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.

Molecular Determination of Tumor Necrosis Factor-alpha, Interleukin-8, Interleukin-10, and C-X-C Chemokine Receptor-2 Genetic Variations and their Association with Disease Susceptibility and Mortality in COVID-19 Patients.

Background: Altered cytokine levels have been associated with poor outcomes among COVID-19 patients. TNF-α, IL-8 and IL-10 are key cytokines in COVID-19 pathogenesis, and CXCR-2 is a major chemokine receptor involved in inflammatory response. Polymorphisms in the genes of these proteins are proposed to influence disease outcomes. In this study, we aimed to find out the association of genetic polymorphisms in TNF-α, IL-8, IL-10 and CXCR-2 genes with susceptibility to and mortality of COVID-19. Methods: The present case-control study was conducted on 230 subjects, among whom 115 were clinically diagnosed and RT-PCR-confirmed COVID-19 patients and 115 healthy control subjects. The polymorphisms in TNFα -308 G>A (rs1800629), IL-8 -251T>A (rs4073), CXCR2 +785 C>T (rs2230054) genes were detected by ARMS -PCR assay whereas for IL-10 (-1082 G>A), rs1800896 G>A allele-specific PCR assay was used and their association with COVID-19 susceptibility and mortality was estimated by multivariate analysis. The results were analyzed for risk of infection and mortality through different inheritance models. Results: Frequencies of TNF-α rs1800629 GA, AA, IL-8 rs4073 TA, AA, IL-10 (-1082 G>A), rs1800896 GA and GG, and CXCR2 rs2230054 CT genotypes were significantly higher in COVID-19 patients compared to the control group (p < 0.05). Furthermore, COVID-19 patients had a higher frequency of the polymorphic A allele of TNF-α, the A allele of IL-8, the G allele of IL-10, and the T allele of CXCR2. The risk of susceptibility to COVID-19 was significantly associated with TNF-α rs1800629 GA, GA+AA genotypes and the A allele, IL-8 rs4073 TA, AA genotypes and A allele, IL-10 rs1800872 GA and CC genotypes and C allele, and CXCR2 rs2230054 CT and CT+CC genotypes. TNF-α-GA and AA genotypes and A allele, IL-8 TA and AA genotypes and A allele and CXCR-2 CC and CT genotypes have significant associations with mortality risk in COVID-19 patients, while GA and GG genotypes of the IL-10 are shown to confer significant protection against mortality from COVID-19. Conclusion: The findings of this study provide important insights into the COVID-19 disease and susceptibility risk. The polymorphisms in TNFα -308 G>A (rs1800629), IL-8 -251T>A (rs4073), IL-10 (-1082 G>A), rs1800896 and CXCR2 +785 C>T (rs2230054) are associated with the risk of susceptibility to COVID-19 and with mortality in COVID-19 patients. Further studies with larger sample sizes are necessary to confirm our findings.

Cancer cell plasticity: from cellular, molecular, and genetic mechanisms to tumor heterogeneity and drug resistance.

Cancer is a complex disease displaying a variety of cell states and phenotypes. This diversity, known as cancer cell plasticity, confers cancer cells the ability to change in response to their environment, leading to increased tumor diversity and drug resistance. This review explores the intricate landscape of cancer cell plasticity, offering a deep dive into the cellular, molecular, and genetic mechanisms that underlie this phenomenon. Cancer cell plasticity is intertwined with processes such as epithelial-mesenchymal transition and the acquisition of stem cell-like features. These processes are pivotal in the development and progression of tumors, contributing to the multifaceted nature of cancer and the challenges associated with its treatment. Despite significant advancements in targeted therapies, cancer cell adaptability and subsequent therapy-induced resistance remain persistent obstacles in achieving consistent, successful cancer treatment outcomes. Our review delves into the array of mechanisms cancer cells exploit to maintain plasticity, including epigenetic modifications, alterations in signaling pathways, and environmental interactions. We discuss strategies to counteract cancer cell plasticity, such as targeting specific cellular pathways and employing combination therapies. These strategies promise to enhance the efficacy of cancer treatments and mitigate therapy resistance. In conclusion, this review offers a holistic, detailed exploration of cancer cell plasticity, aiming to bolster the understanding and approach toward tackling the challenges posed by tumor heterogeneity and drug resistance. As articulated in this review, the delineation of cellular, molecular, and genetic mechanisms underlying tumor heterogeneity and drug resistance seeks to contribute substantially to the progress in cancer therapeutics and the advancement of precision medicine, ultimately enhancing the prospects for effective cancer treatment and patient outcomes.

Extracellular vesicles as tools and targets in therapy for diseases.

Extracellular vesicles (EVs) are nano-sized, membranous structures secreted into the extracellular space. They exhibit diverse sizes, contents, and surface markers and are ubiquitously released from cells under normal and pathological conditions. Human serum is a rich source of these EVs, though their isolation from serum proteins and non-EV lipid particles poses challenges. These vesicles transport various cellular components such as proteins, mRNAs, miRNAs, DNA, and lipids across distances, influencing numerous physiological and pathological events, including those within the tumor microenvironment (TME). Their pivotal roles in cellular communication make EVs promising candidates for therapeutic agents, drug delivery systems, and disease biomarkers. Especially in cancer diagnostics, EV detection can pave the way for early identification and offers potential as diagnostic biomarkers. Moreover, various EV subtypes are emerging as targeted drug delivery tools, highlighting their potential clinical significance. The need for non-invasive biomarkers to monitor biological processes for diagnostic and therapeutic purposes remains unfulfilled. Tapping into the unique composition of EVs could unlock advanced diagnostic and therapeutic avenues in the future. In this review, we discuss in detail the roles of EVs across various conditions, including cancers (encompassing head and neck, lung, gastric, breast, and hepatocellular carcinoma), neurodegenerative disorders, diabetes, viral infections, autoimmune and renal diseases, emphasizing the potential advancements in molecular diagnostics and drug delivery.

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.

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.

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.

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.

Insights on antioxidant therapeutic strategies in type 2 diabetes mellitus: A narrative review of randomized control trials.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is a metabolic disease of impaired glucose utilization. Imbalance in generation and elimination of free radicals generate oxidative stress which modulates glucose metabolism and insulin regulation, resulting in the occurrence and progression of diabetes and associated complications. Antioxidant supplements in T2DM can be seen as a potential preventive and effective therapeutic strategy. AIM: To compare randomized controlled trials (RCTs) in which antioxidants have been shown to have a therapeutic effect in T2DM patients. METHODS: We systematically searched the electronic database PubMed by keywords. RCTs evaluating the effect of antioxidant therapy on glycaemic control as well as oxidant and antioxidant status as primary outcomes were included. The outcomes considered were: A reduction in blood glucose; changes in oxidative stress and antioxidant markers. Full-length papers of the shortlisted articles were assessed for the eligibility criteria and 17 RCTs were included. RESULTS: The administration of fixed-dose antioxidants significantly reduces fasting blood sugar and glycated hemoglobin and is associated with decreased malondialdehyde, advanced oxidation protein products, and increased total antioxidant capacity. CONCLUSION: Antioxidant supplements can be a beneficial approach for the treatment of T2DM.

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.

Molecular Evaluation of the Impact of Polymorphic Variants in Apoptotic (Bcl-2/Bax) and Proinflammatory Cytokine (TNF-α/IL-8) Genes on the Susceptibility and Progression of Myeloproliferative Neoplasms: A Case-Control Biomarker Study.

The regulation of apoptosis (the programmed cell death) is dependent on the crucial involvement of BCL2 and BAX. The Bax-248G>A and Bcl-2-938 C>A polymorphic variations in the promoter sequences of the Bax and Bcl-2 gene have been recently associated with low Bax expression, progression to advanced stages, treatment resistance, and shortened overall survival rate in some hematological malignancies, including chronic myeloid leukemia (CML) and other myeloproliferative neoplasms. Chronic inflammation has been linked to various stages of carcinogenesis wherein pro-inflammatory cytokines play diverse roles in influencing cancer microenvironment leading to cell invasion and cancer progression. Cytokines such as TNF-α and IL-8 have been implicated in cancer growth in both solid and hematological malignancies with studies showing their elevated levels in patients. Genomic approaches have in recent years provided significant knowledge with the regard to the association of certain SNPs (single nucleotide polymerphisms) either in a gene or its promoter that can influence its expression, with the risk and susceptibility to human diseases including cancer. This study has investigated the consequences of promoter SNPs in apoptosis genes Bax-248G>A (rs4645878)/Bcl-2-938C>A (rs2279115) and pro-inflammatory cytokines TNF-α rs1800629 G>A/IL-8 rs4073 T>A on the risk and susceptibility towards hematological cancers. The study design has 235 individuals both male and female enrolled as subjects that had 113 cases of MPDs (myeloproliferative disorders) and 122 healthy individuals as controls. The genotyping studies were conducted through ARMS PCR (amplification-refractory mutation system PCR). The Bcl-2-938 C>A polymorphism showed up in 22% of patients in the study, while it was observed in only 10% of normal controls. This difference in genotype and allele frequency between the two groups was significant (p = 0.025). Similarly, the Bax-248G>A polymorphism was detected in 6.48% of the patients and 4.54% of the normal controls, with a significant difference in genotype and allele frequency between the groups (p = 0.048). The results suggest that the Bcl-2-938 C>A variant is linked to an elevated risk of MPDs in the codominant, dominant, and recessive inheritance models. Moreover, the study indicated allele A as risk allele which can significantly increase the risk of MPDs unlike the C allele. In case of Bax gene covariants, these were associated with an increased risk of MPDs in the codominant inheritance model and dominant inheritance model. It was found that the allele A significantly enhanced the risk of MPDs unlike the G allele. The frequencies of IL-8 rs4073 T>A in patients was found to be TT (16.39%), AT (36.88%) and AA (46.72%), compared to controls who were more likely to have frequencies of TT (39.34%), AT (37.70%) and AA (22.95%) as such, respectively. There was a notable overrepresentation of the AA genotype and GG homozygotes among patients compared to controls in TNF-α polymorphic variants, with 6.55% of patients having the AA genotype and 84% of patients being GG homozygotes, compared to 1.63% and 69%, respectively in controls. The data from the current study provide partial but important evidence that polymorphisms in apoptotic genes Bcl-2-938C>A and Bax-248G>A and pro-inflammatory cytokines IL-8 rs4073 T>A and TNF-α G>A may help predict the clinical outcomes of patients and determine the significance of such polymorphic variations in the risk of myeloproliferative diseases and their role as prognostic markers in disease management using a case-control study approach.

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.

Antiapoptotic Gene Genotype and Allele Variations and the Risk of Lymphoma.

BACKGROUND: The findings of earlier investigations of antiapoptotic gene genotypes and allele variants on lymphoma risk are ambiguous. This study aimed to examine the relationship between the mutation in the antiapoptotic genes and lymphoma risk among Saudi patients. METHODS: This case-control study included 205 patients, 100 of whom had lymphoma (cases) and 105 who were healthy volunteers (controls). We used tetra amplification refractory mutation polymerase chain reaction (PCR) to identify antiapoptotic genes such as B-cell lymphoma-2 (BCL2-938 C > A), MCL1-rs9803935 T > G, and survivin (BIRC5-rs17882312 G > C and BIRC5-rs9904341 G > C). Allelic-specific PCR was used to identify alleles such as BIRC5-C, MCL1-G, and BIRC5-G. RESULTS: The dominant inheritance model among cases showed that mutations in all four antiapoptotic genes were more likely to be associated with the risk of lymphoma by the odds of 2.0-, 1.98-, 3.90-, and 3.29-fold, respectively, compared to controls. Apart from the BCL-2-A allele, all three specified alleles were more likely to be associated with lymphoma by the odds of 2.04-, 1.65-, and 2.11-fold, respectively. CONCLUSION: Unlike healthy individuals, lymphoma patients are more likely to have antiapoptotic gene genotypes and allele variants, apart from BCL-2-A alterations. In the future, these findings could be used to classify and identify patients at risk of lymphoma.

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.

Comparative analysis of Nε-carboxymethyl-lysine and inflammatory markers in diabetic and non-diabetic coronary artery disease patients.

BACKGROUND: Coronary artery disease (CAD) is a major cause of death worldwide, and India contributes to about one-fifth of total CAD deaths. The development of CAD has been linked to the accumulation of Nε-carboxymethyl-lysine (CML) in heart muscle, which correlates with fibrosis. AIM: To assess the impact of CML and inflammatory markers on the biochemical and cardiovascular characteristics of CAD patients with and without diabetes. METHODS: We enrolled 200 consecutive CAD patients who were undergoing coronary angiography and categorized them into two groups based on their serum glycosylated hemoglobin (HbA1c) levels (group I: HbA1c ≥ 6.5; group II: HbA1c < 6.5). We analyzed the levels of lipoproteins, plasma HbA1c levels, CML, interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and nitric oxide. RESULTS: Group I (81 males and 19 females) patients had a mean age of 54.2 ± 10.2 years, with a mean diabetes duration of 4.9 ± 2.2 years. Group II (89 males and 11 females) patients had a mean age of 53.2 ± 10.3 years. Group I had more severe CAD, with a higher percentage of patients with single vessel disease and greater stenosis severity in the left anterior descending coronary artery compared to group II. Group I also exhibited a larger left atrium diameter. Group I patients exhibited significantly higher levels of CML, TNF-α, and IL-6 and lower levels of nitric oxide as compared with group II patients. Additionally, CML showed a significant positive correlation with IL-6 (r = 0.596, P = 0.001) and TNF-α (r = 0.337, P = 0.001) and a negative correlation with nitric oxide (r=-4.16, P = 0.001). Odds ratio analysis revealed that patients with CML in the third quartile (264.43-364.31 ng/mL) were significantly associated with diabetic CAD at unadjusted and adjusted levels with covariates. CONCLUSION: CML and inflammatory markers may play a significant role in the development of CAD, particularly in diabetic individuals, and may serve as potential biomarkers for the prediction of CAD in both diabetic and non-diabetic patients.

In vitro inhibition of biofilm and virulence factor production in azole-resistant strains of Candida albicans isolated from diabetic foot by Artemisia vulgaris stabilized tin (IV) oxide nanoparticles.

The advent of nanotechnology has been instrumental in the development of new drugs with novel targets. Recently, metallic nanoparticles have emerged as potential candidates to combat the threat of drug-resistant infections. Diabetic foot ulcers (DFUs) are one of the dreadful complications of diabetes mellitus due to the colonization of numerous drug-resistant pathogenic microbes leading to biofilm formation. Biofilms are difficult to treat due to limited penetration and non-specificity of drugs. Therefore, in the current investigation, SnO2 nanoparticles were biosynthesized using Artemisia vulgaris (AvTO-NPs) as a stabilizing agent and were characterized using ultraviolet-visible (UV-vis) spectroscopy, Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDX). Furthermore, the efficacy of AvTO-NPs against biofilms and virulence factors of drug-resistant Candida albicans strains isolated from DFUs was assessed. AvTO-NPs displayed minimum inhibitory concentrations (MICs) ranging from 1 mg/mL to 2 mg/mL against four strains of C. albicans. AvTO-NPs significantly inhibited biofilm formation by 54.8%-87%, germ tube formation by 72%-90%, cell surface hydrophobicity by 68.2%-82.8%, and exopolysaccharide (EPS) production by 69%-86.3% in the test strains at respective 1/2xMIC. Biosynthesized NPs were effective in disrupting established mature biofilms of test strains significantly. Elevated levels of reactive oxygen species (ROS) generation in the AvTO-NPs-treated C. albicans could be the possible cause of cell death leading to biofilm inhibition. The useful insights of the present study could be exploited in the current line of treatment to mitigate the threat of biofilm-related persistent DFUs and expedite wound healing.

Genetic Determinants of Cardiovascular Disease: The Endothelial Nitric Oxide Synthase 3 (eNOS3), Krüppel-Like Factor-14 (KLF-14), Methylenetetrahydrofolate Reductase (MTHFR), MiRNAs27a and Their Association with the Predisposition and Susceptibility to Coronary Artery Disease.

Coronary artery disease (CAD) is an important cause of death worldwide. CAD is caused by genetic and other factors including hypertension, hyperlipidemia, obesity, stress, unhealthy diet, physical inactively, smoking and Type 2 diabetes (T2D). The genome wide association studies (GWASs) have revealed the association of many loci with risk to diseases such as cancers, T2D and CAD. Nitric oxide (NO) is a potent vasodilator and is required for normal vascular health. It is produced in the endothelial cells in a reaction catalyzed by the endothelial NO synthase (eNOS). Methylenetetrahydrofolate reductase (MTHFR) is a very important enzyme involved in metabolism of folate and homocysteine, and its reduced function leads to cardiovascular disease. The Krüppel-like factor-14 (KLF-14) is an important transcriptional regulator that has been implicated in metabolic syndrome. MicroRNA (MiRNAs) are short non-coding RNAs that regulate the gene expression of proteins involved in important physiological processes including cell cycle and metabolism. In the present study, we have investigated the potential impact of germline pathogenic variants of endothelial eNOS, KLF-14, MTHFR, MiRNA-27a and their association with risk to CAD in the Saudi population. Methods: Amplification Refractory Mutation System (ARMS) PCR was used to detect MTHFR, KLF-14, miRNA-27a and eNOS3 genotyping in CAD patients and healthy controls. About 125 CAD cases and 125 controls were enrolled in this study and statistical associations were calculated including p-value, risk ratio (RR), and odds ratio (OD). Results: There were statistically significant differences (p < 0.05) in genotype distributions of MTHFR 677 C>T, KLF-14 rs972283 G>A, miRNAs27a rs895819 A>G and eNOS3 rs1799983 G>T between CAD patients and controls. In addition, our results indicated that the MTHFR-TT genotype was associated with increased CAD susceptibility with an OR 2.75 (95%) and p < 0.049, and the KLF14-AA genotype was also associated with increased CAD susceptibility with an OR of 2.24 (95%) and p < 0.024. Moreover, the miRNAs27a-GG genotype protects from CAD risk with an OR = 0.31 (0.016), p = 0.016. Our results also indicated that eNOS3 -GT genotype is associated with CAD susceptibility with an OR = 2.65, and p < 0.0003. Conclusion: The MTHFR 677C>T, KLF14 rs972283 G>A, miRNAs27a A>G, and eNOS3 rs1799983 G>T genotypes were associated with CAD susceptibility (p < 0.05). These findings require verification in future large-scale population based studies before these loci are used for the prediction and identification of individuals at risk to CAD. Weight control, physical activity, and smoking cessation are very influential recommendations given by clinicians to the at risk individuals to reduce or delay the development of CAD.

Protective Effect of Catharanthus roseus Extract on Cadmium-Induced Toxicity in Albino Rats: A Putative Mechanism of Detoxification.

Globally, people are highly affected by Cadmium (Cd), the most hazardous heavy metal. It has been implicated in various pathogeneses. Oxidative stress may be one the main reasons for Cd-induced disorders in the body. This article investigates the protective ability of Catharanthus roseus (CR) extract on oxidative stress in the kidney and liver of rats exposed to Cd. After 21 days, a significant increase in MDA concentration (6.81 ± 0.05), (6.64 ± 0.03) was observed in Cd-treated groups compared to the control (5.54 ± 0.02), (5.39 ± 0.04) for the kidney and liver, respectively, while significant changes were observed in the haematological parameters. Antioxidant enzymes, GPx, CAT, and SOD showed a significant decrease in their activity. We established that increasing the concentration of Cd in the presence of H2O2 was able to cause stand scission in pBR322 plasmid DNA, which may be due to the mediation of ROS generated in the process. The antioxidant ability of CR extract was tested in DPPH and H2O2 scavenging assay, depicted by the increase in the percentage inhibition. Upon treatment of CR extract to rats, MDA concentration was decreased for the kidney and liver compared to the Cd-treated groups. This was again confirmed by comet assay of both tissues, where the degree of cellular DNA breakage caused by Cd toxicity decreased significantly upon treatment with CR extract. Overall, the results suggest that Cd plays a major role as an effector metal ion, causing a decrease in the concentration and activity of AO enzymes and enhanced lipid peroxidation. ROS production resulted in oxidative DNA damage within the cell, whereas CR extract showed potential antioxidant activity against ROS-mediated DNA damage induced by Cd poisoning.

Phase II/III trial of hyperimmune anti-COVID-19 intravenous immunoglobulin (C-IVIG) therapy in severe COVID-19 patients: study protocol for a randomized controlled trial.

BACKGROUND: COVID-19 poses a global health challenge with more than 325 million cumulative cases and above 5 million cumulative deaths reported till January 17, 2022, by the World Health Organization. Several potential treatments to treat COVID-19 are under clinical trials including antivirals, steroids, immunomodulators, non-specific IVIG, monoclonal antibodies, and passive immunization through convalescent plasma. The need to produce anti-COVID-19 IVIG therapy must be continued, alongside the current treatment modalities, considering the virus is still mutating into variants of concern. In this context, as the present study will exploit pooled diversified convalescent plasma collected from recovered COVID-19 patients, the proposed hyperimmune Anti-COVID-19 intravenous immunoglobulin (C-IVIG) therapy would be able to counter new infectious COVID-19 variants by neutralizing the virus particles. After the successful outcome of the phase I/II clinical trial of C-IVIG, the current study aims to further evaluate the safety and efficacy of single low dose C-IVIG in severe COVID-19 patients for its phase II/III clinical trial. METHODS: This is a phase II/III, adaptive, multi-center, single-blinded, randomized controlled superiority trial of SARS-CoV-2 specific polyclonal IVIG (C-IVIG). Patients fulfilling the eligibility criteria will be block-randomized using a sealed envelope system to receive either 0.15 g/Kg C-IVIG with standard of care (SOC) or standard of care alone in 2:1 ratio. The patients will be followed-up for 28 days to assess the primary and secondary outcomes. DISCUSSION: This is a phase II/III clinical trial evaluating safety and efficacy of hyperimmune anti-COVID-19 intravenous immunoglobulin (C-IVIG) in severe COVID-19 patients. This study will provide clinical evidence to use C-IVIG as one of the first-line therapeutic options for severe COVID-19 patients. TRIAL REGISTRATION: Registered at clinicaltrial.gov with NCT number NCT04891172 on May 18, 2021.

IL-2 and IL-1ß Patient Immune Responses Are Critical Factors in SARS-CoV-2 Infection Outcomes.

BACKGROUND: Immune dysregulation has been linked to morbidity and mortality in COVID-19 patients. Understanding the immunology of COVID-19 is critical for developing effective therapies, diagnostics, and prophylactic strategies to control the disease. AIM: The aim of this study was to correlate cytokine and chemokine serum levels with COVID-19 disease severity and mortality. SUBJECTS AND METHODS: A total of 60 hospitalized patients from the Tabuk region of Saudi Arabia with confirmed COVID-19 were included in the study. At hospital admission, the IL-1 ß, IL-2, IL-8, IL-10, LT-B4, and CCL-2 serum levels were measured. The cytokine levels in COVID-19 patients were compared to the levels in 30 healthy matched control subjects. RESULTS: The IL-1 ß, IL-2, LTB-4, CCL-2, and IL-8 levels (but not IL-10) were significantly higher in all COVID-19 patients (47 survivors and 13 non-survivors) compared with the levels in the healthy control group. In the non-survivor COVID-19 patients, patients' age, D-dimer, and creatinine kinase were significantly higher, and IL-1 ß, IL-2, and IL-8 were significantly lower compared with the levels in the survivors. CONCLUSION: Mortality rates in COVID-19 patients are associated with increased age and a failure to mount an effective immune response rather than developing a cytokine storm. These results warrant the personalized treatment of COVID-19 patients based on cytokine profiling.