SOX2 Gene Research Articles

Evaluation of the effect of boron derivatives on cardiac differentiation of mouse pluripotent stem cells

BackgroundThe heart is one of the first organs to form during embryonic development and has a very important place. So much that the formation of a functional heart is completed on the 55th day of human development and the 15th day of mouse development. Myocardial, endocardial and epicardial cells, which are derived from the mesoderm layer, are the cells that form the basis of the heart. Cardiac development, like other embryonic developments, is tightly controlled and regulated by various signaling pathways. The WNT signaling pathway is the most studied of these signaling pathways and the one with the clearest relationship with heart development. It is known that boron compounds and the Wnt/β-catenin pathway are highly correlated. Therefore, this study aimed to investigate the role of boron compounds in heart development as well as its effect on pluripotency of mouse embryonic stem cells for the first time in the literature. MethodsToxicity of boron compounds was evaluated by using MTS analysis and obtained results were supported by morphological pictures, Trypan Blue staining and Annexin V staining. Additionally, the possible boron-related change in pluripotency of embryonic stem cells were analyzed with alkaline phosphatase activity and immunocytochemical staining of Oct4 protein as well as gene expression levels of pluripotency related OCT4, SOX2 and KLF4 genes. The alterations in the embryonic body formation capacity of mouse embryonic stem cells due to the application boron derivatives were also evaluated. Three linage differentiation was conducted to clarify the real impact of boron compounds on embryonic development. Lastly, cardiac differentiation of mESCs was investigated by using morphological pictures, cytosolic calcium measurement, gene expression and immunocytochemical analysis of cardiac differentiation related genes and in the presence of boron compounds. ResultsObtained results show that boron treatment maintains the pluripotency of embryonic stem cells at non-toxic concentrations. Additionally, endodermal, and mesodermal fate was found to be triggered after boron treatment. Also, initiation of cardiomyocyte differentiation by boron derivative treatments caused an increased gene expression levels of cardiac differentiation related TNNT2, Nkx2.5 and ISL-1 gene expression levels. ConclusionThis study indicates that boron application, which is responsible for maintaining pluripotency of mESCs, can be used for increased cardiomyocyte differentiation of mESCs.

The Antiproliferative Effect of Chloroform Fraction of Eleutherine bulbosa (Mill.) Urb. on 2D- and 3D-Human Lung Cancer Cells (A549) Model.

Since lung cancer is the leading cause of cancer-related death worldwide, research is being conducted to discover anticancer agents as its treatment. Eleutherine bulbosa, a Dayak folklore medicine, exhibited anticancer effects against several cancer cells; however, its anticancer potency against lung cancer cells has not been explored yet. This study aims to determine the anticancer potency of E. bulbosa bulbs against lung cancer cells (A549) using 2D and 3D culture models, as well as determine its active compounds using gas chromatography-mass spectrometry (GC-MS) analysis. Three fractions of E. bulbosa bulbs, namely chloroform, n-hexane, and ethyl acetate, were tested for cytotoxicity using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) and CellTiter-Glo. The antiproliferative effects of the most cytotoxic fraction against the 2D culture model were determined by a clonogenic survival assay and propidium iodide/Hoechst 33342 double staining, whereas the effects against the 3D culture model were determined by microscopy, flow cytometry, and gene expression analysis. The chloroform fraction is the most cytotoxic against A549 cells than other fractions, and it inhibited colony formation and induced apoptosis of A549 cells. The chloroform fraction also inhibited the growth of the A549 spheroid by suppressing the spheroid size, inducing apoptosis, reducing the proportion of CD44 lung cancer stem cells, causing arrest at the S phase of the cell cycle, and suppressing the expression of the SOX2 and MYC genes. Furthermore, the GC-MS analysis detected 20 active compounds in the chloroform fraction, including the major compounds of eleutherine and isoeleutherine. In conclusion, the chloroform fraction of E. bulbosa bulbs exhibit its antiproliferative effect on 2D and 3D culture models of A549 cells, suggesting it could be a lung cancer chemopreventive agent.

O-255 microRNA-23a/b-3p regulate expression levels of testis-specific transcripts in men with impaired fecundity

Abstract Study question To investigate whether microRNA-23a/b-3p targets spermatogenesis-related transcripts and whether this targeting impacts their expression contents in patients with subfertility. Summary answer The higher expression of microRNA-23a/b-3p and lower expression of 11 tested target genes are associated with men subfertility What is known already Spermatogenesis is a complex and highly regulated process, many genes are involved, the expression levels of which are strongly or partially coordinated by microRNAs (miRNAs). MiRNAs are small, non-coding RNAs that are involved in the post-transcriptional regulation of gene expression. Transcriptome analysis shows that hundreds of genes are expressed exclusively or predominantly in male germ cells including CEP41, G2E3, GOLGA6B, GOLGA6C, LMLN, NOL4, PAPOLB, PCDHA9, RGPD1, SOX6, and ZNF695 genes, which play a crucial role during spermatogenesis and/or sperm function. However, the expression regulation of these genes is still unclear. Study design, size, duration Reverse transcription-quantitative PCR (RT-qPCR), dual luciferase assay, Western blot, and bioinformatics analysis were used to validate the lower expression of 11 target genes as a result of the known higher expression of microRNA-23a/b-3p in men with subfertility. A total of 82 men were included for RT-qPCR, consisting of 41 oligoasthenozoospermic subfertile men who attended the IVF center for infertility treatment and 41 age-matched normozoospermic volunteers who served as controls. Participants/materials, setting, methods In silico prediction and dual-luciferase assays were performed to evaluate the potential links between the higher expression of microRNA-23a/b-3p and the lower expression of 11 genes. Total RNA, including miRNA, was isolated from the sperm of oligoasthenozoospermic (n = 41) and normozoospermic men (n = 41). RT-qPCR was used to detect the expression levels of 11 target genes. Correlation analyses between the mRNA expression levels and basic semen parameters were carried out. Main results and the role of chance The expression levels of microRNA-23a/b-3p were significantly up-regulated and 11 genes were significantly down-regulated in oligoasthenozoospermic men compared with age-matched normozoospermic men as determined by RT-qPCR. Using dual-luciferase assays, 9 genes including CEP41, G2E3, GOLGA6C, NOL4, PAPOLB, PCDHA9, RGPD1, SOX6, and ZNF695 were identified as direct targets of miR-23a-3p and 4 genes including GOLGA6C, PAPOLB, SOX6, and ZNF695 were identified as direct targets of miR-23b-3p. Mutations in the miR-23a/b-3p binding site within the 3ˊuntranslated regions (3ˊUTRs) of the 9 target genes, which target either miR-23a-3p and/or miR-23b-3p, resulted in abrogated responsiveness to microRNA-23a/b-3p and confirmed that CEP41, GOLGA6C, NOL4, PCDHA9, and SOX6 as direct targets for miR-23a-3p and NOL4, SOX6 and PCDHA9 as direct targets for miR-23b-3p. Correlation analysis highlighted sperm count, motility, and morphology were positively correlated with the lower expression level of these validated genes. Limitations, reasons for caution Despite the correlation between the higher expression of microRNA-23a/b-3p and the lower expression of the validated genes, further validation by Western blotting in human sperm and testicular tissues is needed. Wider implications of the findings Findings suggest that the higher expression of microRNA-23a/b-3p or the lower expression of validated target genes are associated with male subfertility, probably through influencing the basic semen parameters. This study lay the groundwork for future studies focused on investigating therapies for male infertility. Trial registration number Hedwig-Stalter foundation (2016)

SMYD3 induces sorafenib resistance by activating SMAD2/3-mediated epithelial-mesenchymal transition in hepatocellular carcinoma

Drug resistance prominently hampers the effects of systemic therapy of sorafenib to hepatocellular carcinoma (HCC). Epigenetics have critical regulatory roles in drug resistance. However, the contributions of histone methylatransferase SET and MYND domain containing 3 (SMYD3) to sorafenib resistance in HCC remain largely unknown. Here, using our established sorafenib-resistant HCC cell and xenograft models, we found SMYD3 was markedly elevated in sorafenib-resistant tumors and cells. Functionally, loss- and gain-of-function studies showed that SMYD3 promoted the migration, invasion, metastasis and stemness of sorafenib-resistant HCC cells. Mechanistically, SMYD3 is required for SMAD2/3-mediated epithelial-mesenchymal transition (EMT) in sorafenib-resistant HCC cells by interacting with SMAD2/3 and epigenetically promoting the expression of SOX4, ZEB1, SNAIL1 and MMP9 genes. In summary, our data demonstrate that targeting SMYD3 is an effective approach to overcome sorafenib resistance in HCC.

Effect of A. graveolens hydroalcoholic extract on epilepsy-related damaging of Sox2 gene expression in embryos of epileptic mothers

BackgroundAnethum graveolens is a plant species belonging to the Apiaceae family that is commonly found in southwestern Asia (Iran-Khorasan province) and southeastern Europe. Previous studies have shown that A. graveolens extract may have anticonvulsant properties, reducing the frequency or severity of seizures in people with epilepsy. To better understand the mechanism behind how A. graveolens extract improves epilepsy, this study investigated the effects of the hydroalcoholic extract of A. graveolens leaves on the expression of the sox2 gene in the embryos of pregnant, epileptic mice. Sox2 is a transcription factor that plays a crucial role in the development of the nervous system, and its dysregulation has been implicated in various neurological disorders, including epilepsy. By examining the effects of A. graveolens extract on the expression of sox2 in embryonic epileptic mice, this study aimed to provide insights into the potential therapeutic benefits of this natural remedy for epilepsy. MethodsSixty BALB/c mice aged 6–8 weeks and weighing 25–30 g were used for this experiment. Four groups of 12 mice each were randomly selected from fully kindled pregnant mice. A. graveolens extract was administered intraperitoneally for 13 days to three experimental groups at doses of 100, 200, and 400 mg/kg/day, while the control group was given PBS. Following the final injection and completion of the protocol, mice were anesthetized with ether, and the heads of pregnant mice were cut to dissect prenatal mouse brains. Tissues were cut and immediately placed in RNA Later (QIAGEN) solution under sterile conditions for RNA extraction and analysis of sox2 gene expression. ResultsThe expression of the SOX2 gene was significantly reduced in the embryos of pregnant, epileptic mice compared to healthy controls and PBS group (P < 0.05) and (P < 0.0001), respectively. Additionally, intraperitoneal injections of 100 mg/kg/day of A. graveolens for 13 days significantly increased the expression of the sox2 gene in the embryos of pregnant epileptic mice. A similar effect was observed with 200 and 400 mg/kg/day of the extract, but it was less pronounced. Therefore, 100 mg/kg/day of A. graveolens may be optimal for exerting its protective effect on the brain expression of sox2 in embryonic epileptic mice. ConclusionsBased on the findings of this study, A. graveolens hydroalcoholic extract inhibits epilepsy-related damaging effects on Sox2 gene expression in the embryos of epileptic mothers.

A novel mutation in the SOX5 gene c.1627del; p.(Tyr543IlefsTer14) is associated to Lamb–Shaffer syndrome: a case report

BackgroundLamb–Shaffer syndrome (LAMSHF) is a rare neurodevelopmental disorder caused by heterozygous mutation or microdeletion involving the SOX5 gene. LAMSHF is characterize by developmental delay, intellectual disability, poor expressive speech, mild dysmorphic facial features and skeletal abnormalities.Case presentationWe presented a case of a child with delayed psychomotor development in all areas, scoliosis, peculiar facies, and suspicion of intermittent endotropia, alteration in the alignment of one foot and difficulty in standing. These clinical features lead to genetics studies, in which a novel pathogenic variant in the SOX5 gene was detected in association with LAMSHF.ConclusionsLAMSHF should be suspected in patients with developmental delay, speech delay, intellectual disability, behavioural disturbances, ophthalmological alterations and skeletal abnormalities. A novel pathogenic mutation in the SOX5 gene c.1627del p.(Tyr543IlefsTer14) was identified in this patient as responsible of Lamb–Shaffer syndrome. This case contributes to understanding the genetic characteristics, clinical features, and diagnosis of LAMSHF.

HOXA9 gene promotor methylation and copy number variation of SOX2 and HV2 genes in cell free DNA: A potential diagnostic panel for non-small cell lung cancer

BackgroundMost cases of lung cancer are diagnosed at advanced stage. Detection of genetic and epigenetic markers in cell-free DNA (cfDNA) is a promising tool for the diagnosis of lung cancer at an early stage. The aim of this study was to identify non-invasive diagnostic markers in cell free DNA (cfDNA) for non-small cell lung cancer (NSCLC) as it is the most common type of lung cancer.MethodsWe investigated the cfDNA HOXA9 gene promotor methylation by pyrosequencing. Copy number variation of SOX2 and HV2 genes were detected by real-time PCR in cfDNA extracted from plasma samples of 25 newly diagnosed NSCLC patients and 25 age and sex matched controls.ResultsMethylation level of HOXA9 was significantly higher in NSCLC patients than controls (p > 0.001). SOX2 showed significantly higher CNV and HV2 showed lower CNV in patients than controls (p > 0.001, p = 0.001 respectively). Receiver Operating Characteristic (ROC) curve analysis for HOXA9 methylation, SOX2 CNV and HV2 CNV showed a discrimination power of 79.4%, 80% and 77.5% respectively and the area under the curve for the combined analysis of the three genes was 0.958 with 88% sensitivity and 100% specificity.ConclusionsIn this study, we suggest a potentially diagnostic panel that may help in detection of lung cancer with high sensitivity and specificity using cell free DNA. This Panel included HOXA9 gene methylation and the CNV of SOX2 and HV2 genes.

Abstract 6077: Comprehensive multi-omic characterization of genetic changes associated with sinonasal squamous cell carcinoma progression

Abstract Approximately 50-80% of sinonasal tract cancers are sinonasal squamous cell carcinoma (SNSCC), a rare malignancy which occur in the nasal cavity and maxillary sinus. These lesions possess a propensity for local invasion into adjacent structures including the skull base, brain, and orbit. Owing to relatively mild symptoms during early stages, which resemble benign sinonasal disease, most patients are already in clinically advanced stages when diagnosed. Therefore, despite improvements in endoscopic surgical approaches, radiotherapy and imaging techniques, the 5-year overall survival for advanced SNSCC patients remains poor (~40%). Inverted sinonasal papilloma (IP) is a locally aggressive, benign epithelial neoplasm arising in the paranasal sinuses which has a high recurrence rate and transforms to SNSCC in 10%-25% of cases. As some IP evolve to malignant neoplasms, it has been presumed that they represent an intermediate step in SNSCC progression. However, the current tools for detecting altered epithelial cells, such as clinical examination and histological characteristics, have limited prognostic value for predicting which IP will progress to malignancy, and the link between IP and invasive disease remains unclear. Activating mutations in EGFR and PIK3CA genes as well as loss-of-function mutations in TP53 have been reported in a few very small cohorts of IP and SNSCC lesions, suggesting their role in IP pathogenesis and progression to invasive malignancy. Other observed alterations potentially associated with malignant transition include KRAS mutations, loss of heterozygosity (LOH) at the 9p21 locus (containing p16INK4a), mismatch repair genes deficiency, amplification of FGFR1 and SOX2 genes, as well as NFκB, COX2 and HOXA9 overexpression. While these studies provide a snapshot of molecular changes in SNSCC, genetic alterations critical to the development of SNSCC are poorly understood, and the exact mechanism underlying malignant transformation remains unknown. In this study we have used PandaOmic, a multi-omic data analysis and visualization algorithm, to perform a first comprehensive integrative analysis of WES, mitochondrial sequencing and transcriptomic data obtained from 11 IP lesions, matched invasive SNSCC tumors and histologically normal paranasal sinus epithelium collected from the same patients. Our analysis reveals heterogeneous mutational patterns, gene expression changes and signaling pathways features associated with SNSCC pathogenesis, and provides crucial insights that may aid in the development of novel means of prevention, diagnosis, and treatment of this rare, aggressive and poorly characterized malignancy. Citation Format: Alka Singh, Nikita Babushkin, Michael Korzinkin, Viktoria Sarkisova, Vasudha Mishra, Ari Rosenberg, Mark Lingen, Justin Bishop, Alex Zhavoronkov, Nyall London, Nishant Agrawal, Xuanyao Liu, Evgeny Izumchenko. Comprehensive multi-omic characterization of genetic changes associated with sinonasal squamous cell carcinoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6077.

Abstract 2428: Cancer stem cells maintain stemness via autocrine activation of the IFN/JAK/STAT signaling pathway

Abstract Interferons (IFNs) and Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signaling are best known for their roles in immunity. However, recent work has suggested that both IFNs and the JAK/STAT pathways are associated with tumor growth and progression, as well as the maintenance of cancer stem cell (CSC) populations. To better understand the regulation of the CSC population maintenance by the IFN/JAK/STAT signaling pathway, we enriched CSCs from a panel of non-small cell lung cancer (NSCLC) cell lines by using spheroid culture and determined the phosphorylation of STAT1. We found that sphere cells exhibit increased level of pSTAT1-Y701 compared to their corresponding bulk cells. We further determined the expression level of a few interferon-stimulated genes (ISGs) and found that sphere cells possess enhanced expression of ISG54 and ISG56 genes, which are normally induced by type I IFN (IFN-I), as well as IFNA and IFNB. These results indicate that the IFN-I/STAT1 signaling is highly activated in CSCs, which produce and secrete increased amount of IFN-I. To directly show the effect of IFN-I on the stemness of NSCLC cells, we treated NSCLC cells with IFNβ, and found that IFNβ can significantly enhance the expression of SOX2, a stem cell-specific transcription factor; Inhibition of the JAK signaling blocked both basal and IFNβ-induced SOX2 expression. In addition, knockdown of STAT1 also reduced SOX2 expression, indicating that STAT1 activation plays an important role in mediating IFN-induced enhancement of stemness. Finally, the ChIP assay demonstrated that STAT1 protein can bind to the promoter region of the SOX2 gene to serve as a transcription factor. In summary, our results indicate that CSCs produce and secrete IFN-I via their enhanced JAK/STAT1 signaling. The secreted IFN-I can bind to the receptor on the CSCs, further activate their JAK/STAT1 signaling, promoting the expression of SOX2 and maintenance of stemness of CSCs via an autocrine manner. Citation Format: Aidan Li, Xuetao Bai, Na Li, Shurui Cai, Ananya Banerjee, Kousalya Lavudi, Linzhou Wang, Qianyun Ge, Yajing Yang, Qi-En Wang. Cancer stem cells maintain stemness via autocrine activation of the IFN/JAK/STAT signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2428.

Extreme myopia is more susceptible to SOX2 gene than high myopia

PurposeTo explore the association between two single-nucleotide polymorphisms (SNPs) in the SOX2 gene and high and extreme myopia in the Han Chinese population. Materials and methodsA genetic association study using a case-control method was performed with 139 high myopia, 318 extreme myopia, and 918 healthy participants from the Chinese Han population. Two SNPs (rs4459940 and rs4575941) near SOX2 gene were selected for genotyping. We compared the allelic frequencies and haplotypes of the SNPs to assess their association with high and extreme myopia. This study was adjusted for sex and age of participants in the groups. ResultThe mean ages of the extreme myopia and control subjects were 47.44 ± 15.59 and 44.15 ± 14.08 years, respectively. The rs4575941 SNP of the SOX2 gene and the GG and AG genotypes showed no significant association with the risk of high myopia as opposed to the AA genotype (GG, OR = 0.94, 95% CI = 0.55–1.60, P = 0.820, Pc = NS; AG, OR = 0.91, 95% CI = 0.54–1.52, P = 0.708, Pc = NS). However, the frequency of the risk G allele of rs4575941 was significantly higher in the extreme myopia group than in the control group (OR = 1.31, 95% CI = 1.08–1.59; P = 0.007; Pc = 0.014). Furthermore, there were significant differences in the GG genotype frequency between the extreme myopia and control groups (OR = 1.77, 95% CI = 1.45–2.74, P = 0.009, Pc = 0.036). The A-G haplotype frequency was higher in the extreme group (OR = 1.27, 95% CI = 1.05–1.55, P = 0.014), while there were no significant differences found in high myopia group (OR = 1.18, 95% CI = 0.77–1.31, P = 0.979). ConclusionThe SOX2 rs4575941 polymorphism, in Chinese Han population, contributes to the susceptibility of extreme myopia. SOX2 may thus be implicated in extreme myopia rather than in high myopia.

Abstract P2-21-12: Targeting ADAM8 dependent maintenance of the myCAF phenotype in the breast cancer tumor microenvironment

Abstract Background: Cancer associated fibroblasts (CAFs) regulate breast cancer growth, differentiation and metastasis. Targeting the signaling pathways between myofibroblast CAFs (myCAF) and cancer cells required for myCAF maintenance provides a mechanism to reverse engineer the tumor microenvironment to inhibit cancer growth and metastasis. Methods and Results: We co-cultured human triple negative breast cancer (MDA-MB-231) with human mesenchymal stem cells (MSC). After 72h, MSC mRNA expression of the myCAF markers, alpha-smooth muscle actin (α-SMA)/vimentin (Vim)/tenascin C (Ten-C) are all significantly increased. In parallel, MB-231 mRNA expression of stemness markers, SRY-Box Transcription factor 2 (Sox2)/Octamer-binding transcription factor 4 (Oct4)/Nanog, are also significantly increased. Migration assay and morphology imaging confirm the induction of the myCAF phenotype to promote MB-231 cancer stemness. shRNA lentivirus was used to knock down Sox2 gene expression in MB-231. We performed RNA-seq in MB-231 first comparing MB-231 vs. MB-231+MSC and then MB-231(-/-sox2)+MSC vs. MB-231+MSC. There are 104 overlapping genes in MB-231+MSC from the two analyses indicating upregulation with myCAF differentiation and cancer stemness. Using the Human Cancer Secretome Database, 9 genes are either membrane or secreted proteins. Using antibody blockade in co-culture, antibody depleted co-culture medium, and adding activated proteins to culture, A Disintegrin And Metalloprotease 8 (Adam8) gene was found to be required for myCAF maintenance. Using SELEX, we isolated 5 RNA aptamers targeting the human Adam8 soluble MP domain. Using in vitro quantification of Adam8 MP activity with human Adam8 fluorogenic peptide substrate and recombinant human Adam8 protein, Adam8 Apt-1 with IC50=33nM and Kd=29nM was selected. APt-1 does not inhibit human Adam 10 or 17 MP activity and remains extracellular. The core inhibitory domain of Apt-1 aptamer contains 23nt. Following induction of the myCAF phenotype and cancer stemness in MB-231+MSC coculture, addition of Apt-1 significantly decreased myCAF and cancer stemness markers. In murine NOD/scid xenograft model, 10^6 luciferase-MB-231 and 10^6 MSC were implanted in the R3 position at week 0. Apt1 was administered (10mg/kg via tail vein every 2 days) from week 3 to week 6. MB-231 volume (bioluminescence) was significantly decreased following Apt1. (Table) Conclusion: Adam8 is expressed in MB-231 breast cancer cells in the context of MSC/myCAF coculture and MB231 stemness and is required for MB-231 cancer growth. Aptamer ablation of Adam8 extracellular activity reverses MB-231 growth. MB-231 and MSC Murine Xenograft Model In murine NOD/scid xenograft model, 10^6 luciferase-MB-231 and 10^6 MSC were implanted in the R3 position at week 0. Apt1 was administered (10mg/kg via tail vein every 2 days) from week 3 to week 6. Citation Format: Zhiyong Mi, Paul C. Kuo. Targeting ADAM8 dependent maintenance of the myCAF phenotype in the breast cancer tumor microenvironment [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-21-12.

The Dual Roles of Triiodothyronine in Regulating the Morphology of Hair Cells and Supporting Cells during Critical Periods of Mouse Cochlear Development.

Clinically, thyroid-related diseases such as endemic iodine deficiency and congenital hypothyroidism are associated with hearing loss, suggesting that thyroid hormones are essential for the development of normal hearing. Triiodothyronine (T3) is the main active form of thyroid hormone and its effect on the remodeling of the organ of Corti remain unclear. This study aims to explore the effect and mechanism of T3 on the remodeling of the organ of Corti and supporting cells development during early development. In this study, mice treated with T3 at postnatal (P) day 0 or P1 showed severe hearing loss with disordered stereocilia of the outer hair cells (OHCs) and impaired function of mechanoelectrical transduction of OHCs. In addition, we found that treatment with T3 at P0 or P1 resulted in the overproduction of Deiter-like cells. Compared with the control group, the transcription levels of Sox2 and notch pathway-related genes in the cochlea of the T3 group were significantly downregulated. Furthermore, Sox2-haploinsufficient mice treated with T3 not only showed excess numbers of Deiter-like cells but also a large number of ectopic outer pillar cells (OPCs). Our study provides new evidence for the dual roles of T3 in regulating both hair cells and supporting cell development, suggesting that it is possible to increase the reserve of supporting cells.

Comparison of SOX2 and POU5F1 Gene Expression in Leukapheresis-Derived CD34+ Cells before and during Cell Culture

Bone marrow is an abundant source of both hematopoietic as well as non-hematopoietic stem cells. Embryonic, fetal and stem cells located in tissues (adipose tissue, skin, myocardium and dental pulp) express core transcription factors, including the SOX2, POU5F1 and NANOG gene responsible for regeneration, proliferation and differentiation into daughter cells. The aim of the study was to examine the expression of SOX2 and POU5F1 genes in CD34-positive peripheral blood stem cells (CD34+ PBSCs) and to analyze the influence of cell culture on the expression of SOX2 and POU5F1 genes. The study material consisted of bone marrow-derived stem cells isolated by using leukapheresis from 40 hematooncology patients. Cells obtained in this process were subject to cytometric analysis to determine the content of CD34+ cells. CD34-positive cell separation was conducted using MACS separation. Cell cultures were set, and RNA was isolated. Real-time PCR was conducted in order to evaluate the expression of SOX2 and POU5F1 genes and the obtained data were subject to statistical analysis. We identified the expression of SOX2 and POU5F1 genes in the examined cells and demonstrated a statistically significant (p < 0.05) change in their expression in cell cultures. Short-term cell cultures (<6 days) were associated with an increase in the expression of SOX2 and POU5F1 genes. Thus, short-term cultivation of transplanted stem cells could be used to induce pluripotency, leading to better therapeutic effects.

Clinical characterization of Lamb-Shaffer syndrome: a case report and literature review

BackgroundLamb-Shaffer syndrome (LAMSHF, MIM 616,803) is a rare neurodevelopmental disorder due to haploinsufficiency of SOX5. Furthermore, studies about the clinical features of LAMSHF patients with same allele of c.1477C > T (p. R493*) are very limited.Case presentationWe analyzed the phenotypes of one of our cases and two previously reported cases with c.1477C > T (p. R493*), and reviewed the correlating literature. A de novo heterozygous variation c.1477C > T (p. R493*) in SOX5 was identified in a 4 years and 2 months old boy with global development delay by trio-based whole exome sequencing. We compared our case and previously 2 cases reported with recurrent variation, the overlapping clinical features are global developmental delay or intellectual disability, language delay and scoliosis, but their other clinical characteristics are different.ConclusionsThis study suggests that the clinical features of LAMSHF patients with recurrent variations in the SOX5 gene are different. It is suggested that the LAMSHF-related SOX5 gene should be screened and included as one of the candidate genes for neurodevelopmental disorders of unknown etiology.

T-REX17 is a transiently expressed non-coding RNA essential for human endoderm formation.

Long non-coding RNAs (lncRNAs) have emerged as fundamental regulators in various biological processes, including embryonic development and cellular differentiation. Despite much progress over the past decade, the genome-wide annotation of lncRNAs remains incomplete and many known non-coding loci are still poorly characterized. Here, we report the discovery of a previously unannotated lncRNA that is transcribed 230 kb upstream of the SOX17 gene and located within the same topologically associating domain. We termed it T-REX17 (Transcript Regulating Endoderm and activated by soX17) and show that it is induced following SOX17 activation but its expression is more tightly restricted to early definitive endoderm. Loss of T-REX17 affects crucial functions independent of SOX17 and leads to an aberrant endodermal transcriptome, signaling pathway deregulation and epithelial to mesenchymal transition defects. Consequently, cells lacking the lncRNA cannot further differentiate into more mature endodermal cell types. Taken together, our study identified and characterized T-REX17 as a transiently expressed and essential non-coding regulator in early human endoderm differentiation.

Enhancer-promoter interactions can bypass CTCF-mediated boundaries and contribute to phenotypic robustness.

How enhancers activate their distal target promoters remains incompletely understood. Here we dissect how CTCF-mediated loops facilitate and restrict such regulatory interactions. Using an allelic series of mouse mutants, we show that CTCF is neither required for the interaction of the Sox2 gene with distal enhancers, nor for its expression. Insertion of various combinations of CTCF motifs, between Sox2 and its distal enhancers, generated boundaries with varying degrees of insulation that directly correlated with reduced transcriptional output. However, in both epiblast and neural tissues, enhancer contacts and transcriptional induction could not be fully abolished, and insertions failed to disrupt implantation and neurogenesis. In contrast, Sox2 expression was undetectable in the anterior foregut of mutants carrying the strongest boundaries, and these animals fully phenocopied loss of SOX2 in this tissue. We propose that enhancer clusters with a high density of regulatory activity can better overcome physical barriers to maintain faithful gene expression and phenotypic robustness.

Genes Involved in DNA Repair and Mitophagy Protect Embryoid Bodies from the Toxic Effect of Methylmercury Chloride under Physioxia Conditions.

The formation of embryoid bodies (EBs) from human pluripotent stem cells resembles the early stages of human embryo development, mimicking the organization of three germ layers. In our study, EBs were tested for their vulnerability to chronic exposure to low doses of MeHgCl (1 nM) under atmospheric (21%O2) and physioxia (5%O2) conditions. Significant differences were observed in the relative expression of genes associated with DNA repair and mitophagy between the tested oxygen conditions in nontreated EBs. When compared to physioxia conditions, the significant differences recorded in EBs cultured at 21% O2 included: (1) lower expression of genes associated with DNA repair (ATM, OGG1, PARP1, POLG1) and mitophagy (PARK2); (2) higher level of mtDNA copy number; and (3) higher expression of the neuroectodermal gene (NES). Chronic exposure to a low dose of MeHgCl (1 nM) disrupted the development of EBs under both oxygen conditions. However, only EBs exposed to MeHgCl at 21% O2 revealed downregulation of mtDNA copy number, increased oxidative DNA damage and DNA fragmentation, as well as disturbances in SOX17 (endoderm) and TBXT (mesoderm) genes expression. Our data revealed that physioxia conditions protected EBs genome integrity and their further differentiation.

Culture of leukocyte-derived cells from human peripheral blood: Increased expression of pluripotent genes OCT4, NANOG, SOX2, self-renewal gene TERT and plasticity.

There are few stem cells in human peripheral blood (PB). Increasing the population and plasticity of stem cells in PB and applying it to regenerative medicine require suitable culture methods. In this study, leukocyte populations 250 mL of PB were collected using a blood separator before that were cultured in optimal cell culture medium for 4 to 7 days. After culturing, stemness characteristics were analyzed, and red blood cells were removed from the cultured cells. In our results, stemness markers of the leukocyte populations Sca-1+ CD45+, CD117+ CD45+, and very small embryonic-like stem cells CD34+ Lin- CD45- and CXCR4+ Lin- CD45- were significantly increased. Furthermore, the expression of stem cell genes OCT4 (POU5F1), NANOG, SOX2, and the self-renewal gene TERT was analyzed by quantitative real-time polymerase chain reaction in these cells, and it showed a significant increase. These cells could be candidates for multi-potential cells and were further induced using trans-differentiation culture methods. These cells showed multiple differentiation potentials for osteocytes, nerve cells, cardiomyocytes, and hepatocytes. These results indicate that appropriate culture methods can be applied to increase expression of pluripotent genes and plasticity. Leukocytes of human PB can be induced to trans-differentiate into pluripotent potential cells, which will be an important breakthrough in regenerative medicine.

Induced Chondrogenic Differentiation of hESCs by hESC-Derived MSCs Conditioned Medium and Sequential 3D-2D Culture System

Background and Aims: It has been proven that human mesenchymal stem cells (MSCs) conditioned medium (hMSCs-CM) can influence human embryonic stem cells (hESCs) chondrogenic differentiation. In this study, we hypothesized that conditioned medium (CM) from hESCs-derived MSCs in a sequential 3D-2D culture system could facilitate the induction of chondrogenesis in hESCs.&#x0D; Materials and Methods: CM was collected from Yazd2 (hESCs; 46, XY) derived MSCs confluent cultures and stored at -20 °C. Yazd4 hESC line (46, XX) was induced for differentiation using EB formation as 3D culture into SD (spontaneously differentiation) and CM groups (differentiation using conditioned medium) for four days. Cell culture continued in a 2D (monolayer) culture system for both groups till day 14. Ultimately, chondrogenic differentiation was assessed by Alcian blue and masson′s trichrome staining at 4 and 14 days of differentiation, and quantitative real-time polymerase chain reaction (PCR) for NANOG, MEOX1, SOX5, SOX6, SOX9, ACAN, COL2A1 and RUNX2 genes for SD and CM groups on days 0 and 4.&#x0D; Results: The gene expression profile for chondrogenic genes in the CM group was significantly more than the SD group (p&lt; 0.05). Furthermore, chemical staining assessment illustrated a significant GAG and collagen II difference between the CM and SD groups at days 4 and 14 (p&lt; 0.05).&#x0D; Conclusions: Our findings would pave the way for creating an in vitro human chondrogenesis model for further studies in the developmental biology of articular cartilage tissue, which lend itself to cell-based therapy to cure joint diseases such as osteoarthritis.

Candidate genes for height measurements in Campolina horses

Context Genomic tools allow for increased efficiency in animal production and, consequently, greater production gains. Morphometric measurements of height can be used for breed characterisation/registration, and sex distinction, in addition to being associated with balance, posture, and the range of motion of the limbs, influencing the dynamics and quality of the gait. Aims This study was conducted to examine candidate genes related to the morphometric measurement of heights in Campolina horses. Methods The study involved information from 18 876 horses of this breed, containing phenotypic data of the measurements of withers, croup, back, and barrel heights, provided by the Brazilian Association of Campolina Horse Breeders (ABCCCampolina). Of these, 48 horses of the Brazilian Campolina breed were genotyped with the Equine 65 K lllumina® chip. The effects of the single-nucleotide polymorphisms were estimated by the weighted single-step method, using BLUPF90 family programs. Genes in the main chromosomal regions were identified by consultation with the NCBI. Key results A genomic-wide association study for withers height showed that 46% of the variation in this measurement was explained by 20 genomic windows. For croup height, 19 genomic windows were responsible for explaining 44% of the variation. Sixteen genomic windows were responsible for explaining 37.8% of the variation in back height and 18 genomic regions explained 39.7% of the variation in barrel height. Conclusion The ADAM17, SOX11, and KLF11 genes are located in the genomic windows that most explain the variations in the traits under study and have a biological action on the locomotor system. Implications The ADAM17, SOX11, and KLF11 genes are candidates for fine mapping and construction of molecular markers.