Dysregulated Urinary Extracellular Vesicle Small RNAs in Diabetic Nephropathy: Implications for Diagnosis and Therapy.

Background: Diabetic nephropathy (DN) represents a major chronic kidney disorder and a leading cause of end-stage renal disease (ESRD). Small RNAs have been showing great promise as diagnostic markers as well as drug targets. Identifying dysregulated micro RNAs (miRNAs) could help in identifying disease biomarkers and investigation of downstream interactions, shedding light on the molecular pathophysiology of DN. In this study, we analyzed small RNAs within human urinary extracellular vesicles (ECVs) from DN patients using small RNA next-generation sequencing. Method: In this cross-sectional study, urine samples were collected from 88 participants who were divided into 3 groups: type 2 diabetes (T2D) with DN (T2D + DN, n = 20), T2D without DN (T2D - DN, n = 40), and healthy individuals (n = 28). The study focused on isolating urinary ECVs to extract and sequence small RNAs. Differentially expressed small RNAs were identified, and a functional enrichment analysis was conducted. Results: The study revealed a distinct subset of 13 miRNAs and 10 Piwi-interacting RNAs that were significantly dysregulated in urinary ECVs of the DN group when compared to other groups. Notably, miR-151a-3p and miR-182-5p exhibited a unique expression pattern, being downregulated in the T2D - DN group, and upregulated in the T2D + DN group, thus demonstrating their effectiveness in distinguishing patients between the 2 groups. Eight driver genes were identified PTEN, SMAD2, SMAD4, VEGFA, CCND2, CDK6, LIN28B, and CHD1. Conclusion: Our findings contribute valuable insights into the pathogenesis of DN, uncovering novel biomarkers and identifying potential therapeutic targets that may aid in managing and potentially decelerating the progression of the disease.

Gut Dysbiosis Shaped by Cocoa Butter-Based Sucrose-Free HFD Leads to Steatohepatitis, and Insulin Resistance in Mice.

BACKGROUND: High-fat diets cause gut dysbiosis and promote triglyceride accumulation, obesity, gut permeability changes, inflammation, and insulin resistance. Both cocoa butter and fish oil are considered to be a part of healthy diets. However, their differential effects on gut microbiome perturbations in mice fed high concentrations of these fats, in the absence of sucrose, remains to be elucidated. The aim of the study was to test whether the sucrose-free cocoa butter-based high-fat diet (C-HFD) feeding in mice leads to gut dysbiosis that associates with a pathologic phenotype marked by hepatic steatosis, low-grade inflammation, perturbed glucose homeostasis, and insulin resistance, compared with control mice fed the fish oil based high-fat diet (F-HFD). RESULTS: C57BL/6 mice (5-6 mice/group) were fed two types of high fat diets (C-HFD and F-HFD) for 24 weeks. No significant difference was found in the liver weight or total body weight between the two groups. The 16S rRNA sequencing of gut bacterial samples displayed gut dysbiosis in C-HFD group, with differentially-altered microbial diversity or relative abundances. Bacteroidetes, Firmicutes, and Proteobacteria were highly abundant in C-HFD group, while the Verrucomicrobia, Saccharibacteria (TM7), Actinobacteria, and Tenericutes were more abundant in F-HFD group. Other taxa in C-HFD group included the Bacteroides, Odoribacter, Sutterella, Firmicutes bacterium (AF12), Anaeroplasma, Roseburia, and Parabacteroides distasonis. An increased Firmicutes/Bacteroidetes (F/B) ratio in C-HFD group, compared with F-HFD group, indicated the gut dysbiosis. These gut bacterial changes in C-HFD group had predicted associations with fatty liver disease and with lipogenic, inflammatory, glucose metabolic, and insulin signaling pathways. Consistent with its microbiome shift, the C-HFD group showed hepatic inflammation and steatosis, high fasting blood glucose, insulin resistance, increased hepatic de novo lipogenesis (Acetyl CoA carboxylases 1 (Acaca), Fatty acid synthase (Fasn), Stearoyl-CoA desaturase-1 (Scd1), Elongation of long-chain fatty acids family member 6 (Elovl6), Peroxisome proliferator-activated receptor-gamma (Pparg) and cholesterol synthesis (ß-(hydroxy ß-methylglutaryl-CoA reductase (Hmgcr). Non-significant differences were observed regarding fatty acid uptake (Cluster of differentiation 36 (CD36), Fatty acid binding protein-1 (Fabp1) and efflux (ATP-binding cassette G1 (Abcg1), Microsomal TG transfer protein (Mttp) in C-HFD group, compared with F-HFD group. The C-HFD group also displayed increased gene expression of inflammatory markers including Tumor necrosis factor alpha (Tnfa), C-C motif chemokine ligand 2 (Ccl2), and Interleukin-12 (Il12), as well as a tendency for liver fibrosis. CONCLUSION: These findings suggest that the sucrose-free C-HFD feeding in mice induces gut dysbiosis which associates with liver inflammation, steatosis, glucose intolerance and insulin resistance.

Unraveling Verapamil's Multidimensional Role in Diabetes Therapy: From ß-Cell Regeneration to Cholecystokinin Induction in Zebrafish and MIN6 Cell-Line Models.

This study unveils verapamil's compelling cytoprotective and proliferative effects on pancreatic ß-cells amidst diabetic stressors, spotlighting its unforeseen role in augmenting cholecystokinin (CCK) expression. Through rigorous investigations employing MIN6 ß-cells and zebrafish models under type 1 and type 2 diabetic conditions, we demonstrate verapamil's capacity to significantly boost ß-cell proliferation, enhance glucose-stimulated insulin secretion, and fortify cellular resilience. A pivotal revelation of our research is verapamil's induction of CCK, a peptide hormone known for its role in nutrient digestion and insulin secretion, which signifies a novel pathway through which verapamil exerts its therapeutic effects. Furthermore, our mechanistic insights reveal that verapamil orchestrates a broad spectrum of gene and protein expressions pivotal for ß-cell survival and adaptation to immune-metabolic challenges. In vivo validation in a zebrafish larvae model confirms verapamil's efficacy in fostering ß-cell recovery post-metronidazole infliction. Collectively, our findings advocate for verapamil's reevaluation as a multifaceted agent in diabetes therapy, highlighting its novel function in CCK upregulation alongside enhancing ß-cell proliferation, glucose sensing, and oxidative respiration. This research enriches the therapeutic landscape, proposing verapamil not only as a cytoprotector but also as a promoter of ß-cell regeneration, thereby offering fresh avenues for diabetes management strategies aimed at preserving and augmenting ß-cell functionality.

Evaluation of HLA typing content of next-generation sequencing datasets from family trios and individuals of arab ethnicity.

Introduction: HLA typing is a critical tool in both clinical and research applications at the individual and population levels. Benchmarking studies have indicated HLA-HD as the preferred tool for accurate and comprehensive HLA allele calling. The advent of next-generation sequencing (NGS) has revolutionized genetic analysis by providing high-throughput sequencing data. This study aims to evaluate, using the HLA-HD tool, the HLA typing content of whole exome, whole genome, and HLA-targeted panel sequence data from the consanguineous population of Arab ethnicity, which has been underrepresented in prior benchmarking studies. Methods: We utilized sequence data from family trios and individuals, sequenced on one or more of the whole exome, whole genome, and HLA-targeted panel sequencing technologies. The performance and resolution across various HLA genes were evaluated. We incorporated a comparative quality control analysis, assessing the results obtained from HLA-HD by comparing them with those from the HLA-Twin tool to authenticate the accuracy of the findings. Results: Our analysis found that alleles across 29 HLA loci can be successfully and consistently typed from NGS datasets. Clinical-grade whole exome sequencing datasets achieved the highest consistency rate at three-field resolution, followed by targeted HLA panel, research-grade whole exome, and whole genome datasets. Discussion: The study catalogues HLA typing consistency across NGS datasets for a large array of HLA genes and highlights assessments regarding the feasibility of utilizing available NGS datasets in HLA allele studies. These findings underscore the reliability of HLA-HD for HLA typing in underrepresented populations and demonstrate the utility of various NGS technologies in achieving accurate HLA allele calling.

Neutral Sphingomyelinase 2 Inhibition Limits Hepatic Steatosis and Inflammation.

Non-alcoholic fatty liver disease (NAFLD) is manifested by hepatic steatosis, insulin resistance, hepatocyte death, and systemic inflammation. Obesity induces steatosis and chronic inflammation in the liver. However, the precise mechanism underlying hepatic steatosis in the setting of obesity remains unclear. Here, we report studies that address this question. After 14 weeks on a high-fat diet (HFD) with high sucrose, C57BL/6 mice revealed a phenotype of liver steatosis. Transcriptional profiling analysis of the liver tissues was performed using RNA sequencing (RNA-seq). Our RNA-seq data revealed 692 differentially expressed genes involved in processes of lipid metabolism, oxidative stress, immune responses, and cell proliferation. Notably, the gene encoding neutral sphingomyelinase, SMPD3, was predominantly upregulated in the liver tissues of the mice displaying a phenotype of steatosis. Moreover, nSMase2 activity was elevated in these tissues of the liver. Pharmacological and genetic inhibition of nSMase2 prevented intracellular lipid accumulation and TNFα-induced inflammation in in-vitro HepG2-steatosis cellular model. Furthermore, nSMase2 inhibition ameliorates oxidative damage by rescuing PPARα and preventing cell death associated with high glucose/oleic acid-induced fat accumulation in HepG2 cells. Collectively, our findings highlight the prominent role of nSMase2 in hepatic steatosis, which could serve as a potential therapeutic target for NAFLD and other hepatic steatosis-linked disorders.

Glucose intolerance induces anxiety-like behaviors independent of obesity and insulin resistance in a novel model of nutritional metabolic stress.

OBJECTIVES: Type 2 diabetes (T2D) is a metabolic disease of major public health concern. It impacts peripheral tissues and the central nervous system, leading to systemic dysmetabolism and neurocognitive impairments, including memory deficits, anxiety, and depression. The metabolic determinants of these neurocognitive impairments remain unidentified. Here, we sought to address this question by developing a proprietary (P-) high-fat diet (HFD), in which glucose intolerance precedes weight gain and insulin resistance. METHODS: The P-HFD model was nutritionally characterized, and tested in vivo in mice that underwent behavioral and metabolic testing. The diet was benchmarked against reference models. . RESULTS: P-HFD has 42% kcal from fat, high monounsaturated/polyunsaturated fatty acid ratio, and 10% (w/v) sucrose in drinking water. When administered, from the early stages of glucose intolerance alone, animals exhibit anxiety-like behavior, without depression nor recognition memory deficits. Long-term P-HFD feeding leads to weight gain, brain glucose hypometabolism as well as impaired recognition memory. Using an established genetic model of T2D (db/db) and of diet-induced obesity (60% kcal from fat) we show that additional insulin resistance and obesity are associated with depressive-like behaviors and recognition memory deficits. DISCUSSION: Our findings demonstrate that glucose intolerance alone can elicit anxiety-like behavior. Through this study, we also provide a novel nutritional model (P-HFD) to characterize the discrete effects of glucose intolerance on cognition, behavior, and the physiology of metabolic disease.

Global analysis of urinary extracellular vesicle small RNAs in autosomal dominant polycystic kidney disease.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent monogenic renal disease progressing to end-stage renal disease. There is a pressing need for the identification of early ADPKD biomarkers to enable timely intervention and the development of effective therapeutic approaches. Here, we profiled human urinary extracellular vesicles small RNAs by small RNA sequencing in patients with ADPKD and compared their differential expression considering healthy control individuals to identify dysregulated small RNAs and analyze downstream interaction to gain insight about molecular pathophysiology. METHODS: This is a cross-sectional study where urine samples were collected from a total of 23 PKD1-ADPKD patients and 28 healthy individuals. Urinary extracellular vesicles were purified, and small RNA was isolated and sequenced. Differentially expressed Small RNA were identified and functional enrichment analysis of the critical miRNAs was performed to identify driver genes and affected pathways. RESULTS: miR-320b, miR-320c, miR-146a-5p, miR-199b-3p, miR-671-5p, miR-1246, miR-8485, miR-3656, has_piR_020497, has_piR_020496 and has_piR_016271 were significantly upregulated in ADPKD patient urine extracellular vesicles and miRNA-29c was significantly downregulated. Five 'driver' target genes (FBRS, EDC3, FMNL3, CTNNBIP1 and KMT2A) were identified. CONCLUSIONS: The findings of the present study make significant contributions to the understanding of ADPKD pathogenesis and to the identification of novel biomarkers and potential drug targets aimed at slowing disease progression in ADPKD.

Early autoantibody screening for type 1 diabetes: a Kuwaiti perspective on the advantages of multiplexing chemiluminescent assays.

Type 1 diabetes (T1D) incidence has increased globally over the last decades, alongside other autoimmune diseases. Early screening of individuals at risk of developing T1D is vital to facilitate appropriate interventions and improve patient outcomes. This is particularly important to avoid life-threatening diabetic ketoacidosis and hospitalization associated with T1D diagnosis. Additionally, considering that new therapies have been developed for T1D, screening the population and individuals at high risk would be of great benefit. However, adopting such screening approaches may not be feasible due to limitations, such as cost, adaptation of such programs, and sample processing. In this perspective, we explore and highlight the use of multiplexing chemiluminescent assays for T1D screening and emphasize on their advantages in detecting multiple autoantibodies simultaneously, maximizing efficiency, and minimizing sample volume requirements. These assays could be extremely valuable for pediatric populations and large-scale screening initiatives, providing a cost-efficient solution with increased diagnostic accuracy and deeper insights into T1D pathogenesis. Eventually, the adoption of such screening methods can help transform T1D diagnosis, especially in countries with high T1D prevalence, such as Kuwait, which will contribute to the development of novel therapeutic interventions, positively impacting the lives of those affected by T1D and other autoimmune diseases.

ONECUT1 variants beyond type 1 and type 2 diabetes: exploring clinical diversity and epigenetic associations in Arab cohorts.

ONECUT1 gene, encoding hepatocyte nuclear factor 6, is involved in pancreas and liver development. ONECUT1 mutations impair the function of pancreatic ß-cells and control a transcriptional/epigenetic machinery regulating endocrine development. Homozygous nonsense and missense mutations at ONECUT1_p.E231 and a homozygous frameshift mutation at ONECUT1_p.M289 were reported in neonatal diabetes individuals of French, Turkish, and Indian ethnicity, respectively. Additionally, heterozygous variants were observed in Northern European T2D patients, and Italian patients with neonatal diabetes and early-/late-onset T2D. Examining diverse populations, such as Arabs known for consanguinity, can generalize the ONECUT1 involvement in diabetes. Upon screening the cohorts of Kuwaiti T1D and MODY families, and of Kuwaiti and Qatari T2D individuals, we observed two homozygous variants-the deleterious missense rs202151356_p.H33Q in one MODY, one T1D, and two T2D individuals, and the synonymous rs61735385_p.P94P in two T2D individuals. Heterozygous variants were also observed. Examination of GTEx, NephQTL, mQTLdb and HaploReg highlighted the rs61735385_p.P94P variant as eQTL influencing the tissue-specific expression of ONECUT1, as mQTL influencing methylation at CpG sites in and around ONECUT1 with the nearest site at 677-bases 3' to rs61735385_p.P94P; as overlapping predicted binding sites for NF-kappaB and EBF on ONECUT1. DNA methylation profiles of peripheral blood from 19 MODY-X patients versus eight healthy individuals revealed significant hypomethylation at two CpG sites-one located 617-bases 3' to the p.P94P variant and 8,102 bases away from transcription start; and the other located 14,999 bases away from transcription start. Our study generalizes the association of ONECUT1 with clinical diversity in diabetes.

Linkage analysis using whole exome sequencing data implicates SLC17A1, SLC17A3, TATDN2 and TMEM131L in type 1 diabetes in Kuwaiti families.

Type 1 diabetes (T1D) is characterized by the progressive destruction of pancreatic ß-cells, leading to insulin deficiency and lifelong dependency on exogenous insulin. Higher estimates of heritability rates in monozygotic twins, followed by dizygotic twins and sib-pairs, indicate the role of genetics in the pathogenesis of T1D. The incidence and prevalence of T1D are alarmingly high in Kuwait. Consanguineous marriages account for 50-70% of all marriages in Kuwait, leading to an excessive burden of recessive allele enrichment and clustering of familial disorders. Thus, genetic studies from this Arab region are expected to lead to the identification of novel gene loci for T1D. In this study, we performed linkage analyses to identify the recurrent genetic variants segregating in high-risk Kuwaiti families with T1D. We studied 18 unrelated Kuwaiti native T1D families using whole exome sequencing data from 86 individuals, of whom 37 were diagnosed with T1D. The study identified three potential loci with a LOD score of ≥ 3, spanning across four candidate genes, namely SLC17A1 (rs1165196:pT269I), SLC17A3 (rs942379: p.S370S), TATDN2 (rs394558:p.V256I), and TMEM131L (rs6848033:p.R190R). Upon examination of missense variants from these genes in the familial T1D dataset, we observed a significantly increased enrichment of the genotype homozygous for the minor allele at SLC17A3 rs56027330_p.G279R accounting for 16.2% in affected children from 6 unrelated Kuwaiti T1D families compared to 1000 genomes Phase 3 data (0.9%). Data from the NephQTL database revealed that the rs1165196, rs942379, rs394558, and rs56027330 SNPs exhibited genotype-based differential expression in either glomerular or tubular tissues. Data from the GTEx database revealed rs942379 and rs394558 as QTL variants altering the expression of TRIM38 and IRAK2 respectively. Global genome-wide association studies indicated that SLC17A1 rs1165196 and other variants from SLC17A3 are associated with uric acid concentrations and gout. Further evidence from the T1D Knowledge portal supported the role of shortlisted variants in T1D pathogenesis and urate metabolism. Our study suggests the involvement of SLC17A1, SLC17A3, TATDN2, and TMEM131L genes in familial T1D in Kuwait. An enrichment selection of genotype homozygous for the minor allele is observed at SLC17A3 rs56027330_p.G279R variant in affected members of Kuwaiti T1D families. Future studies may focus on replicating the findings in a larger T1D cohort and delineate the mechanistic details of the impact of these novel candidate genes on the pathophysiology of T1D.

Association between alleles, haplotypes, and amino acid variations in HLA class II genes and type 1 diabetes in Kuwaiti children.

Type 1 diabetes (T1D) is a complex autoimmune disorder that is highly prevalent globally. The interactions between genetic and environmental factors may trigger T1D in susceptible individuals. HLA genes play a significant role in T1D pathogenesis, and specific haplotypes are associated with an increased risk of developing the disease. Identifying risk haplotypes can greatly improve the genetic scoring for early diagnosis of T1D in difficult to rank subgroups. This study employed next-generation sequencing to evaluate the association between HLA class II alleles, haplotypes, and amino acids and T1D, by recruiting 95 children with T1D and 150 controls in the Kuwaiti population. Significant associations were identified for alleles at the HLA-DRB1, HLA-DQA1, and HLA-DQB1 loci, including DRB1*03:01:01, DQA1*05:01:01, and DQB1*02:01:01, which conferred high risk, and DRB1*11:04:01, DQA1*05:05:01, and DQB1*03:01:01, which were protective. The DRB1*03:01:01~DQA1*05:01:01~DQB1*02:01:01 haplotype was most strongly associated with the risk of developing T1D, while DRB1*11:04-DQA1*05:05-DQB1*03:01 was the only haplotype that rendered protection against T1D. We also identified 66 amino acid positions across the HLA-DRB1, HLA-DQA1, and HLA-DQB1 genes that were significantly associated with T1D, including novel associations. These results validate and extend our knowledge on the associations between HLA genes and T1D in Kuwaiti children. The identified risk alleles, haplotypes, and amino acid variations may influence disease development through effects on HLA structure and function and may allow early intervention via population-based screening efforts.

Adipose Tissue Caveolin-1 Upregulation in Obesity Involves TNF-α/NF-κB Mediated Signaling.

Obesity is characterized by chronic low-grade inflammation. Obese people have higher levels of caveolin-1 (CAV1), a structural and functional protein present in adipose tissues (ATs). We aimed to define the inflammatory mediators that influence CAV1 gene regulation and the associated mechanisms in obesity. Using subcutaneous AT from 27 (7 lean and 20 obese) normoglycemic individuals, in vitro human adipocyte models, and in vivo mice models, we found elevated CAV1 expression in obese AT and a positive correlation between the gene expression of CAV1, tumor necrosis factor-alpha (TNF-α), and the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). CAV1 gene expression was associated with proinflammatory cytokines and chemokines and their cognate receptors (r ≥ 0.447, p ≤ 0.030), but not with anti-inflammatory markers. CAV1 expression was correlated with CD163, indicating a prospective role for CAV1 in the adipose inflammatory microenvironment. Unlike wild-type animals, mice lacking TNF-α exhibited reduced levels of CAV1 mRNA/proteins, which were elevated by administering exogenous TNF-α. Mechanistically, TNF-α induces CAV1 gene transcription by mediating NF-κB binding to its two regulatory elements located in the CAV1 proximal regulatory region. The interplay between CAV1 and the TNF-α signaling pathway is intriguing and has potential as a target for therapeutic interventions in obesity and metabolic syndromes.

Caveolin-1 rs1997623 Single Nucleotide Polymorphism Creates a New Binding Site for the Early B-Cell Factor 1 That Instigates Adipose Tissue CAV1 Protein Overexpression.

Caveolin-1 (CAV1) is implicated in the pathophysiology of diabetes and obesity. Previously, we demonstrated an association between the CAV1 rs1997623 C > A variant and metabolic syndrome (MetS). Here, we decipher the functional role of rs1997623 in CAV1 gene regulation. A cohort of 38 patients participated in this study. The quantitative MetS scores (siMS) of the participants were computed. CAV1 transcript and protein expression were tested in subcutaneous adipose tissue using RT-PCR and immunohistochemistry. Chromatin immunoprecipitation assays were performed using primary preadipocytes isolated from individuals with different CAV1 rs1997623 genotypes (AA, AC, and CC). The regulatory region flanking the variant was cloned into a luciferase reporter plasmid and expressed in human preadipocytes. Additional knockdown and overexpression assays were carried out. We show a significant correlation between siMS and CAV1 transcript levels and protein levels in human adipose tissue collected from an Arab cohort. We found that the CAV1 rs1997623 A allele generates a transcriptionally active locus and a new transcription factor binding site for early B-cell factor 1 (EBF1), which enhanced CAV1 expression. Our in vivo and in vitro combined study implicates, for the first time, EBF1 in regulating CAV1 expression in individuals harboring the rs1997623 C > A variant.

Caveolin-1 rs1997623 variant and adult metabolic syndrome-Assessing the association in three ethnic cohorts of Arabs, South Asians and South East Asians.

Background: Animal and cell model studies have implicated CAV1 in the pathophysiology of metabolic disorders. Our previous studies demonstrated a potential association of CAV1 rs1997623 C/A variant with pediatric metabolic syndrome (MetS) in Arab children. In the present study, we evaluate whether the CAV1 variant associates with MetS Arab adults as well. The association signal is further examined for ancestry-specific variation by considering cohorts of other ethnicities. Method: The CAV1 rs1997623 was genotyped in three cohorts of Arab (n = 479), South Asian (n = 660), and South East Asian (n = 362) ethnic adults from Kuwait. MetS status of the individuals was diagnosed using the IDF criteria (presence of central obesity and at least two abnormalities out of: elevated TG, low HDL, hypertension, or T2D). The quantitative measure of MetS was calculated as siMS = 2 × WC/Height + FBG/5.6 + TG/1.7 + SBP/130-HDL/1.02 for males or HDL/1.28 for females. Allelic associations with quantitative and dichotomous MetS traits were assessed using linear and logistic regression models adjusted for age and sex. In addition, empirical p-values (P emp ) were generated using max(T) permutation procedure based on 10,000 permutations. Results: The CAV1 variant was significantly associated with MetS status (OR = 1.811 [1.25-2.61]; p-value = 0.0015; P emp = 0.0013) and with siMS (Effect size = 0.206; p-value = 0.0035; P emp = 0.0028) in the cohort of Arab individuals. The association was weak and insignificant in the South Asian and South East Asian cohorts (OR = 1.19 and 1.11; p-values = 0.25 and 0.67, respectively). Conclusion: The reported association of CAV1 rs1997623 C/A with MetS in Arab pediatric population is now demonstrated in an adult Arab cohort as well. The weak association signal seen in the Asian cohorts lead us to propose a certain extent of ethnic-specificity in CAV1 rs1997623 association with MetS.

IFN-γ and LPS Induce Synergistic Expression of CCL2 in Monocytic Cells via H3K27 Acetylation.

Background: Overexpression of CCL2 (MCP-1) has been implicated in pathogenesis of metabolic conditions, such as obesity and T2D. However, the mechanisms leading to increased CCL2 expression in obesity are not fully understood. Since both IFN-γ and LPS levels are found to be elevated in obesity and shown to be involved in the regulation of metabolic inflammation and insulin resistance, we investigated whether these two agents could synergistically trigger the expression of CCL2 in obesity. Methods: Monocytes (Human monocytic THP-1 cells) were stimulated with IFN-γ and LPS. CCL2 gene expression was determined by real-time RT-PCR. CCL2 protein was determined by ELISA. Signaling pathways were identified by using epigenetic inhibitors and STAT1 siRNA. Acetylation of H3K27 was analyzed by Western blotting. The acetylation level of histone H3K27 in the transcriptional initiation region of CCL2 gene was determined by ChIP-qPCR. Results: Our results show that the co-incubation of THP-1 monocytes with IFN-γ and LPS significantly enhanced the expression of CCL2, compared to treatment with IFN-γ or LPS alone. Similar results were obtained using primary monocytes and macrophages. Interestingly, IFN-γ priming was found to be more effective than LPS priming in inducing synergistic expression of CCL2. Moreover, STAT1 deficiency significantly suppressed this synergy for CCL2 expression. Mechanistically, we showed that IFN-γ priming induced acetylation of lysine 27 on histone 3 (H3K27ac) in THP-1 cells. Chromatin immunoprecipitation (ChIP) assay followed by qRT-PCR revealed increased H3K27ac at the CCL2 promoter proximal region, resulting in stabilized gene expression. Furthermore, inhibition of histone acetylation with anacardic acid suppressed this synergistic response, whereas trichostatin A (TSA) could substitute IFN-γ in this synergy. Conclusion: Our findings suggest that IFN-γ, in combination with LPS, has the potential to augment inflammation via the H3K27ac-mediated induction of CCL2 in monocytic cells in the setting of obesity.

Differentially methylated and expressed genes in familial type 1 diabetes.

There has recently been a growing interest in examining the role of epigenetic modifications, such as DNA methylation, in the etiology of type 1 diabetes (T1D). This study aimed to delineate differences in methylation patterns between T1D-affected and healthy individuals by examining the genome-wide methylation of individuals from three Arab families from Kuwait with T1D-affected mono-/dizygotic twins and non-twinned siblings. Bisulfite sequencing of DNA from the peripheral blood of the affected and healthy individuals from each of the three families was performed. Methylation profiles of the affected individuals were compared to those of the healthy individuals Principal component analysis on the observed methylation profiling based on base-pair resolution clustered the T1D-affected twins together family-wide. The sites/regions that were differentially methylated between the T1D and healthy samples harbored 84 genes, of which 18 were known to be differentially methylated in T1D individuals compared to healthy individuals in publicly available gene expression data resources. We further validated two of the 18 genes-namely ICA1 and DRAM1 that were hypermethylated in T1D samples compared to healthy samples-for upregulation in T1D samples from an extended study cohort of familial T1D. The study confirmed that the ICA1 and DRAM1 genes are differentially expressed in T1D samples compared to healthy samples.

Identification of Maturity-Onset-Diabetes of the Young (MODY) mutations in a country where diabetes is endemic.

Genetic variants responsible for Maturity-Onset-Diabetes of the Young (MODY) in Kuwait were investigated. A newly established a National Referral Clinic, the Dasman Diabetes Institute (DDI-NRC), assessed forty-five members from 31 suspected MODY families by whole exome sequencing. Thirty-three of the 45 samples were independently sequenced at the DDI-NRI, Exeter University, UK ( https://www.diabetesgenes.org/ ) using targeted 21-gene panel approach. Pathogenic mutations in GCK, HNF1A, HNF1B, HNF4A, and PDX1 confirmed MODY in 7 families, giving an overall positivity rate of 22.6% in this cohort. Novel variants were identified in three families in PDX1, HNF1B, and HNF1B. In this cohort, Multiplex Ligation-dependent Probe Amplification assay did not add any value to MODY variant detection rate in sequencing negative cases. In highly selected familial autoantibody negative diabetes, known MODY genes represent a minority and 77.3% of the familial cases have yet to have a causal variant described.

Salivary Microbiome Diversity in Kuwaiti Adolescents with Varied Body Mass Index-A Pilot Study.

The potential role of the salivary microbiome in human diseases has increasingly been explored. The salivary microbiome has been characterized in several global populations, except the Arabian Gulf region. Hence, in this pilot study, we profiled the salivary microbiome of Kuwaiti adolescents with varied body mass indexes (BMI). The analyses of core microbiome composition showed Firmicutes, Bacteroidota, Proteobacteria, Patescibacteria, Fusobacteriota, Actinobacteriota, and Campylobacterota as the common phylum found in the Kuwaiti adolescent population. We also illustrated a diverse microbial community among the sampled individuals grouped according to their BMI. Notably, the overweight group was found with a higher number of distinct taxa than other groups. As such, the core microbiome composition was found to be significantly different (p-value < 0.001) across different BMI groups. Overall, this pilot investigation outlined the microbial diversity and suggested that changes in salivary microbiome composition in people with obese or overweight BMI might reflect their susceptibility to oral diseases.

Microarray analysis reveals ONC201 mediated differential mechanisms of CHOP gene regulation in metastatic and nonmetastatic colorectal cancer cells.

The imipramine ONC201 has antiproliferative effects in several cancer cell types and activates integrated stress response pathway associated with the induction of Damage Inducible Transcript 3 (DDIT3, also known as C/EBP homologous protein or CHOP). We investigated the signaling pathways through which ONC201/CHOP crosstalk is regulated in ONC201-treated nonmetastatic and metastatic cancer cell lines (Dukes' type B colorectal adenocarcinoma nonmetastatic SW480 and metastatic LS-174T cells, respectively). Cell proliferation and apoptosis were evaluated by MTT assays and flow cytometry, gene expression was assessed by Affymetrix microarray, signaling pathway perturbations were assessed in silico, and key regulatory proteins were validated by Western blotting. Unlike LS-174T cells, SW480 cells were resistant to ONC201 treatment; Gene Ontology analysis of differentially expressed genes showed that cellular responsiveness to ONC201 treatment also differed substantially. In both ONC201-treated cell lines, CHOP expression was upregulated; however, its upstream regulatory mechanisms were perturbed. Although, PERK, ATF6 and IRE1 ER-stress pathways upregulated CHOP in both cell types, the Bak/Bax pathway regulated CHOP only LS-174T cells. Additionally, CHOP RNA splicing profiles varied between cell lines; these were further modified by ONC201 treatment. In conclusion, we delineated the signaling mechanisms by which CHOP expression is regulated in ONC201-treated non-metastatic and metastatic colorectal cell lines. The observed differences could be related to cellular plasticity and metabolic reprogramming, nevertheless, detailed mechanistic studies are required for further validations.

SARS-CoV-2: Possible recombination and emergence of potentially more virulent strains.

COVID-19 is challenging healthcare preparedness, world economies, and livelihoods. The infection and death rates associated with this pandemic are strikingly variable in different countries. To elucidate this discrepancy, we analyzed 2431 early spread SARS-CoV-2 sequences from GISAID. We estimated continental-wise admixture proportions, assessed haplotype block estimation, and tested for the presence or absence of strains' recombination. Herein, we identified 1010 unique missense mutations and seven different SARS-CoV-2 clusters. In samples from Asia, a small haplotype block was identified, whereas samples from Europe and North America harbored large and different haplotype blocks with nonsynonymous variants. Variant frequency and linkage disequilibrium varied among continents, especially in North America. Recombination between different strains was only observed in North American and European sequences. In addition, we structurally modelled the two most common mutations, Spike_D614G and Nsp12_P314L, which suggested that these linked mutations may enhance viral entry and replication, respectively. Overall, we propose that genomic recombination between different strains may contribute to SARS-CoV-2 virulence and COVID-19 severity and may present additional challenges for current treatment regimens and countermeasures. Furthermore, our study provides a possible explanation for the substantial second wave of COVID-19 presented with higher infection and death rates in many countries.