Long-Term Evaluation of Retinal Morphology and Function in Rosa26-Cas9 Knock-In Mice.

The CRISPR/Cas9 system is a robust, efficient, and cost-effective gene editing tool widely adopted in translational studies of ocular diseases. However, in vivo CRISPR-based editing in animal models poses challenges such as the efficient delivery of the CRISPR components in viral vectors with limited packaging capacity and a Cas9-associated immune response. Using a germline Cas9-expressing mouse model would help to overcome these limitations. Here, we evaluated the long-term effects of SpCas9 expression on retinal morphology and function using Rosa26-Cas9 knock-in mice. We observed abundant SpCas9 expression in the RPE and retina of Rosa26-Cas9 mice using the real-time polymerase chain reaction (RT-PCR), Western blotting, and immunostaining. SD-OCT imaging and histological analysis of the RPE, retinal layers, and vasculature showed no apparent structural abnormalities in adult and aged Cas9 mice. Full-field electroretinogram of adult and aged Cas9 mice showed no long-term functional changes in the retinal tissues because of constitutive Cas9 expression. The current study showed that both the retina and RPE maintain their phenotypic and functional features in Cas9 knock-in mice, establishing this as an ideal animal model for developing therapeutics for retinal diseases.

Biomolecules of mushroom: a recipe of human wellness.

The Indian system of medicine - Ayurveda says "When diet is wrong, medicine is of no use. When diet is correct, medicine is of no use". In this context, mushroom constitutes one of the major resources for nutraceuticals. Biomolecules of mushrooms have attracted the attention of researchers around the globe due to their proven healthy attributes. They have a plenitude of health-giving properties and these range from immunomodulatory, antiviral, antibacterial, antifungal, antioxidant, anti-inflammatory, antitumor, anticancer, anti-HIV, antidiabetic, anticholesterolic to antiarthritic activities.Mushrooms contain both primary and secondary metabolites. The primary metabolites provide energy while the secondary metabolite exhibits medicinal properties. Hence, the mushroom can be a recipe for human wellness and will play a significant role in fighting COVID-19 pandemics and other infectious diseases.The key findings suggested in this paper refer to the exploration of health and the healing traits of biomolecules of mushrooms. This article reviews the current status of the medicinal attributes of mushrooms and their biomolecules in different diseases such as cardiovascular, diabetes, reproductive diseases, cancer, and neurodegenerative diseases. The global malnutrition-related morbidity and mortality among children under five and lactating women presents a frightening picture and also a black spot on the human face. Malnutrition is responsible for more ill-health than any other cause. Mushrooms as a rich source of bioactive compounds can be claimed as "Best from the Waste" since they grow on the most abundant organic wastes of the Earth, the lignocellulosic substrate, and 'Best of the Rest' because they are excellent nutraceutical resources.

ABCB6 mutations cause ocular coloboma.

Ocular coloboma is a developmental defect of the eye and is due to abnormal or incomplete closure of the optic fissure. This disorder displays genetic and clinical heterogeneity. Using a positional cloning approach, we identified a mutation in the ATP-binding cassette (ABC) transporter ABCB6 in a Chinese family affected by autosomal-dominant coloboma. The Leu811Val mutation was identified in seven affected members of the family and was absent in six unaffected members from three generations. A LOD score of 3.2 at θ = 0 was calculated for the mutation identified in this family. Sequence analysis was performed on the ABCB6 exons from 116 sporadic cases of microphthalmia with coloboma (MAC), isolated coloboma, and aniridia, and an additional mutation (A57T) was identified in three patients with MAC. These two mutations were not present in the ethnically matched control populations. Immunostaining of transiently transfected, Myc-tagged ABCB6 in retinal pigment epithelial (RPE) cells showed that it localized to the endoplasmic reticulum and Golgi apparatus of RPE cells. RT-PCR of ABCB6 mRNA in human cell lines and tissue indicated that ABCB6 is expressed in the retinae and RPE cells. Using zebrafish, we show that abcb6 is expressed in the eye and CNS. Morpholino knockdown of abcb6 in zebrafish produces a phenotype characteristic of coloboma and replicates the clinical phenotype observed in our index cases. The knockdown phenotype can be corrected with coinjection of the wild-type, but not mutant, ABCB6 mRNA, suggesting that the phenotypes observed in zebrafish are due to insufficient abcb6 function. Our results demonstrate that ABCB6 mutations cause ocular coloboma.

Mutational analysis and genotype-phenotype correlations in southern Indian patients with sporadic and familial aniridia.

PURPOSE: Aniridia is a rare panocular disorder characterized by iris hypoplasia and other associated eye anomalies. Heterozygous null mutations in paired box gene 6 (PAX6) are the major cause of the classic aniridia phenotype. This study aims to detect the mutational spectrum of PAX6 and associated phenotypes in southern Indian patients with sporadic and familial aniridia. METHODS: Genomic DNA was isolated from peripheral blood from all participants. The coding regions and flanking intronic sequences of PAX6 were screened with Sanger sequencing in 30 probands with aniridia. The identified variations were further evaluated in available family members and 150 healthy controls. The pathogenic potential of the mutations were assessed using bioinformatics tools. RESULTS: Thirteen different mutations were detected in eight sporadic and five familial cases. Eleven novel mutations, including five insertions (c.7_10dupAACA, c.567dupC, c.704dupC, c.868dupA and c.753_754insTA), two deletions (c.242delC and c.249delT), and four splicing variants (c.10+1G>A, c.141G>A, c.141+4A>G and c.764A>G) were identified in this study. Clinical findings of the patients revealed phenotypic heterogeneity with the same or different mutations. CONCLUSIONS: This study reported 11 novel mutations and thus expanded the spectrum of PAX6 mutations. Interestingly, all mutations reported in this study were truncations, which confirms the hypothesis that haploinsufficiency of PAX6 causes the aniridia phenotype. Our observations revealed inter- and intrafamilial phenotypic variability with PAX6 mutations. The common ocular findings associated with PAX6 mutations were iris hypoplasia, nystagmus, and foveal hypoplasia reported in almost all cases, with cataract, glaucoma, and keratopathy reported in approximately 50% of the patients.

Unraveling Histone Loss in Aging and Senescence.

As the global population experiences a notable surge in aging demographics, the need to understand the intricate molecular pathways exacerbated by age-related stresses, including epigenetic dysregulation, becomes a priority. Epigenetic mechanisms play a critical role in driving age-related diseases through altered gene expression, genomic instability, and irregular chromatin remodeling. In this review, we focus on histones, a central component of the epigenome, and consolidate the key findings of histone loss and genome-wide redistribution as fundamental processes contributing to aging and senescence. The review provides insights into novel histone expression profiles, nucleosome occupancy, disruptions in higher-order chromatin architecture, and the emergence of noncanonical histone variants in the aging cellular landscape. Furthermore, we explore the current state of our understanding of the molecular mechanisms of histone deficiency in aging cells. Specific emphasis is placed on highlighting histone degradation pathways in the cell and studies that have explored potential strategies to mitigate histone loss or restore histone levels in aging cells. Finally, in addressing future perspectives, the insights gained from this review hold profound implications for advancing strategies that actively intervene in modulating histone expression profiles in the context of cellular aging and identifying potential therapeutic targets for alleviating a multitude of age-related diseases.

Evaluation of Pleurotus Mushroom Effects on Histopathological Changes in Organs of Diabetic Rats.

Diabetes mellitus (DM) is a metabolic disorder that can be categorized mainly into type 1 and type 2. Diabetes type 1 is caused due to ß-cell destruction, whereas type 2 is caused by the resistance of cell receptors. Many therapies are available for the management of diabetes, but they have some side effects, and as a result of this, people are attracted to natural treatments. Pleurotus mushrooms are well documented for their medicinal attributes and their role in the treatment of diseases like cancer, infectious disease, neurodiseases, and inflammatory disease. The protective mechanism of the Pleurotus fossulatus (P. fossulatus) mushroom and its detailed histological study on kidneys and the liver in diabetic conditions were unexplored. The present study evaluated the effects of P. fossulatus aqueous extract on histological changes in the diabetic rat model. Male Wistar albino rats were used to create the diabetic model by using streptozotocin (STZ) intraperitoneal (IP) injection. The animals were separated into five different groups, with six animals in each. Only group I, animals that did not receive STZ, was considered a normal control. Group II was a diabetic control and received normal saline, and group III was a drug control and received metformin as a standard drug. Groups IV and V were dosing groups, which received the aqueous extract of P. fossulatus in 250 mg/kg and 500 mg/kg of body weight concentrations, labeled as T1 and T2 groups, respectively. The T1 and T2 groups clearly showed their potential to reverse the histopathological changes in the kidney and liver. However, the T2 group was more effective than the T1 group, as results indicate that functions of the glomerulus and its structural deformity were restored to their near-natural form in the T2 group. In the case of the liver, the histological changes like the dilatation of sinusoids, more numbers of the Kupffer cell formation, and necrosis were restored in the T2 group. All these results proved the potential of P. fossulatus against the side effects of diabetes. It could protect the organs from developing diabetic nephropathy (DN) and liver-related diseases like cirrhosis and nonalcoholic fatty liver disease (NAFLD).

Computational exploration of the dual role of the phytochemical fortunellin: Antiviral activities against SARS-CoV-2 and immunomodulatory abilities against the host.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections generate approximately one million virions per day, and the majority of available antivirals are ineffective against it due to the virus's inherent genetic mutability. This necessitates the investigation of concurrent inhibition of multiple SARS-CoV-2 targets. We show that fortunellin (acacetin 7-O-neohesperidoside), a phytochemical, is a promising candidate for preventing and treating coronavirus disease (COVID-19) by targeting multiple key viral target proteins. Fortunellin supports protective immunity while inhibiting pro-inflammatory cytokines and apoptosis pathways and protecting against tissue damage. Fortunellin is a phytochemical found in Gojihwadi kwath, an Indian traditional Ayurvedic formulation with an antiviral activity that is effective in COVID-19 patients. The mechanistic action of its antiviral activity, however, is unknown. The current study comprehensively evaluates the potential therapeutic mechanisms of fortunellin in preventing and treating COVID-19. We have used molecular docking, molecular dynamics simulations, free-energy calculations, host target mining of fortunellin, gene ontology enrichment, pathway analyses, and protein-protein interaction analysis. We discovered that fortunellin reliably binds to key targets that are necessary for viral replication, growth, invasion, and infectivity including Nucleocapsid (N-CTD) (-54.62 kcal/mol), Replicase-monomer at NSP-8 binding site (-34.48 kcal/mol), Replicase-dimer interface (-31.29 kcal/mol), Helicase (-30.02 kcal/mol), Papain-like-protease (-28.12 kcal/mol), 2'-O-methyltransferase (-23.17 kcal/mol), Main-protease (-21.63 kcal/mol), Replicase-monomer at dimer interface (-22.04 kcal/mol), RNA-dependent-RNA-polymerase (-19.98 kcal/mol), Nucleocapsid-NTD (-16.92 kcal/mol), and Endoribonuclease (-16.81 kcal/mol). Furthermore, we identify and evaluate the potential human targets of fortunellin and its effect on the SARS-CoV-2 infected tissues, including normal-human-bronchial-epithelium (NHBE) and lung cells and organoids such as pancreatic, colon, liver, and cornea using a network pharmacology approach. Thus, our findings indicate that fortunellin has a dual role; multi-target antiviral activities against SARS-CoV-2 and immunomodulatory capabilities against the host.

A new commercially viable synthetic route for donepezil hydrochloride: anti-Alzheimer's drug.

An economical new process has been developed for the synthesis of donepezil hydrochloride (1) an anti-Alzheimer's drug. The process involves Darzen reaction of pyridine-4-carboxaldehyde and 2-bromo-5,6-dimethoxy indanone affording epoxide 5,6-dimethoxy-3-(pyridine-4-yl)spiro[indene-2,2'-oxiran]-1(3H)-one (4) as a key intermediate. The one-pot deoxygenation of 4 and hydrogenation of the aryl moiety in high yield improved the overall yield of the process.

Whole mitochondrial genome analysis in South Indian patients with Leber's hereditary optic neuropathy.

Leber's hereditary optic neuropathy (LHON) is a mitochondrial DNA (mtDNA) associated neurodegenerative disorder of retinal ganglion cells. In this study, whole mitochondrial genome sequencing of 75 LHON patients and 40 controls was performed to identify the mutation frequency and haplogroup background of South Indian population. Analysis of mtDNA revealed 559 different variants in LHON patients, including 7 pathogenic mutations, 30 private, and 22 other disease associated variants. A significantly higher (p=0.0008) overall variation load per individual was noted among LHON patients versus controls. We reported for the first time, the association of M haplogroup (p=0.028) with LHON in this cohort.

Lysyl oxidase-like 1 gene in the reversal of promoter risk allele in pseudoexfoliation syndrome.

IMPORTANCE: This study was necessary to establish the association between common genetic variants in the lysyl oxidase-like 1 (LOXL1) gene with pseudoexfoliation (PEX) syndrome and emphasize the reversal of promoter risk allele in a South Indian population. OBJECTIVE: To investigate the potential association of genetic variants across the LOXL1 gene in South Indian patients with PEX syndrome and glaucoma. DESIGN, SETTING, AND PARTICIPANTS: A case-control study of individuals from Madurai, India, with PEX syndrome and glaucoma as well as healthy people serving as controls. Three hundred unrelated people with PEX syndrome and 225 age- and ethnically matched controls were recruited for genetic analysis. MAIN OUTCOMES AND MEASURES: Four single-nucleotide polymorphisms in LOXL1 (rs16958477, rs1048661, rs3825942, and rs2165241) were genotyped by direct sequencing in all participants. Regulatory regions and 7 coding exons of LOXL1 were directly sequenced in 50 patients and 50 controls. A case-control association analysis was performed using the Golden Helix SVS suite. RESULTS: An association between 4 LOXL1 single-nucleotide polymorphisms with PEX syndrome and glaucoma was observed (rs16958477, P = 4.77 × 10-6 [odds ratio, 0.50]; rs1048661, P = 4.28 × 10-5 [1.79]; rs3825942, P = 4.68 × 10-30 [9.19]; and rs2165241, P = 1.98 × 10-15 [2.88]). Sequencing of 7 exons and regulatory regions of LOXL1 identified 11 additional sequence variants; only rs41435250 showed an association (P = 3.80 × 10-5 [0.49]) with PEX syndrome and glaucoma. CONCLUSIONS AND RELEVANCE: Genetic variants in LOXL1 are associated with PEX syndrome and glaucoma in the South Indian population. To our knowledge, this is the first study to demonstrate the association of rs41435250 with PEX as well as reversal of the promoter risk allele. Understanding the role of the LOXL1 gene in PEX pathogenesis will facilitate early detection in individuals at risk for this condition.