LncRNA VEAL2 regulates PRKCB2 to modulate endothelial permeability in diabetic retinopathy.

Long non-coding RNAs (lncRNAs) are emerging as key regulators of endothelial cell function. Here, we investigated the role of a novel vascular endothelial-associated lncRNA (VEAL2) in regulating endothelial permeability. Precise editing of veal2 loci in zebrafish (veal2gib005Δ8/+ ) induced cranial hemorrhage. In vitro and in vivo studies revealed that veal2 competes with diacylglycerol for interaction with protein kinase C beta-b (Prkcbb) and regulates its kinase activity. Using PRKCB2 as bait, we identified functional ortholog of veal2 in humans from HUVECs and named it as VEAL2. Overexpression and knockdown of VEAL2 affected tubulogenesis and permeability in HUVECs. VEAL2 was differentially expressed in choroid tissue in eye and blood from patients with diabetic retinopathy, a disease where PRKCB2 is known to be hyperactivated. Further, VEAL2 could rescue the effects of PRKCB2-mediated turnover of endothelial junctional proteins thus reducing hyperpermeability in hyperglycemic HUVEC model of diabetic retinopathy. Based on evidence from zebrafish and hyperglycemic HUVEC models and diabetic retinopathy patients, we report a hitherto unknown VEAL2 lncRNA-mediated regulation of PRKCB2, for modulating junctional dynamics and maintenance of endothelial permeability.

Novel LINS1 missense mutation in a family with non-syndromic intellectual disability.

Newer sequencing technologies decipher molecular variations and increase the knowledge of pathogenesis of complex diseases like intellectual disability (ID), affecting 2-3% of the population. We report a novel family with a missense mutation in LINS1 as a cause for non-syndromic ID. Clinical exome sequencing for ID related genes carried out for a male with dysmorphism, mutism, and cognitive delay was uninformative. Subsequently, "pathogenic" and "likely pathogenic" variants associated with other inherited disorders were searched for as secondary findings. Further, PCR-RFLP carried out in other family members confirmed the result. A novel missense variant (c.937G>A) in exon 5 of LINS1 was detected in the proband. His affected elder brother was homozygous and the parents were heterozygous respectively, for the mutation. No mutation was observed in his unaffected sister. Mutations in LINS1 were suspected in this non-syndromic ID case with mutism. LINS1 alterations affect ELAV1 expression and result in reduction in the commissural axonal growth, thus affecting peripheral and central neuronal function. LINS1 acts in association with ß-catenin to influence WNT1 signaling. It is hypothesized that mutations in LINS1 may alter HuR expression during neural differentiation, leading to ID in humans. © 2017 Wiley Periodicals, Inc.

A Cross-Sectional Study on Neuropathic Pain Associated With Quality of Sleep in Spondylosis Patients.

INTRODUCTION: Neuropathic pain (NP) is common in spondylosis patients. Cervical and lumbar spondylosis are more common in the elderly population. Spondylosis patients also suffer from poor quality of sleep (QOS). This study aims to find a correlation between NP and QOS in spondylosis patients. METHODS: We conducted a cross-sectional study and analyzed data using the chi-square test to correlate the NP with QOS. The Pittsburgh Sleep Quality Index (PSQI) and the Leeds Assessment of Neuropathic Symptoms and Signs, Self-complete (S-LANSS) questionnaires were used to assess QOS and evaluate neuropathic pain, respectively. Spondylosis was diagnosed based on the history, clinical examination, and radiological findings. RESULTS: A total of 72 spondylosis patients, with a mean age of 47.35 years, were included in this study. Out of 72 subjects, 52 (72.2%) patients had neuropathic pain (NP group), and 20 (27.8%) patients had non-neuropathic pain (non-NP group). In the NP group, 41 patients (78.8%) had poor QOS, while 11 (21.2%) had good QOS. In the non-NP group, eight (40%) had poor QOS, and 12 (60%) had good QOS. CONCLUSION: This study concludes that neuropathic pain is associated with poor quality of sleep in spondylosis patients.

Knowledge and perception regarding adverse drug reactions among undergraduate medical students of Bihar, Eastern India.

Background: Adverse drug reactions (ADRs) have national and international monitoring and are part of teaching-learning of undergraduate medical course and curriculum. Objectives: To find the knowledge and perception of ADRs among undergraduate medical students in a tertiary care teaching institute in eastern India. Materials and Methods: This was an observational cross-sectional study conducted among the MBBS medical students by administration of pre-designed, pre-tested, semi-structured questionnaires. The data on their knowledge and candid reflections on ADRs were analyzed question by question using software and compared with peers. Results: The responses from the participants on knowledge and perception of ADRs varied widely. Final-year students had the most precise response on classification, filing an ADR report, national reporting centers, and the first step in monitoring ADRs; the majority accepted their first- hand experience and legal and professional responsibilities on ADRs. Third-year students responded well on objectives, methods, and scope of patients on direct reporting or drug overdose and monitoring; respect patient confidentiality while reporting; and expect feedback from monitoring centers, with special training on ADR. Second-year students responded well on definitions, pharmacovigilance programs in India, alertness of banned drugs because of ADR, and related capacity building. Conclusions: The awareness and insight on ADRs of the undergraduate medical students were quite reasonable. However, further reinforcement is needed in future to be updated to relevant issues to their practice as primary care physicians.

Mitochondrial calcium signaling mediated transcriptional regulation of keratin filaments is a critical determinant of melanogenesis.

Mitochondria are versatile organelles that regulate several physiological functions. Many mitochondria-controlled processes are driven by mitochondrial Ca2+ signaling. However, role of mitochondrial Ca2+ signaling in melanosome biology remains unknown. Here, we show that pigmentation requires mitochondrial Ca2+ uptake. In vitro gain and loss of function studies demonstrated that Mitochondrial Ca2+ Uniporter (MCU) is crucial for melanogenesis while the MCU rheostats, MCUb and MICU1 negatively control melanogenesis. Zebrafish and mouse models showed that MCU plays a vital role in pigmentation in vivo. Mechanistically, MCU controls activation of transcription factor NFAT2 to induce expression of three keratins (keratin 5, 7 and 8), which we report as positive regulators of melanogenesis. Interestingly, keratin 5 in turn modulates mitochondrial Ca2+ uptake thereby this signaling module acts as a negative feedback loop that fine-tunes both mitochondrial Ca2+ signaling and melanogenesis. Mitoxantrone, an FDA approved drug that inhibits MCU, decreases physiological melanogenesis. Collectively, our data demonstrates a critical role for mitochondrial Ca2+ signaling in vertebrate pigmentation and reveal the therapeutic potential of targeting MCU for clinical management of pigmentary disorders. Given the centrality of mitochondrial Ca2+ signaling and keratin filaments in cellular physiology, this feedback loop may be functional in a variety of other pathophysiological conditions.

Functional long non-coding and circular RNAs in zebrafish.

The utility of model organisms to understand the function of a novel transcript/genes has allowed us to delineate their molecular mechanisms in maintaining cellular homeostasis. Organisms such as zebrafish have contributed a lot in the field of developmental and disease biology. Attributable to advancement and deep transcriptomics, many new transcript isoforms and non-coding RNAs such as long noncoding RNA (lncRNA) and circular RNAs (circRNAs) have been identified and cataloged in multiple databases and many more are yet to be identified. Various methods and tools have been utilized to identify lncRNAs/circRNAs in zebrafish using deep sequencing of transcriptomes as templates. Functional analysis of a few candidates such as tie1-AS, ECAL1 and CDR1as in zebrafish provides a prospective outline to approach other known or novel lncRNA/circRNA. New genetic alteration tools like TALENS and CRISPRs have helped in probing for the molecular function of lncRNA/circRNA in zebrafish. Further latest improvements in experimental and computational techniques offer the identification of lncRNA/circRNA counterparts in humans and zebrafish thereby allowing easy modeling and analysis of function at cellular level.

Rapid and accurate nucleobase detection using FnCas9 and its application in COVID-19 diagnosis.

Rapid detection of DNA/RNA pathogenic sequences or variants through point-of-care diagnostics is valuable for accelerated clinical prognosis, as witnessed during the recent COVID-19 outbreak. Traditional methods relying on qPCR or sequencing are tough to implement with limited resources, necessitating the development of accurate and robust alternative strategies. Here, we report FnCas9 Editor Linked Uniform Detection Assay (FELUDA) that utilizes a direct Cas9 based enzymatic readout for detecting nucleobase and nucleotide sequences without trans-cleavage of reporter molecules. We also demonstrate that FELUDA is 100% accurate in detecting single nucleotide variants (SNVs), including heterozygous carriers, and present a simple web-tool JATAYU to aid end-users. FELUDA is semi-quantitative, can adapt to multiple signal detection platforms, and deploy for versatile applications such as molecular diagnosis during infectious disease outbreaks like COVID-19. Employing a lateral flow readout, FELUDA shows 100% sensitivity and 97% specificity across all ranges of viral loads in clinical samples within 1hr. In combination with RT-RPA and a smartphone application True Outcome Predicted via Strip Evaluation (TOPSE), we present a prototype for FELUDA for CoV-2 detection closer to home.

Initial Insights Into the Genetic Epidemiology of SARS-CoV-2 Isolates From Kerala Suggest Local Spread From Limited Introductions.

Coronavirus disease 2019 (COVID-19) rapidly spread from a city in China to almost every country in the world, affecting millions of individuals. The rapid increase in the COVID-19 cases in the state of Kerala in India has necessitated the understanding of SARS-CoV-2 genetic epidemiology. We sequenced 200 samples from patients in Kerala using COVIDSeq protocol amplicon-based sequencing. The analysis identified 166 high-quality single-nucleotide variants encompassing four novel variants and 89 new variants in the Indian isolated SARS-CoV-2. Phylogenetic and haplotype analysis revealed that the virus was dominated by three distinct introductions followed by local spread suggesting recent outbreaks and that it belongs to the A2a clade. Further analysis of the functional variants revealed that two variants in the S gene associated with increased infectivity and five variants mapped in primer binding sites affect the efficacy of RT-PCR. To the best of our knowledge, this is the first and most comprehensive report of SARS-CoV-2 genetic epidemiology from Kerala.

High throughput detection and genetic epidemiology of SARS-CoV-2 using COVIDSeq next-generation sequencing.

The rapid emergence of coronavirus disease 2019 (COVID-19) as a global pandemic affecting millions of individuals globally has necessitated sensitive and high-throughput approaches for the diagnosis, surveillance, and determining the genetic epidemiology of SARS-CoV-2. In the present study, we used the COVIDSeq protocol, which involves multiplex-PCR, barcoding, and sequencing of samples for high-throughput detection and deciphering the genetic epidemiology of SARS-CoV-2. We used the approach on 752 clinical samples in duplicates, amounting to a total of 1536 samples which could be sequenced on a single S4 sequencing flow cell on NovaSeq 6000. Our analysis suggests a high concordance between technical duplicates and a high concordance of detection of SARS-CoV-2 between the COVIDSeq as well as RT-PCR approaches. An in-depth analysis revealed a total of six samples in which COVIDSeq detected SARS-CoV-2 in high confidence which were negative in RT-PCR. Additionally, the assay could detect SARS-CoV-2 in 21 samples and 16 samples which were classified inconclusive and pan-sarbeco positive respectively suggesting that COVIDSeq could be used as a confirmatory test. The sequencing approach also enabled insights into the evolution and genetic epidemiology of the SARS-CoV-2 samples. The samples were classified into a total of 3 clades. This study reports two lineages B.1.112 and B.1.99 for the first time in India. This study also revealed 1,143 unique single nucleotide variants and added a total of 73 novel variants identified for the first time. To the best of our knowledge, this is the first report of the COVIDSeq approach for detection and genetic epidemiology of SARS-CoV-2. Our analysis suggests that COVIDSeq could be a potential high sensitivity assay for the detection of SARS-CoV-2, with an additional advantage of enabling the genetic epidemiology of SARS-CoV-2.

A rapid and sensitive method to detect SARS-CoV-2 virus using targeted-mass spectrometry.

In the last few months, there has been a global catastrophic outbreak of severe acute respiratory syndrome disease caused by the novel coronavirus SARS-CoV-2 affecting millions of people worldwide. Early diagnosis and isolation are key to contain the rapid spread of the virus. Towards this goal, we report a simple, sensitive and rapid method to detect the virus using a targeted mass spectrometric approach, which can directly detect the presence of virus from naso-oropharyngeal swabs. Using a multiple reaction monitoring we can detect the presence of two peptides specific to SARS-CoV-2 in a 2.3 min gradient run with 100% specificity and 90.5% sensitivity when compared to RT-PCR. Importantly, we further show that these peptides could be detected even in the patients who have recovered from the symptoms and have tested negative for the virus by RT-PCR highlighting the sensitivity of the technique. This method has the translational potential of in terms of the rapid diagnostics of symptomatic and asymptomatic COVID-19 and can augment current methods available for diagnosis of SARS-CoV-2.

Knockdown of calcium-binding calb2a and calb2b genes indicates the key regulator of the early development of the zebrafish, Danio rerio.

The present study initiates our investigation regarding the role of calb2a and calb2b genes that are expressed in the central nervous system, including the multiple tissues during early embryonic development of zebrafish. In this study, we have adopted individual and combined morpholino-mediated inactivation approach to investigate the functions of calb2a and calb2b in early development of the zebrafish. We have found that calb2a and calb2b morpholino alone failed to generate an obvious phenotype; however, morphological inspection in early developmental stages of calb2a and calb2b combined knockdown morphants show abnormal neural plate folding in midbrain-hindbrain region. In addition to this, combinatorial loss of these mRNA leads to severe hydrocephalus, axial curvature defect, and yolk sac edema in later developmental stages. Also, the combined knockdown of calb2a and calb2b are found to be associated with an impaired touchdown and swimming performance in the zebrafish. Co-injection of the calb2a and calb2b morpholino oligonucleotide cocktail with human CALB2 mRNA leads to the rescue of the strong phenotype. This study provided the first comprehensive analyses of the zebrafish Calb2a and Calb2b proteins; we have found that Calb2a and Calb2b are highly conserved across vertebrate species and originated from the same ancestral gene long back in the evolution. Homology modeling and docking with the similar structure and Ca2+ binding sites for both proteins provide the evidence that both the proteins may have similar function and one can compensate for the loss of other. Collectively, these findings confirm the unique and essential functions of calb2a and calb2b genes in the early development of the zebrafish.

Alcohol-related acute-on-chronic liver failure-Comparison of various prognostic scores in predicting outcome.

BACKGROUND AND AIMS: Various prognostic scores are available for predicting outcome in acute-on-chronic liver failure (ACLF). We compared the available prognostic models as predictors of outcome in alcohol-related ACLF patients. METHODS: All consecutive patients with alcohol-related ACLF were included. At admission, prognostic indices-acute physiology and chronic health evaluation score (APACHE II), model for end-stage liver disease (MELD), MELD-Na, Maddrey's discriminant function (DF), age-bilirubin-INR-creatinine (ABIC), and Chronic Liver Failure Consortium (CLIF-C) ACLF score (CLIF-C ACLF) score were calculated. Receiver operator characteristic (ROC) curves were plotted for all prognostic scores with in-hospital, 90-day, and 1-year mortality as outcome. RESULTS: Of the 171 patients, 170 were males, and grade 1 ACLF in 20 (11.7%), grade 2 in 52 (30.4%), and grade 3 in 99 (57.9%) patients. One hundred and nineteen (69.6%) died in-hospital. The median (IQR) Maddrey's score, MELD, MELD-Na, ABIC, APACHE II, and CLIF-C ACLF were 87.8 (66.5-123.0), 33.1 (27.6-40.0), 34.4 (29.5-40.0), 8.5 (7.3-9.6), 15 (12-21), and 51.1 (44.1-56.4), respectively. On multivariate Cox regression analysis, independent predictors of in-hospital outcome were presence of hepatic encephalopathy (early HR, 2.078; 95%CI, 1.173-3.682, p = 0.012 and advanced, HR, 2.330; 95% CI, 1.270-4.276, p = 0.006), elevated serum creatinine (HR, 1.140; 95% CI, 1.023-1.270, p = 0.018), and infection at admission (HR, 1.874; 95% CI, 1.160-23.029, p = 0.010). On comparison of ROC curves, APACHE II and CLIF-C ACLF AUROC were significantly higher than MELD, MELD-Na, DF, and ABIC (p < 0.05) for predicting in-hospital, 90-day, and 1-year mortality. The AUROC was highest for APACHE II followed by CLIF-C ACLF (Hanley and McNeil, p = 0.660). CONCLUSIONS: Alcohol-related ACLF has high in-hospital mortality. Among the available prognostic scores, CLIF-C ACLF and APACHE II perform best.

Prevalence, predictors and impact of bacterial infection in acute on chronic liver failure patients.

BACKGROUND: Acute on chronic liver failure (ACLF) is associated with high short term mortality. We aimed to evaluate the prevalence, predictors and impact of bacterial infection in ACLF. METHODS: Consecutive hospitalized patients with cirrhosis and acute decompensation (AD), from January 2011-March 2017, were included. Predictors of survival and infection were assessed. RESULTS: 572 patients with cirrhosis and AD were classified into 3 groups - no infection (group 1, n = 190, 33.2%), infection at admission/within 48 h (group 2, n = 298, 52.1%) and infection after 48 h (group 3, n = 84, 14.7%). Higher frequency of organ failures - kidney, brain, circulation and respiratory failure - were seen in groups 2 and 3 as compared with group 1 (P < 0.001 for all). Most common site of infection was lungs, followed by spontaneous bacterial peritonitis and urinary tract infection. The frequency of infection increased with higher ACLF grades. Among ACLF patients, on Cox-proportional multivariate analysis, presence of infection was associated with significantly higher mortality [group 2 (HR 2.93; 95%CI, 1.97-4.38, P < 0.001) and group 3 (HR 1.84; 95%CI, 1.16-2.91, P = 0.009)], as compared with group 1. On multivariate logistic regression analysis, advanced hepatic encephalopathy and elevated total leucocyte count were independently associated with development of infection. CONCLUSIONS: Infections are common in ACLF, and associated with poor outcome.

Long-term outcomes of transjugular intrahepatic portosystemic shunt in Indian patients with Budd-Chiari syndrome.

BACKGROUND/AIM: Transjugular intrahepatic portosystemic shunt (TIPS) is the treatment of choice in Budd-Chiari syndrome (BCS) based on current data. Our objective was to evaluate outcomes and assess prognostic factors in BCS patients undergoing TIPS. PATIENTS AND METHODS: In this retrospective analysis of a propectively maintained database, all consecutive BCS patients undergoing TIPS from September 2010 to February 2017 were included. Complete response after TIPS was defined as resolution of symptoms (ascites/pedal edema) with no requirement of diuretics at the end of 4 weeks. The Cox proportional hazard regression model was used to assess predictors of outcome and complications. RESULTS: Eighty patients with BCS who underwent TIPS were included; 40 (50%) were male. The mean age at onset of symptoms was 24.2±8.7 years. The median (range) follow-up was 660 (2-2400) days. The 1-, 3-, and 5-year rates for TIPS stent patency were 89, 81, and 81%, respectively. Cumulative encephalopathy-free rates were 91, 86, and 86%, respectively, and survival rates were 93, 89, and 84%, respectively. Eight (10.0%) patients died during follow-up, five within the first year (three of these five had incomplete response). On univariate analysis, serum bilirubin, response to intervention, serum creatinine, Child class, model for end-stage liver disease, and All India Institute of Medical Sciences-hepatic venous outflow tract obstruction score were significantly different between survivors and nonsurvivors. On multivariate analysis, response to therapy after TIPS (hazard ratio: 8.37; 95% confidence interval: 1.60-43.82) was independently associated with mortality. The 1-year survival was 97% in patients with complete response, compared with 59% in those with incomplete response (P<0.004). CONCLUSION: Incomplete symptom response after TIPS is associated with poor outcome and can be used for selection of patients for liver transplantation.

Prospective analysis of indications and early complications of emergency temporary loop ileostomies for perforation peritonitis.

BACKGROUND: To determine the indications, nature, and rate of early complications of temporary loop ileostomy created in emergency for benign diseases, their management, and to find out the associated risk factors. METHODS: A total of 630 patients undergoing temporary loop ileostomy for benign diseases were studied prospectively over a period of 6 years. Stoma-related early complications occurring within 6-8 weeks were analyzed. Only emergency cases were included in this study. Descriptive statistics were used to summarize the data and statistical significance was evaluated by applying the Pearson's chi-square test. RESULTS: Typhoid perforation (n=402) was the most commonpathology, followed by tuberculosis (n=106); trauma (n=81); and intestinal obstruction with gangrenous bowel (n=41). 299 patients had no stoma-related complications. Skin excoriation was the most commonstoma-related complication. Age more than 50 years; shock at presentation; delay in presentation; delay in surgery; presence of comorbidities; and surgery done out of working hours, were associated with increased complications. CONCLUSION: Temporary loop ileostomy for perforation peritonitis due to benign systemic diseases like typhoid fever and tuberculosis confers a very high morbidity.

Acinar cell carcinoma: a rare pancreatic malignancy.

Acinar cell carcinoma of the pancreas is a rare pancreatic malignancy, constituting only 1-2% of all the pancreatic tumors. A young adult male presented with chronic diarrhea and upper abdominal pain, on investigations was found to have a large pancreatic tumor of size 12×7.5 cm involving the body of the pancreas. Pancreatic body and tail resection with splenectomy was done and final histopathological examination showed acinar cell carcinoma of the pancreas. Prognosis of acinar cell carcinoma is better than adenocarcinoma of the pancreas.

Validation of a scoring system to predict difficult laparoscopic cholecystectomy.

INTRODUCTION: Laparoscopic cholecystectomy (LC) is one of the most common laparoscopic procedures being performed by general surgeons all over the world. Preoperative prediction of the risk of conversion or difficulty of operation is an important aspect of planning laparoscopic surgery. The purpose of our prospective study was to analyze various risk factors and to predict difficulty and degree of difficulty preoperatively by the use of a scoring system. MATERIALS: This prospective study was conducted in the department of surgery, Lady Hardinge Medical College and associated Dr Ram Manohar Lohia Hospital, Delhi, India. The parameters considered in the preoperative scoring method were old age, male sex, history of hospitalization, obesity, previous abdominal surgery scar, palpable gall bladder, wall thickness of gall bladder, pericholecystic collection and impacted stone. A total of 210 patients were included in the study. RESULTS: We found that history of hospitalization, palpable gall bladder, impacted stone and gall bladder wall thickness were statistically significant factors for prediction of difficult laparoscopic cholecystectomy. Sensitivity and specificity of this preoperative scoring method were found to be 95.74% and 73.68% respectively. Positive predictive values of this scoring method were 90% and 88% for easy and difficult cases respectively. Area under ROC curve was 0.86. Conversion rate from laparoscopic to open cholecystectomy was found to be 4.28%. CONCLUSION: With the help of accurate prediction, high risk patient may be informed before hand regarding the probability of conversion and hence they may have a chance to make arrangements accordingly. On the other hand, surgeons also may have to schedule the time and team for the operation appropriately. Surgeons can also be aware about the possible complications that may arise in high risk patients.