Evaluation of CHD5, H3K9me3, and H4K12ac in Human Testes with Spermatogenic Maturation Arrest: A Cross-Sectional Study.

BACKGROUND: Spermatogenic maturation arrest is thought to be caused by epigenetic defects, specifically in chromatin remodeling and histone modification. This study evaluated the status of chromatin remodeling chromodomain helicase DNA binding protein 5 (CHD5) and histone modifications histone 4 lys-12 acetylation (H4K12ac) and histone 3 lys-9 trimethylation (H3K9me3) in human testicular biopsies, based on maturation arrest type. MATERIALS AND METHODS: The cross-sectional study utilized 18 Bouin-fixed paraffin-embedded (BFPE) specimens prepared from residual tissue from routine laboratory tests of infertile patients. The expression of CHD5, H4K12ac, and H3K9me3 was examined through immunohistochemistry (IHC). The intensity was measured using ImageJ with IHC Profiler and StarDist plugins. Statistical analysis was performed using Python with Scipy.Stats module. The data were tested with Shapiro- Wilk for normality and Levene test for homogeneity. The differences in the intensity of spermatogenic cells were assessed using Kruskal-Wallis and Mann-Whitney tests. A difference was considered statistically significant if P<0.05. RESULTS: We found three types of maturation arrest, including Sertoli cell only (n=5), spermatocyte arrest (n=4), and spermatid arrest (n=9). CHD5 was positive in spermatogonia and round spermatids but absent in spermatocytes. The mean grey value (MGV) of CHD5 in spermatogonia was generally weak in spermatocyte arrest (157.4 ± 16.6) and spermatid arrest (155.3 ± 16.8), and there was no significant difference between them [P=0.49, 95% confidence interval (CI): (-4.3, 6), effect size (r): 0.02]. Although there was a significant difference in the expression of H3K9me3 and H4K12ac (P<0.001), both histone modifications were found in all observed spermatogenic cells. CONCLUSION: The expressions of CHD5, H3K9me3, and H4K12ac in different spermatogenic cell types produce similar results, indicating that they cannot be used as markers to determine the type of spermatogenic maturation arrest in humans. The significant finding in this research is the expression of CHD5 in human spermatogonia cells, which requires further study for elaboration.

Prediction of the Signaling Pathway in Polycystic Ovary Syndrome Using an Integrated Bioinformatics Approach.

Objectives The purpose of this study was to define the underlying biological mechanisms of PCOS utilizing the protein-protein interaction networks that were constructed based on the putative disease-causing genes for PCOS. Design No animals were used in this research because this study is an In-Silico studies which mainly uses softwares and online analysis tools. Participants/Materials, Settings Genes datasets related with PCOS were obtained from Genecard. Methods The protein-protein interaction networks (PPIN) of PCOS were created using the String Database after genes related with PCOS were obtained from Genecard. After that, we performed an analysis of the hub-gene clusters extracted from the PPIN using the ShinyGO algorithm. In the final step of this research project, functional enrichment analysis was used to investigate the primary biological activities and signaling pathways that were associated with the hub clusters. Results The Genecard database provided the source for the identification of a total of 1072 potential genes related with PCOS. The PPIN that was generated by using the genes that we collected above contained a total of 82 genes and three different types of cluster interaction interactions. In addition, after conducting research on the PPIN with the shinyGO plug-in, 19 of the most important gene clusters were discovered. The primary biological functions that were enriched in the key clusters that were developed were ovarian stereoidogenesis, breast cancer pathway, regulation of lipid and glucose metabolism by AMPK pathway, and ovarian stereoidogenesis. The integrated analysis that was performed in the current study demonstrated that these hub clusters and their connected genes are closely associated to the pathogenesis of PCOS. Limitations Several of the significant genes that were identified in this study, such as ACVR1, SMAD5, BMP6, SMAD3, SMAD4 and AMH. It is necessary to do additional research using large samples, several centers, and multiple ethnicities in order to verify these findings. Conclusions The integrated analysis that was performed in the current study demonstrated that these hub clusters and their connected genes are closely associated to the pathogenesis of PCOS. This information may possibly bring unique insights for the treatment of PCOS as well as the investigation of its underlying pathogenic mechanism.

Human Milk Oligosaccharide Profiles and the Secretor and Lewis Gene Status of Indonesian Lactating Mothers / 대한소아소화기영양학회지

Purpose@#Human milk oligosaccharides (HMOs) may be genetically determined based on the secretor and Lewis status of the mother. This study aims to determine the HMO profile and the secretor and Lewis gene status of Indonesian lactating mothers. @*Methods@#Baseline data of 120 mother-infant pairs between 0-4 months post-partum obtained from a prospective longitudinal study was used. The concentrations of 2'-fucosyllactose (2’FL), lacto-N-fucopentaose I (LNFP I), lacto-N-tetraose (LNT), lactoN-neotetraose (LNnT), 3'-sialyllactose (3’SL), and 6'-sialyllactose (6’SL) were measured.Genetic analysis was performed for mothers using targeted next-generation sequencing and Sanger sequencing. Wild-type AA with the rs1047781 (A385T) polymorphism was categorized as secretor positive, while heterozygous mutant AT was classified as a weak secretor. The presence of rs28362459 (T59G) heterozygous mutant AC and rs3745635 (G508A) heterozygous mutant CT genes indicated a Lewis negative status, and the absence of these genes indicated a positive status. Subsequently, breast milk was classified into various groups, namely Group 1: Secretor+Lewis+ (Se+Le+), Group 2: Secretor−Lewis+ (Se−Le+), Group 3: Secretor+Lewis− (Se+Le−), and Group 4: Secretor−Lewis− (Se−Le−). Data were analyzed using the Mann–Whitney and Kruskal–Wallis rank tests, and a p-value of 0.05 indicated statistical significance. @*Results@#A total of 58.3% and 41.7% of the samples had positive and weak secretor statuses, respectively. The proportion of those in Group 1 was 85%, while 15% were Group 3.The results showed that only 2'FL significantly differed according to the secretor status (p-value=0.018). @*Conclusion@#All Indonesian lactating mothers in this study were secretor positive, and most of them had a Lewis-positive status.

Expression of programmed cell death ligand-1 protein in germinal center B-cell-like and non-germinal center B-cell-like subtypes of diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma worldwide. However, there is still limited information on the expression of programmed cell death ligand-1 (PD-L1), a type 1 transmembrane protein of immunoglobulin B7/CD28 in the DLBCL subtypes. The present study aimed to identify the expression of PD-L1 in germinal center B-cell-like (GCB) subtype and non-germinal center B-cell-like (non-GCB) subtype of DLBCL. A total of 40 patient samples (formalin-fixed paraffin-embedded tissues) consisting of 20 cases of GCB subtype and 20 cases of non-GCB subtype of DLBCL were examined. The PD-L1 protein expressions were evaluated by using immunohistochemical staining in the tumor cells. The results showed a statistically significant difference (P=0.003) between the expression of PD-L1 in the GCB subtype and the non-GCB subtype of DLBCL. PD-L1 expression in the tumor cells were observed in 13 cases (65%) of non-GCB subtype and in three cases (15%) of the GCB subtype of DLBCL. In conclusion, it was found that the expression of PD-L1 protein in the tumor cells of the non-GCB subtype of DLBCL was significantly higher as compared with the tumor cells of the GCB subtype of DLBCL.

Suppressing HOXA-10 Gene Expression by MicroRNA 135b During the Window of Implantation in Infertile Women.

BACKGROUND: Implantation failure has long been identified as a common problem underlying low success rate of IVF. Currently, endometrial receptivity has gained expert attention as it is demonstrated to contribute to successful embryo implantation. MicroRNAs (miRNAs) is known to affect endometrial receptivity through post-transcriptional gene expression regulation. This study aimed to evaluate the expression of miRNA 135b and HOXA-10 during the implantation window in endometrial tissue of infertile women. METHODS: A total of 14 patients diagnosed with infertility in the gynaecology clinic of Cipto Mangunkusumo and Daya Medika hospitals Jakart, Indonesia were selected as the observed group, and 9 fertile patients were enrolled in the control group. Total RNA was isolated from endometrial tissues collected at the secretory phase of the menstrual cycle. The miRNA 135b and HOXA-10 mRNA expression were measured using quantitative real-time PCR (qPCR). The correlation between these variables was then determined using Pearson's correlation coefficient. RESULTS: The expression of miRNA 135b in the infertile group was significantly higher by 1.81-fold compared to the control group (p<0.01), whereas, expression of HOXA-10 mRNA was significantly lower in the infertile group compared to the controls (p=0.047). Significant negative correlation was observed between the expression of miRNA 135b and HOXA-10 mRNA in infertile women (p=0.021; r=-0.607). CONCLUSION: Taken together, this study provides that alteration of miRNA expression is involved in regulating the implantation process partly via modulation of the expression of gene required for implantation.

The Role of Cytotoxic T-lymphocyte-associated Protein 4 (CTLA-4) Gene, Thyroid Stimulating Hormone Receptor (TSHR) Gene and Regulatory T-cells as Risk Factors for Relapse in Patients with Graves Disease.

BACKGROUND: graves' disease (GD) is the most common condition of thyrotoxicosis. The management of GD is initiated with the administration of antithyroid drugs; however, it requires a long time to achieve remission. In reality more than 50% of patients who had remission may be at risk for relapse after the drug is stopped. This study aimed to evaluate the role of clinical factors such as smoking habit, degree of ophtalmopathy, degree of thyroid enlargement; genetic factors such as CTLA-4 gene on nucleotide 49 at codon 17 of exon 1, CTLA-4 gene of promotor -318, TSHR gene polymorphism rs2268458 of intron 1; and immunological factors such as regulatory T cells (Treg) and thyroid receptor antibody (TRAb); that affecting the relapse of patients with Graves' disease in Indonesia. METHODS: this was a case-control study, that compared 72 subjects who had relapse and 72 subjects without relapse at 12 months after cessation of antithyroid treatment, who met the inclusion criteria. Genetic polymorphism examination was performed using PCR-RFLP. The number of regulatory T cells was counted using flow cytometry analysis and ELISA was used to measure TRAb. The logistic regression was used since the dependent variables were categorical variables. RESULTS: the analysis of this study demonstrated that there was a correlation between relapse of disease and family factors (p=0.008), age at diagnosis (p=0.021), 2nd degree of Graves' ophthalmopathy (p=0.001), enlarged thyroid gland, which exceeded the lateral edge of the sternocleidomastoid muscles (p=0.040), duration of remission period (p=0.029), GG genotype of CTLA-4 gene on the nucleotide 49 at codon 17 of exon 1 (p=0.016), CC genotype of TSHR gene on the rs2268458 of intron 1 (p=0.003), the number of regulatory T cells (p=0.001) and TRAb levels (p=0.002). CONCLUSION: genetic polymorphisms of CTLA-4 gene on the nucleotide 49 at codon 17 of exon 1, TSHR gene SNP rs2268458 of intron 1, number of regulatory T cells and TRAb levels play a role as risk factors for relapse in patients with Graves' disease.

Expression of the Bcl-2 family genes and complexes involved in the mitochondrial transport in prostate cancer cells.

Alteration of molecular pathways triggering apoptosis gives raise to various pathological tissue processes, such as tumorigenesis. The mitochondrial pathway is regulated by both the genes of the Bcl-2 family and the genes encoding mitochondrial transport molecules. Those proteins allow a release of cyctochrome c through the outer mitochondrial membrane. This release activates the caspase cascade resulting in death of cells. There are at least two main transport systems associated with the family of Bcl-2 proteins that are involved in transport of molecules through the outer mitochondrial membrane, i.e., the voltage dependent anion channels (VDACs) and translocases of the outer mitochondrial membrane proteins (TOMs). We investigated the expression of genes of the Bcl-2 family, i.e., pro-apoptotic Bak and Bid, and anti-apoptotic Bcl-2; VDAC gene, i.e., VDAC1, VDAC2 and VDAC3; and TOMM genes, i.e., TOMM20, TOMM22 and TOMM40. This study was performed at the mRNA and the protein level. Fourteen paraffin embedded prostate cancer tissues and five normal prostate tissues were analyzed by the quantitative PCR array and immunohistochemistry. We found a significant increase in both mRNA expression of the anti-apoptotic Bcl-2 gene and VDAC1 gene in prostate cancer tissue in comparison with their normal counterparts. Translation of the anti-apoptotic Bcl-2 and VDAC1 genes in prostate cancer tissue was slightly increased. We observed no significant differences in the mRNA expression of the pro-apoptotic Bak and Bid genes, VDAC2 or VDAC3 genes or the three TOMM genes in these tissues. The pro-apoptotic Bax protein was downtranslated significantly in secretory cells of prostate cancer as compared to normal prostate. We suggest that this protein is a good candidate as biomarker for prostate cancer.