihara color vision chart, transfoveal full thickness hyper-reflective band on SD OCT, unremarkable FFA and normal foveal peak in mfERG. The flash ERG and EOG were unremarkable. A month later, his VA improved to 20/20, he had relative scotoma in Amsler grid, no scotoma in HVF (10-2), restoration of the inner segment of the photoreceptors with sharp defect involving ellipsoid and photoreceptor interdigitation zone in SDOCT and blunting of foveal peaks in mfERG. Three months later, his corrected VA was 20/20 with relative scotoma in Amsler grid, normal color vision, no scotoma in HVF 10-2 and unchanged SDOCT findings. In subsequent examinations at 6, 9, 14, 29, 39 and 60 months, he was symptomless with VA 20/20, unremarkable fundus, normal Amsler grid and HVF (normal foveal threshold), unchanged SDOCT findings and the reduced foveal peaks on mfERG in both eyes got normalized only at 60 months. CONCLUSION: Presented here is a case of bilaterally symmetrical idiopathic foveomacular retinitis that had a clinical appearance similar to solar retinopathy. The fundus changes persisted for 4 weeks, the symptoms and changes in Amsler grid lasted for 3 months, and the foveal threshold in visual fields normalized within 3 months. Maximum change in the SDOCT defect occurred within a month, and the extrafoveal defect in the ellipsoid and photoreceptor interdigitation line persisted despite resolution of symptoms and resolution of the visual field defect and normal distance vision. Probably, the foveal lesion detected on SDOCT was too small to cause a reduction in the distance visual acuity or show up in the visual field and mfERG later.

hsp90 and hsp47 appear to play an important role in minnow Puntius sophore for surviving in the hot spring run-off aquatic ecosystem.

Changes in the expression of a number of hsp genes in minnow Puntius sophore collected from a hot spring run-off (Atri hot spring in Odisha, India; 20o09'N 85°18'E, 36-38 °C) were investigated to study the upper thermal acclimation response under heat stress, using same species from aquaculture ponds (water temperature 27 °C) as control. Expression of hsp genes was analyzed in both groups using RT-qPCR, which showed up-regulation of hsp90 (2.1-fold) and hsp47 (2.5-fold) in hot spring run-off fishes, whereas there was no alteration in expression of other hsps. As the fish inhabit the hot spring run-off area for very long duration, they could have adapted to the environment. To test this hypothesis, fishes collected from hot spring run-off were divided into two groups; one was heat-shocked at 41 °C/24 h, and the other was acclimatized at 27 °C/24 h. Up-regulation of all the hsps (except hsp78) was observed in the heat-shocked fishes, whereas expression of all hsps was found to be down-regulated to the basal level in fishes maintained at 27 °C/24 h. Pathway analysis showed that the expressions of all the hsps except hsp90 are regulated by the transcription factor heat shock factor 1 (Hsf1). This study showed that hsp90 and hsp47 play an important role in Puntius sophore for surviving in the high-temperature environment of the hot spring run-off. Additionally, we show that plasticity in hsp gene expression is not lost in the hot spring run-off population.

Proteomic changes in the liver of Channa striatus in response to high temperature stress.

The present study was undertaken to investigate the proteomic changes in the liver of murrel Channa striatus exposed to high temperature stress. Fishes were exposed to 36°C for 4 days and liver proteome changes were analyzed using gel- based proteomics, i.e. 2DE, MALDI-TOF/TOF-MS, and validation by transcript analysis. The study showed, besides others, increased abundance of two sets of proteins, the antioxidative enzymes superoxide dismutase (SOD), ferritin, cellular retinol binding protein (CRBP), glutathione-S-transferase (GST), and the chaperones HSP60 and protein disulfide isomerase; this was validated by transcript analysis. The proteome data are available via ProteomeXchange with identifier PXD002608. Further, gene expression analysis was also carried out in the fishes exposed to thermal stress for longer durations (30 days experimental exposure in laboratory and for 30 days beyond, taking Channa collected from a hot spring runoff at 36-38°C); sod, gst, crbp, and hsp60 were found to continue to remain upregulated at eight-, 2.5-, 2.4-, and 2.45-fold, respectively, in the hot spring runoff fish. Pathway analysis showed that the upregulations of the antioxidant enzymes as well as molecular chaperones are induced by the transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2). Thus, while short-term heat stress tolerance involves the antioxidative enzymes SOD, ferritin, CRBP, GST, and chaperones HSP60 and protein disulfide isomerase, adaptation under chronic heat stress is associated with SOD, CRBP, GST, and HSP60.