Search

Several studies have confirmed that microglia are involved in neuropathic pain. Inhibition of guanosine-5'-triphosphate cyclohydrolase 1 (GTPCH1) can reduce the inflammation of microglia. However, the precise mechanism by which GTPCH1 regulates neuropathic pain remains unclear. In this study, BV2 microglia were transfected with adenovirus to knockdown GTPCH1 expression. High throughput sequencing analysis revealed that the mitogen-activated protein kinase (MAPK) related pathways and proteins were the most significantly down-regulated molecular function. Co-expression network analysis of Mapk14 mRNA and five long noncoding RNAs (lncRNAs) revealed their correlation. Quantitative reverse transcription-polymerase chain reaction revealed that among five lncRNAs, ENSMUST00000205634, ENSMUST00000218450 and ENSMUST00000156079 were related to the downregulation of Mapk14 mRNA expression. These provide some new potential targets for the involvement of GTPCH1 in neuropathic pain. This study is the first to note the differential expression of lncRNAs and mRNA in GTPCH1 knockdown BV2 microglia. Findings from this study reveal the mechanism by which GTPCH1 activates microglia and provide new potential targets for microglial activation in neuropathic pain.

Androgen insensitivity syndrome (AIS) is an X-linked recessive genetic disease characterized by disorders of sex development, commonly caused by mutations of the androgen receptor (AR) gene. Herein, we identified a novel hemizygous mutation (c.2118T A, p. Asn706Lys) of AR resulting in complete androgen insensitivity syndrome (CAIS) in twins. This missense mutation contributed to significantly decreased mRNA transcription and protein expression. In addition, structure model analysis showed that Asn706Lys resulted in loss of hydrogen bond with Asp891 and reduced protein stability. Furthermore, the mutant AR failed to bind to ligand due to the loss of hydrogen bond with dihydrotestosterone (DHT). This disrupted the translocation of AR protein from cytoplasm to nucleus after hormone stimulation. Our findings firstly demonstrated the novel mutation of c.2118T A in AR directly caused CAIS. This contributed to expanding the AR mutational spectrum and revealed the pathogenic mechanism of AIS, as well as facilitating precise diagnosis and genetic counseling.

Neuropathic pain (NP) is a chronic pain directly caused by injury or disease of the somatosensory nervous system. Previous studies suggest that GTP cyclohydrolase I (GCH1) may play a pivotal role in microglial activation, which has been shown to be essential for NP. However, its underlying mechanisms in microglial activation remain unclear. A wide range of microRNAs (miRNAs) have been found to be involved in microglial activation-induced NP. To identify the miRNAs regulated by GCH1 and predict their functions in the progression of microglial activation, we analyzed the miRNA expression profiles of GCH1-knockdown (KD) BV2 microglial cells. Small RNA-sequencing analysis revealed 13 differentially expressed (DE) miRNAs in GCH1-KD cells. The target genes of DE miRNAs mainly participate in PI3K-Akt signaling pathway, peroxisome and ferroptosis. The miRNA–mRNA regulatory network analysis showed that GCH1, MAP4K5 and YWHAB acted as hub genes. qRT-PCR results further verified the expression levels of mmu-miR-1a-3p, mmu-miR-133a-3p, mmu-miR-7a-5p and mmu-miR-10a-5p in GCH1-KD cells, which were consistent with the sequencing data. In addition, our data indicated that overexpression of mmu-miR-133a-3p alleviated the pro-inflammatory cytokines IL-1β and IL-6 production induced by lipopolysaccharide (LPS), indicating that mmu-miR-133a-3p has a negative effect on microglial activation. Taken together, our findings suggest that many miRNAs regulated by GCH1 may be involved in microglial activation, which may provide new potential targets for GCH1 in the pathogenesis of NP.

Objective: To evaluate the effect of preimplantation genetic testing for aneuploidy (PGT-A) in infertile patients with recurrent pregnancy loss (RPL). Methods: A prospective randomized clinical trial was performed in a university-affiliated fertility center in Shanghai, China. Patients in the PGT-A group underwent blastocyst biopsy followed by single-nucleotide polymorphism microarray-based PGT-A and single euploid blastocyst transfer, whereas patients in the control group underwent routinein vitro fertilization/ICSI procedures and frozen embryo transfer of 1–2 embryos selected according to morphological standards. Results: Two hundred and seven infertile patients with RPL were included in this study and randomly assigned to either the control or the PGT-A group. Baseline variables and cycle characteristics were comparable between the two groups. The results showed that PGT-A significantly improved the ongoing pregnancy rate (55.34% vs. 29.81%) as well as the live birth rate (48.54% vs. 27.88%) and significantly reduced the miscarriage rate (0.00% vs. 14.42%) on a per-patient analysis. A significant increase in cumulative ongoing pregnancy rates over time was observed in the PGT-A group. Subgroup analysis showed that the significant benefit diminished for patients who attempted ≥2 PGT-A cycles. Conclusions: PGT-A significantly improved the ongoing pregnancy and live birth rate, while reduced miscarriage rate in infertile RPL patients. However, the significance diminished in patients attempting ≥2 cycles; thus, further studies are warranted to explore the most cost-effective number of attempts in these patients to avoid overuse.

Intervertebral disc degeneration (IDD) leads to low back pain (LBP). This study aimed to determine the regulation of IDD by competing endogenous RNAs (ceRNAs). We obtained the GSE63492, GSE124272, and GSE129789 datasets from the Gene Expression Omnibus database. The changes of long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and mRNAs in IDD were characterized. The significantly changed mRNAs were subjected to protein-protein interaction analysis using the STRING database, and its functions and involved pathways were analyzed using the DAVID database and gene set enrichment analysis (GSEA). The significant changed lncRNAs, miRNAs and mRNAs were linked in a ceRNA network based on their interactions - predicted by Starbase and miRWalk. Differentially methylated loci of significantly changed mRNAs in early and advanced IDD were compared using the GSE129789 dataset. We identified 245 significantly changed mRNAs, 133 lncRNAs, and 228 miRNAs between patients with IDD and normal individuals. GSEA suggested that 17 pathways related to cell proliferation were activated while 35 cell signaling and immune-related pathways were suppressed in IDD. The following ceRNA network in IDD was built: LINC00665/hsa-miR-7-5p/FZD3, ZNF549; LINC00665/hsa-let-7e-5p/FZD3, ACVR2B; TRG-AS1/hsa-miR-574-5p/ACVR2B, P3H2; TRG-AS1/ hsa-let-7e-5p/FZD3, ACVR2B; and ZNF571-AS1/let-7e-5p/ACVR2B, FZD3. A lncRNA-miRNAmRNA ceRNA network which might regulate the progression of IDD was developed.

Early embryonic arrest is a challenge for in vitro fertilization (IVF). No genetic factors were previously revealed in the sperm-derived arrest of embryonic development. Here, we reported two infertile brothers presenting normal in conventional semen analysis, but both couples had no embryos for transfer after several IVF and intracytoplasmic sperm injection (ICSI). Whole-exome sequencing identified a homozygous missense mutation of ACTL7A in both brothers. This mutation is deleterious and causes sperm acrosomal ultrastructural defects. The Actl7a knock-in mouse model was generated, and male mutated mice showed sperm acrosomal defects, which were completely consistent with the observations in patients. Furthermore, the sperm from ACTL7A/Actl7a-mutated men and mice showed reduced expression and abnormal localization of PLCζ as a potential cause of embryonic arrest and failure of fertilization. Artificial oocyte activation could successfully overcome the Actl7a-mutated sperm-derived infertility, which is meaningful in the future practice of IVF/ICSI for the ACTL7A-associated male infertility.

Neuropathic pain is a severe and chronic neurological disease caused by injury or disease of the somatosensory system. Currently, there are no effective treatments for neuropathic pain. Neuroinflammation, characterized by activation of spinal glial cells and increased production of pro-inflammatory cytokines (for example, IL-1β, TNF-α and IL-6), is a pathophysiological process closely related to neuropathic pain. The anti-inflammatory drug sulfasalazine (SFZ) is approved for inflammatory bowel disease and rheumatoid arthritis. Although the analgesic effect of SFZ has been reported in diabetic mice, its role in neuropathic pain caused by peripheral nerve injury has not been clarified. Here, we show that SFZ significantly alleviated mechanical hypersensitivity and attenuated neuroinflammatory response in neuropathic pain induced by chronic constriction injury (CCI) in rats. Additionally, SFZ inhibited the activation of astrocytes and abolished the CCI-induced increase of NF-κB in the spinal cord. Hence, our results show that SFZ is a potential treatment for neuropathic pain induced by peripheral nerve injury.

Background MicroRNAs (miRNAs) are a class of noncoding small RNAs that play important roles in many physiological processes by regulating gene expression. Previous studies have shown that the expression levels of total miRNAs increase during mouse embryonic development, and some miRNAs control the regulatory network in development progression. However, few studies have focused on the effects of miRNAs on early human embryonic development. The relationship between miRNAs and early human embryogenesis is still unknown. Results In this study, RNA-seq data collected from sperm samples from 102 patients with a normal sperm index but treated with assisted reproductive technology (ART) were analyzed for the relationships between differentially expressed small RNAs and the fertilization rate (FR), blastocyst rate and high-quality embryo rate (HQER). The sperm samples with high hsa-mir-191 expression had a higher FR, effective embryo rate (EER) and HQER. hsa-mir-191 was used as a single indicator to predict the HQER. The receiver operating characteristic (ROC) curve had an area under the ROC curve (AUC) of 0.686. We also found that hsa-mir-191 expression is correlated with an abnormal sperm rate (cor = 0.29, p Conclusions These findings suggest that high hsa-mir-191-5p expression in sperm is associated with early human embryonic quality and that hsa-mir-191-5p could be used as a potential marker to screen high-quality sperm to improve the success rates of in vitro fertilization (IVF).

Background: MicroRNAs (miRNAs) are a class of noncoding small RNAs that play important roles in many physiological processes by regulating gene expression. Previous studies have shown that the expression levels of total miRNAs increase during mouse embryonic development, and some miRNAs control the regulatory network in development progression. However, few studies have focused on the effects of miRNAs on early human embryonic development. The relationship between miRNAs and early human embryogenesis is still unknown. Results: In this study, sperm samples from 102 patients with a normal sperm index but treated with assisted reproductive technology (ART) were collected for small RNA sequencing, and the relationships between differentially expressed small RNAs and the fertilization rate (FR), blastocyst rate and high-quality embryo rate (HQER) were analyzed. The sperm samples with high hsa-mir-191 expression had a higher FR, effective embryo rate (EER) and HQER. hsa-mir-191 was used as a single indicator to predict the HQER. The receiver operating characteristic (ROC) curve had an area under the ROC curve (AUC) of 0.686. We also found that hsa-mir-191 expression is correlated with an abnormal sperm rate (cor = 0.29, p< 0.01). We also evaluated the relationship between hsa-mir-34c and early human embryo development in these 102 sperm samples and obtained negative results. Conclusions: These findings suggest that high hsa-mir-191-5p expression in sperm is associated with early human embryonic quality and that hsa-mir-191-5p could be used as a potential marker to screen high-quality sperm to improve the success rates of in vitro fertilization (IVF). Keywords: miRNA, hsa-mir-191, hsa-mir-34c, RNA-Seq, Embryonic development

ACTL7A deficiency is a novel male factor inducing early embryonic arrest that can be overcome by artificial oocyte activation., Early embryonic arrest is a challenge for in vitro fertilization (IVF). No genetic factors were previously revealed in the sperm-derived arrest of embryonic development. Here, we reported two infertile brothers presenting normal in conventional semen analysis, but both couples had no embryos for transfer after several IVF and intracytoplasmic sperm injection (ICSI). Whole-exome sequencing identified a homozygous missense mutation of ACTL7A in both brothers. This mutation is deleterious and causes sperm acrosomal ultrastructural defects. The Actl7a knock-in mouse model was generated, and male mutated mice showed sperm acrosomal defects, which were completely consistent with the observations in patients. Furthermore, the sperm from ACTL7A/Actl7a-mutated men and mice showed reduced expression and abnormal localization of PLCζ as a potential cause of embryonic arrest and failure of fertilization. Artificial oocyte activation could successfully overcome the Actl7a-mutated sperm-derived infertility, which is meaningful in the future practice of IVF/ICSI for the ACTL7A-associated male infertility.

DEFB126 rs140685149 mutation was shown to cause sperm dysfunction and subfertility. Indel rs11467497 is another 4-nucleotide frame-shift mutation (151bp upstream of rs140685149) that leads to the premature termination of translation and the expression of peptide truncated at the carboxyl terminus. In the present study, we performed a comprehensive association study to check the contribution of rs140685149 and rs11467497 to male infertility. Our results confirmed the previous findings that there was no association between rs140685149 and sperm motility. In contrast, we found a significant association of another indel rs11467497 with male infertility. Moreover, rs11467497 was shown to be associated with higher number of round cells in the infertile males with low sperm motility. Surprisingly, the two mutations commonly existed in the sperm donors (n = 672), suggesting a potential application of the two indels in the screening for eligible sperm donors. Western blotting assays showed the sperms with rs140685149 2-nt deletion tended to have unstable DEFB126 protein in contrast of no DEFB126 protein expressed in the sperms with rs11467497 4-nt deletion, suggesting a more severe consequence caused by rs11467497 mutation. In conclusion, our study presented a significant contribution of another functional frame-shift polymorphism of DEFB126 (rs11467497) to male infertility.

Background Semen cryopreservation has been widely applied in assisted reproductive technologies and sperm bank, but it causes considerable impairments on sperm quality. It is necessary to find an evaluation indicator for determining the sperm-freezing tolerance. Methods The glycocalyx of good freezability ejaculates was compared with poor freezability ejaculates by lectin microarray. The significant different lectins were validated by flow cytometry (FACS). To analyze the relationship between the potential biomarker and the tolerance of sperm to cryopreservation, 60 samples with different recovery rates were collected and detected the lectin-binding intensity by FACS. The receiver operating characteristic (ROC) curve was analyzed to test the capability of the lectin as a potential biomarker for detecting the sperm freezablility. Results ABA and DSL were found to develop significant differences between them. Further validation showed that ABA was significantly negative correlated with the sperm recovery rates (r = − 0.618, P

The role of AMP-activated protein kinase (AMPK) in the regulation of energy metabolism and the control of skeletal muscle regeneration post injury has been described previously. It remains unknown whether this metabolic sensor plays a role in the mechanism of axonal regeneration post injury. In this study, we used a sciatic nerve crushed mouse model to detect the expression of AMPK in sciatic nerve and spinal motor neurons at 1 week, 2 weeks and 3 weeks after injury by immunofluorescence staining. Electrophysiological and histopathological studies were used to confirm the nerve injury and regeneration. Our results showed that frequency of AMPK-positive spinal motor neurons was significantly higher on day 7 after sciatic nerve crush (SNC) and peaked on day 14. No expression of AMPK was detected in axons of the sciatic nerve before and after the injury. Taken together, our study suggested a possible role of AMPK in the mechanism of motor nerve regeneration after injury. Keywords: Sciatic nerve injury, Motor neuron, AMP-Activated protein kinase

Tamoxifen is administered for estrogen receptor positive (ER+) breast cancers, but it can induce uterine endometrial cancer and non-alcoholic fatty liver disease (NAFLD). Importantly, ten years of tamoxifen treatment has greater protective effect against ER+ breast cancer than five years of such treatment. Tamoxifen was also approved by the FDA as a chemopreventive agent for those deemed at high risk for the development of breast cancer. The side effects are of substantial concern because of these extended methods of tamoxifen administration. In this study, we found that anordrin, marketed as an antifertility medicine in China, inhibited tamoxifen-induced endometrial epithelial cell mitosis and NAFLD in mouse uterus and liver as an anti-estrogenic and estrogenic agent, respectively. Additionally, compared with tamoxifen, anordiol, the active metabolite of anordrin, weakly bound to the ligand binding domain of ER-α. Anordrin did not regulate the classic estrogen nuclear pathway; thus, it did not affect the anti-tumor activity of tamoxifen in nude mice. Taken together, these data suggested that anordrin could eliminate the side effects of tamoxifen without affecting its anti-tumor activity.

To construct an adenovirus vector co-expressing human bone morphogenetic protein (hBMP2) and human vascular endothelial growth factor (hVEGF165) as well as green fluorescence protein (GFP) as a marker, with which the intracellular expression of the inserted genes could be identified in Bone marrow mesenchymal stem cells (BM-MSCs). BMP2 and VEGF165 genes were PCR amplified from a cDNA library and inserted to the polyclonal site of adenovirus shuttle plasmid pAd-MCMV-GFP. The virus solution (Ad-BMP2-VEGF165) was generated by co-transfecting HEK293 cells with the constructed recombinant shuttle plasmid pAd-MCMV-BMP2-VEGF165 and adenovirus helper plasmid pBHGloxΔ (delta) E1, 3Cre. The virus solution was further purified and virus titer was determined accordingly. The expression of the target genes was subsequently detected and quantified in rabbit BM-MSCs by using real time PCR, ELISA and Western blotting. The recombinant adenovirus vector containing BMP2 and VEGF165 (Ad-BMP2-VEGF165) was successfully constructed, which was confirmed by Sanger sequencing, colony PCR, as well as visually detection of GFP, and the titer of the adenovirus was 1×10(10)PFU/mL, and the proteins level of BMP2 and VEGF165 secreted in the supernatant are significantly higher than the control. Recombinant adenovirus vector containing hBMP2 and hVEGF165 genes was successfully constructed. The transfection rate of BM-MSCs by the adenovirus was high (95% at 100 MOI) and the BMP2 and VEGF165 genes was highly expressed in the cells. The present study provides a method to efficiently express the target genes in BM-MSCs and an vector for further research of bone defect repair using dual genes of BMP2 and VEGF165.

The angular dependence of anisotropic magnetoresistance (AMR) and planar Hall effect (PHE) were studied as a function of temperatures from the same epitaxial Fe 3 O 4 film on MgO(001) substrates. The PHE contains only a twofold angular dependence, but the AMR below 200 K is constituted with both twofold and fourfold symmetric terms. Our results also prove that the origin of the fourfold symmetry of AMR is related to the lattice symmetry rather than the spin scattering near the antiphase boundaries.

Coating on the sperm surface, glycocalyx, plays a key role in sperm motility, maturation and fertilization. A comprehensive profile of sperm surface glycans will greatly facilitate both basic researches and clinical studies. Because of the capability of recognizing different glycan moieties, lectins are widely used in glycobiology. However, lacking high-throughput technology, limited lectins have been reported for analyzing the glycan of human sperm. In this study, we employed a lectin microarray for profiling the surface glycans of human sperm, on which 54 out of 91 lectins showed positive binding. Based on this technique, we compared lectin binding profiling of sperm with homozygous DEFB126 mutation (del/del) with that of wild type (wt/wt). DEFB126 was reported to contribute to the sialylation on sperm surface and its homozygous mutation was related to male subfertility. Six lectins (Jacalin/AIA, GHA, ACL, MPL, VVL and ABA) were found to develop lower binding affinity to sperm with del/del. Further validation showed that these lectins, especially ABA and MPL, can be potential biomarkers for clinical diagnosis of subfertility due to the mutation of DEFB126. Our research provides insight into the detection of some unexplained male subfertility and the lectin microarray is generally applicable for infertility/subfertility sperm biomarker discovery.

Additional file 1. Additional figures and tables.

Sperm motility is essential for male reproduction or natural fertilization. The cyclic AMP (cAMP)/cAMP-dependent protein kinase A (PKA) signaling pathway is generally recognized as one of the significant signaling pathways in the regulation of mammalian spermatozoan motility. Since Zn-α2-glycoprotein (ZAG) activity in mammalian adipose tissue is mediated via the β3-adrenoreceptor, with upregulation of the cAMP pathway, we hypothesize that ZAG may play the same role in sperm motility regulation, a new factor of regulation of sperm motility. Therefore, the gene encoding human ZAG was cloned and polyclonal antibodies were generated, and then laser scanning confocal microscopy and flow cytometry were employed to identify this protein in human spermatozoa. The results showed that ZAG protein was mostly localized on the pre-equatorial region covering the acrosome, neck, and middle piece of the flagellum of spermatozoa. Furthermore, using computer-assisted sperm analysis, we found that anti-human ZAG antibodies could significantly reduce the motility of human swim-up spermatozoa after 90- or 120-min incubation (PP

Background Polycystic ovary syndrome (PCOS) is a common endocrine disorder in women of reproductive age, and oocyte developmental competence is altered in patients with PCOS. In recent years microRNAs (miRNAs) have emerged as important regulators of gene expression, the aim of the study was to study miRNAs expression patterns of cumulus cells from PCOS patients. Methods The study included 20 patients undergoing in vitro fertilization (IVF) and intra-cytoplasmic sperm injection (ICSI): 10 diagnosed with PCOS and 10 matching controls. We used deep sequencing technology to identify the miRNAs differentially expressed in the cumulus cells of PCOS. Results There were 17 differentially expressed miRNAs in PCOS cumulus cells, including 10 miRNAs increase and 7 miRNAs decrease. These miRNAs were predicted to target a large set of genes with different functions, including Wnt- and MAPK- signaling pathways, oocyte meiosis, progesterone-mediated oocyte maturation and cell cycle. Unsupervised hierarchical clustering analysis demonstrated that there was a specific miRNAs expression pattern in PCOS cumulus cells. Conclusion We found that the miRNAs expression profile was different in cumulus cells isolated from PCOS patients compared with control. This study provided new evidence for understanding the pathogenesis of PCOS.

It is well known that cell surface glycans or glycocalyx play important roles in sperm motility, maturation and fertilization. A comprehensive profile of the sperm surface glycans will greatly facilitate both basic research (sperm glycobiology) and clinical studies, such as diagnostics of infertility. As a group of natural glycan binders, lectin is an ideal tool for cell surface glycan profiling. However, because of the lack of effective technology, only a few lectins have been tested for lectin-sperm binding profiles. To address this challenge, we have developed a procedure for high-throughput probing of mammalian sperm with 91 lectins on lectin microarrays. Normal sperm from human, boar, bull, goat and rabbit were collected and analyzed on the lectin microarrays. Positive bindings of a set of ~50 lectins were observed for all the sperm of 5 species, which indicated a wide range of glycans are on the surface of mammalian sperm. Species specific lectin bindings were also observed. Clustering analysis revealed that the distances of the five species according to the lectin binding profiles are consistent with that of the genome sequence based phylogenetic tree except for rabbit. The procedure that we established in this study could be generally applicable for sperm from other species or defect sperm from the same species. We believe the lectin binding profiles of the mammalian sperm that we established in this study are valuable for both basic research and clinical studies.

It has been reported that the expression of GTP cyclohydrolase I (GCH1) and neuronal apoptosis in dorsal root ganglion (DRG) is related to the generation of neuropathic pain. In this study, we hypothesize that GCH1 protein and neuronal apoptosis in rat spinal dorsal horn may also increase after chronic sciatic nerve injury. To establish the neuropathic pain model, we slightly ligated the right sciatic nerve of experimental rats. Mechanical allodynia was observed in CCI group on the 3rd day postoperatively determined by the Von Frey test, and it lasted until the 14th day after operation. No matter which method we used, western blotting or immunohistochemistry analysis, they all showed the enhancement of GCH1 protein in spinal dorsal horn on the 3rd, 7th, and 14th days after operation due to the sciatic nerve injury (P < 0.05). The apoptosis of nerve cells also increased evidently compared with sham group detected by TUNEL staining (P < 0.05). Therefore, the data suggested that along with mechanical allodynia caused by peripheral nerve injury, both GCH1 level and apoptosis index of nerve cells in spinal dorsal horn was elevated, which might also contribute to the generation of neuropathic pain.

Audio effects processing technology is widely used in various types of music production, live performances and other occasions. Most of traditional technologies produce real-time audio effects with hardware analog circuits. In this paper, focus on the demand of time scaling, pitch shifting and special effects of real-time audio. Improve the algorithm performance, efficiency and effectiveness in subjective base on traditional phase vocoder model in high performance DSP processor BlackFin which be made the use of its structural features. Experiment shows that, new algorithm do well in both computation and hearing effect.