A functional SNP regulates E-cadherin expression by dynamically remodeling the 3D structure of a promoter-associated non-coding RNA transcript.

Transcription of E-cadherin, a tumor suppressor that plays critical roles in cell adhesion and the epithelial-mesenchymal transition, is regulated by a promoter-associated non-coding RNA (paRNA). The sense-oriented paRNA (S-paRNA) includes a functional C/A single nucleotide polymorphism (SNP rs16260). The A-allele leads to decreased transcriptional activity and increased prostate cancer risk. The polymorphic site is known to affect binding of a microRNA-guided Argonaute 1 (AGO1) complex and recruitment of chromatin-modifying enzymes to silence the promoter. Yet the SNP is distant from the microRNA-AGO1 binding domain in both primary sequence and secondary structure, raising the question of how regulation occurs. Here we report the 3D NMR structure of the 104-nucleotide domain of the S-paRNA that encompasses the SNP and the microRNA-binding site. We show that the A to C change alters the locally dynamic and metastable structure of the S-paRNA, revealing how the single nucleotide mutation regulates the E-cadherin promoter through its effect on the non-coding RNA structure.

A seed-like proteome in oil-rich tubers.

There are numerous examples of plant organs or developmental stages that are desiccation-tolerant and can withstand extended periods of severe water loss. One prime example are seeds and pollen of many spermatophytes. However, in some plants, also vegetative organs can be desiccation-tolerant. One example are the tubers of yellow nutsedge (Cyperus esculentus), which also store large amounts of lipids similar to seeds. Interestingly, the closest known relative, purple nutsedge (Cyperus rotundus), generates tubers that do not accumulate oil and are not desiccation-tolerant. We generated nanoLC-MS/MS-based proteomes of yellow nutsedge in five replicates of four stages of tuber development and compared them to the proteomes of roots and leaves, yielding 2257 distinct protein groups. Our data reveal a striking upregulation of hallmark proteins of seeds in the tubers. A deeper comparison to the tuber proteome of the close relative purple nutsedge (C. rotundus) and a previously published proteome of Arabidopsis seeds and seedlings indicates that indeed a seed-like proteome was found in yellow but not purple nutsedge. This was further supported by an analysis of the proteome of a lipid droplet-enriched fraction of yellow nutsedge, which also displayed seed-like characteristics. One reason for the differences between the two nutsedge species might be the expression of certain transcription factors homologous to ABSCISIC ACID INSENSITIVE3, WRINKLED1, and LEAFY COTYLEDON1 that drive gene expression in Arabidopsis seed embryos.

A Study of Association of Plasma Procalcitonin with Various Components of Metabolic Syndrome and Insulin Resistance.

The metabolic syndrome is a cluster of risk factors that are associated with increased risk of cardiovascular diseases and Type 2 diabetes mellitus. The aim was to study the association of plasma procalcitonin with various components of metabolic syndrome (abdominal obsesity, dyslipidemia, hypertension and hyperglycemia) and insulin resistance and compare with healthy controls. In addition, we tried to gauge the association of plasma procalcitonin with complications of metabolic syndrome at presentation. MATERIAL: This was a hospital based observational comparative study on 30 cases of metabolic syndrome in hospital wards and OPD of SMS Medical College, Jaipur and 30 accompanying healthy controls. INCLUSION CRITERIA: Age more than 18 years, and newly diagnosed or old cases of metabolic syndrome using the definition given by International Diabetes Federation (2006). EXCLUSION CRITERIA: Individuals with active infection, trauma, surgery, neoplasms, cirrhosis, pancreatitis and autoimmune diseases or taking medications which can alter the biochemical profile. Detailed history was taken, subjects clinically examined and anthropometric measurements were taken. Required investigations were obtained and statistical analysis done. OBSERVATION: Plasma procalcitonin was significantly higher in cases (mean 0.11 ng/ml) compared to controls (mean 0.002 ng/ml). Waist and neck circumference (102.87±5.19 and 42.03±3.08) values were higher in case group compared to control group (79.67 ±7.98 and 37.10±1.35). Plasma procalcitonin significantly (p<0.05) correlated with level of insulin resistance (HOMA IR), waist circumference, neck circumference, S. Triglycerides, S. VLDL, fasting blood glucose and fasting insulin level in the case group when analyzed by linear regression analysis. We also found that out of 30 subjects in cases, 16.7% had history of MI, whereas in control group no subject was reported with MI. In case of history of CVA, 16.7% had CVA in case group, whereas in control group, no patient was reported with CVA., Conclusion: Our findings based on community-based data showed that higher plasma procalcitonin levels in the normal range are associated with increased measures of obesity, components of the metabolic syndrome, and greater risk of having metabolic syndrome and insulin resistance. Because associations only partly depend on BMI, plasma procalcitonin may serve as a new marker for adipocyte dysfunction, chronic low-grade inflammation, or both.

A Study of Adrenal Insufficiency in Hemodynamically Stable Patients with Cirrhosis.

Adrenal insufficiency (AI) is well entrenched in medical constraints like septic shock, critically ill and multi-morbid hemodynamically unstable patients but its exact prevalence or differences in the cases of chronic liver disease (CLD) at variable grades of severity has recently gained momentum. The eventuality of AI propounding in stable compensated and decompensated cirrhosis without sepsis or in early and late stages of liver desecration are the existing lacunae in popular literature that this study aims to address. MATERIAL: A prospective, analytical study was conducted from March 2021 to December 2021 encompassing 100 hemodynamically stable patients with cirrhosis without infection, admitted at SMS Medical College, Jaipur, who were assessed clinically, biochemically and for adrenal functions. Adrenal insufficiency was defined on multivariable approach including basal 8am cortisol levels, followed by giving 250mcg synthetic adrenocorticotrophic hormone IM injection and retaking serum cortisol levels post-hourly interval to delineate peak and delta cortisol variables. All samples were processed by chemoluminiscence based method on fully automatic immunoassay analyser. OBSERVATION: The study comprised 81 males and 19 females with the mean age being 45.4±12.92 years, with CLD etiology concentrating substantially around alcohol consumption (71%). Viral comorbidities viz. HBV, HCV, both viral and alcohol related and miscellaneous causes were documented in 23, 10, 14 and 12 patients respectively. AI surfaced in 38% patients with CLD being statistically significant with p< 0.001. Inclusively, 10.5% patients with Child-Turcotte-Pugh (CTP) class A, 57.89% with CTP class B and 31.57% cases with CTP class C developed adrenal insufficiency. No statistical differences were found in age, sex; mean arterial pressure, heart rate, HDL, cirrhosis etiology, degree of alcohol consumption and manifestations of portal hypertension between patients with or without AI. For prudence, serum albumin levels were lower (p<0.5) with INR raised (p<0.33) in patients with AI than their counterparts. However, multivariate analysis revealed no direct independent adrenal insufficiency predictor. ROC curve showed that the CTP score may be a good predictor for AI in liver cirrhosis patients as supplemented by significant negative correlations found between CTP score and peak cortisol levels (p=0.001). CONCLUSION: Adrenal insufficiency found frequent even in stable cirrhotic patients form an integral division of the CLD spectra and worsening glucocorticoid levels should be periodically assessed in such patients for preventing parallel comorbidities.

A Comparative Study between Mean Platelet Volume and Hba1c Levels in Patients of Type 2 Diabetes Mellitus.

Diabetes mellitus is a major global health problem, increasingly affecting the population across the world. Diabetic patients have an increased risk of developing micro and macro vascular diseases, and platelets may be involved as a causative agent with respect to altered platelet morphology and function. There are studies evaluating the association between Mean Platelet Volume (MPV) and HbA1c and its role in predicting glycaemic control with conflicting results. Thus the present study was conducted to assess the relationship between HbA1c levels and platelet activity (MPV), determine the association among MPV, glycemic control, and diabetic vascular complications and to evaluate the influence of improved glycemic control on MPV in type 2 diabetic patients. MATERIAL: This was a hospital based observational comparative study on 100 cases of diabetes mellitus divided in 2 groups i.e Group A (HbA1c <7) and Group B (HbA1c >7) and 50 healthy controls in Group C in hospital wards and OPD of SMS Medical College, Jaipur. INCLUSION CRITERIA: Age more than 18 years, and newly diagnosed or old cases of diabetes mellitus using the definition given by American Diabetes Association. EXCLUSION CRITERIA: Abnormal platelet count (<100 and >450×103/µL), Acute febrile illness, Use of drugs affecting platelet function, Male patients with Hb<12.5mg/dl and females with Hb<11.5 mg/dl and Pregnant females. OBSERVATION: It was observed that mean MPV(fl) was maximum in Group B (13.35±1.27), followed by Group A (10.77±.77) and Group C (9.09±.85) and a significant (p-value<0.05) relation was found statistically. We also observed that mean HbA1c (%) was maximum in Group B (8.82±1.41), followed by Group A (6.66±.004) and Group C (5.67±.45) and a significant (p-value<0.05) relation was found statistically in these 3 groups. In group B, at baseline MPV(fl) levels were more (13.35±1.26) than at follow up after glycemic control of 3mths (12.13±1.20) and this was found to be statistically significant. CONCLUSION: We found that Mean platelet volume in diabetic mellitus type 2 patients was significantly higher than non-diabetic group. We also found that the mean platelet volume in uncontrolled diabetic group (HbA1c more than 7 percent) was significantly higher than controlled diabetic group (HbA1c less than 7 percent). Our study showed that in diabetes mellitus, platelets become more reactive and aggregable and their mean volume (MPV) is increased. We also found that increase in HbA1c concentration was directly proportional to increased MPV.

A Phase 3, Randomized, Open-label, Noninferiority Trial Evaluating Anti-Rabies Monoclonal Antibody Cocktail (TwinrabTM) Against Human Rabies Immunoglobulin (HRIG).

BACKGROUND: Limited supply, cost and potential for severe adverse effects observed with the blood derived rabies immunoglobulin products has led to search for alternative therapies. This issue has been addressed by developing an anti-rabies monoclonal antibody cocktail. METHODS: This is a phase 3, randomized, open-label, noninferiority trial conducted in patients with World Health Organization (WHO) category III exposure with suspected rabid animal. Eligible patients were assigned to either the test arm, TwinrabTM (docaravimab and miromavimab) or the reference arm, human rabies immunoglobulin (HRIG; Imogam® Rabies-HT), in a ratio of 1:1. The primary endpoint was the comparison of responder rates between the 2 arms assessed as percentage of those with rabies virus neutralizing antibodies titers ≥0.5 IU/mL on day 14. RESULTS: A total of 308 patients were equally randomized into the 2 arms. In the per-protocol (PP) population, there were 90.21% responders in the TwinrabTM arm and 94.37% in the HRIG arm. The geometric mean of rapid fluorescent foci inhibition test titers in the PP on day 14 were 4.38 and 4.85 IU/mL, for the TwinrabTM and HRIG arms, respectively. There were no deaths or serious adverse events reported. CONCLUSIONS: This study confirmed that TwinrabTM is noninferior to HRIG in terms of providing an unbroken window of protection up to day 84. This trial in healthy adults with WHO category III exposure from suspected rabid animal also establishes the safety of TwinrabTM in patients with 1 WHO approved vaccine regimen (Essen). CLINICAL TRIALS REGISTRATION: CTRI/2017/07/009038.

Anthocyanin synthesis in orange carrot cv. Danvers is activated by transgene expression of the transcription factors DcMYB113_NB and DcEGL1_NB from black carrot cv. Nightbird.

Black carrots are potent sources of anthocyanin for the natural food color industry as their anthocyanins contain very high percentages of acylated anthocyanins which are much more stable than non-acylated anthocyanins. Anthocyanins are synthesized by a specific branch of the phenylpropanoid pathway activated by a triad of R2R3-MYB, bHLH and WD40 transcription factors (TFs). Recent studies in black carrots have elucidated major anthocyanin related structural genes and also regulatory TFs. However, the active TFs responsible for anthocyanin production in black carrots differ between cultivars. We have previously shown by RNAseq that DcMYB113 (LOC108213488), a R2R3-MYB TF, was up-regulated in colored as compared to non-colored tissues of the black carrots 'Superblack' and 'CH05544' and that this upregulation was positively correlated with anthocyanin content. However, this gene showed no upregulation in the black carrot 'Nightbird' also included in that study. In the present study, we present a novel R2R3-MYB DcMYB113_NB (LOC108212072) and a complementary bHLH DcEGL1_NB (LOC108210744) isolated from the RNA of 'Nightbird'. Their functionality as anthocyanin regulators was confirmed by their simultaneous expression under the control of a constitutive promoter in the background of the orange carrot 'Danvers 126'. Transformants showed activation of the structural anthocyanin genes and accumulation of anthocyanins across leaves, stems and taproots. Interestingly, the anthocyanin profile of the transformants showed increases of 20 to 30% in acylated anthocyanins as compared to 'Nightbird' resulting in transformants with almost 100% acylated anthocyanins.

Cyanidin based anthocyanin biosynthesis in orange carrot is restored by expression of AmRosea1 and AmDelila, MYB and bHLH transcription factors.

KEY MESSAGE: The simultaneous expression of AmRosea1 and AmDelila transcription factors from snapdragon can activate the anthocyanin pathway in orange carrots, leading to the synthesis and accumulation of anthocyanins in the taproots. Anthocyanins are phenolic compounds produced in various parts of plants. They are used as natural food dyes and are reported as beneficial antioxidants for humans. Black carrot is an important source for anthocyanins; however, the reason for the lack of anthocyanin production in the orange carrot is unknown. Anthocyanins are synthesized by a specific branch of the phenylpropanoid pathway that has previously been reported to be activated by a triad of R2R3-MYB, basic helix-loop helix (bHLH) and WD40 transcription factors (TFs). In the current study, orange carrots were turned purple by simultaneous expression of R2R3-MYB and bHLH TFs, i.e. AmRosea1 and AmDelila from snapdragon (Antirrhinum majus). Simultaneous transgenic expression of the TFs under a constitutive promoter in the orange carrot cultivar 'Danvers 126' lead to consistent upregulation of anthocyanin-related biosynthetic genes and significant accumulation of anthocyanins in leaves, stems and taproots. Highest overall content of soluble anthocyanins in the taproot among the transformants amounted to 44.38 mg g-1 dry weight. The anthocyanin profile of the transformants were significantly different from the profile in the reference black carrot 'Deep Purple'. The main anthocyanins present in the transformed taproots were cyanidin 3-xylosyl(sinapoylglucosyl)galactoside, whereas the main anthocyanin present in Deep Purple was cyanidin 3-xylosyl(feruloylglucosyl)galactoside. This study confirms the presence of the necessary biosynthetic genes in orange carrots for production of anthocyanins and demonstrates the absence of suitable R2R3-MYB and bHLH TFs for stimulating anthocyanin biosynthesis in the orange carrot.

NMR structure of Dengue West Nile viruses stem-loop B: A key cis-acting element for flavivirus replication.

Flaviviruses are major emerging human pathogenic viruses that pose a persistent and growing menace to global health. They are enveloped single-stranded RNA viruses with positive polarity transmitted by arthropod vectors like mosquitoes or ticks, responsible for a significant and growing number of human deaths and illnesses. The 5'- and 3'-untranslated regions (UTRs) are highly structured and contain conserved cis-acting RNA elements that participate in viral translation, replication and host adaptation. Despite their role in fiaviviruses replication, few high-resolution structural studies have investigated the RNA elements required for viral replication. Here we report the NMR structures of stem-loop B from WNV and DENV4 viruses. Because this element is required for cyclization of the genome and the activity of the replicative viral enzymes, viral replication rates are sensitive to even small changes in these RNAs. Therefore, this work provides structural insight into a new drug target to reduce flavivirus replication rates.

Study of the Association of Epicardial Adiposity with Albuminuria in Patients with Essential Hypertension.

BACKGROUND: Epicardial adipose tissue (EAT), a type of visceral adipose tissue, has been found to be a cardiometabolic risk factor contributing to cardiovascular disease. Albuminuria, a marker of endothelial dysfunction is also associated with an increased risk for cardiovascular disease. The aim of this study was to investigate the relationship of echocardiographic epicardial adipose tissue (EAT) thickness and albuminuria in hypertensive patients. AIMS AND OBJECTIVES: To compare EAT between essential hypertensive patients with normal UACR (<30) and albuminuria (UACR>30). METHODS: 100 essential hypertensive patients were included into the study. Age, body mass index (BMI), blood pressure, hemoglobin, random blood sugar, creatinine, albumin was evaluated. Spot urine sample was collected for the assessment urine albumin creatinine ratio (UACR). Patients were divided into two groups according to UACR; Group A included UACR <30 mg/g; and Group B included UACR > 30mg/g. Thereafter, all subjects underwent transthoracic echocardiography to measure EAT thickness, left ventricular mass (LVM), left ventricular mass index (LVMI) and ejection fraction (EF). MAIN OUTCOMES AND MEASURES: Epidemiological, demographic, clinical, laboratory, radiological, and treatment data were collected and analyzed. The primary composite end-point was admission to an intensive care unit (ICU), the use of mechanical ventilation or death. Patients were categorized as severe pneumonia and non-severe pneumonia at time of admission and outcome data was compared. RESULTS: The mean EAT was found to be significantly higher in patients with significant albumin excretion (UACR>30 mg/g) as compared to group A (6.65 ± 1.944 mm vs. 3.64 ± 1.13 mm, respectively, p<0.001). Upon correlation analysis, EAT was found to be positively correlated with age (r = 0.749, p < 0.001), serum creatinine (r = 0.244, p = 0.014), LVM (r = 0.691, p = 0.001) and LVMI (r = 0.677, p = 0.001) and negatively correlated with EF (r = -0.599, p = 0.001). CONCLUSIONS: Epicardial adipose tissue thickness can serve as a high-risk marker to stratify essential hypertensive patients.

Clinical Profile of Covid-19 Infected Patients Admitted in a Tertiary Care Hospital in North India.

BACKGROUND: The novel coronavirus (Covid-19) continues to wreck havoc across China, European countries, USA and now seems to gain a strong foothold in India. The aim of this report is to describe the clinical profiles of these Covid-19 infected patients admitted in Sawai Mansingh Hospital(S.M.S), Jaipur ranging from their age, sex, travel history, clinical symptoms, laboratory evaluation, radiological characteristics, treatment provided along with common side effects and the final outcome. The described cases are one of the earliest cases of Covid-19 in the Indian subcontinent. METHODS: Epidemiological, clinical, laboratory, and radiological characteristics and treatment and outcomes data were obtained with data collection forms from electronic medical records and history given by 21 Covid-19 infected patients admitted in S.M.S., Jaipur. Patients were tested for Covid-19 by real-time reverse transcription polymerase chain reaction (RT-PCR) assay of 2019-nCoVRNA. RESULTS AND DISCUSSION: During the course of this study 21 Covid-19 positive patients were admitted in S.M.S Hospital, Jaipur. Male patients constituted 66.66% of total patients and majority of the patients (80.90%) were below 60 years of age. Most of the patients (71.40%) were either foreigners or had a history of foreign travel suggesting that these cases were not community acquired except for 4 cases from textile producing district Bhilwara (known as Manchester of India), an epicenter of North India. Approximately 33.33% patients were completely asymptomatic and of those who were symptomatic cough was the most common symptom (85.71%) followed by fever (78.57%), myalgia (64.28%), headache (28.57%) and dyspnea (28.57%). Three patients (14.28 %) had underlying co morbidity in the form of hypertension, diabetes mellitus, hypothyroidism, chronic kidney disease or coronary artery disease. 11 patients (52.38%) had lymphopenia in their hemogram during the course of admission. 3 patients (14.28%) had leucocytosis and 4 patients (19.04%) presented with thrombocytopenia. All 4 patients in the severe category had raised FDP, D-Dimer levels and they needed oxygen support. These patients had deranged liver functions and had elevated pro-calcitonin levels, serum ferritin levels and LDH levels. 1 out of the these 4 cases went into ARDS during the course of treatment. 10 patients yielded negative results for Covid-19. The mean duration from admission to getting 1st Covid-19 sample negative was 8.3 days. 18 patients (85.71%) are still under treatment. CONCLUSION: Clinical investigations in initial Covid-19 patients in the Indian subcontinent reveal lymphopenia as predominant finding in hemogram. Patients with older age and associated comorbid conditions (COPD and diabetes) seem to have greater risk for lung injury thereby requiring oxygen support during the course of disease and these patients also had greater derangement in their biochemical profile.

Clinico-Radiological Evaluation and Correlation of CT Chest Images with Progress of Disease in COVID-19 Patients.

PURPOSE: The present study was undertaken to investigate and quantify the severity of COVID-19 infection on high-resolution chest computed tomography (CT) and to determine its relationship with clinical parameters. This study also aimed to see CT changes with clinical recovery or progression of disease. MATERIALS AND METHODS: In an attempt to provide extensive information pertaining to clinical and radiological characteristics of COVID-19, the present study was undertaken in 80 hospitalized patients. The patients were COVID-19 confirmed positive by genomic analysis through RT-PCR at tertiary care center in Jaipur. Initially all patients were evaluated for their clinical parameters and then correlated with HRCT chest after hospitalization. CT findings correlated with duration of disease to assess progress or recovery. RESULTS: A total of 80 patients of laboratory confirmed COVID-19 test by RT-PCR at SMS Hospitals, Jaipur were assessed. Among the confirmed cases, most of patients were young adults in the fifth and sixth decade of age group with mean age of 50.40 years. There was a male preponderance (59% male and 41% female). Out of total analyzed patients, 39 patients (48.75%) were symptomatic, among them fever (79.47%), cough (74.35%), shortness of breath (36%) and sore throat (17.94%) were the most common presenting clinical manifestations. A few patients (12.82%) also had other symptoms like headache, chest pain, pain abdomen, altered sensorium etc. 54% patients had some underlying co morbid disease in sample population. The most prevalent comorbidities were Diabetes mellitus (56%), Hypertension (48.83%), COPD/K-chest (12%), CAD (9.32%) and others (11.62%) like hypothyroidism, anemia, CVA etc. The lung pathological changes were evaluated by HRCT imaging and by assigning CT severity score. We found Typical COVID findings in 50% patients, Indeterminate in 11%, Atypical in 11% and 28% patients had Negative CT chest for COVID. The clinical status of patients correlated with the CT severity score, with mild cases showing score <15/25 in 45.83% patients and severe cases showing CT severity score >15/25 in 87.50% patients. The CT features varied with duration and course of disease. Proportional GGO was higher (59.37%) in early phase and it was lower (12.5%) in later stage of disease. CONCLUSION: The varied spectra of COVID-19 presentation included fever, cough, shortness of breath, sore throat etc. Diabetes mellitus, hypertension, COPD/K-Chest and CAD were found as major comorbid conditions. Symptomatic presentation of COVID-19 was observed to be higher in patients with co morbid disease, especially if multiple. HRCT chest in COVID-19 patients had a major diagnostic and prognostic importance as positive CT findings were more prominent in symptomatic patients and co-morbid patients. Clinical symptoms of patients directly correlated with CT severity index. CT imaging was found to be useful in predicting clinical recovery of patients or progression of disease.

Characteristics, Treatment Outcomes and Role of Hydroxychloroquine among 522 COVID-19 hospitalized patients in Jaipur City: An Epidemio-Clinical Study.

PURPOSE: The present study was undertaken to investigate epidemiological distribution, clinical manifestation, co morbid status, treatment strategy and case fatality index of emerging COVID-19 infection at SMS Medical College Hospital, Jaipur, Rajasthan. It also evaluated efficacy of hydroxychloroquine (HCQ) in treatment of patients and risk of serious adverse outcomes in patients with COVID-19 in relation to their co morbid status. MATERIALS AND METHODS: In an attempt to provide extensive information pertaining to epidemiological and clinical characteristics of COVID-19, the present study was undertaken on 522 patients. The patients were COVID-19 confirmed positive by genomic analysis through Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) at SMS Medical College and Attached Hospitals, Jaipur. The indoor admitted patient's information inclusive of demographic profile (age, sex, nationality, residence), date of confirmation for positive COVID-19 case, travel/ exposure history, date of recovery/ death, clinical features, co morbidities and treatment plan was recorded. A serial follow-up of recovered patients to evaluate infective period of the disease was also part of the study. RESULTS: A total of 522 patients of laboratory confirmed COVID-19 test by RT-PCR at SMS Hospitals, Jaipur were assessed. Among the confirmed cases, most of patients were young adult in the age group with mean age of 35.42 years. 22.41% patients were below 20 years of age, majority of patients (58.80%) were in the age range of 21 to 50 years and only 18.79% patient population was in the age range of above 50 years. Females (39.08%) were affected less than males (60.91%) with an average sex ratio of female: male being 0.64. Out of the total analyzed patients, only 24.32% patients were symptomatic, among them fever (55.90%), cough (52.75%), sore throat (49.60%) and shortness of breath (46.45%) were the most common presenting clinical manifestations while a few patients also had symptoms of headache (26.77%), chest pain (6.29%) and other symptoms (7.87%) like pain abdomen, fatigue, joints pain, altered sensorium etc. Most of symptomatic patients belonging to older age group. An average of 40.40% patient population of above 50 years of age, were symptomatic while none of the patients below 10 years of age were symptomatic. 13.98% patients had some or the other underlying co morbid disease. The most prevalent co morbidity was hypertension (42.46%) followed by Diabetes mellitus (39.72%), Old k-chest (20.54%), COPD/ Bronchial Asthma (16.43%), Coronary artery disease (13.69%), Chronic kidney disease (13.69%) and Valvular heart disease (6.84%) distributed in co morbid patients of COVID-19. 60.27% of patient population with underlying co morbid conditions were more prone to develop symptomatology complex as compared to that observed in patients with no co morbidity (18.42%). 116 patients had recovered with effective treatment till the date of data analysis. Time of recovery was counted from the date of positive report to 1st negative report of oropharyngeal sample by RT-PCR for COVID-19 with an average recovery time of 8.15 days. 23.27% patients recovered within 5 days, while 52.58% patients took about 6-10 days, 23.27% patients took 11-15 days and remaining 0.86% took more than 16 days to recover. In the present study 15 patients had died till analysis of data, among the deceased, 73.33% were above 50 year of age with a male preponderance (66.6%). Interestingly, all deceased (100%) had presented with clinical manifestations of COVID-19 and all had underlying multiple co morbid conditions. Majority of patients had early mortality after admission to hospital with two third death account in initial three days. Asymptomatic patients (cases) treated with HCQ recovered early (average recovery time =5.4 days) compared to asymptomatic patients who did not receive any treatment (control group) and had longer recovery time (average recovery time =7.6 days). CONCLUSION: The varied spectra of COVID-19 mostly affects young adult age group (third to fifth decades of life). Interestingly, early age group was also affected in significant proportion when compared with similar data from other countries. It was observed that male population seemed to be was more prone to getting infected. Majority of COVID-19 positive patients (nearly three-fourth) were asymptomatic (mostly in young age range) at the time of diagnosis, which poses a major challenge for health care workers. Fever, cough, sore throat and shortness of breath were major symptoms that could be detected in such COVID-19 patients. Symptomatic clinical manifestations were more common in old age population. Infectivity was higher in patients that had underlying co morbid disease, especially in patients with multiple co morbid conditions. Symptomatic presentation of COVID-19 was observed to be higher in patients with co morbid disease. Average recovery time from COVID-19 was 8 days with effective treatment. Mortality in COVID-19 was higher in old age population, male gender, symptomatic and co morbid patients as compared to other similarly matched group. Most of mortality was noted within first few days of admission, suggestive of early mortality due to the primary disease process. Treatment with HCQ had early recovery without effectively influencing the overall mortality.

Structure of G57W mutant of human γS-crystallin and its involvement in cataract formation.

A recently identified mutant of human γS-crystallin, G57W is associated with dominant congenital cataracts, the familial determinate of childhood blindness worldwide. To investigate the structural and functional changes that mediate the effect of this cataract-related mutant to compromise eye lens transparency and cause lens opacification in children, we recently reported complete sequence-specific resonance assignments of γS-G57W using a suite of heteronuclear NMR experiments. As a follow up, we have determined the 3D structure of γS-G57W and studied its conformational dynamics by solution NMR spectroscopy. Our structural dynamics results reveal greater flexibility of the N-terminal domain, which undergoes site-specific structural changes to accommodate W57, than its C-terminal counterpart. Our structural inferences that the unusual solvent exposure of W57 is associated with rearrangement of the N-terminal domain suggest an efficient pathway for increased aggregation in γS-G57W and illuminates the molecular dynamics underlying cataractogenic aggregation of lens crystallins in particular and aggregation of proteins in general.

Structural studies on the individual domains of human γS-crystallin and its G57W mutant unfolds mechanistic insights into childhood cataracts.

Inter-domain interactions tune the exceptional stability of human γS-crystallin (γS-WT) in the eye lens, which lasts a lifetime with no protein turnover. Our recent NMR studies revealed the key role of G57W mutation in γS-WT, as the familial determinate of childhood cataracts. As the unusually exposed W57 is near the inter-domain interface, a recurring theme of this study is the upsetting of inter-domain contacts exposing hydrophobic patches, which may initiate aggregation at crystallin concentrations not so surprising in the eye lens. In this endeavour, to untangle the mechanistic pathways triggering aggregation in the cataract variant γS-G57W, we undertook high-resolution structural characterization of isolated domains vis-a-vis full length γS-crystallin. Here we report for the first time, thermodynamic and kinetic determinants of structural stability with their eccentric shifts under pathological stress employing sophisticated spectroscopy techniques. We propose that domain interface acts as an intrinsic stabilizer for the otherwise floppy N-terminal domain in γS-G57W than in γS-WT where it serves an extrinsic role. Our results present a residue resolved quantitative analysis for differential domain stabilities from non-linear temperature coefficients of 1HN chemical shifts using solution NMR spectroscopy. Consistent with the Ca2+-binding episode that lasted poorly for human lens crystallins, our results show that disease mutants attenuate it further and completely silence it in extreme cases. Overall, our study provides a compelling evidence for the diverse structural evolution of lens crystallins elucidating molecular details to apprehend lens opacification and suggests the scope of therapeutics in reducing the global trauma of cataracts.

On identifying low energy conformational excited states with differential ruggedness in human γS-crystallin promoting severe infantile cataracts.

Transient excited states in proteins can be accurately probed from temperature dependence of amide proton (1HN) chemical shifts displaying significant curvatures. Characterizing these near-native alternative states is of high therapeutic relevance in conformational diseases wherein missense mutations promote structural instability that leads to conformational heterogeneity. Extending the structure-function paradigm from physiology to pathology, we recently reported the solution NMR structure and dynamics of a severe congenital cataract variant, G57W of human γS-crystallin (abbreviated as γS-G57W) which is resistant towards crystallization. In an endeavour to explore the functional consequences of this mutation, here we report for the first time, native state conformational ruggedness in human γS-G57W as compared to its wild-type counterpart from residue resolved nonlinear temperature dependence of backbone 1HN chemical shifts using solution NMR spectroscopy. Our calculations suggest that the simulated chemical shift curvatures are indicative of low energy excited states within 2-4 kcal mol-1 from the native state. Residues accessing alternative conformations populate the N-terminal domain of γS-G57W more than its C-terminal counterpart. Collectively, curvatures retaining native state ensemble on mild denaturation suggest that the free energy landscape in human γS-G57W at the bottom of the folding funnel is sufficiently robust and malleable against such perturbations. Overall, this critical study highlights the functional aspects of such structural malleability promoting infantile cataracts as a global health risk marker.

Enhanced H/D exchange unravels sequential structural excursions in G57W variant of human γS-crystallin with pro-cataractogenic conformations.

Our two recent reports on the high resolution NMR structure and conformational dynamics of G57W variant of human γS-crystallin (abbreviated as γS-G57W) causing severe infantile cataracts, revealed slackening of its N-terminal domain with enhanced local conformational dynamics attributed to mutation. Exploring the biochemistry of infantile cataracts in detail, here we studied structural unfolding in both human γS-WT and γS-G57W at residue level resolution using solution NMR spectroscopy and chemical kinetics and characterized the molecular intermediates with functional consequences. We report, for the first time, that human γS-crystallin unfolds sequentially under H/D exchange. This communication forms the first experimental evidence for non-concerted destabilization of structural foldon units in human γS-G57W. Residues contributing to the compact fold and structural stability exchanged their amide protons with deuterons more readily in γS-G57W compared to γS-WT, displaying differential free energies of exchange. Overall, our results establish a direct conformational link between the structure, dynamics, design and function in human γS-crystallin such that the G57W cataract variant promotes enhanced structural excursions concomitant with increased instability, elucidating very crucial molecular details of cataract formation affecting infants across the globe.

Conformational dynamics study on human γS-crystallin as an efficient route to childhood blindness.

Single point mutants of human γS-crystallin cause dominant congenital cataracts, a recent one of which involves the substitution of highly conserved glycine at 57th position with a bulkier tryptophan. Our high-resolution 3D structure of this G57W mutant (abbreviated hereafter as γS-G57W), reported recently revealed site-specific structural perturbations with higher aggregation and lower stability compared to its wild-type; a structural feature associated with important functional and therapeutic consequences. In this communication, we report for the first time, residue resolved conformational dynamics in both γS-WT and γS-G57W using solution NMR spectroscopy, and suggest how these differences could crucially affect the biochemistry of the mutant. Guided by our critical structural investigations, extensive conformational dynamics and biophysical studies presented here show that loss of structural stability arises from enhanced dynamics in Greek key motif 2 inducing flexibility in the N-terminal domain as opposed to its structurally unperturbed C-terminal counterpart. NMR spectral density correlations and internal dynamics comparisons with the wild-type suggest that the overall thermodynamic instability propagates from the mutated N-terminal ß4-ß5 loop providing a residue level understanding of the structural changes associated with this early onset of lens opacification. Our results highlight the vital role of conserved Greek key motifs in conferring structural stability to crystallins and provide crucial molecular insights into crystallin aggregation in the eye lens, which triggers cataract formation in children. Overall, this critical study provides a residue level understanding of how conformational changes affect the structure and function of crystallins in particular and proteins in general, during health and disease.

Identification of transcription factor genes involved in anthocyanin biosynthesis in carrot (Daucus carota L.) using RNA-Seq.

BACKGROUND: Anthocyanins are water-soluble colored flavonoids present in multiple organs of various plant species including flowers, fruits, leaves, stems and roots. DNA-binding R2R3-MYB transcription factors, basic helix-loop-helix (bHLH) transcription factors, and WD40 repeat proteins are known to form MYB-bHLH-WD repeat (MBW) complexes, which activates the transcription of structural genes in the anthocyanin pathway. Although black cultivars of carrots (Daucus carota L.) can accumulate large quantities of anthocyanin in their storage roots, the regulatory genes responsible for their biosynthesis are not well characterized. The current study aimed to analyze global transcription profiles based on RNA sequencing (RNA-Seq), and mine MYB, bHLH and WD40 genes that may function as positive or negative regulators in the carrot anthocyanin biosynthesis pathways. RESULTS: RNA was isolated from differently colored calli, as well as tissue samples from taproots of various black carrot cultivars across the course of development, and gene expression levels of colored and non-colored tissue and callus samples were compared. The expression of 32 MYB, bHLH and WD40 genes were significantly correlated with anthocyanin content in black carrot taproot. Of those, 11 genes were consistently up- or downregulated in a purple color-specific manner across various calli and cultivar comparisons. The expression of 10 out of these 11 genes was validated using real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). CONCLUSIONS: The results of this study provide insights into regulatory genes that may be responsible for carrot anthocyanin biosynthesis, and suggest that future focus on them may help improve our overall understanding of the anthocyanin synthesis pathway.

Structural and functional characterization of a missense mutant of human γS-crystallin associated with dominant infantile cataracts.

Infantile cataracts constitute one of the most important causes of childhood blindness worldwide. Human γS-crystallin is the most abundant protein in the eye lens cortex. A missense mutant of human γS-crystallin, Y67N (abbreviated hereafter as γS-Y67N) is recently reported to be associated with dominant infantile cataracts. To understand the structural basis for γS-Y67N to cause lens opacification, we constructed, expressed and purified γS-Y67N and its wild-type (abbreviated hereafter as γS-WT) and studied the structural and functional differences between them in solution using circular dichroism (CD), differential scanning calorimetry (DSC), fluorescence spectroscopy and extrinsic spectral probes. Extensive equilibrium characterization indicate that replacement of the highly conserved Tyr at 67th position by Asn distorts the conserved Tyr corner at the second Greek key motif in the N-terminal domain (NTD) and leads to substantial loss of structural stability. Our intrinsic fluorescence quenching results reveal differential in-vitro refolding kinetics identifying partially folded kinetic intermediates for both proteins. Extrinsic fluorescence studies further reveal loosening up of the compact structure of γS-crystallin upon mutation associated with enhanced aggregation. As Ca2+ homeostasis is a crucial regulator of lens transparency, we further investigated the Ca2+-binding properties of γS-WT and γS-Y67N by isothermal titration calorimetry (ITC) to identify lens Ca2+ distribution in health and in disease. Overall, our results highlight the vital role of conserved Tyr corners in stabilizing Greek key motifs and provide useful structural and functional insights into the mechanism of cataract formation in humans.