Evaluation of DNA methylation in BDNF, SLC6A4, NR3C1 and FKBP5 before and after treatment with selective serotonin-reuptake inhibitor in major depressive disorder.

Aim: To identify the DNA methylation status of related genes in major depressive disorder following selective serotonin-reuptake inhibitor treatment. Materials & methods: 45 patients with major depressive disorder and 45 healthy volunteers were considered experimental and control groups, respectively. High-resolution melting real-time PCR was implemented to evaluate DNA methylation. Results: After 100 days of selective serotonin-reuptake inhibitor treatment, methylation of promoter CpG sites of BDNF, NR3C1, FKBP5 and SLC6A4 was significantly reduced. Compared with before treatment, patients' Hamilton Depression Rating Scale scores were significantly reduced after selective serotonin-reuptake inhibitor treatment (p ≤ 0.0001). Conclusion: Based on the proven effect of antidepressants on DNA methylation and gene expression, these medications can improve the treatment process and reduce depression scores after treatment.

Changes in DNA methylation in APOE and ACKR3 genes in multiple sclerosis patients and the relationship with their heavy metal blood levels.

Multiple sclerosis (MS) is a chronic inflammatory disease with demyelinated lesions in the central nervous system caused by genetic and environmental factors. DNA methylation as an epigenetic change influenced by environmental factors, including heavy metals has been implemented in MS disease. We investigated the correlation of DNA methylation changes in APOE and ACKR3 genes in MS patients and the possible association with blood concentration of arsenic (As), cadmium (Cd) and lead (Pb) as major heavy metal pollutants. This study included 69 relapsing-remitting multiple sclerosis (RR-MS) patients and 69 age/gender-matched healthy subjects. The HRM real-time PCR method was used to investigate the changes in DNA methylation and heavy metal concentrations were measured by electrothermal atomic absorption spectrometry. Our results showed that the methylation pattern in the ACKR3 gene of the patient group was more hypomethylated, while in the case of the APOE gene, this pattern was more towards hypermethylation compared to healthy subjects. Moreover, the blood levels of As and Cd metals, but not Pb, were significantly higher in the patient group compare to the control group (p ≤ 0.05). The data indicate that the increase in expression of ACKR3 gene by hypomethylation and the decrease in expression of APOE gene via hypermethylation are possibly involved in the onset and progression of inflammatory processes in MS patients. The level of As can also lead to hypomethylation by disrupting the methylation patterns of the ACKR3 gene, resulting in increased expression in MS patients. Finally, we have shown that epigenetic changes can be an important factor in increasing and decreasing the expression of genes involved in the onset and/or progression of inflammatory processes in MS. Furthermore, exposure to heavy metals, especially As, by changing the natural patterns of DNA methylation can be effective in this disease.

ß-Sitosterol Alters the Inflammatory Response in CLP Rat Model of Sepsis by Modulation of NFκB Signaling.

PURPOSE: Sepsis originates from the host inflammatory response, especially to bacterial infections, and is considered one of the main causes of death in intensive care units. Various agents have been developed to inhibit mediators of the inflammatory response; one prospective agent is ß-sitosterol (ßS), a phytosterol with a structure similar to cholesterol. This study is aimed at evaluating the effects of ßS on the biomarkers of inflammation and liver function in cecal ligation and puncture- (CLP-) induced septic rats. METHODS: Thirty male Wistar rats were divided equally into six groups as follows: sham, CLP, CLP+dexamethasone (DX, 0.2 mg/kg), CLP+ßS (1 mg/kg), CLP+imipenem (IMI, 20 mg/kg), and CLP+IMI (20 mg/kg)+ßS (1 mg/kg). Serum levels of IL-1ß, IL-6, IL-10, AST, ALT, and liver glutathione (GSH) were assessed by ELISA. Liver expression levels of TNF-α and NF-κBi mRNAs were evaluated by RT-qPCR. RESULTS: Serum concentrations of IL-1ß, IL-6, IL-10, ALT, and AST and mRNA levels of TNF-α and NF-κBi were all significantly higher in septic rats than in normal rats (p < 0.05). Liver GSH content was markedly lower in the CLP group than that in the sham group. ßS-treated rats had remarkably lower levels of IL-1ß, IL-6, IL-10, TNF-α, NF-κBi, AST, and ALT (51.79%, 62.63%, 41.46%, 54.35%, 94.37%, 95.30%, 34.87%, and 46.53% lower, respectively) and greater liver GSH content (35.71% greater) compared to the CLP group (p < 0.05). CONCLUSION: ßS may play a protective role in the septic process by mitigating inflammation. This effect is at least partly mediated by inhibition of the NF-κB signaling pathway. Thus, ßS can be considered as a supplementary treatment in septic patients.

Association Between CYP3A5 Genetic Polymorphisms with Tacrolimus Dose Requirement and Allograft Outcomes in Iranian Kidney Transplant Recipients.

INTRODUCTION: Tacrolimus is the cornerstone of immunosuppressive therapy in organ transplantation with variable inter-individual pharmacokinetics. This study assessed the relationship between CYP3A5/3A4 polymorphisms and tacrolimus dose requirement as well as 6-month transplant outcomes in Iranian kidney transplant recipients. METHODS: In this prospective study, 110 adult kidney transplant recipients treated with tacrolimus were genotyped for the presence of common SNPs: rs776746: A > G (CYP3A5*3). Patients who carried at least one CYP3A5*1 allele were known as CYP3A5 expressers while those who were CYP3A5*3/*3 homozygotes were classified as CYP3A5 non-expressers. RESULTS: The daily tacrolimus dose was significantly higher and tacrolimus dose adjusted trough levels (C/D ratio) was significantly lower in CYP3A5 expressers compared with non-expressers (P < .05). Although the incidence of clinically suggested acute allograft rejection was significantly higher (OR = 0.365 [95% CI: 0.14 - 0.93]; P < .05) and median time to first acute rejection was sooner among CYP3A5 expressers compared with non-expressers (12.17 vs. 26.83 days, P < .05); however, estimated glomerular filtration rate, incidence of biopsy proven acute rejection and delayed graft function and 6-month patients' and grafts' survival did not differ between the two groups. CONCLUSION: CYP3A5 genetic polymorphism is significantly associated with required tacrolimus dose. After achieving desired tacrolimus blood level, although some transplant outcomes such as the incidence of clinically suggested acute rejection and time to first rejection were different between CYP3A5 expressers and non-expressers, however, other clinical outcomes did not differ between groups. Therefore, it is not the time to routinely assess kidney transplant recipients for CYP3A5 genetic polymorphism before transplantation.

The Role of microRNAs in Embryonic and Induced Pluripotency.

Research on stem cells is one of the fastest growing areas of regenerative medicine that paves the way for a comprehensive solution to cell therapy. Today, stem cells are precious assets for generating different types of cells derived from either natural embryonic stem (ES) cells or induced pluripotent stem (iPS) cells. The iPS technology can revolutionize the future of clinics by offering personalized medicine, which will provide the future treatment for curing untreatable diseases. Although iPS cell therapy is now at its infancy, promising research has motivated scientists to pursue this therapeutic approach. In this article, we provide information regarding similarities and differences between ES and iPS cells, and focus on the non-integrating methods of iPS generation via RNA molecules, especially microRNAs with an emphasis on the elucidation of their role and importance in pluripotency.

Monomethyl fumarate alleviates sepsis-induced hepatic dysfunction by regulating TLR-4/NF-κB signalling pathway.

AIMS: Sepsis is a potentially fatal illness that can lead to impairment of multiple organs such as liver. The condition is deeply associated with oxidative stress and inflammation. Monomethyl fumarate (MMF) has manifested antioxidant and immunomodulatory properties. The aim of current study was to evaluate protective effects of MMF in sepsis-induced hepatic dysfunction. MAIN METHODS: Sepsis was induced by cecal ligation and puncture (CLP). Wistar rats were assigned to one of sham, CLP, CLP + dexamethasone (as positive control of inflammation) and CLP + MMF groups. Levels of serum IL-1ß, IL-6, IL-10, AST, ALT and γ­GT were quantified. Furthermore, Hepatic levels of GSH and MDA and mRNA expression of TNF and NFKBIA along with hepatic protein level of TLR-4 were assessed. Also, histopathological study of liver was carried out to evaluate hepatic injuries. KEY FINDINGS: Septic rats demonstrated risen levels of IL-1ß, IL-6, IL-10, AST, ALT and γ­GT, while treatment with dexamethasone or MMF attenuated these levels. Moreover, enhancements in protein level of TLR-4 and mRNA levels of TNF and NFKBIA were observed in CLP rats. These elevations were mitigated in CLP-induced rats that were treated with either dexamethasone or MMF. Treatment with dexamethasone or MMF also shifted sepsis-induced disturbance in the levels of GSH and MDA towards sham levels. Hepato-protective effects of dexamethasone and MMF were further confirmed by histopathological observations. SIGNIFICANCE: Our findings imply that MMF alleviates sepsis-induced hepatic dysfunction by mitigating the inflammatory and oxidative state and this effect is at least partly mediated by the inhibition of TLR-4/NF-κB signalling pathway.

Inhibition of miRNA-212/132 improves the reprogramming of fibroblasts into induced pluripotent stem cells by de-repressing important epigenetic remodelling factors.

MicroRNAs (miRNAs) repeatedly have been demonstrated to play important roles in the generation of induced pluripotent stem cells (iPSCs). To further elucidate the molecular mechanisms underlying transcription factor-mediated reprogramming we have established a model, which allows for the efficient screening of whole libraries of miRNAs modulating the generation of iPSCs from murine embryonic fibroblasts. Applying this model, we identified 14 miRNAs effectively inhibiting iPSC generation, including miR-132 and miR-212. Intriguingly, repression of these miRNAs during iPSC generation also resulted in significantly increased reprogramming efficacy. MiRNA target evaluation by qRT-PCR, Western blot, and luciferase assays revealed two crucial epigenetic regulators, the histone acetyl transferase p300 as well as the H3K4 demethylase Jarid1a (KDM5a) to be directly targeted by both miRNAs. Moreover, we demonstrated that siRNA-mediated knockdown of either p300 or Jarid1a recapitulated the miRNA effects and led to a significant decrease in reprogramming efficiency. Thus, conducting a full library miRNA screen we here describe a miRNA family, which markedly reduces generation of iPSC and upon inhibition in turn enhances reprogramming. These miRNAs, at least in part, exert their functions through repression of the epigenetic modulators p300 and Jarid1a, highlighting these two molecules as an endogenous epigenetic roadblock during iPSC generation.

Improved bi-allelic modification of a transcriptionally silent locus in patient-derived iPSC by Cas9 nickase.

Homology directed repair (HDR)-based genome editing via selectable long flanking arm donors can be hampered by local transgene silencing at transcriptionally silent loci. Here, we report efficient bi-allelic modification of a silent locus in patient-derived hiPSC by using Cas9 nickase and a silencing-resistant donor construct that contains an excisable selection/counter-selection cassette. To identify the most active single guide RNA (sgRNA)/nickase combinations, we employed a lentiviral vector-based reporter assay to determine the HDR efficiencies in cella. Next, we used the most efficient pair of sgRNAs for targeted integration of an improved, silencing-resistant plasmid donor harboring a piggyBac-flanked puroΔtk cassette. Moreover, we took advantage of a dual-fluorescence selection strategy for bi-allelic targeting and achieved 100% counter-selection efficiency after bi-allelic excision of the selection/counter-selection cassette. Together, we present an improved system for efficient bi-allelic modification of transcriptionally silent loci in human pluripotent stem cells.

Molecular Cloning and Characterization of P4 Nuclease from Leishmania infantum.

Parasite of the genus Leishmania is reliant on the salvage pathway for recycling of ribonucleotides. A class I nuclease enzyme also known as P4 nuclease is involved in salvage of purines in cutaneous Leishmania species but the relevant enzymes have not been characterized in Leishmania infantum (L. infantum). The aim of this study was to clone and characterize the gene encoding class I nuclease in L. infantum. DNA extracted from L. infantum was used for amplification of P4 nuclease gene (Li-P4) by PCR. The product was cloned, sequenced, and expressed in E. coli for further characterization. Analysis of the sequence of Li-P4 revealed that the gene consists of an ORF of 951 bp. Sequence similarity analysis indicated that Li-P4 has a high homology to relevant enzymes of other kintoplastids with the highest homology (88%) to p1/s1 class I nuclease from L. donovani. Western blotting of antirecombinant Li-P4 with promastigote and amastigote stages of L. infantum showed that this nuclease is present in both stages of parasite with higher expression in amastigote stage. The highly conserved nature of this essential enzyme in Leishmania parasites suggests it as a promising drug target for leishmaniasis.