Spatio-temporal dynamics of intra-host variability in SARS-CoV-2 genomes.

During the course of the COVID-19 pandemic, large-scale genome sequencing of SARS-CoV-2 has been useful in tracking its spread and in identifying variants of concern (VOC). Viral and host factors could contribute to variability within a host that can be captured in next-generation sequencing reads as intra-host single nucleotide variations (iSNVs). Analysing 1347 samples collected till June 2020, we recorded 16 410 iSNV sites throughout the SARS-CoV-2 genome. We found ∼42% of the iSNV sites to be reported as SNVs by 30 September 2020 in consensus sequences submitted to GISAID, which increased to ∼80% by 30th June 2021. Following this, analysis of another set of 1774 samples sequenced in India between November 2020 and May 2021 revealed that majority of the Delta (B.1.617.2) and Kappa (B.1.617.1) lineage-defining variations appeared as iSNVs before getting fixed in the population. Besides, mutations in RdRp as well as RNA-editing by APOBEC and ADAR deaminases seem to contribute to the differential prevalence of iSNVs in hosts. We also observe hyper-variability at functionally critical residues in Spike protein that could alter the antigenicity and may contribute to immune escape. Thus, tracking and functional annotation of iSNVs in ongoing genome surveillance programs could be important for early identification of potential variants of concern and actionable interventions.

Methods to generate and evaluate zebrafish models of human kidney diseases.

Kidney-related disorders affect millions of people worldwide. A survey of chronic kidney disease (CKD) patients showed that the burden of kidney diseases is increasing every year. The global burden of disease (GBD) study 2017 ranked CKD as the 12th leading cause of deaths worldwide. Hence, identification of the causes of kidney diseases, development of accurate diagnostic methods and novel therapeutics is highly relevant. Model organisms that faithfully recapitulate human diseases play important roles in understanding the disease process and provide valuable ground to find their cure. Zebrafish is an excellent model to study the development, pathophysiology and molecular aspects of human kidney diseases. In this review, we summarize various genetic and experimental manipulations that can be carried out in zebrafish to better understand the pathophysiology of human kidney diseases. We suggest that these methods will be helpful in the development of potential therapies to treat kidney diseases.

Expression of two uncharacterized protein coding genes in zebrafish lateral line system.

The lateral line system is a mechanosensory organ of fish and amphibians that detects changes in water flow and is formed by the coordinated action of many signalling pathways. These signalling pathways can easily be targeted in zebrafish using pharmacological inhibitors to decipher their role in lateral line system development at cellular and molecular level. We have identified two uncharacterized proteins, whose mRNA are expressed in the lateral line system of zebrafish. One of these proteins, uncharacterized protein LOC564095 precursor, is conserved across vertebrates and its mRNA is expressed in posterior lateral line primordium (pLLP). The other uncharacterized protein, LOC100536887, is present only in the teleost fishes and its mRNA is expressed in neuromasts. We show that inhibition of retinoic acid (RA) signalling reduces the expression of both of these uncharacterized genes. It is reported that inhibition of RA signalling during gastrulation starting at 7 hours post fertilization (hpf) abrogates pLLP formation, and inhibition of RA signalling at 10 hpf delays the initiation of pLLP migration. Here, we show that inhibition of RA signalling before and during segmentation (9-16 hpf) results in delayed initiation and reduced speed of pLLP migration, as well as inhibition of posterior neuromasts formation.

Enantioselective analysis of D- and l- Serine on a layer-by-layer imprinted electrochemical sensor.

The present work describes a new, simple, and easy method of generating acrylamide functionalised reduced graphene oxide-fullerene layer-by-layer assembled dual imprinted polymers to quantify D- and L-Serine at ultra trace level in aqueous and real samples. Herein, the pencil graphite electrode was initially spin coated with D-Serine imprinted acrylamide functionalized reduced graphene oxide. After 10 min thermal treatment (50 °C), this electrode was again modified with L-Serine imprinted acrylamide functionalized fullerene molecules. This bilayer assembly was finally made thermally stable by 60 °C exposure for 3 h. The proposed sensor showed better electronic properties with an improved synergism. We have compared this modified electrode with other modified pencil graphite electrodes like single layered acrylamide functionalised reduced graphene oxide or fullerene, single layered acrylamide functionalised reduced graphene oxide-fullerene composite and double layered acrylamide functionalised reduced graphene oxide or fullerene molecules, which yielded very inferior sensitivity due to possible agglomeration and decreased synergism. The chosen system demonstrated a very good analytical figures of merit with differential pulse anodic stripping voltammetry and cyclic voltammetry transduction, showing lower limits of detection (0.24 ng mL-1, S/N = 3) for both isomers. The proposed sensor assures practical applications as disease biomarker, manifesting several diseases at very ultra-trace level.

Electrochemical simultaneous analysis of dopamine and epinephrine using double imprinted One MoNomer acryloylated graphene oxide-carbon black composite polymer.

A novel One MoNomer dual imprinted graphene oxide/carbon black composite polymer was developed applying 'surface-grafting from' approach on the screen printed carbon electrode for the electrochemical sensing of dopamine and epinephrine. Acryloylated-graphene oxide/carbon black was synthesized for the first time. This served both as a crosslinker and monomer leading to the fast electron transfer from the redox centre to the electrode. The oxidation peak potentials of both the targets were found separated by 200 mV which enabled their simultaneous analysis in real world samples, without any cross reactivity, interferences, and false-positives. The detection limits realized by the proposed sensor, under optimized analytical conditions, were found to be as low as 0.028, 0.028,0.061 and 0.029 ng mL-1 for dopamine and 0.017, 0.018, 0.019 and 0.020 ng mL-1 for epinephrine (S/N = 3) in aqueous, blood serum, urine and pharmaceutical samples. Such sensor could be considered suitable for the primitive diagnosis of several chronic diseases, manifested at ultra-trace level.

One-by-one imprinting in two eccentric layers of hollow core-shells: Sequential electroanalysis of anti-HIV drugs.

Double layered one-by-one imprinted hollow core-shells@ pencil graphite electrode was fabricated for sequential sensing of anti-HIV drugs. For this, two eccentric layers were developed on the surface of vinylated silica nanospheres to obtain double layered one-by-one imprinted solid core-shells. This yielded hollow core-shells on treatment with hydrofluoric acid. The modified hollow core-shells (single layered dual imprinted) evolved competitive diffusion of probe/analyte molecules. However, the corresponding double layered one-by-one imprinted hollow core-shells (outer layer imprinted with Zidovudine, and inner layer with Lamivudine) were found relatively better owing to their bilateral diffusions into molecular cavities, without any competition. The entire work is based on differential pulse anodic stripping voltammetry at double layered one-by-one imprinted hollow core-shells. This resulted in indirect detection of electro inactive targets with limits of detection as low as 0.91 and 0.12 (aqueous sample), 0.94 and 0.13 (blood serum), and 0.99 and 0.20 ng mL-1 (pharmaceutics) for lamivudine and zidovudine, respectively in anti-HIV drug combination.

Effect of Different Storage Conditions on Analytical and Sensory Quality of Thermally Processed, Milk-Based Germinated Foxtail Millet Porridge.

Foxtail millet porridge was prepared using germinated grains and milk and was evaluated for its storage stability after thermal processing at ultra-high temperatures (UHT) of 142 °C for 5 s and retort processing temperatures of 121.5 °C for 15 min. Various physical, chemical, and microbial changes of the porridge were studied for a storage period of 180 days at 25 ± 1 °C. Using consumer perception and survival analysis, the predicted shelf life of the UHT treated and retort processed foxtail millet porridge samples stored at 25 ± 1 °C was found to be 186 ± 9 days and 245 ± 15 days, respectively. Also, data from consumer liking, profiling, physical, chemical, and microbial parameters showed significant changes (P < 0.05) in the thermally treated packaged porridge samples over time. As the consumer overall acceptability decreased, the detection of positive attributes (thick and uniformly colored texture and appearance; grainy mouth texture; caramel taste and aroma) in the porridge decreased, while the detection of negative attributes (uneven, decolored, and curdled texture and appearance; sticky mouth texture; cooked, sour and off smell; cooked, sour and off taste) increased. The present study could establish a significant difference (P < 0.05) in the storage induced properties of UHT and retort processed porridge samples. The analytical evaluation of foxtail millet porridge found that UHT treated porridge was better in quality, but consumers preferred retort processed porridge. PRACTICAL APPLICATION: The quality and sensory attributes, evaluated for UHT treated and retort processed porridge samples during the storage period of 180 days, were found to be contradictory. Based on the results of CATA sensory analysis, the shelf life of UHT treated and retort processed porridge samples was predicted to be more than 6 months. Therefore, both UHT treatment and retort processing can be effectively applied to prepare a ready to eat milk based porridge using germinated foxtail millet grains.

Inhibiting Effect of Zinc Oxide Nanoparticles on Advanced Glycation Products and Oxidative Modifications: a Potential Tool to Counteract Oxidative Stress in Neurodegenerative Diseases.

Advanced glycation end products (AGEs) are implicated in several central nervous system (CNS) pathologies including Alzheimer and Parkinson's diseases. In the face-off of AGE menace, we have attempted to investigate the zinc oxide nanoparticle (ZnONP) role in inhibition of AGE formation. Synthesized ZnONPs were used to investigate the inhibitory effects on AGE formation. The inhibitory effects of ZnONPs on AGE formation were determined by biophysical immunological and biochemical techniques. The results showed that ZnONP is a potential anti-glycating agent inhibiting AGE formation as well as protecting the protein structure from change. Therefore, our findings suggest ZnONPs may be used as a therapeutic in resolving the AGE role in CNS-related complications.