Significance of Addressal of Clinical Investigations of Kidney Functions in Recovery/Mortality of Certain COVID-19 Patients

Clinical management of COVID-19 patients through a robust protocol is key to the good recovery and reduced mortality of patients. Efficient kidney functions during treatment period can contribute for improvised recovery and reduced mortality of patients. Analysis of the kidney function among Recovered and Dead cases of COVID-19 was made to reveal the degree of association of kidney functions with the two categories of patients. 83.4% of recovered patients did not show hyper values of blood urea whereas 72.5% of dead patients showed hyper-urea level in blood. 88.8% of survivors showed non-hyper creatinine level of blood whereas only 40% of dead cases showed hyper creatine level. Strong degree of association of blood urea with recovery/mortality was observed. Sodium levels were seen to be low while potassium and chloride ions were seen to be high in COVID-19 individuals. Our preliminary study suggests that kidney functions especially the value of blood urea and creatinine need to be addressed during COVID-19 patients to ensure the best recovery and reduced mortality. After more number of case studies, the present observation could sensitize consideration for inclusion of addressal and treatment of kidney functions into treatment protocol against COVID-19. It was also interesting to observe that levels of sodium and potassium ions among Survivors and Dead cases have impacted function of the essential ion channels in patient’s physiology.

Alternative splicing level related to intron size and organism complexity

Background Alternative splicing is the process of selecting different combinations of splice sites to produce variably spliced mRNAs. However, the relationships between alternative splicing prevalence and level (ASP/L) and variations of intron size and organism complexity (OC) remain vague. Here, we developed a robust protocol to analyze the relationships between ASP/L and variations of intron size and OC. Approximately 8 Tb raw RNA-Seq data from 37 eumetazoan species were divided into three sets of species based on variations in intron size and OC. Results We found a strong positive correlation between ASP/L and OC, but no correlation between ASP/L and intron size across species. Surprisingly, ASP/L displayed a positive correlation with mean intron size of genes within individual genomes. Moreover, our results revealed that four ASP/L-related pathways contributed to the differences in ASP/L that were associated with OC. In particular, the spliceosome pathway displayed distinct genomic features, such as the highest gene expression level, conservation level, and fraction of disordered regions. Interestingly, lower or no obvious correlations were observed among these genomic features. Conclusions The positive correlation between ASP/L and OC ubiquitously exists in eukaryotes, and this correlation is not affected by the mean intron size of these species. ASP/L-related splicing factors may play an important role in the evolution of OC.

Observational case studies of the effect of phage laden Ganga water on psoriasis

Water of Ganga river is reported to have more than 200 isolates of phages. This study has used the naturally available cocktail of phages in the Ganga water as a treatment for chronic Psoriasis. In the conventional Phage Therapy (PT) phages that are active against specific bacteria are first identified; then isolated, multiplied and administered to the patient. We have made a novel innovation of administering the naturally available cocktail of phages in the water of the Ganga river without first identifying the target bacteria and isolating specific phages that may be active against them. In doing so, we enable the large numbers of phages to self-identify the bacteria that are present and act against them. This approach shortcuts the tortuous process of conventional PT. Further, the phages act against a number of bacteria simultaneously and provide good results in psoriasis which has multiple causes.Patients who took Ganga water for only 2 weeks showed benefit but the benefit did not sustain after stoppage of the treatment and the disease relapsed to the pre-treatment levels. The same patients showed sustained benefit after they took Ganga water for four weeks. Conclusion is that Ganga water can be used for therapeutic purposes as long as the treatment is continued for at least four weeks. The study underscores the need to establish more robust protocol for treatment of dermatological and possibly other diseases with the cocktail of phages available in the waters of the Ganga river.

Statistical Evaluation of Total Expiratory Breath Samples Collected throughout a Year: Reproducibility and Applicability toward Olfactory Sensor-Based Breath Diagnostics

The endogenous volatile organic compounds (VOCs) in exhaled breath can be promising biomarkers for various diseases including cancers. An olfactory sensor has a possibility for extracting a specific feature from collective variations of the related VOCs with a certain health condition. For this approach, it is important to establish a feasible protocol for sampling exhaled breath in practical conditions to provide reproducible signal features. Here we report a robust protocol for the breath analysis, focusing on total expiratory breath measured by a Membrane-type Surface stress Sensor (MSS), which possesses practical characteristics for artificial olfactory systems. To assess its reproducibility, 83 exhaled breath samples were collected from one subject throughout more than a year. It has been confirmed that the reduction of humidity effects on the sensing signals either by controlling the humidity of purging room air or by normalizing the signal intensities leads to reasonable reproducibility verified by statistical analyses. We have also demonstrated the applicability of the protocol for detecting a target material by discriminating exhaled breaths collected from different subjects with pre- and post-alcohol ingestion on different occasions. This simple yet reproducible protocol based on the total expiratory breath measured by the MSS olfactory sensors will contribute to exploring the possibilities of clinical applications of breath diagnostics.

A Robust Protocol for Decellularized Human Lung Bioink Generation Amenable to 2D and 3D Lung Cell Culture

Decellularization efforts must balance the preservation of the extracellular matrix (ECM) components while eliminating the nucleic acid and cellular components. Following effective removal of nucleic acid and cell components, decellularized ECM (dECM) can be solubilized in an acidic environment with the assistance of various enzymes to develop biological scaffolds in different forms, such as sheets, tubular constructs, or three-dimensional (3D) hydrogels. Each organ or tissue that undergoes decellularization requires a distinct and optimized protocol to ensure that nucleic acids are removed, and the ECM components are preserved. The objective of this study was to optimize the decellularization process for dECM isolation from human lung tissues for downstream 2D and 3D cell culture systems. Following protocol optimization and dECM isolation, we performed experiments with a wide range of dECM concentrations to form human lung dECM hydrogels that were physically stable and biologically responsive. The dECM based-hydrogels supported the growth and proliferation of primary human lung fibroblast cells in 3D cultures. The dECM is also amenable to the coating of polyester membranes in Transwell™ Inserts to improve the cell adhesion, proliferation, and barrier function of primary human bronchial epithelial cells in 2D. In conclusion, we present a robust protocol for human lung decellularization, generation of dECM substrate material, and creation of hydrogels that support primary lung cell viability in 2D and 3D culture systems

Genotyping by Multiplexed Sequencing (GMS) protocol in Barley

Genotyping by sequencing (GBS) and single nucleotide polymorphism (SNP) chip technologies are the primary SNP genotyping technologies used today. However, these genotyping technologies have some drawbacks that limit their usefulness in analysis. We have developed a robust protocol called genotyping by multiplexed sequencing (GMS) using SNP markers, providing informative genotypic data with greater flexibility. The genotypes derived from direct sequence reads reduce ambiguity in genetic analysis. The advantages of this protocol include: (1) This PCR-based direct sequencing protocol generates information from markers of interest and provides a more streamlined and accurate analysis process, by multiplexing hundreds of informative markers into a single sequencing run. (2) The marker sets are easily customized to the species of interest and can readily be changed. In this study we have taken the GMS protocol developed in wheat and adapted it to barley. We have identified 577 SNP markers that work well using this protocol providing adequate genome coverage for genomic selection and tag 267 QTL’s for genes of interest. Good markers have an adequate read depth of at least 5 amplicons and are reliably present across the population.

A cookbook for DNase Hi-C

Background The Hi-C technique is widely employed to study the 3-dimensional chromatin architecture and to assemble genomes. The conventional in situ Hi-C protocol employs restriction enzymes to digest chromatin, which results in nonuniform genomic coverage. Using sequence-agnostic restriction enzymes, such as DNAse I, could help to overcome this limitation. Results In this study, we compare different DNAse Hi-C protocols and identify the critical steps that significantly affect the efficiency of the protocol. In particular, we show that the SDS quenching strategy strongly affects subsequent chromatin digestion. The presence of biotinylated oligonucleotide adapters may lead to ligase reaction by-products, which can be avoided by rational design of the adapter sequences. Moreover, the use of nucleotide-exchange enzymes for biotin fill-in enables simultaneous labelling and repair of DNA ends, similar to the conventional Hi-C protocol. These improvements simplify the protocol, making it less expensive and time-consuming. Conclusions We propose a new robust protocol for the preparation of DNAse Hi-C libraries from cultured human cells and blood samples supplemented with experimental controls and computational tools for the evaluation of library quality.