Effects of Acute Exposure to Methylglyoxal or/and A Diet Rich in Advanced Glycation End Products on Sperm Parameters in Mice.

BACKGROUND: Advanced glycation end products (AGEs) that accompany many metabolic disorders including diabetes, obesity, and a wide range of dyslipidemia conditions, are strongly associated with adverse effects on cell and tissue homeostasis. Accordingly, our objective was to investigate the impact of AGE-promoting diets on mouse models, considering both scenarios with and without methylglyoxal (MGO) as a primary precursor of AGEs. MATERIALS AND METHODS: In this experimental study, 5-week-old C57BL/6 mice were split into four groups as a control group (n=5), AGE (n=5), MGO (n=8), and AGE-MGO-diets (n=8). After five weeks the level of fasting blood sugar (FBS), body weight, food intake, sperm parameters, and functional tests were evaluated. Furthermore, testicular superoxide dismutase (SOD) activity, malondialdehyde, and total antioxidant capacity (TAC) were assessed. RESULTS: After five weeks, AGE, AGE-MGO, and MGO groups showed the highest level of body weight and FBS in comparison to the control group. Mean sperm concentration, sperm malondialdehyde, testicular lipid peroxidation, and TAC did not differ significantly among the study groups. While, AGE, MGO, and AGE-MGO groups showed a significant reduction in sperm motility and progressive motility compared to the control group (P<0.05). The greatest increases in abnormal sperm morphology and intracytoplasmic reactive oxygen species (ROS) were observed in the MGO and AGE-MGO groups than in the control group (P<0.05). Sperm protamine deficiency and residual histone were significantly increased in the three treatment groups compared to the control group (P<0.05). Regarding the DNA damage, the AGE and AGE-MGO groups showed the most severe damage. The lowest amount of testicular superoxide dismutases (SOD, P<0.001) was observed in the AGE-MGO group. CONCLUSION: AGEs and MGO have a negative influence on sperm function and reproductive potential. These effects could be possibly attributed to both increased oxidative stress (OS) and inflammation.

Chitosan, polyethylene oxide/polycaprolactone electrospun core/shell nanofibrous mat containing rosuvastatin as a novel drug delivery system for enhancing human mesenchymal stem cell osteogenesis.

Introduction: Due to the potential positive effects of rosuvastatin (RSV) on human mesenchymal stem cells (MSCs) osteogenesis and new bone regeneration, it is crucial to develop a suitable carrier that can effectively control the release profile of RSV. The primary objective of this study was to introduce a novel drug delivery system based on core/shell nanofibrous structures, enabling a sustained release of RSV. Methods: To achieve this, coaxial electrospinning was employed to fabricate chitosan (CS)+polyethylene oxide (PEO)/polycaprolactone (PCL) nanofibrous mats, wherein RSV was incorporated within the core of nanofibers. By optimizing the relevant parameters of the electrospinning process, the mats' surface was further modified using plasma treatment. The fibers' shape, structure, and thermal stability were characterized. The wettability, and degradation properties of the fabricated mats were also examined. In vitro studies were conducted to examine the release behavior of RSV. Additionally, the capability of MSCs to survive and differentiate into osteocytes when cultured on nanofibers containing RSV was evaluated. Results: Results demonstrated the successful fabrication of CS + PEO + RSV/PCL core/shell mats with a core diameter of approximately 370 nm and a shell thickness of around 70 nm under optimized conditions. Plasma treatment was found to enhance the wettability and drug-release behavior of the mats. The nanofibrous structure, serving as a carrier for RSV, exhibited increased proliferation of MSCs and enhanced osteogenic differentiation. Conclusion: Therefore, it can be concluded that CS + PEO + RSV/PCL core/shell nanofibrous structure can be utilized as a sustained-release platform for RSV over an extended period, making it a promising candidate for guided bone regeneration.

An increase in the expression of circulating miR30d-5p and miR126-3p is associated with intermediate hyperglycaemia in Iranian population.

Type 2 diabetes is the most prevalent metabolic disease worldwide. The disease is characterised by high blood glucose levels and recently it has been shown that changes in the plasma levels of several miRNAs (miRNA) are associated with the disease. Interestingly, alterations in circulating miRNAs occur years before the onset of the disease and demonstrate predictive power. In this study, we carried out RT-qPCR to examine the plasma levels of two type 2 diabetes specific miRNAs, miR-30d-5p and miR-126-3p in an Iranian population of non-diabetic control individuals, subjects with intermediate hyperglycaemia and type 2 diabetic individuals with hyperglycaemia. We found that the plasma levels of miR-30d and miR-126 increase by 3.1 and 11.16 times, respectively, in individuals with intermediate hyperglycaemia compared to non-diabetic controls. However, no significant changes in the expression of these two miRNAs have been observed between type 2 diabetic individuals and non-diabetic subjects. Our results confirm that alterations in the plasma levels of miR-30d-5p and miR-126-3p could be used as diagnostic markers of type 2 diabetes in the Iranian population as well.

Lactase persistence phenotype and genotype in Iranian Mazani-Shahmirzadi and Afghan Hazara ethnicities.

Lactase persistence is an autosomal dominant trait characterized by sustained expression of lactase gene throughout adulthood. This trait is mostly prevalent in populations with pastoral or agro-pastoral ancestry and allows lactase persistent individuals to benefit from milk nutrients. Several genetic variants have been identified to be responsible for lactase persistence in different populations and other genetic variants associated with lactase persistence are expected to be found. In this study, we aimed to investigate the lactase persistence phenotype and genotype in two isolated populations, the Iranian Mazani-Shahmirzadi and Afghan Hazaras living in Iran. For this purpose, we genotyped five single nucleotide polymorphisms -13.907C/G, -13.910C/T, -13.913T/C, -13.915T/G and -22.018A/G in 45 Mazanis from Shahmirzad and 50 Hazaras living in the suburb of Tehran. We also investigated lactase persistence by inquiring about digestive symptoms and measuring blood glucose levels after 50g lactose consumption. Our results show that 24.2% of Mazani-Shahmirzadis and 14% of Hazaras are lactase persistent based on blood glucose levels. Genotype investigation shows that only two SNPs, 13.910 C/T and 22.018 A/G display variation in the studied populations. The -13.910*T allele has a frequency of 7.7% in Mazani-Shahmirzadis and 12.7% in Hazaras. The frequency of -22.018*A was 16.6% in Mazani-Shahmirzadis and 17% in Hazaras. Importantly, we found a new genetic variant at -13.913 single nucleotide polymorphism which has not been previously reported. Given that the -13.913 single nucleotide polymorphism is within the enhancer Oct-1 binding site, the presence of this variant could affect lactase gene expression in adults. Further studies are required to elucidate the impact of this variant on LCT gene enhancer function.

The expression of DNMTs is dramatically decreased in peripheral blood mononuclear cells of male patients with juvenile idiopathic arthritis

INTRODUCTION: Juvenile idiopathic arthritis (JIA) is an autoimmune rheumatic disease, which affects primarily the joints in children under 16 years old. The etiology of JIA is yet unknown but research has shown that JIA is a multifactorial disease implicating several genes and environmental factors. Environmental factors affect immune cells via epigenetic mechanisms. One of the most important epigenetic mechanisms is DNA methylation catalyzed by DNA methyltransferases (DNMTs) and usually associated with gene silencing. In this study, we analyzed the expression of three DNA methyltransferases namely DNMT1, DNMT3a and DNMT3b in peripheral blood mononuclear cells (PBMCs) of patients with JIA and compared it with the expression of these genes in healthy young individuals. MATERIALS AND METHODS: Peripheral blood mononuclear cells of 28 JIA patients and 28 healthy controls were isolated. Total RNA was extracted, cDNA was synthesized and the transcript levels of DNMTs were analyzed by quantitative PCR. RESULTS: Analysis of DNMT1, DNMT3a and DNMT3b relative gene expression in PBMCs of JIA patients and control individuals shows that the expression of DNMT1 and DNMT3a is reduced significantly by 7 folds and 5.5 folds, respectively, in JIA patients compared to healthy controls. Furthermore, the expression of all three DNMTs were significantly and drastically reduced in young affected males compared to healthy males. CONCLUSION: This study shows that the expression of DNMTs is reduced in JIA patients and this reduction is severe in male JIA patients

No disponible

The expression of DNMTs is dramatically decreased in peripheral blood mononuclear cells of male patients with juvenile idiopathic arthritis.

INTRODUCTION: Juvenile idiopathic arthritis (JIA) is an autoimmune rheumatic disease, which affects primarily the joints in children under 16 years old. The etiology of JIA is yet unknown but research has shown that JIA is a multifactorial disease implicating several genes and environmental factors. Environmental factors affect immune cells via epigenetic mechanisms. One of the most important epigenetic mechanisms is DNA methylation catalyzed by DNA methyltransferases (DNMTs) and usually associated with gene silencing. In this study, we analyzed the expression of three DNA methyltransferases namely DNMT1, DNMT3a and DNMT3b in peripheral blood mononuclear cells (PBMCs) of patients with JIA and compared it with the expression of these genes in healthy young individuals. MATERIALS AND METHODS: Peripheral blood mononuclear cells of 28 JIA patients and 28 healthy controls were isolated. Total RNA was extracted, cDNA was synthesized and the transcript levels of DNMTs were analyzed by quantitative PCR. RESULTS: Analysis of DNMT1, DNMT3a and DNMT3b relative gene expression in PBMCs of JIA patients and control individuals shows that the expression of DNMT1 and DNMT3a is reduced significantly by 7 folds and 5.5 folds, respectively, in JIA patients compared to healthy controls. Furthermore, the expression of all three DNMTs were significantly and drastically reduced in young affected males compared to healthy males. CONCLUSION: This study shows that the expression of DNMTs is reduced in JIA patients and this reduction is severe in male JIA patients.

Methylation of Specific CpG Sites in IL-1ß and IL1R1 Genes is Affected by Hyperglycaemia in Type 2 Diabetic Patients.

Objective: Type 2 diabetes (T2D), which is the most common metabolic disorder in the world, results from insulin resistance of target tissues and reduced production of insulin from pancreatic ß cells with genetic and environmental factors both playing roles in the pathogenesis. The aim of this study was to investigate the effect of blood glucose levels on DNA methylation of IL-1ß and IL1R1 genes in the peripheral blood mononuclear cells (PBMCs) of non-diabetic, type 2 pre-diabetic and diabetic individuals.Methods: In this case-control study, 54 non-diabetic, pre-diabetic and type 2 diabetic individuals were enrolled and categorized based on their fasting plasma glucose (FPG) and glycated hemoglobin (A1C) levels. DNA was extracted from PBMCs and subjected to bisulfite treatment. The methylation status of two CpG sites in the IL-1ß gene and three CpG sites in IL1R1 gene was then determined using Sanger sequencing.Results: Our results show that the methylation of IL-1ß gene is decreased and the methylation of IL1R1 gene is increased in diabetic individuals with hyperglycemia. Further analysis revealed that both CpG sites in IL-1ß gene are affected by hyperglycemia and display decreased methylation while only one CpG site in IL1R1 gene is affected by hyperglycemia.Conclusion: We propose that the DNA methylation status of the CpG sites cg18773937 and cg23149881 in IL-1ß gene and the CpG site cg13399261 in IL1R1 gene could serve as an epigenetic marker of chronic inflammation and T2D development. These CpG sites can also be considered for studies on metabolic memory.

The inflammatory effect of epigenetic factors and modifications in type 2 diabetes.

Inflammation has a central role in the etiology of type 2 diabetes (T2D) and its complications. Both genetic and epigenetic factors have been implicated in the development of T2D-associated inflammation. Epigenetic mechanisms regulate the function of several components of the immune system. Diabetic conditions trigger aberrant epigenetic alterations that contribute to the progression of insulin resistance and ß-cell dysfunction by induction of inflammatory responses. Thus, targeting epigenetic factors and modifications, as one of the underlying causes of inflammation, could lead to the development of novel immune-based strategies for the treatment of T2D. The aim of this review is to provide an overview of the epigenetic mechanisms involved in the propagation and perpetuation of chronic inflammation in T2D. We also discuss the possible anti-inflammatory approaches that target epigenetic factors for the treatment of T2D.

The expression of GAS5, THRIL, and RMRP lncRNAs is increased in T cells of patients with rheumatoid arthritis.

OBJECTIVE: Long non-coding RNAs (lncRNAs) comprise a large and diverse group of non-coding RNAs (ncRNAs) with important regulatory roles in various biological processes, including the immune system regulation. Rheumatoid arthritis (RA) as an autoimmune disease initiates inflammation in the synovial joints. T cells infiltrating into the synovial membrane have an important role in the pathogenesis of RA. The aim of the current investigation was to analyze the expression of four lncRNAs in the T cells from RA patients and healthy controls. METHODS: In the current study, we investigated the expression of GAS5, RMRP, IFNϒ-AS1, and THRIL lncRNAs in circulating T cells from 20 patients with RA and 18 healthy matched controls by quantitative real-time PCR. T cell isolation was accomplished using the MAC method. We also analyzed the correlation between lncRNA expression and clinical parameters. Also, the mRNA expression levels of IL-17 and TNF-α and the association between lncRNAs and these cytokines were examined. RESULTS: The results indicate that T cells of RA patients display increased levels of GAS5 (3.31-fold, p = 0.007), RMRP (2.43-fold, p = 0.02), and THRIL (2.14-fold, p = 0.03) lncRNAs compared with those of controls. Furthermore, a positive correlation was found between RMRP expression and disease duration in RA. Receiver operating characteristic (ROC) curve of GAS5, RMRP, and THRIL has a discriminative value in comparing RA patients and controls. CONCLUSION: The results suggest lncRNAs may be involved in T cell dysfunction in RA. Further studies are required to see whether these lncRNAs have an effect on dysregulation of immune responses in RA disease. Key Points • 70% of non-coding sequences in the human genome are transcribed to RNA. • A growing body of evidence shows the importance of lncRNAs in innate and adaptive immune cell differentiation and functions. • Important recent works suggest a key role of immune cell lncRNAs in autoimmune processes and diseases including RA.

Hyperglycemia Affects the Expression of Inflammatory Genes in Peripheral Blood Mononuclear Cells of Patients with Type 2 Diabetes.

OBJECTIVE: Innate immune system dysregulation and chronic low-grade inflammation are associated with the pathogenesis of type 2 diabetes (T2D). The aim of this study was to investigate the effect of hyperglycemia on mRNA expression of four inflammatory genes in peripheral blood mononuclear cells (PBMCs) of pre-diabetic and diabetic individuals. METHODS: In a case-control study, quantitative real-time PCR was used to analyze changes in IL-1ß, IL1R1, IL-6, and IL6ST gene expression in PBMCs of 30 T2D patients with high blood glucose levels, 24 diabetic and nondiabetic individuals with moderately high blood glucose levels and 30 controls with normal blood glucose levels. RESULTS: In T2D patients with high blood glucose, IL-1ß expression showed a 2.69-fold increase (p = 0.0380), while IL-6 expression levels were 3.45 fold lower (p = 0.0045) versus control subjects. Moreover, compared with control group the expression of IL1R1 and IL-6 genes both were downregulated in individuals with moderately high blood glucose levels by 2.38 (p = 0.0365) and 4.34 fold (p = 0.0027), respectively. In addition, hemoglobin A1C (A1C) levels were positively correlated with IL-1ß expression and fasting plasma glucose (FPG) levels showed a positive correlation with IL-1ß and a negative correlation with IL-6 expression. CONCLUSION: The observed changes in both IL-1ß and IL-6 mRNA levels in PBMCs may contribute to the development of inflammatory processes involved in the pathogenesis of T2D. Downregulation of IL1R1 in individuals with mild hyperglycemia may indicate an attempt to reduce the pro-inflammatory effects of IL-1ß via auto-stimulation.

IL-6 signalling pathways and the development of type 2 diabetes.

Interleukin 6 (IL-6), a multifunctional cytokine, has been implicated in the pathophysiology of type 2 diabetes (T2D). The elevated circulating level of IL-6 is an independent predictor of T2D and is considered to be involved in the development of inflammation, insulin resistance and ß-cell dysfunction. On the other hand, an increasing number of evidence suggests that IL-6 has an anti-inflammatory role and improves glucose metabolism. The complex signal transduction mechanism of IL-6 may help explain the pleiotropic nature of the cytokine. IL-6 acts via two distinct signalling pathways called classic signalling and trans-signalling. While both signalling modes lead to activation of the same receptor subunit, their final biological effects are completely different. The aim of this review is to summarize our current knowledge about the role of IL-6 in the development of T2D. We will also discuss the importance of specific blockade of IL-6 trans-signalling rather than inhibiting both signalling pathways as a therapeutic strategy for the treatment of T2D and its associated macrovascular complications.

Histone variants expression in peripheral blood mononuclear cells of patients with rheumatoid arthritis.

AIM: The purpose of the present study was to analyze the expression of four histone variants, implicated in the regulation of gene expression, in peripheral blood mononuclear cell (PBMC) samples of patients with rheumatoid arthritis and healthy controls. METHOD: We analyzed the expression of three genes encoding histone variants H3.3, H2A.Z, macroH2A1.1 and macroH2A1.2 in PBMC samples of 50 patients with RA, diagnosed according to American College of Rheumatology (ACR) criteria, and 51 matched healthy controls using SYBR green real-time polymerase chain reaction. Mann-Whitney U-test and Spearman correlation were used for data analysis. RESULTS: The expression of H2A.Z was increased by 1.51-fold (P < 0.001) and the expression of H3.3 was increased by 1.13-fold (P = 0.048) in PBMCs of patients with RA compared to healthy controls. Furthermore, we found a positive correlation between Disease Activity Score (DAS-28) based on erythrocyte sedimentation rate and the expression of H2A.Z. CONCLUSIONS: Given the role of H3.3 and H2A.Z in nucleosome positioning, chromatin structure and transcription regulation, we suggest that lymphocytes and monocytes, the main cell subtypes in PBMCs of RA patients, possess a more accessible chromatin.

DNA methylation is dispensable for changes in global chromatin architecture but required for chromocentre formation in early stem cell differentiation.

Epiblast stem cells (EpiSCs), which are pluripotent cells isolated from early post-implantation mouse embryos (E5.5), show both similarities and differences compared to mouse embryonic stem cells (mESCs), isolated earlier from the inner cell mass (ICM) of the E3.5 embryo. Previously, we have observed that while chromatin is very dispersed in E3.5 ICM, compact chromatin domains and chromocentres appear in E5.5 epiblasts after embryo implantation. Given that the observed chromatin re-organization in E5.5 epiblasts coincides with an increase in DNA methylation, in this study, we aimed to examine the role of DNA methylation in chromatin re-organization during the in vitro conversion of ESCs to EpiSCs. The requirement for DNA methylation was determined by converting both wild-type and DNA methylation-deficient ESCs to EpiSCs, followed by structural analysis with electron spectroscopic imaging (ESI). We show that the chromatin re-organization which occurs in vivo can be re-capitulated in vitro during the ESC to EpiSC conversion. Indeed, after 7 days in EpiSC media, compact chromatin domains begin to appear throughout the nuclear volume, creating a chromatin organization similar to E5 epiblasts and embryo-derived EpiSCs. Our data demonstrate that DNA methylation is dispensable for this global chromatin re-organization but required for the compaction of pericentromeric chromatin into chromocentres.

Coming to terms with chromatin structure.

Chromatin, once thought to serve only as a means to package DNA, is now recognized as a major regulator of gene activity. As a result of the wide range of methods used to describe the numerous levels of chromatin organization, the terminology that has emerged to describe these organizational states is often imprecise and sometimes misleading. In this review, we discuss our current understanding of chromatin architecture and propose terms to describe the various biochemical and structural states of chromatin.

G4 motifs affect origin positioning and efficiency in two vertebrate replicators.

DNA replication ensures the accurate duplication of the genome at each cell cycle. It begins at specific sites called replication origins. Genome-wide studies in vertebrates have recently identified a consensus G-rich motif potentially able to form G-quadruplexes (G4) in most replication origins. However, there is no experimental evidence to demonstrate that G4 are actually required for replication initiation. We show here, with two model origins, that G4 motifs are required for replication initiation. Two G4 motifs cooperate in one of our model origins. The other contains only one critical G4, and its orientation determines the precise position of the replication start site. Point mutations affecting the stability of this G4 in vitro also impair origin function. Finally, this G4 is not sufficient for origin activity and must cooperate with a 200-bp cis-regulatory element. In conclusion, our study strongly supports the predicted essential role of G4 in replication initiation.

USF binding sequences from the HS4 insulator element impose early replication timing on a vertebrate replicator.

The nuclear genomes of vertebrates show a highly organized program of DNA replication where GC-rich isochores are replicated early in S-phase, while AT-rich isochores are late replicating. GC-rich regions are gene dense and are enriched for active transcription, suggesting a connection between gene regulation and replication timing. Insulator elements can organize independent domains of gene transcription and are suitable candidates for being key regulators of replication timing. We have tested the impact of inserting a strong replication origin flanked by the ß-globin HS4 insulator on the replication timing of naturally late replicating regions in two different avian cell types, DT40 (lymphoid) and 6C2 (erythroid). We find that the HS4 insulator has the capacity to impose a shift to earlier replication. This shift requires the presence of HS4 on both sides of the replication origin and results in an advance of replication timing of the target locus from the second half of S-phase to the first half when a transcribed gene is positioned nearby. Moreover, we find that the USF transcription factor binding site is the key cis-element inside the HS4 insulator that controls replication timing. Taken together, our data identify a combination of cis-elements that might constitute the basic unit of multi-replicon megabase-sized early domains of DNA replication.

Genome-wide studies highlight indirect links between human replication origins and gene regulation.

To get insights into the regulation of replication initiation, we systematically mapped replication origins along 1% of the human genome in HeLa cells. We identified 283 origins, 10 times more than previously known. Origin density is strongly correlated with genomic landscapes, with clusters of closely spaced origins in GC-rich regions and no origins in large GC-poor regions. Origin sequences are evolutionarily conserved, and half of them map within or near CpG islands. Most of the origins overlap transcriptional regulatory elements, providing further evidence of a connection with gene regulation. Moreover, we identify c-JUN and c-FOS as important regulators of origin selection. Half of the identified replication initiation sites do not have an open chromatin configuration, showing the absence of a direct link with gene regulation. Replication timing analyses coupled with our origin mapping suggest that a relatively strict origin-timing program regulates the replication of the human genome.