CRISPR-Based Therapies: Revolutionizing Drug Development and Precision Medicine.

With the discovery of CRISPR-Cas9, drug development and precision medicine have undergone a major change. This review article looks at the new ways that CRISPR-based therapies are being used and how they are changing the way medicine is done. CRISPR technology's ability to precisely and flexibly edit genes has opened up new ways to find, validate, and develop drug targets. Also, it has made way for personalized gene therapies, precise gene editing, and advanced screening techniques, all of which hold great promise for treating a wide range of diseases. In this article, we look at the latest research and clinical trials that show how CRISPR could be used to treat genetic diseases, cancer, infectious diseases, and other hard-to-treat conditions. However, ethical issues and problems with regulations are also discussed in relation to CRISPR-based therapies, which shows how important it is to use them safely and responsibly. As CRISPR continues to change how drugs are made and used, this review shines a light on the amazing things that have been done and what the future might hold in this rapidly changing field.

Lipid-Polymer Hybrid Nanosystems: A Rational Fusion for Advanced Therapeutic Delivery.

Lipid nanoparticles (LNPs) are spherical vesicles composed of ionizable lipids that are neutral at physiological pH. Despite their benefits, unmodified LNP drug delivery systems have substantial drawbacks, including a lack of targeted selectivity, a short blood circulation period, and in vivo instability. lipid-polymer hybrid nanoparticles (LPHNPs) are the next generation of nanoparticles, having the combined benefits of polymeric nanoparticles and liposomes. LPHNPs are being prepared from both natural and synthetic polymers with various techniques, including one- or two-step methods, emulsification solvent evaporation (ESE) method, and the nanoprecipitation method. Varieties of LPHNPs, including monolithic hybrid nanoparticles, core-shell nanoparticles, hollow core-shell nanoparticles, biomimetic lipid-polymer hybrid nanoparticles, and polymer-caged liposomes, have been investigated for various drug delivery applications. However, core-shell nanoparticles having a polymeric core surrounded by a highly biocompatible lipid shell are the most commonly explored LPHNPs for the treatment of various diseases. In this review, we will shed light on the composition, methods of preparation, classification, surface functionalization, release mechanism, advantages and disadvantages, patents, and clinical trials of LPHNPs, with an emphasis on core-shell-structured LPHNPs.

Recent advances in ZnO nanostructure as a gas-sensing element for an acetone sensor: a short review.

Air pollution is a severe concern globally as it disturbs the health conditions of living beings and the environment because of the discharge of acetone molecules. Metal oxide semiconductor (MOS) nanomaterials are crucial for developing efficient sensors because of their outstanding chemical and physical properties, empowering the inclusive developments in gas sensor productivity. This review presents the ZnO nanostructure state of the art and notable growth, and their structural, morphological, electronic, optical, and acetone-sensing properties. The key parameters, such as response, gas detection limit, sensitivity, reproducibility, response and recovery time, selectivity, and stability of the acetone sensor, have been discussed. Furthermore, gas-sensing mechanism models based on MOS for acetone sensing are reported and discussed. Finally, future possibilities and challenges for MOS (ZnO)-based gas sensors for acetone detection have also been explored.

Chemical Biology of Sortase A Inhibition: A Gateway to Anti-infective Therapeutic Agents.

Staphylococcus aureus is the leading cause of hospital-acquired infections. The enzyme sortase A, present on the cell surface of S. aureus, plays a key role in bacterial virulence without affecting the bacterial viability. Inhibition of sortase A activity offers a powerful but clinically less explored therapeutic strategy, as it offers the possibility of not inducing any selective pressure on the bacteria to evolve drug-resistant strains. In this Perspective, we offer a chemical space narrative for the design of sortase A inhibitors, as delineated into three broad domains: peptidomimetics, natural products, and synthetic small molecules. This provides immense opportunities for medicinal chemists to alleviate the ever-growing crisis of antibiotic resistance.

Graphene oxide based electrochemical immunosensor for rapid detection of groundnut bud necrosis orthotospovirus in agricultural crops.

Groundnut bud necrosis orthotospovirus (GBNV) is one of the causative plant viruses responsible for the outbreak of many viral epidemics in food crops across India and other south-Asian countries. Its management is a major challenge due to fast vector transmission, and the non-availability of appropriate agrochemical treatment. The timely detection of GBNV becomes indispensable for the effective management of viral infection and the periodic monitoring of plant health. We report the fabrication of graphene oxide (GO) based electrochemical immunosensor for the rapid and sensitive detection of GBNV. The immunoelectrode is prepared by depositing GO onto indium-tin oxide (ITO) coated glass substrates and functionalized by anti-GBNV antibodies using N-ethyl-N'-(3- dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide (EDC-NHS) conjugation chemistry. The response measurements of the immunoelectrodes revealed a sensitivity of 221 ± 1 µA µg-1 mL-1(n = 3) and limit of detection (LOD) of 5.7 ± 0.7 ng mL-1(n = 3) for the standard concentrations of GBNV antigen. Further, the GBNV detection was carried out in infected leaf extracts of three different host plants i.e., Tomato, Cowpea, and N. benthamiana, and the results have been compared with the conventionally used direct antigen coated enzyme-linked immunosorbent assay (DAC-ELISA) technique. The comparable results obtained for the detection of GBNV in infected plants using electrochemical immunosensing and DAC-ELISA techniques advocated the immense potential of GO based immunosensor as a point-of-care sensing device that is poised to overcome the limitations of the traditional methods of virus detection in field conditions and may transform the diagnostics in agriculture.

Exploring role of probiotics and Ganoderma lucidum extract powder as solid carriers to solidify liquid self-nanoemulsifying delivery systems loaded with curcumin.

Solid self-nanoemulsifying drug delivery system (S-SENDDS) containing Curcumin (CRM) were prepared using combination of Ganoderma lucidum extract powder (GLEP) and probiotics (PB) as carriers. Liquid SNEDDS containing CRM were prepared by mixing Capmul MCM, Labrafil M1944CS, Tween 80 and Transcutol P. These were further spray dried and finally converted into spheroids. The droplet size of reconstituted S-SNEDDS powder and spheroids was found in the range of 35 to 37 nm, zeta potential in the range of - 21.48 to -23.22 mV and drug loading in the range of 95-96%. The release of drug from formulations was found to be more than 90%. Similarly, significant improvement (p < 0.05) in permeability of CRM was observed through SNEDDS using Caco2 cell lines. The non-significant difference (p> 0.05) in drug loading, droplet size, dissolution rate and angle of repose between L-SNEDDS and S-SNEDDS indicated the potential of GLEP-PB to produce stable SNEDDS.

Improving the shelf life of fresh-cut 'Royal Delicious' apple with edible coatings and anti-browning agents.

The objective of this study was to monitor overall quality and to extend the shelf life of fresh-cut apple wedges. Fresh-cut apple wedges were treated with anti-browning agents and edible coatings alone as well as in combination and packed in polypropylene trays. Packed apple wedges were stored at 5 ± 2 °C and monitored for physical (colour, physiological loss in weight and firmness), bio-chemical (ascorbic acid, total antioxidant, phenol, polyphenol oxidase and peroxidase enzymes) and microbial quality. In general carboxymethyl cellulose and aloe vera coatings along with anti-browning agents helped in preserving quality of apple slices during storage. Microbial load was significantly low for wedges coated with carboxymethyl cellulose and aloe vera. Polyphenol oxidase and peroxidase enzyme activity was also low in the coated samples. Firmness of the uncoated apple wedges declined more rapidly than the coated ones during storage. The results showed that carboxymethyl cellulose and aloe vera coating in combination with anti-browning agents improved the quality of stored apple wedges.

Differential response of immune-related genes to peptidoglycan and lipoteichoic acid challenge in vitro.

AIM: To study the effect of Staphylococcus aureus cell wall antigens, peptidoglycan (PGN) and lipoteichoic acid (LTA) challenge on immune cells present in bovine peripheral blood mononuclear cells (PBMCs). MATERIALS AND METHODS: In this study, efforts have been made to investigate the effects of three combinations (10+10, 20+20 and 30+30 µg/ml) of PGN and LTA obtained from S. aureus. These antigens were used to challenge the bovine PBMCs. After 6 h of incubation quantitative, real time-polymerase chain reaction was used to study toll-like receptor 2 (TLR-2) and major cytokine mRNA expression in bovine PBMC challenged with three different antigen blends. RESULTS: The results indicated that mRNA level of interferon gamma is influenced by the expression of TLR-2 gene. Tumor necrosis factor-alpha (TNF-α), interleukin 10 (IL-10), and IL-8 genes showed a maximum response at a dose of 10 µg of PGN and 10 µg of LTA challenge per ml of culture medium. The outcome also suggests that both IL-10 and IL-8 followed the expression pattern of TNF-α. CONCLUSION: A dose of 10 µg of PGN and 10 µg of LTA per ml of culture medium was found to be most suitable for challenging PBMC.

Overexpression of microRNA-30a inhibits hepatitis B virus X protein-induced autophagosome formation in hepatic cells.

Hepatitis B virus (HBV) enters the host and survives by using several mechanisms. One of the ways that HBV survives and replicates in the host cells is by inducing autophagy. Previous reports have shown that microRNA (miRNA)-30a inhibits autophagosome formation in cancer cells. Hence, we hypothesized that overexpression of miRNA-30a could inhibit HBV-induced autophagosome formation in hepatic cells. To study this, both HepG2 cells and HepG2.2.1.5 cells (HBV-expressing stable cell line) were transfected with miRNA-30a, and the cells were collected either for RNA isolation or protein isolation after 72 h of transfection. Beclin-1 expression was significantly higher in untransfected HepG2.2.1.5 cells than in HepG2 cells. Western blots showed that miRNA-30a overexpression resulted in a significant decrease in beclin-1 expression (eight-fold and four-fold in HepG2 and HepG2.2.1.5 cells, respectively) and c-myc expression, whereas the numbers of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells were increased. In contrast, overexpression of HBV X protein (HBx) in HepG2 cells resulted in the enhancement of beclin-1 (six-fold increase as compared with the empty vector-transfected cells) and c-myc expression, whereas the numbers of TUNEL-positive cells were reduced. To confirm these findings, HBx and miRNA-30a were coexpressed in HepG2 cells, and the results showed significant inhibition of autophagosome formation and beclin-1 and c-myc expression, whereas apoptosis increased. These data demonstrate that HBx induces autophagosome formation via beclin-1 expression, whereas miRNA-30a overexpression could successfully inhibit the beclin-1 expression induced by HBx, thereby modulating autophagosome formation in hepatic cells.

Association and expression analysis of single nucleotide polymorphisms of partial tumor necrosis factor alpha gene with mastitis in crossbred cattle.

A total of 129 crossbred cows were selected to explore the genotypic and expression profiling of partial TNF-α gene and its association with mastitis susceptibility. Two exon spanning region of TNF-α gene (221 bp and 239 bp) were amplified by Polymerase Chain Reaction (PCR). The different genotypic analysis by SSCP revealed that 221 bp fragment was monomorphic, whereas 239 bp was polymorphic. Association studies revealed that AA genotypes of 239 bp were more prevalent in mastitis group and the mRNA expression of TNF-α was significantly (P < 0.05) higher in AA genotypic animals compare to AB and BB. This suggested that genotypes AB and BB may be used as candidate markers for mastitis resistance selection in dairy cattle.

Polymorphism of exon 2-3 of bovine major histocompatibility complex class I BoLa-A gene

The exon 2-3 region of bovine major histocompatibility complex (MHC) class I BoLa-A gene was investigated for polymorphisms in three breeds of cattle originated in the Indian subcontinent namely Sahiwal, Tharparkar, Hariana, as well as crossbred (Bos taurus x Bos indicus) cattle and Jersey, the exotic breed (Bos taurus). The PCR amplified fragment of 714 bp showed distinct DdeI-, TaqI- and HinfI- RFLP patterns, thus confirming a higher degree of polymorphism in this region. To our knowledge this is the first report of HinfI restriction patterns for BoLa-A exon 2-3. The sequencing results revealed a number of nucleotide substitutions in this region, which resulted in amino acid changes. The present investigation confirmed that MHC class I BoLa-A exon 2-3 is highly polymorphic in cattle.

DNA polymorphism of leptin gene in Bos indicus and Bos taurus cattle

Leptin plays an important role in the regulation of feed intake, energy metabolism, growth and reproduction of cattle. We used the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique to screen for DNA polymorphisms of the leptin gene in 403 cattle belonging to various breeds of Bos indicus (Hariana, Sahiwal, Gir and Nimari cattle), Bos taurus (Holstein Friesian (HF) and Jersey cattle) as well as Bos taurus x Bos indicus crossbreds (½ HF x ½ Hariana). In all the cattle we amplified two regions of the leptin gene, a 522 bp fragment comprising the partial intron 2 and exon 3 and another 94 bp fragment consisting of part of exon 2. Digestion of 522 bp PCR products with the BsaAI restriction enzyme revealed three genotypes in all the breeds of cattle studied. This is the first report of the presence of leptin gene polymorphism in purebred Bos indicus cattle of Indian origin (indicine cattle). Almost similar gene and genotype frequencies were observed in all the breed groups, while the frequency of mutant homozygotes (AA) was very low (0.03 to 0.07). On digestion of the 94 bp fragment with the Kpn2I restriction enzyme, three genotypes were observed in HF, Jersey and crossbred cattle. The CC genotype had the highest frequency (0.68) in crossbreds whereas the frequency of CT heterozygotes was highest (0.69) in HF cattle. This mutation was absent in all the breeds of indicine cattle. The results suggest that the BsaAI-RFLP mutation has occurred far back in evolution before the divergence of taurine and indicine cattle while the Kpn2I mutation has occurred recently as indicated by the fact that this mutation was only detected in taurine cattle.