2.5A mechanistic study on the tolerance of PAM distal end mismatch by SpCas9.

The therapeutic application of CRISPR-Cas9 is limited due to its off-target activity. To have a better understanding of this off-target effect, we have focused on its mismatch-prone PAM distal end. The off-target activity of SpCas9 depends directly on the nature of mismatches, which in turn results in deviation of the active site of SpCas9 due to structural instability in the RNA-DNA duplex strand. In order to test the hypothesis, we have designed an array of mismatched target sites at the PAM distal end and performed in vitro and cell line-based experiments, which showed a strong correlation for Cas9 activity. We found that target sites having multiple mismatches in the 18th to 15th position upstream of the PAM showed no to little activity. For further mechanistic validation, Molecular Dynamics simulations were performed, which revealed that certain mismatches showed elevated root mean square deviation (RMSD) values that can be attributed to conformational instability within the RNA-DNA duplex. Therefore, for successful prediction of the off-target effect of SpCas9, along with complementation-derived energy, the RNA-DNA duplex stability plays a crucial role.

Exploration of Variable Solvent Directed Self-Healable Supramolecular M(II)-Metallogels (M = Co, Ni, Zn) of Azelaic Acid: Investigating Temperature-Dependent Ion Conductivity and Antibacterial Efficiency.

Molecular self-assembly assisted self-healing supramolecular metallogels of azelaic acid with cobalt(II)-, nickel(II)-, and zinc(II)-based metal acetate salts were successfully fabricated. Individually, N,N'-dimethylformamide and dimethyl sulfoxide were immobilized within these distinctly synthesized soft-scaffolds of metallogels to attain their semisolid viscoelastic nature. Rheological experiments such as amplitude sweep, frequency sweep, and thixotropic measurements were executed for these metallogels to ratify their gel features. The different extents of supramolecular interactions operating within these solvent-directed metallogels were clearly reflected in terms of their distinct morphological patterns as investigated through field emission scanning electron microscopy. Comparative infrared (IR) spectral properties of metallogels along with individual metal salts and azelaic acid were analyzed. These experimental data clearly depict the significant shifting of Fourier transform (FT)-IR peaks of xerogel samples of different metallogels from the gel-forming precursors. The networks present within the soft-scaffold are evidently illustrated by the electrospray ionization-mass experimental data. The temperature-dependent ionic conductivity studies with these solvent-directed versatile metallogel systems were investigated through impedance spectroscopy. The temperature-dependent impedance spectroscopic studies clearly demonstrate that the ion-transportation within the gel matrix depends not only on the types of cations but also on the dielectric properties of the immobilized solvents. The antipathogenic effect of these metallogel systems has also been explored by testing their effectiveness against human pathogenic Gram-negative bacteria Klebsiella pneumoniae (MTCC 109) and Vibrio parahemolyticus, and Gram-positive bacteria like Bacillus cereus (MTCC 1272). These gel soft-scaffolds show no significant cytotoxicity against both the human neuroblastoma cell line-SH-SY5Y and the human embryonic kidney cell line-HEK 293.

A Review on the Current Status of Homeopathy in the Clinical Manage-ment of Cancer.

Homeopathy is a widely practiced alternate system of medicine around the world that employs small doses of various medicines to promote auto-regulation and self-healing. It is among the most commonly used alternative approaches in cancer and other diseases and alternative therapeutic systems. It is widely used as palliative and as supportive therapy in cancer patients. Few cases have been reported on patients using homeopathy after surgery, radiotherapy, and chemotherapy, generally for overcoming side effects. The dose of Homoeopathic medicines and their mechanism of action in cancer has also been documented, while clinical trials on the effects of Homoeopathy in cancer treatment are rare. It is found that the anticancer potential of homeopathic medicines is reported for different cancer types, which show their efficacy through apoptosis and immune system modulation. Homeopathic treatment is an add-on to conventional therapy, with almost no interaction with the conventional drugs due to the small dose, and is largely attributed to improving lives by providing symptomatic relief, increasing survival time and boosting patient immunity. This review explores the accountability of the homeopathic system of medicine by highlighting some of the most commonly used homeopathic drugs for different types of cancers.

An insight into SARS-CoV-2 structure, pathogenesis, target hunting for drug development and vaccine initiatives.

SARS-CoV-2, the virus responsible for the COVID-19 pandemic, has been confirmed to be a new coronavirus having 79% and 50% similarity with SARS-CoV and MERS-CoV, respectively. For a better understanding of the features of the new virus SARS-CoV-2, we have discussed a possible correlation between some unique features of the genome of SARS-CoV-2 in relation to pathogenesis. We have also reviewed structural druggable viral and host targets for possible clinical application if any, as cases of reinfection and compromised protection have been noticed due to the emergence of new variants with increased infectivity even after vaccination. We have also discussed the types of vaccines that are being developed against SARS-CoV-2. In this review, we have tried to give a brief overview of the fundamental factors of COVID-19 research like basic virology, virus variants and the newly emerging techniques that can be applied to develop advanced treatment strategies for the management of COVID-19 disease.

A Short Review on Glucogallin and its Pharmacological Activities.

Plant derived natural products have multifaceted beneficial roles in human pathophysiology. Plant secondary metabolites have been used as an adjunct medicine for a long time and ß- Glucogallin is one such pharmaceutically important plant derived natural product. Β-glucogallin (1-O-galloyl-ß-d-glucopyranose), a plant-derived polyphenolic ester, is regarded as the primary metabolite in the biosynthesis of hydrolyzable tannins. It is majorly found in amla, pomegranate, strawberry etc. Owing to its free radical scavenging properties, ß-glucogallin (BG) is believed to protect against several diseases like diabetes and related complications like retinopathy, glaucoma, inflammation, hepatic damage, skin damage from UV, etc. Several semisynthetic derivatives of ß-Glucogallin are being developed, which have better pharmacokinetic and pharmacodynamic parameters than ß-glucogallin. Studies have shown the prophylactic role of ß-Glucogallin in developing defence mechanisms against the advent and progression of certain diseases. ß- glucogallin formulations have shown a positive effect as a neutraceutical. In this manuscript, we have discussed ß-glucogallin, its natural sources, biosynthetic pathways, its semi-synthetic derivatives, and the plethora of its pharmacological activities like antioxidant-antiinflammatory, antidiabetic, cataract-preventing, anti glaucoma, and UV protectant. We have also highlighted various biological pathways, which are modulated by ß-glucogallin. The manuscript will convey the importance of ß-glucogallin as a compound of natural origin, having multifaceted health benefits.

Cas13d: A New Molecular Scissor for Transcriptome Engineering.

The discovery of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and its associated Cas endonucleases in bacterial and archaeal species allowed scientists to modify, utilized, and revolutionize this tool for genetic alterations in any species. Especially the type II CRISPR-Cas9 system has been extensively studied and utilized for precise and efficient DNA manipulation in plant and mammalian systems over the past few decades. Further, the discovery of the type V CRISPR-Cas12 (Cpf1) system provides more flexibility and precision in DNA manipulation in prokaryotes, plants, and animals. However, much effort has been made to employ and utilize the above CRISPR tools for RNA manipulation but the ability of Cas9 and Cas12 to cut DNA involves the nuisance of off-target effects on genes and thus may not be employed in all RNA-targeting applications. Therefore, the search for new and diverse Cas effectors which can precisely detect and manipulate the targeted RNA begins and this led to the discovery of a novel RNA targeting class 2, type VI CRISPR-Cas13 system. The CRISPR-Cas13 system consists of single RNA-guided Cas13 effector nucleases that solely target single-stranded RNA (ssRNA) in a programmable way without altering the DNA. The Cas13 effectors family comprises four subtypes (a-d) and each subtype has distinctive primary sequence divergence except the two consensuses Higher eukaryotes and prokaryotes nucleotide-binding domain (HEPN) that includes RNase motifs i.e. R-X4-6-H. These two HEPN domains are solely responsible for executing targetable RNA cleavage activity with high efficiency. Further, recent studies have shown that Cas13d exhibits higher efficiency and specificity in cleaving targeted RNA in the mammalian system compared to other Cas13 endonucleases of the Cas13 enzyme family. In addition to that, Cas13d has shown additional advantages over other Cas13 variants, structurally as well as functionally which makes it a prominent and superlative tool for RNA engineering and editing. Therefore considering the advantages of Cas13d over previously characterized Cas13 subtypes, in this review, we encompass the structural and mechanistic properties of type VI CRISPR-Cas13d systems, an overview of the current reported various applications of Cas13d, and the prospects to improve Cas13d based tools for diagnostic and therapeutic purposes.

A Review on CRISPR-mediated Epigenome Editing: A Future Directive for Therapeutic Management of Cancer.

Recent studies have shed light on the role of epigenetic marks in certain diseases like cancer, type II diabetes mellitus (T2DM), obesity, and cardiovascular dysfunction, to name a few. Epigenetic marks like DNA methylation and histone acetylation are randomly altered in the disease state. It has been seen that methylation of DNA and histones can result in down-regulation of gene expression, whereas histone acetylation, ubiquitination, and phosphorylation are linked to enhanced expression of genes. How can we precisely target such epigenetic aberrations to prevent the advent of diseases? The answer lies in the amalgamation of the efficient genome editing technique, CRISPR, with certain effector molecules that can alter the status of epigenetic marks as well as employ certain transcriptional activators or repressors. In this review, we have discussed the rationale of epigenetic editing as a therapeutic strategy and how CRISPR-Cas9 technology coupled with epigenetic effector tags can efficiently edit epigenetic targets. In the later part, we have discussed how certain epigenetic effectors are tagged with dCas9 to elicit epigenetic changes in cancer. Increased interest in exploring the epigenetic background of cancer and non-communicable diseases like type II diabetes mellitus and obesity accompanied with technological breakthroughs has made it possible to perform large-scale epigenome studies.

Application of CRISPR/Cas System in the Metabolic Engineering of Small Molecules.

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated Cas protein technology area is rapidly growing technique for genome editing and modulation of transcription of several microbes. Successful engineering in microbes requires an emphasis on the aspect of efficiency and targeted aiming, which can be employed using CRISPR/Cas system. Hence, this type of system is used to modify the genome of several microbes such as yeast and bacteria. In recent years, CRISPR/Cas systems have been chosen for metabolic engineering in microbes due to their specificity, orthogonality, and efficacy. Therefore, we need to review the scheme which was acquired for the execution of the CRISPR/Cas system for the modification and metabolic engineering in yeast and bacteria. In this review, we highlighted the application of the CRISPR/Cas system which has been used for the production of small molecules in the microbial system that is chemically and biologically important.

A review on potential of natural products in the management of COVID-19.

At the end of 2019, a life threatening viral infection (COVID-19) caused by a novel coronavirus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) was reported. This virus has spread worldwide in a short duration and forced the world to face unprecedented life and economic loss. To date, there are no known specific drugs to combat this virus and the process for new drug development is lengthy. Most promising candidates, which emerged as potential leads, were abandoned in the later phases of clinical trials. Repurposing of already approved drugs for other therapeutic applications can be done only after extensive testing for safety and efficacy. With no definite therapeutics in the horizon, natural products are in extensive use arbitrarily as anti-viral agents and immune boosters. For ages it has been known that most natural products possess potent anti-viral activity and it is no different for SARS-CoV-2. It has been shown that natural products display inhibitory effects on MERS-CoV and SARS-CoV infections. In silico studies have shown that various natural products have strong binding affinity for and inhibitory action on the non-structural proteins of the virus, namely PLPRO, MPRO, and RdRp, and structural proteins such as spike (S) protein. Since the virus utilizes the transmembrane ACE2 receptor of the host cell, it also proves to be a valid target for drug development. In this review promising targets for drug development against SARS-CoV-2 and anti-viral activities of some of the known natural products are discussed.