Selenium represses microRNA-202-5p/MICU1 aixs to attenuate mercuric chloride-induced kidney ferroptosis.

Mercuric chloride (HgCl2) is a nephrotoxic contaminant that is widely present in the environment. Selenium (Se) can effectively antagonize the biological toxicity caused by heavy metals. Here, in vivo and in vitro models of Se antagonism to HgCl2-induced nephrotoxicity in chickens were established, with the aim of exploring the specific mechanism. Morphological observation and kidney function analysis showed that Se alleviated HgCl2-induced kidney tissue injury and cytotoxicity. The results showed that ferroptosis was the primary mechanism for the toxicity of HgCl2, as indicated by iron overload and lipid peroxidation. On the one hand, Se significantly prevented HgCl2-induced iron overload. On the other hand, Se alleviated the intracellular reactive oxygen species (ROS) levels caused by HgCl2. Subsequently, we focused on the sources of ROS during HgCl2-induced ferroptosis. Mechanically, Se reduced ROS overproduction induced by HgCl2 through mitochondrial calcium uniporter (MCU)/mitochondrial calcium uptake 1 (MICU1)-mediated mitochondrial calcium ion (Ca2+) overload. Furthermore, a dual luciferase reporter assay demonstrated that MICU1 was the direct target of miR-202-5p. Overall, Se represses miR-202-5p/MICU1 axis to attenuate HgCl2-induced kidney ferroptosis.

Dietary Iron Overload Triggers Hepatic Metabolic Disorders and Inflammation in Laying Hen.

Iron, an essential trace element, is involved in various physiological processes; however, consumption of excessive iron possesses detrimental effects. In practical feed production, the iron content added to feeds often far exceeds the actual demand, resulting in an excess of iron in the body. The liver as a central regulator of iron homeostasis is susceptible to damage caused by disorders in iron metabolism. A model of hepatic iron overload in laying hens was developed in this study by incorporating iron into their diet, and the specific mechanisms underlying iron overload-induced hepatic injury were investigated. Firstly, this study revealed that a high-iron diet resulted in hepatic iron overload, accompanied by impaired liver function. Next, assessment of oxidative stress markers indicated a decrease in activities of T-SOD and CAT, coupled with an increase in MDA content, pointing to the iron-overloaded liver oxidative stress. Thirdly, the impact of iron overload on hepatic glycolipid and bile acid metabolism-related gene expressions were explored, including PPAR-α, GLUT2, and CYP7A1, highlighting disruptions in hepatic metabolism. Subsequently, analyses of inflammation-related genes such as iNOS and IL-1ß at both protein and mRNA levels demonstrated the presence of inflammation in the liver under conditions of dietary iron overload. Overall, this study provided comprehensive evidence that dietary iron overload contributed to disorders in glycolipid and bile acid metabolism, accompanied by inflammatory responses in laying hens. Further detailing the specific pathways involved and the implications of these findings could offer valuable insights for future research and practical applications in poultry nutrition.

miR-15b-5p promotes HgCl2-induced chicken embryo kidney cells ferroptosis by targeting ß-TrCP-mediated ATF4 ubiquitin degradation.

Mercuric chloride (HgCl2), a widespread environmental pollutant, induces ferroptosis in chicken embryonic kidney (CEK) cells. Whereas activating transcription factor 4 (ATF4), a critical mediator of oxidative homeostasis, plays a dual role in ferroptosis, but its precise mechanisms in HgCl2-induced ferroptosis remain elusive. This study aims to investigate the function and molecular mechanism of ATF4 in HgCl2-induced ferroptosis. Our results revealed that ATF4 was downregulated during HgCl2-induced ferroptosis in CEK cells. Surprisingly, HgCl2 exposure has no significant impact on ATF4 mRNA level. Further investigation indicated that HgCl2 enhanced the expression of the E3 ligase beta-transducin repeat-containing protein (ß-TrCP) and increased ATF4 ubiquitination. Subsequent findings identified that miR-15b-5p as an upstream modulator of ß-TrCP, with miR-15b-5p downregulation observed in HgCl2-exposed CEK cells. Importantly, miR-15b-5p mimics suppressed ß-TrCP expression and reversed HgCl2-induced cellular ferroptosis. Mechanistically, HgCl2 inhibited miR-15b-5p, and promoted ß-TrCP-mediated ubiquitin degradation of ATF4, thereby inhibited the expression of antioxidant-related target genes and promoted ferroptosis. In conclusion, our study highlighted the crucial role of the miR-15b-5p/ß-TrCP/ATF4 axis in HgCl2-induced nephrotoxicity, offering a new therapeutic target for understanding the mechanism of HgCl2 nephrotoxicity.

How human genetic context can inform pathogenicity classification: FGFR1 variation in idiopathic hypogonadotropic hypogonadism.

Precision medicine requires precise genetic variant interpretation, yet many disease-associated genes have unresolved variants of unknown significance (VUS). We analyzed variants in a well-studied gene, FGFR1, a common cause of Idiopathic Hypogonadotropic Hypogonadism (IHH) and examined whether regional genetic enrichment of missense variants could improve variant classification. FGFR1 rare sequence variants (RSVs) were examined in a large cohort to (i) define regional genetic enrichment, (ii) determine pathogenicity based on the American College of Medical Genetics/Association for Molecular Pathology (ACMG/AMP) variant classification framework, and (iii) characterize the phenotype of FGFR1 variant carriers by variant classification. A total of 143 FGFR1 RSVs were identified in 175 IHH probands (n = 95 missense, n = 48 protein-truncating variants). FGFR1 missense RSVs showed regional enrichment across biologically well-defined domains: D1, D2, D3, and TK domains and linker regions (D2-D3, TM-TK). Using these defined regions of enrichment to augment the ACMG/AMP classification reclassifies 37% (20/54) of FGFR1 missense VUS as pathogenic or likely pathogenic (PLP). Non-proband carriers of FGFR1 missense VUS variants that were reclassified as PLP were more likely to express IHH or IHH-associated phenotypes [anosmia or delayed puberty] than non-proband carriers of FGFR1 missense variants that remained as VUS (76.9% vs 34.7%, p = 0.035). Using the largest cohort of FGFR1 variant carriers, we show that integration of regional genetic enrichment as moderate evidence for pathogenicity improves the classification of VUS and that reclassified variants correlated with phenotypic expressivity. The addition of regional genetic enrichment to the ACMG/AMP guidelines may improve clinical variant interpretation.

Selenoprotein K knockdown induces apoptosis in skeletal muscle satellite cells via calcium dyshomeostasis-mediated endoplasmic reticulum stress.

Skeletal muscle satellite cells (SMSCs), known as muscle stem cells, play an important role in muscle embryonic development, post-birth growth, and regeneration after injury. Selenoprotein K (SELENOK), an endoplasmic reticulum (ER) resident selenoprotein, is known to regulate calcium ion (Ca2+) flux and ER stress (ERS). SELENOK deficiency is involved in dietary selenium deficiency-induced muscle injury, but the regulatory mechanisms of SELENOK in SMSCs development remain poorly explored in chicken. Here, we established a SELENOK deficient model to explore the role of SELENOK in SMSCs. SELENOK knockdown inhibited SMSCs proliferation and differentiation by regulating the protein levels of paired box 7 (Pax7), myogenic factor 5 (Myf5), CyclinD1, myogenic differentiation (MyoD), and Myf6. Further analysis exhibited that SELENOK knockdown markedly activated the ERS signaling pathways, which ultimately induced apoptosis in SMSCs. SELENOK knockdown-induced ERS is related with ER Ca2+ ([Ca2+]ER) overload via decreasing the protein levels of STIM2, Orai1, palmitoylation of inositol 1,4,5-trisphosphate receptor 1 (IP3R1), phospholamban (PLN), and plasma membrane Ca2+-ATPase (PMCA) while increasing the protein levels of sarco/endoplasmic Ca2+-ATPase 1 (SERCA1) and Na+/Ca2+ exchanger 1 (NCX1). Moreover, thimerosal, an activator of IP3R1, reversed the overload of [Ca2+]ER, ERS, and subsequent apoptosis caused by SELENOK knockdown. These findings indicated that SELENOK knockdown triggered ERS driven by intracellular Ca2+ dyshomeostasis and further induced apoptosis, which ultimately inhibited SMSCs proliferation and differentiation.

Metabolic Syndrome Rather Than Other Phenotypes in PCOS as a Predictive Indicator for Clinical Outcomes in IVF: Comprehensive Phenotypic Assessment across All PCOS Classifications.

Polycystic ovary syndrome (PCOS) is a well-recognized, multi-system metabolic disorder affecting fertility. Although various classification methods have been proposed to assess the phenotypic heterogeneity of PCOS, there is currently no reliable phenotype for predicting clinical IVF outcomes. This retrospective study, as a comprehensive phenotypic assessment across all PCOS classifications, aimed to identify dependable phenotypes that can serve as predictors for IVF and pregnancy outcomes. The study included 1313 PCOS patients who received their initial IVF treatment between January 2019 and December 2021. The phenotypes reflect the diverse metabolic and hormonal characteristics in this study. Phenotype A, within the Rotterdam criteria classification, exhibited the highest anti-Müllerian hormone levels (AMH), while phenotype D displayed the lowest Homeostasis Model Assessment of Insulin Resistance (HOMA-IR) values. Both the hyperandrogenism (HA) phenotype within HA-based classification and the overweight phenotype within the body-mass-index-based classification showed increased HOMA-IR and metabolic syndrome (MetS). The MetS phenotype had higher free androgen index and a lower AMH. Notably, the MetS-based classification system demonstrated an independent association of MetS with cumulative live birth, preterm birth, and gestational diabetes mellitus as a contributing risk factor for PCOS patients undergoing IVF (p < 0.05). These findings carry noteworthy implications for advancing clinical management strategies for PCOS.

Whole exome sequencing reveals novel variants associated with diminished ovarian reserve in young women.

Background: Diminished ovarian reserve is one of the most important causes of female infertility. In the etiology study of DOR, besides age, it is known that chromosomal abnormality, radiotherapy, chemotherapy and ovarian surgery can result in DOR. For young women without obvious risk factors, gene mutation should be considered as a possible cause. However, the specific molecular mechanism of DOR has not been fully elucidated. Methods: In order to explore the pathogenic variants related to DOR, twenty young women under 35 years old affected by DOR without definite factors damaging ovarian reserve were recruited as the research subjects, and five women with normal ovarian reserve were recruited as the control group. Whole exome sequencing was applied as the genomics research tool. Results: As a result, we obtained a set of mutated genes that may be related to DOR, where the missense variant on GPR84 was selected for further study. It is found that GPR84Y370H variant promotes the expression of proinflammatory cytokines (TNF-α, IL12B, IL-1ß) and chemokines (CCL2, CCL5), as well as the activation of NF-κB signaling pathway. Conclusion: In conclusion, GPR84Y370H variant was identified though analysis for WES results of 20 DOR patients. The deleterious variant of GPR84 could be the potential molecular mechanism of non-age-related pathological DOR through its role in promoting inflammation. The findings of this study can be used as a preliminary research basis for the development of early molecular diagnosis and treatment target selection of DOR.

ART outcomes of patients in women with Isolated Hypogonadotropic Hypogonadism: a retrospective study in China.

BACKGROUND: Isolated Hypogonadotropic Hypogonadism (IHH) is a rare reproductive disorder caused by the dysfunction of the gonadotropin-releasing hormone axis. Patients with IHH typically fail to enter or develop through puberty and retain infertile without an exogenous hormone supplement. This study aimed to investigate the population characteristics and reproductive outcomes in IHH patients undergoing assisted reproductive technology (ART) treatment, and evaluate the best-performed predictor for ovarian response and clinical pregnancy in patients with IHH. METHODS: This retrospective cohort study included 83 women with IHH who underwent fresh ART cycles and non-diagnosed controls (n = 676). The receiver operating characteristic curves were generated to assess the predictor for the ovarian response. Logistic regression analyses were performed to investigate the independent factors for clinical pregnancy in IHH. RESULTS: The basal hormone levels were significantly lower in the IHH group compared to the control group. The fertilization rate and 2PN rate were significantly higher in IHH groups, as was the number of transferable embryos. The study identified that AMH was the best predictor of high ovarian response in IHH, with an AUC of 0.767 (0.573, 0.961). Conversely, the follicle-to-oocyte index (FOI) exhibited the highest AUC of 0.814 (0.642, 0.985) for predicting low ovarian response. Based on FOI values, the IHH patients were divided into two groups, and the study found a significant increase in clinical pregnancy rate (43.8%, 58%; P < 0.001) and live birth rate (37.5%, 58%; P < 0.001) from the low FOI to the normal FOI groups. Moreover, the number of oocytes retrieved, fertilized embryos/rate, 2PN embryos/rate, and number of excellent quality embryos were significantly higher in the normal FOI group (P < 0.001 or P = 0.005) than in the low FOI group. Logistic regression analyses revealed FOI to be the independent factor affecting clinical pregnancy in IHH patients. CONCLUSIONS: The study findings suggest that patients with IHH were good responders to IVF treatment. Although AMH was the best-performed predictor for the high ovarian response, FOI had the best capability in predicting the low ovarian response. FOI was an independent factor affecting clinical pregnancy in IHH undergoing IVF/ICSI.

The p190 RhoGAPs, ARHGAP35, and ARHGAP5 are implicated in GnRH neuronal development: Evidence from patients with idiopathic hypogonadotropic hypogonadism, zebrafish, and in vitro GAP activity assay.

PURPOSE: The study aimed to identify novel genes for idiopathic hypogonadotropic hypogonadism (IHH). METHODS: A cohort of 1387 probands with IHH underwent exome sequencing and de novo, familial, and cohort-wide investigations. Functional studies were performed on 2 p190 Rho GTPase-activating proteins (p190 RhoGAP), ARHGAP35 and ARHGAP5, which involved in vivo modeling in larval zebrafish and an in vitro p190A-GAP activity assay. RESULTS: Rare protein-truncating variants (PTVs; n = 5) and missense variants in the RhoGAP domain (n = 7) in ARHGAP35 were identified in IHH cases (rare variant enrichment: PTV [unadjusted P = 3.1E-06] and missense [adjusted P = 4.9E-03] vs controls). Zebrafish modeling using gnrh3:egfp phenotype assessment showed that mutant larvae with deficient arhgap35a, the predominant ARHGAP35 paralog in the zebrafish brain, display decreased GnRH3-GFP+ neuronal area, a readout for IHH. In vitro GAP activity studies showed that 1 rare missense variant [ARHGAP35 p.(Arg1284Trp)] had decreased GAP activity. Rare PTVs (n = 2) also were discovered in ARHGAP5, a paralog of ARHGAP35; however, arhgap5 zebrafish mutants did not display significant GnRH3-GFP+ abnormalities. CONCLUSION: This study identified ARHGAP35 as a new autosomal dominant genetic driver for IHH and ARHGAP5 as a candidate gene for IHH. These observations suggest a novel role for the p190 RhoGAP proteins in GnRH neuronal development and integrity.

Ovarian aging: mechanisms and intervention strategies.

Ovarian reserve is essential for fertility and influences healthy aging in women. Advanced maternal age correlates with the progressive loss of both the quantity and quality of oocytes. The molecular mechanisms and various contributing factors underlying ovarian aging have been uncovered. In this review, we highlight some of critical factors that impact oocyte quantity and quality during aging. Germ cell and follicle reserve at birth determines reproductive lifespan and timing the menopause in female mammals. Accelerated diminishing ovarian reserve leads to premature ovarian aging or insufficiency. Poor oocyte quality with increasing age could result from chromosomal cohesion deterioration and misaligned chromosomes, telomere shortening, DNA damage and associated genetic mutations, oxidative stress, mitochondrial dysfunction and epigenetic alteration. We also discuss the intervention strategies to delay ovarian aging. Both the efficacy of senotherapies by antioxidants against reproductive aging and mitochondrial therapy are discussed. Functional oocytes and ovarioids could be rejuvenated from pluripotent stem cells or somatic cells. We propose directions for future interventions. As couples increasingly begin delaying parenthood in life worldwide, understanding the molecular mechanisms during female reproductive aging and potential intervention strategies could benefit women in making earlier choices about their reproductive health.

Cytotoxic lymphocytes-related gene ITK from a systematic CRISPR screen could predict prognosis of ovarian cancer patients with distant metastasis.

BACKGROUND: Ovarian cancer, a highly metastatic malignancy, has benefited tremendously from advances in modern human genomics. However, the genomic variations related to the metastasis remains unclear. METHODS: We filtered various significant genes (n = 6722) associated with metastasis within a large-scale functional genomic CRISPR/Cas9 knock-out library including 122,756 single guide RNAs, and identified ITK (IL2 Inducible T Cell Kinase) as a potential cancer suppressor gene for ovarian cancer metastasis. Downstream bioinformatic analysis was performed for ITK using public databases. RESULTS: We found that patients in low-ITK group had poor prognosis and more distant metastasis than those in high-ITK group in TCGA and GEO databases. We also demonstrated that ITK combined with the clinical factors could accurately predict prognosis through multiple Cox regression analysis and ROC analysis. Moreover, alterations correlated with distant metastasis emereged with significantly increased expression in SAMRCD1 in low-ITK group, but CD244 and SOCS1 in high-ITK group. Integrated analysis revealed dysregulated molecular processes including predominantly oncogenic signaling pathways in low-ITK group but immune related pathways in high-ITK group, which suggested ITK might inhibit distant metastasis in ovarian cancer. Furtherly, deconvolution of the cellular composition of all samples validated the close correlation between ITK and immune related function especially for cytotoxic lymphocytes. CONCLUSIONS: Together, these data provide insights into the potential role of ITK, with implications for the future development of tansformative ovarian cancer therapeutics.

Comprehensive Evaluation of Differential Methylation Analysis Methods for Bisulfite Sequencing Data.

Background: With advances in next-generation sequencing technologies, the bisulfite conversion of genomic DNA followed by sequencing has become the predominant technique for quantifying genome-wide DNA methylation at single-base resolution. A large number of computational approaches are available in literature for identifying differentially methylated regions in bisulfite sequencing data, and more are being developed continuously. Results: Here, we focused on a comprehensive evaluation of commonly used differential methylation analysis methods and describe the potential strengths and limitations of each method. We found that there are large differences among methods, and no single method consistently ranked first in all benchmarking. Moreover, smoothing seemed not to improve the performance greatly, and a small number of replicates created more difficulties in the computational analysis of BS-seq data than low sequencing depth. Conclusions: Data analysis and interpretation should be performed with great care, especially when the number of replicates or sequencing depth is limited.

Long-term and large-scale spatiotemporal patterns of soundscape in a tropical habitat of the Indo-Pacific humpback dolphin (Sousa chinensis).

Little is known about the characteristics of ambient sound in shallow waters southwest of Hainan Island, China, a tropical habitat of the Indo-Pacific humpback dolphin. The spatiotemporal patterns of soundscape in this area were thus studied and described here. Acoustic data collected from February 2018 to February 2019 at ten monitoring sites, spanning ~200 km of the coastline, were analyzed. The ambient sound characteristics in the investigated area showed significant spatiotemporal variations. Sound levels centered at 0.5 and 1 kHz were higher during dusk and night than other times of the day at all monitoring sites except for one. Higher sound levels at frequencies above 8 kHz were documented during autumn and winter at all sites except for three of them. Biological and anthropogenic sound sources including soniferous fishes, snapping shrimps, dolphins, ships, pile-driving activities, and explosions were identified during spectrogram analyses of a subsample of the dataset. The shipping noise was frequently detected throughout the monitoring sites. Spatiotemporal variations of the soundscape in the investigated waters provided baseline information on the local marine environment, which will be beneficial to the protection of the vulnerable Indo-Pacific humpback dolphin population recently discovered in the investigated waters.

Influence of acoustic habitat variation on Indo-Pacific humpback dolphin (Sousa chinensis) in shallow waters of Hainan Island, China.

The Indo-Pacific humpback dolphin (IPHD, Sousa chinensis) is a coastal species inhabiting tropical and warm-temperate waters. The presence of this vulnerable dolphin was recently discovered in shallow waters southwest of Hainan Island, China. The influence of the acoustic habitat on the distribution and behavior of IPHD was investigated using an array of passive acoustic platforms (n = 6) that spanned more than 100 km of coastline during a 75-day monitoring period. Its presence was assessed within 19 215 five-min recordings by classifying echolocation clicks using machine learning techniques. Spectrogram analysis was applied to further investigate the acoustic behavior of IPHD and to identify other prominent sound sources. The variation in the ambient noise levels was also measured to describe the spatiotemporal patterns of the acoustic habitat among the different sampling sites. Social and feeding sounds of IPHD (whistles and click-series of pulsed sounds) were identified together with other biological sources (finless porpoise, soniferous fishes, and snapping shrimps) and anthropogenic activities (ship noise, explosions, and sonars). Distribution, acoustic behavior, and habitat use of this nearshore dolphin species were strongly influenced by the abundance of soniferous fishes, and under similar conditions, the species was more acoustically active in locations with lower noise levels.

Hypogonadotropic hypogonadism due to variants in RAB3GAP2: expanding the phenotypic and genotypic spectrum of Martsolf syndrome.

Biallelic pathogenic variants in RAB3GAP2 cause Warburg Micro syndrome (WARBM) and Martsolf syndrome (MS), two rare, phenotypically overlapping disorders characterized by congenital cataracts, intellectual disability, and hypogonadism. Although the initial report documented hypergonadotropic hypogonadism (implying a gonadal defect), an adolescent girl with WARBM/MS was subsequently reported to have hypogonadotropic hypogonadism (implying a central defect in either the hypothalamus or anterior pituitary). However, in adult MS, hypogonadotropism has not been convincingly demonstrated. Additionally, the correlation between the pathogenic severity of variants in RAB3GAP2 and the phenotypic severity also remains unclear. Here we present a clinical report of a woman with congenital cataracts, apparent intellectual disability, and pubertal failure who underwent exome sequencing (ES) to determine a precise molecular diagnosis. Reproductive phenotypes reported previously in individuals with MS and the genotypic spectrum of previous RAB3GAP2 variants were also reviewed. The ES identified pathogenic compound heterozygous RAB3GAP2 variants (c.387-2A > G; p.(Arg428Glu)) combined with her phenotypic features, which enabled a unifying molecular diagnosis of MS. Reproductive evaluation confirmed a normosmic idiopathic hypogonadotropic hypogonadism. Review of the RAB3GAP2 allelic spectrum in WARBM/MS suggests that although variants resulting in complete abrogation of RAB3GAP2 protein function cause severe WARBM, variants associated with partially preserved RAB3GAP2 function cause milder MS. This report expands the genotypic and phenotypic spectrum of MS and demonstrates hypogonadotropic hypogonadism as a key pathophysiologic abnormality in MS. Genotype-phenotype associations of previously reported RAB3GAP2 variants indicate that variants that fully abolish RAB3GAP2 function result in WARBM, whereas MS is associated with variants of lesser severity with residual RAB3GAP2 function.

Integrative analysis of DNA methylation and gene expression identified cervical cancer-specific diagnostic biomarkers.

Cervical cancer is the leading cause of death among women with cancer worldwide. Here, we performed an integrative analysis of Illumina HumanMethylation450K and RNA-seq data from TCGA to identify cervical cancer-specific DNA methylation markers. We first identified differentially methylated and expressed genes and examined the correlation between DNA methylation and gene expression. The DNA methylation profiles of 12 types of cancers, including cervical cancer, were used to generate a candidate set, and machine-learning techniques were adopted to define the final cervical cancer-specific markers in the candidate set. Then, we assessed the protein levels of marker genes by immunohistochemistry by using tissue arrays containing 93 human cervical squamous cell carcinoma samples and cancer-adjacent normal tissues. Promoter methylation was negatively correlated with the local regulation of gene expression. In the distant regulation of gene expression, the methylation of hypermethylated genes was more likely to be negatively correlated with gene expression, while the methylation of hypomethylated genes was more likely to be positively correlated with gene expression. Moreover, we identified four cervical cancer-specific methylation markers, cg07211381 (RAB3C), cg12205729 (GABRA2), cg20708961 (ZNF257), and cg26490054 (SLC5A8), with 96.2% sensitivity and 95.2% specificity by using the tenfold cross-validation of TCGA data. The four markers could distinguish tumors from normal tissues with a 94.2, 100, 100, and 100% AUC in four independent validation sets from the GEO database. Overall, our study demonstrates the potential use of methylation markers in cervical cancer diagnosis and may boost the development of new epigenetic therapies.