In silico prediction of deleterious non-synonymous SNPs in STAT3.

Background: STAT3, a pleiotropic transcription factor, plays a critical role in the pathogenesis of autoimmunity, cancer, and many aspects of the immune system, as well as having a link with inflammatory bowel disease. Changes caused by non-synonymous single nucleotide polymorphisms (nsSNPs) have the potential to damage the protein's structure and function. Objective: We identified disease susceptible single nucleotide polymorphisms (SNPs) in STAT3 and predicted structural changes associated with mutants that disrupt normal protein-protein interactions using different computational algorithms. Methods: Several in silico tools, such as SIFT, PolyPhen v2, PROVEAN, PhD-SNP, and SNPs&GO, were used to determine nsSNPs of the STAT3. Further, the potentially deleterious SNPs were evaluated using I-Mutant, ConSurf, and other computational tools like DynaMut for structural prediction. Result: 417 nsSNPs of STAT3 were identified, 6 of which are considered deleterious by in silico SNP prediction algorithms. Amino acid changes in V507F, R335W, E415K, K591M, F561Y, and Q32K were identified as the most deleterious nsSNPs based on the conservation profile, structural conformation, relative solvent accessibility, secondary structure prediction, and protein-protein interaction tools. Conclusion: The in silico prediction analysis could be beneficial as a diagnostic tool for both genetic counseling and mutation confirmation. The 6 deleterious nsSNPs of STAT3 may serve as potential targets for different proteomic studies, large population-based studies, diagnoses, and therapeutic interventions.

In silico screening of non-synonymous SNPs in human TUFT1 gene.

BACKGROUND: Tuftelin 1 (TUFT1) gene is important in the development and mineralization of dental enamel. The study aimed to identify potential functionally deleterious non-synonymous SNPs (nsSNPs) in the TUFT1 gene by using different in silico tools. The deleterious missense SNPs were identified from SIFT, PolyPhen-2, PROVEAN, SNPs & GO, PANTHER, and SNAP2. The stabilization, conservation, and three-dimensional modeling of mutant proteins were analyzed by I-Mutant 3.0, Consurf, and Project HOPE, respectively. The protein-protein interaction using STRING, GeneMANIA for gene-gene interaction, and DynaMut for evaluating the impact of the mutation on protein stability, conformation, and flexibility. RESULTS: Eight deleterious nsSNPs (E242A, R303W, K182N, K123N, R117W, H289Q, R203W, and Q107R) out of 304 were found to have high-risk damaging effects using six in silico tools. Among them, K182N and K123N alone had increased stability, whereas E242A, R303W, R117W, H289Q, Q107R, and R203W exhibited a decrease in protein stability, based on DDG values. Meanwhile, all the eight deleterious nsSNPs altered the size, charge, hydrophobicity, and spatial organization of the amino acids and predominantly had alpha helix domains. These deleterious variants were located in highly conserved regions except R203W. Protein-protein interaction predicted that TUFT1 interacted with ten proteins that are involved in enamel mineralization and odontogenesis. Gene-gene interaction network showed that TUFT1 is involved in physical interactions, gene co-localization, and pathway interactions. DynaMut ΔΔG values predicted that five nsSNPs were destabilizing the protein, ΔΔG ENCoM values showed a destabilizing effect for all mutants, and seven nsSNPs increased the molecular flexibility of TUFT1. CONCLUSION: Our study predicted eight functional SNPs that had detrimental effects on the structure and function of the TUFT1 gene. This will aid in the development of candidate deleterious markers as a potential target for disease diagnosis and therapeutic interventions.

Molecular docking and dynamics simulation of Orthosiphon stamineus against SGLT1 and SGLT2.

Orthosiphon stamineus Benth a traditional medicine used in the treatment of diabetes and kidney diseases. Sodium-glucose co-transporter (SGLT1 and SGLT2) inhibitors are the novel group of drugs used to treat patients with type 2 diabetes mellitus. In this study 20 phytochemical compounds from Orthosiphon stamineus Benth were obtained from 3 databases viz Dr.Duke's phytochemical, Ethno botanical database and IMPPAT. They were subjected to physiochemical, drug likeliness, and ADMET and toxicity predictions. Homology modeling and molecular docking against SGLT1 and SGLT2 were performed and the stability of the selected drug molecule was validated by molecular dynamic (MD) simulation for 200 ns. Among the 20 compounds, 14-Dexo-14-O-acetylorthosiphol Y alone showed higher binding affinity with SGLT1 and SGLT2 protein with the binding energy of -9.6 and -11.4 Kcal/mol respectively and had highest affinity towards SGLT2 inhibitor. This compound also satisfied Lipinski rule of 5 and had a good ADMET profile. The compound is non-toxic to marine organisms and to normal cell lines and non-mutagenic. The RMSD value attained equilibrium at 150 ns with the stability around 4.8 Å and no significant deviation was reported from 160 to 200 ns for SGLT2. Our study suggests that 14-Dexo-14-O-acetylorthosiphol Y showed promising results against the SGLT2 and could be considered as a potent anti-diabetic drug.Communicated by Ramaswamy H. Sarma.

Genetic Polymorphism and Gene Expression of ß-Defensin-1 in Periodontitis Associated With Type 2 Diabetes Mellitus.

Introduction Periodontitis is a multifactorial disease caused by periodontopathic bacteria and influenced by both genetic and environmental factors. Genetic predispositions are found to play a crucial part in the onset and progression of periodontal disease. There is a two-way relationship between diabetes and periodontitis with severe periodontal tissue destruction seen in diabetic patients. Antimicrobial peptide, ß-defensin-1 (DEFB1 gene), plays an important role in the innate immune responses and forms the first line of host defense against periodontal pathogens. Single nucleotide polymorphisms (SNPs) in the specific genetic loci of the DEFB1 gene and its expression level could confer a degree of risk or protection from periodontitis associated with diabetes. The present study determined the association between SNPs at the 5' untranslated region (UTR) in the DEFB1 gene and susceptibility to periodontitis associated with type 2 diabetes mellitus (T2DM) and analyzed the effect of 5' UTR polymorphisms on DEFB1 gene expression. Methods SNPs in the 5' UTR of the DEFB1 gene (-20G>A (rs11362), -44C>G (rs1800972), and -52G>A (rs1799946)) were genotyped by polymerase chain reaction (PCR) followed by Sanger sequencing. The study group included periodontitis (n = 40), periodontitis with T2DM (n = 20), and periodontally and systemically healthy as controls (n = 40). DEFB1 gene expression was determined by real-time PCR in the study group comprising periodontitis (n = 20), periodontitis with T2DM (n = 15), and healthy controls (n = 20). The effect of 5' UTR polymorphisms on the expression was analyzed by statistical tools. Results Statistically significant higher prevalence of the variant AA genotype of rs11362 was observed in periodontitis (odds ratio (OR) = 3.64, 95% confidence Interval (CI) = 1.16-11.43, p = 0.04) and periodontitis with T2DM (OR = 5.14, 95% CI = 1.29-20.5, p = 0.03) in comparison with healthy controls. Moreover, there was a significant increase of the variant AA genotype of rs1799946 in periodontitis (OR = 3.88, 95% CI = 1.19-12.68, p = 0.04) compared to healthy controls. DEFB1 gene expression was downregulated in periodontitis and upregulated in periodontitis with T2DM patients when compared to healthy controls but was not statistically significant. No significant association was found for the effect of SNPs of the DEFB1 gene on its expression. Conclusion From the SNP analysis, it can be inferred that the presence of SNPs at the 5' UTR (rs11362 and rs1799946) in the DEFB1 gene may be an important predictive factor for periodontitis.

The Role of ß-Defensin 1 Against Porphyromonas gingivalis Lipopolysaccharide-Mediated Inflammation in the THP-1 Cell Line.

Introduction Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) is one of the crucial virulence factors of periodontitis. Antimicrobial peptides (AMPs) are emerging as alternatives or adjuncts to antibiotics in the treatment of microbial infections. In this study, cytotoxicity, anti-inflammatory activity, anti-oxidative stress, cell cycle analysis, and apoptosis properties of AMP, ß-defensin 1, were studied in Pg-LPS-stimulated THP-1 (Tohoku Hospital Pediatrics - 1) cell line. Methods The cytotoxic nature of Pg-LPS and ß-defensin 1 was studied by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method. The cytotoxic effect of ß-defensin 1 on Pg-LPS-stimulated THP-1 cells was also studied by the same method. The anti-inflammatory role of ß-defensin 1 against cyclooxygenase (COX), lipoxygenase (LOX), myeloperoxidase (MPO), and inducible nitric oxide synthase activities were studied. The anti-oxidative nature of ß-defensin 1 was analyzed by measuring reactive oxygen species (ROS) generation by dichlorodihydrofluorescein diacetate (DCFDA) assay. Cell cycle distribution and apoptosis were studied by flow cytometry. The hemolytic nature of ß-defensin 1 was predicted using the HemoPred web tool. Results The results of the study demonstrated that Pg-LPS showed dose-dependent cytotoxicity to THP-1 cells. ß-Defensin 1 had dose-dependent cytotoxicity to THP-1 cells and showed a protective effect on THP-cells up to 1 µg/mL of Pg-LPS, beyond which cell viability decreased. ß-Defensin 1 inhibited COX, LOX, MPO, and inducible nitric oxide synthase activities in a concentration-dependent manner. ß-Defensin 1 showed anti-oxidative activity by suppressing the generation of ROS measured through fluorescence intensity. From the cell cycle analysis, it was found that ß-defensin 1 was able to reduce the Pg-LPS-induced cell cycle arrest at the G0/G1 phase. From the apoptosis profile, ß-defensin 1 was found to increase the live cells when compared to THP-1 cells stimulated only with Pg-LPS, indicating that ß-defensin 1 provided a protective role to THP-1 cells. ß-Defensin 1 was found to be hemolytic in nature by the HemoPred web tool. Conclusion ß-Defensin 1 exerted multifunctional activities and can be considered a promising agent for controlling periodontitis.

Determination of deleterious single-nucleotide polymorphisms of human LYZ C gene: an in silico study.

BACKGROUND: Single-nucleotide polymorphisms (SNPs) have a crucial function in affecting the susceptibility of individuals to diseases and also determine how an individual responds to different treatment options. The present study aimed to predict and characterize deleterious missense nonsynonymous SNPs (nsSNPs) of lysozyme C (LYZ C) gene using different computational methods. Lyz C is an important antimicrobial peptide capable of damaging the peptidoglycan layer of bacteria leading to osmotic shock and cell death. The nsSNPs were first analyzed by SIFT and PolyPhen v2 tools. The nsSNPs predicted as deleterious were then assessed by other in silico tools - SNAP, PROVEAN, PhD-SNP, and SNPs & GO. These SNPs were further examined by I-Mutant 3.0 and ConSurf. GeneMANIA and STRING tools were used to study the interaction network of the LYZ C gene. NetSurfP 2.0 was used to predict the secondary structure of Lyz C protein. The impact of variations on the structural characteristics of the protein was studied by HOPE analysis. The structures of wild type and variants were predicted by SWISS-MODEL web server, and energy minimization was carried out using XenoPlot software. TM-align tool was used to predict root-mean-square deviation (RMSD) and template modeling (TM) scores. RESULTS: Eight missense nsSNPs (T88N, I74T, F75I, D67H, W82R, D85H, R80C, and R116S) were found to be potentially deleterious. I-Mutant 3.0 determined that the variants decreased the stability of the protein. ConSurf predicted rs121913547, rs121913549, and rs387906536 nsSNPs to be conserved. Interaction network tools showed that LYZ C protein interacted with lactoferrin (LTF). HOPE tool analyzed differences in physicochemical properties between wild type and variants. TM-align tool predicted the alignment score, and the protein folding was found to be identical. PyMOL was used to visualize the superimposition of variants over wild type. CONCLUSION: This study ascertained the deleterious missense nsSNPs of the LYZ C gene and could be used in further experimental analysis. These high-risk nsSNPs could be used as molecular targets for diagnostic and therapeutic interventions.

In silico targeting of red complex bacteria virulence factors of periodontitis with ß-defensin 1.

BACKGROUND: Periodontitis is a multi-factorial infection with red complex bacteria playing a crucial role in the pathogenesis. As bacteria are tending to develop resistance against conventional antibiotics, new treatment modalities need to be developed. Antimicrobial peptides (AMPs) are potential tools for drug development and are gaining widespread interest. ß-defensin 1 is an important AMP and forms the first-line host defense mechanism. The present study analyzed the structure and molecular docking of ß-defensin 1 with the virulence factors of red complex bacteria of periodontitis. The physico-chemical properties of ß-defensin 1 were determined by various online tools such as ProtParam, ProteinPredict, ToxinPred, and BioPep web servers. The structure of ß-defensin 1was predicted by the SWISS-MODEL web server and the structure was evaluated by different web tools. The structure of lipopolysaccharide of Porphyromonas gingivalis was drawn using Chem3D ultra 11.0 software. The structure of important protein virulence factors of red complex bacteria of periodontitis was determined by the SWISS-MODEL web server. The interaction study between ß-defensin 1 and virulence factors was carried out by molecular docking using Auto dock version 4.0 software and pyDock WEB server. RESULTS: Using online tools, ß-defensin 1 was predicted to be stable and non-toxic. SWISS-MODEL web server predicted Ramachandran score as 94.12% and clash score 0.0 for ß-defensin 1. Auto dock version 4.0 software and pyDock WEB server analyzed the interaction to have low binding energies and hydrogen bonds were formed between the peptide and virulence factors. CONCLUSION: ß-defensin 1 was found to have good binding interaction with the disease-causing factors of red complex bacteria of periodontitis and in turn could play a role in reducing the severity of infection. ß-defensin 1 could be a potential candidate for drug development for periodontitis.

Selenium Influences Trace Elements Homeostasis, Cancer Biomarkers in Squamous Cell Carcinoma Patients Administered with Cancerocidal Radiotherapy

In the perspective of selenium as an antioxidant and anti-carcinogen, so far no strong intervention trials with selenium over radiation-treated oral squamous cell carcinoma cases have been conducted, to examine the response of the disease and the subsequent biochemical alterations. In the present study, untreated oral cancer cases (Gp II) were compared with radiation-treated groups with and without selenium (Gp IIa, IIb), forward to find the trace elements and cancer biomarkers status, at a follow-up of 6 months. Severe alteration in the trace elements levels of Se, Cu, Fe, Zn, Na, K, Ca, Cl, were noticed in Gp II. Though Gp IIa showed slight improvement, administration of selenium (Gp IIb) improved the level of all these elements to a greater extent (p<0.001). GpII and IIa showed increased level of bio markers 5'-nucleotidase, PschE, LAP, γ-GTP, LDH, SGOT, SGPT, ACP, ALP, CPK, TNF, CEA, AFP, Scc-Ag. The greater extent of restitution to near normalcy was observed in patients given selenium (Gp IIb) (p<0.001). Owing to the fact that selenium scavengers oxidants and hence decelerate carcinogenesis by eliminating tumors, so the tumor released constituents into the systemic circulation declined significantly. Therefore, the outcome of the study suggests selenium as a valuable therapeutic measure as adjuvant for oral cancer patients undergoing cancerocidal radiotherapy.

Differential Behaviour of Selenium Analogs against Anticancer Drug Induced Apoptosis of Lymphocytes in Human Peripheral Blood.

Sensitising cancer cells and at the same time desensitizing normal cells is a double task in cancer management. Agents which can combat the debilitating side effects of cancer therapeutics and simultaneously synergize with anticancer agents in specifically targeting cancer cells are needed. Selenium, a proven anticarcinogen, gains due importance in terms of its efficacy to combat the side effects of cancer therapy. This study is a comparative analysis of the chemoprotective effects of selenium compounds, methyl selenol (generated from organic selenomethionine (5mmol/L ; METase 40U/L)) and sodium selenite (inorganic form)(30µM) in peripheral blood human lymphocytes exposed to cisplatin and mitomycin. Biochemical alterations occurring in many cells during apoptosis include loss of plasma membrane phospholipid asymmetry, DNA fragmentation, and activation of caspase-3. The present study demonstrated that the selenium metabolite and selenite are efficient in protecting lymphocytes undergoing DNA damage and exerted their activity by reducing caspase 3 expression. Interestingly organic methylselenol (MeSe) was found to offer more protective effects compared to inorganic selenite (SeL), by reducing the induction of apoptosis by the cytotoxic agents. This suggests that MeSe and to a lesser extent selenite might have potential for assessment in clinical trials and could be considered as strong candidates in pharmacogenomics or in the nutriprotective arena.

Influence of selenium on radiogenic collagen destruction and the degree of collagen tissue maturation in stage III oral squamous cell carcinoma patients undergoing therapeutic irradiation.

INTRODUCTION: We set out to assess whether selenium, an antioxidant mineral could influence radiogenic collagen maturation. MATERIALS AND METHODS: The study comprise of normal (Group I), untreated oral carcinoma cases (Group II) (n = 20), cases who underwent radiotherapy (Group IIa) n = 10 and cases supplemented with selenium along with radiotherapy (Group IIb) n = 10. RESULTS: Spectrophotometric estimation and luminescence spectral assignment of collagen showed improved collagen maturation status. Measurement of the mature collagen cross-links hydroxylysylpyridinoline and lysylpyridinoline by high-performance liquid chromatography on irradiated tissues showed a considerable decrease in the selenium Group IIb (P < 0.05) indicating a decrease in collagen fragments. Electron microscopic studies showed significant morphological alteration in the selenium group. The micro nucleus frequency, decreased in radiation group (P < 0.05) compared with untreated (P < 0.05). While much more decrease observed in the selenium group (P < 0.05). DISCUSSION: The results represent the effect of selenium treatment with a bearing on carcinogenic process to curtail it, thus enhancing the maturity of collagen.

Chemoprotective action of resveratrol and genistein from apoptosis induced in human peripheral blood lymphocytes.

Extensive research is being carried out to analyse the importance of plant products such as resveratrol and genistein, which are known to exert a variety of pharmacological effects. This study aims at evaluating the protective role of these compounds against the apoptosis induced in normal cells by cytotoxic anticancer agents such as cisplatin and mytomycin C during therapy. Despite the broad antineoplastic action of cisplatin and mitomycin C, their genotoxicity in normal cell might lead to the induction of secondary malignancies. Therefore, the problem of identifying plant compounds, which might exert protective action in normal cells, gains lot of significance. We have analyzed the chemoprotective effect of plant compounds on peripheral blood human lymphocytes when exposed to cisplatin and mitomycin C by pre-treating and post-treating them with resveratrol and genistein at 100 microM concentration Biochemical alterations occurring in many cells during apoptosis include loss of plasma membrane phospholipid asymmetry, DNA fragmentation, and activation of caspase-3, et cetera, and have been assessed. Fluorescence microscopy, flow cytometric techniques have clearly demonstrated that resveratrol and genistein are efficient in protecting lymphocytes undergoing DNA damage when exposed to cisplatin and mitomycin C and exerted their activity by reducing the caspase 3 expression. An interesting observation is that, these compounds offered their protective effect by reducing their apoptotic potential on membrane and nucleic acids against cytotoxic agents, cisplatin, and mitomycin C. These results suggest that resveratrol and genistein might be useful for risk assessments in advance of clinical trials and could be considered as a strong candidate in pharmacogenomics or nutriprotective arena.