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Objectives Despite of strenuous research in the past decades, the etiology of schizophrenia (SCZ) still remains incredibly controversial. Previous genetic analysis has uncovered a close association of Unc-51 like kinase 4 (ULK4), a family member of Unc-51-like serine/threonine kinase, with SCZ. However, animal behavior data which may connect Ulk4 deficiency with psychiatric disorders, particularly SCZ are still missing. Methods We generated Emx1-Cre:Ulk4flox/flox conditional knockout (CKO) mice, in which Ulk4 was deleted in the excitatory neurons of cerebral cortex and hippocampus. Results The cerebral cellular architecture was maintained but the spine density of pyramidal neurons was reduced in Ulk4 CKO mice. CKO mice showed deficits in the spatial and working memories and sensorimotor gating. Levels of p-Akt and p-GSK-3α/β were markedly reduced in the CKO mice indicating an elevation of GSK-3 signaling. Mechanistically, Ulk4 may regulate the GSK-3 signaling via putative protein complex comprising of two phosphatases, protein phosphatase 2A (PP2A) and 1α (PP1α). Indeed, the reduction of p-Akt and p-GSK-3α/β was rescued by administration of inhibitor acting on PP2A and PP1α in CKO mice. Conclusions Our data identified potential downstream signaling pathway of Ulk4, which plays important roles in the cognitive functions and when defective, may promote SCZ-like pathogenesis and behavioral phenotypes in mice.

Recent studies indicate existence of beige adipocytes in adults. Upon activation, beige adipocytes burn energy for thermogenesis and contribute to regulation of energy balance. In this study, we have analyzed whether Jinlida granules (JLD) could activate beige adipocytes. JLD suspended in 0.5% carboxymethyl cellulose (CMC) was gavage fed to db/db mice at a daily dose of 3.8 g/kg. After 10 weeks, body weight, biochemical, and histological analyses were performed. In situ hybridization, immunofluorescence, and western blotting were conducted to test beige adipocyte activation in mice. X9 cells were induced with induction medium and maintenance medium containing 400 μg/mL of JLD. After completion of induction, cells were analyzed by Nile red staining, time polymerase chain reaction (PCR), western blotting, and immunofluorescence to understand the effect of JLD on the activation of beige adipocytes. A molecular docking method was used to preliminarily identify compounds in JLD, which hold the potential activation effect on uncoupling protein 1 (UCP1). JLD treatment significantly improved obesity in db/db mice. Biochemical results showed that JLD reduced blood glucose (GLU), triglyceride (TG), and low-density lipoprotein cholesterol (LDL) levels as well as liver aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in mice. Hematoxylin and eosin staining (H&E) showed that JLD reduced hepatocyte ballooning changes in the liver. Immunofluorescence showed that JLD increased the expression of the thermogenic protein, UCP1, in the beige adipose tissue of mice. JLD also increased the expression of UCP1 and inhibited the expression of miR-27a in X9 cells. Molecular docking results showed that epmedin B, epmedin C, icariin, puerarin, and salvianolic acid B had potential activation effects on UCP1. The results suggest that JLD may activate beige adipocytes by inhibiting miR-27a expression, thereby promoting thermogenesis in beige adipocytes. This study provides a new pharmacological basis for the clinical use of JLD.

Obtaining high-quality sperm is key to improving the success rate of assisted reproductive technology (ART). Although cytokines secreted by cumulus-oocyte complexes (COCs) bind to sperm surface receptors to improve sperm quality, the effects of adding mouse COCs to human tubal fluid (HTF) medium on sperm capacitation have not yet been explored. Eight-week-old ICR mouse COCs were added to HTF medium and crushed to obtain the post-modified HTF medium. Compared with using HTF medium, the fertilisation rate and number of sperm combined with the zona pellucida significantly increased after in vitro capacitation using the post-modified HTF medium (P 0.01). Proteomic and Western blotting analyses showed that the level of SERPINA5 in sperm increased significantly following in vitro capacitation with the post-modified HTF medium (P 0.05). Immunohistochemical staining analysis demonstrated that SERPINA5 protein was expressed in mouse cumulus cells. A SERPINA5 antibody was added in the post-modified HTF medium to block the effects of SERPINA5 after in vitro capacitation, which significantly decreased the fertilisation rate and the number of sperm combined with the zona pellucida (P 0.05). Recombinant mouse SERPINA5 protein (1 ~ 2 μg/ml) was added to HTF medium and the fertilisation rate and the number of sperm combined with the zona pellucida significantly increased (P 0.01). Moreover, recombinant human SERPINA5 protein (5 μg/ml) was added before human semen freezing. Compared with adding no SERPINA5 protein, the percentage of normal sperm morphology and the intact acrosome significantly increased (P 0.05). Our study provides a reference method for optimising sperm quality in the process of in vitro capacitation.

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Erectile dysfunction (ED) is closely associated with smooth muscle dysfunction, but its underlying mechanisms remains incompletely understood. We here reported that the reduced expression of myosin phosphatase target subunit 1 (MYPT1), the main regulatory unit of myosin light chain phosphatase, was critical for the development of vasculogenic ED. Male MYPT1 knockout mice had reduced fertility and the penises displayed impaired erections as evidenced by reduced intracavernous pressure (ICP). The penile smooth muscles of the knockout mice displayed enhanced response to G-Protein Couple Receptor agonism and depolarization contractility and resistant relaxation. We further identified a natural compound lotusine that increased the MYPT1 expression by inhibiting SIAH1/2 E3 ligases-mediated protein degradation. This compound sufficiently restored the ICP and improved histological characters of the penile artery of Mypt1 haploinsufficiency mice. In diabetic ED mice (db/db), the decreased expression of MYPT1 was measured, and ICP was improved by lotusine treatment. We conclude that the reduction of MYPT1 is the major pathogenic factor of vasculogenic ED. The restoration of MYPT1 by lotusine improved the function of injured penile smooth muscles, and could be a novel strategy for ED therapy.

Endometrial cancer (EC) is the most common gynecological malignancy worldwide. However, the molecular mechanisms underlying EC progression are still largely unknown, and chemotherapeutic options for EC patients are currently very limited. In this study, we found that histone methyltransferase EZH2 and DNA methyltransferase DNMT3B were upregulated in EC samples from patients, and promoted EC cell proliferation as evidenced by assays of cell viability, cell cycle, colony formation. Mechanistically, we found that EZH2 promoted EC cell proliferation by epigenetically repressing TCF3, a direct transcriptional activator of CCKN1A (p21WAF1/Cip1), in vitro and in vivo. In addition, we found that DNMT3B specifically methylated the TCF3 promoter, repressing TCF3 expression and accelerating EC cell proliferation independently of EZH2. Importantly, elevated expression of EZH2 or DNMT3B in EC patients inversely correlated with expression of TCF3 and p21, and was associated with shorter overall survival. We show that combined treatment with GSK126 and 5-Aza-2d treatment wit synergistically inhibited methyltransferase activity of EZH2 and DNMT3B, resulting in a profound block of EC cell proliferation as well as EC tumor progression in cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) mouse models. These findings reveal that TCF3 functions as a tumor suppressor epigenetically silenced by EZH2 and DNMT3B in EC, and support the notion that targeting the EZH2/DNMT3B/TCF3/p21 axis may be a novel and effective therapeutic strategy for treatment of EC.

Osteoporosis is a metabolic disorder characterized by low bone mass and deteriorated microarchitecture, with an increased risk of fracture. Some miRNAs have been confirmed as potential modulators of osteoblast differentiation to maintain bone mass. Our miRNA sequencing results showed that miR‐664‐3p was significantly down‐regulated during the osteogenic differentiation of the preosteoblast MC3T3‐E1 cells. However, whether miR‐664‐3p has an impact on bone homeostasis remains unknown. In this study, we identified overexpression of miR‐664‐3p inhibited the osteoblast activity and matrix mineralization in vitro. Osteoblastic miR‐664‐3p transgenic mice exhibited reduced bone mass due to suppressed osteoblast function. Target prediction analysis and experimental validation confirmed Smad4 and Osterix (Osx) are the direct targets of miR‐664‐3p. Furthermore, specific inhibition of miR‐664‐3p by subperiosteal injection with miR‐664‐3p antagomir protected against ovariectomy‐induced bone loss. In addition, miR‐664‐3p expression was markedly higher in the serum from patients with osteoporosis compared to that from normal subjects. Taken together, this study revealed that miR‐664‐3p suppressed osteogenesis and bone formation via targeting Smad4 and Osx. It also highlights the potential of miR‐664‐3p as a novel diagnostic and therapeutic target for osteoporotic patients.

Background Although using a blockade of programmed death-ligand 1 (PD-L1) to enhance T cell immune responses shows great promise in tumor immunotherapy, the immune-checkpoint inhibition strategy is limited for patients with solid tumors. The mechanism and efficacy of such immune-checkpoint inhibition strategies in solid tumors remains unclear. Results Employing qRT-PCR, Sanger sequencing, and RNA BaseScope analysis, we show that human lung adenocarcinoma (LUAD) all produce a long non-coding RNA isoform of PD-L1 (PD-L1-lnc) by alternative splicing, regardless if the tumor is positive or negative for the protein PD-L1. Similar to PD-L1 mRNA, PD-L1-lnc in various lung adenocarcinoma cells is significantly upregulated by IFNγ. Both in vitro and in vivo studies demonstrate that PD-L1-lnc increases proliferation and invasion but decreases apoptosis of lung adenocarcinoma cells. Mechanistically, PD-L1-lnc promotes lung adenocarcinoma progression through directly binding to c-Myc and enhancing c-Myc transcriptional activity. Conclusions In summary, the PD-L1 gene can generate a long non-coding RNA through alternative splicing to promote lung adenocarcinoma progression by enhancing c-Myc activity. Our results argue in favor of investigating PD-L1-lnc depletion in combination with PD-L1 blockade in lung cancer therapy.

Background Human brucellosis has become one of the major public health problems in China, and increases atypical manifestations, such as fever of unknown origin (FUO), and misdiagnosis rates has complicated the diagnosis of brucellosis. To date, no relevant study on the relationship between brucellosis and FUO has been conducted. Methods We retrospectively reviewed the medical charts of 35 patients with confirmed human brucellosis and prospectively recorded their outcomes by telephone interview. The patients were admitted to the Second Affiliated Hospital of Nanchang University between January 01, 2013 and October 31, 2019. Patient data were collected from hospital medical records. Results The percentage of males was significantly higher than that of female in FUO (78.95% vs. 21.05%, P P P P Conclusion Brucellosis is predominantly FUO admission in a non-endemic area of China, accompanied by irregular fever and toxic hepatitis. Careful examination of the epidemiological history and timely improvement of blood and bone marrow cultures can facilitate early diagnosis and prevent misdiagnosis.

The formation and transmission of antibiotic resistance genes (ARGs) have attracted increasing attention. It is unclear whether the internal mechanisms by which antibiotics affect horizontal gene transfer (HGT) of ARGs during anaerobic digestion (AD) were influenced by dose and type. We investigated the effects of two major antibiotics (oxytetracycline, OTC, and sulfamethoxazole, SMX) on ARGs during AD according to antibiotic concentration in livestock wastewater influent. The low-dose antibiotic (0.5 mg/L) increased ROS and SOS responses, promoting the formation of ARGs. Meanwhile, low-dose antibiotics could also promote the spread of ARGs by promoting pili, communication responses, and the type IV secretion system (T4SS). However, different types and doses of antibiotics would lead to changes in the above functional modules and then affect the enrichment of ARGs. With the increasing dose of SMX, the advantages of pili and communication responses would gradually change. In the OTC system, low-dose has the strongest promoting ability in both pili and communication responses. Similarly, an increase in the dose of SMX would change T4SS from facilitation to inhibition, while OTC completely inhibits T4SS. Microbial and network analysis also revealed that low-dose antibiotics were more favorable for the growth of host bacteria.

Hydrogen sulfide (H2S), a gaseous intracellular signal transducer, participates in multiple physiological and pathological conditions, including reproductive conditions, and disrupts spermatogenesis. The blood-testis barrier (BTB) plays a vital role in spermatogenesis. However, the effect of H2S on the BTB and the underlying mechanism remain unclear. Herein, we examined the effect of H2S and omega-3 polyunsaturated fatty acids (ω-3 PUFAs) on the BTB and testicular functions. ICR male mice were randomly divided into the following groups: control, H2S exposure, and H2S exposure with ω-3 PUFAs intervention. The sperm parameters (sperm concentration and sperm motility) declined in the H2S group and improved in the ω-3 intervention group. BTB integrity was severely disrupted by H2S, and the BTB-related gene levels (ZO-1, Occludin, Claudin 11) decreased; ω-3 supplementation could alleviate BTB disruption by upregulating BTB-related genes, and TM4 Sertoli cells had a similar trend in vitro. p38 MAPK phosphorylation was upregulated in the Na2S treatment group and downregulated after ω-3 cotreatment. These findings suggest that H2S can impair the BTB and that ω-3 PUFAs supplementation can attenuate H2S toxicity in the male reproductive system. Our study elucidated the relationship between a gasotransmitter (H2S) and the BTB and identified the potential therapeutic effect of ω-3 PUFAs.

The mechanism underlying the association between age and depletion of the human ovarian follicle reserves remains uncertain. Many identified that impaired DNA polymerase β (Pol β)-mediated DNA base-excision repair (BER) drives to mouse oocyte aging. With aging, DNA lesions accumulate in primordial follicles. However, the expression of most DNA BER genes, including APE1, OGG1, XRCC1, Ligase I, Ligase α, PCNA and FEN1, remains unchanged during aging in mouse oocytes. Also, the reproductive capacity of Pol β+/- heterozygote mice was impaired, and the primordial follicle counts were lower than that of wild type (wt) mice. The DNA lesions of heterozygous mice increased. Moreover, the Pol β knockdown leads to increased DNA damage in oocytes and decreased survival rate of oocytes. Oocytes over-expressing Pol β showed that the vitality of senescent cells enhances significantly. Furthermore, serum concentrations of anti-Müllerian hormone (AMH) indicated that the ovarian reserves of young mice with Pol β germline mutations were lower than those in wt. These data show that Pol β-related DNA BER efficiency is a major factor governing oocyte aging in mice.

The main objective of this work was to demonstrate which kind of payload is the suitable choice for antibody-drug conjugates directed to widely expressed tumor-associated antigen. Trop-2 is overexpressed in various solid tumors, but it is also present on the epithelium of several normal tissues. A well-designed anti-Trop-2 ADC demands a good balance of efficacy and toxicity. In this research, MMAE, SN-38, and DXd were selected as candidates for payloads of the anti-Trop-2 mAb SY02. The antitumor activities and safety profiles of these ADCs were investigated to compare the therapeutic windows. Robust in vitro cytotoxicity was observed on human pancreatic cancer cell CFPAC-1 and breast cancer cell MDA-MB-468 with IC

Previous studies have revealed the critical roles of the N6-methyladenosine (m6A) modification of long non-coding RNAs (lncRNAs) in cancers, but the relationship between the oncogenic role of the lncRNA THOR (a representative of cancer/testis lncRNAs) and m6A modification remains unclear. Here, we show that the internal m6A modification of the lncRNA THOR via an m6A-reader-dependent modality regulates the proliferation of cancer cells. Our findings demonstrated that the loss of the lncRNA THOR inhibits the proliferation, migration, and invasion of cancer cells in vitro and in vivo. In addition, m6A is highly enriched on lncRNA THOR transcripts, which contain GA (m6A) CA, GG (m6A) CU, and UG (m6A) CU sequence motifs. RIP-qRT-PCR and RNA pull-down assay results revealed that the specific m6A readers YTHDF1 and YTHDF2 can read the m6A motifs and regulate the stability of the lncRNA THOR (stabilization and decay). These m6A-dependent RNA-protein interactions can maintain the oncogenic role of the lncRNA THOR. Collectively, these findings highlight the critical role of the m6A modification in oncogenic lncRNA THOR and reveal a novel long non-coding RNA regulatory mechanism, providing a new way to explore RNA epigenetic regulatory patterns in the future.

ObjectiveHigh-fat diet (HFD)-induced metabolic disorders can lead to impaired sperm production. We aim to investigate if HFD-induced gut microbiota dysbiosis can functionally influence spermatogenesis and sperm motility.DesignFaecal microbes derived from the HFD-fed or normal diet (ND)-fed male mice were transplanted to the mice maintained on ND. The gut microbes, sperm count and motility were analysed. Human faecal/semen/blood samples were collected to assess microbiota, sperm quality and endotoxin.ResultsTransplantation of the HFD gut microbes into the ND-maintained (HFD-FMT) mice resulted in a significant decrease in spermatogenesis and sperm motility, whereas similar transplantation with the microbes from the ND-fed mice failed to do so. Analysis of the microbiota showed a profound increase in genus Bacteroides and Prevotella, both of which likely contributed to the metabolic endotoxaemia in the HFD-FMT mice. Interestingly, the gut microbes from clinical subjects revealed a strong negative correlation between the abundance of Bacteroides-Prevotella and sperm motility, and a positive correlation between blood endotoxin and Bacteroides abundance. Transplantation with HFD microbes also led to intestinal infiltration of T cells and macrophages as well as a significant increase of pro-inflammatory cytokines in the epididymis, suggesting that epididymal inflammation have likely contributed to the impairment of sperm motility. RNA-sequencing revealed significant reduction in the expression of those genes involved in gamete meiosis and testicular mitochondrial functions in the HFD-FMT mice.ConclusionWe revealed an intimate linkage between HFD-induced microbiota dysbiosis and defect in spermatogenesis with elevated endotoxin, dysregulation of testicular gene expression and localised epididymal inflammation as the potential causes.Trial registration numberNCT03634644.

Sertoli cells are essential for spermatogenesis in the testicular seminiferous tubules by forming blood-testis barrier (BTB) and creating a unique microenvironment for spermatogenesis. Many lncRNAs have been reported to participate in spermatogenesis. However, the role of long noncoding RNAs (lncRNAs) in Sertoli cells has rarely been examined. Herein, we found that a high-fat diet (HFD) decreased sperm quality, impaired BTB integrity and resulted in accumulation of saturated fatty acids (SFAs), especially palmitic acid (PA), in mouse testes. PA decreased the expression of tight junction (TJ)-related proteins, increased permeability and decreased transepithelial electrical resistance (TER) in primary Sertoli cells and TM4 cells. Moreover, lncRNA Tug1 was found to be involved in PA-induced BTB disruption by RNA-seq. Tug1 depletion distinctly impaired the TJs of Sertoli cells and overexpression of Tug1 alleviated the disruption of BTB integrity induced by PA. Moreover, Ccl2 was found to be a downstream target of Tug1, and decreased TJ-related protein levels and TER and increased FITC-dextran permeability in vitro. Furthermore, the addition of Ccl2 damaged BTB integrity after overexpression of Tug1 in the presence of PA. Mechanistically, we found that Tug1 could directly bind to EZH2 and regulate H3K27me3 occupancy in the Ccl2 promoter region by RNA immunoprecipitation and chromatin immunoprecipitation assays. Our study revealed an important role of Tug1 in the BTB integrity of Sertoli cells and provided a new view of the role of lncRNAs in male infertility.

Aberrant changes in epigenetic mechanisms such as histone modifications play an important role in cancer progression. PRMT1 which triggers asymmetric dimethylation of histone H4 on arginine 3 (H4R3me2a) is upregulated in human colorectal cancer (CRC) and is essential for cell proliferation. However, how this dysregulated modification might contribute to malignant transitions of CRC remains poorly understood.In this study, we integrated biochemical assays including protein interaction studies and chromatin immunoprecipitation (ChIP), cellular analysis including cell viability, proliferation, colony formation, and migration assays, clinical sample analysis, microarray experiments, and ChIP-Seq data to investigate the potential genomic recognition pattern of H4R3me2s in CRC cells and its effect on CRC progression.We show that PRMT1 and SMARCA4, an ATPase subunit of the SWI/SNF chromatin remodeling complex, act cooperatively to promote colorectal cancer (CRC) progression. We find that SMARCA4 is a novel effector molecule of PRMT1-mediated H4R3me2a. Mechanistically, we show that H4R3me2a directly recruited SMARCA4 to promote the proliferative, colony-formative, and migratory abilities of CRC cells by enhancing EGFR signaling. We found that EGFR and TNS4 were major direct downstream transcriptional targets of PRMT1 and SMARCA4 in colon cells, and acted in a PRMT1 methyltransferase activity-dependent manner to promote CRC cell proliferation. In vivo, knockdown or inhibition of PRMT1 profoundly attenuated the growth of CRC cells in the C57BL/6 J-ApcPRMT1-mediated H4R3me2a recruits SMARCA4, which promotes colorectal cancer progression by enhancing EGFR signaling.

Grim-19 (gene associated with retinoid-IFN-induced mortality 19), the essential component of complex I of mitochondrial respiratory chain, functions as a noncanonical tumor suppressor by controlling apoptosis and energy metabolism. However, additional biological actions of Grim-19 have been recently suggested in male reproduction. We investigated here the expression and functional role of Grim-19 in murine testis. Testicular Grim-19 expression was detected from mouse puberty and increased progressively thereafter, and GRIM-19 protein was observed to be expressed exclusively in interstitial Leydig cells (LCs), with a prominent mitochondrial localization. In vivo lentiviral vector-mediated knockdown of Grim-19 resulted in a significant decrease in testosterone production and triggered aberrant oxidative stress in testis, thus impairing male fertility by inducing germ cell apoptosis and oligozoospermia. The control of testicular steroidogenesis by GRIM-19 was validated using the in vivo knockdown model with isolated primary LCs and in vitro experiments with MA-10 mouse Leydig tumor cells. Mechanistically, we suggest that the negative regulation exerted by GRIM-19 deficiency-induced oxidative stress on steroidogenesis may be the result of two phenomena: a direct effect through inhibition of phosphorylation of steroidogenic acute regulatory protein (StAR) and subsequent impediment to StAR localization in mitochondria and an indirect pathway that is to facilitate the inhibiting role exerted by the extracellular matrix on the steroidogenic capacity of LCs via promotion of integrin activation. Altogether, our observations suggest that Grim-19 plays a potent role in testicular steroidogenesis and that its alterations may contribute to testosterone deficiency-related disorders linked to metabolic stress and male infertility.

Yi Guan Jian decoction (YGD) may induce the differentiation of bone marrow mesenchymal stem cells (BMSCs) into hepatocyte-like cells (HLCs); however, the underlying mechanisms remain to be elucidated. The present study aimed to investigate this process. To do this, a dimethylnitrosamine (DMN)-induced liver cirrhosis mouse model was established. The mice from the model group were randomly divided into three subgroups: i) Negative control, ii) hepatocyte growth factor and iii) YGD. The overall health, liver function and histological alterations were monitored. The expression of α‑smooth muscle actin (α‑SMA), C‑X‑C chemokine receptor type 4 (CXCR4), extracellular signal‑regulated kinase (ERK1/2), nuclear factor κB p65 subunit (NF‑κB p65) and β‑catenin were measured by immunohistochemistry, western blotting and reverse transcription‑quantitative polymerase chain reaction. Following administration of DMN, the overall health of the mice significantly decreased, with an increase in pathological developments and liver damage resulting in a decrease in liver function. Immunohistochemistry revealed that the expression of α‑SMA, CXCR4, ERK1/2, NF‑κB p65 and β‑catenin was upregulated. Following treatment with YGD, the overall health, liver function and pathology improved. The mRNA and protein expression levels of CXCR4 and ERK1/2 were upregulated, where as α‑SMA, NF‑κB p65 and β‑catenin levels were downregulated. The results demonstrated that YGD may induce the differentiation of BMSCs into HLCs to reverse DMN‑induced liver cirrhosis; this may be achieved via an upregulation of the SDF‑1/CXCR4 axis to activate the mitogen activated protein kinase/ERK1/2 signaling pathway.

Endometrial receptivity is crucial for successful embryo implantation. It is regulated by multiple factors which include ovarian steroid hormones and the immune microenvironment among others. Nod-Like Receptor Pyrins-3 (NLRP3) is a key intracellular pattern-recognition receptor and a critical component of the inflammasome, which plays an essential role in the development of inflammation and of immune responses. However, the physiological functions of NLRP3 in the endometrium remain largely unclear. This study investigated the physiological and pathological significance of NLRP3 in human endometrial epithelial cell during the implantation window. NLRP3 is highly expressed during the mid-proliferative and mid-secretory phases of the human endometrium and transcriptionally up-regulated by estradiol (E2) through estrogen receptor β (ERβ). In addition, NLRP3 promotes embryo implantation and enhances epithelial-mesenchymal transition (EMT) of Ishikawa (IK) cells via both inflammasome-dependent and inflammasome-independent pathways, which might provide a novel insight into endometrial receptivity and embryo implantation. Our findings suggest that NLRP3, which is transcriptionally regulated by E2, induces epithelial–mesenchymal transition of endometrial epithelial cells and promotes embryo adhesion.

PTEN is known as a tumor suppressor and plays essential roles in brain development. Here, we report that PTEN in primary sensory neurons is involved in processing itch and thermal information in adult mice. Deletion of PTEN in the dorsal root ganglia (DRG) is achieved in adult Drg11-Cre

Non-obstructive azoospermia (NOA) severely affects male infertility, however, the deep mechanisms of this disease are rarely interpreted. In this study, we find that undifferentiated spermatogonial stem cells (SSCs) still exist in the basal compartment of the seminiferous tubules and the blood–testis barrier (BTB) formed by the interaction of neighbor Sertoli cells (SCs) is incomplete in NOA patients with spermatogenic maturation arrest. The adhesions between SCs and germ cells (GCs) are also broken in NOA patients. Meanwhile, the expression level of geranylgeranyl diphosphate synthase (Ggpps), a key enzyme in mevalonate metabolic pathway, is lower in NOA patients than that in obstructive azoospermia (OA) patients. After Ggpps deletion specifically in SCs, the mice are infertile and the phenotype of the SC-Ggpps−/− mice is similar to the NOA patients, where the BTB and the SC–GC adhesions are severely destroyed. Although SSCs are still found in the basal compartment of the seminiferous tubules, fewer mature spermatocyte and spermatid are found in SC-Ggpps−/− mice. Further examination suggests that the defect is mediated by the aberrant protein isoprenylation of RhoA and Ras family after Ggpps deletion. The exciting finding is that when the knockout mice are injected with berberine, the abnormal cell adhesions are ameliorated and spermatogenesis is partially restored. Our data suggest that the reconstruction of disrupted BTB is an effective treatment strategy for NOA patients with spermatogenic maturation arrest and hypospermatogenesis.

SUMMARY 5-hydroxymethylcytosine (5hmC) is enriched in brain and has been recognized as an important DNA modification. However, the roles of 5hmC and its writers, ten-eleven translocation (Tet) proteins, in stress-induced response have yet to be elucidated. Here, we show that chronic restraint stress (CRS) induced depression-like behavior in mice and resulted in a 5hmC reduction in prefrontal cortex (PFC). We found that loss of Tet1 (Tet1 KO) led to resistance to CRS, whereas loss of Tet2 (Tet2 KO) increased the susceptibility of mice to CRS. Genome-wide 5hmC profiling identified the phenotype-associated stress-induced dynamically hydroxymethylated loci (PA-SI-DhMLs), which are strongly enriched with hypoxia-induced factor (HIF) binding motifs. We demonstrated the physical interaction between TET1 and HIF1α induced by CRS and revealed that the increased HIF1α binding under CRS is associated with SI-DhMLs. These results suggest that TET1 could regulate stress-induced response by interacting with HIFIα., Graphical Abstract, In Brief The roles of 5-hydroxymethylcytosine (5hmC) and its writers, Tet proteins, in stress-induced response remain unclear. Cheng et al. show that Tet1 knockout mice exhibit resistance, whereas Tet2 knockout mice have increased susceptibility to stress. Biochemical and genome-wide analyses suggest that Tet1 could regulate stress-induced response by interacting with Hif1α.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is capable of inducing a variety of biological effects, and the regulation of the Nrf2 signaling pathway is closely related to longevity. To find out whether the nuclear factor erythroid 2-related factor 2 (Nrf2) is involved in oocyte aging or not which may cause reduced female fertility, a series of biological methods was applied, including oocyte collection and culture, micro injection, RNA interference, western blotting, immunofluorescence and confocal microscopy, and quantitative real-time PCR.Our data demonstrated that Nrf2 depletion disrupted oocyte maturation and spindle/chromosome organization by suppressing Cyclin B1 expression. Sirtuin 1 (Sirt1) depletion reduced Nrf2 expression, which indicated the existence of the Sirt1-Nrf2-Cyclin B1 signaling pathway in mouse oocytes. Additionally, immunoblotting results reflected a lower Nrf2 protein level in oocytes from aged mice, and maternal age-associated meiotic defects can be ameliorated through overexpression of Nrf2, which supported the hypothesis that decreased Nrf2 is an important factor contributing toward oocyte age-dependent deficits. Furthermore, we show that the expression of Nrf2 is related to female age in ovarian granular cells, suggesting that the decreased expression of Nrf2 may be related to the decline in the reproductive capacity of older women.

The Sertoli cell is the only somatic cell within the seminiferous tubules, and is vital for testis development and spermatogenesis. Rosiglitazone (RSG) is a member of the thiazolidinedione family and is a peroxisome proliferator-activated receptor-γ (PPARγ) agonist. It has been reported that RSG protects various types of cells from fatty acid-induced damage. However, whether RSG serves a protective role in Sertoli cells against palmitic acid (PA)-induced toxicity remains to be elucidated. Therefore, the aim of the present study was to investigate the effect of RSG on PA-induced cytotoxicity in Sertoli cells. MTT assay and Oil Red O staining revealed that RSG ameliorated the PA-induced decrease in TM4 cell viability, which was accompanied by an alleviation of PA-induced lipid accumulation in cells. In primary mouse Sertoli cells, RSG also showed similar protective effects against PA-induced lipotoxicity. Knockdown of PPARγ verified that RSG exerted its protective role in TM4 cells through a PPARγ-dependent pathway. To evaluate the mechanism underlying the protective role of RSG on PA-induced lipotoxicity, the present study analyzed the effects of RSG on PA uptake, and the expression of genes associated with both fatty acid oxidation and triglyceride synthesis. The results demonstrated that although RSG did not affect the endocytosis of PA, it significantly elevated the expression of carnitine palmitoyltransferase (CPT)-1A, a key enzyme involved in fatty acid oxidation, which indicated that the protective effect of RSG may have an important role in fatty acid oxidation. On the other hand, the expression of CPT1B was not affected by RSG. Moreover, the expression levels of diacylglycerol O-acyltransferase (DGAT)-1 and DGAT2, both of which encode enzymes catalyzing the synthesis of triglycerides, were not suppressed by RSG. The results indicated that RSG reduced PA-induced lipid accumulation by promoting fatty acid oxidation mediated by CPT1A. The effect of RSG in protecting cells from lipotoxicity was also found to be specific to Sertoli cells and hepatocytes, and not to other cell types that do not store excess lipid in large quantities, such as human umbilical vein endothelial cells. These findings provide insights into the cytoprotective effects of RSG on Sertoli cells and suggest that PPARγ activation may be a useful therapeutic method for the treatment of Sertoli cell dysfunction caused by dyslipidemia. Electronic supplementary material The online version of this article (10.1186/s12958-018-0416-0) contains supplementary material, which is available to authorized users.

Forkhead box O (FoxO) protein, a major downstream transcription factor of insulin/insulin-like growth factor signaling/target of rapamycin pathway (IIS/TOR), is involved in the regulation of larval growth and the determination of organ size. FoxO also interacts with 20-hydroxyecdysone (20E) and juvenile hormone (JH) signal transduction pathways, and hence is critical for larval development in holometabolans. However, whether FoxO plays a critical role during larval metamorphosis needs to be further determined in Leptinotarsa decemlineata. We found that 20E stimulated the expression of LdFoxO. RNA interference (RNAi)-aided knockdown of LdFoxO at the third-instar stage repressed 20E signaling and reduced larval weight. Although the resultant larvae survived through the third-fourth instar ecdysis, around 70% of the LdFoxO depleted moribund beetles developmentally arrested at prepupae stage. These LdFoxO depleted beetles were completely wrapped in the larval exuviae, gradually darkened and finally died. Moreover, approximately 12% of the LdFoxO RNAi beetles died as pharate adults. Ingestion of either 20E or JH by the LdFoxO depletion beetles excessively rescued the corresponding hormonal signals, but could not alleviate larval performance and restore defective phenotypes. Therefore, FoxO plays an important role in regulation of larval-pupal-adult transformation in L. decemlineata, in addition to mediation of IIS/TOR pathway and stimulation of ecdysteroidogenesis.

BackgroundN6-methyladenosine (m6A) modification is known to impact many aspects of RNA metabolism, including mRNA stability and translation, and is highly prevalent in the brain.ResultsWe show that m6A modification displays temporal and spatial dynamics during neurodevelopment and aging. Genes that are temporally differentially methylated are more prone to have mRNA expression changes and affect many pathways associated with nervous system development. Furthermore, m6A shows a distinct tissue-specific methylation profile, which is most pronounced in the hypothalamus. Tissue-specific methylation is associated with an increase in mRNA expression and is associated with tissue-specific developmental processes. During the aging process, we observe significantly more m6A sites as age increases, in both mouse and human. We show a high level of overlap between mouse and human; however, humans at both young and old ages consistently show more m6A sites compared to mice. Differential m6A sites are found to be enriched in alternative untranslated regions of genes that affect aging-related pathways. These m6A sites are associated with a strong negative effect on mRNA expression. We also show that many Alzheimer-related transcripts exhibit decreased m6A methylation in a mouse model of Alzheimer’s disease, which is correlated with reduced protein levels.ConclusionsOur results suggest that m6A exerts a critical function in both early and late brain development in a spatio-temporal fashion. Furthermore, m6A controls protein levels of key genes involved in Alzheimer’s disease-associated pathways, suggesting that m6A plays an important role in aging and neurodegenerative disease.

Abnormal lipid/lipoprotein metabolism induced by obesity may affect spermatogenesis by inhibiting testosterone synthesis in Leydig cells. It is crucial to determine which components of lipoproteins inhibit testosterone synthesis. Circulating oxidized low-density lipoprotein (oxLDL), the oxidized form of LDL, has been reported to be an independent risk factor for decreased serum testosterone levels. However, whether oxLDL has a damaging effect on Leydig cell function and the detailed mechanisms have been rarely studied. This study first showed the specific localization of oxLDL and mitochondrial structural damage in testicular Leydig cells of high-fat diet-fed mice in vivo. We also found that oxLDL reduced the mitochondrial membrane potential (MMP) by disrupting electron transport chain and inhibited testosterone synthesis-related proteins and enzymes (StAR, P450scc, and 3β‑HSD), which ultimately led to mitochondrial dysfunction and decreased testosterone synthesis in Leydig cells. Further experiments demonstrated that oxLDL promoted lipid uptake and mitochondrial dysfunction by inducing CD36 transcription. Meanwhile, oxLDL facilitated COX2 expression through the p38 MAPK signaling pathway in Leydig cells. Blockade of COX-2 attenuated the oxLDL-induced decrease in StAR and P450scc. Our clinical results clarified that the increased serum oxLDL level was associated with a decline in circulating testosterone levels. Our findings amplify the damaging effects of oxLDL and provide the first evidence that oxLDL is a novel metabolic biomarker of male-acquired hypogonadism caused by abnormal lipid metabolism.

The previous report shows the minimal promoter (P1) contributes to the Xist RNA activation in cells, while the role of the Xist P1 has not yet been investigated in animal individuals. Here, female Xist P1 knockout rabbits (Xist P1(−/-)) were generated for the studies. The results showed that there is no significant difference in transmission ratio, Xist and X-linked genes expression, and Xist RNA localization between the female wild type (WT) and Xist P1(−/-) rabbits, suggesting that P1 is non-essential for Xist expression and XCI in rabbits. Our study has explored the function of Xist P1 in animal level for the first time, and the results provide new ideas for future studies of XCI mechanisms.

Background: During disc degeneration, inflammatory cytokine tumor necrosis factor (TNF)-α is correlated with nucleus pulposus (NP) cell apoptosis. Transforming growth factor (TGF)-β1 has the potential to regenerate degenerative disc. Objective: To investigate the protective role of TGF-β1 against TNF-α-mediated NP cell apoptosis and the underlying mechanism. Methods: Rat NP cells were treated with TNF-α (100 ng/ml) for 48 h. TGF-β1 was added into the culture medium to investigate its protective effects against TNF-α-induced NP cell apoptosis. Exogenous FasL was used to investigate the potential role of the Fas/FasL pathway in this process. Flow cytometry assay was used to analyze NP cell apoptosis. Real-time PCR and Western blotting were used to analyze gene and protein expression of apoptosis-related molecules. Results: In TNF-α-treated NP cells, TGF-β1 significantly decreased NP cell apoptosis, declined caspase-3 and -8 activity, and decreased expression of Bax and caspase-3 (cleaved-caspase-3) but increased expression of Bcl-2. However, exogenous FasL partly reversed these effects of TGF-β1 in NP cells treated with TNF-α. Additionally, expression of Fas and FasL in TNF-α-treated NP cells partly decreased by TGF-β1, whereas exogenous FasL increased expression of Fas and FasL in NP cells treated with TGF-β1 and TNF-α. Conclusion: TGF-β1 helps to inhibit TNF-α-induced NP cell apoptosis and the Fas/FasL pathway may be involved in this process. The present study suggests that TGF-β1 may be effective to retard inflammation-mediated disc degeneration.

3-Phosphoinositide-dependent protein kinase-1 (PDK1) plays a critical role in the development of mammalian brain. Here, we investigated the role of PDK1 in Purkinje cells (PCs) by generating the PDK1-conditional knock-out mice (cKO) through crossing PV-cre or Pcp2-cre mice with Pdk1(fl/fl) mice. The male mice were used in the behavioral testing, and the other experiments were performed on mice of both sexes. These PDK1-cKO mice displayed decreased cerebellar size and impaired motor balance and coordination. By the electrophysiological recording, we observed the reduced spontaneous firing of PCs from the cerebellar slices of the PDK1-cKO mice. Moreover, the cell body size of PCs in the PDK1-cKO mice was time dependently reduced compared with that in the control mice. And the morphologic complexity of PCs was also decreased after PDK1 deletion. These effects may have contributed to the reduction of the rpS6 (reduced ribosomal protein S6) phosphorylation and the PKCγ expression in PDK1-cKO mice since the upregulation of pS6 by treatment of 3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-1, the agonist of mTOR1, partly rescued the reduction in the cell body size of the PCs, and the delivery of recombinant adeno-associated virus-PKCγ through cerebellar injection rescued the reduced complexity of the dendritic arbor in PDK1-cKO mice. Together, our data suggest that PDK1, by regulating rpS6 phosphorylation and PKCγ expression, controls the cell body maintenance and the dendritic development in PCs and is critical for cerebellar motor coordination. SIGNIFICANCE STATEMENT Here, we show the role of 3-phosphoinositide-dependent protein kinase-1 (PDK1) in Purkinje cells (PCs). The ablation of PDK1 in PCs resulted in a reduction of cell body size, and dendritic complexity and abnormal spontaneous firing, which attributes to the motor defects in PDK1-conditional knock-out (cKO) mice. Moreover, the ribosomal protein S6 (rpS6) phosphorylation and the expression of PKCγ are downregulated after the ablation of PDK1. Additionally, upregulation of rpS6 phosphorylation by3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-1 partly rescued the reduction in cell body size of PCs, and the overexpression of PKCγ in PDK1-KO PCs rescued the reduction in the dendritic complexity. These findings indicate that PDK1 contributes to the maintenance of the cell body and the dendritic development of PCs by regulating rpS6 phosphorylation and PKCγ expression.

Cancer cells frequently exhibit aerobic glycolysis, known as the Warburg effect. An et al. identify a novel RNA regulatory activity of BAG3, a protein implicated in various cancers. BAG3 enhances posttranscriptionally the expression of Hexokinase 2, the first enzyme involved in glycolysis, and it reprograms glucose metabolism by pancreatic cancer cells., Aerobic glycolysis, a phenomenon known historically as the Warburg effect, is one of the hallmarks of cancer cells. In this study, we characterized the role of BAG3 in aerobic glycolysis of pancreatic ductal adenocarcinoma (PDAC) and its molecular mechanisms. Our data show that aberrant expression of BAG3 significantly contributes to the reprogramming of glucose metabolism in PDAC cells. Mechanistically, BAG3 increased Hexokinase 2 (HK2) expression, the first key enzyme involved in glycolysis, at the posttranscriptional level. BAG3 interacted with HK2 mRNA, and the degree of BAG3 expression altered recruitment of the RNA-binding proteins Roquin and IMP3 to the HK2 mRNA. BAG3 knockdown destabilized HK2 mRNA via promotion of Roquin recruitment, whereas BAG3 overexpression stabilized HK2 mRNA via promotion of IMP3 recruitment. Collectively, our results show that BAG3 promotes reprogramming of glucose metabolism via interaction with HK2 mRNA in PDAC cells, suggesting that BAG3 may be a potential target in the aerobic glycolysis pathway for developing novel anticancer agents.

Chemical modifications on DNA molecules, such as 5-methylcytosine and 5-hydroxymethylcytosine, play important roles in the mammalian brain. A novel DNA adenine modification, N(6)-methyladenine (6mA), has recently been found in mammalian cells. However, the presence and function(s) of 6mA in the mammalian brain remain unclear. Here we demonstrate 6mA dynamics in the mouse brain in response to environmental stress. We find that overall 6mA levels are significantly elevated upon stress. Genome-wide 6mA and transcriptome profiling reveal an inverse association between 6mA dynamic changes and a set of upregulated neuronal genes or downregulated LINE transposon expression. Genes bearing stress-induced 6mA changes significantly overlap with loci associated with neuropsychiatric disorders. These results suggest an epigenetic role for 6mA in the mammalian brain as well as its potential involvement in neuropsychiatric disorders., N6-methyladenine is a covalent epigenetic modification of the genome. Here, Yao and colleagues show that N6-methyladenine level in the mouse brain is dynamic following environmental stress, and the subsequent differential gene expression is correlated with LINE transposon expression.

N-α-acetyltransferase D (NatD) mediates N-α-terminal acetylation (Nt-acetylation) of histone H4 known to be involved in cell growth. Here we report that NatD promotes the migratory and invasive capabilities of lung cancer cells in vitro and in vivo. Depletion of NatD suppresses the epithelial-to-mesenchymal transition (EMT) of lung cancer cells by directly repressing the expression of transcription factor Slug, a key regulator of EMT. We found that Nt-acetylation of histone H4 antagonizes histone H4 serine 1 phosphorylation (H4S1ph), and that downregulation of Nt-acetylation of histone H4 facilitates CK2α binding to histone H4 in lung cancer cells, resulting in increased H4S1ph and epigenetic reprogramming to suppress Slug transcription to inhibit EMT. Importantly, NatD is commonly upregulated in primary human lung cancer tissues where its expression level correlates with Slug expression, enhanced invasiveness, and poor clinical outcomes. These findings indicate that NatD is a crucial epigenetic modulator of cell invasion during lung cancer progression., NatD is an acetyltransferase responsible for N-α-terminal acetylation of the histone H4 and H2A and has been linked to cell growth. Here the authors show that NatD-mediated acetylation of histone H4 serine 1 competes with the phosphorylation by CK2α at the same residue thus leading to the upregulation of Slug and tumor progression.

SUMMARY 5-hydroxymethylcytosine (5hmC) undergoes dynamic changes during mammalian brain development, and its dysregulation is associated with Alzheimer’s disease (AD). The dynamics of 5hmC during early human brain development and how they contribute to AD pathologies remain largely unexplored. We generate 5hmC and transcriptome profiles encompassing several developmental time points of healthy forebrain organoids and organoids derived from several familial AD patients. Stage-specific differentially hydroxymethylated regions demonstrate an acquisition or depletion of 5hmC modifications across developmental stages. Additionally, genes concomitantly increasing or decreasing in 5hmC and gene expression are enriched in neurobiological or early developmental processes, respectively. Importantly, our AD organoids corroborate cellular and molecular phenotypes previously observed in human AD brains. 5hmC is significantly altered in developmentally programmed 5hmC intragenic regions in defined fetal histone marks and enhancers in AD organoids. These data suggest a highly coordinated molecular system that may be dysregulated in these early developing AD organoids., Graphical Abstract, In brief Kuehner et al. use forebrain organoids derived from healthy controls to study the dynamics of 5hmC across early brain development. In addition, organoids derived from several AD patients reveal aberrant 5hmC patterns that could disrupt early neuronal networks and contribute to the onset of AD later in life.

Epidermal growth factor receptor (EGFR) has been implicated in the pathogenesis of diabetic nephropathy and renal fibrosis; however, the causative role of sustained EGFR activation is unclear. Here, we generated a novel kidney fibrotic mouse model of persistent EGFR activation by selectively expressing the EGFR ligand, human heparin-binding EGF-like growth factor (hHB-EGF), in renal proximal tubule epithelium. hHB-EGF expression increased tyrosine kinase phosphorylation of EGFR and the subsequent activation of downstream signaling pathways, including ERK and AKT, as well as the profibrotic TGF-β1/SMAD pathway. Epithelial-specific activation of EGFR was sufficient to promote spontaneous and progressive renal tubulointerstitial fibrosis, as characterized by increased collagen deposition, immune cell infiltration, and α-smooth muscle actin (α-SMA)–positive myofibroblasts. Tubule-specific EGFR activation promoted epithelial dedifferentiation and cell-cycle arrest. Furthermore, EGFR activation in epithelial cells promoted the proliferation of α-SMA+ myofibroblasts in a paracrine manner. Genetic or pharmacologic inhibition of EGFR tyrosine kinase activity or downstream MEK activity attenuated the fibrotic phenotype. This study provides definitive evidence that sustained activation of EGFR in proximal epithelia is sufficient to cause spontaneous, progressive renal tubulointerstitial fibrosis, evident by epithelial dedifferentiation, increased myofibroblasts, immune cell infiltration, and increased matrix deposition.—Overstreet, J. M., Wang, Y., Wang, X., Niu, A., Gewin, L. S., Yao, B., Harris, R. C., Zhang, M.-Z. Selective activation of epidermal growth factor receptor in renal proximal tubule induces tubulointerstitial fibrosis.

Gestational diabetes mellitus (GDM) is characterized by an initial diagnosis of glucose intolerance during pregnancy. There is increasing evidence supporting the association between GDM and the inhibited development of several organs in offspring. In the present study, a murine GDM model was established in mice by intraperitoneal injection of streptozotocin to evaluate the effect of maternal diabetes on the initiation of meiosis in female germ cells of offspring. The effect of GDM on the initiation of meiosis in the offspring was evaluated by reverse transcription-quantitative polymerase chain reaction, flow cytometry and hematoxylin and eosin staining. The results showed that, compared with the control group, fetal ovary growth was inhibited, the expression levels of meiosis-specific genes, stimulated by retinoic acid gene 8, synaptonemal complex protein, and DNA meiotic recombinase were inhibited, and the number of primordial/primary follicles was reduced in the GDM group. These may have been induced by an increase of apoptosis and inhibition of growth, as the mRNA levels of p21, a vital G1 cell cycle inhibitor, and apoptotic genes were upregulated, whereas the expression levels of genes important in folliculogenesis were decreased in the GDM group. In conclusion, the data obtained in the present study suggested that maternal diabetes may impair the initiation of meiosis and ovarian growth via growth inhibition, cell cycle arrest and the induction of apoptosis.

Controlled releasing of regulations remains the most convenient method to deliver various drugs. In the present study, we precipitated gold nanoparticles with chrysophanol. The gold-chrysophanol into poly (DL-lactide-co-glycolide) nanoparticles was loaded and the biological activity of chrysophanol nanoparticles on human LNCap prostate cancer cells, was tested to acquire the sustained releasing property. The circular dichroism spectroscopy indicated that chrysophanol nanoparticles effectively resulted in conformational alterations in DNA and regulated different proteins associated with cell cycle arrest. The reactive oxygen species (ROS), apoptosis, cell cycle, DNA damage, Cyto-c and caspase-3 activity were analyzed, and the expression levels of different anti- and pro-apoptotic were studied using immunoblotting analysis. The cytotoxicity assay suggested that chrysophanol nanoparticles preferentially killed prostate cancer cells in comparison to the normal cells. Chrysophanol nanoparticles reduced histone deacetylases (HDACs) to suppress cell proliferation and induce apoptosis by arresting the cell cycle in sub-G phase. In addition, the cell cycle-related proteins, including p27, CHK1, cyclin D1, CDK1, p-AMP-activated protein kinase (AMPK) and p-protein kinase B (AKT), were regulated by chrysophanol nanoparticles to prevent human prostate cancer cell progression. Chrysophanol nanoparticles induced apoptosis in LNCap cells by promoting p53/ROS crosstalk to prevent proliferation. Pharmacokinetic study in mice indicated that chrysophanol nanoparticle injection showed high bioavailability compared to the free chrysophanol. Also, in vivo study revealed that chrysophanol nanoparticles obviously reduced tumor volume and weight. In conclusion, the data above suggested that chrysophanol nanoparticles might be effective to prevent human prostate cancer progression.

Insufficient sialylation can result in rapid clearance of therapeutic glycoproteins by intracellular degradation, which is mainly mediated by asialoglycoprotein receptors (ASGPRs) on hepatic cells. In contrast, for glycoproteins, a long half-life is often related to high level of terminal sialic acid. These could be extremely important for insufficient sialylated biomedicines in clinic, and development of therapeutic glycoproteins in laboratory. However, how the desialylated glycoproteins are removed and how to evaluate the ASGPRs mediated endocytosis in vitro needs further investigate. Herein we described an integrative characterization of ASGPRs in vitro to elucidate its endocytosis properties. The endocytosis was determined by a fluorescence-based quantization method. The results showed that the ASGPRs could bind to poorly sialylated glycoproteins including asialofetuin and low sialylated recombinant Factor VIIa with a relatively higher ASGPRs binding affinity, and induce a more rapid endocytosis in vitro. Moreover, the mechanism under the internalization of ASGPRs was also investigated, which was found to depend on clathrin and caveolin. Utilizing the relative fluorescence quantification can be suitable for measurement of insufficient sialylated glycoprotein endocytosis and quality control of therapeutic glycoproteins, which could be useful for the understanding of the development of therapeutic glycoproteins.

A previous study reported that Yi Guan Jian (YGJ) may increase the proliferation and differentiation of hepatic oval cells in a rat liver cirrhosis model. The aim of the present study was to investigate the effect and mechanism of action of YGJ on inducing hepatic differentiation in bone marrow‑derived mesenchymal stem cells (BM‑MSCs) via stromal‑cell derived factor‑1 (SDF‑1). Murine BM‑MSCs were isolated with whole bone marrow adherence, then identified by immunocytochemical staining and flow cytometry. Passage 2 cells were divided into 8 groups and their differentiation was induced by cell factors added to the medium, including hepatocyte growth factor (HGF), SDF‑1 and YGJ. Each of the cell factors was used alone and any two or three of them were combined to establish different cell microenvironments in the different treatment groups. Albumin (ALB) was selected as a hepatocellular marker and cytokeratin‑18 (CK‑18) as a cholangiocellular marker. The protein and mRNA expression levels of ALB and CK‑18 were used to determine the differentiation of BM‑MSCs using immunocytochemical staining, western blotting and reverse transcription‑quantitative polymerase chain reaction on days 7, 14, 21 and 28 during induction. The relative expression levels of ALB and CK‑18 resulted in time‑dependent increases in the groups supplemented only with HGF, SDF‑1 or YGJ. Combination treatment of any two HGF, SDF‑1 and YGJ led to a higher expression of ALB and CK‑18 compared with only one cell factor treatment. Additionally, when all three were used in a combined treatment the expression levels of ALB and CK‑18 occurred at an earlier time and was higher overall. Therefore, the present study suggested that YGJ had an effect on inducing hepatic differentiation in BM‑MSCs via SDF‑1 and may act in a synergistic manner with HGF and SDF‑1.

// Bing Yao 1, * , Bingqian Liu 1, * , Lei Shi 1, * , Xiang Li 2 , Chuanchuan Ren 1 , Mingbo Cai 3 , Wen Wang 3 , Jianhua Li 1 , Yongde Sun 1 , Yudong Wu 1 , Jianguo Wen 1 1 Department of Urology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China 2 Department of Plastic Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China 3 Department of Gynaecology and Obstetrics, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China * These authors contributed equally to this work Correspondence to: Yudong Wu, email: yudongwu_zhd@163.com Jianguo Wen, email: wenjg_zhdu@163.com Keywords: platelet activating factor receptor, prostatic neoplasms, radiotherapy, autophagy, drug sensitivity Received: July 22, 2016 Accepted: January 03, 2017 Published: January 14, 2017 ABSTRACT Platelet-activating factor receptor (PAFR) promotes tumorigenesis, angiogenesis and metastasis. Here, we defined the PAFR as a yielding new inhibiting target to selectively enhance the sensitivity of prostate cancer (PCa) cells to radiation. The selective responding to PAFR inhibiter may be caused by the differential expression pattern of PAFR in PCa cells. In this study, we also determined PAFR as a molecular basis by which the radiation induces autophagy suppression independent of activating mTOR pathway. PAFR can bind to the autophagy-indispensable protein Beclin 1, leading to the disability in its serine phosphorylation. The PAFR antagonist Ginkgolide B (GB) can sensitize radiotherapy by disrupting the formation of PAFR/Beclin 1 complex in PC3 and LNCaP cells, which have elevated PAFR expression after radiation exposure. Most importantly, GB efficiently radiosensitized PC3 and LNCaP tumor xenografts in vivo , and significantly reduced tumor burden. Overall, our results elucidated a significant role of GB in selectively improving the outcomes of PCa receiving radiation therapy.

Diabetic nephropathy (DN) is characterized by increased macrophage infiltration, and proinflammatory M1 macrophages contribute to development of DN. Previous studies by us and others have reported that macrophage cyclooxygenase-2 (COX-2) plays a role in polarization and maintenance of a macrophage tissue-reparative M2 phenotype. We examined the effects of macrophage COX-2 on development of DN in type 1 diabetes. Cultured macrophages with COX-2 deletion exhibited an M1 phenotype, as demonstrated by higher inducible nitric oxide synthase and nuclear factor-κB levels but lower interleukin-4 receptor-α levels. Compared with corresponding wild-type diabetic mice, mice with COX-2 deletion in hematopoietic cells (COX-2 knockout bone marrow transplantation) or macrophages (CD11b-Cre COX2f/f) developed severe DN, as indicated by increased albuminuria, fibrosis, and renal infiltration of T cells, neutrophils, and macrophages. Although diabetic kidneys with macrophage COX-2 deletion had more macrophage infiltration, they had fewer renal M2 macrophages. Diabetic kidneys with macrophage COX-2 deletion also had increased endoplasmic reticulum stress and decreased number of podocytes. Similar results were found in diabetic mice with macrophage PGE2 receptor subtype 4 deletion. In summary, these studies have demonstrated an important but unexpected role for macrophage COX-2/prostaglandin E2/PGE2 receptor subtype 4 signaling to lessen progression of diabetic kidney disease, unlike the pathogenic effects of increased COX-2 expression in intrinsic renal cells.

Particle preloading method by first loading proteins onto nano- or microparticles and then integrating these particles into electrospun polyester nanofibers has been widely used to encapsulate therapeutic proteins into polyester nanofibers. However, poor method design has resulted in unsatisfactory protein delivery performance. For example, the harsh conditions involved in preloading procedures damage the bioactivities of proteins, the improper integration leads to an uneven distribution of particles in nanofibers or insecure attachment of particles to nanofibers, producing uncontrolled protein release profiles. This study aimed to improve the protein delivery performance of polyester nanofibers by rationally designing a particle preloading method. Positively charged chitosan nanoparticles (CNPs) were used as carriers to adsorb negatively charged proteins in mild conditions and as primary barriers for protein release. The polar CNPs were then homogeneously dispersed in a polar polyester solution and subjected to electrospinning. Microscope observations indicated that CNPs were homogeneously embedded within polyester nanofibers. In vitro release behaviour and cell studies showed that proteins retained their bioactivity and could release from polyester nanofibers in a sustained manner for more than 4 weeks without any initial burst. Epidermal growth factor encapsulated in polyester nanofibers enhanced diabetic wound healing in vivo, demonstrating an application potential in biomedicine. Other properties of the nanofibers, including composition, wettability, cytotoxicity, and cell adhesion and spreading, were examined in detail as well.

To observe the repairing effects of estrogen and wheat-grain moxibustion on thin-type endometrium in rats and to explore its possible mechanism.Forty healthy SPF-grade adult female SD rats were randomly divided into a normal group, a model group, an estrogen group and a moxibustion group according to random number table method, 10 rats in each group. The model of thin-type endometrium was established during estrous period in all the groups except for the normal group. No intervention was given in the normal group. The intragastric administration of 2 mL of 0.9% sodium chloride solution was applied the next day after modeling in the model group. The intragastric administration of 2 mL of estradiol was given the next day after modeling in the estrogen group. The wheat-grain moxibustion was given at "Guanyuan" (CV 4) and "Shenshu" (BL 23) the next day after modeling in the moxibustion group, 7 moxa cones for each acupoint. The treatment in 3 groups was given once a day. After three estrous cycles, the samples were collected during estrous period; the thickness and morphology of endometrium were observed by HE staining; the expressions of vimentin, keratin and vascular endothelial growth factor (VEGF) in endometrium tissue were detected by immunohistochemistry; the expressions of HOXA10 and LIF in endometrium tissue were detected by Western blot.The endometrial thickness in the model group was significantly thinner than that in the normal group (The wheat-grain moxibustion could up-regulate the expressions of keratin, vimentin and VEGF in endometrium to improve the endometrial thickness; in addition, it could increase the levels of factors related to endometrial receptivity including HOXA10, LIF, which improves endometrial receptivity and play a repair role.

Summary The Drosophila Taiman (Tai) protein is homologous to the human steroid-receptor coactivators SRC1–3 and activates transcription in complex with the 20-hydroxyecdysone (20E) receptor (EcR). Tai has roles in intestinal homeostasis, germline maintenance, cell motility, and proliferation through interactions with EcR and the coactivator Yorkie (Yki). Tai also promotes invasion of tumor cells in adjacent organs, but this pro-invasive mechanism is undefined. Here, we show that Tai expression transforms sessile pupal wing cells into an invasive mass that penetrates the adjacent thorax during a period of high 20E. Candidate analysis confirms a reliance on elements of the 20E and Hippo pathways, such as Yki and the Yki-Tai target dilp8. Screening the Tai-induced wing transcriptome detects enrichment for innate immune factors, including the Spatzle (Spz) family of secreted Toll ligands that induce apoptosis during cell competition. Tai-expressing wing cells induce immune signaling and apoptosis among adjacent thoracic cells, and genetic reduction of spz, Toll, or the rpr/hid/grim pro-apoptotic factors each suppresses invasion, suggesting an intercellular Spz-Toll circuit supports killing-mediated invasion. Modeling these interactions in larval epithelia confirms that Tai kills neighboring cells via a mechanism involving Toll, Spz factors, and the Spz inhibitor Necrotic. Tai-expressing cells evade death signals by repressing the immune deficiency (IMD) pathway, which operates in parallel to Toll to control nuclear factor κB (NF-κB) activity and independently regulates JNK activity. In sum, these findings suggest that Tai promotes competitive cell killing via Spz-Toll and that this killing mechanism supports pathologic intertissue invasion in Drosophila.

DNA methylation is dynamically modulated during postnatal brain development, and plays a key role in neuronal lineage commitment. This epigenetic mark has also recently been implicated in the development of neural sex differences, many of which are found in the hypothalamus. The level of DNA methylation depends on a balance between the placement of methyl marks by DNA methyltransferases (Dnmts) and their removal, which is catalyzed by ten-eleven translocation (Tet) methylcytosine dioxygenases. Here, we examined developmental changes and sex differences in the expression of Tet and Dnmt enzymes from birth to adulthood in two hypothalamic regions (the preoptic area and ventromedial nucleus) and the hippocampus of mice. We found highest expression of all Tet enzymes (Tet1, Tet2, Tet3) and Dnmts (Dnmt1, Dnmt3a, Dnmt3b) in newborns, despite the fact that global methylation and hydroxymethylation were at their lowest levels at birth. Expression of the Dnmt co-activator, Dnmt3l, followed a pattern opposite to that of the canonical Dnmts (i.e., was very low in newborns and increased with age). Tet enzyme activity was much higher at birth than at weaning in both the hypothalamus and hippocampus, mirroring developmental changes in gene expression. Sex differences in Tet enzyme expression were seen in all brain regions examined during the first week of life, whereas Dnmt expression was more balanced between the sexes. Neonatal testosterone treatment of females only partially masculinized enzyme expression. Thus, Tet expression and activity are elevated during neonatal brain development, and may play important roles in sexual differentiation of the brain. Fil: Cisternas, Carla Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina. Georgia State University; Estados Unidos Fil: Cortes, Laura R.. Georgia State University; Estados Unidos Fil: Bruggeman, Emily C.. Emory University School Of Medicine; Estados Unidos Fil: Yao, Bing. Emory University School Of Medicine; Estados Unidos Fil: Forger, Nancy G.. Georgia State University; Estados Unidos

Aims Activation of brown adipose tissue (BAT) thermogenesis could contribute to energy expenditure, which is critical for the treatment of obesity and type 2 diabetes mellitus (T2DM). In the present study, we aimed to systematically investigate whether traditional Chinese medication Jinlida (JLD) granules could improve metabolic disorders and activate BAT thermogenesis in C57BL/6 J mice fed with a high-fat diet (HFD). Methods In the present study, JLD (3.8 g/kg) in 0.5% of carboxymethyl cellulose (CMC) solution was administrated daily by oral gavage to HFD-induced mice for 15 weeks. The body weight, biochemical analysis, histology analysis, intraperitoneal glucose and insulin tolerance (OGTT and ITT) tests were measured to explore metabolic disorders. Cold tolerance test, real-time PCR (qRT-PCR), immunohistochemistry, and western blot were performed to evaluate BAT function. Results As results, JLD treatment significantly ameliorated HFD-induced obesity and fat mass gain, maintained glucose and lipid homeostasis, and improved hepatic steatosis and inflammation. More importantly, we observed that JLD markedly activated BAT thermogenesis in HFD-induced obese mice. Moreover, our data confirmed that JLD promoted mitochondrial biogenesis and fatty acid oxidation metabolism in BAT. Conclusions These data suggested that JLD could improve metabolic disorders in associated with activation of BAT thermogenesis via enhancement of mitochondrial biogenesis and fatty acid oxidation metabolism, thus providing a new pharmacological evidence for the clinical usage of JLD in T2DM treatment.

Approximately 70% of patients with advanced breast cancer develop bone metastases, accompanied by complications, such as bone pain, fracture, and hypercalcemia. However, our understanding of the molecular mechanisms that govern this process remains fragmentary. Osterix (Osx) is a zinc finger-containing transcription factor essential for osteoblast differentiation and bone formation. Here, we identified the functional roles of Osx in facilitating breast cancer invasion and bone metastasis. Osx upregulation was associated with lymph node metastasis and was negatively prognostic for overall survival. Knockdown of Osx inhibited invasion of breast cancer and osteolytic metastasis by downregulating MMP9, MMP13, VEGF, IL-8, and PTHrP, which are involved in invasion, angiogenesis, and osteolysis; overexpression of Osx had the opposite effect. Moreover, MMP9 was a direct target of Osx and mediated the Osx-driven invasion of breast cancer cells. Together, our data showed that Osx facilitates bone metastasis of breast cancer by upregulating the expression of a cohort of genes that contribute to steps in the metastatic cascade. These findings suggest that Osx is an attractive target for the control of bone metastasis of breast cancers., Osx facilitates bone metastasis of breast cancer by upregulating the expression of a cohort of genes that contribute to steps in the metastatic cascade. These genes include MMP9, VEGF, as well as IL-8, PTHrP, and MMP13. MMP9 is a newly identified target of Osx. .

This study aimed to elucidate the effect of electroacupuncture treatment on preventing early ovarian hyperstimulation syndrome (OHSS) and the potential mechanisms involved using an induced rat model. The ovarian response was examined by measuring ovary weight, vascular permeability, levels of inflammation (interleukin-6), tumour necrosis factor alpha, chemokine ligand 2 (also known as monocyte chemoactic protein 1), vascular endothelial growth factor and hormone concentrations (oestradiol, progesterone, testosterone and prolactin). Sprague–Dawley female rats underwent ovarian stimulation to induce OHSS. Hyperstimulated rats received consecutive electroacupuncture treatment from 3 days before the beginning of pregnant mare serum gonadotrophin treatment or the time point of pregnant mare serum gonadotrophin treatment respectively, and last until 3 days after HCG administration. Electroacupuncture treatment reduced ovary weight and vascular permeability in hyperstimulated rats. Electroacupuncture treatment also reduced the levels of serum steroid hormones (progesterone and testosterone), inflammatory cytokines (interleukin-6, tumour necrosis factor alpha and monocyte chemotactic protein 1 and vascular endothelial growth factor in hyperstimulated rats. The results indicate that electroacupuncture can modulate endocrine hormone secretion and affect the secretion of inflammatory cytokines and vascular endothelial growth factor, and thus prevent the progress of OHSS. Electroacupuncture may provide a simple and effective method for the prevention and treatment of OHSS.

Late‑onset hypogonadism is defined as a condition caused by a decline in the levels of testosterone with aging. One of the major factors contributing to the low levels of testosterone is the accumulation of reactive oxygen species (ROS) in Leydig cells during the ageing process. Peroxiredoxin 2 (Prdx2), a member of the peroxiredoxin family, is an antioxidant protein, the predominant function of which is to neutralize ROS. However, its role in Leydig cells remains to be elucidated. In the present study, primary Leydig cells were exposed to low concentrations of hydrogen peroxide (H2O2) to induce oxidative stress. Cell apoptosis was measured using an Annexin V fluorescein isothiocyanate/propidium iodide apoptosis detection kit and flow cytometry. The level of testosterone was determined by radioimmunoassay, and the mRNA and protein expression levels of Prdx2 were detected by reverse transcription‑polymerase chain reaction and western blotting, respectively. The results revealed a significant increase in cell apoptosis and decrease in testosterone production. In addition, the expression of Prdx2 was decreased by H2O2 in a dose‑ and time‑dependent manner, and this decrease may have been caused by the induction of its molecular structure transformation due to H2O2 elimination. The above findings indicated that Prdx2 may prevent H2O2 accumulation in Leydig cells, and may be important in oxidative stress‑induced apoptosis and decreased testosterone production.