Active immunization with recombinant GnRH6-CRM197 inhibits reproductive function of male rats.

Gonadotropin-releasing hormone (GnRH) vaccines have been successfully used for the inhibition of gonadal development and function, but current GnRH-based vaccines often present variability in the response. Cross-reactive material 197 (CRM197) has been used as carrier molecules to enhance an immune response to associated antigens. So, the synthetic mammalian tandem-repeated GnRH hexamer (GnRH6) gene was integrated into the expression plasmid pET-21a. Recombinant GnRH6-CRM197 protein was subsequently overexpressed in Escherichia coli strain BL21 and purified through Nickel column affinity chromatography and the antigenicity and biological effects of GnRH6-CRM197 were evaluated in rats. Sixteen 4-month-old adult male rats were randomly divided into two groups: the GnRH6-CRM197 group (n = 8) and the control group (n = 8). The GnRH6-CRM197 group rats were subcutaneously immunized with 100 µg of GnRH6-CRM197, administered thrice at 2-week intervals with GnRH6-CRM197.The control group received only a white oil adjuvant. Following the initial immunization, the weights of animals were recorded, and blood samples were collected from the orbital sinus at 4, 4.5, 5, 5.5, 6, 6.5, and 7 months. Serum antibody titers and testosterone concentrations were quantified using ELISA and CLIA, respectively. Additionally, testicular tissues were collected for morphological examination. The results revealed a significant increase in serum GnRH antibody titers (p < 0.05), but a significant decrease in serum testosterone concentrations (p < 0.05), and the weight, length, width, and girth of the testis, and the number of spermatogonia cells, spermatocytes, and sperm cells in the immunized rats. Furthermore, seminiferous tubules revealed significant atrophy and no sperm were observed in the immunized animals. Thus, GnRH6-CRM197 may be an effective antigen and a potential immunocastration vaccine.

Grid1 regulates the onset of puberty in female rats.

The study aimed to investigate the effect of Grid1, encoding the glutamate ionotropic receptor delta type subunit 1 (GluD1), on puberty onset in female rats. Grid1 mRNA and protein expression was detected in the hypothalamus of female rats at prepuberty and puberty. The levels of Grid1 mRNA in the hypothalamus, the fluorescence intensity in the arcuate nucleus and paraventricular nucleus of the prepubertal rats was significantly lower than pubertal. Additionally, the expression of Grid1 was suppressed in primary hypothalamus cells and prepubertal rat. Finally, investigated the effect of Grid1 knockdown on puberty onset and reproductive performance. Treatment of hypothalamic neurons with LV-Grid1 decreased the level of Grid1 and Rfrp-3 (encoding RFamide-related peptide 3) mRNA expression, but increased the Gnrh (encoding gonadotropin-releasing hormone) mRNA levels. After an ICV injection, the time for the rat vaginal opening occurred earlier. Moreover, Gnrh mRNA expression was increased, whereas Rfrp-3 mRNA expression was decreased in the hypothalamus. The concentration of progesterone (P4) in the serum was significantly decreased compare with control group. Ovary hematoxylin-eosin staining revealed that the LV-Grid1 group mainly contained primary and secondary follicles. The reproductive performance of the rats was not affected by the Grid1 knockdown. Therefore, Grid1 may affect the onset of puberty in female rats by regulating the levels of Gnrh, and Rfrp-3 in the hypothalamus, as well as the concentrations of P4, but not reproduction performance.

The effect of semaglutide on blood pressure in patients with type-2 diabetes: a systematic review and meta-analysis.

OBJECTIVE: To evaluate the blood pressure (BP) lowering ability of semaglutide, a glucagon-like peptide-1 receptor agonist (GLP-1 RA), in individuals with type-2 diabetes (T2D). METHODS: Randomized controlled trials (RCTs) comparing subcutaneous or oral semaglutide with placebo or other antihyperglycemic agents (AHAs) in T2D patients were identified by searching PubMed, Embase, Web of Science, ClinicalTrials.gov and Cochrane Library. These screened studies included the outcomes of interest: systolic and/or diastolic BP. Weighted mean differences (WMDs) and 95 % confidence intervals (CIs) were used to present the meta-analysis results. Pooled and sensitivity analyses were performed, and the risk of bias was evaluated. RESULTS: Twenty-nine RCTs with a total of 26985 participants were recruited in the final analysis. The WMD in change from baseline in systolic BP (SBP) of semaglutide versus placebo or other AHAs was -2.31 mmHg (95% CI: -3.11 to -1.51), while that for diastolic BP (DBP) was 0.09 mmHg (95% CI: -0.16 to 0.33). It also reduced glycated hemoglobin A1c (HbA1c) by 0.75% (95% CI: -0.92 to -0.58) and body weight loss by 2.80 kg (95% CI: -3.51 to -2.08). The reduction in SBP was similar for subcutaneous and oral administration of semaglutide, with -2.36 (95% CI: -3.38 to -1.35) and -2.50 (95% CI: -3.48 to -1.53), respectively. CONCLUSIONS: In T2D, SBP decreased significantly in the semaglutide group compared with placebo or other active controls. According to the efficacy results from this meta-analysis, subcutaneous and oral semaglutide have similar SBP-reducing effects. Therefore, the treatment of T2D patients with subcutaneous semaglutide or oral preparations is beneficial for reducing SBP.

Integrative proteomic and phosphoproteomic analysis in the female goat hypothalamus to study the onset of puberty.

BACKGROUND: Puberty marks the end of childhood and achieve sexual maturation and fertility. The role of hypothalamic proteins in regulating puberty onset is unclear. We performed a comprehensive differential proteomics and phosphoproteomics analysis in prepubertal and pubertal goats to determine the roles of hypothalamic proteins and phosphoproteins during the onset of puberty. RESULTS: We used peptide and posttranslational modifications peptide quantification and statistical analyses, and identified 69 differentially expressed proteins from 5,057 proteins and 576 differentially expressed phosphopeptides from 1574 phosphorylated proteins. Combined proteomic and phosphoproteomics, 759 correlated proteins were identified, of which 5 were differentially expressed only at the protein level, and 201 were only differentially expressed at the phosphoprotein level. Pathway enrichment analyses revealed that the majority of correlated proteins were associated with glycolysis/gluconeogenesis, Fc gamma R-mediated phagocytosis, focal adhesion, GABAergic synapse, and Rap1 signaling pathway. These pathways are related to cell proliferation, neurocyte migration, and promoting the release of gonadotropin-releasing hormone in the hypothalamus. CTNNB1 occupied important locations in the protein-protein interaction network and is involved in focal adhesion. CONCLUSION: The results demonstrate that the proteins differentially expression only at the protein level or only differentially expressed at the phosphoprotein level and their related signalling pathways are crucial in regulating puberty in goats. These differentially expressed proteins and phosphorylated proteins may constitute the proteomic backgrounds between the two different stages.

Knockdown of lncRNA Meg3 delays the onset of puberty in female rats.

This study investigated how lncRNA Meg3 affects the onset of puberty in female rats. We determined Meg3 expression in the hypothalamus-pituitary-ovary axis of female rats at the infancy, prepubertal, pubertal, and adult life stages, using quantitative reverse transcription polymerase chain reaction (qRT-PCR). We also assessed the effects of Meg3 knockdown on the expression levels of puberty-related genes and Wnt/ß-catenin proteins in the hypothalamus, time of puberty onset, levels of reproductive genes and hormones, and ovarian morphology in female rats. Meg3 expression in the ovary varied significantly between prepuberty and puberty (P < 0.01). Meg3 knockdown decreased the expression of Gnrh, and Kiss1 mRNA (P < 0.05) and increased the expression of Wnt (P < 0.01) and ß-catenin proteins (P < 0.05) in the hypothalamic cells. Onset of puberty in Meg3 knockdown rats was delayed compared to the control group (P < 0.05). Meg3 knockdown decreased Gnrh mRNA levels (P < 0.05) and increased Rfrp-3 mRNA levels (P < 0.05) in the hypothalamus. The serum concentrations of progesterone (P4) and estradiol (E2) of Meg3 knockdown rats were lower than those in the control animals (P < 0.05). Higher longitudinal diameter and ovary weight were found in Meg3 knockdown rats (P < 0.05). These findings suggest that Meg3 regulates the expression of Gnrh, Kiss-1 mRNA and Wnt/ß-catenin proteins in the hypothalamic cells, and Gnrh, Rfrp-3 mRNA of the hypothalamus and the serum concentration of P4 and E2, and its knockdown delays the onset of puberty in female rats.

Analysis of serum reproductive hormones and ovarian genes in pubertal female goats.

BACKGROUND: Age at puberty is an important factor affecting goat fertility, with endocrine and genetic factors playing a crucial role in the onset of puberty. To better understand the relationship between endocrine and genetic factors and mechanisms underlying puberty onset in goats, reproductive hormone levels were analyzed by ELISA and ultraperformance liquid chromatography-multiple reaction monitoring-multistage/mass spectrometry and RNA sequencing was performed to analyze ovarian genes. RESULTS: Serum follicle stimulating hormone, luteinizing hormone, estradiol, 11-deoxycortisol, 11-deoxycorticosterone, corticosterone, cortisone, and cortisol levels were found to be higher but progesterone were lower in pubertal goats as compared to those in prepubertal goats (P < 0.05). A total of 18,139 genes were identified in cDNA libraries, and 75 differentially expressed genes (DEGs) were identified (|log2 fold change|≥ 1, P ≤ 0.05), of which 32 were significantly up- and 43 were down-regulated in pubertal goats. Gene ontology enrichment analyses indicated that DEGs were mainly involved in "metabolic process," "signaling," "reproduction," and "growth." Further, DEGs were significantly enriched in 91 Kyoto Encyclopedia of Genes and Genomes pathways, including estrogen signaling pathway, steroid hormone biosynthesis, and cAMP signaling pathway. Bioinformatics analysis showed that PRLR and THBS1 were highly expressed in pubertal ovaries, and ZP3, ZP4, and ASTL showed low expression, suggesting their involvement in follicular development and lutealization. CONCLUSIONS: To summarize, serum hormone changes and ovarian DEGs expression were investigated in our study. Further studies are warranted to comprehensively explore the functions of DEGs in goat puberty.

MicroRNA21 inhibition affects porcine oocyte maturation and alters protein expression critical for metabolic pathway function.

MicroRNA21 (MIR21) abundance in porcine oocytes and cumulus cells increases during in vitro maturation. The mechanism by which MIR21 regulates oocyte maturation and the effect on the developmental competence of subsequent embryos remains unclear. The objective of this study was to assess the function of MIR21 during porcine oocyte maturation and its effect on embryonic development. Treatment with peptide nucleic acid MIR21 inhibitor (MIR21-PNA), designed to specifically bind to and prevent MIR21 activity during in vitro oocyte maturation, decreased cumulus cell expansion, and the oocyte ability to achieve metaphase II maturation stage when compared to control groups. Following parthenogenetic activation, the cleavage rate at 48 h in the MIR21-PNA group was decreased (p ≤ 0.03) relative to the control groups. Additionally, liquid chromatography-mass spectrometry (LC-MS/MS) of oocyte and cumulus cell total protein following MIR21-PNA treatment during in vitro maturation identified changes in signaling pathways with primary involvement of glucose metabolism (GM) pathways. Furthermore, there was no difference (p = 0.21) in oocyte maturation of control and MIR21-PNA treated oocytes when cultured in pyruvate lacking medium. Finally, MIR21-PNA treatment decreased (p = 0.04) glutathione and increased (p = 0.07) reactive oxygen species production in the oocyte. These data suggest that MIR21 influences porcine oocyte maturation by regulating GM pathways in the cumulus-oocyte complex.

The expression of IGFBP-5 in the reproductive axis and effect on the onset of puberty in female rats.

Insulin-like growth factor-binding protein-5 (IGFBP-5) has recently been shown to alter the reproductive capacity by regulating insulin-like growth factor (IGF) bioavailability or IGF-independent effects. The present study aimed to investigate the effect and mechanism of IGFBP-5 on the onset of puberty in female rats. Immunofluorescence and real-time quantitative PCR were used to determine the expression and location of IGFBP-5 mRNA and protein distribution in the infant's hypothalamus-pituitary-ovary (HPO) axis prepuberty, peripuberty, puberty and adult female rats. Prepubertal rats with IGFBP-5 intracerebroventricular (ICV) were injected to determine the puberty-related genes expression and the concentrations of reproductive hormones. Primary hypothalamic cells were treated with IGFBP-5 to determine the expression of puberty-related genes and the Akt and mTOR proteins. Results showed that Igfbp-5 mRNA and protein were present on the HPO axis. The addition of IGFBP-5 to primary hypothalamic cells inhibited the expression of Gnrh and Igf-1 mRNAs (P < 0.05) and increased the expression of AKT and mTOR protein (P < 0.01). IGFBP-5 ICV-injection delayed the onset of puberty, reduced Gnrh, Igf-1, and Fshß mRNAs, and decreased the concentrations of E2, P4, FSH,serum LH levels and the ovaries weight (P < 0.05). More corpus luteum and fewer primary follicles were found after IGFBP-5 injection (P < 0.05).

Quantitative proteomics analysis to assess protein expression levels in the ovaries of pubescent goats.

BACKGROUND: Changes in the abundance of ovarian proteins play a key role in the regulation of reproduction. However, to date, no studies have investigated such changes in pubescent goats. Herein we applied isobaric tags for relative and absolute quantitation (iTRAQ) and liquid chromatography-tandem mass spectrometry to analyze the expression levels of ovarian proteins in pre-pubertal (n = 3) and pubertal (n = 3) goats. RESULTS: Overall, 7,550 proteins were recognized; 301 (176 up- and 125 downregulated) were identified as differentially abundant proteins (DAPs). Five DAPs were randomly selected for expression level validation by Western blotting; the results of Western blotting and iTRAQ analysis were consistent. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis indicated that DAPs were enriched in olfactory transduction, glutathione metabolism, and calcium signaling pathways. Besides, gene ontology functional enrichment analysis revealed that several DAPs enriched in biological processes were associated with cellular process, biological regulation, metabolic process, and response to stimulus. Protein-protein interaction network showed that proteins interacting with CDK1, HSPA1A, and UCK2 were the most abundant. CONCLUSIONS: We identified 301 DAPs, which were enriched in olfactory transduction, glutathione metabolism, and calcium signaling pathways, suggesting the involvement of these processes in the onset of puberty. Further studies are warranted to more comprehensively explore the function of the identified DAPs and aforementioned signaling pathways to gain novel, deeper insights into the mechanisms underlying the onset of puberty.

Effect of GnRH immunocastration on immune function in male rats.

The present study aimed to reveal the effects of immunocastration on the development of the immune system in rats. Seventy rats were randomly assigned into two groups: Control (n = 35) and immunized (n = 35). Twenty-day-old rats were immunized with gonadotropin-releasing hormone (GnRH) and booster immunization was administered every two weeks (three immunizations in total). From 20-day-old rats, we collected samples every two weeks, including five immunized rats and five control rats (seven collections in total). We collected blood samples, testicles, thymuses, and spleens. The results showed that GnRH immunization increased the GnRH antibody titers and reduced the testosterone concentration (both P < 0.05). Compared with the control group, the number of CD4+CD8- cells, CD4-CD8+ cells, and CD4+CD8+ cells increased (P < 0.05) whereas the number of CD4-CD8- cells and CD4+CD25+ cells reduced in the immunized group (P < 0.05) over time. GnRH immunization also increased the relative weights of thymus and spleen (P < 0.05), serum concentrations of interleukin (IL)-2, IL-4, IL-6, IL-10, IL-17 and Interferon-γ (IFN-γ) over time (P < 0.05), and changed the mRNA levels of IL-2, IL-4, IL-6. IL-10, IL-17, IFN-γ, CD4, D8, CD19 GnRH, and GnRH receptor (GnRH-R) in thymus and spleen. Thus, GnRH immunization enhanced the immune markers in thymus, spleen, and blood immune cytokines in rats.

Knockdown of Ptprn-2 delays the onset of puberty in female rats.

In the present study, we evaluated how Ptprn-2 (encoding tyrosine phosphatase, receptor type, N2 polypeptide protein) affects the onset of puberty in female rats. We evaluated the expression of Ptprn-2 mRNA and protein in the hypothalamus-pituitary-ovary axis of female rats using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunofluorescence at infancy, prepuberty, puberty, peripuberty, and adulthood. We evaluated the effects of Ptprn-2 gene knockdown on different aspects of reproduction-related biology in female rats, including the expression levels of puberty-related genes in vivo and in vitro, the time to onset of puberty, the concentration of serum reproductive hormones, the morphology of ovaries, and the ultrastructure of pituitary gonadotropin cells. Our results demonstrated that PTPRN-2 was primarily distributed in the arcuate nucleus (ARC), periventricular nucleus (PeN), adenohypophysis, and the ovarian follicular theca, stroma, and granulosa cells of female rats at various stages. Ptprn-2 mRNA levels significantly varied between peripuberty and puberty (P < 0.05) in the hypothalamus and pituitary gland. In hypothalamic cells, Ptprn-2 knockdown decreased the expression of Ptprn-2 and Rfrp-3 mRNA (P < 0.05) and increased the levels of Gnrh and Kiss-1 mRNA (P < 0.05). Ptprn-2 knockdown in the hypothalamus resulted in delayed vaginal opening compared to the control group (n = 12, P < 0.01), and Ptprn-2, Gnrh, and Kiss-1 mRNA levels (P < 0.05) all decreased, while the expression of Igf-1 (P < 0.05) and Rfrp-3 mRNA (P < 0.01) increased. The concentrations of FSH and P4 in the serum of Ptprn-2 knockdown rats were lower than in control animals (P < 0.05). Large transverse perimeters and longitudinal perimeters (P < 0.05) were found in the ovaries of Ptprn-2 knockdown rats. There were fewer large secretory particles from gonadotropin cells in adenohypophysis tissue of the Ptprn-2 knockdown group compared to the control group. This indicates that Ptprn-2 knockdown can regulate levels of Gnrh, Kiss-1, and Rfrp-3 mRNA in the hypothalamus, regulate the concentration of serum FSH and P4, and alter the morphology of ovarian and gonadotropin cells, delaying the onset of puberty in female rats.

[Salvianolic acid B promotes the proliferation, migration and osteogenic differentiation of human gingival mesenchymal stem cells by activating the PI3K/AKT pathway].

Objective To investigate the effect of salvianolic acid B (SalB) on the proliferation, migration and differentiation of human gingival mesenchymal stem cells (hGMSCs). Methods The hGMSCs were isolated and cultured, and the expressions of CD73, CD90, CD34, and CD45 were detected by flow cytometry; logarithmic phase cells were selected and hGMSCs were treated with 0, 5, 10 µmol/L SalB for 24 hours. The proliferation activity of cells in each group was detected by CCK-8 assay, the migration ability of cells was detected by TranswellTM assay and scratch test, and the osteogenic differentiation ability and the adipogenic differentiation ability were detected by alkaline phosphatase (ALP) staining and oil red O staining, respectively; the mRNA and protein expressions of cell differentiation proteins and genes as well as proteins related to the PI3K/AKT signal pathway were detected by Real-time quantiative PCR and Western blotting. Results The proliferation and migration ability of SalB-treated hGMSCs were significantly increased in a dose-dependent manner; the ability of osteogenic differentiation of hGMSCs and the expressions of osteogenesis associated proteins and PI3K/AKT signal pathway related proteins were up-regulated, while the adipogenic differentiation decreased, and the expression of adipogenesis related proteins was significantly down-regulated. Conclusion SalB promotes the proliferation, migration, and osteogenic differentiation of hGMSCs and inhibits the adipogenic differentiation, which may be related to the activation of PI3K/AKT signal pathway.

Oxycodone versus morphine for analgesia after laparoscopic endometriosis resection.

BACKGROUND: The objective of this study was to compare the analgesic potency of oxycodone versus morphine after laparoscopic deep infiltrating endometriosis resection. METHODS: Fifty patients undergoing laparoscopic deep infiltrating endometriosis resection were randomized to receive oxycodone or morphine intravenous-PCA after surgery. The primary outcome was opioid consumption during the 24 h after surgery. Secondary outcomes included time to first request for analgesia, the number of bolus, pain, sedation, nausea, vomiting, respiratory depression, and bradycardia. The prominent pain that caused patients to press the analgesic device was also recorded. RESULTS: Oxycodone consumption (14.42 ± 2.83) was less than morphine consumption (20.14 ± 3.83). Compared with the morphine group, the total number of bolus (78 vs 123) was less and the average time to first request for analgesia (97.27 ± 59.79 vs 142.17 ± 51) was longer in the oxycodone group. The incidence of nausea was higher in the morphine group than in the oxycodone group at 0-2 h (45.45% vs 17.19%), 2-4 h (50% vs 17.19%),12-24 h (40.91% vs 13.04%) and 0-24 h (39.17% vs 19.13%). The overall incidence of vomiting was higher in the morphine group (27.27% vs 13.92%). There was no difference in visual analogue scale score, the incidence of respiratory depression, and bradycardia between groups. Of the three types of pain that prompted patients to request analgesia, the incidence of visceral pain was highest (59.9%, P < 0.01). CONCLUSION: Oxycodone was more potent than morphine for analgesia after laparoscopic endometriosis resection, and oxycodone has fewer side effects than morphine. Name of the registry: Chinese Clinical Trial Registry Trial registration number: ChiCTR1900021870 URL of trial registry record: http://www.chictr.org.cn/edit.aspx?pid=35799&htm=4 Date of registration: 2019/3/13 0:00:00.

Fluorescence Microscopy for ATP Internalization Mediated by Macropinocytosis in Human Tumor Cells and Tumor-xenografted Mice.

Adenosine triphosphate (ATP), including extracellular ATP (eATP), has been shown to play significant roles in various aspects of tumorigenesis, such as drug resistance, epithelial-mesenchymal transition (EMT), and metastasis. Intratumoral eATP is 103 to 104 times higher in concentration than in normal tissues. While eATP functions as a messenger to activate purinergic signaling for EMT induction, it is also internalized by cancer cells through upregulated macropinocytosis, a specific type of endocytosis, to perform a wide variety of biological functions. These functions include providing energy to ATP-requiring biochemical reactions, donating phosphate groups during signal transduction, and facilitating or accelerating gene expression as a transcriptional cofactor. ATP is readily available, and its study in cancer and other fields will undoubtedly increase. However, eATP study remains at an early stage, and unresolved questions remain unanswered before the important and versatile activities played by eATP and internalized intracellular ATP can be fully unraveled. These authors' laboratories' contributions to these early eATP studies include microscopic imaging of non-hydrolysable fluorescent ATP, coupled with high- and low-molecular weight fluorescent dextrans, which serve as macropinocytosis and endocytosis tracers, as well as various endocytosis inhibitors, to monitor and characterize the eATP internalization process. This imaging modality was applied to tumor cell lines and to immunodeficient mice, xenografted with human cancer tumors, to study eATP internalization in vitro and in vivo. This paper describes these in vitro and in vivo protocols, with an emphasis on modifying and finetuning assay conditions so that the macropinocytosis-/endocytosis-mediated eATP internalization assays can be successfully performed in different systems.

Characterization of the effects of heat stress on autophagy induction in the pig oocyte.

BACKGROUND: Heat stress (HS) occurs when body heat accumulation exceeds heat dissipation and is associated with swine seasonal infertility. HS contributes to compromised oocyte integrity and reduced embryo development. Autophagy is a potential mechanism for the oocyte to mitigate the detrimental effects of HS by recycling damaged cellular components. METHODS: To characterize the effect of HS on autophagy in oocyte maturation, we utilized an in vitro maturation (IVM) system where oocytes underwent thermal neutral (TN) conditions throughout the entire maturation period (TN/TN), HS conditions during the first half of IVM (HS/TN), or HS conditions during the second half of IVM (TN/HS). RESULTS: To determine the effect of HS on autophagy induction within the oocyte, we compared the relative abundance and localization of autophagy-related proteins. Heat stress treatment affected the abundance of two well described markers of autophagy induction: autophagy related gene 12 (ATG12) in complex with ATG5 and the cleaved form of microtubule-associated protein 1 light chain 3 beta (LC3B-II). The HS/TN IVM treatment increased the abundance of the ATG12-ATG5 complex and exacerbated the loss of LC3B-II in oocytes. The B-cell lymphoma 2 like 1 protein (BCL2L1) can inhibit autophagy or apoptosis through its interaction with either beclin1 (BECN1) or BCL2 associated X, apoptosis regulator (BAX), respectively. We detected colocalization of BCL2L1 with BAX but not BCL2L1 with BECN1, suggesting that apoptosis is inhibited under the HS/TN treatment but not autophagy. Interestingly, low doses of the autophagy inducer, rapamycin, increased oocyte maturation. CONCLUSIONS: Our results here suggest that HS increases autophagy induction in the oocyte during IVM, and that artificial induction of autophagy increases the maturation rate of oocytes during IVM. These data support autophagy as a potential mechanism activated in the oocyte during HS to recycle damaged cellular components and maintain developmental competence.

A small-molecule pan-class I glucose transporter inhibitor reduces cancer cell proliferation in vitro and tumor growth in vivo by targeting glucose-based metabolism.

BACKGROUND: Cancer cells drastically increase the uptake of glucose and glucose metabolism by overexpressing class I glucose transporters (GLUT1-4) to meet their energy and biomass synthesis needs and are very sensitive and vulnerable to glucose deprivation. Although targeting glucose uptake via GLUTs has been an attractive anticancer strategy, the relative anticancer efficacy of multi-GLUT targeting or single GLUT targeting is unclear. Here, we report DRB18, a synthetic small molecule, is a potent anticancer compound whose pan-class I GLUT inhibition is superior to single GLUT targeting. METHODS: Glucose uptake and MTT/resazurin assays were used to measure DRB18's inhibitory activities of glucose transport and cell viability/proliferation in human lung cancer and other cancer cell lines. Four HEK293 cell lines expressing GLUT1-4 individually were used to determine the IC50 values of DRB18's inhibitory activity of glucose transport. Docking studies were performed to investigate the potential direct interaction of DRB18 with GLUT1-4. Metabolomics analysis was performed to identify metabolite changes in A549 lung cancer cells treated with DRB18. DRB18 was used to treat A549 tumor-bearing nude mice. The GLUT1 gene was knocked out to determine how the KO of the gene affected tumor growth. RESULTS: DRB18 reduced glucose uptake mediated via each of GLUT1-4 with different IC50s, which match with the docking glidescores with a correlation coefficient of 0.858. Metabolomics analysis revealed that DRB18 altered energy-related metabolism in A549 cells by changing the abundance of metabolites in glucose-related pathways in vitro and in vivo. DRB18 eventually led to G1/S phase arrest and increased oxidative stress and necrotic cell death. IP injection of DRB18 in A549 tumor-bearing nude mice at 10 mg/kg body weight thrice a week led to a significant reduction in the tumor volume compared with mock-treated tumors. In contrast, the knockout of the GLUT1 gene did not reduce tumor volume. CONCLUSIONS: DRB18 is a potent pan-class I GLUT inhibitor in vitro and in vivo in cancer cells. Mechanistically, it is likely to bind the outward open conformation of GLUT1-4, reducing tumor growth through inhibiting GLUT1-4-mediated glucose transport and metabolisms. Pan-class I GLUT inhibition is a better strategy than single GLUT targeting for inhibiting tumor growth.

miR-130a-3p regulates steroid hormone synthesis in goat ovarian granulosa cells by targeting the PMEPA1 gene.

MicroRNAs (miRNAs) are key epigenomic regulators of proliferation, differentiation, and secretion in cells involved in follicular development. We here studied the functional role of one such molecule, miR-130a-3p, in goat ovarian granulosa cells (GCs). High expression of this miRNA was evident in goat GCs by fluorescence in situ hybridization and suppressed estradiol and progesterone secretion from these cells, as determined by ELISA. miR-130a-3p was predicted to have a binding site for the 3' UTR of the prostate transmembrane protein androgen induced 1 gene (PMEPA1), and this was verified by a dual-luciferase reporter assay. PMEPA1 mRNA and protein expression were both found to be regulated by miR-130a-3p in GCs. Moreover, the overexpression or knockdown of PMEPA1 enhanced or suppressed estradiol and progesterone secretion from these cells, respectively. Furthermore, the secretion of estradiol and progesterone did not change significantly after the offsetting of PMEPA1 overexpression in GCs by miR-130a-3p. In summary, our present data indicate that miR-130a-3p inhibits the secretion of estradiol and progesterone in GCs by targeting PMEPA1. Our study thus provides seminal data and important new insights into the regulation of reproductive mechanisms in the nanny goat and other female mammals.

The efficacy of transversus abdominis plane block with or without dexmedetomidine for postoperative analgesia in renal transplantation. A randomized controlled trial.

BACKGROUND: Current options for effective postoperative analgesia after renal transplantation are limited, due to altered renal clearance and the risk of renal damage. This study compared the analgesic effect of the transversus abdominis plane block, with or without dexmedetomidine, in renal transplant recipients. MATERIALS AND METHODS: This prospective randomized double-blinded clinical trial was performed from November 2014 to March 2017. Patients were randomly divided into group C (morphine intravenous patient-controlled analgesia), group R (morphine intravenous patient-controlled analgesia and transversus abdominis plane block), and group RD (morphine intravenous patient-controlled analgesia and transversus abdominis plane block with 1 µg/kg dexmedetomidine). Morphine consumption, time to first request for analgesia, pain, sedation, nausea, vomiting, respiratory depression, and bradycardia were measured at 2, 4, 6, 12 and 24 h after surgery. RESULTS: The visual analogue pain score in group C was the highest among the three groups at the 2nd and 4th hour. Morphine consumption was the highest in group C at all assessed time intervals (p < 0.01). By the 12th hour and 24th hour, morphine consumption (calculated by time interval) was the lowest in group RD (p < 0.05), while no statistical difference was found between groups C and R. The average time to first request of analgesia was the longest and shortest in group RD and group C, respectively (p < 0.01). The overall incidence of nausea and vomiting was the highest in group C (p < 0.05). CONCLUSIONS: The transversus abdominis plane block reduced morphine consumption in the first 24 h following renal transplantation, and the addition of dexmedetomidine provided a more effective analgesic effect.

Effect of GABA-T on Reproductive Function in Female Rats.

This study explored the role of γ-aminobutyric acid transaminase (GABA-T) in the puberty and reproductive performance of female rats. Immunofluorescence technique, quantitative real-time PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect the distribution of GABA-T and the expression of genes and hormones in female rats, respectively. The results showed that GABA-T was mainly distributed in the arcuate nucleus (ARC), paraventricular nucleus (PVN) and periventricular nucleus (PeN) of the hypothalamus, and in the adenohypophysis, ovarian granulosa cells and oocytes. Abat mRNA level at 28 d was lowest in the hypothalamus and the pituitary; at puberty, it was lowest in the ovary. Abat mRNA level was highest in adults in the hypothalamus; at infancy and puberty, it was highest in the pituitary; and at 21 d it was highest in the ovary. After vigabatrin (GABA-T irreversible inhibitor) was added to hypothalamus cells, the levels of Abat mRNA and Rfrp-3 mRNA were significantly reduced, but Gnrh mRNA increased at the dose of 25 and 50 µg/mL; Kiss1 mRNA was significantly increased but Gabbr1 mRNA was reduced at the 50 µg/mL dose. In prepubertal rats injected with vigabatrin, puberty onset was delayed. Abat mRNA, Kiss1 mRNA and Gnrh mRNA levels were significantly reduced, but Rfrp-3 mRNA level increased in the hypothalamus. Vigabatrin reduced the concentrations of GABA-T, luteinizing hormone (LH) and progesterone (P4), and the ovarian index. Lactation performance was reduced in adult rats with vigabatrin treatment. Four hours after vigabatrin injection, the concentrations of GABA-T and LH were significantly reduced in adult and 25 d rats, but follicle-stimulating hormone (FSH) increased in 25 d rats. In conclusion, GABA-T affects the reproductive function of female rats by regulating the levels of Gnrh, Kiss1 and Rfrp-3 in the hypothalamus as well as the concentrations of LH and P4.

Novel Engraftment and T Cell Differentiation of Human Hematopoietic Cells in ART-/-IL2RG-/Y SCID Pigs.

Pigs with severe combined immunodeficiency (SCID) are an emerging biomedical animal model. Swine are anatomically and physiologically more similar to humans than mice, making them an invaluable tool for preclinical regenerative medicine and cancer research. One essential step in further developing this model is the immunological humanization of SCID pigs. In this work we have generated T- B- NK- SCID pigs through site directed CRISPR/Cas9 mutagenesis of IL2RG within a naturally occurring DCLRE1C (ARTEMIS)-/- genetic background. We confirmed ART-/-IL2RG-/Y pigs lacked T, B, and NK cells in both peripheral blood and lymphoid tissues. Additionally, we successfully performed a bone marrow transplant on one ART-/-IL2RG-/Y male SCID pig with bone marrow from a complete swine leukocyte antigen (SLA) matched donor without conditioning to reconstitute porcine T and NK cells. Next, we performed in utero injections of cultured human CD34+ selected cord blood cells into the fetal ART-/-IL2RG-/Y SCID pigs. At birth, human CD45+ CD3ε+ cells were detected in cord and peripheral blood of in utero injected SCID piglets. Human leukocytes were also detected within the bone marrow, spleen, liver, thymus, and mesenteric lymph nodes of these animals. Taken together, we describe critical steps forwards the development of an immunologically humanized SCID pig model.