Determination of IL-6 Gene Promoter Polymorphism in Patients with Hepatitis C and Its Impact on RNA Secondary Structure.

Background and Objectives: A polymorphism in the promoter region of the IL-6 gene would influence the level of IL-6 expression in patients with HCV, resulting in a pro-inflammatory response. Few studies have shown the association between -174G>C (rs1800795) and -1363G>T (rs2069827) polymorphisms and HCV infection, and their results have been contradictory. There are no data published in our population to study such an IL-6 stimulus against HCV infection and its impact on RNA secondary structure. Therefore, we isolated human subjects from the province of Punjab, Pakistan. The objective was to screen for IL-6 gene promoter polymorphisms -174G/C and -1363G/T and those correlated with serum concentrations of IL-6 in patients with HCV and compared with a control. Materials and Methods: In conventional PCR, measurement of serum IL-6 by CLIA and statistical analysis were performed to observe the genotype association studies. By integrating bioinformatics and computational tools, our study aimed to provide a comprehensive understanding of how variations in the promoter region of IL-6 may have functional implications on gene expression. Results: The -174G>C and -1363G>T genotypes in the promoter region of patients with HCV were in strong allelic association (Δ = 0.97, p < 0.001). Interestingly, the bioinformatics analysis was well aligned with our experimental data. Conclusions: Based on the data, it can be inferred that IL-6 gene promoter polymorphisms are important in the dysregulation of IL-6 levels in patients with HCV.

Exploring the association of ESR1 and ESR2 gene SNPs with polycystic ovary syndrome in human females: a comprehensive association study.

BACKGROUND: Polycystic Ovary Syndrome (PCOS) affects a significant proportion of human females worldwide and is characterized by hormonal, metabolic, and reproductive dysfunctions, including infertility, irregular menstrual cycles, acanthosis nigricans, and hirsutism. Mutations in the estrogen receptor genes ESR1 and ESR2, involved in normal follicular development and ovulation, can contribute to development of the PCOS. The present study focuses on investigating the potential correlation between single nucleotide polymorphisms (SNPs) of ESR1 and ESR2 genes and the incidence of this syndrome. METHODS: For this study, SNPs in ESR1 and ESR2 genes were retrieved from the ENSEMBL database and analyzed for their effect on mutated proteins using different bioinformatics tools including SIFT, PolyPhen, CADD, REVEL, MetaLR, I-Mutant, CELLO2GO, ProtParam, SOPMA, SWISS-MODEL and HDDOCK. RESULTS: All the SNPs documented in the present study were deleterious. All the SNPs except rs1583384537, rs1450198518, and rs78255744 decreased protein stability. Two variants rs1463893698 and rs766843910 in the ESR2 gene altered the localization of mutated proteins i.e. in addition to the nucleus, proteins were also found in mitochondria and extracellular, respectively. SNPs rs104893956 in ESR1 and rs140630557, rs140630557, rs1596423459, rs766843910, rs1596405923, rs762454979 and rs1384121511 in ESR2 gene significantly changed the secondary structure of proteins (2D). SNPs that markedly changed 3D configuration included rs1554259481, rs188957694 and rs755667747 in ESR1 gene and rs1463893698, rs140630557, rs1596423459, rs766843910, rs1596405923, rs762454979 and rs1384121511 in ESR2 gene. Variants rs1467954450 (ESR1) and rs140630557 (ESR2) were identified to reduce the binding tendency of ESRα and ß receptors with estradiol as reflected by the docking scores i.e. -164.97 and -173.23, respectively. CONCLUSION: Due to the significant impact on the encoded proteins, these variants might be proposed as biomarkers to predict the likelihood of developing PCOS in the future and for diagnostic purposes.

Possible prognostic impact of PKCι genetic variants in prostate cancer.

BACKGROUND: Single nucleotide polymorphisms (SNPs) have been linked with prostate cancer (PCa) and have shown potential as prognostic markers for advanced stages. Loss of function mutations in PKCι have been linked with increased risk of malignancy by enhancing tumor cell motility and invasion. We have evaluated the impact of two coding region SNPs on the PKCι gene (PRKCI) and their prognostic potential. METHODS: Genotypic association of non-synonymous PKCι SNPs rs1197750201 and rs1199520604 with PCa was determined through tetra-ARMS PCR. PKCι was docked with interacting partner Par-6 to determine the effect of these variants on PKCι binding capabilities. Molecular dynamic simulations of PKCι docked with Par-6 were performed to determine variant effects on PKCι protein interactions. The possible impact of changes in PKCι protein interactions on epithelial cell polarity was hypothesized. RESULTS: PKCι rs1199520604 mutant genotype TT showed association with PCa (p = 0.0055), while rs1197750201 mutant genotype AA also showed significant association with PCa (P = 0.0006). The binding interaction of PKCι with Par-6 was altered for both variants, with changes in Van der Waals energy and electrostatic energy of docked structures. CONCLUSION: Genotypic analysis of two non-synonymous PKCι variants in association with PCa prognosis was performed. Both variants in the PB1 domain showed potential as a prognostic marker for PCa. In silico analysis of the effect of the variants on PKCι protein interactions indicated they may be involved in PCa progression through aberration of epithelial cell polarity pathways.

In silico assessment of diterpenes as potential inhibitors of SARS-COV-2 main protease.

Aim: We aimed to investigate the potential inhibitory effects of diterpenes on SARS-CoV-2 main protease (Mpro). Materials & methods: We performed a virtual screening of diterpenoids against Mpro using molecular docking, molecular dynamics simulation and absorption, distribution, metabolism and excretion) analysis. Results: Some tested compounds followed Lipinski's rule and showed drug-like properties. Some diterpenoids possessed remarkable binding affinities with SARS-CoV-2 Mpro and drug-like pharmacokinetic properties. Three derivatives exhibited structural deviations lower than 1 Å. Conclusion: The findings of the study suggest that some of the diterpenes could be candidates as potential inhibitors for Mpro of SARS-CoV-2.

Cross talk of tumor protein D52 (TPD52) with KLF9, PKCε, and MicroRNA 223 in ovarian cancer.

BACKGROUND: Gynecologic cancers comprise malignancies in the female reproductive organs. Ovarian cancer ranks sixth in terms of incidence rates while seventh in terms of mortality rates. The stage at which ovarian cancer is diagnosed mainly determines the survival outcomes of patients. Various screening approaches are presently employed for diagnosing ovarian cancer; however, these techniques have low accuracy and are non-specific, resulting in high mortality rates of patients due to this disease. Hence, it is crucial to identify improved screening and diagnostic markers to overcome this cancer. This study aimed to find new biomarkers to facilitate the prognosis and diagnosis of ovarian cancer. METHODS: Bioinformatics approaches were used to predict the tertiary structure and cellular localization along with phylogenetic analysis of TPD52. Its molecular interactions were determined through KEGG analysis, and real-time PCR-based expression analysis was performed to assess its co-expression with another oncogenic cellular pathway (miR-223, KLF9, and PKCε) proteins in ovarian cancer. RESULTS: Bioinformatics analysis depicted the cytoplasmic localization of TPD52 and the high conservation of its coiled-coil domains. Further study revealed that TPD52 mRNA and miRNA-223 expression was elevated, while the expression of KLF 9 and PKCε was reduced in the blood of ovarian cancer patients. Furthermore, TPD52 and miR-223 expression were upregulated in the early stages of cancer and non-metastatic cancers. CONCLUSION: TPD52, miR-223, PKCε, and KLF9, can be used as a blood based markers for disease prognosis, metastasis, and treatment response. The study outcomes hold great potential to be translated at the clinical level after further validation on larger cohorts.

Prediction of Phytochemicals for Their Potential to Inhibit New Delhi Metallo ß-Lactamase (NDM-1).

The effectiveness of all antibiotics in the ß-lactam group to cure bacterial infections has been impaired by the introduction of the New Delhi Metallo-ß-lactamase (NDM-1) enzyme. Attempts have been made to discover a potent chemical as an inhibitor to this enzyme in order to restore the efficacy of antibiotics. However, it has been a challenging task to develop broad-spectrum inhibitors of metallo-ß-lactamases. Lack of sequence homology across metallo-ß-lactamases (MBLs), the rapidly evolving active site of the enzyme, and structural similarities between human enzymes and metallo-ß-lactamases, are the primary causes for the difficulty in the development of these inhibitors. Therefore, it is imperative to concentrate on the discovery of an effective NDM-1 inhibitor. This study used various in silico approaches, including molecular docking and molecular dynamics simulations, to investigate the potential of phytochemicals to inhibit the NDM-1 enzyme. For this purpose, a library of about 59,000 phytochemicals was created from the literature and other databases, including FoodB, IMPPAT, and Phenol-Explorer. A physiochemical and pharmacokinetics analysis was performed to determine possible toxicity and mutagenicity of the ligands. Following the virtual screening, phytochemicals were assessed for their binding with NDM-1using docking scores, RMSD values, and other critical parameters. The docking score was determined by selecting the best conformation of the protein-ligand complex. Three phytochemicals, i.e., butein (polyphenol), monodemethylcurcumin (polyphenol), and rosmarinic acid (polyphenol) were identified as result of pharmacokinetics and molecular docking studies. Furthermore, molecular dynamics simulations were performed to determine structural stabilities of the protein-ligand complexes. Monodemethylcurcumin, butein, and rosmarinic acid were identified as potential inhibitors of NDM-1 based on their low RMSD, RMSF, hydrogen bond count, average Coulomb-Schrödinger interaction energy, and Lennard-Jones-Schrödinger interaction energy. The present investigation suggested that these phytochemicals might be promising candidates for future NDM-1 medication development to respond to antibiotic resistance.

Multiplex Cas9-based excision of CLCuV betasatellite and DNA-A revealed reduction of viral load with asymptomatic cotton plants.

MAIN CONCLUSION: Multiplexed Cas9-based genome editing of cotton resulted in reduction of viral load with asymptomatic cotton plants. In depth imaging of proteomic dynamics of resulting CLCuV betasatellite and DNA-A protein was also performed. The notorious  cotton leaf curl virus (CLCuV), which is transmitted by the sap-sucking insect whitefly, continuously damages cotton crops. Although the application of various toxins and RNAi has shown some promise, sustained control has not been achieved. Consequently, CRISPR_Cas9 was applied by designing multiplex targets against DNA-A (AC2 and AC3) and betasatellite (ßC1) of CLCuV using CRISPR direct and ligating into the destination vector of the plant using gateway ligation method. The successful ligation of targets into the destination vector was confirmed by the amplification of 1049 bp using a primer created from the promoter and target, while restriction digestion using the AflII and Asc1 enzymes determined how compact the plasmid developed and the nucleotide specificity of the plasmid was achieved through Sanger sequencing. PCR confirmed the successful introduction of plasmid into CKC-1 cotton variety. Through Sanger sequencing and correlation with the mRNA expression of DNA-A and betasatellite in genome-edited cotton plants subjected to agroinfiltration of CLCuV infectious clone, the effectiveness of knockout was established. The genome-edited cotton plants demonstrated edited efficacy of 72% for AC2 and AC3 and 90% for the (ßC1) through amplicon sequencing, Molecular dynamics (MD) simulations were used to further validate the results. Higher RMSD values for the edited ßC1 and AC3 proteins indicated functional loss caused by denaturation. Thus, CRISPR_Cas9 constructs can be rationally designed using high-throughput MD simulation technique. The confidence in using this technology to control plant virus and its vector was determined by the knockout efficiency and the virus inoculation assay.

Exploring the prognostic significance of PKCε variants in cervical cancer.

BACKGROUND: Protein Kinase C-epsilon (PKCε) is a member of the novel subfamily of PKCs (nPKCs) that plays a role in cancer development. Studies have revealed that its elevated expression levels are associated with cervical cancer. Previously, we identified pathogenic variations in its different domains through various bioinformatics tools and molecular dynamic simulation. In the present study, the aim was to find the association of its variants rs1553369874 and rs1345511001 with cervical cancer and to determine the influence of these variants on the protein-protein interactions of PKCε, which can lead towards cancer development and poor survival rates. METHODS: The association of the variants with cervical cancer and its clinicopathological features was determined through genotyping analysis. Odds ratio and relative risk along with Fisher exact test were calculated to evaluate variants significance and disease risk. Protein-protein docking was performed and docked complexes were subjected to molecular dynamics simulation to gauge the variants impact on PKCε's molecular interactions. RESULTS: This study revealed that genetic variants rs1553369874 and rs1345511001 were associated with cervical cancer. Smad3 interacts with PKCε and this interaction promotes cervical cancer angiogenesis; therefore, Smad3 was selected for protein-protein docking. The analysis revealed PKCε variants promoted aberrant interactions with Smad3 that might lead to the activation of oncogenic pathways. The data obtained from this study suggested the prognostic significance of PRKCE gene variants rs1553369874 and rs1345511001. CONCLUSION: Through further in vitro and in vivo validation, these variants can be used at the clinical level as novel prognostic markers and therapeutic targets against cervical cancer.

Investigating pathogenic SNP of PKCι in HCV-induced hepatocellular carcinoma.

Hepatocellular carcinoma is a leading cause of cancer-related deaths due to its complexity in diagnosis, chemo-resistance, and aggressive nature. Identifying pathogenic single nucleotide polymorphism (SNP) in protein kinase C iota (PKCι) can be a potential biomarker in the prognosis and treatment of HCC. This study investigated the association between a SNP in PRKCI and the Pakistani population's hepatocellular carcinoma (HCC) risk. Obtained samples were first evaluated for ALT measurements and viral load quantification through reverse transcriptase-PCR. The PKCι nsSNP rs1199520604 was evaluated computationally by multiple consensus bioinformatics tools for predicting its potential deleterious effects. Its association with hepatitis C virus- (HCV) mediated HCC was then investigated through ARMS-PCR (Amplification Refractory Mutation System Polymerase Chain Reaction). SNP analysis of rs1199520604 was performed in 100 cases and 100 controls. Variant rs1199520604's homozygous T genotype is a risk factor allele for the HCV-induced HCC (odds ratio: 4.13, relative risk: 2.01, P-value < 0.0001). The heterozygous genotype is determined to protect HCV patients from HCC development (P < 0.001). The study highlighted the disease association of variant rs1199520604 with HCV-induced HCC in the Pakistani populations. This variant, after further validation through high-throughput investigation on a larger cohort, has the potential to be translated at the clinical level.

Non-synonymous SNPs variants of PRKCG and its association with oncogenes predispose to hepatocellular carcinoma.

BACKGROUND: PRKCG encodes PKC γ, which is categorized under the classical protein kinase C family. No studies have specifically established the relationship between PRKCG nsSNPs with structural and functional variations in PKC γ in the context of hepatocellular carcinoma (HCC). The present study aims to uncover this link through in-silico and experimental studies. METHODS: The 3D structure of PKC γ was predicted. Molecular Dynamic (MD) Simulations were run and estimates were made for interactions, stability, conservation and post-translational alterations between wild and mutant structures. The association of PRKCG levels with HCC survival rate was determined. Genotyping analyses were conducted to investigate the deleterious PRKCG nsSNP association with HCC. mRNA expression of PKC γ, HIF-1 alpha, AKT, SOCS3 and VEGF in the blood of controls and HCC patients was analyzed and a genetic cascade was constructed depicting these interactions. RESULTS: The expression level of studied oncogenes was compared to tumour suppressor genes. Through Alphafold, the 3D structure of PKC γ was explored. Fifteen SNPs were narrowed down for in-silico analyses that were identified in exons 5, 10 and 18 and the regulatory and kinase domain of PKC γ. Root mean square deviation and fluctuation along with the radius of gyration unveiled potential changes between the wild and mutated variant structures. Mutant genotype AA (homozygous) corresponding to nsSNP, rs386134171 had more frequency in patients with OR (2.446), RR (1.564) and P-values (< 0.0029) that highlights its significant association with HCC compared to controls in which the wild genotype GG was found more prevalent. CONCLUSION: nsSNP rs386134171 can be a genetic marker for HCC diagnosis and therapeutic studies. This study has laid down a road map for future studies to be conducted on HCC.

Elucidating the role of missense SNP of protein kinase C epsilon in HCV-induced hepatocellular carcinoma.

BACKGROUND: The protein kinase C (PKC) family of serine/threonine kinases contains more than ten isozymes that are involved in multiple signaling pathways, including cell cycle regulation and carcinogenesis. The PKCε isozyme is an oncogene known to be upregulated in various signaling pathways involved in hepatitis C virus (HCV)-induced hepatocellular carcinoma (HCC). However, there is no known association of missense SNPs in PKCε with this disease, which can be a potential biomarker for early diagnosis and treatment. This research reveals a novel missense SNP in PKCε that is associated with HCV-induced HCC in the Pakistani population. METHODS: The PKCε SNP with amino acid substitution of E14K was chosen for wet lab analysis. Tetra ARMS-PCR was employed for the identification of high-risk SNP in PKCε of HCV-induced HCC patients. Liver function testing was also performed for comparison between the liver condition of the HCC patient and control group, and the viral load of HCC patient samples was evaluated to determine any alteration in the viral infectivity between different genotypes of the selected high-risk PKCε variant SNP. RESULTS: Frequency distribution of the homozygous GG genotype was found to be highest among HCV-induced HCC patients and was also found to be significantly associated with disease development and progression. The p values of comparative data obtained for the other two genotypes, heterozygous AG and homozygous AA, of the SNP also showed the significance of the data for these alleles. Still, their odds ratio and relative risk analysis did not indicate their association with HCV-induced HCC. CONCLUSION: The distribution of a genotype GG of PKCε has been found in HCV- induced HCC patients. Therefore, these PKCε SNP have the potential to be biomarkers for HCV-induced HCC. Further investigation using a larger sample size would provide additional insight into these initial data and open a new avenue for a better prognosis of this disease.

Molecular Dynamic Simulations Unravel the Underlying Impact of Missense Mutation in Autoimmunity Gene PTPN22 on Predisposition to Rheumatoid Arthritis.

Genetic mutations in various proteins have been implicated with increased risk or severity of rheumatoid arthritis (RA) in different population groups. In the present case-control study, we have investigated the risk association of single nucleotide mutations present in some of the highly reported anti-inflammatory proteins and/or cytokines, with RA susceptibility in the Pakistani subjects. The study involves 310 ethnically and demographically similar participants from whom blood samples were taken and processed for DNA extraction. Through extensive data mining, 5 hotspot mutations reported in 4 genes, that is, interleukin (IL)-4 (-590; rs2243250), IL-10 (-592; rs1800872), IL-10 (-1082; rs1800896), PTPN22 (C1858T; rs2476601), and TNFAIP3 (T380G; rs2230926), were selected for RA susceptibility analyses using genotyping assays. The results demonstrated the association of only 2 DNA variants [rs2243250 (odds ratio, OR = 2.025, 95% confidence interval, CI = 1.357-3.002, P = 0.0005 Allelic) and rs2476601 (OR = 4.25, 95% CI = 1.569-11.55, P = 0.004 Allelic)] with RA susceptibility in the local population. The former single nucleotide mutation was nonfunctional, whereas the latter, residing in the exonic region of a linkage-proven autoimmunity gene PTPN22, was involved in R620→W620 substitution. Comparative molecular dynamic simulations and free-energy calculations revealed a radical impact on the geometry/confirmation of key functional moieties in the mutant protein leading to a rather weak binding of W620 variant with the interacting receptor (SRC kinase). The interaction imbalance and binding instabilities provide convincing clues about the insufficient inhibition of T cell activation and/or ineffective clearance of autoimmune clones-a hallmark of several autoimmune disorders. In conclusion, the present research describes the association of 2 hotspot mutations in IL-4 promoter and PTPN22 gene with RA susceptibility in the Pakistani study cohort. It also details how a functional mutation in PTPN22 impacts the overall protein geometry, charge, and/or receptor interactions to contribute to RA susceptibility.

Analyzing PKC Gamma (+ 19,506 A/G) polymorphism as a promising genetic marker for HCV-induced hepatocellular carcinoma.

BACKGROUND: HCC is a major health concern worldwide. PKC gamma, a member of the conventional PKC subclass, is involved in many cancer types, but the protein has received little attention in the context of single nucleotide polymorphisms and HCC. Therefore, the study aims to investigate the association of PKC gamma missense SNP with HCV-induced hepatocellular carcinoma. METHODS: The PKC gamma nsSNPs were retrieved from the ENSEMBL genome browser and the deleterious nsSNPs were filtered out through involvingPredictSNP2, CADD, DANN, FATHMM, FunSeq2 and GWAVA. Among the filtered nsSNPs, nsSNP rs1331262028 was identified to be the most pathogenic one. Through involving I-TASSER, ProjectHOPE, I-Mutant, MUpro, mCSM, SDM, DynaMut and MutPred, the influence of SNP rs1331262028 on protein structure, function and stability was estimated. A molecular Dynamic simulation was run to determine the conformational changes in mutant protein structure compared to wild. The blood samples were collected for genotyping analysis and for assessing ALT levels in the blood. RESULTS: The study identified for the first time an SNP (rs1331262028) of PRKCG to strongly decrease protein stability and induce HCC. The RMSD, RMSF, and Rg values of mutant and wild types found were significantly different. Based on OR and RR values of 5.194 and 2.287, respectively, genotype analysis revealed a higher correlation between the SNP homozygous wild Typeform, AA, and the disease while patients with genotype AG have higher viral load. CONCLUSION: Outcomes of the current study delineated PKC gamma SNP rs1331262028 as a genetic marker for HCV-induced HCC that could facilitate disease management after further validation.

Screening of Pathogenic Missense Single Nucleotide Variants From LHPP Gene Associated With the Hepatocellular Carcinoma: An In silico Approach.

LHPP gene encodes a phospholysine phosphohistidine inorganic pyrophosphate phosphatase, which functions as a tumor-suppressor protein. The tumor suppression by this protein has been confirmed in various cancers, including hepatocellular carcinoma (HCC). LHPP downregulation promotes cell growth and proliferation by modulating the PI3K/AKT signaling pathway. This study identifies potentially deleterious missense single nucleotide variants (SNVs) associated with the LHPP gene using multiple computational tools based on different algorithms. A total of 4 destabilizing mutants are identified as L22P, I212T, G227R, and G236R, from the conserved region of the phosphatase. The 3-dimensional (3D) modeling and structural comparison of variants with the native protein reveals significant structural and conformational variations after mutations, suggesting disruption in the function of phospholysine phosphohistidine inorganic pyrophosphate phosphatase. The identified mutations might, therefore, participate in the cause of HCC.

Determination of Pleiotropic Effect of Warfarin in VKORC1 and CYP2C9 Genotypes in Patients With Heart Valve Replacement.

Warfarin has been widely used as an oral anticoagulant agent. In past, efforts have been done to study the contribution of genetic variation on warfarin dose requirements. The possible therapeutic dose determination of warfarin is very challenging, i.e., extremely low dose leading to unusable antithrombotic therapy or high dose causes particularly bleeding complications. Our study aimed to investigate these observations in more detail, we determined the correlation of interleukin-6 (IL-6), cyclooxygenase-2 (COX-2), and tumor necrosis factor-α (TNF-α) among VKORC1 and CYP2C9 genetic variants in patients with heart valve replacement who were treated with a range of warfarin doses and compared with levels in healthy controls. A total of 107 human subjects were recruited with low < 5 mg, medium 5-10 mg/day, and high > 10 mg/day warfarin doses. The genetic study of VKORC1-1639G/A, C1173T, 3730G > A, CYP2C9*2, and CYP2C9*3 was performed using TaqMan genotyping and DNA sequencing. The gene expression of IL-6, TNF-α, and COX-2 mRNA was analyzed. IL-6, TNF-α, and COX-2 protein expressions were determined by ELISA and Western blot analysis to evaluate the pro- and anti-inflammatory effects of warfarin. A statistically significant difference was found among the haplotypes of VKORC1 rs9934438 (C1173T), rs9923231 (-1639G > A), rs7294 (3730G > A) and CYP2C9 *2 p. Arg144 Cys (rs28371674), CYP2C9 *3 p. Ile359Leu (rs1057910) genotypes with warfarin dose requirements (p = 0.001). The increased levels of COX-2, IL-6, and TNF-α proteins were observed when a high dose of warfarin (>10 mg/ml) was administered. However, a lower concentration (1.0 mg/ml) was observed with decreased warfarin dose (<5 mg/day). The present study reported that in addition to its anticoagulant action, the genetic variants of warfarin may have a pleiotropic effect by influencing IL-6 depending on the dosing regimen and inducing the expression of COX-2.

Association of CTLA-4 and IL-4 polymorphisms in viral induced liver cancer.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most prevalent types of cancer and is responsible for close to one million annual deaths globally. In Pakistan, HCC accounts for 10.7% of cancer incidence. Prior studies indicated an association between interleukin 4 (IL-4) and cytotoxic T lymphocyte protein 4 (CTLA-4) gene polymorphisms in many types of cancers, including HCC that are either hepatitis B virus (HBV)- or hepatitis C Virus (HCV)-induced. The association of IL-4 and CTLA-4 genetic polymorphisms with HCV-induced HCC is not yet determined in the Pakistani population. Therefore, this research is designed to investigate the implication of IL-4 and CTLA-4 gene polymorphisms by determining the association of IL-4 -590 C/T (rs2243250) and CTLA-4 + 49 A/G (rs231775) with HCC in Pakistan. METHODS: Different bioinformatics tools were employed to determine the pathogenicity of these polymorphisms. Samples were collected from HCV-induced HCC patients, followed by DNA extraction and ARMS-PCR analysis. RESULTS: The SNP analysis results indicated a positive association of IL-4 -590C/T and CTLA-4 + 49A/G gene polymorphisms with HCV-induced HCC in Pakistan. The CTLA-4 polymorphism might enhance therapeutic efficiency of HCC chemotherapy medicines. The IL-4 polymorphism might introduce new transcription factor binding site in IL-4 promoter region. CONCLUSION: This study delineated risk factor alleles in CTLA-4 and IL-4 genes associated with HCV-mediated HCC among Pakistani patients that may have application to serve as genetic markers for pre- and early diagnosis and prognosis of HCC in HCV patients.

Targeted gene sequencing of FYCO1 identified a novel mutation in a Pakistani family for autosomal recessive congenital cataract.

BACKGROUND: Congenital cataract is causing one-third of blindness worldwide. Congenital cataract is heterogeneous in its inheritance patterns. The current study is aimed to explore the unknown genetic causes underlying congenital cataracts. METHODS: Blood samples from affected and normal individuals of n = 25 Pakistani families identified with congenital cataracts were collected. Genomic DNA was extracted and Sanger sequencing was performed to identify novel pathogenic variants in the FYCO1 (MIM#607182) gene. Later structural bioinformatics tools and molecular dynamics simulations were performed to analyze the impact of these variants on protein structure and function. RESULTS: Sanger sequencing resulted in the identification of a novel splice site mutation (NM_024513.3: c.3151-29_3151-7del) segregating in an autosomal recessive manner. This novel variant was confirmed to be absent in the n = 300 population controls. Further, bioinformatics tools revealed the formation of a mutant protein with a loss of the Znf domain. In addition, we also found a previously known (c.4127 T > C; p.Leu1376Pro) mutation in four families. We also report a novel heterozygous variant (c.3419G > A; p.Arg1140Gln) in another family. CONCLUSIONS: In conclusion, we report a novel deletion (NM_024513.3: c.3151-29_3151-7del) in one family and a frequent homozygous missense mutation (c.4127 T > C; p.Leu1376Pro) in four Pakistani families. The current research highlights the importance of autophagy in lens development and maintaining its transparency.

Impact of deleterious missense PRKCI variants on structural and functional dynamics of protein.

Protein kinase C iota (PKCÉ©) is a novel protein containing 596 amino acids and is also a member of atypical kinase family. The role of PKCÉ© has been explored in neurodegenerative diseases, neuroblastoma, ovarian and pancreatic cancers. Single nucleotide polymorphisms (SNPs) have not been studied in PKCÉ© till date. The purpose of the current study is to scrutinize the deleterious missense variants in PKCÉ© and determine the effect of these variants on stability and dynamics of the protein. The structure of protein PKCÉ© was predicted for the first time and post translational modifications were determined. Genetic variants of PKCÉ© were retrieved from ENSEMBL and only missense variants were further analyzed because of its linkage with diseases. The pathogenicity of missense variants, effect on structure and function of protein, association with cancer and conservancy of the protein residues were determined through computational approaches. It is observed that C1 and the pseudo substrate region has the highest number of pathogenic SNPs. Variations in the kinase domain of the protein are predicted to alter overall phosphorylation of the protein. Molecular dynamic simulations predicted noteworthy change in structural and functional dynamics of the protein because of these variants. The study revealed that nine deleterious variants can possibly contribute to malfunctioning of the protein and can be associated with diseases. This can be useful in diagnostics and developing therapeutics for diseases related to these polymorphisms.

Identification of the Genes of the Plant Pathogen Pseudomonas syringae MB03 Required for the Nematicidal Activity Against Caenorhabditis elegans Through an Integrated Approach.

Nematicidal potential of the common plant pathogen Pseudomonas syringae has been recently identified against Caenorhabditis elegans. The current study was designed to investigate the detailed genetic mechanism of the bacterial pathogenicity by applying comparative genomics, transcriptomics, mutant library screening, and protein expression. Results showed that P. syringae strain MB03 could kill C. elegans in the liquid assay by gut colonization. The genome of P. syringae MB03 was sequenced and comparative analysis including multi locus sequence typing, and genome-to-genome distance placed MB03 in phylogroup II of P. syringae. Furthermore, comparative genomics of MB03 with nematicidal strains of Pseudomonas aeruginosa (PAO1 and PA14) predicted 115 potential virulence factors in MB03. However, genes for previously reported nematicidal metabolites, such as phenazine, pyochelin, and pyrrolnitrin, were found absent in the MB03 genome. Transcriptomics analysis showed that the growth phase of the pathogen considerably affected the expression of virulence factors, as genes for the flagellum, glutamate ABC transporter, phoP/phoQ, fleS/fleR, type VI secretion system, and serralysin were highly up-regulated when stationary phase MB03 cells interacted with C. elegans. Additionally, screening of a transposon insertion mutant library led to the identification of other nematicidal genes such as acnA, gltP, oprD, and zapE. Finally, the nematicidal activity of selected proteins was confirmed by heterologous expression in Escherichia coli.