Supramolecular polyplexes from Janus peptide nucleic acids (bm-PNA-G5): self-assembled bm-PNA G-quadruplex and its tetraduplex with DNA.

Nucleic acids (DNA and RNA) can form diverse secondary structures ranging from hairpins to duplex, triplex, G4-tetraplex and C4-i-motifs. Many of the DNA analogues designed as antisense oligonucleotides (ASO) are also adept at embracing such folded structures, although to different extents with altered stabilities. One such analogue, peptide nucleic acid (PNA), which is uncharged and achiral, forms hybrids with complementary DNA/RNA with greater stability and specificity than DNA:DNA/RNA hybrids. Like DNAs, these single-stranded PNAs can form PNA:DNA/RNA duplexes, PNA:DNA:PNA triplexes, PNA-G4 tetraplexes and PNA-C4-i-motifs. We have recently designed Janus-like bimodal PNAs endowed with two different nucleobase sequences on either side of a single aminoethylglycyl (aeg) PNA backbone and shown that these can simultaneously bind to two complementary DNA sequences from both faces of PNA. This leads to the formation of supramolecular polyplexes such as double duplexes, triple duplexes and triplexes of double duplexes with appropriate complementary DNA/RNA. Herein, we demonstrate that Janus/bimodal PNA with a poly G-sequence on the triazole side of the PNA backbone and mixed bases on the t-amide side, templates the initial formation of a (PNA-G5)4 tetraplex (triazole side), followed by the formation of a PNA:DNA duplex (t-amide side). Such a polyplex shows synergistic overall stabilisation compared to the isolated duplexes/quadruplex. The assembly of polyplexes with a shared backbone for duplexes and tetraplexes is programmable and may have potential applications in the self-assembly of nucleic acid nano- and origami structures. It is also shown that Janus PNAs enter the cells better than the standard aeg-PNA oligomers, and hence have implications for in vivo applications as well.

Discovery of Free Glycated Amines and Glycated Urea in Diabetic Plasma: Potential Implications in Diabetes.

The role of protein glycation in the pathogenesis of diabetes has been well established. Akin to proteins, free amino acids and other small-molecule amines are also susceptible to glycation in hyperglycemic conditions and may have a role in the pathogenesis of the disease. However, information about glycation of free amino acids and other small-molecule amines is relatively obscure. In the quest to discover small-molecule glycated amines in the plasma, we have synthesized glycated amino acids, glycated creatine, and glycated urea, and by using a high-resolution accurate mass spectrometer, a mass spectral library was developed comprising the precursor and predominant fragment masses of glycated amines. Using this information, we report the discovery of the glycation of free lysine, arginine, and leucine/isoleucine from the plasma of diabetic patients. This has great physiological significance as glycation of these amino acids may create their deficiency and affect vital physiological processes such as protein synthesis, cell signaling, and insulin secretion. Also, these glycated amino acids could serve as potential markers of diabetes and its complications. While other amines, such as creatinine and urea, accumulate in the plasma and act as biomarkers of diabetic nephropathy. For the first time, we report the detection of glycated urea in diabetic plasma, which is confirmed by matching the precursor and fragment masses with the in vitro synthesized glycated urea by using 12C6 and 13C6-glucose. Further, we quantified glycated urea detected in two forms, monoglycated urea (MGU) and diglycated urea (DGU), by a targeted mass spectrometric approach in the plasma of healthy, diabetic, and diabetic nephropathy subjects. Both MGU and DGU showed a positive correlation with clinical parameters, such as blood glucose and HbA1c. Given that urea gets converted to glycated urea in hyperglycemic conditions, it is crucial to quantify MGU and DGU along with the urea for the diagnosis of diabetic nephropathy and study their physiological role in diabetes.

The multi-dimensional impact of captopril modification on human serum albumin.

Captopril is a thiol drug, widely used for the management of hypertension and cardiovascular diseases. Reactive thiols are found to covalently modify the cysteines of plasma proteins and affect their structure and function. Human serum albumin (HSA) is prone to undergo modification by various low molecular weight compounds, including drugs. Cysteine34 (Cys34) in HSA has a free thiol group with antioxidant properties, considered to be the most redox-sensitive amino acid in plasma. Through mass-spectrometric analysis, we demonstrate for the first time that captopril forms a disulfide adduct at Cys34 residue and increases the protease susceptibility of HSA to trypsin. As evidenced by our biophysical and electron microscopy studies, HSA undergoes structural alteration, aggregation and morphological changes when treated with different captopril concentrations. Molecular dynamics studies further revealed the regions of secondary structural changes in HSA due to disulfide adduct formation by captopril at Cys34. It also elucidated the residues involved in the noncovalent interactions with captopril. It is envisaged that structural change in HSA may influence the efficacy of drug delivery as well as its own biological function. These findings may thus provide significant insights into the field of pharmacology intriguing further investigation into the effects of long-term captopril treatment.

SWATH-MS reveals that bisphenol A and its analogs regulate pathways leading to disruption in insulin signaling and fatty acid metabolism.

Bisphenol A (BPA) is an endocrine-disrupting chemical (EDC), associated with obesity and insulin resistance. The FDA prohibited the use of BPA-based polycarbonate resins in infant formula packaging; thus, its analogs, viz. Bisphenol S (BPS) and Bisphenol F (BPF) were considered alternatives in epoxy resins, plastics, and food cans. As these analogs might evoke a similar response, we investigated the role of Bisphenols (BPA, BPF, and BPS), on insulin signaling in CHO-HIRc-myc-GLUT4eGFP cells at environmentally relevant concentrations of 2 nM and 200 nM. Insulin signaling demonstrated that Bisphenols reduced phosphorylation of IR and AKT2, GLUT4 translocation, and glucose uptake. This was accompanied by increased oxidative stress. Furthermore, SWATH-MS-based proteomics of 3T3-L1 cells demonstrated that Bisphenol-treated cells regulate proteins in insulin resistance, adipogenesis, and fatty acid metabolism pathways differently. All three Bisphenols induced differentially expressed proteins enriched similar pathways, although their abundance differed for each Bisphenol. This might be due to their varying toxicity level, structural differences, and estrogen-mimetic activity. This study has important implications in addressing health concerns related to EDCs. Given that the analogs of BPA are considered alternatives to BPA, the findings of this study suggest they are equally potent in altering fatty acid metabolism and inducing insulin resistance.

Reintroduction of Hip and Knee Arthroplasty Through COVID-19 Pandemic: Experience at a High-Volume Hip and Knee Arthroplasty Centre in India.

Background: COVID-19 was declared as a pandemic in March 2020. Government of India declared a countrywide lockdown on 24 March 2020. All elective surgeries including Hip and Knee arthroplasty were postponed in view of pandemic. Gradually cases were resumed after stepwise unlock measures. The aim of this study is to assess how hip and knee arthroplasty surgeries were affected during first wave of pandemic, and how situation was tackled by an arthroplasty unit of a tertiary-care hospital in India. Methods: This study was a single-centre retrospective observational study. Data pertaining to patient demographic details, surgery, preoperative screening for COVID-19, duration of hospital stay, and post-op 30-day complications were collected from hospital records and analyzed. These data were compared with 2019 data. Results: There was significant decrease (88.45%) in total number of hip and knee arthroplasty cases between March 2020 and November 2020 as compared to the same duration in 2019. 30-day mortality was only 2 deaths both who died due to COVID-19-related complications. Duration of stay in hospital and post-operative complications were not statistically and significantly affected. There was a statistically significant increase in tourniquet time compared to the previous year. If deaths due to COVID-19 are excluded, there was statistically no significance difference in 30-day mortality rate. Conclusion: Following strict local policy for patient selection and reducing the number of post-operative patient visits to the hospital allowed us to perform hip and knee arthroplasty safely with minimum COVID-19-related mortality and morbidity. Supplementary Information: The online version contains supplementary material available at 10.1007/s43465-023-00930-6.

Nanopore Sequencing of RAGE Gene Polymorphisms and Their Association with Type 2 Diabetes.

The receptor for advanced glycation end products (RAGE) is a transmembrane protein that interacts with its ligands, advanced glycation end products (AGEs). AGEs are elevated in diabetes and diabetic complications, leading to increased oxidative stress and activation of pro-inflammatory pathways facilitated by AGE-RAGE signaling. Polymorphisms in the RAGE gene can potentially affect AGE-RAGE interaction and its downstream signaling, which plays a crucial role in the progression of diabetes and its complications. In this study, we used nanopore sequencing for genotyping of RAGE polymorphism and identified a maximum number of 33 polymorphisms, including two previously unreported novel mutations in a cohort of healthy, type 2 diabetics without nephropathy and type 2 diabetics with nephropathy in order to identify associations. Two novel RAGE polymorphisms in the intron 8 and 3'UTR region at genomic locations 32181834 and 32181132, respectively, were detected with a low frequency. For four previously reported polymorphisms, cross-validation by PCR-RFLP showed 99.75% concordance with nanopore sequencing. Analysis of genotype distribution and allele frequencies revealed that five single nucleotide polymorphisms, i.e., rs1800625, rs3131300, rs3134940, rs2070600, and rs9391855, were associated with an increased risk for type 2 diabetes.

De novo structure prediction of meteorin and meteorin-like protein for identification of domains, functional receptor binding regions, and their high-risk missense variants.

Meteorin (Metrn) and Meteorin-like (Metrnl) are homologous secreted proteins involved in neural development and metabolic regulation. In this study, we have performed de novo structure prediction and analysis of both Metrn and Metrnl using Alphafold2 (AF2) and RoseTTAfold (RF). Based on the domain and structural homology analysis of the predicted structures, we have identified that these proteins are composed of two functional domains, a CUB domain and an NTR domain, connected by a hinge/loop region. We have identified the receptor binding regions of Metrn and Metrnl using the machine-learning tools ScanNet and Masif. These were further validated by docking Metrnl with its reported KIT receptor, thus establishing the role of each domain in the receptor interaction. Also, we have studied the effect of non-synonymous SNPs on the structure and function of these proteins using an array of bioinformatics tools and selected 16 missense variants in Metrn and 10 in Metrnl that can affect the protein stability. This is the first study to comprehensively characterize the functional domains of Metrn and Metrnl at their structural level and identify the functional domains, and protein binding regions. This study also highlights the interaction mechanism of the KIT receptor and Metrnl. The predicted deleterious SNPs will allow further understanding of the role of these variants in modulating the plasma levels of these proteins in disease conditions such as diabetes.Communicated by Ramaswamy H. Sarma.

Elevated Level of Glycated KQTALVELVK Peptide of Albumin Is Associated with the Risk of Diabetic Nephropathy.

Diabetic nephropathy is a leading cause of end-stage renal disease. Hence, early detection of diabetic nephropathy is essential to mitigate the disease burden. Microalbuminuria, the currently used diagnostic marker of diabetic nephropathy, is not efficient in detecting it at an early stage. Therefore, we explored the utility of glycated human serum albumin (HSA) peptides for risk prediction of diabetic nephropathy. Three glycation-sensitive HSA peptides, namely, FKDLGEENFK, KQTALVELVK, and KVPQVSTPTLVEVSR, with deoxyfructosyllysine (DFL) modification were quantified by targeted mass spectrometry (MS) in a study population comprising healthy and type II diabetes subjects with and without nephropathy. Mass spectrometry, receiver operating characteristic (ROC) curve, and correlation analysis revealed that the DFL-modified KQTALVELVK peptide was better than other glycated HSA peptides and HbA1c for identifying diabetic nephropathy. DFL-modified KQTALVELVK could be a potential marker for risk prediction of diabetic nephropathy.

Prevalence of road traffic injuries in South East and South Asian region - A systematic review.

Background: South and South-East Asian countries report a great liability for the world's road traffic injuries (RTIs) and deaths. A vast number of research studies tested various interventions including specific protective devices to prevent accidents, but no review papers have been conducted to find out the prevalence of RTIs in South-East and South Asian countries. Objective: This review paper was an attempt to find out the prevalence of RTIs and their associated factors in South-East and South Asian countries. Methods: Following the guidelines of Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA), we searched the articles in the electronic databases of PubMed/Medline, Scopus, CINAHL, ProQuest, and Web of Science. Articles were selected if they reported the prevalence of RTI, or road traffic accident (RTA) deaths. In addition, a data quality assessment was done. Results: Out of the 10,818 article hits from the literature search, ten articles found the eligibility and inclusion criteria. Most of the studies reported that males are involved in the RTIs more than females. The male mortality rate is more than the female mortality in RTI mortality. Young adult males are the major victims when compared with the different age groups of male victims. Two-wheelers are the major contributors to the accident rate. Religious or national festivals are not free from accident-prone times. Climatic seasons and nighttime have a major influence on the RTIs. RTIs are increasing due to the sudden and huge increase in the number of motor vehicles and the development of cities and towns. Conclusion: Accidents are non-predictable but controllable disasters in society. Overspeeding, bad conditions on road, the vulnerability of the vehicles, and careless driving are the major reported reasons for RTIs. Making and implementing strict laws can help us to control RTAs. The major effect on the reduction of RTI can be assured only with the presence of responsible people. That can be achieved only by creating awareness in society about traffic rules and responsibilities.

A Comprehensive Molecular and Clinical Investigation of Approved Anti-HCV Drugs Repurposing against SARS-CoV-2 Infection: A Glaring Gap between Benchside and Bedside Medicine.

The limited availability of effective treatment against SARS-CoV-2 infection is a major challenge in managing COVID-19. This scenario has augmented the need for repurposing anti-virals for COVID-19 mitigation. In this report, the anti-SARS-CoV-2 potential of anti-HCV drugs such as daclatasvir (DCV) or ledipasvir (LDP) in combination with sofosbuvir (SOF) was evaluated. The binding mode and higher affinity of these molecules with RNA-dependent-RNA-polymerase of SARS-CoV-2 were apparent by computational analysis. In vitro anti-SARS-CoV-2 activity depicted that SOF/DCV and SOF/LDP combination has IC50 of 1.8 and 2.0 µM, respectively, comparable to remdesivir, an approved drug for COVID-19. Furthermore, the clinical trial was conducted in 183 mild COVID-19 patients for 14 days to check the efficacy and safety of SOF/DCV and SOF/LDP compared to standard of care (SOC) in a parallel-group, hybrid, individually randomized, controlled clinical study. The primary outcomes of the study suggested no significant difference in negativity after 3, 7 and 14 days in both treatments. None of the patients displayed any worsening in the disease severity, and no mortality was observed in the study. Although, the post hoc exploratory analysis indicated significant normalization of the pulse rate showed in SOF/DCV and SOF/LDP treatment vs. SOC. The current study highlights the limitations of bench side models in predicting the clinical efficacy of drugs that are planned for repurposing.

Protective Effects of Rifampicin and Its Analog Rifampicin Quinone in a Mouse Model of Obesity-Induced Type 2 Diabetes.

Advanced glycation end-products (AGEs) form when glucose reacts non-enzymatically with proteins, leading to abnormal protein function, oxidative stress, and inflammation. AGEs are associated with aging and age-related diseases; their formation is aggravated during diabetes. Therefore, drugs preventing AGE formation can potentially treat diabetic complications, positively affecting health. Earlier, we demonstrated that rifampicin and its analogs have potent anti-glycating activities and increase the life span of Caenorhabditis elegans. This study aimed to investigate the effects of rifampicin during hyperglycemia in C. elegans and in a mouse model of obesity-induced type 2 diabetes. The effects of rifampicin were assessed by determining the life span of C. elegans cultured in the presence of glucose and by measuring HbA1c, AGE levels, and glucose excursions in the diabetic mouse model. Our results show that rifampicin protects C. elegans from glucose-induced toxicity and increases life span. In mice, rifampicin reduces HbA1c and AGEs, improves insulin sensitivity, and reduces indications of diabetic nephropathy without inducing hepatotoxicity. Rifampicin quinone, an analog with lower anti-microbial activity, also reduces HbA1c levels, improves glucose homeostasis and insulin sensitivity, and lowers indications of diabetic nephropathy, without adversely affecting the liver of the diabetic mice. Altogether, our results indicate that rifampicin and its analog have protective roles during diabetes without inflicting hepatic damage and may potentially be considered for repositioning to treat hyperglycemia-related complications in patients.

Behavioral and Proteomic Studies Reveal Methylglyoxal Activate Pathways Associated with Alzheimer's Disease.

Diabetes is one of the major risk factors for Alzheimer's disease (AD) development. The role of elevated levels of glucose, methylglyoxal (MGO), and advanced glycation end products (AGEs) in the pathogenesis of AD is not well understood. In this pursuit, we studied the role of methylglyoxal in the pathogenesis of AD in rat models. The elevated plus-maze (EPM) behavioral study indicated that MGO induces anxiety. Treatment of telmisartan (RAGE expression inhibitor) and aminoguanidine (MGO quencher) attenuated MGO induced anxiety. Further, hippocampal proteomics demonstrated that MGO treated rats differentially regulate proteins involved in calcium homeostasis, mitochondrial functioning, and apoptosis, which may affect neurotransmission and neuronal plasticity. The hippocampal tau phosphorylation level was increased in MGO treated rats, which was reduced in the presence of aminoguanidine and telmisartan. The plasma fructosamine level was increased upon MGO treatment. Hippocampal histochemistry showed vascular degeneration and neuronal loss upon MGO treatment. This study provides mechanistic insight into the role of MGO in the diabetes-associated development of AD.

Novel fluconazole-resistant zoonotic yeast isolated from mastitis.

A hospital-based cross-sectional study was conducted during 2018-2019 to decipher the prevalence of yeast mastitis. The results indicated a 19.68% prevalence of clinical mastitis in bovines. Among them, 5.51% of samples revealed yeasts constituting 1.09% overall prevalence. Candida albicans was recorded as a significant fungal agent involved in clinical bovine mastitis. We record the association of Kodamaea ohmeri in clinical bovine mastitis. On proteomic and molecular confirmation, K. ohmeri isolates were re-identified from phenotypically identified Candida isolates associated with bovine mastitis. After conventional identification, the yeast isolates were re-identified by MALDI-TOF MS-based proteomic approaches. The D1/D2 domains of 26S-rRNA gene and 5.8S-internal transcribed spacer (ITS) rDNA regions based molecular phylogenetic analysis identified the isolates as K. ohmeri. The isolates were resistant to fluconazole. This study reports the first systemic study of K. ohmeri isolates recovered from bovine clinical mastitis, utilizing conventional, automated, proteomic, and genomic approaches followed by antifungal susceptibility. The findings suggest K. ohmeri as a potent opportunistic emerging pathogen of veterinary and public health concern, need for accurate identification of fungal agents from mycotic mastitis, and use of validated antifungal susceptibility assay because of developing resistance to antimycotic agents. Our findings suggest judicious use of fluconazole and alternative antifungal agents may be considered in case of an outbreak.

The investigation on allelic, genotypic frequencies and gene expression study in coding region of tyrosinase gene in Deoni cattle breed of western India.

An investigation was carried out on Deoni animals of western India to study the allelic and genotypic frequencies in coding region of TYR gene as well as gene expression profile. The animals were grouped according to age, gender, strain and intensity of partial albinism (low, medium and high). The present study revealed that the genotypic frequency of TYR gene across different strains, gender, age group and level of partial albinism was found to be non-significant for both exon-I and exon-II. The AB genotype in Balankya (0.70) was observed highest genotypic frequency followed by Wanera (0.55) and Shewara (0.55) strains. The genotypic frequency of AB and BB genotypes were observed highest in male and female, respectively. In exon-I, genotype frequency of AA genotype was found highest (0.55) in low level of partial albinism. The allelic frequencies in Shewara strain, male and low level of partial albinism were 0.75, 0.63 and 0.73, respectively. However, in exon-II genotype frequency of AB and BB was observed highest (0.70) in Wanera and Balankya strains followed by AA genotype in Shewara (0.50). The highest genotypic frequency of AA (0.87) and BB (0.50) were in male and female, respectively. The genotype frequency of AB genotype was found highest in all level of partial albinism. The allelic frequency was highest (0.85 for B allele) in Wanera strain, male (0.80 for A allele) and high level (0.60 for A allele) of particle albinism. The highly significant (p = 0.002) expression of tyrosinase gene was observed in young animals as compared to adult animals. The TYR gene expression was significantly (p = 0.047) higher in animals with low intensity of partial albinism followed by in the animals with medium and high intensity. Therefore, it is inferred that the TYR gene expression in young animals were high and as compared to the old animals of Deoni cattle breed.

Molecular phylodynamics of fowl adenovirus serotype 11 and 8b from inclusion body hepatitis outbreaks.

Fowl adenovirus (FAdV) serotypes are involved in a variety of clinical manifestations in poultry and has resulted in substantial economic loss to the poultry farmers. Despite the endemicity of Inclusion body hepatitis (IBH) in South Asian countries, including India, its etiology is not well studied. In western India, the rural poultry flocks obtained from the vaccinated parents were experiencing disease outbreaks with substantial economic losses due to heavy outbreaks and mortality. Therefore, the study was conducted to decipher the molecular epidemiology of the FAdV from field outbreaks in western India. A total of 37 commercial broiler poultry flocks and 29 village poultry flocks of western India were visited during 2019 to 2021. Out of these, 19.14% flocks showed incidence of IBH during the age of 15 to 35 days. The mortality ranged from 3.3 percent to 55.28 percent. The samples were subjected for amplification of partial hexon gene covering loop 1 and loop 2. The results revealed 48.28% positivity by PCR. The sequence analysis identified 14 isolates as species D serotype 11 with 0.97 to 0.99% divergence and two as species E serotype 8b with 0.99% divergence. The FAdV-11 isolates showed amino acid substitutions D195N, T399A, N417S, and N496H. The amino acids I188 and N195 were conserved in FAdV-11. The molecular clock in Bayesian methods was used to determine most common ancestor. The isolates MH379249 and MH379248 were determined the most recent common ancestor for FAdV-11 and FAdV-8b isolates. The analysis suggested evolution of 10 FAdV-11 strains in 2012, and four FAdV-11 strains and two FAdV-8b strains in 2018.

Mechanistic insights on anserine hydrolyzing activities of human carnosinases.

Anserine and carnosine represent histidine-containing dipeptides that exert a pluripotent protective effect on human physiology. Anserine is known to protect against oxidative stress in diabetes and cardiovascular diseases. Human carnosinases (CN1 and CN2) are dipeptidases involved in the homeostasis of carnosine. In poikilothermic vertebrates, the anserinase enzyme is responsible for hydrolyzing anserine. However, there is no specific anserine hydrolyzing enzyme present in humans. In this study, we have systematically investigated the anserine hydrolyzing activity of human CN1 and CN2. A targeted multiple reaction monitoring (MRM) based approach was employed for studying the enzyme kinetics of CN1 and CN2 using carnosine and anserine as substrates. Surprisingly, both CN1 and CN2 can hydrolyze anserine effectively. The observed catalytic turnover rate (Vmax/[E]t) was 21.6 s-1 and 2.8 s-1 for CN1 and CN2, respectively. CN1 is almost eight-fold more efficient in hydrolyzing anserine compared to CN2, which is comparable to the efficiency of the carnosine hydrolyzing activity of CN2. The Michaelis constant (Km) value for CN1 (1.96 mM) is almost three-fold lower compared to CN2 (6.33 mM), representing higher substrate affinity for anserine-CN1 interactions. Molecular docking studies showed that anserine binds at the catalytic site of the carnosinases with an affinity similar to carnosine. Overall, the present study elucidated the inherent promiscuity of human carnosinases in hydrolyzing anserine using a sensitive LC-MS/MS approach.

Water, sanitation, and hygiene (WaSH) practices among people living with HIV/AIDS (PLHA) - need to relook, in the era of COVID-19 pandemic.

Background: Water, sanitation, and hygiene (WaSH) practices always have been neglected among HIV/AIDS (Human immunodeficiency virus/ Acquired immunodeficiency syndrome) programs, even when HIV and WaSH services have robust bearing on each other. With COVID-19 pandemic on the go, it is utmost necessary for the people living with HIV/AIDS (PLHA) to ensure adequate WaSH practices. Objective: This study was carried out with an objective to assess baseline WaSH practices among PLHA and to find out if any association between nutritional status and WaSH parameters so as to identify the shortcomings and highlight the importance of WaSH practices among PLHAs and give suitable recommendations to program managers. Methodology: A cross-sectional study was carried out among PLHA registered in ART centres of western Maharashtra. A sample size of 378 consented to be part of the study were included in the study, by means of systematic random sampling. Data were collected by means of pretested questionnaire prepared from guidelines and previous studies. Institutional ethical clearance was obtained and informed consent was taken from study participants before data collection. Strict confidentiality was maintained throughout the study period. Results: When asked about the water processing method, 76% of them told they do not treat/process the water supplied to them. In contrast, only a few PLHA told they would filter (17%), boil (7%), and use aquaguard (1%). The majority (67%) had their latrines, and while 29% of them were using public latrines and its hygienic sanitation was a concern for them and 4% were still practising open-air defecation in rural areas. Almost all study participants were handwashing after toilet use and handwashing before consuming food. The majority (79%) told they used soap and water, while 20% were using only water, soil and water (1%) and ash and water (1%) which was a concern. Of all the households, 87% cooked their food twice and consumed it thrice a day, while 4% prepared thrice. 10% of them cooked only once and consumed thrice a day; 56% practised consumption of leftover food of the previous night which was a concern. A majority (54%) did not consume street food while remaining said that they used to consume street food. But majority (59%) said they did not follow the habit of checking hygiene or sanitation of hotel where they used to consume food while the remaining 41% did not check the hotel before ordering the food; 50% of them bought packaged milk while 40.9% brought from unpasteurized dairy. On analysis, hygiene and sanitation factors had no statistically significant association with the nutritional status of study participants. Conclusion: WaSH factors act synergistically with other factors to affect the holistic health of PLHA. Information, Education and Communication (IEC) activities (continuous and regular), that focus on improving awareness level on WaSH practices, need to be promoted and integrated into HIV program, including providing basic care packages to PLHA like household water treatment products and soap, etc., Such measures will go a long way in maintaining health of PLHA even during ongoing COVID-19 pandemic scenario.

Stacking herbicide detoxification and resistant genes improves glyphosate tolerance and reduces phytotoxicity in tobacco (Nicotiana tabacum L.) and rice (Oryza sativa L.).

Glyphosate residues retained in the growing meristematic tissues or in grains of glyphosate-resistant crops affect the plants physiological functions and crop yield. Removing glyphosate residues in the plants is desirable with no penalty on crop yield and quality. We report a new combination of scientific strategy to detoxify glyphosate that reduces the residual levels and improve crop resistance. The glyphosate detoxifying enzymes Aldo-keto reductase (AKR1) and mutated glycine oxidase (mGO) with different modes of action were co-expressed with modified EPSPS, which is insensitive to glyphosate in tobacco (Nicotiana tabacum L.) and rice (Oryza sativa L.). The transgenic tobacco plants expressing individual PsAKR1, mGO, CP4-EPSPS, combinations of PsAKR1:CP4EPSPS, PsAKR1:mGO, and multigene with PsAKR1: mGO: CP4EPSPS genes were developed. The bio-efficacy studies of in-vitro leaf regeneration on different concentrations of glyphosate, seedling bioassay, and spray on transgenic tobacco plants demonstrate that glyphosate detoxification with enhanced resistance. Comparative analysis of the transgenic tobacco plants reveals that double and multigene expressing transgenics had reduced accumulation of shikimic acid, glyphosate, and its primary residue AMPA, and increased levels of sarcosine were observed in all PsAKR1 expressing transgenics. The multigene expressing rice transgenics showed improved glyphosate resistance with yield maintenance. In summary, results suggest that stacking genes with two different detoxification mechanisms and insensitive EPSPS is a potential approach for developing glyphosate-resistant plants with less residual content.

Investigation of the Captopril-Insulin Interaction by Mass Spectrometry and Computational Approaches Reveals that Captopril Induces Structural Changes in Insulin.

Post-translational modifications remarkably regulate proteins' biological function. Small molecules such as reactive thiols, metabolites, and drugs may covalently modify the proteins and cause structural changes. This study reports the covalent modification and noncovalent interaction of insulin and captopril, an FDA-approved antihypertensive drug, through mass spectrometric and computation-based approaches. Mass spectrometric analysis shows that captopril modifies intact insulin, reduces it into its "A" and "B" chains, and covalently modifies them by forming adducts. Since captopril has a reactive thiol group, it might reduce the insulin dimer or modify it by reacting with cysteine residues. This was proven with dithiothreitol treatment, which reduced the abundance of captopril adducts of insulin A and B chains and intact Insulin. Liquid chromatography tandem mass spectrometric analysis identified the modification of a total of four cysteine residues, two in each of the A and B chains of insulin. These modifications were identified to be Cys6 and Cys7 of the A chain and Cys7 and Cys19 of the B chain. Mass spectrometric analysis indicated that captopril may simultaneously modify the cysteine residues of intact insulin or its subunits A and B chains. Biophysical studies involving light scattering and thioflavin T assay suggested that the binding of captopril to the protein leads to the formation of aggregates. Docking and molecular dynamics studies provided insights into the noncovalent interactions and associated structural changes in insulin. This work is a maiden attempt to understand the detailed molecular interactions between captopril and insulin. These findings suggest that further investigations are required to understand the long-term effect of drugs like captopril.

Knee Osteoarthritis with Old Untreated Patella Fracture Treated with Single Stage Total Knee Arthroplasty - A Case Series of Two Cases.

Introduction: Total knee arthroplasty (TKA) is one of the most successful procedures for osteoarthritis (OA), but when associated with an old patella fracture, the knee arthroplasty becomes complex due to discontinuity in extensor mechanism. Moreover, in elderly patients, early mobilization and avoiding multiple surgeries are important to prevent associated morbidity. This is the first instance of a case series being reported where an untreated patella fracture >3-months-old with knee OA has been managed with single stage TKA. Case Report: Two patients, both with OA of knee and untreated patellar fractures, presented to us with knee pain. A 74-year-old lady having 6-months-old untreated right patella fracture without extensor lag was our first patient and a 57-years-old lady having 2-years-old patella fracture non-union with extensor lag of 30° was our second patient. Both were assessed and a decision to perform total knee replacement was taken. Patient with extensor lag required reconstruction of the extensor mechanism whereas the one without extensor lag was treated as a normal routine total knee replacement. Conclusion: Elderly patients with untreated patella fracture and degenerative knee OA can be considered for single stage TKA, with decision on patella management based on the presence or absence of extensor lag. Rehabilitation is faster with single surgery and also there are decreased risks and complications associated with multiple surgeries.