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PURPOSE: Multiple morphological abnormalities in the sperm flagella (MMAF) comprise a severe phenotype of asthenoteratozoospermia with reduced or absent spermatozoa motility. Whereas dozens of candidate pathogenic genes for MMAF have been identified, the genetic cause in a large proportion of patients is unknown. We attempted to identify novel genetic explanations for MMAF. METHODS: We performed whole-exome sequencing of patients with MMAF to identify pathogenic variants. The phenotypes of spermatozoa in patients carrying DNAH10 variants were investigated using haematoxylin and eosin staining, scanning electron microscopy, and transmission electron microscopy. The expression and location of DNAH10 and other spermatozoa structure–related proteins were analyzed using immunofluorescence assays. RESULTS: We found one homozygous frameshift DNAH10 variant (NM_207437: c.2514delG:p.L839*) and one compound heterozygous DNAH10 variant (NM_207437: c.10820 T > C:p.M3607T; c.12692C > T:p.T4231I) in two patients with MMAF. These variants were absent or rare in the general population. Haematoxylin and eosin staining and scanning electron microscopy revealed the significant disruption of sperm flagella in the patients. In addition, ultrastructural analysis by transmission electron microscopy showed significant inner dynein arm (IDA) deficiency in sperm flagella. Using immunofluorescence assays, we found a significant reduction in IDA-related proteins including DNAH10 and DNAH1. CONCLUSIONS: We identified putative novel pathogenic variants in DNAH10 for MMAF, which might advance the genetic diagnosis and clinical genetic counselling for male infertility. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10815-021-02306-x.

Rationale: Brain arteriovenous malformations (bAVMs) are abnormal entanglement of blood vessels in brain, with direct connections from arteries to veins, lacking functional capillary bed. Although several somatic mutations were reported, the molecular mechanism and genetic disposition of bAVM remain poorly understood. Objective: We aim to identify transcriptional anomalies and critical functional pathways in bAVM lesions and explore their association with key de novo germline and somatic variants in bAVM patients. Methods and Results: We established a comprehensive bAVM dataset from 269 patients, by performing single-cell sequencing of 17 bAVM lesions, whole-exome sequencing of germline DNA from 60 case-unaffected-parental trios, and genomic/transcriptomic sequencing of 231 bAVM lesions. We found abnormal expression of endothelial and mesenchymal markers in bAVM at both bulk and single-cell level, which was validated by flow cytometric analysis and immunofluorescence staining, suggesting an involvement of endothelial-to-mesenchymal transition (EndMT) process in AVM (arteriovenous-malformation). Using data from the 60 trios, we identified nonsynonymous de novo germline mutations affecting 46 genes, including EXPH5 (detected in 2 independent cases), and vessel-related genes, such as EPAS1 and ENG . Interestingly, knockdown of epas1 in zebrafish embryo showed AVM-like phenotype exclusively in brain. Subsequent computational and experimental analyses demonstrated that expression of genes affected by de novo germline mutations was enriched in vascular cell types and was involved in EndMT-relevant behaviors including cell migration, angiogenesis, and cell marker transition. Moreover, we detected somatic KRAS mutations in 129 of 179 (72%) cases and showed that KRAS mutations were associated with bleeding as the first symptom ( P =0.0072). Following experimental studies demonstrated that KRAS mutations independently regulated EndMT features, consolidating the involvement of EndMT in this disease. Lastly, we showed that lovastatin reversed EndMT features in vitro and ex vivo. Conclusions: Our results suggest the convergent role of de novo germline mutations and somatic mutations in regulating EndMT in bAVM and provided a potential therapeutic option.

Tubulin beta eight class VIII (TUBB8) is a subtype of β-tubulin that only exists in primates. TUBB8 mutations have been reported to cause arrest of oocyte maturation and embryonic development. We aim to further investigate the mutational spectrum of TUBB8 and its relevance with female infertility. In our study, infertile patients were recruited, and their basal and clinical characteristics were analyzed. Genomic DNA was extracted from peripheral blood donated by patients. Candidate variants were identified by whole-exome sequencing, selected by relevant criteria, and validated by Sanger sequencing. We found five heterozygous variants: c.C208A(p.P70T), c.T907C(p.C303R), c.G173A(p.R58K), c.G326T(p.G109V), and c.C916T(p.R306C) in TUBB8 among six infertile patients characterized by abnormal phenotypes in oocyte maturation, fertilization, or embryo development. Most of oocytes retrieved from affected individuals were arrested at GV (germinal vesicle) stage and early embryos were arrested at variable stages. In vitro experiments were performed, and the relationship between variant c.G173A(p.R58K), c.C208A(p.P70T), and infertility phenotype was confirmed. We also discussed the possibility about patient II-1 from family 4 is affected by germinal/germline mosaicism. These results expand the kinds of variants and phenotypic spectrum of TUBB8 variants with regard to female infertility.

Summary Cerebral cavernous malformations (CCMs) are vascular disorders that affect up to 0.5% of the total population. About 20% of CCMs are inherited because of familial mutations in CCM genes, including CCM1/KRIT1, CCM2/MGC4607, and CCM3/PDCD10, whereas the etiology of a majority of simplex CCM-affected individuals remains unclear. Here, we report somatic mutations of MAP3K3, PIK3CA, MAP2K7, and CCM genes in CCM lesions. In particular, somatic hotspot mutations of PIK3CA are found in 11 of 38 individuals with CCMs, and a MAP3K3 somatic mutation (c.1323C>G [p.Ile441Met]) is detected in 37.0% (34 of 92) of the simplex CCM-affected individuals. Strikingly, the MAP3K3 c.1323C>G mutation presents in 95.7% (22 of 23) of the popcorn-like lesions but only 2.5% (1 of 40) of the subacute-bleeding or multifocal lesions that are predominantly attributed to mutations in the CCM1/2/3 signaling complex. Leveraging mini-bulk sequencing, we demonstrate the enrichment of MAP3K3 c.1323C>G mutation in CCM endothelium. Mechanistically, beyond the activation of CCM1/2/3-inhibited ERK5 signaling, MEKK3 p.Ile441Met (MAP3K3 encodes MEKK3) also activates ERK1/2, JNK, and p38 pathways because of mutation-induced MEKK3 kinase activity enhancement. Collectively, we identified several somatic activating mutations in CCM endothelium, and the MAP3K3 c.1323C>G mutation defines a primary CCM subtype with distinct characteristics in signaling activation and magnetic resonance imaging appearance.

The human zona pellucida (ZP) is a highly organized glycoprotein matrix that encircles oocytes and plays an essential role in successful reproduction. Previous studies have reported that mutations in human ZP1, ZP2 and ZP3 influence their functions and result in a lack of ZP or in an abnormal oocytes and empty follicle syndrome, which leads to female infertility. Here, we performed whole‐exome sequencing in two probands with primary infertility whose oocytes lacked a ZP, and we identified a heterozygous mutation in ZP1 (NM_207341:c.326G>A p.Arg109His), which is situated in the N‐terminus, and a heterozygous mutation in ZP3 (NM_001110354:c.400G>A p.Ala134Thr), which is situated in the ZP domain. The effects of the mutations were investigated through structure prediction and in vitro studies in HeLa cells. The results, which were in line with the phenotype, suggested that these mutations might impede the function of cross‐linking and secretion of ZP proteins. Our study showed that the two mutations in ZP1 and ZP3 influenced the formation of the ZP, causing female infertility. Meanwhile, these data highlight the importance of the ZP1 N‐terminus in addition to the conserved domains for ZP1 function and ZP formation. Additionally, the patient with the ZP1 mutation delivered a baby following intracytoplasmic sperm injection (ICSI); thus, we suggest the targeted genetic diagnosis of ZP genes to choose appropriate fertilization methods and improve the success rate of assisted reproductive technology (ART) treatments.

Background The identification of epidermal growth factor receptor (EGFR) mutations represents a milestone in the treatment of advanced non-small cell lung cancer. Patients with lung adenosquamous carcinomas (ASCs) rarely present with germline EGFR T790M mutation. The optimal treatment for cancers with germline EGFR T790M mutation (especially ASC) is not clear. Materials and Methods Using next-generation sequencing, we tested 450 cancer-related genes in a 27-year-old patient's lung adenosquamous carcinoma and matched blood samples. Germline mutations in samples from the patient's available relatives were identified by Sanger sequencing. Results We identified germline EGFR T790M mutation in the patient's lung adenosquamous carcinoma. He was treated with osimertinib and achieved complete response for more than 30 months, without significant drug-related adverse events. Genetic testing showed that germline EGFR T790M mutation might be a characteristic of inherited lung cancer. Conclusion Osimertinib can be a treatment option for patients with lung ASC harboring germline EGFR T790M mutation. Key Points A patient with adenosquamous carcinoma harboring a germline T790M mutation responded well to osimertinib with a progression-free survival of more than 30 months and without any unexpected toxicities. Osimertinib is effective for patients with lung squamous cell carcinoma with T790M and L858R mutations. The germline EGFR T790M mutation is associated with genetic susceptibility to lung cancer. The clinical use of next-generation sequencing could maximize the benefits of precision medicine in patients with cancer.

Aim: This study was conducted to reveal epigenetic landscape in infantile hemangiomas (IHs) and identify transcription factors (TFs) and their downstream genes active in IHs. Materials & methods: We performed Assay for Transposase Accessible Chromatin (ATAC-seq) with RNA-seq in three pairs of IHs and their adjacent normal tissues. Functions of candidate TFs were investigated in human umbilical vein endothelial cells (HUVECs). Results: Chromatin of IH tissues is less compact. Some candidate genes and TFs were identified. In HUVECs, SPDEF inhibited cell viability and tube formation, and promoted apoptosis; SOX4 exerted the opposite effect. SPDEF may act through EPHA5, ZBTB46 and SASH1; SOX4 may act through MMP12 and HIVEP3. Conclusion: Epigenetics plays a role in IHs. SPDEF and SOX4 may act in IHs.

Background Cerebral arteriovenous malformation (AVM) is a serious life-threatening congenital cerebrovascular disease. Specific anatomical features, such as nidus size, location, and venous drainage, have been validated to affect treatment outcomes. Until recently, molecular biomarkers and corresponding molecular mechanism related to anatomical features and treatment outcomes remain unknown. Methods RNA N6-methyladenosine (m6A) Methyltransferase METTL3 was identified as a differentially expressed gene in groups with different lesion sizes by analyzing the transcriptome sequencing (RNA-seq) data. Tube formation and wound healing assays were performed to investigate the effect of METTL3 on angiogenesis. In addition, Methylated RNA Immunoprecipitation Sequencing technology (MeRIP-seq) was performed to screen downstream targets of METTL3 in endothelial cells and to fully clarify the specific underlying molecular mechanisms affecting the phenotype of cerebral AVM. Results In the current study, we found that the expression level of METTL3 was reduced in the larger pathological tissues of cerebral AVMs. Moreover, knockdown of METTL3 significantly affected angiogenesis of the human endothelial cells. Mechanistically, down-regulation of METTL3 reduced the level of heterodimeric Notch E3 ubiquitin ligase formed by DTX1 and DTX3L, thereby continuously activating the Notch signaling pathway. Ultimately, the up-regulated downstream genes of Notch signaling pathway dramatically affected the angiogenesis of endothelial cells. In addition, we demonstrated that blocking Notch pathway with DAPT could restore the phenotype of METTL3 deficient endothelial cells. Conclusions Our findings revealed the mechanism by which m6A modification regulated the angiogenesis and might provide potential biomarkers to predict the outcome of treatment, as well as provide suitable pharmacological targets for preventing the formation and progression of cerebral AVM.

Brain arteriovenous malformations (AVMs) are congenital vascular abnormality in which arteries and veins connect directly without an intervening capillary bed. So far, the pathogenesis of brain AVMs remains unclear. Here, we found that Wilms' tumour 1‐associating protein (WTAP), which has been identified as a key subunit of the m6A methyltransferase complex, was down‐regulated in brain AVM lesions. Furthermore, the lack of WTAP could inhibit endothelial cell angiogenesis in vitro. In order to screen for downstream targets of WTAP, we performed RNA transcriptome sequencing (RNA‐seq) and Methylated RNA Immunoprecipitation Sequencing technology (MeRIP‐seq) using WTAP‐deficient and control endothelial cells. Finally, we determined that WTAP regulated Desmoplakin (DSP) expression through m6A modification, thereby affecting angiogenesis of endothelial cells. In addition, an increase in Wilms' tumour 1 (WT1) activity caused by WTAP deficiency resulted in substantial degradation of β‐catenin, which might also inhibit angiogenesis of endothelial cells. Collectively, our findings revealed the critical function of WTAP in angiogenesis and laid a solid foundation for the elucidation of the pathogenesis of brain AVMs.

KIAA1429 (also known as vir-like m(6)A methyltransferase-associated protein (VIRMA)), a newly identified component of the RNA m(6)A methyltransferase complex, plays critical roles in guiding region-selective m(6)A deposition. However, in mammals, whether KIAA1429 mediates RNA m(6)A regulatory pathway functions in vivo remains unknown. Here, we show that the Kiaa1429-specific deficiency in oocytes resulted in female infertility with defective follicular development and fully grown germinal vesicle (GV) oocytes failing to undergo germinal vesicle breakdown (GVBD) and consequently losing the ability to resume meiosis. The oocyte growth is accompanied by the accumulation of abundant RNAs and posttranscriptional regulation. We found that the loss of Kiaa1429 could also lead to abnormal RNA metabolism in GV oocytes. RNA-seq profiling revealed that Kiaa1429 deletion altered the expression pattern of the oocyte-derived factors essential for follicular development. In addition, our data show that the conditional depletion of Kiaa1429 decreased the m(6)A levels in oocytes and mainly affected the alternative splicing of genes associated with oogenesis. In summary, the m(6)A methyltransferase KIAA1429-mediated RNA metabolism plays critical roles in folliculogenesis and the maintenance of oocyte competence.

N6-methyladenosine (m6A) is the most prevalent epigenetic modification of messenger RNA (mRNA) in higher eukaryotes; this modification is mainly catalyzed by a methyltransferase complex including methyltransferase-like 3 (METTL3) as a key factor. Although m6A modification has been proven to play an essential role in diverse biological processes, our knowledge of Mettl3 is still limited because Mettl3 mutations are lethal to embryos in both mammals and plants. In this study, we knocked down Mettl3 by microinjection of its specific short interfering RNAs (siRNAs) or morpholino into fully grown germinal vesicle (GV) oocytes. As a result, we demonstrated that knocking down Mettl3 in female germ cells severely inhibited oocyte maturation by decreasing mRNA translation efficiency and led to defects in the maternal-to-zygotic transition, probably due to its interference in disrupting mRNA degradation. The discovery from this study suggests that the reversible m6A modification has vital functions in mammalian oocyte maturation and pre-implantation embryonic development processes.

Background Intestinal mucosa barrier dysfunction after burn injury is an important factor for causing mortality of burn patients. The current study established a burn model in rats and used a free radical scavenger edaravone (ED) to treat the rats, so as to investigate the effect of edaravone on intestinal mucosa barrier after burn injury. Methods Anesthetized rats were subjected to 40% total body surface area water burn immediately, followed by treatment with ED, scrambled antagomir, or antagomiR-320. Intestinal mucosa damage was observed by hematoxylin-eosin staining and graded by colon mucosal damage index (CMDI) score. The contents of total sulfhydryl (TSH), superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) were determined by spectrophotometry. Cell apoptosis, protein relative expression,and the in situ expressions of p-Akt and p-Bad were detected by flow cytometry, Western blotting and immunohistochemistry, respectively. The miR-320 expression was determined by quantitative real-time polymerase chain reaction. Results ED alleviated intestinal mucosal damage caused by burn injury, down-regulated the levels of MDA, cytochrome C, cleaved caspase-9 and cleaved caspase-3, but up-regulated the levels of TSH, SOD, CAT and Bcl-2. We also found that ED could reduce oxidative stress, inhibit cell apoptosis, increase the expressions of p-Akt, p-Bad and miR-320, and decrease PTEN expression. PTEN was predicted to be the target gene for miR-320, and cell apoptosis could be promoted by inhibiting miR-320 expression. Conclusion ED regulates Akt/Bad/Caspase signaling cascade to reduce apoptosis and oxidative stress through up-regulating miR-320 expression and down-regulating PTEN expression, thus protecting the intestinal mucosal barrier of rats from burn injury.

Objectives: Proteomics and high connotation functional gene screening (HCS) were used to screen key functional genes that play important roles in the pathogenesis of venous malformation. Furthermore, this study was conducted to analyze and explore their possible functions, establish a gene mutation zebrafish model, and perform a preliminary study to explore their possible pathogenic mechanisms in venous malformation.Methods: Pathological and normal tissues from patients with disseminated venous malformation were selected for Tandem Mass Tag (TMT) proteomics analysis to identify proteins that were differentially expressed. Based on bioinformatics analysis, 20 proteins with significant differential expression were selected for HCS to find key driver genes and characterize the expression of these genes in patients with venous malformations. In vitro experiments were then performed using human microvascular endothelial cells (HMEC-1). A gene mutant zebrafish model was also constructed for in vivo experiments to explore gene functions and pathogenic mechanisms.Results: The TMT results showed a total of 71 proteins that were differentially expressed as required, with five of them upregulated and 66 downregulated. Based on bioinformatics and proteomics results, five highly expressed genes and 15 poorly expressed genes were selected for functional screening by RNAi technology. HCS screening identified ACTA2 as the driver gene. Quantitative polymerase chain reaction (qPCR) and western blot were used to detect the expression of ACTA2 in the pathological tissues of patients with venous malformations and in control tissues, and the experimental results showed a significantly lower expression of ACTA2 in venous malformation tissues (P < 0.05). Cell assays on the human microvascular endothelial cells (HMEC-1) model showed that cell proliferation, migration, invasion, and angiogenic ability were all significantly increased in the ACTA2 over-expression group (P < 0.05), and that overexpression of ACTA2 could improve the inhibitory effect on vascular endothelial cell proliferation. We constructed an ACTA2-knockdown zebrafish model and found that the knockdown of ACTA2 resulted in defective vascular development, disruption of vascular integrity, and malformation of micro vein development in zebrafish. Further qPCR assays revealed that the knockdown of ACTA2 inhibited the Dll4/notch1 signaling pathway, Ephrin-B2 signaling pathway, and vascular integrity-related molecules and activated the Hedgehog signaling pathway.Conclusion: This study revealed that ACTA2 deficiency is an important factor in the pathogenesis of venous malformation, resulting in the disruption of vascular integrity and malformed vascular development. ACTA2 can be used as a potential biomarker for the treatment and prognosis of venous malformations.

Zygotic genomic activation (ZGA) is a landmark event in the maternal-to-zygotic transition (MZT), and the regulation of ZGA by maternal factors remains to be elucidated. In this study, the depletion of maternal ring finger protein 114 (RNF114), a ubiquitin E3 ligase, led to developmental arrest of two-cell mouse embryos. Using immunofluorescence and transcriptome analysis, RNF114 was proven to play a crucial role in major ZGA. To study the underlying mechanism, we performed protein profiling in mature oocytes and found a potential substrate for RNF114, chromobox 5 (CBX5), ubiquitylation and degradation of which was regulated by RNF114. The overexpression of CBX5 prevented embryonic development and impeded major ZGA. Furthermore, TAB1 was abnormally accumulated in mutant two-cell embryos, which was consistent with the result of in vitro knockdown of Rnf114. Knockdown of Cbx5 or Tab1 in maternal RNF114-depleted embryos partially rescued developmental arrest and the defect of major ZGA. In summary, our study reveals that maternal RNF114 plays a precise role in degrading some important substrates during the MZT, the misregulation of which may impede the appropriate activation of major ZGA in mouse embryos.

Valproate (VPA), an effective clinical approved anti-epileptic drug and mood stabilizer, has been believed to induce neuronal differentiation at the expense of inhibiting astrocytic and oligodendrocytic differentiation. Nevertheless, the involving mechanisms of it remain unclear yet. In the present study, we explored the global gene expression changes of fetus rat hippocampal neural stem cells following VPA treatment by high-throughput microarray. We obtained 874 significantly upregulated genes and 258 obviously downregulated genes (fold change > 2 and P < 0.05). Then, we performed gene ontology and pathway analyses of these differentially expressed genes and chose several genes associated with nervous system according to gene ontology analysis to conduct expression analysis to validate the reliability of the array results as well as reveal possible mechanisms of VPA. To get a better comprehension of the differentially regulated genes by VPA, we conducted protein-protein association analysis of these genes, which offered a source for further studies. In addition, we made the overlap between the VPA-downregulated genes and the predicted target genes of VPA-upregulated microRNAs (miRNAs), which were previously demonstrated. These overlapped genes may provide a source to find functional VPA/miRNA/mRNA axes during neuronal differentiation. This study first constructed a comprehensive potential downstream gene map of VPA in the process of neuronal differentiation.

Background Pancreatic cancer is a deadly malignancy with aggressive properties. MicroRNAs (miRNAs) participate in the pathogenesis of a variety of diseases and molecular processes by targeting functional mRNAs. Nevertheless, the regulatory role of miRNAs in signaling pathways involved in pancreatic cancer remains largely unknown. Aim To explore the molecular regulation involved in pancreatic cancer and potential mechanisms of miR-205. Methods Microarray analysis was performed to investigate the expression profile of miRNAs in pancreatic cancer. Expression of miR-205 was validated by qRT-PCR. Target prediction and functional enrichment analysis were employed to seek potential target genes of miR-205 and potential functions of these genes. The target binding of miR-205 and adenomatous polyposis coli (APC) was validated by luciferase reporter assay. APC protein expression in pancreatic cancer was validated by qRT-PCR and Western blot. Proliferation was evaluated by MTT and colony formation assays. Results A large number of miRNAs with altered expression were identified in pancreatic cancer. MiR-205 was significantly up-regulated. APC was found to be a validated target of miR-205 and down-regulated in pancreatic cancer. Proliferation experiments showed that miR-205 could promote cell proliferation in pancreatic cancer by targeting APC. Conclusion The above findings suggested that miR-205 mediated APC regulation contributes to pancreatic cancer development, which could be considered as a novel prognostic biomarker for clinical care.

Objective To compare the differences of brain apparent diffusion coefficient (ADC) values in surviving fetuses, twin controls and single fetus controls using diffusion weighted imaging (DWI) sequence, and to perform follow-up study to reveal the underlying cerebral microstructure changes. Materials and methods Thirty-two twins with demise of one fetus, 25 twins, and 20 single fetuses were included. DWI was performed and ADC map was reconstructed automatically. ADC values of certain regions were compared among three-groups, and between left- and right-hemisphere in surviving fetuses. ROC was generated to identify ADC values in surviving fetuses and twin controls. Results ADC values were lower in bilateral white matter of frontal lobes (left: 1.68 ± 0.19 vs. 1.91 ± 0.26, 1.88 ± 0.19, P P = 0.002), parietal lobes (left: 1.80 ± 0.25 vs. 2.00 ± 0.21, 1.92 ± 0.28, P = 0.012; right: 1.81 ± 0.19 vs. 1.99 ± 0.24, 1.92 ± 0.26, P = 0.012) and occipital lobes (left: 1.72 ± 0.24 vs. 1.87 ± 0.20, 1.70 ± 0.24, P = 0.017; right: 1.73 ± 0.23 vs. 1.91 ± 0.22, 1.70 ± 0.27, P = 0.005) in surviving fetuses compared with twin controls and single fetus, respectively. In discriminating surviving fetuses and twin controls, the area-under-the-curve of ADC values in frontal, parietal and occipital lobes were range from 0.677 to 0.737. The combination of frontal and parietal lobes and gestational age had highest area-under-the-curve (0.771, 95% CI 0.648–0.894). Conclusions DWI is a very useful sequence for detecting underlying changes. ADC values might be effective indicators of subtle anomalies in surviving fetuses. Trial registration: None.

Circular RNAs (circRNAs) are noncoding RNAs that have important roles in tumor progression. Previous studies have examined the circRNAs involved in infantile hemangioma (IH) tumors. The present study compared the circRNA levels in plasma samples from patients with IH and control individuals. The circRNA expression profiles were determined using microarray in three pairs of plasma samples from patients with proliferative IH and healthy control subjects. Expression of the identified differentially expressed circRNAs was verified using reverse transcription-quantitative PCR (RT-qPCR) and a bioinformatics analysis was performed to predict the microRNAs targeted by the validated circRNAs. From the circRNA expression profiles in the plasma of patients with IHs, 128 differentially expressed circRNAs were identified, of which 72 were upregulated and 56 were downregulated. The downregulated expression of three circRNAs [Homo sapiens (hsa)_circRNA_101566, hsa_circRNA_103546 and hsa_circRNA_103573] was verified using RT-qPCR. Gene Ontology term and Kyoto Encyclopedia of Genes and Genomes pathway analyses indicated that all identified networks participated in angiogenesis and tumor formation and progression. It was determined that hsa_circRNA_101566, which is able to regulate the mTOR signaling pathway, may be an important regulatory molecule in IH development and that targeting of hsa_miR_520c is able to indirectly regulate the vascular endothelial growth factor signaling pathway. Further studies are required to clarify these effects and the underlying mechanisms.

PAT1 homolog 2 (PATL2), encoding an RNA-binding protein, is a repressor involved in the translational regulation of maternal mRNAs during oocyte maturation. Previous studies have reported mutations inPATL2those led to female infertility with oocyte maturation arrest; however, the mechanisms by which mutations affected meiotic maturation remained unclear. Here, we identified several novel and recurrent mutations ofPATL2in patients with similar phenotype, and chose the missense mutation c.649 T>A p.Tyr217Asn inPATL2(PATL2Y217N) as a typical to investigate the underlying mechanisms. We confirmed that this mutation disturbed oocyte maturation and observed morphological defects of large polar body, symmetrical division and abnormal spindle after microinjection of corresponding mutated mRNA. We further evaluated the effect of the PATL2Y217Nmutation in 293T cells, and found this mutation decreased the ubiquitination level and degradation of PATL2. Then, abnormally increased PATL2 bound mRNAs of Mos, an upstream activator of mitogen activated protein kinase (MAPK), to regulate its translational activity and subsequently impaired MAPK signaling pathway and oocyte meiosis. These results dissented from the previous view thatPATL2mutations reduced their expression and highlight the role of PATL2 in translational regulation of Mos and its association with MAPK signaling pathway during oocyte meiotic maturation.

The T-cell immunoglobulin and mucin domain containing molecule 3 (TIM-3), a crucial immune regulatory molecule, is an emerging immune checkpoint target for cancer therapy. Our study aimed to investigate the association between TIM-3 polymorphisms (rs10053538 C > A, rs10515746 C > A, and rs1036199 A > C) and the susceptibility and prognosis of esophageal squamous cell carcinoma (ESCC). We further detect the effects of polymorphisms on TIM-3 expression. Two independent case-control sets (population-based and hospital-based sets) were performed in total 994 ESCC patients and 998 controls. TIM-3 polymorphisms were genotyped by polymerase chain reaction-ligase detection reaction (PCR). Survival data were available for 198 patients who received platinum-based chemotherapy after surgery. The regulation on TIM-3 expression by the polymorphisms was investigated in 35 patients using real-time quantitative PCR. The association between mRNA level of TIM-3 and survival was detected by using Kaplan-Meier plotter database. We found that for rs10053538 C > A polymorphisms, A allele was associated with significant increased risk of ESCC (odds ratios [OR] = 1.34, 95%CI = 1.05-1.72), and CA/AA genotypes enhanced susceptibility to ESCC for smokers (adjusted OR = 1.61, 95%CI = 1.00-2.59). The patients with AA genotypes had significantly poor prognosis (adjusted HR = 4.98, 95%CI = 1.14-21.71). The patients carrying CA/AA genotypes had significantly higher mRNA levels of TIM-3 than those carrying the CC genotype. Furthermore, high mRNA level of TIM-3 had a shorter overall survival in patients (HR = 2.56, 95%CI = 1.04-6.28). For rs10515746 C > A and rs1036199 A > C polymorphisms, there were no statistical correlation with the progression of ESCC. These data demonstrate that rs10053538 C > A polymorphisms may be associated with the susceptibility and prognosis of ESCC patients through regulating expression of TIM-3.

Keloid is an extremely common and often overlooked benign neoplastic disease, but its consequences should not be underestimated. Therefore, a deep exploration of the pathological mechanism of keloid becomes very essential. After 22 samples were collected from each patient's keloid tissues and normal skin tissues, circ_0008450 and Runx3 expression was tested by qRT-PCR. When primary human keratinized epithelial cells were transfected by sh-circ_0008450 or sh-Runx3, cell proliferation, apoptosis, migration, and EMT process were assessed by CCK-8, BrdU assay, apoptosis assay, migration assay, and Western blot. Finally, transfection was performed to explore the effect of circ_0008450 on the TGF-β/Smad signal pathway by adopting western blot. Circ_0008450 was highly expressed in keratinized epithelial tissues. After the transfection of sh-circ_0008450 into primary human keratinized epithelial cells, cell proliferation, migration, and EMT process were inhibited, and apoptosis was stimulated. Moreover, circ_0008450 silence-induced above changes were partly reversed by transfecting sh-Runx3. In addition, transfecting sh-circ_0008450 could repress TGF-β/Smad pathway, while transfecting sh-Runx3 activated the above pathway. Circ_0008450 down-regulated Runx3 to promote the proliferation and EMT process of human keratinized epithelial cells. This discovery may be related to the activation of the TGF-β/Smad pathway.

The Glioblastoma (GBM) immune microenvironment plays a critical role in tumor development, progression, and prognosis. A comprehensive understanding of the intricate milieu and its interactions remains unclear, and single-cell analysis is crucially needed. Leveraging mass cytometry (CyTOF), we analyzed immunocytes from 13 initial and three recurrent GBM samples and their matched peripheral blood mononuclear cells (pPBMCs). Using a panel of 30 markers, we provide a high-dimensional view of the complex GBM immune microenvironment. Hematoxylin and eosin staining and polychromatic immunofluorescence were used for verification of the key findings. In the initial and recurrent GBMs, glioma-associated microglia/macrophages (GAMs) constituted 59.05 and 27.87% of the immunocytes, respectively; programmed cell death-ligand 1 (PD-L1), T cell immunoglobulin domain and mucin domain-3 (TIM-3), lymphocyte activation gene-3 (LAG-3), interleukin-10 (IL-10) and transforming growth factor-β (TGFβ) demonstrated different expression levels in the GAMs among the patients. GAMs could be subdivided into different subgroups with different phenotypes. Both the exhausted T cell and regulatory T (Treg) cell percentages were significantly higher in tumors than in pPBMCs. The natural killer (NK) cells that infiltrated into the tumor lesions expressed higher levels of CXC chemokine receptor 3 (CXCR3), as these cells expressed lower levels of interferon-γ (IFNγ). The immune microenvironment in the initial and recurrent GBMs displayed similar suppressive changes. Our study confirmed that GAMs, as the dominant infiltrating immunocytes, present great inter- and intra-tumoral heterogeneity and that GAMs, increased exhausted T cells, infiltrating Tregs, and nonfunctional NK cells contribute to local immune suppressive characteristics. Recurrent GBMs share similar immune signatures with the initial GBMs except the proportion of GAMs decreases.

Our previous work found cancer-testis (CT) genes as a new source of epi-driver candidates of cancer. LIN28B was a CT gene, but the "driver" ability and the activation mechanism in lung adenocarcinoma (LUAD) remain unclear. We observed that LIN28B expression was restricted in testis. It was re-activated in LUAD patients without known genomic alterations in oncogenes and was related to poorer survival. In vitro and In vivo experiments confirmed that the activation of LIN28B could promote the proliferation and metastasis of LUAD cells and can influence cell cycle, DNA damage repair, and genome instability. In addition to the known let-7-LIN28B regulation loop, our results further revealed a let-7-independent Cis-regulator of LIN28B: LIN28B-AS1. LIN28B-AS1 is a CT long non-coding RNA (CT-lncRNA). It altered the messenger RNA stability of LIN28B by directly interacting with another CT protein IGF2BP1 but not with LIN28B and constituted a novel regulation network. In sum, we identify that LIN28B is an "epi-driver" of LUAD and clarify a new lncRNA-activated mechanism of LIN28B, which provide new candidate targets for precise anticancer therapy in the future.

What is the function of CAV1 in folliculogenesis and female reproduction?CAV1 regulates germline cyst breakdown and primordial follicle (PF) formation in mice, and CAV1 mutation may be related to premature ovarian insufficiency (POI).Pre-granulosa cells are essential for the establishment of the PF pool, which determines female fertility and reproductive lifespan. Cav1 participates in vascularization in fetal mouse ovaries. However, the role of CAV1 in early folliculogenesis and POI pathogenesis remains unclear.Cav1 function was investigated in mice and Human Embryonic Kidney 293 cells. Ovaries (six per group) were randomly assigned to Cav1-vivo-morpholino, control and control-morpholino groups, and all experiments were repeated at least three times. To investigate CAV1 mutations in women, 200 Chinese women with POI and 200 control individuals with regular menstrual cycles and normal endocrine profiles were recruited from the Center for Reproductive Medicine of Shandong University between September 2012 and December 2013.Wild-type CD1 mice, Lgr5-EGFP-ires-CreERT2 (Lgr5-KI) reporter mice and Human Embryonic Kidney 293 cells were used for these experiments. Protein expression was detected by Western blot, and quantitative RT-PCR was used to detect gene expression. The expression pattern of CAV1 in mouse ovaries and the phenotype of Cav1 deficiency in mice were detected by immunofluorescence. Pre-granulosa cell proliferation in ovaries was detected by bromodeoxyuridine (BrdU) assay and immunofluorescence. The coding region of the CAV1 gene was sequenced in 200 women with POI and 200 controls. The functional effect of the novel mutation c.142 GC (p.Glu48Gln) was investigated by Cell Counting Kit-8 (CCK8) assays and Western blot.We confirmed that Cav1 deficiency in mouse ovary induced by CAV1-vivo-morpholino resulted in more multi-oocyte follicles than in the control and control-morpholino groups (P0.01). Suppression of Cav1 decreased Leucine rich repeat containing G protein coupled receptor 5 (Lgr5)-positive cell proliferation (P0.01) and reduced the number of Lgr5 and Forkhead box L2 (Foxl2) double-positive cells (P0.01). Furthermore, suppression of Cav1 inhibited ovarian epithelial Lgr5-positive cell proliferation and differentiation through the Notch2 signalling pathway. Two of the POI women carried novel CAV1 mutations (c.45 CG synonymous and c.142 GC [Glu48Gln]). The deleterious effect of p.Glu48Gln was corroborated by showing that it adversely affected the function of CAV1 in cell proliferation and NOTCH2 expression in HEK293FT cells.N/A.The novel Glu48Gln mutation was only detected in one of 200 POI patients and we were unable to investigate its effects in the ovary.The identification of CAV1 as a potentially causative gene for POI provides a theoretical basis to devise treatments for POI in women.This work was supported by the National Basic Research Program of China (973 Programs: 2012CB944700; 2013CB945501; 2013CB911400; 2014CB943202), the National Key Research and Development Program of China (2016YFC1000604, 2017YFC1001301), the State Key Program of National Natural Science Foundation of China (81430029), and the National Natural Science Foundation of China (31571540, 81522018, 81471509, 81601245, 81701406, 81571406). The authors declare no competing financial interests.

Gut microbiota has an important role in the immune system, metabolism, and digestion, and has a significant effect on the nervous system. Recent studies have revealed that abnormal gut microbiota induces abnormal behaviors, which may be associated with the hypothalamic–pituitary–adrenal (HPA) axis. Therefore, we investigated the behavioral changes in germ-free (GF) mice by behavioral tests, quantified the basal serum cortisol levels, and examined glucocorticoid receptor pathway genes in hippocampus using microarray analysis followed by real-time PCR validation, to explore the molecular mechanisms by which the gut microbiota influences the host’s behaviors and brain function. Moreover, we quantified the basal serum cortisol levels and validated the differential genes in an Escherichia coli-derived lipopolysaccharide (LPS) treatment mouse model and fecal “depression microbiota” transplantation mouse model by real-time PCR. We found that GF mice showed antianxiety- and antidepressant-like behaviors, whereas E. coli LPS-treated mice showed antidepressant-like behavior, but did not show antianxiety-like behavior. However, “depression microbiota” recipient mice exhibited anxiety- and depressive-like behaviors. In addition, six glucocorticoid receptor pathway genes (Slc22a5, Aqp1, Stat5a, Ampd3, Plekhf1, and Cyb561) were upregulated in GF mice, and of these only two (Stat5a and Ampd3) were upregulated in LPS-treated mice, whereas the shared gene, Stat5a, was downregulated in “depression microbiota” recipient mice. Furthermore, basal serum cortisol levels were decreased in E. coli LPS-treated mice but not in GF mice and “depression microbiota” recipient mice. These results indicated that the gut microbiota may lead to behavioral abnormalities in mice through the downstream pathway of the glucocorticoid receptor. Herein, we proposed a new insight into the molecular mechanisms by which gut microbiota influence depressive-like behavior.

Germinal center (GC) B cell response is critical for pathogen protection. Chen et al. find that Uhrf1 is up-regulated by the c-Myc–AP4 axis and plays an essential role in GC B cell expansion and affinity maturation., The production of high-affinity antibody is essential for pathogen clearance. Antibody affinity is increased through germinal center (GC) affinity maturation, which relies on BCR somatic hypermutation (SHM) followed by antigen-based selection. GC B cell proliferation is essentially involved in these processes; it provides enough templates for SHM and also serves as a critical mechanism of positive selection. In this study, we show that expression of epigenetic regulator ubiquitin-like with PHD and RING finger domains 1 (Uhrf1) was markedly up-regulated by c-Myc–AP4 in GC B cells, and it was required for GC response. Uhrf1 regulates cell proliferation–associated genes including cdkn1a, slfn1, and slfn2 by DNA methylation, and its deficiency inhibited the GC B cell cycle at G1-S phase. Subsequently, GC B cell SHM and affinity maturation were impaired, and Uhrf1 GC B knockout mice were unable to control chronic virus infection. Collectively, our data suggest that Uhrf1 regulates GC B cell proliferation and affinity maturation, and its expression in GC B cells is required for virus clearance.

The maternal-to-zygotic transition (MZT) is essential for the developmental control handed from maternal products to newly synthesized zygotic genome in the earliest stages of embryogenesis, including maternal component (mRNAs and proteins) degradation and zygotic genome activation (ZGA). Various protein post-translational modifications have been identified during the MZT, such as phosphorylation, methylation and ubiquitination. Precise post-translational regulation mechanisms are essential for the timely transition of early embryonic development. In this review, we summarize recent progress regarding the molecular mechanisms underlying post-translational regulation of maternal component degradation and ZGA during the MZT and discuss some important issues in the field.

Major depressive disorder (MDD) is a common mood disorder. Gut microbiota may be involved in the pathogenesis of depression via the microbe–gut–brain axis. Liver is vulnerable to exposure of bacterial products translocated from the gut via the portal vein and may be involved in the axis. In this study, germ-free mice underwent fecal microbiota transplantation from MDD patients and healthy controls. Behavioral tests verified the depression model. Metabolomics using gas chromatography–mass spectrometry, nuclear magnetic resonance, and liquid chromatography–mass spectrometry determined the influence of microbes on liver metabolism. With multivariate statistical analysis, 191 metabolites were distinguishable in MDD mice from control (CON) mice. Compared with CON mice, MDD mice showed lower levels for 106 metabolites and higher levels for 85 metabolites. These metabolites are associated with lipid and energy metabolism and oxidative stress. Combined analyses of significantly changed proteins in livers from another depression model induced by chronic unpredictive mild stress returned a high score for the Lipid Metabolism, Free Radical Scavenging, and Molecule Transports network, and canonical pathways were involved in energy metabolism and tryptophan degradation. The two mouse models of depression suggest that changes in liver metabolism might be involved in the pathogenesis of MDD. Conjoint analyses of fecal, serum, liver, and hippocampal metabolites from fecal microbiota transplantation mice suggested that aminoacyl-tRNA biosynthesis significantly changed and fecal metabolites showed a close relationship with the liver. These findings may help determine the biological mechanisms of depression and provide evidence about “depression microbes” impacting on liver metabolism.

This study was conducted in order to elucidate the role microRNA-708 (miR-708) plays between proliferation, invasion, migration, and epithelial-mesenchymal transition (EMT) involving melanoma cells by targeting using LEF1 through the Wnt signaling pathway. Male Kunming mice were selected and subsequently divided into normal and model groups to take part in this study. Following cell line selection, the B16 cells with the highest miR-708 expression were selected and assigned into the control, blank, negative control (NC), miR-708 mimic, miR-708 inhibitor, siRNA-LEF1, and miR-708 inhibitor + siRNA-LEF1 groups. A Bioinformatics Web service and dual-luciferase reporter assay were conducted in order to determine the relationship between LEF1 and miR-708. The RT-qPCR method was performed in order to detect the miR-708 expression and mRNA expressions of LEF1, β-catenin, Wnt3a, N-cadherin, Bcl-2, Bax, Caspase3, E-cadherin, and western blotting was used in order to detect the protein expressions of these genes. MTT assay, scratch test, Transwell assay, and flow cytometry were all conducted in order to detect the cell proliferation, migration, invasion, and cycle/apoptosis, respectively. LEF1 was verified as the target gene of miR-708. In comparison with the normal group, the model group had reduced expressions of miR-708, Bax, Caspase3, and E-cadherin, while showing elevated expressions of LEF1, β-catenin, Bcl-2, Wnt3a, and N-cadherin. In comparison to the blank and control groups, the miR-708, mimic, and siRNA-LEF1 groups had elevated expressions of Bax, Caspase3, and E-cadherin, while also showing enhanced cell apoptosis. The miR-708, mimic, and siRNA-LEF1 groups also had decreased expressions of LEF1, β-catenin, Bcl-2, Wnt3a, and N-cadherin, and reduced optical density value 48 h and 72 h after transfection. Besides, these two groups showed declined cell migration and invasion, as well as lengthened G0/G1 phase (increased cell number) and shortened S phase (decreased cell number). Our findings demonstrated that an overexpressed miR-708 inhibits the proliferation, invasion, migration, and EMT, but also promotes the apoptosis of melanoma cells by targeting LEF1 through the suppression of the Wnt signaling pathway.

Piwi‐like RNA‐mediated gene silencing 1 (PIWIL1) has been identified as a novel extremely highly expressed cancer‐testis (CT) gene in lung adenocarcinoma. However, the exact function and mechanism of PIWIL1 in lung adenocarcinoma remains unclear. Herein, we sought to investigate the role of PIWIL1 in the occurrence and development of lung adenocarcinoma. We examined the expression pattern of PIWIL1 in The Cancer Genome Atlas (TCGA) lung adenocarcinoma samples, and validated it by Real‐Time PCR (RT‐PCR) in additional 21 paired lung adenocarcinoma tissues and 16 normal tissues. Subsequently, we explored the biological function of PIWIL1 in A549 and H1299 cell lines by gain and loss‐of‐function analyses. Using TCGA lung adenocarcinoma data, we further performed coexpression and Gene Ontology (GO) analyses, and analyzed the association of DNA methylation levels in PIWIL1 promoter region with its expression. Finally, we evaluated its expression in different mutation status of significantly mutated genes (SMGs) in TCGA lung adenocarcinoma data. We observed that PIWIL1 was expressed in testis and lung adenocarcinoma but not in other normal tissues, and its high expression was associated with shortened survival of lung cancer patients. Overexpression of PIWIL1 could facilitate the proliferation, invasion and migration of lung adenocarcinoma cells and vice versa. GO analysis revealed that PIWIL1 upregulated genes were enriched in embryonic development, cell proliferation and regulation of transcription. Moreover, promoter DNA hypomethylation of PIWIL1 could contribute to its aberrant expression in tumors. Interestingly, PIWIL1 expression was significantly higher in patients without hepatocyte growth factor (HGF) or serine/threonine kinase 11 (STK11) mutation (P = 0.006 and 0.005, respectively). PIWIL1 is an epidriver gene in lung adenocarcinoma, indicating a potential target for further therapy.

APOBEC3G (A3G) is a human enzyme that inhibits human immunodeficiency virus type 1 (HIV-1) infectivity, in the absence of the viral infectivity factor Vif, through deoxycytidine deamination and a deamination-independent mechanism. A3G converts from a fast to a slow binding state through oligomerization, which suggests that large A3G oligomers could block HIV-1 reverse transcriptase-mediated DNA synthesis, thereby inhibiting HIV-1 replication. However, it is unclear how the small number of A3G molecules found in the virus could form large oligomers. Here we measure the single-stranded DNA binding and oligomerization kinetics of wild-type and oligomerization-deficient A3G, and find that A3G first transiently binds DNA as a monomer. Subsequently, A3G forms N-terminal domain-mediated dimers, whose dissociation from DNA is reduced and their deaminase activity inhibited. Overall, our results suggest that the A3G molecules packaged in the virion first deaminate viral DNA as monomers before dimerizing to form multiple enzymatically deficient roadblocks that may inhibit reverse transcription., APOBEC3G inhibits HIV-1 viral replication via catalytic and non-catalytic processes. Here the authors show that APOBEC3G binds single-stranded DNA as an active deaminase monomer, subsequently forming catalytic-inactive dimers that block reverse transcriptase-mediated DNA synthesis.

The aim of the present study was to identify differentially expressed genes (DEGs) and their related functions and pathways of major burn injuries, and to prevent the occurrence of complications. The expression profiling of E-GEOD-37069 was downloaded from ArrayExpress Archive. The DEGs of major burn injuries were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG) functional enrichment analysis were then performed for the DEGs. Based on the KEGG database, a pathway relationship network was constructed, and DEGs in significant GO terms and pathways were investigated. Gene signal network and gene co-expression network of these inserted DEGs were constructed. A total of 3,328 DEGs of major burn injuries were identified, including 1,337 up- and 1,991 downregulated DEGs. These DEGs were mainly enriched into various GO terms, including transcription, DNA-dependent, signal transduction and blood coagulation. Moreover, they were also enriched into different pathways, such as hematopoietic cell lineage, metabolic pathway and chemokine signaling pathway. The pathway relationship network was constructed with 72 nodes. The MAPK signaling pathway was the hub node. Based on the same gene symbol, 702 DEGs were obtained, identified in both GO terms and pathways. Finally, the gene signaling network and gene co-expression network were constructed with 391 and 128 nodes, respectively. These identified DEGs, including GNB2, LILRA2, ARRB2 and ARHGEF2, may be potential key genes involved in the treatment of major burn injuries and prevention of complications.

The functional role of the ubiquitin‐proteasome pathway during maternal‐to‐zygotic transition (MZT) remains to be elucidated. Here we show that the E3 ubiquitin ligase, Rnf114, is highly expressed in mouse oocytes and that knockdown of Rnf114 inhibits development beyond the two‐cell stage. To study the underlying mechanism, we identify its candidate substrates using a 9,000‐protein microarray and validate them using an in vitro ubiquitination system. We show that five substrates could be degraded by RNF114‐mediated ubiquitination, including TAB1. Furthermore, the degradation of TAB1 in mouse early embryos is required for MZT, most likely because it activates the NF‐κB pathway. Taken together, our study uncovers that RNF114‐mediated ubiquitination and degradation of TAB1 activate the NF‐κB pathway during MZT, and thus directly link maternal clearance to early embryo development.

OBJECTIVEThe dominant inferior parietal lobe (IPL) contains cortical and subcortical structures that serve language processing. A high incidence of postoperative short-term aphasia and good potential for language reorganization have been observed. The authors’ goal was to study the plasticity of the language cortex and language-related fibers in patients with brain arteriovenous malformations (BAVMs) located in the IPL.METHODSA total of 6 patients who underwent microsurgical treatment of an IPL BAVM were prospectively recruited between September 2016 and May 2018. Blood oxygen level–dependent functional MRI (BOLD-fMRI) and diffusion tensor imaging (DTI) were performed within 1 week before and 6 months after microsurgery. Language-related white matter (WM) eloquent fiber tracts and their contralateral homologous fiber tracts were tracked. The Western Aphasia Battery was administered to assess language function. The authors determined the total number of fibers and mean fractional anisotropy (FA) indices for each individual tract. In addition, they calculated the laterality index (LI) between the activated language cortex voxels in the lesional and contralesional hemispheres and compared these indices between the preoperative and postoperative fMR and DT images.RESULTSOf the 6 patients with IPL BAVMs, all experienced postoperative short-term language deficits, and 5 (83.3%) recovered completely at 6 months after surgery. Five patients (83.3%) had right homologous reorganization of BOLD signal activations in both Broca’s and Wernicke’s areas. More fibers were observed in the arcuate fasciculus (AF) in the lesional hemisphere than in the contralesional hemisphere (1905 vs 254 fibers, p = 0.035). Six months after surgery, a significantly increased number of fibers was seen in the right hemispheric AF (249 fibers preoperatively vs 485 postoperatively, p = 0.026). There were significantly more nerve fibers in the postoperative left inferior frontooccipital fasciculus (IFOF) (874 fibers preoperatively vs 1186 postoperatively, p = 0.010). A statistically significant increase in right hemispheric dominance of Wernicke’s area was observed. The overall functional LI showed functional lateralization of Wernicke’s area in the right hemisphere (LI ≤ −0.20) in all patients.CONCLUSIONSThe authors’ findings provide evidence for the functional reorganization by recruiting the right hemispheric homologous region of Broca’s and Wernicke’s areas, right hemispheric AFs, and left hemispheric IFOFs following resection of IPL BAVMs.Clinical trial registration no.: NCT02868008 (clinicaltrials.gov)

The ubiquitin-like, containing PHD and RING finger domains, 1 (UHRF1) protein recognizes DNA methylation and histone modification and plays a critical role in epigenetic regulation. Recently, UHRF1 was shown to have a role in DNA methylation in oocytes and early embryos. Here, we reveal that maternal UHRF1 determines the quality of mouse oocytes. We generated oocyte-specific Uhrf1-knockout mice and found that females were sterile, and few maternal UHRF1-null embryos developed into blastocysts. The UHRF1-null oocytes had an increased incidence of aneuploidy and DNA damage. In addition to defective DNA methylation, histone modification was affected during oogenesis, with UHRF1-null germinal vesicle and metaphase II-stage oocytes exhibiting reduced global histone H3 lysine 9 dimethylation levels and elevated acetylation of histone H4 lysine 12. Taken together, our results suggest that UHRF1 plays an important role in determining oocyte quality and affects epigenetic regulation of oocyte maturation as a maternal protein, which is crucial for embryo developmental potential. Further exploration of the biologic function and underlying mechanisms of maternal UHRF1 will enhance our understanding of the maternal control of the oocyte and early embryonic development.-Cao, Y., Li, M., Liu, F., Ni, X., Wang, S., Zhang, H., Sui, X., Huo, R. Deletion of maternal UHRF1 severely reduces mouse oocyte quality and causes developmental defects in preimplantation embryos.

Polymorphisms in DNA repair genes can alter an individual's DNA repair capability and contribute to the risk of various cancers.This study was designed to evaluate the association of single-nucleotide polymorphisms (SNPs) in the XPG gene with the risk of gastric cardia adenocarcinoma (GCA) in a high-incidence population in northern China.Two SNPs from 431 GCA patients and 432 healthy controls were genotyped using the polymerase chain reaction/ligase detection reaction (PCR-LDR) method.The rs751402 C/T SNP T allele and the T/T genotype were associated with an increased risk of GCA in younger individuals (≤61 years) (odds ratio [OR] = 1.33 and 1.77, 95% confidence interval [CI] = 1.00-1.76 and 1.12-3.30, respectively). The rs873601 G/A SNP was not associated with susceptibility to GCA.Our findings indicate that the rs751402 C/T SNP has potential as a predictive marker for the risk of GCA and that carriers of the T/T genotype should receive periodic upper gastrointestinal fiber tests to facilitate the early detection and early treatment of GCA.

Triptolide is used in traditional Chinese medicine. It has the advantages of a unique mechanism of action, a wide antitumor spectrum, multiple targets, multi-channel effects and low toxicity. The current study was conducted to evaluate whether the potential anticancer effects of triptolide reduces proliferation and enhances apoptosis of human non‑small cell lung cancer (NSCLC) cells, and to assess the underlying anticancer mechanisms. In PC‑9 cells, treatment with triptolide reduced cell proliferation and increased cell apoptosis and caspase‑3 and 9 activity. Triptolide treatment reduced miR‑21 expression and enhanced phosphatase and tensin homolog (PTEN) protein expression levels in the PC‑9 cells. Furthermore, the upregulation of miR‑21 expression levels suppressed the effect of triptolide on cell viability and PTEN protein expression levels in PC‑9 cells. To the best of our knowledge, the present study is the first to demonstrate that triptolide reduced the proliferation and enhanced the apoptosis of human NSCLC cells through PTEN by targeting miR-21.

Primordial germ cells (PGCs) are embryonic founders of germ cells that ultimately differentiate into oocytes and spermatogonia. Embryonic proliferation of PGCs starting from E11.5 ensures the presence of germ cells in adulthood, especially in female mammals whose total number of oocytes declines after this initial proliferation period. To better understand mechanisms underlying PGC proliferation in female mice, we constructed a proteome profile of female mouse gonads at E11.5. Subsequent KEGG pathway analysis of the 3,662 proteins profiled showed significant enrichment of pathways involved in fatty acid degradation. Further, the number of PGCs found in in vitro cultured fetal gonads significantly decreased with application of etomoxir, an inhibitor of the key rate-limiting enzyme of fatty acid degradation carnitine acyltransferase I (CPT1). Decrease in PGCs was further determined to be the result of reduced proliferation rather than apoptosis. The inhibition of fatty acid degradation by etomoxir has the potential to activate the Ca(2+)/CamKII/5'-adenosine monophosphate-activated protein kinase (AMPK) pathway; while as an upstream activator, activated AMPK can function as activator of p53 to induce cell cycle arrest. Thus, we detected the expressional level of AMPK, phosphorylated AMPK (P-AMPK), phosphorylated p53 (P-p53) and cyclin-dependent kinase inhibitor 1 (p21) by Western blots, the results showed increased expression of them after treatment with etomoxir, suggested the activation of p53 pathway was the reason for reduced proliferation of PGCs. Finally, the involvement of p53-dependent G1 cell cycle arrest in defective proliferation of PGCs was verified by rescue experiments. Our results demonstrate that fatty acid degradation plays an important role in proliferation of female PGCs via the p53-dependent cell cycle regulation.

Adenomyosis, defined as ectopic endometrial tissue within the myometrium, can often be misdiagnosed as multiple uterine leiomyomata or endometrial thickening. We therefore performed a combined mRNA and long noncoding (lnc)RNA microarray and bioinformatic analysis of eutopic and ectopic endometria in women with adenomyosis to better understand its pathogenesis and help in the development of a semi-invasive diagnostic test. A total of 586 mRNAs were increased and 305 mRNAs decreased in the ectopic endometrium of adenomyosis compared with the eutopic endometrium, while 388 lncRNA transcripts were up-regulated and 188 down-regulated in ectopic compared with paired eutopic endometrial tissue. Bioinformatic analysis suggested a series of metabolic and molecular abnormalities in adenomyosis, which have many similarities with endometriosis. Furthermore, our study constitutes the first known report of lncRNA expression patterns in human adenomyosis ectopic and eutopic endometrial tissue.

The vascular endothelial growth factor (VEGF) and Notch signaling pathways have been identified to be involved in the neovascularization, and angiogenesis of various tumor types. However, there is little data regarding their roles and association in infantile hemangiomas (IHs). In the present study, the significance and association of the VEGF-VEGF receptor (R)2, and δ like canonical Notch ligand 4 (Dll4)-Notch1 pathways in different clinical phases of IHs were investigated. Specimens in the proliferating phase (n=15) and involuting phase (n=12) were collected. Quantitative polymerase chain reaction was used to analyze the mRNA levels of VEGF, VEGFR2, Dll4 and Notch1. A further 61 paraffin-embedded IHs (26 in the proliferating group and 35 in the involuting group) specimens were collected to investigate the protein levels of Notch1 and Dll4 using immunohistochemistry, and then analyzed for the association between these factors and microvessel density (MVD). The relative expression levels of VEGF, VEGFR2 and Dll4 mRNA in the proliferating group were significantly higher compared with that in the involuting group. In addition, the relative levels of Notch1 mRNA were similar in the proliferating and involuting phases. Expression levels of VEGFR2 and Dll4 mRNA were positively correlated with Notch1 expression in the proliferating IHs. The relative expression of VEGFR mRNA was positively correlated with Dll4 in the proliferating and involuting groups. Immunohistochemical staining demonstrated that Notch1 and Dll4 protein were upregulated in the proliferating IHs compared with the control group. In the proliferating IHs, Notch1 and Dll4 protein expression levels were positively correlated with MVD, while in the involuting phase, only Dll4 was positively correlated with MVD. The expression levels of related factors in the VEGF and Notch pathways, and the associations among them suggest that they may be involved in the regulation of IH neovascularization and angiogenesis.

Background Preeclampsia, a serious pregnancy-associated syndrome, is the leading cause of maternal and perinatal morbidity and mortality. Significant exacerbation of the hypercoagulation status as well as imbalanced steroid hormones have been reported in developed preeclampsia. However, it remains unclear whether the two pathological changes are directly associated. Method and results Our proteomic analysis revealed a significantly elevated SerpinF2/α2-antiplasmin level in preeclampsia plasma. Measurement of the longitudinally gestational change of plasmin-α2-antiplasmin (PAP) complex, testosterone, estradiol in preeclampsia patients and normal pregnant women demonstrated that the circulating PAP and testosterone levels in the early-onset preeclampsia (E-PE) patients were substantially higher, whereas estradiol concentration was significantly lower than that in normal pregnant controls from early pregnancy throughout gestation. Correlation analysis revealed that circulating PAP is in positive correlation with the concentration of testosterone, and in negative correlation with estradiol in E-PE patients. In E-PE placenta, the productions and activities of 17β-hydroxysteroid dehydrogenases 3 and aromatase, the essential enzymes for testosterone and estradiol synthesis, were compromised. In human renal and trophoblastic cells, testosterone and estradiol could regulate SerpinF2 expression in opposite ways. In addition, obvious fibrin deposition was colocalized with SerpinF2 in intervillous spaces and the area surrounding syncytiotrophoblasts in E-PE placenta. Conclusion The findings reveal a tight correlation between the imbalanced steroid hormone production and the procoagulation factor in E-PE patients, which provide potential biomarkers to predict preeclampsia, and bring new insight into the pathogenesis of preeclampsia.

In mammals, pronucleus formation, a landmark event for egg activation and fertilization, is critical for embryonic development. However, the mechanisms underlying pronucleus formation remain unclear. Increasing evidence has shown that the transition from a mature egg to a developing embryo and the early steps of development are driven by the control of maternal cytoplasmic factors. Herein, a two-dimensional-electrophoresis-based proteomic approach was used in metaphase II and parthenogenetically activated mouse eggs to search for maternal proteins involved in egg activation, one of which was poly(rC)-binding protein 1 (PCBP1). Phosphoprotein staining indicated that PCBP1 displayed dephosphorylation in parthenogenetically activated egg, which possibly boosts its ability to bind to mRNAs. We identified 75 mRNAs expressed in mouse eggs that contained the characteristic PCBP1-binding CU-rich sequence in the 3'-UTR. Among them, we focused on H2a.x mRNA, as it was closely related to pronucleus formation in Xenopus oocytes. Further studies suggested that PCBP1 could bind to H2a.x mRNA and enhance its stability, thus promoting mouse pronucleus formation during parthenogenetic activation of murine eggs, while the inhibition of PCBP1 evidently retarded pronucleus formation. In summary, these data propose that PCBP1 may serve as a novel maternal factor that is required for determining the normal timing of pronucleus formation.

Generation of haploid gametes by meiosis is a unique property of germ cells and is critical for sexual reproduction. Leaving mitosis and entering meiosis is a key step in germ cell development. Several inducers or intrinsic genes are known to be important for meiotic initiation, but the regulation of meiotic initiation, especially at the protein level, is still not well understood. We constructed a comparative proteome profile of female mouse fetal gonads at specific time points (11.5, 12.5, and 13.5 days post coitum), spanning a critical window for initiation of meiosis in female germ cells. We identified 3666 proteins, of which 473 were differentially expressed. Further bioinformatics analysis showed that these differentially expressed proteins were enriched in the mitochondria, especially in the electron transport chain and, notably, 9 proteins in electron transport chain Complex I were differentially expressed. We disrupted the mitochondrial electron transport chain function by adding the complex I inhibitor, rotenone to 11.5 days post coitum female gonads cultured in vitro. This treatment resulted in a decreased proportion of meiotic germ cells, as assessed by staining for histone γH2AX. Rotenone treatment also caused decreased ATP levels, increased reactive oxygen species levels and failure of the germ cells to undergo premeiotic DNA replication. These effects were partially rescued by adding Coenzyme Q10. Taken together, our results suggested that a functional electron transport chain is important for meiosis initiation. Our characterization of the quantitative proteome of female gonads provides an inventory of proteins, useful for understanding the mechanisms of meiosis initiation and female fertility.

Intestinal microbes are an important system in the human body, with significant effects on behavior. An increasing body of research indicates that intestinal microbes affect brain function and neurogenesis, including sensitivity to stress. To investigate the effects of microbial colonization on behavior, we examined behavioral changes associated with hormones and hormone receptors in the hypothalamic-pituitary-adrenal (HPA) axis under stress. We tested germ-free (GF) mice and specific pathogen-free (SPF) mice, divided into four groups. A chronic restraint stress (CRS) protocol was utilized to induce external pressure in two stress groups by restraining mice in a conical centrifuge tube for 4 h per day for 21 days. After CRS, Initially, GF restraint-stressed mice explored more time than SPF restraint-stressed mice in the center and total distance of the OFT. Moreover, the CRH, ACTH, CORT, and ALD levels in HPA axis of GF restraint-stressed mice exhibited a significantly greater increase than those of SPF restraint-stressed mice. Finally, the Crhr1 mRNA levels of GF CRS mice were increased compared with SPF CRS mice. However, the Nr3c2 mRNA levels of GF CRS mice were decreased compared with SPF CRS mice. All results revealed that SPF mice exhibited more anxiety-like behavior than GF mice under the same external stress. Moreover, we also found that GF mice exhibited significant differences in, hormones, and hormone receptors compared with SPF mice. In conclusion, Imbalances of the HPA axis caused by intestinal microbes could affect the neuroendocrine system in the brain, resulting in an anxiety-like behavioral phenotype. This study suggested that intervention into intestinal microflora may provide a new approach for treating stress-related diseases.

The aim of the study was to identify key long non-coding RNAs (lncRNA) and related subpathways following severe burn injuries and research their functions. The miRNA-mRNA and lncRNA-miRNA interactions were downloaded from starBase v2.0 database. In addition, mRNA-miRNA interactions were obtained from TarBase, mirTarBase, mir2Disease, miRecords (V4.0) databases. The relationships of lncRNA-miRNA-mRNA were constructed. Genes of expression profiling were intersected with mRNA and lncRNA in lncRNA-mRNA interaction. Screened mRNAs were enriched into various pathways and screened lncRNAs were embedded into candidate pathways. Wallenius approximation methods were used to calculate the false discovery rate value of each sub-pathway. Based on the results of significant sub-pathways, the related lncRNA-mRNA network was constructed. A total of 18,081 genes were obtained. The lncRNA-mRNA intersections including 835 lncRNAs, 1,749 mRNAs and 7,693 interacting pairs were constructed. The enriched mRNAs were further enriched into various candidate pathways such as ribosome biogenesis in eukaryotes. Several sub-pathways were screened, including ribosome biogenesis in eukaryotes and MAPK signaling pathway. The network of pathway-lncRNA-mRNA was constructed. Hub-genes were identified, including C14orf169 and YLPM1. Several hub-lncRNAs were obtained, including PRKAG2 antisense RNA 1 and LEF1 antisense RNA 1. Several hub-lncRNAs including C14orf169, YLPM1, TTTY15, and PCBP1-AS1 were screened. The sub-pathways regulated by these lncRNAs were identified, and functions were predicted.

MicroRNAs (miRNA) are considered to be potential therapeutic targets for the treatment of various cardiovascular diseases (CVDs). To understand the underlying mechanism of miRNAs and target genes associated with CVD, deep sequencing of blood samples from three patients with CVD and three controls was performed using the Illumina HiSeq 2000 system. The results of the present study revealed that 65 abnormal hsa‑miRNAs targeted 2,784 putative genes in patients with CVD; 59 upregulated miRNAs targeted 2,401 genes and six downregulated miRNAs targeted 383 genes. In addition, a total of 49 Gene Ontology (GO) biological processes and were enriched, and the target genes of downregulated miRNAs were enriched in 12 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Most of these pathways are responsible for lipid and glycan metabolism. In particular, three downregulated miRNAs, hsa‑miR‑1268b, hsa‑miR‑1273d, hsa‑miR‑3187‑5p, were involved in a‑linolenic acid metabolism. The target genes of upregulated miRNAs were enriched in 15 KEGG pathways, mainly in the 'neurodegenerative diseases and cancers' class. In the present study five novel upregulated miRNAs, including m0499‑5p, m0970‑5p, m1042‑5p, m1061‑5p and m1953‑5p, and a downregulated miRNA, novel‑m1627‑5p, were identified in patients with CVD. Novel‑m1627‑5p was demonstrated to target 146 human genes. Additionally, Novel‑m1061‑5p targeted four genes, including fumarylacetoacetate hydrolase domain containing 2A, potassium voltage‑gated channel, Shaw‑related subfamily, member 4, coiled‑coil domain containing 85C and solute carrier family 35 member E3 (SLC35E3). The GO term, 'carbohydrate derivative transport involving in biological process', was associated with SLC35E3. Novel‑m1061‑5p in patients with CVD may repress the expression levels of SLC35E3, a member of the nucleoside sugar transporter subfamily E, which is known to cause defective glycol‑conjugation in the Golgi complex and/or the endoplasmic reticulum. Further investigation is required to understand the underlying mechanisms of the novel miRNAs. Novel‑m1061‑5p may serve as a marker for prognosis or a potential target for the treatment of CVD.

Background Circular RNAs (circRNAs) are a recently identified class of noncoding RNAs that participate in several physiological processes. However, the expression of circRNAs in infantile hemangioma (IH) remains unknown. Methods The profile of circRNAs was assessed by microarray in four pairs of IH and adjacent skin tissues. The expression of circRNAs was validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, circRNA-microRNAs (miRNA)-mRNA networks were constructed using bioinformatics tools. Results 234 up- and 374 down- regulated circRNAs were identified in IH by microarray. Among them, the expression of two up-regulated circRNAs (hsa_circRNA_100933 and hsa_circRNA_100709) and one down-regulated circRNA (hsa_circRNA_104310) was confirmed by qRT-PCR. In addition, 3,019 miRNA response elements (MREs) of circRNAs were predicted, and two circRNA-miRNA-mRNA networks were constructed, including 100 and 94 target genes of hsa_circRNA_100933 and hsa_circRNA_104310, respectively. GO and pathway analysis showed that both networks participated in angiogenesis and vascular development-related biological processes. Conclusions This is the first study to reveal the profiling of circRNAs in IH and pave the way for further characterization of the role of circRNAs in the pathogenesis of IH.

Hemangiomas are the most common vascular tumors that occur frequently in prematures and females. microRNA (miR)-130a is associated with the growth and invasion in many tumors, and its role in hemangiomas has not been addressed so far. The present study revealed that miR‑130a was overexpressed in infantile hemangioma tissues compared with matched tumor-adjacent tissues. The inhibitor of miR-130a restrained cell growth and induced cell apoptosis in vitro. miR‑130a inhibitor also induced a cell cycle arrest at G2/M phase. Further studies revealed that tissue factor pathway inhibitor 2 (TFPI2) was a novel miR-130a target, due to miR-130a bound directly to its 3'-untranslated region and miR-130a inhibitor enhanced the expression of TFPI2. Contrary to the effects of miR-130a inhibitor, TFPI2 siRNA strongly promoted cell growth and colony formation, whereas TFPI2 overexpression contributed to the suppressing effect of miR-130a inhibitor in cell viability. Furthermore, miR-130a inhibitor reduced the activation of focal adhesion kinase (FAK)/phosphoinositide 3-kinase (PI3K)/Rac1/anti-mouse double minute (mdm2) pathway proteins, inhibited the expression and nuclear translocation of mdm2. Moreover, FAK overexpression prevented miR-130a inhibitor-induced cell cycle arrest and decrease of cell viability. In vivo experiments, miR-130a inhibition effectively suppressed the tumor growth, restrained angiogenesis by decreasing the expression of angiogenesis markers and the percentage of CD31+ and CD34+. Taken together, our research indicated that miR-130a functions as an oncogene by targeting TFPI2, miR-130a inhibition reduced the growth and angiogenesis of hemangioma by inactivating the FAK/PI3K/Rac1/mdm2 pathway. Thus, miR-130a may serve as a potential therapeutic strategy for the treatment of hemangioma.

The vascular endothelial growth factor (VEGF) and Notch signaling pathways have been identified to be involved in the neovascularization, and angiogenesis of various tumor types. However, there is little data regarding their roles and association in infantile hemangiomas (IHs). In the present study, the significance and association of the VEGF-VEGF receptor (R)2, and δ like canonical Notch ligand 4 (Dll4)-Notch1 pathways in different clinical phases of IHs were investigated. Specimens in the proliferating phase (n=15) and involuting phase (n=12) were collected. Quantitative polymerase chain reaction was used to analyze the mRNA levels of VEGF, VEGFR2, Dll4 and Notch1. A further 61 paraffin-embedded IHs (26 in the proliferating group and 35 in the involuting group) specimens were collected to investigate the protein levels of Notch1 and Dll4 using immunohistochemistry, and then analyzed for the association between these factors and microvessel density (MVD). The relative expression levels of VEGF, VEGFR2 and Dll4 mRNA in the proliferating group were significantly higher compared with that in the involuting group. In addition, the relative levels of Notch1 mRNA were similar in the proliferating and involuting phases. Expression levels of VEGFR2 and Dll4 mRNA were positively correlated with Notch1 expression in the proliferating IHs. The relative expression of VEGFR mRNA was positively correlated with Dll4 in the proliferating and involuting groups. Immunohistochemical staining demonstrated that Notch1 and Dll4 protein were upregulated in the proliferating IHs compared with the control group. In the proliferating IHs, Notch1 and Dll4 protein expression levels were positively correlated with MVD, while in the involuting phase, only Dll4 was positively correlated with MVD. The expression levels of related factors in the VEGF and Notch pathways, and the associations among them suggest that they may be involved in the regulation of IH neovascularization and angiogenesis.