Deficiency of the HGF/Met pathway leads to thyroid dysgenesis by impeding late thyroid expansion.

The mechanisms of bifurcation, a key step in thyroid development, are largely unknown. Here we find three zebrafish lines from a forward genetic screening with similar thyroid dysgenesis phenotypes and identify a stop-gain mutation in hgfa and two missense mutations in met by positional cloning from these zebrafish lines. The elongation of the thyroid primordium along the pharyngeal midline was dramatically disrupted in these zebrafish lines carrying a mutation in hgfa or met. Further studies show that MAPK inhibitor U0126 could mimic thyroid dysgenesis in zebrafish, and the phenotypes are rescued by overexpression of constitutively active MEK or Snail, downstream molecules of the HGF/Met pathway, in thyrocytes. Moreover, HGF promotes thyrocyte migration, which is probably mediated by downregulation of E-cadherin expression. The delayed bifurcation of the thyroid primordium is also observed in thyroid-specific Met knockout mice. Together, our findings reveal that HGF/Met is indispensable for the bifurcation of the thyroid primordium during thyroid development mediated by downregulation of E-cadherin in thyrocytes via MAPK-snail pathway.

Identification of Eukaryotic Translation Initiation Factor 4B as a Novel Candidate Gene for Congenital Hypothyroidism.

CONTEXT: Congenital hypothyroidism (CH) is the most common endocrine disorder in neonates, but its etiology is still poorly understood. OBJECTIVE: We performed whole exome sequencing to identify novel causative gene for CH and functional studies to validate its role in the occurrence of CH. METHODS: Whole exome sequencing in 98 CH patients not harboring known CH candidate genes and bioinformatic analysis were performed. Functional analysis was performed using morpholino, a synthetic short antisense oligonucleotide that contains 25 DNA bases on a methylene morpholine backbone, in zebrafish and CRISPR‒Cas9-mediated gene knockout in mice. RESULTS: Eukaryotic translation initiation factor 4B (EIF4B) was identified as the most promising candidate gene. The EIF4B gene was inherited in an autosomal recessive model, and one patient with thyroid dysgenesis carried EIF4B biallelic variants (p.S430F/p.P328L). In zebrafish, the knockdown of eif4ba/b expression caused thyroid dysgenesis and growth retardation. Thyroid hormone levels were significantly decreased in morphants compared with controls. Thyroxine treatment in morphants partially rescued growth retardation. In mice, the homozygous conceptuses of Eif4b+/- parents did not survive. Eif4b knockout embryos showed severe growth retardation, including thyroid dysgenesis and embryonic lethality before E18.5. CONCLUSION: These experimental data supported a role for EIF4B function in the pathogenesis of the hypothyroid phenotype seen in CH patients. Our work indicated that EIF4B was identified as a novel candidate gene in CH. EIF4B is essential for animal survival, but further studies are needed to validate its role in the pathogenesis of CH.

TAF1 is needed for the proliferation and maturation of thyroid follicle cells via Notch signaling.

Thyroid dysgenesis (TD) is the common pathogenic mechanism of congenital hypothyroidism (CH). In addition, known pathogenic genes are limited to those that are directly involved in thyroid development. To identify additional candidate pathogenetic genes, we performed forward genetic screening for TD in zebrafish, followed by positional cloning. The candidate gene was confirmed in vitro using the Nthy-ori 3.1 cell line and in vivo using a zebrafish model organism. We obtained a novel zebrafish line with thyroid dysgenesis and identified the candidate pathogenetic gene taf1 by positional cloning. Further molecular studies revealed that taf1 was needed for the proliferation of thyroid follicular cells by binding to the NOTCH1 promoter region. Knockdown of TAF1 impaired the proliferation and maturation of thyroid cells, thereby leading to thyroid dysplasia. This study showed that TAF1 promoted Notch signaling and that this association played a pivotal role in thyroid development.

TSHR Variant Screening and Phenotype Analysis in 367 Chinese Patients With Congenital Hypothyroidism.

Background: Genetic defects in the human thyroid-stimulating hormone (TSH) receptor (TSHR) gene can cause congenital hypothyroidism (CH). However, the biological functions and comprehensive genotype-phenotype relationships for most TSHR variants associated with CH remain unexplored. We aimed to identify TSHR variants in Chinese patients with CH, analyze the functions of the variants, and explore the relationships between TSHR genotypes and clinical phenotypes. Methods: In total, 367 patients with CH were recruited for TSHR variant screening using whole-exome sequencing. The effects of the variants were evaluated by in-silico programs such as SIFT and polyphen2. Furthermore, these variants were transfected into 293T cells to detect their Gs/cyclic AMP and Gq/11 signaling activity. Results: Among the 367 patients with CH, 17 TSHR variants, including three novel variants, were identified in 45 patients, and 18 patients carried biallelic TSHR variants. In vitro experiments showed that 10 variants were associated with Gs/cyclic AMP and Gq/11 signaling pathway impairment to varying degrees. Patients with TSHR biallelic variants had lower serum TSH levels and higher free triiodothyronine and thyroxine levels at diagnosis than those with DUOX2 biallelic variants. Conclusions: We found a high frequency of TSHR variants in Chinese patients with CH (12.3%), and 4.9% of cases were caused by TSHR biallelic variants. Ten variants were identified as loss-of-function variants. The data suggest that the clinical phenotype of CH patients caused by TSHR biallelic variants is relatively mild. Our study expands the TSHR variant spectrum and provides further evidence for the elucidation of the genetic etiology of CH.

ALYREF-mediated RNA 5-Methylcytosine modification Promotes Hepatocellular Carcinoma Progression Via Stabilizing EGFR mRNA and pSTAT3 activation.

5-Methylcytosine (m5C) is one of the most ubiquitous modifications of mRNA and contributes to cancer pathogenesis. Aly/REF export factor (ALYREF), an m5C reader, is associated with the prognosis of liver hepatocellular carcinoma (LIHC). However, the effects of ALYREF on the progression of LIHC and the underlying molecular mechanisms remains elusive. Through an analysis of an online database and 3 independent LIHC cohorts, we found that ALYREF was markedly elevated in human liver cancer tissues and was significantly correlated with LIHC clinicopathological parameters, including Ki67+ cell rate, high-grade TNM stage, and poor prognosis. Several experiments were conducted to investigate the molecular basis and functional role of ALYREF-related progression in this study. ALYREF could enhance LIHC cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) in vitro and tumor formation in vivo. Mechanistically, ALYREF promoted the progression of human LIHC through EGFR pathways. Furthermore, ALYREF could directly bind to the m5C modification site of EGFR 3' untranslated region (3' UTR) to stabilize EGFR mRNA. Collectively, ALYREF played a crucial oncogenic role in LIHC via the stabilization of EGFR mRNA and subsequent activation of the STAT3 signaling pathway. Our results may help to elucidate the potential mechanisms of ALYREF-induced m5C modification in the progression of human LIHC.

Contactin 6, A Novel Causative Gene for Congenital Hypothyroidism, Mediates Thyroid Hormone Biosynthesis Through Notch Signaling.

Background: Congenital hypothyroidism (CH) is the most common neonatal metabolic disorder. In patients with CH in China, thyroid dyshormonogenesis is more common than thyroid dysgenesis; however, the genetic causes of CH due to thyroid dyshormonogenesis remain largely unknown. Therefore, we aimed at identifying novel candidate causative genes for CH. Methods: To identify novel CH candidate genes, a total of 599 patients with CH were enrolled and next-generation sequencing was performed. The functions of the identified variants were confirmed using HEK293T and FTC-133 cell lines in vitro and in a mouse model organism in vivo. Results: Three pathogenic contactin 6 (CNTN6) variants were identified in two patients with CH. Pedigree analysis showed that CH caused by CNTN6 variants was inherited in an autosomal recessive pattern. The CNTN6 gene was highly expressed in the thyroid in humans and mice. Cntn6 knockout mice presented with thyroid dyshormonogenesis and CH due to the decreased expression of crucial genes for thyroid hormone biosynthesis (Slc5a5, Tpo, and Duox2). All three CNTN6 variants resulted in the blocking of the release of the Notch intracellular domain, which could not translocate into the nucleus, impaired NOTCH1 transcriptional activity, and decreased expression of SLC5A5, TPO, and DUOX2. Further, we found that DTX1 was required for CNTN6 to promote thyroid hormone biosynthesis through Notch signaling. Conclusions: This study demonstrated that CNTN6 is a novel causative gene for CH through the mediation of thyroid hormone biosynthesis via Notch signaling, which provides new insights into the genetic background and mechanisms involved in CH and thyroid dyshormonogenesis.

Myeloid cells interact with a subset of thyrocytes to promote their migration and follicle formation through NF-κB.

The pathogenesis of thyroid dysgenesis (TD) is not well understood. Here, using a combination of single-cell RNA and spatial transcriptome sequencing, we identify a subgroup of NF-κB-activated thyrocytes located at the center of thyroid tissues in postnatal mice, which maintained a partially mesenchymal phenotype. These cells actively protruded out of the thyroid primordium and generated new follicles in zebrafish embryos through continuous tracing. Suppressing NF-κB signaling affected thyrocyte migration and follicle formation, leading to a TD-like phenotype in both mice and zebrafish. Interestingly, during thyroid folliculogenesis, myeloid cells played a crucial role in promoting thyrocyte migration by maintaining close contact and secreting TNF-α. We found that cebpa mutant zebrafish, in which all myeloid cells were depleted, exhibited thyrocyte migration defects. Taken together, our results suggest that myeloid-derived TNF-α-induced NF-κB activation plays a critical role in promoting the migration of vertebrate thyrocytes for follicle generation.

The Promotion of Humoral Immune Responses in Humans via SOCS1-Mediated Th2-Bias Following SARS-CoV-2 Vaccination.

The effectiveness of SARS-CoV-2 vaccines varies among individuals. During the COVID-19 global pandemic, SARS-CoV-2 infection showed significant Th1 characteristics, suggesting that the immune disorder and production of SARS-CoV-2 antibodies may be related to Th1/Th2 bias. However, the molecular mechanisms underlying Th1/Th2 bias effects on host immune responses to viruses remain unclear. In this study, the top three subjects with the highest and lowest changes in anti-SARS-CoV-2 antibodies after receiving three doses of SARS-CoV-2 vaccination were selected and defined as the elevated group (E) and the control group (C), respectively. Peripheral blood was collected, single-cell sequencing was performed before and after the third dose of the SARS-CoV-2 vaccine, and the changes in T cell clusters were analyzed. Compared with the C group, the Treg pre-vaccination proportion was lower in E, while the post-vaccination proportion was higher, suggesting that Tregs may be crucial in this process. Differential analysis results of Tregs between the two groups revealed that differentially expressed genes (DEGs) were significantly enriched in the IL4 pathway. Correlation analysis between DEGs and serum antibody showed that the expression of NR4A2, SOCS1, and SOCS3 in Tregs was significantly correlated with serum antibodies, suggesting that the immune response in E group changed to Th2 bias, thereby promoting host humoral immune responses. On the other hand, antibody-related genes SOCS1 and NR4A2, as well as lnc-RNA MALAT1 and NEAT1, were highly expressed in the CD4-MALAT1 subclusters. In summary, our study revealed that Th2 bias promotes humoral immune responses in humans by increasing SOCS1 in T cells after SARS-CoV-2 vaccination. Moreover, NR4A2, SOCS1, MALAT1, and NEAT1 were identified as the potential key biomarkers or treatment targets for enhanced SARS-CoV-2 antibody production by influencing the Th1/Th2 balance in T cells. Our findings have important implications for population stratification and tailored therapeutics for more effective SARS-CoV-2 vaccines.

Genetic screening and functional analysis of TPO variants in Chinese patients with congenital hypothyroidism.

INTRODUCTION: Congenital hypothyroidism (CH), the most common neonatal endocrine disorder world-wide, can be caused by variants in the thyroid peroxidase (TPO) gene. This study aimed to identify TPO variants in Chinese patients with CH, analyze their impact on TPO function, and establish relationships between TPO genotypes and clinical characteristics. METHODS: A total of 328 patients with CH were screened for TPO variants by performing whole exome sequencing. The function of the detected TPO variants was investigated via transfection assays in vitro. The pathogenic effect of five novel variants was further assessed in silico. RESULTS: Among 328 patients with CH, 19 TPO variants, including six novel ones, were identified in 43 patients. Eighteen patients (5.5%) carried biallelic TPO variants. In vitro experiments showed that TPO activity was impaired to varying degrees in 17 variants. Furthermore, we determined that a residual TPO enzyme activity threshold of 15% may serve as a criterion for differentiating CH severity. CONCLUSIONS: According to our study, the prevalence of TPO variants among Chinese patients with CH was 13.1 %. Five novel variants led to impaired TPO function by altering its structure or by affecting its expression or cellular localization, which should result in impaired thyroid hormone synthesis.

YTHDF3as a prognostic predictive biomarker of thyroid cancer and its correlation with immune infiltration.

PURPOSE: Thyroid cancer (TC) is one of the most common endocrine malignancies, and its morbidity continues to rise. N6-methyladenosine (m6A) RNA methylation, an epigenetic modification, is an important regulator of gene expression in TC. Therefore, it's worth finding the characteristics and predictive value of the m6A RNA methylation regulators in thyroid cancer (TC). METHOD: RNA-seq data of TC was downloaded from the Cancer Genome Atlas (TCGA) database to screen out the differential expressed regulators. The absolute contraction selection operator (Lasso) Cox regression was used to construct the risk model of m6A methylation regulators. The predictive value of the risk scoring model was evaluated by Kaplan Meier (K-M) analysis and receiver operating characteristic (ROC) curves. The underlying mechanism of m6A methylation regulators in TC was predicted by gene set enrichment analysis (GSEA). Further validation was performed by using immunohistochemistry (IHC) and q-PCR. The correlation between risk-related gene and immune infiltration was evaluated by Tumour Immune Estimation Resource (TIMER). RESULTS: IGF2BP2, YTHDF1 and YTHDF3 were screened out as strong independent prognostic factors of TC. Then a risk score model was established to further screen the predictors. Finally, according to the results of overall survival (OS) and clinical characteristics of TC, YTHDF3 was screened out as a potential predictor. Meanwhile, IHC and qPCR confirmed that YTHDF3 was expressed differential in TC. The expression of YTHDF3 was positively associated with the infiltration level of CD4+ T cells and macrophages. It was strongly correlated with a variety of immune markers in TC. CONCLUSION: We confirmed that YTHDF3 can be used as a potential prognostic biomarker of TC. It not only plays a decisive role in the initiation and development of TC, but also provides a new perspective for understanding the modification of m6A RNA in TC.

Influence of two anti-tumor drugs, pazopanib, and axitinib, on the development and thyroid-axis of zebrafish (Danio rerio) embryos/larvae.

Introduction: In recent years, the potential toxicities of different pharmaceuticals toward the thyroid system have received increasing attention. In this study, we aim to evaluate the toxic effects of pazopanib and axitinib, two anti-tumor drugs with widespread clinical use, on thyroid function in the zebrafish model. Methods: We measured levels of thyroid-related hormones using the commercial Enzyme-Linked Immunosorbent Assay (ELISA) kit. Whole-mount in situ hybridization (WISH) analysis was employed to detect target gene expression changes. Morphology of the thyroid were evaluated by using transgenic Tg (tg: EGFP) fish line under a confocal microscope. The relative mRNA expression of key genes was verified through quantitative real-time polymerase chain reaction (RT‒qPCR). The size and number of the follicles was quantified whereby Hematoxylin-Eosin (H & E) staining under a light microscope. Results: The results revealed that fertilized zebrafish embryos were incubated in pazopanib or axitinib for 96 hours, development and survival were significantly affected, which was accompanied by significant disturbances in thyroid endocrine system (e.g., increased thyroid-stimulating hormone (TSH) content and decreased triiodothyronine (T3) and thyroxine (T4) content, as well as transcription changes of genes associated with the hypothalamus-pituitary-thyroid (HPT) axis. Moreover, based on whole-mount in situ hybridization staining of tg and histopathological examination of zebrafish embryos treated with pazopanib and axitinib, we observed a significantly abnormal development of thyroid follicles in the Tg (tg: EGFP) zebrafish transgenic line. Conclusion: Collectively, these findings indicate that pazopanib and axitinib may have toxic effects on thyroid development and function, at least partially, by influencing the regulation of the HPT axis. Thus, we believe that the potential thyroid toxicities of pazopanib and axitinib in their clinical applications should receive greater attention.

Identification of SERPINA1 promoting better prognosis in papillary thyroid carcinoma along with Hashimoto's thyroiditis through WGCNA analysis.

Background: Hashimoto's thyroiditis (HT) is an autoimmune thyroid disease. Papillary thyroid carcinoma (PTC) is the most common endocrine cancer. In recent years the rate of coexistence between PTC and HT has increased but the relationship between them remains unclear, meaning it is necessary to find potential biomarkers for PTC coexistence with HT to predict its potential pathways. Method: A co-expression network was constructed using the weighted gene co-expression network analysis (WGCNA) in the R package. The modules of PTC associated with HT (PTC-W) were identified from the GSE138198 dataset. Protein-protein interaction network (PPI) was used to screen the hub genes. Immunohistochemical (IHC) analysis was performed to validate the expression of the hub genes in tissues. Clinical data from The Cancer Genome Atlas (TCGA) datasets were used to analyse the prognosis of the hub genes. Gene set enrichment analysis (GSEA) was used to screen potential pathways of PTC-W. Result: The MEbrown module representing the most significant module, with 958 differentially expressed genes (DEGs), was screened in PTC-W, based on WGCNA analysis. Through PPI, SERPINA1 was identified as a hub gene. Immunostaining validated that SERPINA1 was highly expressed in PTC-W. Moreover, PTC-W expressing SERPINA1 exhibits a better prognosis than PTC without HT (PTC-WO). Conclusion: Our study demonstrates that SERPINA1 promotes the occurrence of PTC-W, and its prognosis is better than PTC-WO. SERPINA1 promotes a better prognosis for PTC-W, possibly through a tumour inhibition signalling pathway.

Pathogenic variations in MAML2 and MAMLD1 contribute to congenital hypothyroidism due to dyshormonogenesis by regulating the Notch signalling pathway.

BACKGROUND: In several countries, thyroid dyshormonogenesis is more common than thyroid dysgenesis in patients with congenital hypothyroidism (CH). However, known pathogenic genes are limited to those directly involved in hormone biosynthesis. The aetiology and pathogenesis of thyroid dyshormonogenesis remain unknown in many patients. METHODS: To identify additional candidate pathogenetic genes, we performed next-generation sequencing in 538 patients with CH and then confirmed the functions of the identified genes in vitro using HEK293T and Nthy-ori 3.1 cells, and in vivo using zebrafish and mouse model organisms. RESULTS: We identified one pathogenic MAML2 variant and two pathogenic MAMLD1 variants that downregulated canonical Notch signalling in three patients with CH. Zebrafish and mice treated with N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butylester, a γ-secretase inhibitor exhibited clinical manifestations of hypothyroidism and thyroid dyshormonogenesis. Through organoid culture of primary mouse thyroid cells and transcriptome sequencing, we demonstrated that Notch signalling within thyroid cells directly affects thyroid hormone biosynthesis rather than follicular formation. Additionally, these three variants blocked the expression of genes associated with thyroid hormone biosynthesis, which was restored by HES1 expression. The MAML2 variant exerted a dominant-negative effect on both the canonical pathway and thyroid hormone biosynthesis. MAMLD1 also regulated hormone biosynthesis through the expression of HES3, the target gene of the non-canonical pathway. CONCLUSIONS: This study identified three mastermind-like family gene variants in CH and revealed that both canonical and non-canonical Notch signalling affected thyroid hormone biosynthesis.

The isl2a transcription factor regulates pituitary development in zebrafish.

Background: ISL LIM homeobox 2, also known as insulin gene enhancer protein ISL-2 (ISL2), is a transcription factor gene that participates in a wide range of developmental events. However, the role of ISL2 in the hypothalamus-pituitary-thyroid axis is largely unknown. In the present study, we characterized the expression patterns of ISL2 and revealed its regulative role during embryogenesis using zebrafish. Methods: We used the CRISPR/Cas9 system to successfully establish homozygous ISL2-orthologue (isl2a and isl2b) knockout zebrafish. Moreover, we utilized these knockout zebrafish to analyze the pituitary and thyroid phenotypes in vivo. For further molecular characterization, in situ hybridization and immunofluorescence were performed. Results: The isl2a mutant zebrafish presented with thyroid hypoplasia, reduced whole-body levels of thyroid hormones, increased early mortality, gender imbalance, and morphological retardation during maturity. Additionally, thyrotropes, a pituitary cell type, was notably decreased during development. Importantly, the transcriptional levels of pituitary-thyroid axis hormones-encoding genes, such as tshba, cga, and tg, were significantly decreased in isl2a mutants. Finally, the thyroid dysplasia in isl2a mutant larvae may be attributed to a reduction in proliferation rather than changes in apoptosis. Conclusions: In summary, isl2a regulates the transcriptional levels of marker genes in hypothalamus-pituitary-thyroid axis, and isl2a knockout causing low thyroid hormone levels in zebrafish. Thus, isl2a identified by the present study, is a novel regulator for pituitary cell differentiation in zebrafish, resulting in thyroid gland hypoplasia and phenotypes of hypothyroidism.

Exposure to bisphenol A alternatives bisphenol AF and fluorene-9-bisphenol induces gonadal injuries in male zebrafish.

Bisphenol A (BPA), present in many household products, can damage the male reproductive system. Accordingly, we summarized urine samples from 6921 human in National Health and Nutrition Examination Survey and found urinary BPA levels were inversely linked with blood testosterone in the children group. Currently, BPA replacements, such as fluorene-9-bisphenol (BHPF) and Bisphenol AF (BPAF), have been introduced to produce "BPA-free" products. Here we demonstrated that BPAF and BHPF could induce delayed gonadal migration and reduce the number of progenitors of germ cell lineage in zebrafish larvae. A close receptor analysis study reveals that BHPF and BPAF can strongly bind to androgen receptors, leading to the downregulation of meiosis-related genes and the overexpression of inflammatory markers. Furthermore, BPAF and BPHF can induce activation of the gonadal axis via negative feedback, leading to the hypersecretion of some upstream hormones and an increase in the expression of upstream hormone receptors. Our findings call for further research on the toxicological effects of BHPF and BPAF on human health and recommend that BPA replacements be investigated for anti-estrogenic action.

In-frame deletion of SMC5 related with the phenotype of primordial dwarfism, chromosomal instability and insulin resistance.

BACKGROUND: SMC5/6 complex plays a vital role in maintaining genome stability, yet the relationship with human diseases has not been described. METHODS: SMC5 variation was identified through whole-exome sequencing (WES) and verified by Sanger sequencing. Immunoprecipitation, cytogenetic analysis, fluorescence activated cell sorting (FACS) and electron microscopy were used to elucidate the cellular consequences of patient's cells. smc5 knockout (KO) zebrafish and Smc5K371del knock-in mouse models were generated by CRISPR-Cas9. RNA-seq, quantitative real-time PCR (qPCR), western blot, microquantitative computed tomography (microCT) and histology were used to explore phenotypic characteristics and potential mechanisms of the animal models. The effects of Smc5 knockdown on mitotic clonal expansion (MCE) during adipogenesis were investigated through Oil Red O staining, proliferation and apoptosis assays in vitro. RESULTS: We identified a homozygous in-frame deletion of Arg372 in SMC5, one of the core subunits of the SMC5/6 complex, from an adult patient with microcephalic primordial dwarfism, chromosomal instability and insulin resistance. SMC5 mutation disrupted its interaction with its interacting protein NSMCE2, leading to defects in DNA repair and chromosomal instability in patient fibroblasts. Smc5 KO zebrafish showed microcephaly, short length and disturbed glucose metabolism. Smc5 depletion triggers a p53-related apoptosis, as concomitant deletion of the p53 rescued growth defects phenotype in zebrafish. An smc5K371del knock-in mouse model exhibited high mortality, severe growth restriction and fat loss. In 3T3-L1 cells, the knockdown of smc5 results in impaired MCE, a crucial step in adipogenesis. This finding implies that defective cell survival and differentiation is an important mechanism linking growth disorders and metabolic homeostasis imbalance.

Large-scale forward genetic screening of zebrafish affecting thyroid development.

The thyroid follicular cells originate from the foregut endoderm and elucidating which genes and signaling pathways regulate their development is crucial for understanding developmental disorders as well as diseases in adulthood. We exploited unique advantages of the zebrafish model to carry an ENU-based forward mutagenesis screen aiming at identifying genes involved in the development and function of the thyroid follicular cells. ENU is an excellent chemical mutagen due to its high mutation efficiency and an indiscriminate selection of genes. A total of 1606 F2 families from 36 ENU treated founders was raised and embryos from F3 generation were collected at 5dpf to perform the whole embryo in situ hybridization with a cocktail probe of thyroid marker thyroglobulin(tg), pituitary marker thyroid stimulating hormone (tshba) to determine the mutagenic phenotype. Among the 1606 F2 families, 112 F2 mutant families with normal development stages except for thyroid dysfunction were identified and divided into three different groups according to their phenotypic characteristics. Further studies of the mutants are likely to shed more insights into the molecular basis of both the thyroid development and function in the zebrafish and vertebrate.

Lessons from 17ß-HSD3 deficiency: Clinical spectrum and complex molecular basis in Chinese patients.

17ß-Hydroxysteroid dehydrogenase type 3 (17ß-HSD3) deficiency is rarely reported in Chinese patients with 46, XY disorders of sexual development (DSD). Seven subjects with 17ß-HSD3 deficiency were identified from 206 Chinese 46, XY DSD patients using targeted next-generation sequencing (NGS). Serum AD and T levels were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In silico and functional studies were performed to evaluate the enzymatic activity impairment of HSD17B3 variants. A minigene assay was performed in an exonic splicing variant. Our results showed that four novel and five reported HSD17B3 variants were identified in 7 unrelated patients. The patients showed cryptic presentation during childhood and classical virilization after puberty with T/AD ratio< 0.4. A heterozygous large deletion from the 5'UTR to exon 1 was identified in a patient with a monoallelic variant of p.N130S. Although predicted to be 'likely pathogenic', only p. S232P and p. S160F drastically reduced the enzymatic activity of 17ß-HSD3. A previously reported 'missense' variant c 0.277 G>A (p. E93K) was revealed to have no impact on enzyme activity but resulted in aberrant splicing of exon 3 and was reclassified as an exonic splicing variant. In our study, one nonsense, one exonic splicing, one deletion, one large deletion and five missense variants were detected in patients with 17ß-HSD3 deficiency, expanding the clinical and molecular profile of this disorder. In silico analysis should be cautiously interpreted when the heredity pattern and functional study are inconsistent.

Osteoglycin: An ECM Factor Regulating Fibrosis and Tumorigenesis.

The extracellular matrix (ECM) is made up of noncellular components that have special properties for influencing cell behavior and tissue structure. Small leucine-rich proteoglycans (SLRPs) are nonfibrillar ECM components that serve as structural scaffolds and signaling molecules. osteoglycin (OGN), a class III SLRP, is a ubiquitous ECM component that not only helps to organize the extracellular matrix but also regulates a number of important biological processes. As a glycosylated protein in the ECM, OGN was originally considered to be involved in fiber assembly and was reported to have a connection with fibrosis. In addition to these functions, OGN is found in a variety of cancer tissues and is implicated in cellular processes linked to tumorigenesis, including cell proliferation, invasion, metastasis, and epithelial-mesenchymal transition (EMT). In this review, we summarize the structure and functions of OGN as well as its biological and clinical importance in the context of fibrotic illness and tumorigenesis. This review aims to improve our understanding of OGN and provide some new strategies for the treatment of fibrosis and cancer.

Corilagin prevents non-alcoholic fatty liver disease via improving lipid metabolism and glucose homeostasis in high fat diet-fed mice.

Non-alcoholic fatty liver disease (NAFLD) has been considered to be one of the most common chronic liver diseases. However, no validated pharmacological therapies have been officially proved in clinic due to its complex pathogenesis. The purpose of this study was to examine the protective effects of Corilagin (referred to Cori) against NAFLD in mice under a high fat diet (HFD) condition. Mice were fed either a normal control diet (NCD) or HFD with or without Cori (5 or 10 mg/kg body weight) for 15 weeks. In our results, Cori treatment significantly attenuated HFD-induced hepatic steatosis, high NAFLD activity score (NAD) and liver injury. Consistently, Cori treatment remarkably alleviated HFD-induced hepatic lipid accumulation (e.g., triglycerides (TG) and total cholesterol (TC) contents in liver), and improved plasma lipid concentrations (e.g., plasma TG, TC, low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c)). Moreover, Cori treatment ameliorated NAFLD associated metabolic disorders such as glucose intolerance and insulin resistance in HFD-fed mice. Additionally, Cori treatment dramatically changed HFD-induced liver gene expression profiles, and identified overlapped differentially expressed genes (DEGs) between NCD vs. HFD group and HFD vs. HCR (high fat diet plus treatment with Cori) group. With these DEGs, we observed a marked enrichment of Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, which were closely associated with the metabolic balance in liver. Particularly, we found several potential hub proteins against NAFLD development with analyses of protein-protein interaction (PPI) network and qPCR assays. Collectively, our results revealed the important protective effects of Cori against the progress of NAFLD, which was probably mediated through improving dysregulated lipid metabolism and insulin resistance in HFD-fed mice. Additionally, Cori-dependent overlapped DEGs might serve as a featured NAFLD-associated gene expression signature for the diagnosis, treatment, as well as drug discovery and development of NAFLD in the near future.