Decoding Molecular Bases of Rodent Social Hetero-Grooming Behavior Using in Silico Analyses and Bioinformatics Tools.

Highly prevalent in laboratory rodents, 'social' hetero-grooming behavior is translationally relevant to modeling a wide range of neuropsychiatric disorders. Here, we comprehensively evaluated all known to date mouse genes linked to aberrant hetero-grooming phenotype, and applied bioinformatics tools to construct a network of their established protein-protein interactions (PPI). We next identified several distinct molecular clusters within this complex network, including neuronal differentiation, cytoskeletal, WNT-signaling and synapsins-associated pathways. Using additional bioinformatics analyses, we further identified 'central' (hub) proteins within these molecular clusters, likely key for mouse hetero-grooming behavior. Overall, a more comprehensive characterization of intricate molecular pathways linked to aberrant rodent grooming may markedly advance our understanding of underlying cellular mechanisms and related neurological disorders, eventually helping discover novel targets for their pharmacological or gene therapy interventions.

In silico prospective analysis of the medicinal plants activity on the CagA oncoprotein from Helicobacter pylori.

BACKGROUND: Colonization with Helicobacter pylori (H. pylori) has a strong correlation with gastric cancer, and the virulence factor CagA is implicated in carcinogenesis. Studies have been conducted using medicinal plants with the aim of eliminating the pathogen; however, the possibility of blocking H. pylori-induced cell differentiation to prevent the onset and/or progression of tumors has not been addressed. This type of study is expensive and time-consuming, requiring in vitro and/or in vivo tests, which can be solved using bioinformatics. Therefore, prospective computational analyses were conducted to assess the feasibility of interaction between phenolic compounds from medicinal plants and the CagA oncoprotein. AIM: To perform a computational prospecting of the interactions between phenolic compounds from medicinal plants and the CagA oncoprotein of H. pylori. METHODS: In this in silico study, the structures of the phenolic compounds (ligands) kaempferol, myricetin, quercetin, ponciretin (flavonoids), and chlorogenic acid (phenolic acid) were selected from the PubChem database. These phenolic compounds were chosen based on previous studies that suggested medicinal plants as non-drug treatments to eliminate H. pylori infection. The three-dimensional structure model of the CagA oncoprotein of H. pylori (receptor) was obtained through molecular modeling using computational tools from the I-Tasser platform, employing the threading methodology. The primary sequence of CagA was sourced from GenBank (BAK52797.1). A screening was conducted to identify binding sites in the structure of the CagA oncoprotein that could potentially interact with the ligands, utilizing the GRaSP online platform. Both the ligands and receptor were prepared for molecular docking using AutoDock Tools 4 (ADT) software, and the simulations were carried out using a combination of ADT and AutoDock Vina v.1.2.0 software. Two sets of simulations were performed: One involving the central region of CagA with phenolic compounds, and another involving the carboxy-terminus region of CagA with phenolic compounds. The receptor-ligand complexes were then analyzed using PyMol and BIOVIA Discovery Studio software. RESULTS: The structure model obtained for the CagA oncoprotein exhibited high quality (C-score = 0.09) and was validated using parameters from the MolProbity platform. The GRaSP online platform identified 24 residues (phenylalanine and leucine) as potential binding sites on the CagA oncoprotein. Molecular docking simulations were conducted with the three-dimensional model of the CagA oncoprotein. No complexes were observed in the simulations between the carboxy-terminus region of CagA and the phenolic compounds; however, all phenolic compounds interacted with the central region of the oncoprotein. Phenolic compounds and CagA exhibited significant affinity energy (-7.9 to -9.1 kcal/mol): CagA/kaempferol formed 28 chemical bonds, CagA/myricetin formed 18 chemical bonds, CagA/quercetin formed 16 chemical bonds, CagA/ponciretin formed 13 chemical bonds, and CagA/chlorogenic acid formed 17 chemical bonds. Although none of the phenolic compounds directly bound to the amino acid residues of the K-Xn-R-X-R membrane binding motif, all of them bound to residues, mostly positively or negatively charged, located near this region. CONCLUSION: In silico, the tested phenolic compounds formed stable complexes with CagA. Therefore, they could be tested in vitro and/or in vivo to validate the findings, and to assess interference in CagA/cellular target interactions and in the oncogenic differentiation of gastric cells.

The continuously evolving phenotype of succinic semialdehyde dehydrogenase deficiency.

The objective of the study is to evaluate the evolving phenotype and genetic spectrum of patients with succinic semialdehyde dehydrogenase deficiency (SSADHD) in long-term follow-up. Longitudinal clinical and biochemical data of 22 pediatric and 9 adult individuals with SSADHD from the patient registry of the International Working Group on Neurotransmitter related Disorders (iNTD) were studied with in silico analyses, pathogenicity scores and molecular modeling of ALDH5A1 variants. Leading initial symptoms, with onset in infancy, were developmental delay and hypotonia. Year of birth and specific initial symptoms influenced the diagnostic delay. Clinical phenotype of 26 individuals (median 12 years, range 1.8-33.4 years) showed a diversifying course in follow-up: 77% behavioral problems, 76% coordination problems, 73% speech disorders, 58% epileptic seizures and 40% movement disorders. After ataxia, dystonia (19%), chorea (11%) and hypokinesia (15%) were the most frequent movement disorders. Involvement of the dentate nucleus in brain imaging was observed together with movement disorders or coordination problems. Short attention span (78.6%) and distractibility (71.4%) were the most frequently behavior traits mentioned by parents while impulsiveness, problems communicating wishes or needs and compulsive behavior were addressed as strongly interfering with family life. Treatment was mainly aimed to control epileptic seizures and psychiatric symptoms. Four new pathogenic variants were identified. In silico scoring system, protein activity and pathogenicity score revealed a high correlation. A genotype/phenotype correlation was not observed, even in siblings. This study presents the diversifying characteristics of disease phenotype during the disease course, highlighting movement disorders, widens the knowledge on the genotypic spectrum of SSADHD and emphasizes a reliable application of in silico approaches.

Genetic and epigenetic factors of arterial hypertension: a bibliometric- and in-silico-based analyses.

Introduction: Arterial hypertension (AH) is a pervasive global health concern with multifaceted origins encompassing both genetic and environmental components. Previous research has firmly established the association between AH and diverse genetic factors. Consequently, scientists have conducted extensive genetic investigations in recent years to unravel the intricate pathophysiology of AH. Methods: In this study, we conducted a comprehensive bibliometric analysis employing VOSviewer software to identify the most noteworthy genetic factors that have been the focal point of numerous investigations within the AH field in recent years. Our analysis revealed genes and microRNAs intricately linked to AH, underscoring their pivotal roles in this condition. Additionally, we performed molecular docking analyses to ascertain microRNAs with the highest binding affinity to these identified genes. Furthermore, we constructed a network to elucidate the in-silico-based functional interactions between the identified microRNAs and genes, shedding light on their potential roles in AH pathogenesis. Results: Notably, this pioneering in silico examination of genetic factors associated with AH promises novel insights into our understanding of this complex condition. Our findings prominently highlight miR-7110-5p, miR-7110-3p, miR-663, miR-328-3p, and miR-140-5p as microRNAs exhibiting a remarkable affinity for target genes. These microRNAs hold promise as valuable diagnostic and therapeutic factors, offering new avenues for the diagnosis and treatment of AH in the foreseeable future. Conclusion: In summary, this research underscores the critical importance of genetic factors in AH and, through in silico analyses, identifies specific microRNAs with significant potential for further investigation and clinical applications in AH management.

Computational and experimental analysis of foxtail millet under salt stress and selenium supplementation.

Salinity stress is a major threat to crop growth and productivity. Millets are stress-tolerant crops that can withstand the environmental constraints. Foxtail millet is widely recognized as a drought and salinity-tolerant crop owing to its efficient ROS scavenging mechanism. Ascorbate peroxidase (APX) is one of the reactive oxygen species (ROS) scavenging enzymes that leads to hydrogen peroxide (H2O2) detoxification and stabilization of the internal biochemical state of the cell under stress. This inherent capacity of the APX enzyme can further be enhanced by the application of an external mitigant. This study focuses on the impact of salt (NaCl) and selenium (Se) application on the APX enzyme activity of foxtail millet using in silico and in-vitro techniques and mRNA expression studies. The NaCl was applied in the concentrations, i.e., 150 mM and 200 mM, while the Se was applied in 1 µM, 5 µM, and 10 µM concentrations. The in silico studies involved three-dimensional structure modeling and molecular docking. The in vitro studies comprised the morphological and biochemical parameters, alongside mRNA expression studies in foxtail millet under NaCl stress and Se applications. The in silico studies revealed that the APX enzyme showed better interaction with Se as compared to NaCl, thus suggesting the enzyme-modulating role of Se. The morphological and biochemical analysis indicated that Se alleviated the NaCl (150 mM and 200 mM) and induced symptoms at 1 µM as compared to 5 and 10 µM by enhancing the morphological parameters, upregulating the gene expression and enzyme activity of APX, and ultimately reducing the H2O2 content significantly. The transcriptomic studies confirmed the upregulation of chloroplastic APX in response to salt stress and selenium supplementation. Hence, it can be concluded that Se as a mitigant at lower concentrations can alleviate NaCl stress in foxtail millet.

Promising Antifungal Molecules against Mucormycosis Agents Identified from Pandemic Response Box®: In Vitro and In Silico Analyses.

Mucormycosis is considered concerning invasive fungal infections due to its high mortality rates, difficult diagnosis and limited treatment approaches. Mucorales species are highly resistant to many antifungal agents and the search for alternatives is an urgent need. In the present study, a library with 400 compounds called the Pandemic Response Box® was used and four compounds were identified: alexidine and three non-commercial molecules. These compounds showed anti-biofilm activity, as well as alterations in fungal morphology and cell wall and plasma membrane structure. They also induced oxidative stress and mitochondrial membrane depolarization. In silico analysis revealed promising pharmacological parameters. These results suggest that these four compounds are potent candidates to be considered in future studies for the development of new approaches to treat mucormycosis.

Inhibitory Effect of Curcumin-Inspired Derivatives on Tyrosinase Activity and Melanogenesis.

Tyrosinase is a well-known copper-containing metalloenzyme typically involved in the synthesis of melanin. Recently, curcumin and several synthetic derivatives have been recognized as tyrosinase inhibitors with interesting anti-melanogenic therapeutic activity. In this study, three curcumin-inspired compounds 1, 6 and 7 were prepared in yields ranging from 60 to 88 % and spectrophotometric, electrochemical, in vitro and in silico analyses were carried out. The viability of PC12 cells, a rat pheochromocytoma derived-cell line, with compounds 1, 6 and 7, showed values around 80% at 5 µM concentration. In cell proliferation assays, compounds 1, 6 and 7 did not show significant toxicity on fibroblasts nor melanoma cells up to 10 µM with viability values over 90%. The inhibition of tyrosinase activity was evaluated both by a UV-Vis spectroscopic method at two different concentrations, 0.2 and 2.0 µM, and by amperometric assay with IC50 for compounds 1, 6 and 7 ranging from 11 to 24 nM. Melanin content assays on human melanoma cells were performed to test the capability of compounds to inhibit melanin biosynthesis. All compounds exerted a decrease in melanin content, with compound 7 being the most effective by showing a melanogenesis inhibition up to four times greater than arbutin at 100 µM. Moreover, the antioxidant activity of the selected inhibitors was evaluated against H2O2 in amperometric experiments, whereby compound 7 was about three times more effective compared to compounds 1 and 6. The tyrosinase X-ray structure of Bacterium megaterium crystal was used to carry out molecular docking studies in the presence of compounds 1, 6 and 7 in comparison with that of kojic acid and arbutin, two conventional tyrosinase inhibitors. Molecular docking of compounds 6 and 7 confirmed the high affinity of these compounds to tyrosinase protein.

Identification of Novel Natural Inhibitors to Human 3-Phosphoglycerate Dehydrogenase (PHGDH) for Cancer Treatment.

Targeting the serine biosynthesis pathway enzymes has turned up as a novel strategy for anti-cancer therapeutics. 3- Phosphoglycerate dehydrogenase (PHGDH) is the rate-limiting enzyme that catalyzes the conversion of 3-Phosphoglyceric acid (3-PG) into 3-Phosphohydroxy pyruvate (3-PPyr) in the first step of serine synthesis pathway and perform a critical role in cancer progression. PHGDH has been reported to be overexpressed in different types of cancers and emerged as a novel target for cancer therapeutics. During this study, virtual screening tools were used for the identification of inhibitors of PHGDH. A library of phenolic compounds was docked against two binding sites of PHGDH using Molegro Virtual Docker (MVD) software. Out of 169 virtually tested compounds, Salvianolic acid C and Schizotenuin F possess good binding potential to co-factor binding site of PHGDH while Salvianolic acid I and Chicoric acid were identified as the best binding compounds toward the substrate binding site of PHGDH. The top selected compounds were evaluated for different physiochemical and ADMET properties, the obtained results showed that none of these hit compounds violated the Pfizer Rule and they possess acceptable ADMET profiles. Further, a commercially available hit compound, Chicoric acid, was evaluated for its anti-cancer potential against PHGDH-expressing gastric cancer cell lines (MGC-803 and SGC-7901) as well as cell lines with low expression of PHGDH (MCF-7 and MDA-MB2-31), which demonstrated that Chicoric acid possesses selective cytotoxicity toward PHGDH expressing cancer cell lines. Thus, this study has unveiled the potential of phenolic compounds, which could serve as novel candidates for the development of PHGDH inhibitors as anti-cancer agents.

Mitochondrial iron transporter (MIT) gene in potato (Solanum tuberosum): comparative bioinformatics, physiological and expression analyses in response to drought and salinity.

Mitochondrial iron transporter (MIT) genes are essential for mitochondrial acquisition/import of iron and vital to proper functioning of mitochondria. Unlike other organisms, research on the MITs in plants is limited. The present study provides comparative bioinformatics assays for the potato MIT gene (StMIT) as well as gene expression analyses. The phylogenetic analyses revealed monocots-dicot divergence in MIT proteins and it was also found clade specific motif diversity. In addition, docking analyses indicated that Asp172 and Gly100 residues to be identified as the closest residues binding to ferrous iron. The percentage of structure overlap of the StMIT 3D protein model with Arabidopsis, maize and rice MIT proteins was found between 80.18% and 85.71%. The transcript analyses exhibited that the expression of StMIT was triggered under drought and salinity stresses. The findings of the present study would provide valuable leads for further studies targeting specifically the MIT gene and generally the plant iron metabolism.

Pharmacogenetic determinants of warfarin in the Indian population.

BACKGROUND: Several studies optimized the warfarin dose based on CYP2C9*2, CYP2C9*3, VKORC1 -1639 G > A, CYP4F2 V433M. But, the information on the rare variants is lacking. In this study, we have explored the prevalence of common and rare pharmacogenetic determinants of warfarin and determined their damaging nature. METHODS: We have analyzed 2000 healthy adults using the Infinium global screening array (GSA) for 15 pharmacogenetic determinants of warfarin. In addition, we have elucidated the impact of these variants on protein function, stability, dynamics, evolutionary preservation, and ligand binding propensity. RESULTS: The GSA Analysis has revealed that CYP4F2 V433M (MAF: 39.425%), VKORC1 -1639 G > A (MAF: 20.5%), CYP2C9*3 (MAF:9.925%), and CYP2C9*2 (MAF:4.575%) are common, while CYP2C9*14 (MAF: 1.475%), CYP2C9*4 (0.175%), CYP2C9*5 (0.125%), and CYP2C9*11 (0.125%) are rare. Position-specific evolutionary preservation (PSEP) analysis has revealed that CYP2C9*4 is possibly damaging, while CYP2C9*5, CYP2C9*11, and CYP2C9*14 are probably damaging. CYP2C9*4 has high thermolability (-10.14 kcal/mol). Among the rare CYP2C9 variants, CYP2C9*4 and CYP2C9*11 exert destabilizing effects and may have increased molecular flexibility, while CYP2C9*5 and CYP2C9*14 exert stabilizing effects and may have decreased molecular flexibility. DNase I footprint analysis has revealed the loss of the E-box consensus sequence due to VKORC1 -1639 G > A polymorphism. CONCLUSION: CYP2C9*2, CYP2C9*3, VKORC1 -1639 G > A and CYP4F2 V433M are common; CYP2C9*4, CYP2C9*5, CYP2C9*11, and CYP2C9*14 variants are rare in Indian subjects. All the CYP2C9 variants are found to be damaging. DNase I footprint analysis provided the mechanistic rationale for the association of VKORC1 -1639 G > A with warfarin sensitivity.

In-Silico Analyses of Nonsynonymous Variants in the BRCA1 Gene.

BReast CAncer gene 1 (BRCA1)-a tumor suppressor gene plays an important role in the DNA repair mechanism. Several BRCA1 variants perturb its structure and function, including synonymous and nonsynonymous single nucleotide polymorphisms (SNPs). In the present study, we performed in-silico analyses of nonsynonymous SNPs (nsSNPs) of the BRCA1 gene. In total, 122 nsSNPs were retrieved from the NCBI SNP database and in-silico analyses were performed using computational prediction tools: SIFT, PROVEAN, Mutation Taster, PolyPhen-2, MutPred, and ConSurf. Of these tools, SIFT, PROVEAN, and Mutation Taster predicted 61 out of 122 nsSNPs as "damaging", based on structural homology analysis. PolyPhen-2 classified 22 nsSNPs as "probably damaging". These nsSNPs were further analyzed by MutPred to predict basic molecular mechanisms of amino acid alteration. ConSurf analysis predicted eleven conserved amino acid residues with structural and functional consequences. We identified five amino acid residues in the RING finger domain (L22, C39, H41, C44, and C47) and two in the BRCT domain (P1771 and I1707) with the potential to deter the BRCA1 protein function. This study provides insights into the effect of nsSNPs and amino acid substitutions in BRCA1.

Therapeutic Efficacy of Anti-Bestrophin Antibodies against Experimental Filariasis: Immunological, Immune-Informatics and Immune Simulation Investigations.

Lymphatic filariasis (LF) is a debilitating parasitic disease caused by filarial parasites and it is prevalent across the underprivileged population throughout the globe. The inadequate efficacy of the existing treatment options has provoked the conception of alternative strategies, among which immunotherapy is steadily emerging as a promising option. Herein, we demonstrate the efficacy of an antibody-based immunotherapeutic approach in an experimental model of filariasis, i.e., Wistar rat infected with Setaria cervi (a model filarial parasite). The polyclonal antibodies were raised against filarial surface antigen bestrophin protein (FSAg) in mice using the purified Wuchereria bancrofti FSAg. The adoptive transfer of anti-FSAg antibody-containing serum resulted in the significant reduction of parasite burden in filaria-infected rats. Intriguingly, anti-FSAg sera-treated animals also displayed a reduction in the level of proinflammatory cytokines as compared to the infected but untreated group. Furthermore, our in silico immunoinformatics data revealed eight B-cell epitopes and several T-cell epitopes in FSAg and these epitopes were linked to form a refined antigen in silico. The immune simulation suggested IgM and IgG1 as the predominant immunoglobulins induced in response to FSAg. Taken together, our experimental and simulation data collectively indicated a therapeutic potential of anti-FSAg sera against LF.

The COVID-19 puzzle: a global nightmare.

In December 2019, WHO was informed with several unknown pneumonia cases and later it was found as highly contagious, transmittable and pathogenic viral infection. The novel coronavirus (nCoV-19) was firstly reported from Wuhan city in China. COVID-19 has raised the concern of the world since its emergence from China. The WHO has declared an ongoing COVID-19 outbreak as a pandemic. Till now 6,057,853 confirmed cases with 371,166 deaths have been reported from approximately 213 countries of the world. The aim of this study is to discuss all the aspects related to recently discovered novel coronavirus. The article, therefore, provides a comprehensive study on the genomic, epidemiological, social, clinical and environmental aspects of SARS-CoV-2. SARS-CoV-2 uses human ACE2 receptor as a ligand to bind and transmit its genome just like the SARS-CoV. The clinical symptoms of SARS-CoV-2 are very non-specific and include fever, sore throat, wheezing, rales, headache and rhinorrhoea with round-glass pulmonary opacifications shadowing in X-ray. Many antiviral drugs show efficacy but only in mild to moderate infection levels. Though efforts on development of SARS-CoV-2 vaccine have been started earlier as soon as the pandemic was emerged, till date no effective drug or vaccine has been validated with significant efficacy against the disease; therefore, there is a dire need to design effective vaccine against SARS-CoV-2. Multiple vaccine candidates are still in evaluation and exploratory stages on different clinical models with potential results on different animals and human models. mRNA-1273, ChAdOx1, Ad5-nCoV, INO-4800, LV-SMENP-DC and pathogen-specific aAPC are the most advanced and potential drug candidates against COVID-19. Recent studies have revealed any attractive vaccine candidates as promising therapeutic agents based on different strategies of vaccines. Here, the rationale of this review was also to provide an overview of the pathogenesis of the virus and summarize the updated potential vaccine candidates against SARS-CoV-2.

Antitumor effect of Cymbopogon densiflorus (Linneu) essential oil in bladder cancer cells.

The aim of this study was to analyse the antitumor effect of the Cymbopogon densiflorus essential oil in silico and in vitro on bladder cancer cells RT4 and T24, with different TP53 status. The oil was extracted by hydrodistillation and the gas chromatography coupled to the mass spectrometry was used for characterisation. In silico analysis was carried out by Pass online software. Cytotoxicity, cell proliferation, cell cycle progression, apoptosis and wound healing assays were performed. Five major compounds were identified. In silico analysis showed that major compounds present high potential for antitumor activities. The treatment with C. densiflorus essential oil reduced cell viability of bladder cancer cells. Only in wild-type cells, the increase of apoptosis rates and the decrease of cell migration were observed. In conclusion, the C. densiflorus essential oil presents antitumor effects on TP53 wild-type and mutated bladder cancer cells, however, the mechanism of action is TP53 status-dependent.[Figure: see text].

Recombinant Expression and Characterization of Lemon (Citrus limon) Peroxidase.

BACKGROUND: Class III plant peroxidases play important role in a number of physiological processes in plants such as lignin biosynthesis, suberization, cell wall biosynthesis, reactive oxygen species metabolism and plant defense against pathogens. Peroxidases are also of significance in several industrial applications. In view of this, the production and identification of novel peroxidases having resistance towards temperature, pH, salts is desirable. OBJECTIVE: The objective of the present work was to clone and characterize a novel plant peroxidase suitable for industrial application. METHODS: A full length cDNA clone of lemon peroxidase was isolated using PCR and RACE approaches, characterized and heterologously expressed in Escherichia coli using standard protocols. The expressed peroxidase was purified using Ni-NTA agarose column and biochemically characterized using standard protocols. The peroxidase was also in-silico characterized at nucleotide as well as protein levels using standard protocols. RESULTS: A full length cDNA clone of lemon peroxidase was isolated and expressed heterologously in E. coli. The expressed recombinant lemon peroxidase (LPRX) was activated by in-vitro refolding and purified. The purified LPRX exhibited pH and temperature optima of pH 7.0 and 50°C, respectively. The LPRX was found to be activated by metal ions (Na+, Ca2+, Mg2+ and Mn2+) at lower concentration. The expressional analysis of the transcripts suggested involvement of lemon peroxidase in plant defense. The lemon peroxidase was in silico modelled and docked with the substrates guaiacol, and pyrogallol and shown the favourability of pyrogallol over guaiacol, which is in agreement with the in-vitro findings. The protein function annotation analyses suggested the involvement of lemon peroxidase in the phenylpropanoid biosynthesis pathway and plant defense mechanisms. CONCLUSION: Based on the biochemical characterization, the purified peroxidase was found to be resistant towards the salts and thus, might be a good candidate for industrial exploitation. The in-silico protein function annotation and transcript analyses highlighted the possible involvement of the lemon peroxidase in plant defense response.

Efficient detection of eukaryotic calcium-sensing receptor (CaSR) by polyclonal antibody against prokaryotic expressed truncated CaSR.

Calcium-sensing receptor (CaSR), which is better known for its action as regulating calcium homeostasis, can bind various ligands. To facilitate research on CaSR and understand the receptor's function further, an in silico designed truncated protein was developed. The resulting protein folding indicated that 99% of predicted three dimensional (3D) structure residues are located in favored and allowed Ramachandran plots. However, it was found that such protein does not fold properly when expressed in prokaryotic host cells. Thioredoxin (Trx) tag was conjugated to increase the final protein's solubility, which could help obtain the soluble antigen with better immunogenic properties. The truncated recombinant proteins were expressed and purified in two forms (Trx-CaSR: RR19 and CaSR: RRJ19). The polyclonal antibody was induced by the rabbit immunization with the form of RR19. Western blot on mouse kidney lysates evidenced the proper immune recognition of the receptor by the produced antibody. The specificity and sensitivity of antibodies were also assayed by immunohistofluorescence. These experiments affirmed antibody's ability to indicate the receptor on the cell surface in native form and the possibility of applying such antibodies in further cellular and tissue assays.

Esomeprazole reduces sperm motility index by targeting the spermic cholinergic machinery: A mechanistic study for the association between use of proton pump inhibitors and reduced sperm motility index.

Recent studies have linked prolonged use of the most commonly prescribed proton pump inhibitors (PPIs) with declined human sperm function and infertility. Here, we report for the first time the most plausible underlying mechanism for this unwarranted secondary mode of action. We followed up on a recent serendipitous discovery in our laboratory regarding PPIs' off-target action and performed detailed pharmacodynamic analyses by combining in silico and in vitro studies to determine the off-target effect of one of the most commonly used PPI, esomeprazole, on the key human acetylcholine biosynthesizing enzyme, choline acetyltransferase (ChAT; EC 2.3.1.6). A pivotal enzyme in the spermic cholinergic system that governs the sperm motility, concentration and quality. Our results were conclusive and showed that both the racemic form, omeprazole and its pure S-enantiomer, esomeprazole, acted as potent mixed-competitive inhibitor of human ChAT with a global inhibition constant (Ki) of 88 nM (95%CI: 10-167 nM) for esomeprazole and 178 nM (95%CI: 140-230 nM) for the racemic drug omeprazole. Most importantly, esomeprazole substantially reduces both total number of motile sperm (by 36%, p < 0.001; and 21% p < 0.0001, at 10 and 100 nM, respectively) as well as the total number of sperm with progressive motility (by 42% p < 0.0016 and by 26% p < 0.0001, respectively) after 60 min relative to 20 min incubation in our ex vivo functional assay performed on ejaculated human sperm. In conclusion, this study presents a completely new perspective regarding PPIs secondary mode of action/unwarranted side effects and calls for further mechanistic and larger clinical studies to elucidate the role of PPIs in infertility.

In-Silico Characterization of Glycosyl Hydrolase Family 1 ß-Glucosidase from Trichoderma asperellum UPM1.

ß-glucosidases (Bgl) are widely utilized for releasing non-reducing terminal glucosyl residues. Nevertheless, feedback inhibition by glucose end product has limited its application. A noticeable exception has been found for ß-glucosidases of the glycoside hydrolase (GH) family 1, which exhibit tolerance and even stimulation by glucose. In this study, using local isolate Trichoderma asperellum UPM1, the gene encoding ß-glucosidase from GH family 1, hereafter designated as TaBgl2, was isolated and characterized via in-silico analyses. A comparison of enzyme activity was subsequently made by heterologous expression in Escherichia coli BL21(DE3). The presence of N-terminal signature, cis-peptide bonds, conserved active site motifs, non-proline cis peptide bonds, substrate binding, and a lone conserved stabilizing tryptophan (W) residue confirms the identity of Trichoderma sp. GH family 1 ß-glucosidase isolated. Glucose tolerance was suggested by the presence of 14 of 22 known consensus residues, along with corresponding residues L167 and P172, crucial in the retention of the active site's narrow cavity. Retention of 40% of relative hydrolytic activity on ρ-nitrophenyl-ß-D-glucopyranoside (ρNPG) in a concentration of 0.2 M glucose was comparable to that of GH family 1 ß-glucosidase (Cel1A) from Trichoderma reesei. This research thus underlines the potential in the prediction of enzymatic function, and of industrial importance, glucose tolerance of family 1 ß-glucosidases following relevant in-silico analyses.

Characterization and in silico analyses of the BRCA1/2 variants identified in individuals with personal and/or family history of BRCA-related cancers.

Pathogenic variants in the coding regions of the BRCA1/2 lead dysfunctional or nonfunctional BRCA proteins however the contribution of non-coding BRCA1/2 variants to BRCA-related disease risk has not been fully elucidated. Thus, we characterized the functional impact of both coding and non-coding BRCA1/2 variants identified in individuals with personal and/or family history of BRCA-related cancers. The data were produced by resequencing the exons and exon-intron junctions of the BRCA1/2 in 125 individuals and were comprehensively analyzed by using bioinformatics tools and databases. A total of 96 variants (59 coding and 37 non-coding) including 7 novel variants were identified and analyzed for their functional importance. We identified 11 missense variants that potentially affect protein function; 22 variants were likely to alter different types of posttranslational modifications. Also, multiple non-coding BRCA1/2 variants were found to reside in the critical regulatory regions that have the potential to act as eQTLs and affect alternative splicing. The results of our study shed light on the possible contributions of not only coding variants but also non-coding BRCA1/2 variants in BRCRA-related cancers. Further investigation is required to fully understand their potential associations with phenotypes which may ultimately lead their utilization on cancer management as a biomarker.

Proton pump inhibitors act with unprecedented potencies as inhibitors of the acetylcholine biosynthesizing enzyme-A plausible missing link for their association with incidence of dementia.

INTRODUCTION: Several pharmacoepidemiological studies indicate that proton pump inhibitors (PPIs) significantly increase the risk of dementia. Yet, the underlying mechanism is not known. Here, we report the discovery of an unprecedented mode of action of PPIs that explains how PPIs may increase the risk of dementia. METHODS: Advanced in silico docking analyses and detailed enzymological assessments were performed on PPIs against the core-cholinergic enzyme, choline-acetyltransferase (ChAT), responsible for biosynthesis of acetylcholine (ACh). RESULTS: This report shows compelling evidence that PPIs act as inhibitors of ChAT, with high selectivity and unprecedented potencies that lie far below their in vivo plasma and brain concentrations. DISCUSSION: Given that accumulating evidence points at cholinergic dysfunction as a driving force of major dementia disorders, our findings mechanistically explain how prolonged use of PPIs may increase incidence of dementia. This call for restrictions for prolonged use of PPIs in elderly, and in patients with dementia or amyotrophic lateral sclerosis.