Performing ICSI within 4 hours after denudation optimizes clinical outcomes in ICSI cycles.

BACKGROUND: The study aimed to investigate whether and how general and partial time intervals between processes, from human chorionic gonadotrophin (HCG) trigger to intracytoplasmic sperm injection (ICSI), affected the laboratory and reproductive outcomes in ICSI cycles. METHODS: This was a retrospective data analysis of 3602 women who underwent ICSI treatment cycles using partner or donor sperms, performed at Reproduction Medicine Center of Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology (Wuhan, China) between October 2016 and September 2018. The clinical pregnancy rate was the major outcome in the study. The fertilization and available embryo rates were secondary outcomes. RESULTS: Data from 3602 consecutive fresh ICSI cycles was analysed. Multivariate linear regression and logistic regression analysis of factors related to fertilization and clinical pregnancy rates showed that fertilization rate (P = 0.001) and clinical pregnancy rate (P = 0.037) were significantly associated with denudation (DN)-ICSI interval. Long DN-ICSI interval was associated with higher rate of fertilization than short DN-ICSI interval but significantly decreased clinical pregnancy rate when the interval is over 4 h (P < 0.05). CONCLUSIONS: DN-ICSI time interval can act as an independent predictor for clinical outcomes in ICSI cycles. The optimal time for ICSI is within 4 h after oocyte denudation for excellent laboratory and reproductive outcomes in ICSI cycles.

Clinical and genetic analysis of an isolated follicle-stimulating hormone deficiency female patient.

OBJECTIVE: To characterize the clinical features of a female patient with isolated follicle-stimulating hormone (FSH) deficiency and to investigate the underlying mechanisms of FSH inactivation. METHODS: The proband was a 29-year-old woman with primary amenorrhea, impaired pubertal development, and infertility. Subsequently, reproductive endocrine was screened. DNA sequencing was conducted for the identification of FSHß mutation. RT-PCR, western blots, in vitro immunometric assay, and bioassay were performed to confirm the impact of the mutation on FSH expression and biological activity. Molecular model consisting of FSHα and mutant FSHß subunit was built for the structural analysis of FSH protein. RESULTS: The evaluation of reproductive endocrine revealed undetectable basal and GnRH-stimulated serum FSH. Sequencing of the FSHß gene identified a homozygous nonsense mutation at codon 97 (Arg97X). RT-PCR and western blot analysis revealed the mutation Arg97X did not affect FSHß mRNA and protein expression. But in vitro immunometric assay and bioassay demonstrated the production of normal bioactive FSH protein was disturbed by the mutation Arg97X. Structural analysis showed the surface structure of the resulting mutant FSH presented with lock-and-key, mosaic binding pattern, while the native structure was an encircling binding mode. CONCLUSION: The mutation Arg97X could disturb structural stability of the resulting FSH protein consisting of FSHα and mutant FSHß subunit, which may lead to FSH deficiency.

The effect and mechanism of inhibiting glucose-6-phosphate dehydrogenase activity on the proliferation of Plasmodium falciparum.

We screened >40,000 patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency and found that the G6PD Kaiping allele was under the most positive selection for fighting against malaria in the Chinese population. However, the mechanism is unknown. The current study was designed to investigate the anti-malarial effect and mechanism of G6PD deficiency. Dehydroepiandrosterone (DHEA) was utilised for inhibiting the G6PD activity of erythrocytes. Giemsa staining of blood smears and quantitative real-time PCR were used for the detection and quantification of Plasmodium falciparum infection. A transmission electron microscope was used to observe the structural changes of P. falciparum. An atomic force microscopy was used for the analyses of morphology, roughness and Young's Modulus of the infective erythrocyte membrane. When G6PD activity was inhibited by DHEA, the infection rate of P. falciparum decreased, its cell nucleus shrank, the cell organelles and metabolites were reduced gradually and the Young's Modulus of the erythrocyte membrane increased with increasing DHEA concentrations. These data indicated that Plasmodium multiplication would be inhibited in G6PD deficient erythrocytes because the Plasmodium organelles could not obtain enough nutrients, including ribose-5-phosphate and the reducing equivalent, NADPH. Moreover, the Young's Modulus of the erythrocyte membrane increased, which resulted in an increased membrane stiffness and decreased deformation. It was difficult for the merozoites to invade erythrocytes through endocytosis. Understanding these points will have a major effect on searching for new anti-malarial drug targets.

Novel insights into the functional metabolic impact of an apparent de novo m.8993T>G variant in the MT-ATP6 gene associated with maternally inherited form of Leigh Syndrome.

In this study, we report a novel perpective of metabolic consequences for the m.8993T>G variant using fibroblasts from a proband with clinical symptoms compatible with Maternally Inherited Leigh Syndrome (MILS). Definitive diagnosis was corroborated by mitochondrial DNA testing for the pathogenic variant m.8993T>G in MT-ATP6 subunit by Sanger sequencing. The long-range PCR followed by massively parallel sequencing method detected the near homoplasmic m.8993T>G variant at 83% in the proband's fibroblasts and at 0.4% in the mother's fibroblasts. Our results are compatible with very low levels of germline heteroplasmy or an apparent de novo mutation. Our mitochondrial morphometric analysis reveals severe defects in mitochondrial cristae structure in the proband's fibroblasts. Our live-cell mitochondrial respiratory analyses show impaired oxidative phosphorylation with decreased spare respiratory capacity in response to energy stress in the proband's fibroblasts. We detected a diminished glycolysis with a lessened glycolytic capacity and reserve, revealing a stunted ability to switch to glycolysis upon full inhibition of OXPHOS activities. This dysregulated energy reprogramming results in a defective interplay between OXPHOS and glycolysis during an energy crisis. Our study sheds light on the potential pathophysiologic mechanism leading to chronic energy crisis in this MILS patient harboring the m.8993T>G variant.

Effects of G6PD activity inhibition on the viability, ROS generation and mechanical properties of cervical cancer cells.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency has been revealed to be involved in the efficacy to anti-cancer therapy but the mechanism remains unclear. We aimed to investigate the anti-cancer mechanism of G6PD deficiency. In our study, dehydroepiandrosterone (DHEA) and shRNA technology were used for inhibiting the activity of G6PD of cervical cancer cells. Peak Force QNM Atomic Force Microscopy was used to assess the changes of topography and biomechanical properties of cells and detect the effects on living cells in a natural aqueous environment. Flow cytometry was used to detect the apoptosis and reactive oxygen species (ROS) generation. Scanning electron microscopy was used to observe cell morphology. Moreover, a laser scanning confocal microscope was used to observe the alterations in cytoskeleton to explore the involved mechanism. When G6PD was inhibited by DHEA or RNA interference, the abnormal Young's modulus and increased roughness of cell membrane were observed in HeLa cells, as well as the idioblasts. Simultaneously, G6PD deficiency resulted in decreased HeLa cells migration and proliferation ability but increased ROS generation inducing apoptosis. What's more, the inhibition of G6PD activity caused the disorganization of microfilaments and microtubules of cytoskeletons and cell shrinkage. Our results indicated the anti-cervix cancer mechanism of G6PD deficiency may be involved with the decreased cancer cells migration and proliferation ability as a result of abnormal reorganization of cell cytoskeleton and abnormal biomechanical properties caused by the increased ROS. Suppression of G6PD may be a promising strategy in developing novel therapeutic methods for cervical cancer.

A comprehensive analysis of membrane and morphology of erythrocytes from patients with glucose-6-phosphate dehydrogenase deficiency.

Acute hemolytic anemia could be triggered by oxidative stress in the patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. However, the underlying hemolytic mechanism is unknown. To make clear the hemolytic mechanisms, a systematic study on membrane ultrastructure had been undertaken. A comprehensive method was used including atomic force microscopy, scanning electron microscopy, flow cytometer and fluorescence microscopy to analyze the membrane ultrastructure, externalized phosphatidylserine (PS), intracellular Ca(2+) concentration, morphology and the distributions of band 3 protein in G6PD deficient red blood cells (RBCs) after tert-butyl-hydroperoxide (t-BHP) oxidation. The results showed that erythrocyte shrinkage, annexin-V binding to externalized PS on the membrane of early-stage apoptotic cells, the increased membrane roughness and intracellular Ca(2+) concentration, as well as the change of distributions of band 3 protein in RBCs. Compared with the control RBCs, as the concentration of t-BHP up to 0.1mM, the membrane roughness of G6PD deficient RBCs showed significant difference (p<0.05) and as the concentration of t-BHP up to 0.3mM, externalized PS showed significant difference (p<0.05). Furthermore, the population types of RBCs showed dramatic difference between control groups and G6PD deficient groups. Oxidative stress induced more serious erythrocyte apoptosis and resulted in increased roughness of erythrocyte membrane and abnormal distributed band 3 protein in G6PD deficient RBCs. Echinocytes are the predominant abnormal erythrocyte shape occurring in the peripheral blood from patients with G6PD deficiency, which may shorten the RBCs lifespan. The results in the present study will give an increased understanding for the hemolytic mechanism of G6PD deficiency.

Identification and analysis of microplastics in para-tumor and tumor of human prostate.

BACKGROUND: While microplastics are widely found in various human organs and tissues, the relationship between microplastics and human health, especially prostate health, remains unclear. This study aims to identify and quantify the properties, types, and abundance of microplastics in paired para-tumor and tumor tissues of human prostate. Additionally, the potential correlation between microplastics abundance and prostate cancer are investigated. METHODS: Paired para-tumor and tumor samples of the prostate were collected from 22 patients who underwent robot-assisted radical prostatectomy. A combination of laser direct infrared spectroscopy, scanning electron microscopy and pyrolysis-gas chromatography-mass spectrometry was utilized to analyse the properties, type and abundance of microplastics. Correlations between microplastics abundance, demographic characteristics and clinical features of patients were also examined. FINDINGS: Laser direct infrared analysis revealed the presence of microplastics, including polyamide, polyethylene terephthalate, and polyvinyl chloride, in both para-tumor and tumor tissues of human prostate. However, polystyrene was exclusively detected in tumor tissues. The particle size distribution in the prostate tissue mainly ranged from 20 to 100 µm. Approximately 31.58% of para-tumor samples exhibited sizes between 20 and 30 µm, while 35.21% of tumor samples displayed sizes between 50 and 100 µm. The shapes of these microplastics varied considerably with irregular forms being predominant. Additionally, microplastics were detected by pyrolysis-gas chromatography-mass spectrometry in 20 paired prostate tissues. The mean abundance of microplastics was found to be 181.0 µg/g and 290.3 µg/g in para-tumor and tumor of human prostate samples, respectively. Among the 11 target types microplastics polymers, only polystyrene, polypropylene, polyethylene, and polyvinyl chloride were detected. Notably, polystyrene, polyethylene, and polyvinyl chloride, except for polypropylene, demonstrated significantly higher abundance in tumor tissues compared to their respective paired para-tumor. Furthermore, a positive correlation was observed between polystyrene abundance in the tumor samples of human prostate and frequency of take-out food consumption. INTERPRETATION: This research provides both qualitative and quantitative evidence of the microplastics presence as well as their properties, types, and abundance in paired para-tumor and tumor samples of human prostate. Correlations between microplastics abundance, demographics, and clinical characteristics of patients need to be further validated in future studies with a larger sample size. FUNDING: This work was supported by the National Key Research and Development Program of China (2022YFC2702600) and the National Natural Science Foundation of China (Grant No. 82071698, No. 82101676, and No. 82271630).

Roles of ferroptosis in type 1 diabetes induced spermatogenic dysfunction.

Diabetes mellitus (DM) is a widespread metabolic disease presenting with various complications, including spermatogenic dysfunction. However, the underlying mechanisms are still unclear. Ferroptosis, a novel type of programmed cell death, is associated with much metabolic diseases. Here, we investigated the role of ferroptosis in spermatogenic dysfunction of streptozotocin (STZ)-induced type 1 diabetic mice (diabetic mice), high glucose (HG)-treated GC-2 cells (HG cells) as well as testicular tissues of diabetic patients. We found an accumulation of iron, elevated malondialdehyde level and reduced glutathione level in the testis tissues of diabetic mice and HG cells. Histological examination showed a decrease in spermatogenic cells and spermatids within the seminiferous tubules as well as mitochondrial shrinkage in the testis tissues of diabetic mice. Ferrostatin-1 (Fer-1), the inhibitor of ferroptosis, mitigated ferroptosis-associated iron overload, lipid peroxidation accumulation and spermatogenic dysfunction of diabetic mice. Furthermore, we observed a downregulation of GPX4, FTL and SLC7A11 in diabetic mice and HG cells. Fer-1 treatment and GPX4 overexpression counteracted the effects of HG on cell viability, reactive oxygen species, lipid peroxidation and glutathione via inhibition of ferroptosis. Moreover, we found an elevation of ferroptosis in testicular tissues of diabetic patients. Taken together, our results identify the crucial role of ferroptosis in diabetic spermatogenic dysfunction and ferroptosis may be a promising therapeutic target to improve spermatogenesis in diabetic patients.

Which endometrial preparation protocol provides better pregnancy and perinatal outcomes for endometriosis patients in frozen-thawed embryo transfer cycles? A retrospective study on 1413 patients.

OBJECTIVE: To determine the optimal endometrial preparation protocol for a frozen embryo transfer in patients with endometriosis. DESIGN: Retrospective cohort study. SETTING: Tertiary care academic medical center. PATIENT(S): One thousand four hundred thirteen patients with endometriosis who underwent oocyte aspiration from 2015 to 2020 and frozen embryo transfer from 2016 to 2020 and received natural cycle, hormone replacement treatment with or without GnRHa pretreatment endometrial preparation. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Clinical pregnancy rate, live birth rate, miscarriage rate, multiple pregnancy rate, biochemical pregnancy rate and ectopic pregnancy rate. Singleton live births were assessed for perinatal outcomes and obstetric complications. RESULT(S): There were no differences in clinical pregnancy outcomes or prenatal outcomes among the three commonly used endometrial preparation protocols for frozen embryo transfer cycles in patients with endometriosis. Results remained after screening variables using univariate logistic regression into multivariate logistic regression. No advantages or disadvantages were found among the three endometrial preparation protocols in patients with endometriosis. CONCLUSION(S): Natural cycle, hormone replacement cycle, or hormone replacement treatment with GnRHa pretreatment showed no superiority or inferiority in pregnancy and perinatal outcomes in patients with endometriosis.

Identification of novel variations of oculocutaneous albinism type 2 with Prader-Willi syndrome/Angelman syndrome in two Chinese families.

Objective: Oculocutaneous albinism (OCA) is an autosomal recessive disorder caused by a variety of genomic variations. Our aim is to identify the molecular basis of OCA in two families and lay the foundation for prenatal diagnosis. Methods: Four types of OCA-causing mutations in the TYR, p, TYRP1, or SLC45A2 genes were screened. Linkage analysis was performed because the mutations found in the p gene violated the laws of classical Mendelian heredity. Primer-walking sequencing combined with microsatellite and single-nucleotide polymorphism analysis was used to ascertain deletion ranges. Bioinformatics methods were used to assess the pathogenicity of the new mutations. Results: Proband 1 was diagnosed as OCA2 with Prader-Willi syndrome (PWS) due to a novel atypical paternal deletion (chromosome 15: 22330347-26089649) and a pathogenic mutation, c.1327G>A (Val443Ile), in the p gene of the maternal chromosome. The prenatal diagnosis results for family 1 indicated the fetus was a heterozygous carrier (c.1327G>A in the p gene) with a normal phenotype. Proband 2 was diagnosed as OCA2 with Angelman syndrome (AS) due to a typical maternal deletion of chromosome 15q11-q13 and a novel mutation, c.1514T>C (Phe505Ser), in the p gene of the paternal chromosome. This novel mutation c.1514T>C (Phe505Ser) in the p gene was predicted as a pathogenic mutation. Conclusion: Our study has shown clear genotype-phenotype correlations in patients affected by distinct deletions of the PWS or AS region and missense mutations in the p gene. Our results have enriched the mutation spectrum of albinism diseases and provided insights for more accurate diagnosis and genetic counseling.

Metformin Exhibits an Attractive Antineoplastic Effect on Human Endometrial Cancer by Regulating the Hippo Signaling Pathway.

Metformin, the first-line oral antidiabetic medicine, has shown great antineoplastic potential in various cancer types, despite an unclear mechanism. This study aimed to elucidate the possible mechanism of metformin as a chemotherapy agent with less reproductive and genetic toxicity in human endometrial cancer. The type I endometrial carcinoma cell lines Ishikawa and RL95-2 were treated with metformin. Cell functions, such as proliferation, migration, and invasion, were analyzed. Flow cytometry was performed for cell cycle and apoptosis analyses. Simultaneously, RT-qPCR and western blotting were performed to explore the possible mechanism. Moreover, YAP1 knockout Ishikawa cells were established via lentivirus to demonstrate the underlying mechanism. The results showed that metformin mediated Ishikawa and RL95-2 cell growth inhibition in a dose- and time-dependent manner. The IC50 values of metformin in Ishikawa and RL95-2 cells were 10 mM and 8 mM, respectively. The migration and invasion abilities were also inhibited in the metformin-treated group using wound healing assays and transwell migration and invasion assays, and Ishikawa and RL95-2 cells were arrested in the G1 or G2 phase, respectively. Moreover, the cell proportions of cells in both early and late apoptosis stages were dramatically elevated when treated with metformin, as was the ratio of Bax/Bcl-2 expression. Additionally, the expression levels of YAP1 mRNA and protein in the treatment group were much lower than those in the control group. The cellular behaviors of YAP1 knockout Ishikawa cells were similar to those in the metformin-treated group. Our results demonstrated that it is an attractive alternative to cytotoxic chemotherapy in human endometrial cancer, and YAP of the Hippo pathway may be a potential molecular target. This study provides novel ideas for the adjuvant therapy of endometrial cancer patients, especially for women with strong fertility desires and demands.

Update Knowledge Assessment and Influencing Predictor of Female Fertility Preservation in Oncologists.

OBJECTIVE: This study aims to offer an update assessment of the knowledge of Chinese oncologists on female fertility preservation, and identify the determinants that influence the implementation of fertility preservation. METHODS: A total of 713 Chinese oncologists with different specialties completed the online self-report questionnaire to assess their understanding of fertility risks in cancer treatment, knowledge on female fertility preservation, and perceptions on the barriers in referring patients for fertility preservation. RESULTS: Although most oncologists were familiar with fertility risk in cancer treatment, half of them lacked the knowledge for reproduction and preservation methods. In the multivariable model, oncologists in a hospital with a specialized reproductive institution, positive precaution for fertility risk, and fertility preservation discussion with patients were significantly correlated with the possibility of fertility preservation referral. CONCLUSIONS: The intervention targets based on the update evaluation and identified influencing determinants will be helpful for all the oncofertility researchers, oncologists and institutions in future efforts for well-established female fertility preservation services.

Single-Cell Transcriptomics of Proliferative Phase Endometrium: Systems Analysis of Cell-Cell Communication Network Using CellChat.

The endometrium thickness increases by which endometrial angiogenesis occurs in parallel with the rapid growth of endometrium during the proliferative phase, which is orchestrated by complex cell-cell interactions and cytokine networks. However, the intercellular communication has not been fully delineated. In the present work, we studied the cell-cell interactome among cells of human proliferative phase endometrium using single-cell transcriptomics. The transcriptomes of 33,240 primary endometrial cells were profiled at single-cell resolution. CellChat was used to infer the cell-cell interactome by assessing the gene expression of receptor-ligand pairs across cell types. In total, nine cell types and 88 functionally related signaling pathways were found. Among them, growth factors and angiogenic factor signaling pathways, including EGF, FGF, IGF, PDGF, TGFb, VEGF, ANGPT, and ANGPTL that are highly associated with endometrial growth, were further analyzed and verified. The results showed that stromal cells and proliferating stromal cells represented cell-cell interaction hubs with a large number of EGF, PDGF incoming signals, and FGF outgoing signals. Endothelial cells exhibited cell-cell interaction hubs with a plenty of VEGF, TGFb incoming signals, and ANGPT outgoing signals. Unciliated epithelial cells, ciliated epithelial cells, and macrophages exhibited cell-cell interaction hubs with substantial EGF outgoing signals. Ciliated epithelial cells represented cell-cell interaction hubs with a large number of IGF and TGFb incoming signals. Smooth muscle cells represented lots of PDGF incoming signals and ANGPT and ANGPTL outgoing signals. This study deconvoluted complex intercellular communications at the single-cell level and predicted meaningful biological discoveries, which deepened the understanding of communications among endometrial cells.

Normalized Mitochondrial DNA Copy Number Can Optimize Pregnancy Outcome Prediction in IVF.

The aim of this study is to explore the relationship between mitochondrial DNA (mtDNA) copy number and embryo implantation potential in in vitro fertilization (IVF). A retrospective study of 319 blastocysts from patients undergoing preimplantation genetic testing (PGT) at Reproductive Medicine Center in Tongji Hospital from January 2016 to February 2018 was conducted. We used multiple annealing- and looping-based amplification cycles (MALBAC) technology to amplify the genetic materials from the trophectoderm cells of blastocysts, and next-generation sequencing (NGS) technology to test mitochondrial DNA copy number. Box-Cox transformation was introduced to eliminate the skewness distribution of mtDNA copy number, and the transformed data were defined as adjusted mtDNA. Subsequently, associations between adjusted mtDNA and the clinical characteristics of patients were assessed by univariate analysis and multiple linear regression. In addition, Gaussian Naive Bayes classifier was also used to predict pregnancy outcomes. We observed that only antral follicle count (AFC) was significantly associated with adjusted mtDNA without the influence of multicollinearity. What's more, the distribution of the adjusted mtDNA of blastocysts resulting in live birth was more concentrated than that of others. The area under the curve (AUC) of the prediction model that combined adjusted mtDNA with other clinical characteristics of patients was up to 0.81, higher than that excluded adjusted mtDNA. Among patient clinical characteristics, AFC was significantly associated with adjusted mtDNA. Mitochondrial DNA copy number may help to optimize the pregnancy outcome prediction in IVF.

RNA Sequencing of Decidua Reveals Differentially Expressed Genes in Recurrent Pregnancy Loss.

Recurrent pregnancy loss (RPL) is affecting 3-5% of the women who are expecting to have babies annually. However, the molecular changes of RPL patients have not been well characterized before. In the current study, we aimed to discover the differentially expressed genes (DEGs) in decidua from RPL patients by utilizing RNA sequencing (RNA-seq) technology. Four RPL patients and three control women were recruited to conduct RNA-seq, and 153 genes comprising 69 were upregulated and 84 downregulated showed different expression levels between the health control and RPL groups. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed to identify potential biological processes associated with RPL. We recognized that type I interferon signaling pathway and TNF signaling pathway are significantly upregulated in women with RPL. Upregulated expression of MX1, IFI27, and ISG15 in type I interferon signaling pathway and TNFRSF21 in TNF signaling pathway were validated by an extended sample of 15 RPL patients and 12 control women, by quantitative real-time polymerase chain reaction (qRT-PCR). In conclusion, the results of the current study revealed molecular changes in decidua which may reflect the intrauterine circumstance when pregnancy loss happens and potentially contributes to RPL.

Verteporfin Is a Promising Anti-Tumor Agent for Cervical Carcinoma by Targeting Endoplasmic Reticulum Stress Pathway.

Accumulated evidence has shown that the photosensitizer Verteporfin (VP) may be an ideal agent for various cancer types. However, the effect and mechanism of VP on human cervical carcinoma remain rudimentary. The aim of this study was to investigate the effect of VP on human cervical carcinoma cells (HeLa and SiHa cells) and to elucidate the possible mechanism. CCK-8, wound healing assay, flow cytometry analysis, western blotting, TUNEL staining were performed to evaluate the effects of VP on HeLa and SiHa cells in vitro as well as in vivo on a xenograft model. In addition, the role of endoplasmic reticulum (ER) stress in VP-induced apoptosis was investigated using RT-qPCR and western blotting. The results showed that the viability of HeLa and SiHa cells was suppressed by VP in dose- and time-dependent manners. Compared with the control group, apoptosis rates were higher with stronger TUNEL fluorescence signals in the experimental group, which substantiated that VP induced apoptosis at both 2D and 3D cell levels. Besides, VP can squelch the growth of tumors in both sizes and weights on the xenograft models without impairing ovarian reserve. Mechanism studies demonstrated that VP activated ER stress by upregulating the expression of GRP78, CHOP, and Caspase-12, and VP-induced apoptosis can be alleviated when ER stress pathway was inhibited. Our results provided a foundation for repurposing VP as a promising agent for cervical cancer patients without obvious reproductive toxicity by targeting ER stress pathway, and more researches are required to support its application in clinical practice.

The m.11778 A > G variant associated with the coexistence of Leber's hereditary optic neuropathy and multiple sclerosis-like illness dysregulates the metabolic interplay between mitochondrial oxidative phosphorylation and glycolysis.

Little is known about the molecular mechanism of the rare coexistence of Leber's Hereditary Optic Neuropathy (LHON) and multiple sclerosis (MS), also known as the Harding's syndrome. In this study, we provide novel evidence that the m.11778A > G variant causes a defective metabolic interplay between mitochondrial oxidative phosphorylation and glycolysis. We used dermal fibroblasts derived from a female proband exhibiting clinical symptoms compatible with LHON-MS due to the presence of the pathogenic m.11778A > G variant at near homoplasmic levels. Our mitochondrial morphometric analysis reveals abnormal cristae architecture. Live-cell respiratory studies show stunted metabolic potential and spare respiratory capacity, vital for cell survival upon a sudden energy demand. The m.11778 A > G variant also alters glycolytic activities with a diminished compensatory glycolysis, thereby preventing an efficient metabolic reprogramming during a mitochondrial ATP crisis. Our collective results provide evidence of limited bioenergetic flexibility in the presence of the m.11778 A > G variant. Our study sheds light on the potential pathophysiologic mechanism of the m.11778 A > G variant leading to energy crisis in this patient with the LHON-MS disease.

Adiponectin exerts a potent anti-arthritic effect and insulin resistance in collagen-induced arthritic rats.

AIM: Previous research has shown that adiponectin (AD) induces severe insulin resistance (IR) and exhibits pro-inflammatory effect, so it could serve as a useful risk biomarker in rheumatoid arthritis (RA). The present study aims to evaluate the effect of AD on IR and anti-arthritis in collagen-induced arthritic (CIA) rats. METHOD: After immunization with bovine type II collagen (CII), Wistar rats were administered with AD (60 µg/kg/day) or saline into the ankle joint cavity of the left hind leg for 15 days. The severity of arthritis was clinically and histologically assessed. Arthritis score was recorded every other day for each paw. Paw volume was measured on alternate days to monitor the progression of the disease in the arthritic control group. Tumor necrosis factor (TNF)-α, interleukin (IL)-1, AD, insulin and fasting glucose were measured in sera. Histopathology of joint synovial tissues was also examined. RESULTS: Treatment with AD resulted in significantly delayed onset of arthritis as well as decreased clinical arthritis and histopathological severity scores. AD reduced both serum fasting glucose, TNF-α, IL-1 and IR. Histological analysis confirmed treatment with AD suppressed joint synovial inflammation and immunohistochemical expression of TNF-α compared to the CIA group. Surprisingly, adiponectin levels measured by enzyme-linked immunosorbent assay in serum were significantly increased in CIA rats compared to the normal group. CONCLUSIONS: Adiponectin might display anti-inflammatory effects. These results suggest that AD may be a potential immunosuppressant for the treatment of RA linked to metabolic disease.

Nicotinamide induces mitochondrial-mediated apoptosis through oxidative stress in human cervical cancer HeLa cells.

AIMS: Nicotinamide participates in energy metabolism and influences cellular redox status and modulates multiple pathways related with both cellular survival and death. Recent studies have shown that it induced proliferation inhibition and apoptosis in many cancer cells. However, little is known about the effects of nicotinamide on human cervical cancer cells. We aimed to evaluate the effects of the indicated concentrations nicotinamide on cell proliferation, apoptosis and redox-related parameters in HeLa cells and investigated the apoptotic mechanism. MATERIALS AND METHODS: After the treatment of the indicated concentrations nicotinamide, HeLa cell proliferation was evaluated by the CCK-8 assay and the production of ROS (reactive oxygen species) was measured using 2',7'-Dichlorofluorescin diacetate. The apoptotic effect was confirmed by observing the cellular and nuclear morphologies with fluorescence microscope and apoptotic rate of HeLa cell apoptosis was measured by flow cytometry using Annexin-V method. Moreover, we examined the mitochondrial membrane potential by JC-1 method and measured the expression of apoptosis related genes using qRT-PCR and immunoblotting. KEY FINDINGS: Nicotinamide restrained the HeLa cell proliferation and significantly increased the accumulation of ROS and depletion of GSH at relatively high concentrations. Furthermore, nicotinamide promoted HeLa cell apoptosis via the intrinsic mitochondrial apoptotic pathway. SIGNIFICANCE: Our study revealed that nicotinamide induced the apoptosis through oxidative stress and intrinsic mitochondrial apoptotic pathways in HeLa cell. The results emerge that nicotinamide may be an inexpensive, safe and promising therapeutic agent or a neoadjuvant chemotherapy for cervical cancer patients, as well useful to find new drugs for cervical cancer therapy.

Pathogenicity analysis of novel variations in Chinese Han patients with polycystic kidney disease.

OBJECTIVE: Locus and allellic heterogeneity in polycystic kidney disease (PKD) is a great challenge in precision diagnosis. We aim to establish comprehensive methods to distinguish the pathogenic mutations from the variations in PKD1, PKD2 and PKHD1 genes in a limited time and lay the foundation for precisely prenatal diagnosis, preimplantation genetic diagnosis and presymptom diagnosis of PKD. METHODS: Nested PCR combined with direct DNA sequencing were used to screen variations in PKD1, PKD2 and PKHD1 genes. The pathogenicity of de novel variations was assessed by the comprehensive methods including clinic data and literature review, databases query, analysis of co-segregation of the variants with the disease, variant frequency screening in the population, evolution conservation comparison, protein structure analysis and splice sites predictions. RESULTS: 17 novel mutations from 15 Chinese Han families were clarified including 10 mutations in PKD1 gene and 7 mutations in PKHD1 gene. The novel mutations were classified as 4 definite pathogenic, 2 highly likely pathogenic, 4 likely pathogenic, 7 indeterminate by the comprehensive analysis. The results were verified the truth by the follow-up visits. CONCLUSIONS: The comprehensive methods may be useful in distinguishing the pathogenic mutations from the variations in PKD1, PKD2 and PKHD1 genes for prenatal diagnosis and presymptom diagnosis of PKD. Our results also enriched PKD genes mutation spectrum and evolved possible genotype-phenotype correlations of Chinese Han population.