Improved Genetic Characterization of Congenital Adrenal Hyperplasia by Long-Read Sequencing Compared with Multiplex Ligation-Dependent Probe Amplification Plus Sanger Sequencing.

Genetic analysis of congenital adrenal hyperplasia (CAH) has been challenging because of high homology between CYP21A2 and its pseudogene CYP21A1P. This study aimed to evaluate the clinical utility of long-read sequencing (LRS) in diagnosis of CAH attributable to 21-hydroxylase deficiency by comparing with multiplex ligation-dependent probe amplification plus Sanger sequencing. In this retrospective study, 69 samples, including 49 probands from 47 families with high-risk of CAH, were enrolled and blindly subjected to detection of CAH by LRS. The genotype results were compared with control methods, and discordant samples were validated by additional Sanger sequencing. LRS successfully identified biallelic variants of CYP21A2 in the 39 probands diagnosed as having CAH. The remaining 10 probands were not patients with CAH. Additionally, LRS directly identified two pathogenic single-nucleotide variations (SNVs; c.293-13C/A>G and c.955C>T) in the presence of interference caused by nearby insertions/deletions (indels). The cis-trans configuration of two or more SNVs and indels identified in 18 samples was directly determined by LRS without family analysis. Eight CYP21A1P/A2 or TNXA/B deletion chimeras, composed of five subtypes, were identified; and the junction sites were precisely determined. Moreover, LRS determined the exact genotype in two probands who had three heterozygous SNVs/indels and duplication, which could not be clarified by control methods. These findings highlight that LRS could assist in more accurate genotype imputation and more precise CAH diagnosis.

d-galactose causes embryonic development arrest and placental development disorders in mice by increasing ROS and inhibiting SIRT1/FOXO3a axis.

INTRODUCTION: Does an elevation in d-Galactose (D-Gal) levels within the body contribute to abnormal embryonic development and placental dysfunction during pregnancy? METHODS: Mouse embryos were cultivated to the blastocyst stage under varying concentrations of D-Gal. The blastocyst formation rate was measured, and the levels of reactive oxygen species (ROS), sirtuin 1 (SIRT1), and forkhead box O3a (FOXO3a) in blastocysts were assessed. Mice were intraperitoneally injected with either saline or D-Gal with or without SRT1720. On the 14th day of pregnancy, the fetal absorption rate and placental weight were recorded. Placental levels of superoxide dismutase (SOD) and malondialdehyde (MDA) were determined. The expression of senescence-related factors, such as senescence-associated ß-galactosidase (SA-ß-gal) in the placenta was examined, and the expression of placental SIRT1, FOXO3a and p21 was evaluated by immunohistochemistry and Western blotting. RESULTS: D-Gal adversely affects early embryonic development in vitro, resulting in a decreased blastocyst formation rate. Furthermore, D-Gal downregulates SIRT1 and FOXO3a while increasing ROS levels in blastocysts. Concurrently, D-Gal induces placental dysfunction, characterized by an elevated fetal absorption rate, reduced placental weight, diminished SOD activity, and increased MDA content. The senescence-related factor SA-ß-gal was detected in the placenta, along with altered expression of placental SIRT1, FOXO3a, and p21. The SIRT1 agonist SRT1720 mitigated this damage by increasing SIRT1 and FOXO3a expression. DISCUSSION: The inhibition of early embryonic development and placental dysfunction induced by D-Gal may be attributed to the dysregulation of SIRT1. Activating SIRT1 emerges as a potentially effective strategy for alleviating the adverse effects of D-Gal exposure.

Effects of phthalates on human chorionic trophoblast cells and mouse embryonic development.

Phthalate exposure is associated with reproductive health, but the mechanism is unclear. This study used human chorionic trophoblast epithelial cells (HTR8/Svneo cells) and mouse embryos as objects aims to explore the effects of phthalate plasticizers on germ cells and fertility and the possible signalling pathways. In the present study, high concentrations of MEHP for 24 h significantly inhibited the proliferation and viability of HTR8/SVneo cells. Compared with the negative control (NC) group, the MEHP medium and high concentration groups promoted the apoptosis of HTR8/SVneo cells and inhibited the cell cycle, HTR8/SVneo cells were blocked in G1/G0 phase and could not enter S phase, and cell meiosis was inhibited. Western blot experiments showed that there was no difference in the protein expression of wnt inhibitory factor 1 (WIF1) and ß-catenin in HTR8/SVneo cells between the MEHP exposure groups and the NC groups. In vitro embryo culture experiments found that there was no difference in blastocyst formation rate among groups after exposure to DEHP for 2 h. Immunofluorescence showed that the expression of WIF1 decreased in the low concentration group, and there was no difference in the medium and high concentration groups, while the expression of ß-catenin was increased in both the low concentration group and the high concentration group. Our data suggest that exposure to phthalate plasticizers can affect the viability, cell cycle and apoptosis of trophoblast cells, resulting in abnormal expression of the embryonic WIF1/ß-catenin signalling pathway and impaired fertility.

SRT1720 plays a role in oxidative stress and the senescence of human trophoblast HTR8/SVneo cells induced by D-galactose through the SIRT1/FOXO3a/ROS signalling pathway.

D-galactose (D-gal) is a reducing sugar widely distributed in food. In a pregnant animal model exposed to D-gal, D-gal was found to have toxic effects on both the mother and foetus through oxidative stress. However, little is known about the effect of D-gal exposure on the placenta and its underlying mechanism. In this study, we evaluated the effects of D-gal on HTR8/SVneo cells and the mechanisms in vitro. In the present study, the activity of HTR8/SVneo human trophoblasts decreased in a time- and concentration-dependent manner after exposure to D-gal. D-gal resulted in premature senescence of HTR8/SVneo cells, as confirmed by assessing ß-galactosidase (SA-ß-gal) activity and the expression of senescence-related factor p21. We also verified the damage of oxidative stress induced by D-gal by measuring the expression of reactive oxygen species (ROS), sirtuin 1 (SIRT1) and forkhead box O (FOXO) 3a. SRT1720, as a SIRT1 activator, mitigated D-gal-induced oxidative stress and senescence by upregulating SIRT1 and FOXO3a expression and reducing ROS production. Our data suggest that D-gal may induce HTR8/SVneo premature ageing through the SIRT1/FOXO3a/ROS signalling pathway mediated by oxidative stress and that SIRT1 protects cells from this damage.

The molecular chaperone Hsp90 maintains Golgi organization and vesicular trafficking by regulating microtubule stability.

Hsp90 is an abundant and special molecular chaperone considered to be the regulator of many transcription factors and signaling kinases. Its high abundance is indicative of its involvement in some more fundamental processes. In this study, we provide evidence that Hsp90 is required for microtubule stabilization, Golgi organization, and vesicular trafficking. We showed that Hsp90 is bound to microtubule-associated protein 4 (MAP4), which is essential for maintaining microtubule acetylation and stabilization. Hsp90 depletion led to the decrease in MAP4, causing microtubule deacetylation and destabilization. Furthermore, in Hsp90-depleted cells, the Golgi apparatus was fragmented and anterograde vesicle trafficking was impaired, with phenotypes similar to those induced by silencing MAP4. These disruptive effects of Hsp90 depletion could be rescued by the expression of exogenous MAP4 or the treatment of trichostatin A that increases microtubule acetylation as well as stability. Thus, microtubule stability is an essential cellular event regulated by Hsp90.

The molecular chaperone Hsp90α deficiency causes retinal degeneration by disrupting Golgi organization and vesicle transportation in photoreceptors.

Heat shock protein 90 (Hsp90) is an abundant molecular chaperone with two isoforms, Hsp90α and Hsp90ß. Hsp90ß deficiency causes embryonic lethality, whereas Hsp90α deficiency causes few abnormities except male sterility. In this paper, we reported that Hsp90α was exclusively expressed in the retina, testis, and brain. Its deficiency caused retinitis pigmentosa (RP), a disease leading to blindness. In Hsp90α-deficient mice, the retina was deteriorated and the outer segment of photoreceptor was deformed. Immunofluorescence staining and electron microscopic analysis revealed disintegrated Golgi and aberrant intersegmental vesicle transportation in Hsp90α-deficient photoreceptors. Proteomic analysis identified microtubule-associated protein 1B (MAP1B) as an Hsp90α-associated protein in photoreceptors. Hspα deficiency increased degradation of MAP1B by inducing its ubiquitination, causing α-tubulin deacetylation and microtubule destabilization. Furthermore, the treatment of wild-type mice with 17-DMAG, an Hsp90 inhibitor of geldanamycin derivative, induced the same retinal degeneration as Hsp90α deficiency. Taken together, the microtubule destabilization could be the underlying reason for Hsp90α deficiency-induced RP.

The basic amino acids in the coiled-coil domain of CIN85 regulate its interaction with c-Cbl and phosphatidic acid during epidermal growth factor receptor (EGFR) endocytosis.

BACKGROUND: During EGFR internalization CIN85 bridges EGFR-Cbl complex, endocytic machinery and fusible membrane through the interactions of CIN85 with c-Cbl, endophilins and phosphatidic acid. These protein-protein and protein-lipid interactions are mediated or regulated by the positively charged C-terminal coiled-coil domain of CIN85. However, the details of CIN85-lipid interaction remain unknown. The present study suggested a possible electric interaction between the negative charge of phosphatidic acid and the positive charge of basic amino acids in coiled-coil domain. RESULTS: Mutations of the basic amino acids in the coiled-coil domain, especially K645, K646, R648 and R650, into neutral amino acid alanine completely blocked the interaction of CIN85 with c-Cbl or phosphatidic acid. However, they did not affect CIN85-endophilin interaction. In addition, CIN85 was found to associate with the internalized EGFR endosomes. It interacted with several ESCRT (Endosomal Sorting Complex Required for Transport) component proteins for ESCRT assembly on endosomal membrane. Mutations in the coiled-coil domain (deletion of the coiled-coil domain or point mutations of the basic amino acids) dissociated CIN85 from endosomes. These mutants bound the ESCRT components in cytoplasm to prevent them from assembly on endosomal membrane and inhibited EGFR sorting for degradation. CONCLUSIONS: As an adaptor protein, CIN85 interacts with variety of partners through several domains. The positive charges of basic amino acids in the coiled-coil domain are not only involved in the interaction with phosphatidic acid, but also regulate the interaction of CIN85 with c-Cbl. CIN85 also interacts with ESCRT components for protein sorting in endosomes. These CIN85-protein and CIN85-lipid interactions enable CIN85 to link EGFR-Cbl endocytic complex with fusible membrane during EGFR endocytosis and subsequently to facilitate ESCRT formation on endosomal membrane for EGFR sorting and degradation.

Berberine promotes the development of atherosclerosis and foam cell formation by inducing scavenger receptor A expression in macrophage.

Berberine is identified to lower the serum cholesterol level in human and hamster through the induction of low density lipoproteins (LDL) receptor in hepatic cells. To evaluate its potential in preventing atherosclerosis, the effect of berberine on atherosclerosis development in apolipoprotein E-deficient (apoE(-/-)) mice was investigated. In apoE(-/-) mice, berberine induced in vivo foam cell formation and promoted atherosclerosis development. The foam cell formation induced by berberine was also observed in mouse RAW264.7 cells, as well as in mouse and human primary macrophages. By inducing scavenger receptor A (SR-A) expression in macrophages, berberine increased the uptake of modified LDL (DiO-Ac-LDL). Berberine-induced SR-A expression was also observed in macrophage foam cells in vivo and in the cells at atherosclerotic lesion. Analysis in RAW264.7 cells indicated that berberine induced SR-A expression by suppressing PTEN expression, which led to sustained Akt activation. Our results suggest that to evaluate the potential of a cholesterol-reducing compound in alleviating atherosclerosis, its effect on the cells involved in atherosclerosis development, such as macrophages, should also be considered. Promotion of foam cell formation could counter-balance the beneficial effect of lowering serum cholesterol.

CIN85 associates with endosomal membrane and binds phosphatidic acid.

CIN85 (Cbl-interacting protein of 85 kDa) is an important molecule involved in receptor tyrosine kinase endocytosis. Here we report that through its positively charged C-terminus, CIN85 associates with a fusogenic lipid - phosphatidic acid. Its coiled-coil domain plays an important role in mediating this protein-lipid interaction. Deletion of the coiled-coil domain results in loss of membrane association, and reduced interaction with c-cbl, finally causing the blockage of epidermal growth factor receptor downregulation. In addition, a significant portion of CIN85 is located on the endosomal compartment and is related to endocytic cargo sorting, characterized by CIN85's localization on the "E class" compartment and EGF degradation blockage in CIN85 knockdown cells. Taken together, our results suggest that CIN85 may function as a scaffold molecule in both the internalization and endocytic cargo sorting processes through its association with the endosomal membrane.

Association of Dnmt3a and thymine DNA glycosylase links DNA methylation with base-excision repair.

While methylcytosines serve as the fifth base encoding epigenetic information, they are also a dangerous endogenous mutagen due to their intrinsic instability. Methylcytosine undergoes spontaneous deamination, at a rate much higher than cytosine, to generate thymine. In mammals, two repair enzymes, thymine DNA glycosylase (TDG) and methyl-CpG binding domain 4 (MBD4), have evolved to counteract the mutagenic effect of methylcytosines. Both recognize G/T mismatches arising from methylcytosine deamination and initiate base-excision repair that corrects them to G/C pairs. However, the mechanism by which the methylation status of the repaired cytosines is restored has remained unknown. We show here that the DNA methyltransferase Dnmt3a interacts with TDG. Both the PWWP domain and the catalytic domain of Dnmt3a are able to mediate the interaction with TDG at its N-terminus. The interaction affects the enzymatic activity of both proteins: Dnmt3a positively regulates the glycosylase activity of TDG, while TDG inhibits the methylation activity of Dnmt3a in vitro. These data suggest a mechanistic link between DNA repair and remethylation at sites affected by methylcytosine deamination.