Hepatic IL22RA1 deficiency promotes hepatic steatosis by modulating oxysterol in the liver.

BACKGROUND AIMS: Imbalance in lipid metabolism is the main cause of nonalcoholic fatty liver disease (NAFLD). While the pathogenesis of lipid accumulation mediated by extrahepatic regulators has been extensively studied, the intrahepatic regulators modulating lipid homeostasis remain unclear. Previous studies have shown that systemic administration of interleukin-22 (IL-22) protects against NAFLD; however, the role of IL-22/IL22RA1 signaling in modulating hepatic lipid metabolism remains uncertain. APPROACH RESULTS: This study shows hepatic IL22RA1 is vital in hepatic lipid regulation. IL22RA1 is downregulated in palmitic acid-treated mouse primary hepatocytes, as well as in the livers of NAFLD model mice and patients. Hepatocyte-specific Il22ra1 knockout (HKO) mice display diet-induced hepatic steatosis, insulin resistance, impaired glucose tolerance, increased inflammation, and fibrosis compared with flox/flox mice. This is attributed to increased lipogenesis mediated by the accumulation of hepatic oxysterols, particularly, 3 beta-hydroxy-5-cholestenoic acid (3ß HCA). Mechanistically, hepatic IL22RA1 deficiency facilitates 3ß HCA deposition via the activating transcription factor 3 (ATF3)/oxysterol 7 alpha-hydroxylase (CYP7B1) axis. Notably, 3ß HCA facilitates lipogenesis in MPHs and human liver organoids (HLOs) by activating LXR-alpha signaling, but IL-22 treatment attenuates this effect. Additionally, restoring CYP7B1 or silencing hepatic ATF3 reduces both hepatic 3ß HCA and lipid contents in HKO mice. CONCLUSIONS: These findings indicate that IL22RA1 plays a crucial role in maintaining hepatic lipid homeostasis in an ATF3/CYP7B1-dependent manner, and establish a link between 3ß HCA and hepatic lipid homeostasis.

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.

Chlordane exposure impairs the growth and behavior of Drosophila.

Chlordane, a previously extensively utilized insecticidal pesticide, has since been prohibited, however, owing to its limited degradability, it continues to persist significantly in soil and water reservoirs, subsequently accumulating within plant and animal organisms, representing a substantial threat to human health. Despite extensive research conducted over the past few decades to investigate the toxic effects of chlordane, there remains a notable dearth of studies focusing on its impact on sleep activity. Therefore, in this study, the effects of short-term and long-term exposure to chlordane on the activity and sleep of Drosophila were investigated. When exposed to chlordane at a concentration of 1 µM, Drosophila lost body weight, decreased body size and resulted in lipid metabolism disorders. In addition, chlordane exposure altered the arousal and sleep behaviors of Drosophila. Short-term exposure to chlordane resulted in an increase in night-time sleep duration, while long-term exposure to chlordane resulted in an increase in activity and a decrease in sleep, as evidenced by a decrease in the duration of each sleep session and the appearance of sleep fragmentation. Under conditions of long-term chlordane exposure, reactive oxygen species levels were significantly up-regulated in Drosophila. Our results suggest that long-term chlordane exposure triggers oxidative stress damage in Drosophila, leading to sleep disruption. This study offers novel insights into the harmful impacts of environmental pollutants on human sleep patterns and proposes that mitigating the presence of chlordane in the environment could potentially contribute to the reduction of global sleep disorder prevalence.

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.

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.

Mutation Screening and Functional Study of SLC26A4 in Chinese Patients with Congenital Hypothyroidism

Objective: Defects in the human solute carrier family 26 member 4 (SLC26A4) gene are reported to be one of the causes of congenital hypothyroidism (CH). We aimed to identify SLC26A4 mutations in Chinese patients with CH and analyze the function of the mutations. Methods: Patients with primary CH were screened for 21 CH candidate genes mutations by targeted next-generation sequencing. All the exons and exon-intron boundaries of SLC26A4 were identified and analyzed. The function of six missense mutation in SLC26A4 were further investigated in vitro. Results: Among 273 patients with CH, seven distinct SLC26A4 heterozygous mutations (p.S49R, p.I363L, p.R409H, p.T485M, p.D661E, p.H723R, c.919-2A>G) were identified in 10 patients (3.66%, 10/273). In vitro experiments showed that mutation p.I363L, p.R409H, p.H723R affect the membrane location and ion transport of SLC26A4, while p.S49R did not. Mutation p.T485M and p.D661E only affected ion transport, but had no effect on the membrane location. Conclusion: The prevalence of SLC26A4 mutations was 3.66% in Chinese patients with CH. Five mutations (p.I363L, p.R409H, p.T485M, p.D661E and p.H723R) impaired the membrane location or ion transport function of SLC26A4, suggesting important roles for Ile363, Arg409, Thr485, Asp661, and His723 residues in SLC26A4 function. As all variants identified were heterozygous, the pathogenesis of these patients cannot be explained, and the pathogenesis of these patients needs further study.

Comparative Performance of Body Composition Parameters in Prediction of Death in Hospitalized Patients on Maintenance Hemodialysis: A Cohort Study.

We compared the prognostic value of nutritional or volumetric parameters measured by body composition in hospitalized patients on maintenance hemodialysis. We conducted a cohort study to assess the association of different parameters of body composition with all-cause mortality in inpatients admitted to our nephrology department from January 2014 to December 2016. Of the 704 study patients, 160 (22.7%) died during a median follow-up of 33 months. In multivariate adjusted Cox models, higher ratio of extracellular water to body cell mass (ECW/BCM) (adjusted HR per 1-SD, 1.49; 95% CI, 1.19 to 1.85), lower lean tissue index (LTI) (adjusted HR per 1-SD, 0.70; 95% CI, 0.57 to 0.86) and lower body cell mass index (BCMI) (adjusted HR per 1-SD, 0.70; 95% CI, 0.58 to 0.85) were associated with a significantly greater risk of death. When these parameters were added to the fully adjusted model, BCMI performed best in improving the predictability for all-cause mortality (integrated discrimination improvement = 0.02, P = 0.04; net reclassification index = 0.11, P = 0.04). Among body composition indexes, ECW/BCM was the most relevant fluid volume indices to mortality and BCMI and LTI were the most relevant nutritional status indices to mortality in maintenance hemodialysis patients.

NUCB2 polymorphisms are associated with an increased risk for type 2 diabetes in the Chinese population.

BACKGROUND: The nucleobindin 2 (NUCB2) gene encodes the NUCB2 protein, which plays a critical role in glucose metabolism and diabetes. This study explored the correlation between NUCB2 genetic variants and type 2 diabetes mellitus (T2DM). The study further examined the different NUCB2 variants that confer risk to T2DM in Chinese Han populations. METHODS: This study evaluated the anthropometric and glycemic profiles of 578 T2DM patients and 1,609 healthy controls. Subsequently, we genotyped five single nucleotide polymorphisms (SNPs) (rs10832756, rs1330, rs10766383, rs10832757, and rs11024251) in all the study participants using a Sequenom Mass ARRAY SNP genotyping platform. RESULTS: The distribution of polymorphisms was significantly different between the T2DM patients and healthy controls. Our logistic regression analysis results showed that the five NUCB2 SNPs are significantly correlated with the risk for T2DM, especially rs11024251(P=2.97×10-6). Interestingly, analysis of male and female sub-populations separately showed that only two of the SNPs (rs10832757 and rs11024251) have significant correlation to T2DM in males [P=0.0244, odds ratio (OR) 1.28 and P=0.0062, OR 1.35, respectively). In females however, we identified four significant SNPs (rs1330, rs10766383, rs10832757, and rs11024251; P<0.05, OR 1.31-1.42). Furthermore, we found that rs1330 is associated with body mass index of female subpopulation only (P=0.0174, ß =0.0060). CONCLUSIONS: NUCB2 polymorphisms could have a pivotal role in the presence of T2DM. Sex-specific SNPs of NUCB2 could account for the differences in clinical features of T2DM between male and female subpopulations. Nevertheless, our results should be replicated using larger sample sizes, and experimental investigations are needed to elucidate the molecular mechanisms of the associations observed in this study.

Candidate gene associations reveal sex-specific Graves' disease risk alleles among Chinese Han populations.

BACKGROUND: With several susceptibility single nucleotide polymorphisms identified by case-control association studies, Graves' disease is one of the most common forms of autoimmune thyroid disease. In this study, we aimed to determine whether any observed differences in genetic associations are influenced by sex in Chinese Han populations. METHODS: A total of 8,835 patients with Graves' disease and 9,936 sex-matched healthy controls were enrolled in the study. Confirmed by a two-staged association analysis, sex-specific analyses among 20 Graves' disease susceptibility loci were conducted. RESULTS: A significant sex-gene interaction was detected primarily at rs5912838 on Xq21.1 between the GPR174 and ITM2A genes, whereby male Graves' disease patients possessed a significantly higher frequency of risk alleles than their female counterparts. Interestingly, compared to women, male patients with Graves' disease had a higher cumulative genetic risk and higher persistent thyroid stimulating hormone receptor antibody-positive rate after receiving antithyroid drug therapy for at least 1 year. CONCLUSION: The findings of this study suggest the existence of one potential sex-specific Graves' disease variant on Xq21.1. This could increase our understanding of the pivotal mechanism behind Graves' disease and ultimately aid in identifying possible therapeutic targets.

The TPO mutation screening and genotype-phenotype analysis in 230 Chinese patients with congenital hypothyroidism.

Inborn defects in thyroid hormone biosynthesis contribute to nearly half of congenital hypothyroidism (CH) cases in China. The thyroid peroxidase (TPO) mutation is one of the most frequent mutations that results in thyroid dyshormonogenesis. In this study, 35 non-synonymous mutations in 15 TPO sites, including 6 novel mutations, were identified in 230 Chinese patients with CH. The enzyme activity of the mutations in TPO was investigated in vitro, and patients with less than 15% residual enzyme activity showed severe CH, such as markedly increased thyroid-stimulating hormone (TSH) at diagnosis (>100 µIU/mL) and pronounced goiter, and required a higher dose of L-thyroxine to maintain the euthyroid. However, CH patients with greater than 16% TPO activity showed mild CH, a typical childhood socially without L-thyroxine treatment before 3 years of age, and the appearance of a macroscopic goiter at childhood. The findings indicated that the residual enzymatic activity of TPO was correlated with clinical phenotypes of CH patients with TPO biallelic mutations.

Fine mapping of thyroglobulin gene identifies two independent risk loci for Graves' disease in Chinese Han population.

BACKGROUND: This study aimed to determine independent risk loci of Graves' disease (GD) in the thyroglobulin (TG) region. METHODS: In this two-staged association study, a total of 9,757 patients with GD and 10,626 sex-matched controls were recruited from Chinese Han population. Illumina Human660-Quad BeadChips in the discovery stage and TaqMan SNP Genotyping Assays in the replication stage were used for genotyping. Trend test and logistic regression analysis were performed in this association study. RESULTS: In the discovery stage, rs2294025 and rs7005834 were the most highly associated susceptibility loci with GD in TG. In the replication phase, 7 SNPs, including rs2294025 and rs7005834, were selected for fine-mapping. Finally, we confirmed that rs2294025 and rs7005834 were the independent risk loci of GD in the combined populations. At the same time, there was no significant difference between the risk allele frequencies of rs2294025 and rs7005834 in different clinical phenotypes of GD. CONCLUSIONS: The fine mapping study of thyroglobulin identified two independent SNPs (rs2294025 and rs7005834) for GD susceptibility. However, no significant differences for rs2294025 and rs7005834 were observed, between the different clinical phenotypes of GD, including gender, Graves' ophthalmopathy (GO), and serum levels of thyrotropin receptor antibody, thyroid peroxidase antibody, and thyroglobulin antibody. These results provide a deeper understanding of the association mechanism of thyroglobulin and GD risk.

Mutation screening of the TSHR gene in 220 Chinese patients with congenital hypothyroidism.

BACKGROUND: Defects in the human thyroid stimulating hormone receptor (TSHR) gene are reported to be one of the causes of congenital hypothyroidism (CH). We aimed to identify mutations in Chinese patients with CH and analyze the relationships between TSHR phenotypes and clinical phenotypes. METHODS: 220 patients with primary CH were screened for TSHR mutations by performing next-generation sequencing. All the exons and exon-intron boundaries of TSHR were analyzed. The function of 8 mutants in TSHR were further investigated in vitro. RESULTS: Among 220 patients with CH, 15 distinct TSHR mutations were identified in 13 patients (5.91%, 13/220, including our previous reported 110 patients, carried with 10 mutations in 8 patients). We found five distinct mutations in the additional cohort of 110 CH patients and identified 7 mutations (including a novel mutation, p.S567R) were loss-of-function mutations. CONCLUSION: Our study indicated that the prevalence of TSHR mutations was 5.91% among studied Chinese patients with CH. One novel TSHR variant was found and four genetic alterations revealed important role of the Ile216, Ala275, Asn372, Ser567 residues in signaling.

Comparison of different equations for estimated glomerular filtration rate in Han Chinese patients with chronic kidney disease.

AIM: To understand the agreement, precision, and accuracy between other estimated glomerular filtration rate (eGFR) equations and the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) creatinine-cystatin C equation (EPI_Cr_CysC). MATERIALS AND METHODS: We conducted a cross-sectional study of 1,913 CKD patients. The eGFRs were calculated separately by creatinine clearance rate and Cockcroft-Gault equation corrected for standard body surface area (Ccr_BSA and eCcr_BSA); CKD-EPI creatinine equation (EPI_Cr); CKD-EPI cystatin C equation (EPI_CysC); EPI_Cr_CysC equation; Modification of Diet in Renal Disease (MDRD) Study equation with standardized serum creatinine; and full-age spectrum creatinine equation (FAS). The EPI_Cr_CysC equation was used as the reference. RESULTS: When compared with the EPI_Cr_CysC equation, the EPI_Cr equation achieved the highest agreement in eGFRs (Lin's concordance correlation coefficient = 0.936, 95% confidence interval (CI) = 0.930, 0.941). eCcr_BSA and EPI_Cr equations achieved the first and second highest percentage agreement in the accurate classification of CKD stage (72.55 vs. 71.25%). The MDRD equation had minimal bias and was closely followed by the EPI_Cr equation (median difference = -1.3, 95% CI = -2.0, -0.8 vs. median difference = 2.5, 95% CI = 1.7, 3.3 mL/min/1.73m2). The EPI_CysC and EPI_Cr equations achieved the first and second highest precision (interquartile range (IQR) of the difference = 12.2, 95% CI = 11.6, 12.9 vs. IQR of the difference = 15.5, 95% CI = 14.7, 16.3 mL/min/1.73m2). The EPI_Cr and MDRD equations performed similarly and both had the highest accuracy at 30% (1 - P30 = 18.6, 95% CI = 16.9, 20.4 vs. 1 - P30 = 18.6, 95% CI = 16.8, 20.3%). CONCLUSION: For assessment of renal function, the EPI_Cr equation performed the best and remained an acceptable alternative to the EPI_Cr_CysC equation in the absence of cystatin C.
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Preparation of biocompatible wound dressings with long-term antimicrobial activity through covalent bonding of antibiotic agents to natural polymers.

Wound dressings with long-term antimicrobial activity are highly desired for treatment of chronic wound infections. Herein, the sustained antimicrobial wound dressings were developed by using antibiotic agents, ciprofloxacin HCL (CIP) and gentamicin sulfate (GS), covalent bonding to natural polymer matrix composites, carboxymethyl chitosan (CMC) and collagen (COL). By amide bond formation between antibiotic agents and polymer chains, two antimicrobial wound dressings CMC-COL-CIP and CMC-COL-GS were prepared. The presented wound dressings exhibited high water absorption capacity, excellent water vapor transmission rate (WVTR), appropriate mechanical properties, and impressive stability. Cytocompatibility of the dressings was demonstrated by in vitro human skin fibroblast (HSF) cells culture study. The results of in vitro and in vivo studies indicated that the two antimicrobial wound dressings have effective antimicrobial activity and prolonged antimicrobial period. Furthermore, the antimicrobial dressings could promote the wound healing, reepithelialization, collagen deposition, and angiogenesis. It also displays superiority wound healing effects compared to commercially available silver-based dressings (Aguacel Ag). This work indicates that the prepared antimicrobial wound dressings have great potential application in chronic wound healing, such as severe wound cure and diabetic foot ulcers.