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Here, we communicate the draft genome sequence of an ocular Mycobacterium tuberculosis strain (SNMICRO 2047-20) that was isolated from the vitreous fluid of a patient diagnosed with endophthalmitis. The genome sequence was 4,391,538 bp long with 3,898 protein-encoding genes and clustered to the East African-Indian lineage.

HIGHLIGHTSChemoprofiling of

Objective Retinoschisis and Norrie disease are X-linked recessive retinal disorders caused by mutations in RS1 and NDP genes respectively. Both are likely to be monogenic and no locus heterogeneity has been reported. However, there are reports showing overlapping features of Norrie disease and retinoschisis in a NDP knock-out mouse model and also the involvement of both the genes in retinoschisis patients. Yet, the exact molecular relationships between the two disorders have still not been understood. The study investigated the association between retinoschisin (RS1) and norrin (NDP) using in vitro and in silico approaches. Specific protein–protein interaction between RS1 and NDP was analyzed in human retina by co-immunoprecipitation assay and MALDI-TOF mass spectrometry. STRING database was used to explore the functional relationship. Result Co-immunoprecipitation demonstrated lack of a direct interaction between RS1 and NDP and was further substantiated by mass spectrometry. However, STRING revealed a potential indirect functional association between the two proteins. Progressively, our analyses indicate that FZD4 protein interactome via PLIN2 as well as the MAP kinase signaling pathway to be a likely link bridging the functional relationship between retinoschisis and Norrie disease.

Early growth response-1 (Egr-1) is one of the key zinc finger transcription factors involved in controlling cell growth, proliferation, differentiation, angiogenesis and apoptosis. It also plays a pivotal role in conditions like cancer, neurological disorders and ocular disorders. The structural features of Egr-1 DNA-binding domain (DBD) are well documented, however, the functional role of its intrinsically disordered regions (IDRs) is yet to be explored. Hence, in this study, 3D structure of Egr-1 was modelled in conjunction with its IDR and was probed for conformational changes at microsecond scale in order to explore the structural plasticity and its functional impact. The results revealed the distortion of Zinc binding was due to the secondary structure transitions and flexibility of the DBD residues. Moreover, the increased distance of the flap residues that contributes to the proper orientation of Zinc interactions were also inferred, which shall aid in proper binding of DNA. This study also portrays the conformations that can be considered as optimal near-natives for further protein–protein interaction studies to delineate its interacting partners involved in various pathological conditions. This study demonstrates the impact of IDRs in Egr-1 on structure–function studies and also provides insights on optimal druggable cavities..

Kinases are key targets for many of the pathological conditions. Inverse screening of ligands serves as an essential mode to identify potential kinase targets in modern drug discovery research. Hence, we intend to develop KinomeRun, a robust pipeline for inverse screening and kinome tree visualization through the seamless integration of kinome structures, docking and kinome-drug interaction fingerprint analysis. In this pipeline, the hurdle of residue numbering in kinome is also resolved by creating a common index file with the conserved kinase pocket residues for comparative interaction analysis. KinomeRun can be used to screen the ligands of interest docked against multiple kinase structures in parallel around the kinase binding site and also to filter out the targets with unique interaction patterns. This automation is essential for prioritization of kinase targets that show specificity for a given drug and will also serve as a crucial tool kit for holistic approaches in kinase drug discovery. KinomeRun is developed using python and bash programming language and is distributed freely under the GNU GPL licence-3.0 and can be downloaded at https://github.com/inpacdb/KinomeRun. The tutorial videos for installation, target screening and customized filtration are available at https://www.youtube.com/playlist?list=PLuIaEFtMVgQ7v__WigQH9ilGVxrfI1LKs and also be downloaded for offline viewing from the github link.

The conventional use of antibiotics for the treatment of infectious keratitis currently faces two major challenges: poor drug penetration and the emergence of antibiotic resistance in microbial strains. Cell-penetrating peptides (CPPs) with antimicrobial properties have the potential to address these challenges. However, their mode of action, mechanism of uptake, and interaction potential have not been explored in detail. In this study, we probed the mechanism of uptake and interaction potential of our previously designed peptides (VRF005 and VRF007). Our results showed that VRF005 undergoes direct translocation and induces a rough membrane surface, whereas VRF007 undergoes clathrin-mediated endocytic uptake. The gel shift assay showed that VRF005 is bound to genomic DNA, whereas VRF007 is bound to chitin and β-d-glucan. Gene expression studies revealed the effect of peptide VRF005 on Candida albicans transcription. Molecular docking and simulations showed that VRF005 forms noncovalent interactions (such as H-bonding and water bridges) with natamycin. It exhibited synergistic antifungal activity in the colony-forming assay. VRF005, functionalized in the polycaprolactone fiber matrix, showed sustained delivery and antifungal activity.

Carbonic anhydrase IX (CAIX) is a membrane-bound enzyme associated with tumor hypoxia and found to be over expressed in various tumor conditions. Targeting CAIX catalytic activity is proven to be efficient modality in modulating pH homeostasis in cancer cells. Proteoglycan-like (PG) region is unique to CAIX and is proposed to serve as an antenna enhancing the export of protons in conjunction with facilitated efflux of lactate ions via monocarboxylate transporters. Moreover, the PG region is also reported to contribute to the assembly and maturation of focal adhesion links during cellular attachment and dispersion on solid supports. Thus, drug targeting of this region shall efficiently modulate pH homeostasis and cell adhesion in cancer cells. As the PG region is intrinsically disordered, the complete crystal structure is not elucidated. Hence, in this study, we intend to sample the conformational landscape of the PG region at microsecond scale simulation in order to sample the most probable conformations that shall be utilized for structure-based drug design. In addition, the sampled conformations were subjected to high-throughput virtual screening against NCI and Maybridge datasets to identify potential hits based on consensus scoring and validation by molecular dynamics simulation. Further, the identified hits were experimentally validated for efficacy by in vitro and direct enzymatic assays. The results reveal 5-(2-aminoethyl)-1,2,3-benzenetriol to be the most promising hit as it showed significant CAIX inhibition at all levels of in silico and experimental validation.

Diabetic Retinopathy is prevalent among patients with uncontrolled hyperglycemia resulting in vision loss. Despite numerous challenges to create a link among these conditions, the characterization of pathological neovascularization causing retinal damage due to the prognosis of early non-proliferative diabetic retinopathy to late proliferative diabetic retinopathy needs deep understanding. In this study, meta-analysis-based integration of gene expression datasets for the fibrovascular membrane of PDR and neural retina of NPDR were compared, to investigate the differentially expressed genes involved in retinal angiogenesis. Human samples with gene expression profiling of the same experiment type and platform with sufficient information for analysis were included in the study. The studies from cell lines and non-human studies, human samples that include serum, cornea, lens, and/or other ocular tissues or fluids, and studies that lack basic information for analysis were excluded. The microarray datasets available in the Gene Expression Omnibus database of the early and late stages in DR were screened to find common gene expression profiles. Using the INMEX bioinformatics tool, significantly upregulated and downregulated genes in the neural retina of Non-Proliferative Diabetic Retinopathy and fibrovascular membrane of Proliferative Diabetic Retinopathy were compared and studied by the combine effect size method. Using the STRING database PPI network, 50 upregulated and 50 downregulated genes were used to find the key candidate genes involved in retinal disease/degeneration in eye/retinal tissues. In the extensive gene expression meta-analysis performed using INMEX bioinformatics tool, overall, 7935 differentially expressed genes were identified and the respective heatmap was created by using the visualization tools of INVEX. STRING database PPI network identified Retinol Binding Protein 3, Neural Retina Leucine Zipper, S-Antigen Visual Arrestin, Peripherin 2, and Aryl Hydrocarbon Receptor Interacting Protein Like-1 to be the most highly ranked hub genes. The newly discovered potential genes related to retinal angiogenesis causing FVM formation in DR may provide insight into the cellular pathogenesis of NPDR to PDR.

Cell-penetrating peptides (CPPs) have been widely used as vehicles for delivering therapeutic molecules to the site of action. Apart from their delivering potential, the biological effects of CPPs have not been explored in detail. JTS-1 is a CPP that has been reported to have gene delivery functions, although its biological role is yet to be determined. Hence, in this study, we revealed the biological mechanism such as its uptake mechanism and immunogenic potential and function using primary human tenon fibroblast (TF) cells collected from patients undergoing glaucoma trabeculectomy surgery. Our results showed that the JTS-1 peptide has an α-helical structure and is nontoxic up to 1 μM concentration. It was found to be colocalized with early endosome (Rab5), recycling endosome (Rab7), and Rab11 and interacted with major histocompatibility complex (MHC) class I and II. The peptide also affected actin polymerization, which is regulated by cofilin phosphorylation and ROCK1 localization. It also inhibited TF cell proliferation. Therefore, the JTS-1 peptide could be used as a possible therapeutic agent for modifying the fibrosis process, where TF proliferation is a key cause of surgery failure.

Herbs are widely utilized in the Western Ghats region of India to treat liver diseases and viral infections. However, such practices lack scientific evidence at the molecular level and may often pose adverse drug reactions. Thus, by this study we intend to identify phytocompounds having druggability and non-toxic profiles with potential activity against HBV-induced HCC. To startwith, the details of phytocompounds in traditionally utilized herbs in Western Ghats region were collated from chemical databases and publications. The druggability and toxicity of these compounds were predicted using MolSoft and ADVERpred, respectively. The probable targets of these phytocompounds were predicted using BindingDB. Moreover, compound-gene set pathways, cellular processes, and functional enrichment analysis were also performed using STRING and KEGG pathway databases. Subsequently, Herb-compound-target-disease pathway networks were constructed using Cytoscape 3.6.1. The potential hub protein was virtually screened against ligand dataset using POAP pipeline. Finally, molecular dynamics (MD) simulations of the most potential protein-ligand complexes were performed in triplicate using Desmond 6.1v. Amongst 274 compounds from 16 herbs studied, 36 showed drug likeness with nontoxic properties, and were also predicted to modulate 16 potential targets involved in the pathogenesis of HBV-induced HCC. Among all the molecules screened, flavonoids and diterpenoids from Andrographis paniculata and Thespesia populnea scored the highest edge count via modulating multiple targets and pathways. Moreover, molecular docking and MD simulation (100ns) also inferred that the top ranking Andrographin and Gossypetin to exhibit stable intermolecular interactions with EGFR protein, which was identified as highly connected hub protein in the constructed network. All these findings are suggestive of these moieties as potential therapeutics for targeting HBV-associated HCC sans adverse drug reactions.

CD147/Basigin/EMMPRIN (Extracellular Matrix Metalloproteinase inducer) is a single pass type1 transmembrane protein playing a central role in developmental process, wound healing, nutrient transport, inflammation, arthritis and also in microbial pathologies. It is also found to be a potent stimulator of MMP (matrix metalloproteinases) and has been considered as a prognostic marker in cancer. Dysregulation of CD147 is reported in several types of cancer. It activates cell proliferation, invasion, metastasis and inhibits tumor cell apoptosis under hypoxic condition. Thus, CD147 serves as a hub protein in cancer, as it is involved in several homophilic and heterophilic cellular interactions spanning the major hallmarks of cancer. Targeting these interactions is considered to be an efficient therapeutic modality in cancerous conditions. Hence, by this review we intend to collate the structure-function relationships of CD147, with an exclusive thrust on potential druggable hotspots based on its intra and inter molecular interactions.

Functional characterisation of proteins often depends on specific interactions with other molecules. In the drug discovery scenario, the ability of a protein to bind with drug-like molecule with a high affinity is referred as druggability. Deciphering such druggable binding pockets on proteins plays an important role in structure-based drug designing studies. Moreover, availability of plethora of structural data poses a need automated pipelines which can efficiently integrate robust algorithms towards large-scale pocket identification and comparison. These pipelines have direct applicability on off-target analysis, drug repurposing and structural prioritization of drug targets in pathogenic microbes. However, currently there is a paucity of such efficient pipelines. Hence, by this study a highly optimized shell script based pipeline (PocketPipe) has been developed with seamless integration of robust algorithms namely, P2Rank (predicts binding sites based on machine learning) and PocketMatch-v2.1 (compares binding pockets by residue-based method), for pocketome generation and comparison, respectively. The process of input workflow and various steps carried out by PocketPipe and the output results are well documented in the operating manual. On execution, the pipeline features seamless operability, high scalability, dynamic file handling and results parsing. PocketPipe is distributed freely under GNU GPL license and can be downloaded at https://github.com/inpacdb/PocketPipe.

Rho-associated, coiled-coil-containing protein kinase (ROCK1) regulates cell contraction, morphology, and motility by phosphorylating its downstream targets. ROCK1 is a proven target for many pathological conditions like cancer, atherosclerosis, glaucoma, neuro-degeneration, etc. Though many kinase inhibitors are available, there is a dearth of studies on repurposing approved drugs and novel peptide inhibitors that could potentially target ROCK1. Hence, in this study, an extensive integration of open-source pipelines was employed to probe the potential inhibitors (ligand/peptide) for targeting ROCK1. To start with, a systematic enrichment analysis was performed to delineate the most optimal ROCK1 crystal structure that can be harnessed for drug design. A comparative analysis of conformational flexibility between monomeric and dimeric forms was also performed to prioritize the optimal assembly for structural studies. Subsequently, Virtual screening of FDA-approved drugs in Drugbank was performed using POAP pipeline. Further, the top hits were probed for binding affinity, crucial interaction fingerprints, and complex stability during MD simulation. In parallel, a combinatorial tetrapeptide library was also virtually screened against ROCK1 using the PepVis pipeline. Following which, all these shortlisted inhibitors (compounds/peptides) were subjected to Kinomerun analysis to infer other potential kinase targets. Finally, Polydatin and conivaptan were prioritized as the most potential repurposable inhibitors, and WWWF, WWVW as potential inhibitory peptides for targeting ROCK1. The prioritized inhibitors are highly promising for use in therapeutics, as these are resultants of the multilevel stringent filtration process. The computational strategies implemented in this study could potentially serve as a scaffold towards selective inhibitor design for other kinases.Communicated by Ramaswamy H. Sarma.

We announce the coding-complete genome sequences of two isolates of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from two coronavirus disease 2019 (COVID-19)-positive samples (RNA isolated from nasopharyngeal swabs) from Belagavi District, Karnataka State, India. Mutational analysis revealed the presence of the D614G substitution in both the isolates.

Mycotic keratitis is a fungal infection of corneal epithelium. It is more common in tropical and subtropical countries and is one of the leading causes of blindness. Many of the antifungal drugs have been less effective in treating this condition and certain drugs which are efficient and yet limited in use due to its extreme side effects. Hence, in this study an attempt is made to identify potential and least toxic antifungal inhibitors that targets thiamine thiazole synthase, a novel target for suppressing Fusarium solani subsp.pisi (Nectria haematococca MPVI) infections, to combat mycotic keratitis. Integrative computational approaches involving model refinement, molecular dynamics simulation and High throughput virtual screening (HTVS) were applied through integrative multi precision mode in order to identify potential inhibitors. Moreover, machine learning approach was also implemented to prioritize potential inhibitors that are ophthalmic adaptive, as well as antifungal molecule. From the NCI and Maybridge datasets, for HTVS only 209,872 compounds that surpassed ligand property filtration were considered, which resulted in 209 compounds after XP docking. Among the top 5 compounds from XP docking, on cumulative analysis only 2-(1-hydroxyethyl)-1,3-thiazole-4-carboxamide was prioritized as the most potential hit, as it showed higher order of significance in terms of binding affinity, structural stability and therapeutic relevance for the treatment of Mycotic keratitis. Thus, widening the scope for novel antifungal therapy in ophthalmic infections.

Aims Toxoplasma gondii (T. gondii) is an intracellular Apicomplexan parasite and a causative agent of toxoplasmosis in human. Its parasitic invasion leads to encephalitis, uveitis, chorioretinitis and congenital infection. Host invasion by T. gondii is mediated by Moving junction (MJ) complex formed by secretory proteins such as micronemes and Rhoptry Neck proteins (RONs). Among these secretory proteins, RON4 shows major interactions with RON2 and RON5 of MJ complex and is also found to facilitate invasion by interacting with β-tubulin of the host. Therefore, drug targeting of RON4 is considered to be an effective modality to inhibit host invasion. Main methods Hence, in this study, we have implemented High throughput virtual screening (HTVS) against predicted novel druggable cavity on RON4, in order to prioritize potential inhibitors. This study also specifically focuses on identifying potential drugs that are ocular accommodative towards targeting ocular toxoplasmosis. Thus, the small molecules resulting from HTVS were subjected to stringent multi-level precision screening which involves PASS prediction, Density functional theory analysis, Binding Free Energy Calculations and stability of complex formation during molecular dynamics simulation to prioritize the potential hits. Key findings Among the compounds from NCI chemical library, five (338683, 333739, 686585, 159706, 405935) were found to surpass all the stringent filtration criteria. Significance Based on comparatively analysis, it was inferred that 333739 (benzyl 2-((2-(((benzyloxy) carbonyl) amino)-3-hydroxybutanoyl) amino) propanoate) to be highly potential in comparison to other compounds and shall prove to be an efficient inhibitor of RON4 in ocular toxoplasmosis.

Background & objectives: The enzyme paraoxonase (PON), an antioxidant enzyme that has both arylesterase and thiolactonase activity, is well studied in cardiovascular diseases. Although a few studies have shown altered PON activity in ocular diseases such as age-related macular degeneration and diabetic retinopathy, but the tissue-wise expression of PON in its three gene forms has not been studied. This study was conducted to see the ocular distribution of PON for any altered expression in ocular pathologies such as in cataract and diabetes mellitus. Methods: Immunohistochemistry (IHC) of the ocular tissues was done for localizing all three forms of the PON in the human donor eyeballs. The PON arylesterase (PON-AREase) and thiolactonase (PON-HCTLase) activities were determined by spectrophotometry in kinetic mode, and the mRNA expression of the PON genes (PON1-3) was determined by reverse transcription-polymerase chain reaction. Results: IHC showed the presence of both PON1 and 2 in all the ocular tissues and PON3 was seen only in retina. The mRNA expression analysis showed that PON2 and PON3 were present in all the tissues, whereas PON1 was seen only in ciliary and retina. Both the PON-AREase and PON-HCTLase activities were detected in all ocular tissues and was in the order of lens>retina>choroid>ciliary body>iris. The expression and activity were studied in cataractous lens and in diabetic retina of the donor eyes. A significant decrease in PON-AREase activity was seen in cataractous lens (P

Methylene tetrahydrofolate reductase (MTHFR) is a flavoprotein, involved in one-carbon pathway and is responsible for folate and homocysteine metabolism. Regulation of MTHFR is pivotal for maintaining the cellular concentrations of methionine and SAM (S-adenosyl methionine) which are essential for the synthesis of nucleotides and amino acids, respectively. Therefore, mutations in MTHFR leads to its dysfunction resulting in conditions like homocystinuria, cardiovascular diseases, and neural tube defects in infants. Among these conditions, homocystinuria has been highly explored, as it manifests ocular disorders, cognitive disorders and skeletal abnormalities. Hence, in this study, we intend to explore the mutational landscape of human MTHFR isoform-1 (h.MTHFR-1) to decipher the most pathogenic variants pertaining to homocystinuria. Thus, a multilevel stringent prioritization of non-synonymous mutations in h.MTHFR-1 by integrative machine learning approaches was implemented to delineate highly deleterious variants based on its pathogenicity, impact on structural stability and functionality. Subsequently, extended molecular dynamics simulations and molecular docking studies were also integrated in order to prioritize the mutations that perturbs structural stability and functionality of h.MTHFR-1. In addition, displacement of Loop (Arg157-Tyr174) and helix α9 (His263-Ser272) involved in open/closed conformation of substrate binding domain were also probed to confirm the functional loss. On juxtaposed analysis, it was inferred that among 126 missense mutations screened, along with known pathogenic mutations (H127 T, A222 V, T227 M, F257 V and G387D) predicted that W500C, P254S and D585 N variants could be potentially driving homocystinuria. Thus, uncovering the prospects for inclusion of these mutations in diagnostic panels based on further experimental validations.