Understanding the Molecular Basis of Fragile X Syndrome Using Differentiated Mesenchymal Stem Cells

Abstract Objectives Fragile X syndrome (FXS) has been known as the most common cause of inherited intellectual disability and autism. This disease results from the loss of fragile X mental retardation protein expression due to the expansion of CGG repeats located on the 5’ untranslated region of the fragile X mental retardation 1 (FMR1) gene. Materials & Methods In the present study, the peripheral blood-mesenchymal stem cells (PB-MSCs) of two female full mutation carriers were differentiated into neuronal cells by the suppression of bone morphogenesis pathwaysignaling. Then, the expression of genes adjacent to CGG repeats expansion, including SLIT and NTRK-like protein 2 (SLITRK2), SLIT and NTRK-like protein 4 (SLITRK4), methyl CpG binding protein 2 (MECP2), and gamma-aminobutyric acid receptor subunit alpha-3 (GABRA3), were evaluated in these cells using SYBR Green real-time polymerase chain reaction. Results The obtained results indicated that the expression of SLITRK2 and SLITRK4 were upregulated and downregulated in the neuron-like cells differentiated from the PB-MSCs of females with FMR1 full mutation, compared to that of the normal females, respectively. Furthermore, the expression of MECP2 and GABRA3 genes were observed to be related to the phenotypic differences observed in the female FMR1full mutation carriers Conclusion The observed association of expression of genes located upstream of the FMR1 gene with phenotypic differences in the female carriers could increase the understanding of novel therapeutic targets for patients with mild symptoms of FXS and the patients affected by other FMR1-related disorder

Investigating the Genetic Characteristics of Cochlear Implant Candidates with Hearing Impairment

Background: Hearing loss, especially at a young age, has severe personal and social consequences for a person and brings enormous costs to the treatment system. Considering the vital role of genetics in hearing loss, genetics research creates a suitable platform for progress in the treatment of these patients, so we decided to conduct a study with the aim of early diagnosis and even before symptoms appear in order to reduce possible complications. Aim: In this study early diagnosis of hearing loss and even before symptoms appear in order to reduce possible complications. Methods: Based on the history and phenotype and examination of the medical records of 1249 patients who are candidates for cochlear implantation, genetic testing among the patients suspected of non-syndromic genetic hearing loss, a request for genetic testing of stage one or two or both was made and according to the willingness of the families and their cooperation A total of 138 genetic tests were performed and subjected to genetic analysis. Results: Among 138 tested cases, 71 women and 67 men, NSHL inheritance autosomal recessive pattern was 84/78% and autosomal dominant, 5/07 which is very close to previous studies. There were genetic mutations in the Gjb2 gene in ten cases of patients. Ninety-one patients were negative for GJB2 involvement and were candidates for WES, but unfortunately, many families refused to perform the test due to the cost of this test. Seven patients underwent WES, and several genetic mutations were identified in the thesis. WES was performed for 34 patients according to the investigations carried out directly. Conclusion: Iranian society has played an essential role in improving our understanding of the genes involved in proper hearing functioning and how these genes' variants cause hearing loss. Researchers have worked tirelessly to solve the genetic mystery of hearing loss in Iran, which has been very successful. However, more work is still needed

Multiple Sclerosis Gene Therapy Using Recombinant Viral Vectors: Overexpression of IL-4, IL-10 and Leukemia Inhibitory Factor in Wharton's Jelly Stem Cells in The EAE Mice Model

Objective: Immunotherapy and gene therapy play important roles in modern medicine. The aim of this study is to evaluate the overexpression of interleukin-4 (IL-4), IL-10 and leukemia inhibitory factor (LIF) in Wharton's jelly stem cells (WJSCs) in the experimental autoimmune encephalomyelitis (EAE) mice model. Materials and Methods: In this experimental study, a DNA construction containing IL-4, IL-10 and LIF was assembled to make a polycistronic vector (as the transfer vector). Transfer and control vectors were co-transfected into Human Embryonic Kidney 293 (HEK-293T) cells with helper plasmids which produced recombinant lentiviral viruses (rLV). WJSCs were transduced with rLV to make recombinant WJSC (rWJSC). In vitro protein and mRNA overexpression of IL-4, LIF, and IL-10 were evaluated using quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA) and western blot (WB) analysis. EAE was induced in mice by MOG-CFA and pertussis toxin. EAE mice were injected twice with 2x10(5) rWJSCs. The in vivo level of IL-4, LIF, IL-10 cytokines and IL-17 were measured by ELISA. Brain tissues were analyzed histologically for evaluation of EAE lesions. Results: Isolated WJSCs were performed to characterize by in vitro differentiation and surface markers were analyzed by flow cytometry method. Cloning of a single lentiviral vector with five genes was done successfully. Transfection of transfer and control vectors were processed based on CaPO4 method with > 90% efficiency. Recombinant viruses were produced and results of titration showed 2-3x10(7) infection-unit/ml. WJSCs were transduced using recombinant viruses. IL-4, IL-10 and LIF overexpression were confirmed by ELISA, WB and qPCR. The EAE mice treated with rWJSC showed reduction of Il-17, and brain lesions as well as brain cellular infiltration, in vivo. Weights and physical activity were improved in gene-treated group. Conclusion: These results showed that gene therapy using anti-inflammatory cytokines can be a promising approach against multiple sclerosis (MS). In addition, considering the immunomodulatory potential of WJSCs, an approach using a combination of WJSCs and gene therapy will enhance the treatment efficacy

Multiple Sclerosis Gene Therapy with Recombinant Viral Vectors: Overexpression of IL-4, Leukemia Inhibitory Factor, and IL-10 in Wharton's Jelly Stem Cells Used in EAE Mice Model.

OBJECTIVES: Immunotherapy and gene therapy play important roles in modern medicine. The aim of this study is to evaluate the overexpression of interleukin-4 (IL-4), IL-10 and leukemia inhibitory factor (LIF) in Wharton's jelly stem cells (WJSCs) in the experimental autoimmune encephalomyelitis (EAE) mice model. MATERIALS AND METHODS: In this experimental study, a DNA construction containing IL- 4, IL-10 and LIF was assembled to make a polycistronic vector (as the transfer vector). Transfer and control vectors were co-transfected into Human Embryonic Kidney 293 (HEK-293T) cells with helper plasmids which produced recombinant lentiviral viruses (rLV). WJSCs were transduced with rLV to make recombinant WJSC (rWJSC). In vitro protein and mRNA overexpression of IL-4, LIF, and IL-10 were evaluated using quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA) and western blot (WB) analysis. EAE was induced in mice by MOG-CFA and pertussis toxin. EAE mice were injected twice with 2×105 rWJSCs. The in vivo level of IL-4, LIF, IL-10 cytokines and IL-17 were measured by ELISA. Brain tissues were analyzed histologically for evaluation of EAE lesions. RESULTS: Isolated WJSCs were performed to characterize by in vitro differentiation and surface markers were analyzed by flow cytometry method. Cloning of a single lentiviral vector with five genes was done successfully. Transfection of transfer and control vectors were processed based on CaPO4 method with >90% efficiency. Recombinant viruses were produced and results of titration showed 2-3×107 infection-unit/ml. WJSCs were transduced using recombinant viruses. IL-4, IL-10 and LIF overexpression were confirmed by ELISA, WB and qPCR. The EAE mice treated with rWJSC showed reduction of Il-17, and brain lesions as well as brain cellular infiltration, in vivo. Weights and physical activity were improved in gene-treated group. CONCLUSIONS: These results showed that gene therapy using anti-inflammatory cytokines can be a promising approach against multiple sclerosis (MS). In addition, considering the immunomodulatory potential of WJSCs, an approach using a combination of WJSCs and gene therapy will enhance the treatment efficacy

Evaluation of Important Molecular Pathways and Candidate Diagnostic Biomarkers of Noninvasive to Invasive Stages in Gastric Cancer by In Silico Analysis

Gastric cancer affects millions of people each year; it is the fifth deadliest cancer globally. Due to failure to perform routine tests such as endoscopy, it is usually diagnosed in the invasive stages. Therefore, finding diagnostic biomarkers in blood can help to speed up the initial diagnosis of cancer. This study aimed to find appropriate diagnostic biomarkers in the extracellular matrix of noninvasive to invasive stages of gastric cancer patients, using bioinformatics analysis. First, we selected the appropriate datasets from the GEO database. We evaluated the genes’ signaling pathways, biological processes, and molecular functions. More accurately, we assessed the genes, in which their protein products are released into the extracellular matrix; we evaluated their protein network. Then, we validated the candidate proteins in the GEPIA and TCGA databases. The extracellular matrix, tyrosine kinase receptors, and immune response pathways are effective factors, which are related to the highly expressed genes and metabolism; cell cycle pathways are also impressive on low-expression genes. 69 highly expressed proteins are released into the extracellular matrix. After drawing the protein network, 5 proteins were selected as more suitable candidates for further studies. These proteins’ expression significantly increases in the human samples, and the survival chart showed up to about 80% mortality in the individuals over time. With integrated bioinformatics analysis, BGN, LOX, MMP-9, SERPINE1, and TGFB1 proteins have been selected as suitable diagnostic biomarkers for noninvasive to invasive stages of gastric cancer. Further studies are needed to evaluate more precise mechanisms between these proteins

Lentiviral Mediating Genetic Engineered Mesenchymal Stem Cells for Releasing IL-27 as a Gene Therapy Approach for Autoimmune Diseases

Objective: Autoimmune diseases precede a complex dysregulation of the immune system. T helper17 (Th17) and interleukin (IL)-17 have central roles in initiation of inflammation and subsequent autoimmune diseases. IL-27 significantly controls autoimmune diseases by Th17 and IL-17 suppression. In the present study we have created genetic engineered mesenchymal stem cells (MSCs) that mediate with lentiviral vectors to release IL-27 as an adequate vehicle for ex vivo gene therapy in the reduction of inflammation and autoimmune diseases. Materials and Methods: In this experimental study, we isolated adipose-derived MSCs (AD-MSCs) from lipoaspirate and subsequently characterized them by differentiation. Two subunits of IL-27 (p28 and EBI3) were cloned in a pCDH-513B-1 lentiviral vector. Expressions of p28 and EBI3 (Epstein-Barr virus induced gene 3) were determined by real time polymerase chain reaction (PCR). MSCs were transduced by a pCDH-CMV-p28-IRESEBI3- EF-copGFP-Pur lentiviral vector and the bioassay of IL-27 was evaluated by IL-10 expression. Results: Cell differentiation confirmed true isolation of MSCs from lipoaspirate. Restriction enzyme digestion and sequencing verified successful cloning of both p28 and EBI3 in the pCDH-513B-1 lentiviral vector. Real time PCR showed high expressions level of IL-27 and IL-10 as well as accurate activity of IL-27. Conclusion: The results showed transduction of functional IL-27 to AD-MSCs by means of a lentiviral vector. The lentiviral vector did not impact MSC characteristics

Additional file 1 of ZIF-8 Nanoparticle: A Valuable Tool for Improving Gene Delivery in Sperm-Mediated Gene Transfer

Additional file 1: Supplementary Table S1. RLU values between the control samples and blanks and psitive control. Supplementary Table S2. The autocorrelation data of DLS. Supplementary Table S3. Dynamic Light Scattering (DLS). Supplementary Table S4. Zeta potential distribution of ZIF-8

Genetic variant association of PTPN22, CTLA4, IL2RA, as well as HLA frequencies in susceptibility to alopecia areata

<p>Alopecia areata (AA) is characterized by a genetically complex inheritance. HLA frequencies, as well as the single nucleotide polymorphism (SNP) in <i>PTPN22, CTLA4</i>, and <i>IL2RA</i> genes, have been described to be associated with AA susceptibility. So far, no independent replication of these studies has been reported, and no data exist on a possible association between AA disease and these SNPs or influence of HLA frequencies in Iranian population. A possible association between HLA-DRB1*11 alleles as well as a single variation in <i>PTPN22, CTLA4</i>, and <i>IL2RA</i> genes and patchy AA disease have been investigated in a cohort from Iran. Patient and control subjects were genotyped for <i>PTPN22</i> (rs2476601), <i>CTLA4</i> (rs3087243), and <i>IL2RA</i> (rs3118470) variations as well as HLA frequencies. Gene expression levels were analyzed by real-time RT-PCR. In contrast to <i>PTPN22</i> and <i>CTLA4</i> gene polymorphisms, a significant association was found between <i>IL2RA</i> SNP and susceptibility to AA in Iranian cohort. While gene expression levels of <i>IL2RA</i> and <i>PTPN22</i> were higher in the patients than that of controls, <i>CTLA4</i> expression levels found significantly lower in the patients. Despite a significant association between AA and HLA-DRB1*11 frequencies, the presence of DRB1*11 is not associated with <i>PTPN22, CTLA4</i>, or <i>IL2RA</i> gene SNPs. Although the minor allele in <i>IL2RA</i> SNP can be a significant determinant of AA disease development in Iranian population, reported an association between the <i>PTPN22</i> and <i>CTLA4</i> variations was not confirmed by our study. Furthermore, these genetic risk factors might act independently from HLA alleles.</p