The potential role of the prolyl isomerase Pin1 during blastocyst implantation and uterine decidualization in mice.

During early pregnancy in mice, the establishment of uterine receptivity and endometrial decidualization require the extensive proliferation and differentiation of endometrial epithelial cells or stromal cells. Pin1 has been suggested to act as a molecular 'timer' of the cell cycle and is involved in the regulation of cellular proliferation and differentiation by binding many cell-cycle regulatory proteins. However, its physiological role during early pregnancy is still not fully understood. Here, we employed immunohistochemistry to determine the spatiotemporal pattern of Pin1 expression during early pregnancy. We found that Pin1 was mainly localized in subluminal stromal cells on day 4, in the decidual zone on days 5 to 8 of pregnancy and in artificial decidualization. Using a uterine stromal cell culture system, we found that progesterone, but not estrogen, induced the expression of Pin1 in a progesterone receptor-dependent manner. Inhibition of Pin1 in the uterus leads to impaired embryo implantation and decidualization in mice. Notably, a decrease in Pin1 activation affected the functional execution of several implantation- or decidualization-related factors. These findings provide new evidence for a previously unknown function of Pin1 in mediating embryo implantation and decidualization during successful pregnancy establishment and maintenance.

Unraveling the Impact of the PROCA1 Mutation in Male Infertility: Incorporating Whole Exome Sequencing in Teratozoospermia Patients and Analyzing Proca1 Knockout Mice.

In the world, about 15% of couples are infertile, and nearly half of all infertility was caused by men. A large number of genetic mutations are thought to affect spermatogenesis by regulating acrosome formation. Here, we identified three patients harbouring the protein interacting with cyclin A1 (PROCA1) mutation by whole exome sequencing (WES) and Sanger sequencing among patients with predominantly acrosome-deficient teratozoospermia. However, the expression and roles of PROCA1 in infertile men remain unclear. We found that PROCA1 is predominantly expressed in the testis, where it is specifically localized to the acrosome of normal human sperm. Proca1 knockout (KO) mice were subsequently generated using CRISPR-Cas9 technology. However, Proca1 KO adult male mice were fertile, with testis-to-body weight ratios comparable to those of wild-type (WT) mice. Testicular tissue or sperm morphology were not significantly different in Proca1 KO mice compared to WT mice. Expression of the acrosome markers PNA and SP56 in the acrosome was comparable between Proca1 KO and WT mice. In summary, these findings suggested that the PROCA1 mutation identified in humans does not affect acrosome biogenesis in mice.

Neuroprotective effects of chlorogenic acid: Modulation of Akt/Erk1/2 signaling to prevent neuronal apoptosis in Parkinson's disease.

As a prevalent neurodegenerative disorder, Parkinson's disease is associated with oxidative stress. Our recent investigations revealed that reactive oxygen species (ROS) and PD-toxins like 6-hydroxydopamine (6-OHDA) can induce neuronal apoptosis through over-activation of Akt signaling. Chlorogenic acid (CGA), a natural acid phenol abundant in the human diet, is well-documented for its ability to mitigate intracellular ROS. In this study, we utilized CGA to treat experimental models of PD both in vitro and in vivo. Our study results demonstrated that SH-SY5Y and primary neurons exhibited cell apoptosis in response to 6-OHDA. Pretreatment with CGA significantly attenuated PD toxins-induced large amount of ROS, inhibiting Erk1/2 activation, preventing Akt inhibition, and hindering neuronal cell death. Combining the Erk1/2 inhibitor U0126 with CGA could reverse 6-OHDA-induced Akt inhibition, ROS, and apoptosis in the cells. Crucially, the Akt activator SC79 and ROS scavenger NAC both could eliminate excessive ROS via Akt and Erk1/2 signaling pathways, and CGA further potentiated these effects in PD models. Behavioral experiments revealed that CGA could alleviate gait abnormalities in PD model mice. The neuroprotective effects have been demonstrated in several endocrine regions and in the substantia nigra tissue, which shows the positive tyrosine hydroxylase (TH). Overall, our results suggest that CGA prevents the activation of Erk1/2 and inactivation of Akt by removing excess ROS in PD models. These findings propose a potential strategy for mitigating neuronal degeneration in Parkinson's disease by modulating the Akt/Erk1/2 signaling pathway through the administration of CGA and/or the use of antioxidants to alleviate oxidative stress.

GSE1 promotes the proliferation and migration of lung adenocarcinoma cells by downregulating KLF6 expression.

Lung cancer is one of the most prevalent human cancers with a high lethality rate worldwide. In this study, we demonstrated that GSE1 (genetic suppressor element 1) expression is aberrantly upregulated in lung adenocarcinoma and that GSE1 depletion inhibits the proliferation and migration of both A549 and H1299 cells. Immunoprecipitation assays demonstrated that GSE1 interacts with histone deacetylase 1 (HDAC1) and other BRAF-HDAC complex (BHC) components in cells. The transcriptome of GSE1-knockdown A549 cells indicated that 207 genes were upregulated and 159 were downregulated based on a p-value < .05 and fold change ≥ 1.5. Bioinformatics analysis suggested that 140 differentially expressed genes harbor binding sites for HDAC1, including the tumor suppressor gene KLF6 (Kruppel-like factor 6). Indeed, quantitative reverse-transcription polymerase chain reaction and western blot analysis revealed that GSE1 could inhibit the transcription of KLF6 in lung cancer cells. In conclusion, GSE1 cooperates with HDAC1 to promote the proliferation and metastasis of non-small cell lung cancer cells through the downregulation of KLF6 expression.

The absence of the ribosomal protein Rpl2702 elicits the MAPK-mTOR signaling to modulate mitochondrial morphology and functions.

Ribosomes mediate protein synthesis, which is one of the most energy-demanding activities within the cell, and mitochondria are one of the main sources generating energy. How mitochondrial morphology and functions are adjusted to cope with ribosomal defects, which can impair protein synthesis and affect cell viability, is poorly understood. Here, we used the fission yeast Schizosaccharomyces Pombe as a model organism to investigate the interplay between ribosome and mitochondria. We found that a ribosomal insult, caused by the absence of Rpl2702, activates a signaling pathway involving Sty1/MAPK and mTOR to modulate mitochondrial morphology and functions. Specifically, we demonstrated that Sty1/MAPK induces mitochondrial fragmentation in a mTOR-independent manner while both Sty1/MAPK and mTOR increases the levels of mitochondrial membrane potential and mitochondrial reactive oxygen species (mROS). Moreover, we demonstrated that Sty1/MAPK acts upstream of Tor1/TORC2 and Tor1/TORC2 and is required to activate Tor2/TORC1. The enhancements of mitochondrial membrane potential and mROS function to promote proliferation of cells bearing ribosomal defects. Hence, our study reveals a previously uncharacterized Sty1/MAPK-mTOR signaling axis that regulates mitochondrial morphology and functions in response to ribosomal insults and provides new insights into the molecular and physiological adaptations of cells to impaired protein synthesis.

Loss of PMFBP1 Disturbs Mouse Spermatogenesis by Downregulating HDAC3 Expression.

PURPOSE: Polyamine modulating factor 1 binding protein (PMFBP1) acts as a scaffold protein for the maintenance of sperm structure. The aim of this study was further to identify the new role and molecular mechanism of PMFBP1 during mouse spermatogenesis. METHODS AND RESULTS: We identified a profile of proteins interacting with PMFBP1 by immunoprecipitation combined with mass spectrometry and demonstrated that class I histone deacetylases, particularly HDAC3 and chaperonin-containing TCP1 subunit 3 (CCT3), were potential interaction partners of PMFBP1 based on network analysis of protein-protein interactions and co-immunoprecipitation. Immunoblotting and immunochemistry assays showed that loss of Pmfbp1 would result in a decline in HDACs and change the proteomic profile of mouse testis, in which differently expressed proteins are associated with spermatogenesis and assembly of flagella, which was proved by proteomic analysis of testis tissue obtained from Pmfbp1-/- mice. After integrating with transcriptome data for Hdac3-/- and Sox30-/- round sperm obtained from a public database, RT-qPCR confirmed ring finger protein 151 (Rnf151) and ring finger protein 133 (Rnf133) were key downstream response factors of the Pmfbp1-Hdac axis affecting mouse spermatogenesis. CONCLUSION: Taken together, this study indicates a previously unidentified molecular mechanism of PMFBP1 in spermatogenesis whereby PMFBP1 interacts with CCT3, affecting the expression of HDAC3, followed by the downregulation of RNF151 and RNF133, resulting in an abnormal phenotype of sperm beyond the headless sperm tails. These findings not only advance our understanding of the function of Pmfbp1 in mouse spermatogenesis but also provide a typical case for multi-omics analysis used in the functional annotation of specific genes.

Biallelic variants in MAD2L1BP (p31comet) cause female infertility characterized by oocyte maturation arrest.

Human oocyte maturation arrest represents one of the severe conditions for female patients with primary infertility. However, the genetic factors underlying this human disease remain largely unknown. The spindle assembly checkpoint (SAC) is an intricate surveillance mechanism that ensures accurate segregation of chromosomes throughout cell cycles. Once the kinetochores of chromosomes are correctly attached to bipolar spindles and the SAC is satisfied, the MAD2L1BP, best known as p31comet, binds mitosis arrest deficient 2 (MAD2) and recruits the AAA+-ATPase TRIP13 to disassemble the mitotic checkpoint complex (MCC), leading to the cell-cycle progression. In this study, by whole-exome sequencing (WES), we identified homozygous and compound heterozygous MAD2L1BP variants in three families with female patients diagnosed with primary infertility owing to oocyte metaphase I (MI) arrest. Functional studies revealed that the protein variants resulting from the C-terminal truncation of MAD2L1BP lost their binding ability to MAD2. cRNA microinjection of full-length or truncated MAD2L1BP uncovered their discordant roles in driving the extrusion of polar body 1 (PB1) in mouse oocytes. Furthermore, the patient's oocytes carrying the mutated MAD2L1BP resumed polar body extrusion (PBE) when rescued by microinjection of full-length MAD2L1BP cRNAs. Together, our studies identified and characterized novel biallelic variants in MAD2L1BP responsible for human oocyte maturation arrest at MI, and thus prompted new therapeutic avenues for curing female primary infertility.

An apparatus for qualitative assessment of the shading ratio of oblique illumination and real-time high-contrast imaging.

Oblique illumination imaging can significantly improve the contrast of transparent thin samples. However, in traditional oblique illumination methods, either the condenser is offset or a block is added to the condenser, which makes it complicated and challenged to build a stable oblique illumination imaging. Herein, we present a method to measure the optimal shading ratio of oblique illumination in an inverted microscope, and develop an apparatus for stable high-speed high-contrast imaging with uniform brightness. At optimal shading ratio, the oblique illumination imaging has better imaging quality than differential interference contrast, which characteristic is independent on sample. In oblique illumination with low magnification objective, the images have uneven brightness. According to target brightness, we have developed a brightness unevenness correction algorithm to form uniform background brightness for oblique illumination. Integrating the algorithm with imaging acquisition, corrected oblique illumination microscopy is appropriate to observe living cells with high contrast.

Pharmacological mechanisms of chitotriose as a redox regulator in the treatment of rat inflammatory bowel disease.

Although extensive development has been made in the treatment of inflammatory bowel disease (IBD), adverse effects and incomplete efficacy of currently used medications are continuous challenge. Accumulated reports on the benefits of chitosan oligosaccharides in intestinal disorders make chitotriose (COS) a breakthrough in the development of new IBD drugs. This study aimed to investigate the biosafety, efficacy and pharmacological mechanisms of COS in the treatment of experimental IBD in compare with the commercial 5-Aminosalicylic acid (5-ASA). In this study, COS effectively relieved active inflammation, restored epithelial function, and reduced intestinal fibrosis. Further investigation demonstrated that COS treatment regulated redox state of the colon tissue by stimulating the transcription factor nuclear factor E2-related factor 2 (Nrf2), increasing production of endogenous antioxidants, and alleviating oxidative stress. The offset of oxidative stress shut down the nuclear factor kappa-B (NF-ĸB) inflammatory pathway, mitophagy of epithelial cells, M2 macrophage polarization in pre-fibrotic inflammation, and myofibroblast activation in intestinal fibrogenesis. In conclusion, COS is a safe and effective therapeutic agent for experimental IBD as a redox regulator. Our results expand the current understanding of the pharmacology of chitosan oligosaccharides for IBD treatment and provides experimental basis for the medicinal development of small molecule carbohydrates.

A novel homozygous missense mutation in AK7 causes multiple morphological anomalies of the flagella and oligoasthenoteratozoospermia.

PURPOSE: To identify the genetic causes of multiple morphological anomalies of the flagella (MMAF) and oligoasthenoteratozoospermia (OAT). METHODS: Whole-exome sequencing (WES) was performed on the proband to identify pathogenic mutation for infertility. Western blotting and immunofluorescence analysis detected the expression level and localization of adenylate kinase 7 (AK7). RESULTS: We identified a novel homozygous missense mutation (NM_152327: c.1846G > A; p.E616K) in AK7 in two brothers with MMAF and OAT from a consanguineous family by WES. Western blotting and immunofluorescence experiments determined that the expression level of AK7 decreased in the sperm from the proband. The proband and his wife underwent two cycles of intracytoplasmic sperm injection (ICSI) treatment but got unfavorable outcomes. CONCLUSION: This study could provide precise genetic diagnosis for the patient and expand the spectrum of AK7 mutations.

Novel Mutation and Deletion in SUN5 Cause Male Infertility with Acephalic Spermatozoa Syndrome.

Acephalic spermatozoa syndrome (ASS) is a severe form of teratozoospermia, previous studies have shown that SUN5 mutations are the major cause of acephalic spermatozoa syndrome. This study is to identify the pathogenic mutations in SUN5 leading to ASS. PCR and Sanger sequence were performed to define the breakpoints and mutations in SUN5. Whole genome sequencing (WGS) was performed to detect heterozygous deletion. Western blotting and immunofluorescence analysis detected the expression level and localization of SUN5. Furthermore, the pathogenicity of the mutant SUN5 was predicted in silico and was verified by the experiments in vitro. We identified one novel homozygous missense mutation (c.775G>A; p.G259S) and one compound heterozygous including one reported missense mutation (c.1043A>T; p.N348I) and a large deletion that contains partial EFCAB8 ( NM_001143967 .1) and BPIFB2 ( NM_025227 ) and complete SUN5 ( NM_080675 ), and one recurrent homozygous splice-site mutation (c.340G>A; p.G114R) in SUN5 in three patients with ASS. Our results showed that SUN5 could not be detected in the patients' spermatozoa and the exogenous expression level of the mutant protein was decreased in transfected HEK-293T cells. This study expands the mutational spectrum of SUN5. We recommended a clinical diagnostic strategy for SUN5 genomic deletion to screen heterozygous deletions and indicated that the diagnostic value of screening for SUN5 mutations and deletions in infertile men with ASS.

Pathogenesis of acephalic spermatozoa syndrome caused by splicing mutation and de novo deletion in TSGA10.

PURPOSE: To identify the genetic causes for acephalic spermatozoa syndrome. METHODS: Whole-exome sequencing was performed on the proband from a non-consanguineous to identify pathogenic mutations for acephalic spermatozoa syndrome. Quantitative real-time polymerase chain reaction and whole genome sequencing were subjected to detect deletion. The functional effect of the identified splicing mutation was investigated by minigene assay. Western blot and immunofluorescence were performed to detect the expression level and localization of mutant TSGA10 protein. RESULTS: Here, we identified a novel heterozygous splicing mutation in TSGA10 (NM_025244: c.1108-1G > T), while we confirmed that there was a de novo large deletion in the proband. The splicing mutation led to the skipping of the exon15 of TSGA10, which resulted in a truncated protein (p. A370Efs*293). Therefore, we speculated that the splicing mutation might affect transcription and translation without the dosage compensation of a normal allele, which possesses a large deletion including intact TSGA10. Western blot and immunofluorescence demonstrated that the very low expression level of truncated TSGA10 protein led the proband to present the acephalic spermatozoa phenotype. CONCLUSION: Our finding expands the spectrum of pathogenic TSGA10 mutations that are responsible for ASS and male infertility. It is also important to remind us of paying attention to the compound heterozygous deletion in patients from non-consanguineous families, so that we can provide more precise genetic counseling for patients.

A homozygous missense mutation in TBPL2 is associated with oocyte maturation arrest and degeneration.

The genetic causes in most of patients with oocyte maturation arrest remain largely unknown. In this study, we identified a homozygous missense mutation (c.895T>C; p.C299R) in TBPL2 (TATA box binding protein like 2) in two infertile sisters with oocyte maturation arrest and degeneration from a consanguineous family by whole-exome sequencing. The TBPL2 mutation is rare and pathogenic, and impaired the transcription initiation function of the protein. Our results showed that TBPL2 mutation might be associated with female infertility due to oocyte maturation arrest and degeneration.

Novel Mutations in CDC20 Are Associated with Female Infertility Due to Oocyte Maturation Abnormality and Early Embryonic Arrest.

The cell division cycle 20 (CDC20) protein is a co-activator of anaphase-promoting complex/cyclosome (APC/C), required for mitotic exit and also meiotic exit, containing seven WD40 repeats in the C-terminus responsible for protein-protein interactions. Recently, a previous study has shown that biallelic mutations in CDC20 are causative for female infertility with abnormalities in oocyte maturation and embryonic development. This study is to further identify new mutations of CDC20 and the prevalence of variants in our cohort. A cohort of 50 primary infertile females with oocyte maturation abnormality and early embryonic arrest were recruited. Genomic DNA was isolated from peripheral blood samples. Mutation screening of all the coding regions of CDC20 was performed by Sanger sequencing. The pathogenicity of the identified variants on the CDC20 protein was accessed in silico. Two CDC20 variants, a nonsense mutation p.R262* and a missense mutation p.A211T, identified in one female of 50 unrelated affected individuals, accounting for a relative small proportion of this cohort (2%). In silico analysis revealed that the p.R262* would cause no production of protein or a truncated protein lacking five WD40 repeats in the C-terminus; and that p.A211T may interfere with the formation of a deep hydrophobic pocket and thus disturb the binding of CDC20 protein to the substrates of APC/C. This study identified two novel mutations in CDC20, further expanding the mutation spectrum of this gene. Our findings further confirm that biallelic mutations in CDC20 occur in a proportion of infertile females with oocyte maturation abnormality and early embryonic arrest.

Global analysis of lysine acetylome reveals the potential role of CCL18 in non-small cell lung cancer.

C-C motif chemokine 18 (CCL18) belongs to the chemokine CC family and is predominantly secreted by M2-tumor-associated macrophages. It has been reported to be associated with various diseases and malignancies. Previous studies showed that CCL18 promotes metastasis by activating downstream kinases. However, it remains unknown whether CCL18 regulates post-translational modifications, other than phosphorylation, during tumorigenesis. Here, we demonstrate that CCL18 is up-regulated in non-small cell lung cancer (NSCLC) and is involved in regulating the lysine acetylome in A549 cells. Using the combination of SILAC labeling and high-efficiency acetylation enrichment methods, we identified 1372 lysine acetylation (Kac) sites on 796 proteins in CCL18-treated A549 cells. Among the identified Kac sites, 147 from 126 proteins were down-regulated and seven from five proteins were up-regulated with fold changes more than two and the p-value less than 0.05. Bioinformatics analysis further showed that the proteins with down-regulated acetylation play critical roles in glycolysis, oxidative phosphorylation, tricarboxylic acid cycle, and pentose phosphate pathway in A549 cells. These results suggest that CCL18 may be involved in the development of NSCLC by regulating acetylation of the proteins in many fundamental cellular processes, especially the metabolic reprogramming of tumor cells.

A novel homozygous missense mutation of PMFBP1 causes acephalic spermatozoa syndrome.

PURPOSE: To identify the pathogenic mutation in PMFBP1 leading to acephalic spermatozoa syndrome. METHODS: Sanger sequencing was used to screen for mutations in the known pathogenic genes SUN5 and PMFBP1 in a patient with acephalic spermatozoa syndrome. Western blotting and immunofluorescence were used to detect the expression and localization of PMFBP1 in sperm. At the same time, a PMFBP1 mutant was constructed, and the expression level of PMFBP1 protein was further verified by in vitro experiments. RESULTS: We identified a novel homozygous PMFBP1 missense mutation, c.301A>C (p.T101P), in an infertile male from a consanguineous family. Our results showed that the expression of PMFBP1 mutant protein was decreased obviously in sperm of the patient. CONCLUSION: Our results showed that the novel homozygous missense mutation of PMFBP1 may be a cause of acephalic spermatozoa syndrome, which provided a basis for genetic counseling for the patient.

Celastrol inhibits ezrin-mediated migration of hepatocellular carcinoma cells.

Progression of hepatocellular carcinoma involves multiple genetic and epigenetic alterations that promote cancer invasion and metastasis. Our recent study revealed that hyperphosphorylation of ezrin promotes intrahepatic metastasis in vivo and cell migration in vitro. Celastrol is a natural product from traditional Chinese medicine which has been used in treating liver cancer. However, the mechanism of action underlying celastrol treatment was less clear. Here we show that ROCK2 is a novel target of celastrol and inhibition of ROCK2 suppresses elicited ezrin activation and liver cancer cell migration. Using cell monolayer wound healing, we carried out a phenotype-based screen of natural products and discovered the efficacy of celastrol in inhibiting cell migration. The molecular target of celastrol was identified as ROCK2 using celastrol affinity pull-down assay. Our molecular docking analyses indicated celastrol binds to the active site of ROCK2 kinase. Mechanistically, celastrol inhibits the ROCK2-mediated phosphorylation of ezrin at Thr567 which harnesses liver cancer cell migration. Our findings suggest that targeting ROCK2-ezrin signaling is a potential therapeutic niche for celastrol-based intervention of cancer progression in hepatocellular carcinoma.

A novel homozygous nonsense ZP1 variant causes human female infertility associated with empty follicle syndrome (EFS).

BACKGROUND: Empty follicle syndrome (EFS) is a rare but severe condition in which no oocyte is recovered in female patients undergoing in vitro fertilization (IVF) after sufficient ovarian response to hormonal trigger. Accumulating evidence highlights the genetic basis of EFS occurrence. METHODS: In this study, we report a patient with primary infertility showing the characteristics of EFS from a consanguineous family. Under the treatment of assisted reproductive technique (ART), no oocyte was retrieved following the aspiration of mature follicles. Through whole-exome sequencing (WES), we discovered a novel recessively transmitted mutation in ZP1 (c.769 C>T, p. Q257*). RESULTS: In vitro Co-immunoprecipitation assays showed that mutant ZP1 protein failed to interact with either ZP2 or ZP3, which explains the degenerated oocytes in the patient with EFS. CONCLUSION: Together, our data further expand the spectrum of ZP1 mutations that are associated with human EFS and thus provide novel insight into the diagnosis of EFS patients.

PHTF2 regulates lipids metabolism in gastric cancer.

Identification of hub genes and key pathways of gastric cancer was recognized to be essential to elucidate the tumorigenesis of GC. This study was aimed to identify the differentially expressed genes (DEGs) in GC via bioinformatics methods and their related pathways involved in the pathological process of GC. Gene expression profile datasets acquired by microarray chips or RNA-seq were downloaded from GEO dataset and TCGA, and 298 differentially expressed genes was identified. The Gene Ontology (GO) and Kyoto Gene and Genomic Encyclopedia (KEGG) pathways of DEGs were then analyzed by the DAVID database to elucidate the potential molecular functions of DEGs. The protein-protein interaction (PPI) network of DEGs was further analyzed with the STRING database and PHTF2 was identified as a hub gene in the PPI network. Subsequently, PHTF2 was found to be highly expressed in different subtypes of gastric cancer tissues obtained from TCGA database or clinical patients, resulting with a poor prognosis. By GSEA, PHTF2 was found to significantly enrich the fatty acid metabolism pathway in gastric cancer. Moreover, PHTF2-regulated lipids metabolism significantly affected the tumorigenesis of GC cells. In summary, this work identified a new mechanism by which PHTF2 precipitated in the pathological process of GC by regulating cellular lipid metabolism.