Pou5f1 and Nanog Are Reliable Germ Cell-Specific Genes in Gonad of a Protogynous Hermaphroditic Fish, Orange-Spotted Grouper (Epinephelus coioides)

Pluripotency markers Pou5f1 and Nanog are core transcription factors regulating early embryonic development and maintaining the pluripotency and self-renewal of stem cells. Pou5f1 and Nanog also play important roles in germ cell development and gametogenesis. In this study, Pou5f1 (EcPou5f1) and Nanog (EcNanog) were cloned from orange-spotted grouper, Epinephelus coioides. The full-length cDNAs of EcPou5f1 and EcNanog were 2790 and 1820 bp, and encoded 475 and 432 amino acids, respectively. EcPou5f1 exhibited a specific expression in gonads, whereas EcNanog was expressed highly in gonads and weakly in some somatic tissues. In situ hybridization analyses showed that the mRNA signals of EcNanog and EcPou5f1 were exclusively restricted to germ cells in gonads. Likewise, immunohistofluorescence staining revealed that EcNanog protein was limited to germ cells. Moreover, both EcPou5f1 and EcNanog mRNAs were discovered to be co-localized with Vasa mRNA, a well-known germ cell maker, in male and female germ cells. These results implied that EcPou5f1 and EcNanog could be also regarded as reliable germ cell marker genes. Therefore, the findings of this study would pave the way for elucidating the mechanism whereby EcPou5f1 and EcNanog regulate germ cell development and gametogenesis in grouper fish, and even in other protogynous hermaphroditic species.

Effect of Passage Number on Human Induced Pluripotent Stem Cell Derived Neurons Culture Contaminants with Non-Neuronal Cell Types

Background: The study of live human neurons has been hindered due to the complexity and potential irreversible damage to the patient during biopsy. However, reprogramming of adult human somatic cells into induced pluripotent stem cells (iPSCs) has proved to be a novel method in the study of the pathophysiology of disease and therapeutic targets of the human nervous system. There are several approaches, and the optimum time (i.e., passage number) to generate highly pure cultures is being studied. Therefore, our laboratory has investigated the effect of passage number on culture contaminants with non-neuronal cell types. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood from three cell lines and reprogrammed into iPSCs. Each cell line consisted of three samples that were analyzed after low (5-10), middle (20-26), and high (30-38) number of passages. Cells were maintained in an induction medium for eight days. On day nine, cells were dissociated and replated in a maintenance medium. On day 33, total RNA was extracted from cells. Normalized values for non-neuronal cell marker genes were compared using paired Student’s t-tests and two-way ANOVA, with the cell line and passage number as independent variables. P-values less than 0.05 were considered significant. Results: Our results showed that lower passage number was associated with decreased astrocyte and chondrocyte marker expression. High passage number was associated with decreased oligodendrocyte and glial precursor marker expression. Of the fibroblast markers evaluated, there were similar trends of expression between all three groups. There was no significant difference in microglial cell marker gene expression between all three groups. Conclusion and Potential Impact: Low gene expression suggests a purer culture. According to these results, as passage number increases, there is more contaminants with oligodendrocytes and glial precursor cells. Conversely, with low passage numbers, there are more contaminants with astrocytes and chondrocytes. Future studies will identify the impact of these non-neuronal contaminants and implications on research.

Reduction in trabecular meshwork stem cell content in donor eyes with primary open angle glaucoma

We previously identified and characterized human trabecular meshwork stem cells (TMSCs) based on high expression of ABCG2/p75 positivity and high nucleus to cytoplasmic ratio. These TMSCs expressing high ABCG2 and p75 were located to the insert region of the human TM. Additionally, we demonstrated an age-related reduction in the TMSC content which was significantly associated with TM cell loss. In continuation, this study was aimed to determine the TMSC content in glaucomatous donor eyes wherein a drastic reduction in TM cellularity has already been reported. Anterior segments from known glaucomatous (n = 6) and age-matched normal (n = 8) donors were dissected into four quadrants. A minimum of three sections from each quadrant were used for histopathological analysis as well as immunostaining. Analysis of hematoxylin and eosin-stained sections from glaucomatous tissues revealed a decrease in total TM cellularity, thickening of trabecular beams, fusion of trabeculae, absence of patent Schlemm’s canal compared to age-matched controls. In addition, the TM thickness at various positions of the meshwork and the coronal as well as the meridional diameters of the Schlemm’s canal were observed to be significantly reduced in glaucomatous eyes. Further, sections from both the groups were immunostained for universal stem cell marker ABCG2 and neural crest derived stem cell marker p75. The images were acquired using Leica SP8 confocal microscope. Quantification of total TM cellularity based on nuclear counterstain (mean ± SD) using ImageJ identified 69.33 ± 12.77 cells/section in control eyes. In glaucomatous donors, the TM cellularity was found to be reduced significantly to 41.83 ± 9.0 (p = 0.0007). In addition, a reduction in the percentage of TMSCs (cells with high ABCG2 expression and p75 positivity) was evident in glaucomatous donors (0.14 ± 0.17%) compared to age-matched controls (4.73 ± 5.46%) (p = 0.064). Thus, the present study confirmed the significant decline in TM cellularity and a reducing trend in the TMSC content, though this reduction was non-significant in glaucomatous donor eyes. Further studies are essential to elucidate the role of TMSCs in the pathogenesis of primary open angle glaucoma.

Regulatory roles of lncRNA PANDAR in breast cancer cell proliferation

Background Breast cancer represents the second most deadly malignancy in women, and long noncoding RNAs (lncRNAs) have crucial functions in its development. Objective To investigate effects of the promoter of CDKN1A antisense DNA damage-activated RNA (PANDAR) on epithelial-mesenchymal transition (EMT) in breast cancer cells and their proliferation. Methods lncRNAs potentially regulating the transcriptional activity of the E-cadherin (E-cad, an epithelial cell marker) gene promoter were screened using a dual-luciferase reporter assay. PANDAR was overexpressed in Michigan cancer foundation 7 (MCF-7) breast cancer cells. E-cad and N-cadherin (N-cad, a mesenchymal cell marker) levels were detected by immunoblotting. Cell viability was assessed using a cell counting kit-8. Results PANDAR and TCONS00068220/LOC105375819 conservatively regulated the promoter activity of E-cad. PANDAR overexpression in MCF-7 inhibited E-cad expression, but upregulated N-cad. The enhanced expression of PANDAR promoted cell proliferation. Conclusion PANDAR is a key transcriptional repressor of E-cad and has regulatory effects on the promotion of cell proliferation. PANDAR is an oncogene in breast cancer, potentially facilitating the EMT process and promoting cell proliferation.

The possible role of SRMS in colorectal cancer by bioinformatics analysis

Background Src-related kinase lacking C-terminal regulatory tyrosine and N-terminal myristoylation sites (SRMS) is a non-receptor tyrosine kinase that has been found to be overexpressed in various tumors. However, the role of SRMS in colorectal cancer (CRC) has not been well established. Methods We evaluated the expression levels of SRMS in CRC using GEPIA, Oncomine, and HPA datasets. Survival information and gene expression data of CRC were obtained from The Cancer Genome Atlas (TCGA). Then, the association between SRMS and clinicopathological features was analyzed using UALCAN dataset. LinkedOmics was used to determine co-expression and functional networks associated with SRMS. Besides, we used TISIDB to assess the correlation between SRMS and immune signatures, including tumor-infiltrating immune cells and immunomodulators. Lastly, protein-protein interaction network (PPI) was established and the function enrichment analysis of the SRMS-associated immunomodulators and immune cell marker genes were performed using the STRING portal. Results Compared to normal colorectal tissues, SRMS was found to be overexpressed in CRC tissues, which was correlated with a poor prognosis. In colon adenocarcinoma (COAD), the expression levels of SRMS are significantly correlated with pathological stages and nodal metastasis status. Functional network analysis suggested that SRMS regulates intermediate filament-based processes, protein autophosphorylation, translational initiation, and elongation signaling through pathways involving ribosomes, proteasomes, oxidative phosphorylation, and DNA replication. In addition, SRMS expression was correlated with infiltrating levels of CD4+ T cells, CD56dim, MEM B, Neutrophils, Th2, Th17, and Act DC. The gene ontology (GO) analysis of SRMS-associated immunomodulators and immune cell marker genes showed that they were mainly enriched in the immune microenvironment molecule-related signals. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of these genes indicated that they are involved in multiple cancer-related pathways. Conclusions SRMS is a promising prognostic biomarker and potential therapeutic target for CRC patients. In particular, SRMS regulates CRC progression by modulating cytokine-cytokine receptor interaction, chemokines, IL-17, and intestinal immune networks for IgA production signaling pathways among others. However, more studies are needed to validate these findings.