Molecular Dynamics Simulation of Kir6.2 Variants Reveals Potential Association with Diabetes Mellitus.

Diabetes mellitus (DM) represents a problem for the healthcare system worldwide. DM has very serious complications such as blindness, kidney failure, and cardiovascular disease. In addition to the very bad socioeconomic impacts, it influences patients and their families and communities. The global costs of DM and its complications are huge and expected to rise by the year 2030. DM is caused by genetic and environmental risk factors. Genetic testing will aid in early diagnosis and identification of susceptible individuals or populations using ATP-sensitive potassium (KATP) channels present in different tissues such as the pancreas, myocardium, myocytes, and nervous tissues. The channels respond to different concentrations of blood sugar, stimulation by hormones, or ischemic conditions. In pancreatic cells, they regulate the secretion of insulin and glucagon. Mutations in the KCNJ11 gene that encodes the Kir6.2 protein (a major constituent of KATP channels) were reported to be associated with Type 2 DM, neonatal diabetes mellitus (NDM), and maturity-onset diabetes of the young (MODY). Kir6.2 harbors binding sites for ATP and phosphatidylinositol 4,5-diphosphate (PIP2). The ATP inhibits the KATP channel, while the (PIP2) activates it. A Kir6.2 mutation at tyrosine330 (Y330) was demonstrated to reduce ATP inhibition and predisposes to NDM. In this study, we examined the effect of mutations on the Kir6.2 structure using bioinformatics tools and molecular dynamic simulations (SIFT, PolyPhen, SNAP2, PANTHER, PhD&SNP, SNP&Go, I-Mutant, MuPro, MutPred, ConSurf, HOPE, and GROMACS). Our results indicated that M199R, R201H, R206H, and Y330H mutations influence Kir6.2 structure and function and therefore may cause DM. We conclude that MD simulations are useful techniques to predict the effects of mutations on protein structure. In addition, the M199R, R201H, R206H, and Y330H variant in the Kir6.2 protein may be associated with DM. These results require further verification in protein-protein interactions, Kir6.2 function, and case-control studies.

In Silico Investigation of AKT2 Gene and Protein Abnormalities Reveals Potential Association with Insulin Resistance and Type 2 Diabetes.

Type 2 diabetes (T2D) develops from insulin resistance (IR) and the dysfunction of pancreatic beta cells. The AKT2 protein is very important for the protein signaling pathway, and the non-synonymous SNP (nsSNPs) in AKT2 gene may be associated with T2D. nsSNPs can result in alterations in protein stability, enzymatic activity, or binding specificity. The objective of this study was to investigate the effect of nsSNPs on the AKT2 protein structure and function that may result in the induction of IR and T2D. The study identified 20 variants that were considered to be the most deleterious based on a range of analytical tools included (SIFT, PolyPhen2, Mut-pred, SNAP2, PANTHER, PhD-SNP, SNP&Go, MUpro, Cosurf, and I-Mut). Two mutations, p.A179T and p.L183Q, were selected for further investigation based on their location within the protein as determined by PyMol. The results indicated that mutations, p.A179T and p.L183Q alter the protein stability and functional characteristics, which could potentially affect its function. In order to conduct a more in-depth analysis of these effects, a molecular dynamics simulation was performed for wildtype AKT2 and the two mutants (p.A179T and p.L183Q). The simulation evaluated various parameters, including temperature, pressure, density, RMSD, RMSF, SASA, and Region, over a period of 100 ps. According to the simulation results, the wildtype AKT2 protein demonstrated higher stability in comparison to the mutant variants. The mutations p.A179T and p.L183Q were found to cause a reduction in both protein stability and functionality. These findings underscore the significance of the effects of nsSNPs (mutations p.A179T and p.L183Q) on the structure and function of AKT2 that may lead to IR and T2D. Nevertheless, they require further verifications in future protein functional, protein-protein interaction, and large-scale case-control studies. When verified, these results will help in the identification and stratification of individuals who are at risk of IR and T2D for the purpose of prevention and treatment.

Identification and Characterization of Novel Mutations in Chronic Kidney Disease (CKD) and Autosomal Dominant Polycystic Kidney Disease (ADPKD) in Saudi Subjects by Whole-Exome Sequencing.

Background: Autosomal dominant polycystic kidney disease (ADPKD) is a condition usually caused by a single gene mutation and manifested by both renal and extrarenal features, eventually leading to end-stage renal disease (ESRD) by the median age of 60 years worldwide. Approximately 89% of ADPKD patients had either PKD1 or PKD2 gene mutations. The majority (85%) of the mutations are in the PKD1 gene, especially in the context of family history. Objectives: This study investigated the genetic basis and the undiscovered genes that are involved in ADPKD development among the Saudi population. Materials and Methods: In this study, 11 patients with chronic kidney disease were enrolled. The diagnosis of ADPKD was based on history and diagnostic images: CT images include enlargement of renal outlines, renal echogenicity, and presence of multiple renal cysts with dilated collecting ducts, loss of corticomedullary differentiation, and changes in GFR and serum creatinine levels. Next-generation whole-exome sequencing was conducted using the Ion Torrent PGM platform. Results: Of the 11 Saudi patients diagnosed with chronic kidney disease (CKD) and ADPKD, the most common heterozygote nonsynonymous variant in the PKD1 gene was exon15: (c.4264G > A). Two missense mutations were identified with a PKD1 (c.1758A > C and c.9774T > G), and one patient had a PKD2 mutation (c.1445T > G). Three detected variants were novel, identified at PKD1 (c.1758A > C), PKD2L2 (c.1364A > T), and TSC2 (deletion of a'a at the 3'UTR, R1680C) genes. Other variants in PKD1L1 (c.3813_381 4delinsTG) and PKD1L2 (c.404C > T) were also detected. The median age of end-stage renal disease for ADPK patients in Saudi Arabia was 30 years. Conclusion: This study reported a common variant in the PKD1 gene in Saudi patients with typical ADPKD. We also reported (to our knowledge) for the first time two novel missense variants in PKD1 and PKD2L2 genes and one indel mutation at the 3'UTR of the TSC2 gene. This study establishes that the reported mutations in the affected genes resulted in ADPKD development in the Saudi population by a median age of 30. Nevertheless, future protein−protein interaction studies to investigate the influence of these mutations on PKD1 and PKD2 functions are required. Furthermore, large-scale population-based studies to verify these findings are recommended.

Clinical Implications of MiR128, Angiotensin I Converting Enzyme and Vascular Endothelial Growth Factor Gene Abnormalities and Their Association with T2D.

Type 2 DM (T2D) results from the interaction of the genetic and environmental risk factors. Vascular endothelial growth factor (VEGF), angiotensin I-converting enzyme (ACE), and MicroRNAs (MiRNAs) are involved in important physiological processes. Gene variations in VEGF, ACE and MiRNA genes are associated with diseases. In this study we investigated the associations of the VEGF-2578 C/A (rs699947), VEGF-2549 insertion/deletion (I/D), and ACE I/D rs4646994 and Mir128a (rs11888095) gene variations with T2D using the amplification refractory mutation system PCR (ARMS-PCR) and mutation specific PCR (MSP). We screened 122 T2D cases and 126 healthy controls (HCs) for the rs699947, and 133 T2D cases and 133 HCs for the VEGF I/D polymorphism. For the ACE I/D we screened 152 cases and 150 HCs, and we screened 129 cases and 112 HCs for the Mir128a (rs11888095). The results showed that the CA genotype of the VEGF rs699947 and D allele of the VEGF I/D polymorphisms were associated with T2D with OR =2.01, p-value = 0.011, and OR = 2.42, p-value = 0.010, respectively. The result indicated the D allele of the ACE ID was protective against T2D with OR = 0.10, p-value = 0.0001, whereas the TC genotype and the T allele of the Mir128a (rs11888095) were associated with increased risk to T2D with OR = 3.16, p-value = 0.0001, and OR = 1.68, p-value = 0.01, respectively. We conclude that the VEGF (rs699947), VEGF I/D and Mir128a (rs11888095) are potential risk loci for T2D, and that the D allele of the ACE ID polymorphism may be protective against T2D. These results help in identification and stratification for the individuals that at risk for T2D. However, future well-designed studies in different populations and with larger sample sizes are required. Moreover, studies to examine the effects of these polymorphisms on VEGF and ACE proteins are recommended.

Role of non-coding RNA networks in leukemia progression, metastasis and drug resistance.

Early-stage detection of leukemia is a critical determinant for successful treatment of the disease and can increase the survival rate of leukemia patients. The factors limiting the current screening approaches to leukemia include low sensitivity and specificity, high costs, and a low participation rate. An approach based on novel and innovative biomarkers with high accuracy from peripheral blood offers a comfortable and appealing alternative to patients, potentially leading to a higher participation rate.Recently, non-coding RNAs due to their involvement in vital oncogenic processes such as differentiation, proliferation, migration, angiogenesis and apoptosis have attracted much attention as potential diagnostic and prognostic biomarkers in leukemia. Emerging lines of evidence have shown that the mutational spectrum and dysregulated expression of non-coding RNA genes are closely associated with the development and progression of various cancers, including leukemia. In this review, we highlight the expression and functional roles of different types of non-coding RNAs in leukemia and discuss their potential clinical applications as diagnostic or prognostic biomarkers and therapeutic targets.

Identification and characterization of peptides that bind the PPIase domain of Parvulin17.

Peptidyl-prolyl cis-trans isomerases (PPIases) are the enzymes that increase the rate of isomerization of the peptide bond N-terminal to the proline substrate. Par14 and its isoform Par17 belong to the Parvulin family of PPIases. Par14 can bind AT-rich double-stranded DNA and was shown to be part of the pre-ribosomal ribonucleoprotein (pre-rRNP) complexes, where it functions as an RNA processing factor that is involved in ribosome biogenesis. Its longer isoform Par17 is expressed only in cells of hominids, where it is targeted to the mitochondria. To find binding partners (peptides or proteins) for Par17, we applied the phage display technology. We panned 7-mer and 12-mer peptide libraries against Par17. The consensus sequence XHSXVHØ, where X can be any amino acid and Ø is a hydrophobic amino acid, was enriched from both libraries. We demonstrate the binding of this motif to the PPIase domain of Par17 using phage ELISA and NMR spectroscopy. We propose that residues Met90, Val91, Phe94, Gln95, Glu96, and Ala98 of Par17 are involved in substrate recognition, and that the phage display-selected motif XHSXVHØ can be recognized by Par17 PPIase domain in vivo.

Potential Association of The Pathogenic Kruppel-like Factor 14 (KLF14) and Adiponectin (ADIPOQ) SNVs with Susceptibility to T2DM.

AIM: To evaluate the associations of the pathogenic variants in Kruppel-like Factor 14 (KLF 14) and Adiponectin (ADIPOQ) with susceptibility to type 2 diabetes mellitus (T2DM). BACKGROUND: Type 2 diabetes mellitus (T2DM) is a pandemic metabolic disease characterized by increased blood sugar and caused by resistance to insulin in peripheral tissues and damage to pancreatic beta cells. Kruppel-like Factor 14 (KLF-14) is proposed to be a regulator of metabolic diseases, such as diabetes mellitus (DM) and obesity. Adiponectin (ADIPOQ) is an adipocytokine produced by the adipocytes and other tissues and was reported to be involved in T2DM. OBJECTIVES: To study the possible association of the KLF-14 rs972283 and ADIPOQ-rs266729 with the risk of T2DM in the Saudi population. METHODS: We have evaluated the association of KLF-14 rs972283 C>T and ADIPOQ-rs266729 C>G SNV with the risk to T2D in the Saudi population using the Amplification Refractory Mutation System PCR (ARMS-PCR), and blood biochemistry analysis. For the KLF-14 rs972283 C>T SNV we included 115 cases and 116 healthy controls, and ADIPOQ-rs266729 C>G SNV, 103 cases and 104 healthy controls were included. RESULTS: Results indicated that the KLF-14 rs972283 GA genotype and A allele were associated with T2D risk with OR=2.14, p-value= 0.014 and OR=1.99, p-value=0.0003, respectively. Results also ADIPOQ-rs266729 CG genotype and C allele were associated with an elevated T2D risk with an OR=2.53, p=0.003 and OR=1.66, p-value =0.012, respectively. CONCLUSION: We conclude that SNVs in KLF-14 and ADIPOQ are potential loci for T2D risk. Future large-scale studies to verify these findings are recommended. These results need further verifications in protein functional and large-scale case control studies before being introduced for genetic testing.

Potential Impact of PI3K-AKT Signaling Pathway Genes, KLF-14, MDM4, miRNAs 27a, miRNA-196a Genetic Alterations in the Predisposition and Progression of Breast Cancer Patients.

Genome-wide association studies have reported link between SNPs and risk of breast cancer. This study investigated the association of the selected gene variants by predicting them as possible target genes. Molecular technique advances with the availability of whole-exome sequencing (WES), now offer opportunities for simultaneous investigations of many genes. The experimental protocol for PI3K, AKT-1, KLF-14, MDM4, miRNAs 27a, and miR-196a genotyping was done by ARMS-PCR and sanger sequencing. The novel and known gene variants were studied by Whole-exome sequencing using Illumina NovaSeq 6000 platform. This case control study reports significant association between BC patients, healthy controls with the polymorphic variants of PI3K C > T, AKT-1 G > A KLF 14 C > T, MDM4 A > G, miR-27a A > G, miR-196a-2 C > T genes (p < 0.05). MDM4 A > G genotypes were strongly associated with BC predisposition with OR 2.08 & 2.15, p < 0.05) in codominant and dominant models respectively. MDM4 A allele show the same effective (OR1.76, p < 0.05) whereas it remains protective in recessive model for BC risk. AKT1G > A genotypes were strongly associated with the BC susceptibility in all genetic models whereas PI3K C > T genotypes were associated with breast cancer predisposition in recessive model OR 6.96. Polymorphic variants of KLF-14 A > G, MDM4G > A, MiR-27aA >G, miR-196a-C > T were strongly associated with stage, tamoxifen treatment. Risk variants have been reported by whole exome sequencing in our BC patients. It was concluded that a strong association between the PI3K-AKT signaling pathway gene variants with the breast cancer susceptibility and progression. Similarly, KLF 14-AA, MDM4-GA, miR27a-GG and miR-196a-CT gene variants were associated with the higher risk probability of BC and were strongly correlated with staging of the BC patients. This study also reported Low, novel, and intermediate-genetic-risk variants of PI3K, AKT-1, MDM4G & KLF-14 by utilizing whole-exome sequencing. These variants should be further investigated in larger cohorts' studies.

Comprehensive Molecular Evaluation of HNF-1 Alpha, miR-27a, and miR-146 Gene Variants and Their Link with Predisposition and Progression in Type 2 Diabetes Patients.

BACKGROUND: Type 2 diabetes (T2D) is a metabolic condition induced by insulin resistance and pancreatic beta cell dysfunction. MicroRNAs (miRNAs) have biological significance because they regulate processes such as the molecular signaling pathways involved in the pathophysiology of diabetes mellitus. The hepatocyte nuclear factor-1 alpha (HNF-1 alpha) is a transcription factor found in hepatocytes and the pancreas. Mutations in the HNF-1 alpha gene were reportedly associated with maturity-onset diabetes of the young (MODY). The objective of the present study was to examine the associations between MiR-27a, MiR-146, and HNF-1 alpha single-nucleotide variations (SNVs) with T2D risk in the Saudi population. METHODOLOGY: We evaluated the association of SNVs of miR-27a rs895819 A>G, 146a-rs2910164 C>G, and HNF-1 alpha rs1169288 G>T (I27L) with the risk of T2D in Saudi patients with the Amplification Refractory Mutation System PCR (ARMS-PCR). For the miR-27a SNVs, we used 115 cases (82 males, 33 females) and 117 matched healthy controls (HCs); for the Mir-146 SNVs, we used 103 cases (70 males, 33 females) and 108 matched HCs; and for the HNF-1 alpha, we employed 110 patients (80 males, 30 females) and 110 HCs. The blood biochemistry of the participants was essayed using commercial kits, and the methods of statistical analysis used were the Chi-square test, the Fisher exact test, and a multivariate analysis based on logistic regression, like the odds ratio (OD) and risk ratio (RR), with 95% confidence intervals (CIs). RESULTS: The MiR-27a rs895819 AG genotype was linked to increased T2D susceptibility, with OR = 2.01 and p-value = 0.011, and the miR-146 rs2910164 CG genotype and C allele were linked to an elevated risk of T2D, with OR = 2.75, p-value < 0.0016, OR = 1.77, and p-value = 0.004. The results also showed that the GT genotype and T allele of the HNF-1 alpha (rs1169288) G>T is linked to T2D, with OR = 2.18, p-value = 0.0061, and 1.77, p-value = 0.0059. CONCLUSIONS: The SNVs in miR-27a, miR-146, and HNF-1 alpha can be potential loci for T2D risk. The limitations of this study include the relatively small sample size and the fact that it was a cross-sectional study. To our knowledge, this is the first study to highlight the association between miR-27a, miR-146, and HNF-1 alpha SNVs and the risk of T2D in the Saudi population. Future large-scale case-control studies, as well as studies on the functions of the proteins and protein interaction studies for HNF-1 alpha, are required to verify our findings. Furthermore, these findings can be used for the identification and stratification of at-risk populations via genetic testing for T2D-prevention strategies.

Long non-coding RNAs modulate tumor microenvironment to promote metastasis: novel avenue for therapeutic intervention.

Cancer is a devastating disease and the primary cause of morbidity and mortality worldwide, with cancer metastasis responsible for 90% of cancer-related deaths. Cancer metastasis is a multistep process characterized by spreading of cancer cells from the primary tumor and acquiring molecular and phenotypic changes that enable them to expand and colonize in distant organs. Despite recent advancements, the underlying molecular mechanism(s) of cancer metastasis is limited and requires further exploration. In addition to genetic alterations, epigenetic changes have been demonstrated to play an important role in the development of cancer metastasis. Long non-coding RNAs (lncRNAs) are considered one of the most critical epigenetic regulators. By regulating signaling pathways and acting as decoys, guides, and scaffolds, they modulate key molecules in every step of cancer metastasis such as dissemination of carcinoma cells, intravascular transit, and metastatic colonization. Gaining a good knowledge of the detailed molecular basis underlying lncRNAs regulating cancer metastasis may provide previously unknown therapeutic and diagnostic lncRNAs for patients with metastatic disease. In this review, we concentrate on the molecular mechanisms underlying lncRNAs in the regulation of cancer metastasis, the cross-talk with metabolic reprogramming, modulating cancer cell anoikis resistance, influencing metastatic microenvironment, and the interaction with pre-metastatic niche formation. In addition, we also discuss the clinical utility and therapeutic potential of lncRNAs for cancer treatment. Finally, we also represent areas for future research in this rapidly developing field.

Association of Genetic and Allelic Variants of Von Willebrand Factor (VWF), Glutathione S-Transferase and Tumor Necrosis Factor Alpha with Ischemic Stroke Susceptibility and Progression in the Saudi Population.

Stroke is a key cerebrovascular disease and important cause of death and disability worldwide, including in the kingdom of Saudi Arabia (KSA). It has a large economic burden and serious socioeconomic impacts on patients, their families and the community. The incidence of ischemic stroke is probably increased by the interaction of GSTT1 and GSTM1 null genotypes with high blood pressure, diabetes and cigarette smoking. The roles of VWF, GSTs and TNF-alpha gene variations in the induction of stroke are still uncertain and require further examination. In the current study, we studied the associations of SNPs in the genes VWF, GSTs and TNF-alpha with stroke in the Saudi population. Genotyping was performed using the ARMS -PCR for TNF-alpha, AS-PCR for VWF and multiplex PCR for GSTs. The study included 210 study subjects: 100 stroke cases and 110 healthy controls. We obtained significant distributions of VWF rs61748511 T > C, TNF-alpha rs1800629 G > A and GST rs4025935 and rs71748309 genotypes between stroke cases and the healthy controls (p < 0.05). The results also indicated that the TNF-alpha A allele was associated with risk of stroke with odd ratio (OR) = 2.22 and risk ratio = RR 2.47, p < 0.05. Similarly, the VWF-TC genotype and C allele were strongly linked with stroke with OR = 8.12 and RR 4.7, p < 0.05. In addition, GSTT1 and GSTT1 null genotype was strongly associated with stroke predisposition with OR = 8.30 and RR = 2.25, p < 0.0001. We conclude that there is a possible strong association between the VWF-T > C, TNF-alpha G > A, GSTT1 gene variants and ischemic stroke susceptibility in the Saudi population. However, future well-designed and large-scale case-control studies on protein-protein interactions and protein functional studies are required to verify these findings and examine the effects of these SNPs on these proteins.

Differential Expression of Serum Proinflammatory Cytokine TNF-α and Genetic Determinants of TNF-α, CYP2C19*17, miR-423 Genes and Their Effect on Coronary Artery Disease Predisposition and Progression.

Coronary artery disease (CAD) is the leading cause of death and hospitalization worldwide and represents a problem for public health systems everywhere. In Saudi Arabia, the prevalence of CAD is estimated to be 5.5%. Risk factors for CAD include older age, male gender, obesity, high blood pressure, smoking, diabetes, hyperlipidemia, and genetic factors. Reducing the risk factors in susceptible individuals will decrease the prevalence of CAD. Genome wide association studies have helped to reveal the association of many loci with diseases like CAD. In this study, we examined the link between single nucleotide variations (SNVs) of TNF-α-rs1800629 G>A, CYP2C19*17 (rs12248560) C>T, and miR-423 rs6505162 C>A and the expression of TNF-α with CAD. We used the mutation specific PCR, ARMS-PCR, and ELISA. The results showed that the A allele of the TNF-α rs1800629 G>A SNP is linked to CAD with odd ratio (OR) (95% CI) = 2.10, p-value = 0.0013. The T allele of the CYP2C19*17 (rs12248560) C>T is linked to CAD with OR (95% CI) = 2.02, p-value = 0.003. In addition, the A allele of the miR-423 rs6505162 C>A SNV is linked to CAD with OR (95% CI) = 1.49, p-value = 0.036. The ELISA results indicated that the TNF-α serum levels are significantly increased in CAD patients compared to healthy controls. We conclude the TNF-α rs1800629 G>A, CYP2C19*17, and miR-423 rs6505162 C>A are potential genetic loci for CAD in the Saudi population. These findings require further verification in future studies. After being verified, our results might be utilized in genetic testing to identify individuals that are susceptible to CAD and, therefore, for whom reducing modifiable risk factors (e.g., poor diet, diabetes, obesity, and smoking) would result in prevention or delay of CAD.

In Silico Evaluation of the Potential Association of the Pathogenic Mutations of Alpha Synuclein Protein with Induction of Synucleinopathies.

Alpha synuclein (α-Syn) is a neuronal protein encoded by the SNCA gene and is involved in the development of Parkinson's disease (PD). The objective of this study was to examine in silico the functional implications of non-synonymous single nucleotide polymorphisms (nsSNPs) in the SNCA gene. We used a range of computational algorithms such as sequence conservation, structural analysis, physicochemical properties, and machine learning. The sequence of the SNCA gene was analyzed, resulting in the mapping of 42,272 SNPs that are classified into different functional categories. A total of 177 nsSNPs were identified within the coding region; there were 20 variants that may influence the α-Syn protein structure and function. This identification was made by employing different analytical tools including SIFT, PolyPhen2, Mut-pred, SNAP2, PANTHER, PhD-SNP, SNP&Go, MUpro, Cosurf, I-Mut, and HOPE. Three mutations, V82A, K80E, and E46K, were selected for further examinations due to their spatial positioning within the α-Syn as determined by PyMol. Results indicated that these mutations may affect the stability and function of α-Syn. Then, a molecular dynamics simulation was conducted for the SNCA wildtype and the four mutant variants (p.A18G, p.V82A, p.K80E, and p.E46K). The simulation examined temperature, pressure, density, root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), solvent-accessible surface area (SASA), and radius of gyration (Rg). The data indicate that the mutations p.V82A, p.K80E, and p.E46K reduce the stability and functionality of α-Syn. These findings highlight the importance of understanding the impact of nsSNPs on α-syn structure and function. Our results required verifications in further protein functional and case-control studies. After being verified these findings can be used in genetic testing for the early diagnosis of PD, the evaluation of the risk factors, and therapeutic approaches.

Structure and dynamics of the first archaeal parvulin reveal a new functionally important loop in parvulin-type prolyl isomerases.

Parvulins are a group of peptidyl-prolyl isomerases (PPIases) responsible for important biological processes in all kingdoms of life. The PinA protein from the psychrophilic archaeon Cenarchaeum symbiosum is a parvulin-like PPIase. Due to its striking similarity to the human parvulins Pin1 and Par14, PinA constitutes an interesting subject for structural and functional studies. Here, we present the first high resolution NMR structure of an archaeal parvulin, PinA, based on 1798 conformational restraints. Structure calculation yields an ensemble of 20 convergent low energy structures with a backbone r.m.s.d. value of 0.6 Å within the secondary structure elements. The overall fold of PinA comprises the ß-α(3)-ß-α-ß(2) fold typical for all parvulin structures known so far, but with helix III being a short 3(10)-helix. A detailed comparison of this high resolution structure of the first archaeal PinA protein with bacterial and eukaryotic parvulin PPIase structures reveals an atypically large catalytic binding site. This feature provides an explanation for cold-adapted protein function. Moreover, the residues in and around 3(10)-helix III exhibit strong intramolecular dynamics on a microsecond to millisecond timescale and display structural heterogeneity within the NMR ensemble. A putative peptide ligand was found for PinA by phage display and was used for (1)H-(15)N-HSQC titrations. Again, the flexible region around 3(10)-helix III as well as residues of the peptide binding pocket showed the strongest chemical shift perturbations upon peptide binding. The local flexibility of this region also was modulated by ligand binding. A glycine and two positively charged residues are conserved in most parvulin proteins in this flexible loop region, which may be of general functional importance for parvulin-type PPIases.

The Impact of the Coronavirus (COVID-19) Infection on the Drug-Metabolizing Enzymes Cytochrome P450s.

Coronaviruses cause disease in human and animals. In 2019 a novel coronavirus was first characterized in Wuhan, China. It causes acute respiratory disease and designated the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or COVID-19. The COVID-19 spread to all cities of China, and in 2020 to the whole world. Patients with COVID-19 may recover without medical treatment. However, some patients need medical care. The Cytochrome p450s (CYP450s) are large superfamily of enzymes catalyze the metabolism of endogenous substrates and xenobiotics. CYP450s catalyze the biotransformation of 80% of the drug in clinical use. The CYP450 present in liver, lungs, intestine and other tissues. COVID-19 has been reported to decrease the activity of certain isoforms of CYP450s in an isoform specific manner. Furthermore, the COVID-19 infection decreases the liver functions including the drug clearance or detoxification medicated by the CYP450s. The healthcare providers should be aware of this disease-drug interaction when prescribing drugs for treatment of COVID-19 and other comorbidities.

Clinical Correlations of Lipid Profiles with the Age and Gender in the Coronary Artery Disease Patients: A Study of 3878 CAD Patients from India.

BACKGROUND: Cardiovascular diseases (CVDs) are crucial cause of death and hospitalization all over the world including India. The CVDs including the coronary artery disease (CAD) are developed by the interaction of genetic and environmental factors. Hyperlipidemia is a traditional risk factor for CVD. AIM: The aim of this study was to study the clinical correlations of lipid profiles with the age and gender in the Coronary Artery Disease Patients: Methods: In this study, we have investigated the effect of age and sex on in lipid profile in 3878 (1171 females and 2707 males) CAD patients from India. RESULTS: The plasma TG was higher in males than in females regardless of the age. Results showed that CAD female patients had significantly increased HDL-C than their aged matched males. Moreover, the plasma TC and LDL-C were significantly higher in males than females until age 40 years. Then after the age of 40 years, TC and LDL-C become significantly higher in females than in males. In addition, we found that more than 85% of CAD cases were <55 years old, and about 30% of CAD cases had normal lipid profile. CONCLUSION: We conclude that elderly females are at a greater risk for CAD than males. Moreover, there were no significant differences in CVDs causes between nonelderly and elderly females. In addition, a higher percentage of cases were premature CAD, and 30% of CAD may be caused by loci that are not related to lipid metabolism.

Anticancer Activity of Extremely Effective Recombinant L-Asparaginase from Burkholderia pseudomallei.

L-asparaginase (E.C. 3.5.1.1) purified from bacterial cells is widely used in the food industry, as well as in the treatment of childhood acute lymphoblastic leukemia. In the present study, the Burkholderia pseudomallei L-asparaginase gene was cloned into the pGEX-2T DNA plasmid, expressed in E. coli BL21 (DE3) pLysS, and purified to homogeneity using Glutathione Sepharose chromatography with 7.26 purification fold and 16.01% recovery. The purified enzyme exhibited a molecular weight of ~33.6 kDa with SDS-PAGE and showed maximal activity at 50°C and pH 8.0. It retained 95.1, 89.6%, and 70.2% initial activity after 60 min at 30°C, 40°C, and 50°C, respectively. The enzyme reserved its activity at 30°C and 37°C up to 24 h. The enzyme had optimum pH of 8 and reserved 50% activity up to 24 h. The recombinant enzyme showed the highest substrate specificity towards L-asparaginase substrate, while no detectable specificity was observed for L-glutamine, urea, and acrylamide at 10 mM concentration. THP-1, a human leukemia cell line, displayed significant morphological alterations after being treated with recombinant L-asparaginase and the IC50 of the purified enzyme was recorded as 0.8 IU. Furthermore, the purified recombinant L-asparaginase improved cytotoxicity in liver cancer HepG2 and breast cancer MCF-7 cell lines, with IC50 values of 1.53 and 18 IU, respectively.

Differential impact of the angiotensin-converting enzyme-2 (ACE2 rs4343 G>A) and miR-196a2 rs11614913 C>T gene alterations in COVID-19 disease severity and mortality.

The recent coronavirus outbreak from Wuhan China in late 2019 caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) resulted in a global pandemic of coronavirus-19 disease (COVID-19). Understating the underlying mechanism of the pathogenesis of coronavirus infection is important not only because it will help in accurate diagnosis and treatment of the infection but also in the production of effective vaccines. The infection begins when SARS-CoV-2 enters the cells through binding of its envelope glycoprotein to angiotensin-converting enzyme2 (ACE2). Gene variations of ACE2 and microRNA (miR)-196 are associated with viral infection and other diseases. The present study investigated the association of the ACE2 rs4343 G>A and miR-196a2 rs11614913 C>T gene polymorphisms with severity and mortality of COVID-19 using amplification refractory mutation system PCR in 117 COVID-19 patients and 103 healthy controls from three regions of Saudi Arabia. The results showed that ACE2 rs4343 GA genotype was associated with severity of COVID-19 (OR=2.10, P-value 0.0028) and ACE2 rs4343 GA was associated with increased mortality with OR=3.44, P-value 0.0028. A strong correlation between the ACE2 rs4343 G>A genotype distribution among COVID-19 patients was reported with respect to their comorbid conditions including sex (P<0.023), coronary artery disease (P<0.0001), oxygen saturation <60 mm Hg (P<0.0009) and antiviral therapy (0.003). The results also showed that the CT genotype and T allele of the miR-196a2 rs11614913 C>T were associated with decreased risk to COVID-19 with OR=0.76, P=0.006 and OR=0.54, P=0.005, respectively. These results need to be validated with future molecular genetic studies in a larger sample size and different populations.

Clinical Implications of Glyoxalase1 Gene Polymorphism and Elevated Levels of the Reactive Metabolite Methylglyoxal in the Susceptibility of Type 2 Diabetes Mellitus in the Patients from Asir and Tabuk Regions of Saudi Arabia.

Diabetes mellitus constitutes a big challenge to the global health care system due to its socioeconomic impacts and very serious complications. The incidence and the prevalence rate are increased in the Gulf region including the KSA. Type 2 diabetes mellitus (T2DM) is caused by diverse risk factors including obesity, unhealthy dietary habits, physical inactivity, smoking and genetic factors. The molecular genetic studies have helped in the detection of many single nucleotide polymorphisms (SNP) with different diseases including cancers, cardiovascular diseases and T2DM. The glyoxalase 1 (GLO1) is a detoxifying enzyme and catalyzes the elimination of the cytotoxic product methylglyoxal (MG) by converting it to D-lactate, which is not toxic to tissues. MG accumulation is associated with the pathogenesis of different diseases including T2DM. In this study, we have investigated the association of the glyoxalase 1 SNPs (rs2736654) rs4746 C>A and rs1130534 T>A with T2DM using the amplification refractory mutation system PCR. We also measured the concentration of MG by ELISA in T2DM patients and matched heathy controls. Results show that the CA genotype of the GLO rs4647 A>C was associated with T2DM with OR = 2.57, p-value 0.0008 and the C allele was also associated with increased risk to T2DM with OR = 2.24, p-value = 0.0001. It was also observed that AT genotype of the rs1130534 was associated with decreased susceptibility to T2DM with OR = 0.3, p-value = 0.02. The A allele of rs1130534 was also associated with reduced risk to T2DM with PR = 0.27 = 0.006. In addition, our ELISA results demonstrate significantly increased MG concentrations in serum of the T2DM patients. We conclude that the GLO1 SNP may be associated with decreased enzyme activity and a resultant susceptibility to T2DM. Further well-designed studies in different and large patient populations are recommended to verify these findings.