Dysanapsis Genetic Risk Predicts Lung Function Across the Lifespan.

Rationale Dysanapsis refers to a mismatch between airway tree caliber and lung size arising early in life. Dysanapsis assessed by computed tomography (CT) is evident by early adulthood and associated with chronic obstructive pulmonary disease (COPD) risk later in life. Objective By examining the genetic factors associated with CT-assessed dysanapsis, we aimed to elucidate its molecular underpinnings and physiological significance across the lifespan. Methods We performed a genome-wide association study (GWAS) of CT-assessed dysanapsis in 11,951 adults, including individuals from two population-based and two COPD-enriched studies. We applied colocalization analysis to integrate GWAS and gene expression data from whole blood and lung. Genetic variants associated with dysanapsis were combined into a genetic risk score that was applied to examine association with lung function in children from a population-based birth cohort (n=1,278) and adults from the UK Biobank (n=369,157). Measurements and Main Results CT-assessed dysanapsis was associated with genetic variants from 21 independent signals in 19 gene regions, implicating HHIP, DSP, and NPNT as potential molecular targets based on colocalization of their expression. Higher dysanapsis genetic risk score was associated with obstructive spirometry among 5 year old children and among adults in the 5th, 6th and 7th decades of life. Conclusions CT-assessed dysanapsis is associated with variation in genes previously implicated in lung development and dysanapsis genetic risk is associated with obstructive lung function from early life through older adulthood. Dysanapsis may represent an endo-phenotype link between the genetic variations associated with lung function and COPD.

Lysine acetyltransferase 14 mediates TGF-ß-induced fibrosis in ovarian endometrioma via co-operation with serum response factor.

BACKGROUND: Fibrogenesis within ovarian endometrioma (endometrioma), mainly induced by transforming growth factor-ß (TGF-ß), is characterized by myofibroblast over-activation and excessive extracellular matrix (ECM) deposition, contributing to endometrioma-associated symptoms such as infertility by impairing ovarian reserve and oocyte quality. However, the precise molecular mechanisms that underpin the endometrioma- associated fibrosis progression induced by TGF-ß remain poorly understood. METHODS: The expression level of lysine acetyltransferase 14 (KAT14) was validated in endometrium biopsies from patients with endometrioma and healthy controls, and the transcription level of KAT14 was further confirmed by analyzing a published single-cell transcriptome (scRNA-seq) dataset of endometriosis. We used overexpression, knockout, and knockdown approaches in immortalized human endometrial stromal cells (HESCs) or human primary ectopic endometrial stromal cells (EcESCs) to determine the role of KAT14 in TGF-ß-induced fibrosis. Furthermore, an adeno-associated virus (AAV) carrying KAT14-shRNA was used in an endometriosis mice model to assess the role of KAT14 in vivo. RESULTS: KAT14 was upregulated in ectopic lesions from endometrioma patients and predominantly expressed in activated fibroblasts. In vitro studies showed that KAT14 overexpression significantly promoted a TGF-ß-induced profibrotic response in endometrial stromal cells, while KAT14 silencing showed adverse effects that could be rescued by KAT14 re-enhancement. In vivo, Kat14 knockdown ameliorated fibrosis in the ectopic lesions of the endometriosis mouse model. Mechanistically, we showed that KAT14 directly interacted with serum response factor (SRF) to promote the expression of α-smooth muscle actin (α-SMA) by increasing histone H4 acetylation at promoter regions; this is necessary for TGF-ß-induced ECM production and myofibroblast differentiation. In addition, the knockdown or pharmacological inhibition of SRF significantly attenuated KAT14-mediating profibrotic effects under TGF-ß treatment. Notably, the KAT14/SRF complex was abundant in endometrioma samples and positively correlated with α-SMA expression, further supporting the key role of KAT14/SRF complex in the progression of endometrioma-associated fibrogenesis. CONCLUSION: Our results shed light on KAT14 as a key effector of TGF-ß-induced ECM production and myofibroblast differentiation in EcESCs by promoting histone H4 acetylation via co-operating with SRF, representing a potential therapeutic target for endometrioma-associated fibrosis.

Variant analysis and PGT-M of OTC gene in a Chinese family with ornithine carbamoyltransferase deficiency.

BACKGROUND: Ornithine carbamoyltransferase deficiency (OTCD) is a kind of X-linked metabolic disease caused by a deficiency in ornithine transcarbamylase leading to urea cycle disorders. The main reason is that the OTC gene variants lead to the loss or decrease of OTC enzyme function, which hinders the ammonia conversion to urea, resulting in hyperammonemia and severe neurological dysfunction. Here, we studied one Chinese family of three generations who consecutively gave birth to two babies with OTCD. This study aims to explore the pathogenicity of two missense variants in the OTC gene and investigate the application of preimplantation genetic testing for monogenic (PGT-M) for a family troubled by Ornithine carbamoyltransferase deficiency (OTCD). METHODS: The retrospective method was used to classify the pathogenicity of two missense variants in the OTC gene in a family tortured by OTCD. Sanger sequencing was used to validate the variants in the OTC gene, and then the pathogenicity of variants was confirmed through family analysis and bioinformatics software. We used PGT-M to target the OTC gene and select a suitable embryo for transplantation. Prenatal diagnosis was recommended to confirm previous results using Sanger sequencing and karyotyping at an appropriate gestational stage. Tandem mass spectrometry (MS-MS) and gas chromatography-mass spectrometry (GC-MS) were used to detect fetal metabolism after birth. The number of the study cohort is ChiCTR2100053616. RESULTS: Two missense variants, c.959G > C (p.Arg320Pro) and c.634G > A (p.Gly212Arg), were validated in the OTC gene in this family. According to the ACMG genetic variation classification criteria, the missense variant c.959G > C can be considered as "pathogenic", and the missense variant c.634G > A can be regarded as "likely benign." PGT-M identified a female embryo carrying the heterozygous variant c.959G > C (p.Arg320Pro), which was selected for transplantation. Prenatal diagnosis revealed the same variant in the fetus, and continued pregnancy was recommended. A female baby was born, and her blood amino acid testing and urine organic acid testing were regular. Follow-up was conducted after six months and indicated the girl was healthy. CONCLUSION: Our research first validated the segregation of both c.959G > C and c.634G > A variants in the OTC gene in a Chinese OTCD family. Then, we classified variant c.959G > C as "pathogenic" and variant c.634G > A as "likely benign", providing corresponding theoretical support for genetic counseling and fertility guidance in this family. PGT-M and prenatal diagnosis were recommended to help the couple receive a female baby successfully with a six-month follow-up.

Detection of AZF microdeletions and analysis of reproductive hormonal profiles in Hainan men undergoing assisted reproductive technology.

BACKGROUND: Male infertility has become a global health problem, and genetic factors are one of the essential causes. Y chromosome microdeletion is the leading genetic factor cause of male infertility. The objective of this study is to investigate the correlation between male infertility and Y chromosome microdeletions in Hainan, the sole tropical island province of China. METHODS: We analyzed the semen of 897 infertile men from Hainan in this study. Semen analysis was measured according to WHO criteria by professionals at the Department of Reproductive Medicine, the First Affiliated Hospital of Hainan Medical University, where samples were collected. Y chromosome AZF microdeletions were confirmed by detecting six STS markers using multiple polymerase chain reactions on peripheral blood DNA. The levels of reproductive hormones, including FSH, LH, PRL, T, and E2, were quantified using the enzyme-linked immunosorbent assay (ELISA). RESULTS: The incidence of Y chromosome microdeletion in Hainan infertile men was 7.13%. The occurrence rate of Y chromosome microdeletion was 6.69% (34/508) in the oligozoospermia group and 7.71% (30/389) in the azoospermia group. The deletion of various types in the AZF subregion was observed in the group with azoospermia, whereas no AZFb deletion was detected in the oligozoospermia group. Among all patients with microdeletions, the deletion rate of the AZFc region was the higher at 68.75% (44 out of 64), followed by a deletion rate of 6.25% (4 out of 64) for the AZFa region and a deletion rate of 4.69% (3 out of 64) for the AZFb region. The deletion rate of the AZFa region was significantly higher in patients with azoospermia than in patients with oligozoospermia (0.51% vs. 0.39%, p < 0.001). In comparison, the deletion rate of the AZFc region was significantly higher in patients with oligozoospermia (3.08% vs. 6.30%, p < 0.001). Additionally, the AZFb + c subregion association deletion was observed in the highest proportion among all patients (0.89%, 8/897), followed by AZFa + b + c deletion (0.56%, 5/897), and exclusively occurred in patients with azoospermia. Hormone analysis revealed FSH (21.63 ± 2.01 U/L vs. 10.15 ± 0.96 U/L, p = 0.001), LH (8.96 ± 0.90 U/L vs. 4.58 ± 0.42 U/L, p < 0.001) and PRL (263.45 ± 21.84 mIU/L vs. 170.76 ± 17.10 mIU/L, p = 0.002) were significantly increased in azoospermia patients with microdeletions. Still, P and E2 levels were not significantly different between the two groups. CONCLUSIONS: The incidence of AZF microdeletion can reach 7.13% in infertile men in Hainan province, and the deletion of the AZFc subregion is the highest. Although the Y chromosome microdeletion rate is distinct in different regions or populations, the regions mentioned above of the Y chromosome may serve an indispensable role in regulating spermatogenesis. The analysis of Y chromosome microdeletion plays a crucial role in the clinical assessment and diagnosis of male infertility.

Pan-cancer assessment of mutational landscape in intrinsically disordered hotspots reveals potential driver genes.

Large-scale cancer genome sequencing has enabled the catalogs of somatic mutations; however, the mutational impact on intrinsically disordered protein regions (IDRs) has not been systematically investigated to date. Here, we comprehensively characterized the mutational landscapes of IDRs and found that IDRs have higher mutation frequencies across diverse cancers. We thus developed a computational method, ROI-Driver, to identify putative driver genes enriching IDR and domain hotspots in cancer. Numerous well-known cancer-related oncogenes or tumor suppressors that play important roles in cancer signaling regulation, development and immune response were identified at a higher resolution. In particular, the incorporation of IDR structures helps in the identification of novel potential driver genes that play central roles in human protein-protein interaction networks. Interestingly, we found that the putative driver genes with IDR hotspots were significantly enriched with predicted phase separation propensities, suggesting that IDR mutations disrupt phase separation in key cellular pathways. We also identified an appreciable number of clinically relevant genes enriching IDR mutational hotspots that exhibited differential expression patterns and are associated with cancer patient survival. In summary, combinations of mutational effects on IDRs significantly increase the sensitivity of driver detection and are likely to open new therapeutic avenues for various cancers.

CsA promotes trophoblast invasion accompanied by changes in leukaemic inhibitory factor and fibroblast growth factor in peri-implantation blastocysts.

During the early stages of human pregnancy, successful implantation of embryonic trophoblast cells into the endometrium depends on good communication between trophoblast cells and the endometrium. Abnormal trophoblast cell function can cause embryo implantation failure. In this study, we added cyclosporine A (CsA) to the culture medium to observe the effect of CsA on embryonic trophoblast cells and the related mechanism. We observed that CsA promoted the migration and invasion of embryonic trophoblast cells. CsA promoted the expression of leukaemic inhibitory factor (LIF) and fibroblast growth factor (FGF). In addition, CsA promoted the secretion and volume increase in vesicles in the CsA-treated group compared with the control group. Therefore, CsA may promote the adhesion and invasion of trophoblast cells through LIF and FGF and promote the vesicle dynamic process, which is conducive to embryo implantation.

MtESN2 is a subgroup II sulphate transporter required for symbiotic nitrogen fixation and prevention of nodule early senescence in Medicago truncatula.

Adequate distribution of mineral sulphur (S) nutrition to nodules mediated by sulphate transporters is crucial for nitrogen fixation in symbiosis establishment process. However, the molecular mechanisms underlying this process remain largely unknown. In this study, we characterized the function of Early Senescent Nodule 2 (MtESN2), a gene crucial to nitrogen fixation in Medicago truncatula. Mutations in MtESN2 resulted in severe developmental and functional defects including dwarf shoots, early senescent nodules, and lower nitrogenase activity under symbiotic conditions compared to wild-type plants. MtESN2 encodes an M. truncatula sulphate transporter that is expressed only in roots and nodules, with the highest expression levels in the transition zone and nitrogen-fixing zone of nodules. MtESN2 exhibited sulphate transport activity when expressed in yeast. Immunolocalization analysis showed that MtESN2-yellow fluorescent protein fusion protein was localized to the plasma membranes of both uninfected and infected cells of nodules, where it might transport sulphate into both rhizobia-infected and uninfected cells within the nodules. Our results reveal an unreported sulphate transporter that contributes to effective symbiosis and prevents nodule early senescence in M. truncatula.

Downregulated ETV4 inhibits the proliferation, migration, and invasion of trophoblast cells in preeclampsia.

In brief: Preeclampsia is a pregnancy complication that can lead to severe adverse maternal and fetal outcomes, but the mechanisms underlying the development of preeclampsia are not fully understood. This study shows that ETV4 plays an essential role in the proliferation, invasion, and migration of trophoblast cells by regulating MMP-2 and MMP-9 and is involved in the pathogenesis of preeclampsia. Abstract: Preeclampsia (PE) is a pregnancy complication that can lead to severe adverse maternal and fetal outcomes. However, the mechanisms underlying the development of PE are not fully understood. ETS Variant Transcription Factor 4 (ETV4) plays an important role in cell proliferation, migration, and invasion. In this study, we aimed to explore the potential function of ETV4 in placental trophoblast cells. We analyzed the expression and location of ETV4 in PE and uncomplicated placental tissues using RT-qPCR, Western blotting, immunohistochemistry, and immunofluorescence staining. The results showed that both the mRNA and protein levels of ETV4 were markedly decreased in PE placental tissues compared with placental tissues from women with uncomplicated pregnancies (P < 0.05). Then, the effects of ETV4 on HTR-8/SVneo and Bewo cell proliferation, migration, and invasion were evaluated by MTT, 5-ethynyl-2-deoxyuridine (EdU), wound healing, and Transwell assays, respectively. The results showed that ETV4 knockdown inhibited both HTR-8/SVneo and Bewo cell proliferation, migration, and invasion (P < 0.05). Conversely, overexpression of ETV4 promoted both HTR-8/SVneo and Bewo cell proliferation, migration, and invasion (P < 0.05). We then measured the expression of MMP-2 and MMP-9 in HTR8/SVneo cells. We found that ETV4 knockdown decreased the mRNA and protein expression of MMP-2 and MMP-9, while ETV4 overexpression increased MMP-2 and MMP-9 mRNA and protein expression (P < 0.05). In conclusion, ETV4 plays an essential role in the proliferation, invasion, and migration of trophoblast cells by regulating MMP-2 and MMP-9. Our findings provide novel insight into the mechanisms underlying the occurrence of PE.

Pan-cancer illumination of TRIM gene family reveals immunology regulation and potential therapeutic implications.

BACKGROUND: The tripartite motif (TRIM) proteins function as important regulators in innate immunity, tumorigenesis, cell differentiation and ontogenetic development. However, we still lack knowledge about the genetic and transcriptome alterations landscape of TRIM proteins across cancer types. METHODS: We comprehensively reviewed and characterized the perturbations of TRIM genes across > 10,000 samples across 33 cancer types. Genetic mutations and transcriptome of TRIM genes were analyzed by diverse computational methods. A TRIMs score index was calculated based on the expression of TRIM genes. The correlation between TRIMs scores and clinical associations, immune cell infiltrations and immunotherapy response were analyzed by correlation coefficients and gene set enrichment analysis. RESULTS: Alterations in TRIM genes and protein levels frequently emerge in a wide range of tumors and affect expression of TRIM genes. In particular, mutations located in domains are likely to be deleterious mutations. Perturbations of TRIM genes are correlated with expressions of immune checkpoints and immune cell infiltrations, which further regulated the cancer- and immune-related pathways. Moreover, we proposed a TRIMs score index, which can accurately predict the clinical outcome of cancer patients. TRIMs scores of patients are correlated with clinical survival and immune therapy response across cancer types. Identifying the TRIM genes with genetic and transcriptome alterations will directly contribute to cancer therapy in the context of predictive, preventive, and personalized medicine. CONCLUSIONS: Our study provided a comprehensive analysis and resource for guiding both mechanistic and therapeutic analyses of the roles of TRIM genes in cancer.

Impacts of different culture times on pregnancy outcomes after thawing of cleavage stage embryos.

OBJECTIVE: This study assessed the impacts of in vitro culture times of cleavage embryos on clinical pregnancy outcomes. METHODS: This retrospective cohort study was performed at the Reproductive Medicine Department of Hainan Modern Women and Children's Hospital in China between January 2018 and December 2022. Patients who first underwent frozen embryo transfer with in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles on day 3 were included. According to the time of embryo culture after thawing, the embryos were divided into long-term culture group(18-20 h) and short-term culture group (2-4 h). The clinical pregnancy rate was regarded as he primary outcome. To minimize confounding factors and reduce selection bias, the propensity score matching was used to balance the effects of known confounding factors and to reduce selection bias. Stratified analyses and multiple logistic regression analyses were used to evaluate the risk factors affecting the clinical pregnancy outcomes after matching. RESULTS: General characteristics between two groups were comparable after matching. In the long-term culture group, 266/381 (69.81%) embryos had more than 10 blastomeres, and 75/381 (19.68%) reached the morula stage. After overnight culture, the implantation rate (27.97% vs. 14.28%, P = 0.018) and clinical pregnancy rate (38.46% vs. 22.5%, P = 0.05) were increased in the group with proliferating blastomeres. The long-term culture group trended to have a higher clinical pregnancy rate compared with the short-term culture group (35.74% vs. 29.79%). No statistical differences in clinical pregnancy outcomes between the two groups were observed after matching, including the rates of implantation (25.46% vs23.98%), miscarriages (25% vs. 22.85%), ongoing pregnancy rate (76.2% vs. 77.15%) and live birth rate (26.8% vs. 22.98%). Stratified analyses were performed according to the age of the patients. After matching, there were no significant differences in the clinical pregnancy, implantation and miscarriage rates between the two groups for patients > 35 or ≤ 35 years of age. Subgroup analyses were performed according to the quality of the transferred embryos. There were no significant differences in the clinical outcomes, between two groups after embryos transferred with the same quality. Multivariate Logistic regression analysis was used to evaluate the influencing factors of clinical pregnancy outcomes after matching. Culture time was not found to be an independent predictor for clinical pregnancy [OR 0.742, 95%CI 0.487 ~ 1.13; P = 0.165]. The age of oocyte retrieval [OR 0.906, 95%CI 0.865 ~ 0.949; P <0.001] and the number of high-quality embryos transferred [OR 1.787, 95%CI 1.256 ~ 2.543; P = 0.001] were independent factors affecting clinical pregnancy outcomes. CONCLUSIONS: In vitro 18-20 h culture of embryos with either good-or non-good-quality will not adversely affect the clinical pregnancy.

Sestrin2 mediates FOXM1 expression to block the EMT process in non-small cell lung cancer through the AMPK/YAP pathway.

Non-small cell lung cancer (NSCLC) is characterized by high incidence and mortality, severely threatening human health. The infinite growth and metastasis of NSCLC cells result in a poor prognosis. Therefore, our study was to investigate the mechanism of Sestrin2 on the epithelial-mesenchymal transition (EMT) process of NSCLC cells. Human embryonic lung fibroblasts, NSCLC cell lines, and nude mice were experimental subjects in this study. qRT-PCR and western blot were performed to evaluate the mRNA and protein expression of genes. CCK-8 and EdU assay were conducted to detect cell proliferation. The scratch test and Transwell assay were applied to examine cell migration and invasion. The bioinformatics analysis and Co-IP assay were employed to predict and consolidate the interaction between YAP and TEAD. We found the expression of Sestrin2 was declined but the expression of YAP was elevated in NSCLC cells. Sestrin2 sufficiency or YAP silencing could effectively impair cell growth and metastasis. Mechanistically, YAP interacted with TEAD to enhance FOXM1 expression. Additionally, the elevation of FOXM1 abolished the inhibitory influences of Sestrin2 sufficiency on NSCLC cell growth, invasion, and EMT process. Eventually, Sestrin2 elevation attenuated tumor growth in mice via modulation of the AMPK/YAP/FOXM1 axis, which was reversed by FOXM1 overexpression. Our consequences suggested Sestrin2 could inhibit the activation of YAP via prompting AMPK phosphorylation and then suppress FOXM1 expression through the interplay between YAP and TEAD to impair the capacities of NSCLC cell proliferation, migration, invasion, and EMT. This study provided a novel mechanism of Sestrin2 in NSCLC.

TUSC3 inhibits cell proliferation and invasion in cervical squamous cell carcinoma via suppression of the AKT signalling pathway.

The decreased expression of tumour suppressor candidate 3 (TUSC3) is associated with proliferation in several types of cancer, leading to an unfavourable prognosis. The present study aimed to assess the cellular and molecular function of TUSC3 in patients with cervical squamous cell carcinoma (CSCC). Levels of mRNA expressions of TUSC3 were analysed in CSCC tissues and six cell lines using qRT-PCR. Immunohistochemistry(IHC) was used to evaluate the protein expression level of TUSC3 in four paired specimens, 220 paraffin-embedded CSCC specimens and 60 cases of normal cervical tissues(NCTs), respectively. Short hairpin RNA interference was employed for TUSC3 knockdown. Cell proliferation, migration and invasion were evaluated using growth curve, MTT assay, wound healing, transwell assay and xenograft tumour model, respectively. The results demonstrated that TUSC3 mRNA and protein expression levels were downregulated in CSCC samples. Multivariate and univariate analyses indicated that TUSC3 was an independent prognostic factor for patients with CSCC. Decreased TUSC3 expression levels were significantly associated with proliferation and an aggressive phenotype of cervical cancer cells both in vitro and in vivo. Moreover, the knockdown of TUSC3 promoted migration and invasion of cancer cells, while the increased expression of TUSC3 exhibited the opposite effects. The downregulation of TUSC3 facilitated proliferation and invasion of CSCC cells through the activation of the AKT signalling pathway. Our data demonstrated that the downregulation of TUSC3 promoted CSCC cell metastasis via the AKT signalling pathway. Therefore, TUSC3 may serve as a novel prognostic marker and potential target for CSCC.

Metabolic plasticity and regulation of T cell exhaustion.

The metabolic reprogramming during T cell activation and differentiation affects T cell fate and immune responses. Cell metabolism may serve as the driving force that induces epigenetic modifications, contributing to regulating T cell differentiation. Persistent pathogen infection leads to T cell exhaustion, which is composed of two main subsets and with distinct metabolic characteristics. The progenitor exhausted T cells utilize mitochondrial fatty acid oxidation (FAO) and oxidative phosphorylation (OXPHOS) for energy, while terminally exhausted T cells mainly rely on glycolytic metabolism with impaired glycolysis and OXPHOS. Here, we compiled the latest research on how T cell metabolism defines differentiation, focusing on T cell exhaustion during chronic infections. In addition, metabolic-related factors including antigen stimulation signals strength, cytokines and epigenetics affecting T cell exhaustion were also reviewed. Furthermore, the intervention strategies on metabolism and epigenetics to reverse T cell exhaustion were discussed in detail, which may contribute to achieving the goal of prevention and treatment of T cell exhaustion.

A Bayesian hierarchical model to estimate DNA methylation conservation in colorectal tumors.

MOTIVATION: Conservation is broadly used to identify biologically important (epi)genomic regions. In the case of tumor growth, preferential conservation of DNA methylation can be used to identify areas of particular functional importance to the tumor. However, reliable assessment of methylation conservation based on multiple tissue samples per patient requires the decomposition of methylation variation at multiple levels. RESULTS: We developed a Bayesian hierarchical model that allows for variance decomposition of methylation on three levels: between-patient normal tissue variation, between-patient tumor-effect variation and within-patient tumor variation. We then defined a model-based conservation score to identify loci of reduced within-tumor methylation variation relative to between-patient variation. We fit the model to multi-sample methylation array data from 21 colorectal cancer (CRC) patients using a Monte Carlo Markov Chain algorithm (Stan). Sets of genes implicated in CRC tumorigenesis exhibited preferential conservation, demonstrating the model's ability to identify functionally relevant genes based on methylation conservation. A pathway analysis of preferentially conserved genes implicated several CRC relevant pathways and pathways related to neoantigen presentation and immune evasion. Our findings suggest that preferential methylation conservation may be used to identify novel gene targets that are not consistently mutated in CRC. The flexible structure makes the model amenable to the analysis of more complex multi-sample data structures. AVAILABILITY AND IMPLEMENTATION: The data underlying this article are available in the NCBI GEO Database, under accession code GSE166212. The R analysis code is available at https://github.com/kevin-murgas/DNAmethylation-hierarchicalmodel. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Maternal RND3/RhoE deficiency impairs placental mitochondrial function in preeclampsia by modulating the PPARγ-UCP2 cascade.

Preeclampsia (PE) is a life-threatening disease of pregnant women associated with severe hypertension, proteinuria, or multi-organ injuries. Mitochondrial-mediated placental oxidative stress plays a key role in the pathogenesis of PE. However, the underlying mechanism remains to be revealed. Here, we identify Rnd3, a small Rho GTPase, regulating placental mitochondrial reactive oxygen species (ROS). We showed that Rnd3 is down-regulated in primary trophoblasts isolated from PE patients. Loss of Rnd3 in trophoblasts resulted in excessive ROS generation, cell apoptosis, mitochondrial injury, and proton leakage from the respiratory chain. Moreover, Rnd3 overexpression partially rescues the mitochondrial defects and oxidative stress in human PE primary trophoblasts. Rnd3 physically interacts with the peroxisome proliferators-activated receptor γ (PPARγ) and promotes the PPARγ-mitochondrial uncoupling protein 2 (UCP2) cascade. Forced expression of PPARγ rescues deficiency of Rnd3-mediated mitochondrial dysfunction. We conclude that Rnd3 acts as a novel protective factor in placental mitochondria through PPARγ-UCP2 signaling and highlight that downregulation of Rnd3 is a potential factor involved in PE pathogenesis.

Downregulated ribosomal protein L39 inhibits trophoblast cell migration and invasion by targeting E-cadherin in the placenta of patients with preeclampsia.

Early-onset preeclampsia (PE) is a pregnancy complication that can lead to severe adverse maternal and fetal outcomes. However, the mechanisms underlying the development of early-onset PE are not fully understood. Ribosomal protein L39 (RPL39) is a member of the S39E family of ribosomal proteins that plays an important role in stem cell self-renewal, cancer metastasis, and chemoresistance. In this study, we aimed to explore the potential function of RPL39 in placental trophoblast cells. We analyzed the expression of RPL39 in early-onset PE and normal placental tissues using real-time PCR, western blot analysis, and immunohistochemistry. The results showed that RPL39 was markedly downregulated in early-onset PE placental tissues. RPL39 knockdown inhibited trophoblast cell proliferation, migration, and invasion, as well as placental explant outgrowth. Flow cytometry analysis suggested that knockdown of RPL39 resulted in cell cycle arrest at the G0/G1 phase, but had no significant effect on cell apoptosis. We also found that RPL39 knockdown could alter cell morphology. We then measured the expression of the epithelial cell marker E-cadherin following knockdown of RPL39 in Bewo and HTR8/SVneo cells. RPL39 knockdown increased the expression of E-cadherin. Furthermore, E-cadherin expression was upregulated in placental explant outgrowth tissues transfected with RPL39 small interfering RNA. In conclusion, RPL39 plays an essential role in proliferation, invasion, and migration of trophoblast cells by targeting E-cadherin. Our findings provide novel insight into the mechanisms underlying the occurrence of early-onset PE.

Establishment of a non-integrated induced pluripotent stem cell line derived from human chorionic villi cells.

BACKGROUND: Few studies have investigated the generation of induced pluripotent stem cells (iPSCs) derived from human primary chorionic villi (CV) cells. The present study aimed to explore an optimal electroporation (EP) condition for generating non-integrated iPSCs from CV cells (CV-iPSCs). METHODS: The EGFP plasmid was transfected into CV cells under different EP conditions to evaluate cell adherence and the rate of EGFP positive cells. Subsequently, CV cells were transfected with the pEP4-E02S-ET2K and pCEP4-miR-302-367 plasmids under optimal EP conditions. Finally, CV-iPSC pluripotency, karyotype analysis, and differentiation ability were investigated. RESULTS: Following EP for 48 h under different conditions, different confluency, and transfection efficiency were observed in CV cells. Higher cell density was observed in CV cells exposed to 200 V for 100 s, while higher transfection efficiency was obtained in cells electroporated at a pulse of 300 V for 300 s. To generate typical primitive iPSCs, CV cells were transfected with pEP4-E02S-ET2K and pCEP4-miR-302-367 plasmids using EP and were then cultured in induction medium for 20 days under selected conditions. Subsequently, monoclonal iPSCs were isolated and were evaluated pluripotency with AP positive staining, the expression of OCT4, SOX2, and NANOG in vitro and the formation of three germ layer teratomas in vivo. CONCLUSION: CV-iPSCs were successfully established under the conditions of 100 µl shock cup and EP pulse of 200 V for 300 s for two times. This may provide a novel strategy for investigating the pathogenesis of several diseases and gene therapy.

Cryopreservation of human induced pluripotent stem cells by using a new CryoLogic vitrification method.

Human induced pluripotent stem cells (hiPSCs) have the properties of differentiation potential and unlimited self-renewal. Developing efficient and highly safe methods to preserve hiPSCs is important due to they have demonstrated tremendous promise in disease etiology, drug discovery, and regenerative medicine applications. Traditionally, open systems for cell cryopreservation, such as conventional slow freezing and vitrification methods, were widespread application in the storage and transportation of hiPSCs. However, these two methods have such problems of low recovery rate and the risk of cross-contamination. Recently, closed systems for cell cryopreservation, such as CryoLogic Vitrification Method (CVM), were introduced to store and transport embryos. In this study, we developed a new friendly CVM by loading a small piece of hiPSCs colonies in the vitrification solution to the hook of Fiberplug to increase the cooling rate. To warm them, the CVM Fiberplug was immersed directly in a 37 °C warming solution for 1 min, and hiPSCs were then transferred to mTeSR1 medium. The result revealed that the new CVM had a high recovery rate and maintained the stemness and differentiation potential of hiPSCs. Our new CVM not only provide a safe way for hiPSCs preservation but also has a high survival rate in the storage of hiPSCs.

HER-2/neu-positive breast cancer neoadjuvant chemotherapy response after implementation of 2018 ASCO/CAP focused update.

Human Epidermal Growth Factor Receptor 2 (HER2), a routinely tested breast cancer marker, is associated with worse prognosis yet increased sensitivity to targeted neoadjuvant therapy (NAT) in breast cancer patients. The presence of HER2 in breast carcinoma can be detected with either immunohistochemistry (IHC) or in situ hybridization (ISH). In this study, we examine the relationship between clinicopathological features, HER2 detection method (IHC vs ISH), and prognostic outcomes in NAT-treated HER2-positive breast cancer patients. We included 99 HER2-positive patients from three academic institutions following 2018 HER2 testing updates and conducted a retrospective correlational study. Seventy-one (72%) were HER2-positive by IHC and 28 (28%) were positive following reflexive ISH. Multivariate analysis showed biomarker status to be significantly associated with pathologic complete response (pCR) (p = 0.003), Residual Cancer Burden (RCB) (p = 0.007), and tumor size downstaging (p = 0.002) and HER2 detection method of IHC to be significantly associated with pCR (p = 0.05), RCB (p = 0.004), and nodal downstaging (p= 0.03). In conclusion, HER2 detection method and biomarker subtype allow for further prognostic stratification of HER2-positive patients when 2018 American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guideline updates are applied.

Abscisic acid receptors maintain abscisic acid homeostasis by modulating UGT71C5 glycosylation activity.

Uridine diphosphate-glucosyltransferases (UGTs) maintain abscisic acid (ABA) homeostasis in Arabidopsis thaliana by converting ABA to abscisic acid-glucose ester (ABA-GE). UGT71C5 plays an important role in the generation of ABA-GE. Abscisic acid receptors are crucial upstream components of the ABA signaling pathway, but how UGTs and ABA receptors function together to modulate ABA levels is unknown. Here, we demonstrated that the ABA receptors RCAR12/13 and UGT71C5 maintain ABA homeostasis in Arabidopsis following rehydration under drought stress. Biochemical analyses show that UGT71C5 directly interacted with RCAR8/12/13 in yeast cells, and the interactions between UGT71C5 and RCAR12/13 were enhanced by ABA treatment. Enzyme activity analysis showed that ABA-GE contents were significantly elevated in the presence of RCAR12 or RCAR13, suggesting that these ABA receptors enhance the activity of UGT71C5. Determination of the content of ABA and ABA-GE in Arabidopsis following rehydration under drought stress revealed that ABA-GE contents were significantly higher in Arabidopsis plants overexpressing RCAR12 and RCAR13 than in non-transformed plants and plants overexpressing RCAR11 following rehydration under drought stress. These observations suggest that RCAR12 and RCAR13 enhance the activity of UGT71C5 to glycosylate excess ABA into ABA-GE following rehydration under drought stress, representing a rapid mechanism for regulating plant growth and development.