iTRAQ-based comparative proteomics reveal an enhancing role of PRDX6 in the freezability of Mediterranean buffalo sperm.

BACKGROUND: Semen cryopreservation is a critical tool for breed improvement and preservation of biodiversity. However, instability of sperm freezability affects its application. The Mediterranean buffalo is one of the river-type buffaloes with the capacity for high milk production. Until now, there is no specific cryopreservation system for Mediterranean buffalo, which influences the promotion of excellent cultivars. To improve the semen freezing extender used in cryopreservation of Mediterranean buffalo, different protein datasets relating to freezability sperm were analyzed by iTRAQ-based proteomics. This study will be beneficial for further understanding the sperm freezability mechanism and developing new cryopreservation strategy for buffalo semen. RESULTS: 2652 quantified proteins were identified, including 248 significantly differentially expressed proteins (DEP). Gene Ontology (GO) analysis indicated that many these were mitochondrial proteins, enriched in the molecular function of phospholipase A2 activity and enzyme binding, and biological processes of regulation of protein kinase A signaling and motile cilium assembly. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis identified 17 significant pathways, including oxidative phosphorylation (OXPHOS). Furthermore, 7 DEPs were verified using parallel reaction monitoring or western blot, which confirmed the accuracy of the iTRAQ data. Peroxiredoxin 6 (PRDX6), which expressed 1.72-fold higher in good freezability ejaculate (GFE) compared to poor freezability ejaculate (PFE) sperms, was selected to explore the function in sperm freezability by adding recombinant PRDX6 protein into the semen freezing extender. The results showed that the motility, mitochondrial function and in vitro fertilization capacity of frozen-thawed sperm were significantly increased, while the oxidation level was significantly decreased when 0.1 mg/L PRDX6 was added compared with blank control. CONCLUSIONS: Above results revealed the metabolic pattern of freezability of Mediterranean buffalo sperms was negatively associated with OXPHOS, and PRDX6 had protective effect on cryo-damage of frozen-thawed sperms.

The freezability of Mediterranean buffalo sperm is associated with lysine succinylation and lipid metabolism.

Semen cryopreservation is used for the propagation of variety among species and domestic breeding. Mitochondria are implicated in sperm freezability, and their proteins are prone to succinylation, but the relationship between sperm freezability and mitochondrial protein succinylation is unclear. In this study, six bulls were classified as having good or poor freezability ejaculates (GFE or PFE, each 3 bulls). The fresh sperm mitochondrial membrane potential (MMP) and pan succinylation level of the two groups were first detected. Then the lysine succinylome and fatty acid content of the two groups were analyzed using label-free LC-MS/MS and GC-MS/MS in multiple reaction monitoring (MRM) modes, respectively. The results indicated that the GFE sperm had significantly higher MMPs than the PFE group (p < 0.05). A total of 1393 succinylation sites corresponding to 426 proteins were assessed and 5 succinylated peptides of the GFE group were markedly upregulated, while 3 were significantly downregulated (FC > 2.0 - < 0.5 and p-value < 0.05) when compared to the PFE group. Forty-six succinylated proteins were identified to have consistent presence/absence expression. The upregulated succinylated proteins in the GFE sperm were enriched in lipid metabolic processes. A total of 31 fatty acids were further subjected to quantitative analysis of which 23 including arachidic (C20:0), linolenic (C18:3n3), and docosahexaenoic acids (C22:6n3) were decreased in GFE sperm when compared with PFE (p < 0.05). These results suggest that lysine succinylation can potentially influence the sperm freezability of Mediterranean buffaloes through mitochondrial lipid metabolism. This novel study provides our understanding of sperm succinylation and the molecular basis for the mechanism of sperm freezability.

Seminal plasma lipid profiles of differential cryotolerance of semen in Mediterranean Buffalo bulls.

The cryotolerance of semen obtained from Mediterranean buffalo bulls usually is more likely to deteriorate during the summer. To obtain the optimal sperm for fertility, the physiological status and reproductive performance of Mediterranean buffalo bulls in the summer and spring were first analysed by assessing blood serum and seminal plasma samples; then, the lipid profiles of seminal plasma were investigated by LC-MS/MS. The T, T3 and SOD levels of serum and seminal plasma in the spring were significantly higher than in the summer (p < .05). The results suggest that T3 level is positively correlated with semen cryotolerance; sphingolipids are potential markers for semen cryotolerance of Mediterranean buffalo. To our knowledge, this is the first report of targeted lipidomics in semen cryotolerance.

Effect of environmental temperature on semen quality and seminal plasma metabolites of Mediterranean buffalo bulls.

High-quality semen with high viability is critical to improving the in-vitro fertilization efficiency. This study aimed to understand the effect of ambient temperature and humidity on semen quality and seminal plasma biochemical parameters of Mediterranean buffalo in March and July. The metabolites of seminal plasma in two seasons were detected using the UPLC-MS/MS method. The results showed that temperature and humidity index (THI) in March were 66.86 ± 2.98, and 82.94 ± 3.52 in July. Compared with in March, breath frequency, rectal temperature, and heat shock protein 70 expressions of seminal plasma were significantly increased in July (p < 0.05), motility of sperm was dramatically reduced, and sperm deformity rate was significantly increased (p < 0.05). Fructose, acid phosphatase and α-glucosidase in seminal plasma were significantly increased (p < 0.05) in July, while testosterone level was significantly reduced (p < 0.05). Six different metabolites were found in the two groups, which involved in three metabolic pathways, the tricarboxylic acid cycle, glycerophospholipid, glyoxylic acid and dicarboxylic acid. The above results indicate that the increased ambient temperature has obvious side effects on the semen quality of Mediterranean buffalo, and the compromised quality is associated with the change of metabolites related to male hormone secretion, energy metabolism and fatty acid oxidation.

Chromatin openness of donor cells is directly correlated with the in vitro developmental capabilities of cloned buffalo embryos.

The Switch/sucrose nonfermentable (SWI/SNF) chromatin remodelling complex is closely related to chromatin openness and gene transcriptional activity. To understand if the chromatin openness of donor cells was related to the development efficiency of somatic cell cloning embryos, two buffalo fetal fibroblasts (BFF), BFF1 and BFF3, with significantly different cloned blastocyst development rates (18.4% and 30.9% respectively), were selected in this study. The expression of SWI/SNF complex genes, chromatin openness, and transcript level of these two cell lines were analysed, and the effect of ATP on the expression of the SWI/SNF complex genes was further explored. The results showed that compared with BFF1, the expression of SWI/SNF complex family genes was higher in BFF3 at the G0/G1 phase, where SMARCC1, SMARCC2 and SMARCE1 were significantly different (p < .05). Assay of Transposase Accessible Chromatin sequencing (ATAC-seq) results showed that, at the genome-wide level, BFF3 had more open chromatin, especially which having more open chromatin peaks at SMARCA4, SMARCA2, and RBPMS2 (RNA Binding Protein, mRNA Processing Factor 2) sites. In total, 2,712 differentially expressed genes (DEGs) were identified by the RNA-Seq method, with 1380 up- and 1332 down-regulated genes in BFF3. Interestingly, the ATPase-related genes ATP1B1 and ATP11A were extreme significantly up-regulated in BFF3 (p < .01). The ATP content and the expression of SWI/SNF complex genes in both BFF1 and BFF3 decreased when treated with rotenone. The above results demonstrated that the SWI/SNF complex contributed to chromatin opening, and chromatin opening of donor cells was essential for cloned embryo development.

AQP8 participates in oestrogen-mediated buffalo follicular development by regulating apoptosis of granulosa cells.

Aquaporins (AQPs), a family of small membrane-spanning proteins, are involved in fluid transport, cell signalling and reproduction. Regulating AQP8 expression influences apoptosis of granulosa cells (GCs), ovarian folliculogenesis, oogenesis and early embryonic development in mice, but its role has never been investigated in other species. The aim of the present study was to characterize the AQP8 function in buffalo follicular development. The expression pattern of AQP8 in buffalo follicle was analysed by immunohistochemistry method. 17ß-Estradiol (E2) or oestrogen receptor antagonist ICI182780 was used to treat GCs cultured in vitro, and the expression of AQP8 was detected using qRT-PCR. Its roles in apoptosis of buffalo GCs were investigated by shRNA technology. AQP8 was found to be expressed higher in secondary follicles (p < .05), and its mRNA level in GCs was upregulated by E2 via receptor-mediated mechanism in a dose-dependent manner. A 732-bp buffalo AQP8 coding region was obtained, which was highly conserved at the amino acid level among different species. AQP8-shRNA2 had more effective inhibition on target gene than AQP8-shRNA1 (66.49% vs. 58.31%) (p < .05). Knockdown of AQP8 induced GCs arrested at G2/M stage and occurred apoptosis. Compared with the control group, higher Caspase9 expression were observed in AQP8-shRNA2 lentivirus infected GCs (p < .05), while Bcl-2 and Bax expression levels had no obvious change (p > .05). Altogether, the above results indicate that AQP8 is involved in oestrogen-mediated regulation of buffalo follicular development by regulating cell cycle progression and apoptosis of GCs.

Inhibition of Suv39h1/2 expression improves the early development of Debao porcine somatic cell nuclear transfer embryos.

Suppressor of variegation 3-9 homolog (Suv39h)1 and 2, Histone H3 lysine 9 trimethylation (H3K9me3)-specific methyltransferases, are mainly involved in regulating the dynamic changes of H3K9me3. Regulating Suv39h expression influences the early development of mice somatic cell nuclear transfer (SCNT) embryos, there are few reports concerning their features in domestic animals. The aim of the present study was to characterize the Suv39h function in early development of Debao porcine SCNT embryos. The global level of H3K9me3 and the expression profiles of Suv39h1/2 in porcine early embryos were analysed by immunohistochemistry and qRT-PCR methods, respectively. Their roles in cell proliferation and histone modification of Debao porcine foetal fibroblast cells (PFFs), and developmental competence of porcine SCNT embryos were investigated by shRNA technology. The methylation levels of H3K9me3 and the expression patterns of Suv39h1 and Suv39h2 were similar (p < .05), and both of them displayed higher levels in Debao porcine SCNT embryos compared with that in PA embryos. The global levels of H3K9me3 and the expressions of G9a, HDAC1 and DNMT1 were decreased by combined inhibition of Suv39h1 and Suv39h2 (p < .05), while the expression of HAT1 was increased (p < .05). Downregulation of Suv39h1/2 also promoted cell proliferation and resulted in a significant increase in the expression of CyclinA2, CyclinB and PCNA in PFFs (p < .05). Furthermore, the use of donor somatic nuclei which depleted H3K9me3 by inhibiting Suv39h1/2 expression markedly increased the cleavage rate, the blastocyst rate and the total cell number of blastocysts of Debao porcine SCNT embryos (p < .05). Altogether, the above results indicate that H3K9me3 levels and Suv39h1/2 expressions display similar patterns in porcine early embryo, and low levels of them are critical to cell proliferation of PFFs and early development of SCNT embryos.

Cloning and functional characterization of STAT5a and STAT5b genes in buffalo mammary epithelial cells.

Signal transducer and activator 5 (STAT5) plays important roles in regulating mammary glandular cell proliferation and milk-protein gene expression. However, the functions of STAT5a and STAT5b genes in lactation of buffalo remain uninvestigated. In this study, full-length STAT5a (2502 bp) and STAT5b (2515 bp) coding sequences were isolated for the first time. The highest STAT5a gene expression was found in buffalo mammary glands and the highest STAT5b gene expression was found in buffalo brains and mammary glands. H&E staining indicated that STAT5a and STAT5b are mainly expressed in epithelial cells of buffalo mammary glands. Next, we investigated the functions of STAT5 by knocking down and overexpressing STAT5 in buffalo mammary epithelial cells (BuMECs). According to our results, STAT5 knockdown resulted in inhibited G1/S transition of BuMECs and significantly lower expression of milk-protein genes, whereas overexpression of STAT5 resulted in significantly higher expression of milk-protein genes. In summary, our results demonstrate that STAT5 can regulate the cell cycle transition of BuMECs and affect the expression of milk-protein genes. Our research lays a foundation for further study of the role of STAT5 in mammary gland development and lactation.

Inhibition of Suv39H1 enhances transgenic IFNα-2b gene expression in Bcap-37 cells.

The low expression of exogenous transferred gene limited the application of transgenic animal technology. Suppressor of variegation 3 ∼ 9 homolog 1(SUV39H1) gene plays a prominent role on repressive heterochromatin and transcription. To understand if exogenous transgenic gene expression was affected by SUV39H1 epigenetic modification, in this paper, the effective shRNA fragments targeting SUV39H1 gene were first screened, their roles on expression of exogenous transgenic genes were determined by using Bcap-37 cell line with stable expressing IFNα-2b gene as a model, the preliminary regulation mechanism of SUV39H1 gene was investigated. The results showed that the designed shRNA1/2 fragments of SUV39H1 gene had an obvious inhibition effect on the expression of SUV39H1 gene, reached 53.07 and 31.28%, respectively by qRT-PCR analysis. Compared with the control group, the expression of IFNα-2b gene in transgenic Bcap-37 cells infected with shRNA1 and 2 viruses significantly increased by 96.25 and 121.08%, respectively (p < 0.05). In addition, the expression of DNMT1, HDAC1 and G9a gene in the shRNA infected cells reduced significantly, and the expression of the HAT1 gene increased significantly (p < 0.05). The above results indicated that the expression of exogenous transgenic gene could be promoted by suppressing SUV39H1 gene at the cell level.

Production of D-alanine from DL-alanine using immobilized cells of Bacillus subtilis HLZ-68.

Immobilized cells of Bacillus subtilis HLZ-68 were used to produce D-alanine from DL-alanine by asymmetric degradation. Different compounds such as polyvinyl alcohol and calcium alginate were employed for immobilizing the B. subtilis HLZ-68 cells, and the results showed that cells immobilized using a mixture of these two compounds presented higher L-alanine degradation activity, when compared with free cells. Subsequently, the effects of different concentrations of polyvinyl alcohol and calcium alginate on L-alanine consumption were examined. Maximum L-alanine degradation was exhibited by cells immobilized with 8% (w/v) polyvinyl alcohol and 2% (w/v) calcium alginate. Addition of 400 g of DL-alanine (200 g at the beginning of the reaction and 200 g after 30 h of incubation) into the reaction solution at 30 °C, pH 6.0, aeration of 1.0 vvm, and agitation of 400 rpm resulted in complete L-alanine degradation within 60 h, leaving 185 g of D-alanine in the reaction solution. The immobilized cells were applied for more than 15 cycles of degradation and a maximum utilization rate was achieved at the third cycle. D-alanine was easily extracted from the reaction solution using cation-exchange resin, and the chemical and optical purity of the extracted D-alanine was 99.1 and 99.6%, respectively.

Establishment of a mouse model to express bovine CD14 short hairpin RNA.

BACKGROUND: Cluster of differentiation 14 (CD14) functions as a co-receptor for Toll-like receptor (TLR)-4 and myeloid differentiation factor (MD)-2 in detecting bacterial lipopolysaccharide. Together, these complexes promote the phagocytosis and digestion of Gram-negative bacteria, and initiate immune responses. To date, much of our understanding of CD14 function during Gram-negative bacterial inflammation comes from studies on mouse knockout models and cell transfection. To identify the effect of CD14 knockdown in this process in large livestock animals, we established a mouse model expressing bovine CD14 short hairpin (sh) RNA. shRNA fragments targeting bovine CD14 were screened by co-transfection in HEK 293 cells, and the most effective CD14 shRNA fragment was cloned into the eukaryotic expression vector pSilencer4.1-CD14 shRNA-IRES (internal ribosome entry site) and transferred into mouse zygotes by pronuclear microinjection to obtain transgenic mice. Expression of the enhanced green fluorescent protein (EGFP) reporter and genes related to the TLR4 signaling pathway was detected by immunohistochemistry (IHC) and quantitative polymerase chain reaction (PCR), respectively. RESULTS: One effective shRNA fragment (shRNA-674) targeting bovine CD14 was obtained, the sequence of which was shown to be conserved between cows, buffalos, sheep, and humans. Thirty-seven founder pups were obtained by pronuclear microinjection, of which three were positive for the transgene. In the F(1) generation, 11 of 33 mice (33%) were positive for the transgene as detected by PCR. IHC analysis detected exogenous EGFP expression in the liver, kidney, and spleen of transgenic F(1) mice, indicating that they were chimeric. The expression of endogenous CD14 mRNA in the heart, liver, spleen, lung, and kidney of transgenic F(1) mice was decreased 8-, 3-, 19.5-, 6-, and 11-fold, respectively. The expression patterns of endogenous MD-2, TLR4, interleukin-6 and tumor necrosis factor-α genes in transgenic mice also varied. CONCLUSIONS: This study confirms that transgenic mice expressing bovine CD14 shRNA can be generated by pronuclear microinjection, and demonstrates inhibited endogenous mouse CD14 expression that alters gene expression related to the TLR4 signaling pathway.

The effect of buffalo CD14 shRNA on the gene expression of TLR4 signal pathway in buffalo monocyte/macrophages.

CD14 plays a crucial role in the inflammatory response to lipopolysaccharide (LPS), which interacts with TLR4 and MD-2 to enable cell activation, resulting in inflammation. Upstream inhibition of the inflammation pathway mediated by bacterial LPS, toll-like receptor 4 (TLR4) and cluster of differentiation antigen 14 (CD14) was proven to be an effective therapeutic approach for attenuating harmful immune activation. To explore the effect of CD14 downregulation on the expression of TLR4 signaling pathway-related genes after LPS stimulation in buffalo (Bubalus bubalis) monocyte/macrophages, effective CD14 shRNA sequences were screened using qRT-PCR and FACS analysis with buffalo CD14 shRNA lentiviral recombinant plasmids (pSicoRGFP-shRNA) and buffalo CD14 fusion expression plasmids (pDsRed-N1-buffalo CD14) co-transfected into HEK293T cells via liposomes. Of the tested shRNAs, shRNA-1041 revealed the highest knockdown efficiency (p < 0.01). When buffalo peripheral blood monocyte/macrophages were infected with shRNA-1041 lentivirus and stimulated with LPS, the expression of endogenous CD14 was significantly decreased by CD14 shRNA (p < 0.01), and the mRNA expression levels of TLR4, IL-6 and TNF-α were also significantly downregulated compared to the control groups (p < 0.01). These results demonstrated that the knockdown of endogenous CD14 had clear regulatory effects on the signal transduction of TLR4 after stimulation with LPS. These results may provide a better understanding of the molecular mechanisms of CD14 regulation in the development of several buffalo diseases.

Expression pattern of ATG4C and its effect on early embryonic development of porcine oocytes.

Autophagy is essential for oocyte maturation and preimplantation embryo development. ATG4C, a member of the ATG4 family, plays a crucial role in the autophagy process. The effect of ATG4C on the early embryonic development in pig has not been studied. In this study, the expression patterns of ATG4C were explored using qRT-PCR and immunofluorescence staining. Different concentrations of serum were added to in vitro maturation (IVM) medium to investigate its effects on oocyte maturation and embryonic development. Finally, the developmental potential of parthenogenetic embryos was detected by downregulating ATG4C in MII stage oocytes under 0 % serum condition. The results revealed that ATG4C was highly expressed in porcine oocytes matured in vitro and in parthenogenetic embryos. Compared with the 10 % serum group, the cumulus cell expansion, first polar body (PB1) extrusion rate, and subsequent developmental competence of embryos were reduced in the 0 % and 5 % serum groups. The mRNA levels of LC3, ATG5, BECLIN1, TFAM, PGC1α, and PINK1 were significantly increased (P < 0.05) in the 0 % serum group. ATG4C was significantly upregulated in the embryos at the 1-cell, 2-cell, 8-cell, and 16-cell stages in the 0 % serum group (P < 0.05). Compared with the negative control group, downregulation of ATG4C significantly decreased the 4-cell, 8-cell, and blastocyst rates (P < 0.05), and the expression of genes related to autophagy, mitochondria, and zygotic genome activation (ZGA) was significantly decreased (P < 0.05). The relative fluorescence intensity of LC3 and mitochondrial content in the ATG4C siRNA group was significantly reduced (P < 0.05). Collectively, the results indicate that ATG4C is highly expressed in porcine oocytes matured in vitro and in early embryos, and inhibition of ATG4C effects embryonic developmental competence by decreasing autophagy, mitochondrial content, and ZGA under serum-free condition.

Overexpression of BRG1 improves early development of porcine somatic cell nuclear transfer embryos.

The epigenetic modification levels of donor cells directly affect the developmental potential of somatic cell nuclear transfer (SCNT) embryos. BRG1, as an epigenetic modifying enzyme, has not yet been studied in donor cells and SCNT embryos. In this study, BRG1 was overexpressed in porcine fetal fibroblasts (PFFs), its effect on chromatin openness and gene transcription was examined, subsequently, the development potential of porcine SCNT embryos was investigated. The results showed that compared with the control group, the percentage of G1 phase cells was significantly increased (32.3 % ± 0.87 vs 25.7 % ± 0.81, P < 0.05) in the experimental group. The qRT-PCR results showed that the expression of H3K9me3-related genes was significantly decreased (P < 0.05), HAT1 was significantly increased (P < 0.05). Assay of Transposase Accessible Chromatin sequencing (ATAC-seq) results revealed that SMARCA4、NANOG、SOX2、MAP2K6 and HIF1A loci had more open chromatin peaks in the experimental group. The RNA-seq results showed that the upregulated genes were mainly enriched in PI3K/AKT and WNT signaling pathways, and the downregulated genes were largely focused on disease development. Interestingly, the developmental rate of porcine SCNT embryos was improved (27.33 % ± 1.40 vs 17.83 % ± 2.02, P < 0.05), the expression of zygotic gene activation-related genes in 4-cell embryos, and embryonic development-related genes in blastocysts was significantly upregulated in the experimental group (P < 0.05). These results suggest that overexpression of BRG1 in donor cells is benefit for the developmental potential of porcine SCNT embryos.

The effects of melatonin, glutathione and vitamin E on semen cryopreservation of Mediterranean buffalo.

The aim of this study was to investigate the effects of melatonin (MLT), reduced glutathione (GSH) and vitamin E (Vit. E) or their combinations on semen cryopreservation of Mediterranean buffalo. The quality parameters such as viability, abnormality rate, motility, structural integrity and the antioxidant capacity of frozen-thawed sperm were evaluated. The efficiency of frozen-thawed sperms in performing their functions was further analyzed by in vitro fertilization (IVF). In those separately supplemented groups, 0.2 mg/mL MLT, 0.2 mM GSH and 0.4 mg/mL Vit. E had the best effect on antioxidant capacity, kinetics and morphology, respectively. In addition, the cleavage, blastocyst and hatching blastocyst rates of IVF embryos were higher in 0.2 mg/mL MLT, 0.2 mM GSH, 0.2 and 0.4 mg/mL Vit. E groups than the blank control. Among the three combination groups, the kinetics and structure integrity of frozen-thawed sperms, cleavage, blastocyst and hatching blastocyst rates of IVF embryos were higher in 0.4 mg/mL Vit. E plus 0.2 mg/mL MLT group than the blank control group, revealed that this combination had comprehensive protection on frozen-thawed sperm of Mediterranean buffalo. These results support to develop special semen freezing extender containing an optimal choice of MLT, GSH and Vit. E, and to enhance the efficiency of frozen-thawed sperm of Mediterranean buffalo for IVF.

The clinical significance and mechanism of microRNA-22-3p targeting TP53 in lung adenocarcinoma.

BACKGROUND: At present, studies on MircoRNA-22-3p (miR-22-3p) in lung adenocarcinoma use a single method, lack multi-center validation and multi-method validation, and there is no big data concept to predict and validate target genes. OBJECTIVE: To investigate the expression, potential targets and clinicopathological significance of miR-22-3p in lung adenocarcinoma (LUAD) tissues. METHODS: LUAD formalin-fixed paraffin-embedded (FFPE) tumors and adjacent normal lung tissues were collected for real-time quantitative polymerase chain reaction (RT-qPCR). Collect miR-22-3p in LUAD and non-cancer lung tissue from high-throughput datasets, standardized mean difference (SMD) and area under the curve (AUC) of the comprehensive receiver operating curve (summary receiver operating characteristic cure, sROC curve) were calculated. Cell function experiments on A549 cells transfected with LV-hsa-miR-22-3p. Target genes were predicted by the miRwalk2.0 website and the resulting target genes were subjected to Gene Ontology (GO) pathway enrichment analysis and constructed to protein-protein interaction network. Finally, the protein expression level of the key gene TP53 was validated by searching The Human Protein Atlas (THPA) database to incorporate TP53 immunohistochemical results in LUAD. RESULTS: RT-qPCR result from 41 pairs of LUAD and adjacent lung tissues showed that miR-22-3p was downregulated in LUAD (AUC = 0.6597, p= 0.0128). Globally, a total of 838 LUADs and 494 non-cancerous lung tissues were included, and were finally combined into 14 platforms. Compared with noncancerous tissue, miR-22-3p expression level was significantly reduced in LUAD tissue (SMD =-0.32, AUC = 0.72l); cell function experiments showed that miR-22-3p has inhibitory effects on cell proliferation, migration and invasion, and has promotion effect on apoptosis. Moreover, target genes prediction, GO pathway enrichment analysis and PPI network exhibited TP53 as a key gene of target gene of miR-22-3p; at last, a total of 114 high-throughput datasets were included, including 3897 LUADs and 2993 non-cancerous lung tissues, and were finally combined into 37 platforms. Compared with noncancerous tissue, TP53 expression level was significantly increased in LUAD (SMD = 0.39, p< 0.01) and it was verified by the protein expression data from THPA. CONCLUSION: Overexpression of miR-22-3p may inhibit LUAD cell proliferation, migration and invasion through TP53, and promote cell apoptosis.

Rapamycin Treatment Is Beneficial for the Generation of Rabbit-Induced Pluripotent Stem-Like Cells.

Autophagy could promote the generation of induced pluripotency stem cells (iPSCs) in humans and mice. However, little was known whether it had similar effects in other species, the detailed mechanism and the features of formed iPSC colonies were also not clear. In this study, we first established the doxycycline (DOX)-inducible tetO lentiviral vector system suitable for the generation of rabbit iPSCs. Rapamycin, a mechanistic target of rapamycin (mTOR) inhibitor, was added during rabbit embryonic fibroblasts induction to improve the autophagy level. The colony formation efficiency and the expression of autophagy- and pluripotent-related genes were detected. The results showed that the established DOX-inducible tetO lentiviral system was successfully used to induce rabbit iPS-like cells. Compared with the untreated group, the number of alkaline phosphatase (AP)-positive colonies was increased 5.5-fold, when 0.5 nM rapamycin was added on days 1-3 after transduction, the colony morphology was improved and the iPS-like cells could be passaged >10 generations. The expression of autophagy-related genes (ATG), ATG5, ATG7, LC3, and ULK1 was increased with different patterns during the induction process, expression of OCT4, SOX2, and KLF4 significantly increased (p < 0.05). The mentioned results indicate that rapamycin treatment is beneficial for the generation of rabbit iPSCs by regulating autophagy and pluripotency-related gene expression.

Characterization of a Group of UDP-Glycosyltransferases Involved in the Biosynthesis of Triterpenoid Saponins of Panax notoginseng.

UDP-glycosyltransferase (UGT)-mediated glycosylation is a common modification in triterpene saponins, which exhibit a wide range of bioactivities and important pharmacological effects. However, few UGTs involved in saponin biosynthesis have been identified, limiting the biosynthesis of saponins. In this study, an efficient heterologous expression system was established for evaluating the UGT-mediated glycosylation process of triterpene saponins. Six UGTs (UGTPn17, UGTPn42, UGTPn35, UGTPn87, UGTPn19, and UGTPn12) from Panax notoginseng were predicted and found to be responsible for efficient and direct enzymatic biotransformation of 21 triterpenoid saponins via 26 various glycosylation reactions. Among them, UGTPn87 exhibited promiscuous sugar-donor specificity of UDP-glucose (UDP-Glc) and UDP-xylose (UDP-Xyl) by catalyzing the elongation of the second sugar chain at the C3 or/and C20 sites of protopanaxadiol-type saponins with a UDP-Glc or UDP-Xyl donor, as well as at the C20 site of protopanaxadiol-type saponins with a UDP-Glc donor. Two new saponins, Fd-Xyl and Fe-Xyl, were generated by catalyzing the C3-O-Glc xylosylations of notoginsenoside Fd and notoginsenoside Fe when incubated with UGTPn87. Moreover, the complete biosynthetic pathways of 17 saponins were elucidated, among which notoginsenoside L, vinaginsenoside R16, gypenoside LXXV, and gypenoside XVII were revealed in Panax for the first time. A yeast cell factory was constructed with a yield of Rh2 at 354.69 mg/L and a glycosylation ratio of 60.40% in flasks. Our results reveal the biosynthetic pathway of a group of saponins in P. notoginseng and provide a theoretical basis for producing rare and valuable saponins, promoting their industrial application in medicine and functional foods.

Expression patterns of ZO-1/2 and their effects on porcine oocyte in vitro maturation and early embryonic development.

Zonula occludens (ZO)-1 and ZO-2 are involved in epithelial polarity maintenance, gene transcription, cell proliferation and tumor cell metastasis. Regulating ZO-1/2 expression influences the early embryonic development of mice, but whether they are involved in oocyte maturation is still poorly understood. In the present study, the expression patterns of ZO-1 and ZO-2 in porcine cumulus cells and oocytes matured in vitro and early embryos from parthenogenetic activation were detected by qRT-PCR or Western blot, and then their roles in porcine oocyte maturation and early embryo development were investigated by shRNA technology. ZO-1 and ZO-2 were found to be expressed in cumulus cells, oocytes and early embryos, while ZO-1α+ was expressed only in cumulus cells, morula and blastocysts. During in vitro maturation (IVM), the abundance of ZO-1 and ZO-2 in oocytes was significantly higher than that in cumulus cells at 0 h (P < 0.01), and their mRNA and protein levels displayed relatively higher expression at 0 and 18 h, respectively. Compared with the control groups, cumulus cell expansion, oocyte nucleus maturation, and subsequent cleavage were not influenced by treatment of the cumulus-oocyte complexes (COCs) with ZO-1-shRNA1, ZO-2-shRNA2 or combined ZO-1-shRNA1 and ZO-2-shRNA2 lentivirus (P > 0.05). However, the blastocyst rate was reduced by treatment of COCs with ZO-1-shRNA1 but not ZO-2-shRNA2. The total cell number of blastocysts was decreased by downregulation of ZO-1 and ZO-2 (P < 0.05). Downregulation of ZO-1 and ZO-2 also resulted in a significant decrease (P < 0.05) in the expression of Cx43, Cx45, PTX3 and PTGS2 in cumulus cells, Cx45, BMP15, ZP3 and C-KIT in MII oocytes, and Nanog in blastocysts, with the exception of HAS2 expression in cumulus cells and Oct4 expression in blastocysts (P > 0.05). Altogether, the above results indicate that ZO-1 and ZO-2 display similar expression patterns during porcine oocyte IVM and are critical to porcine oocyte maturation and early embryonic development.

Comparison of Alinity m HPV and cobas HPV assays on cervical specimens in diverse storage media.

OBJECTIVE: To assess the concordance of high-risk HPV (HR-HPV) testing with the Alinity assay on cervical samples collected with diverse collection/storage protocols (ThinPrep, SurePath, Cervicollect) and to assess inter-assay concordance of HR-HPV testing of cervical cell specimens with Alinity m HR HPV assay (Alinity) vs cobas® 4800 HPV assay (cobas). METHODS: Specimens were obtained from 560 women attending a Women's Health clinic. Two specimens were obtained from each woman with combinations of two of the three collection devices and aliquots were tested by the two assays. RESULTS: Alinity showed an agreement of 93.9%, Kappa = 0.89 (263/280) between ThinPrep and SurePath specimens; 97.5%, Kappa = 0.95 (347/356) and 92.9%, Kappa = 0.85 (104/112) between ThinPrep and SurePath aliquots taken before or after cytology processing, respectively. Cervi-Collect specimens showed an agreement of 94.6%, Kappa = 0.89 (265/280) with ThinPrep specimens. Compared to cobas, Alinity showed agreements of 94.3%, Kappa = 0.88 (395/419) and 91.8%, Kappa = 0.82 (257/280) between ThinPrep and SurePath specimens, respectively. Alinity and cobas detected genotypes 16/18 and other high-risk HPV types at similar rates and showed similar correlations with cytology grades. CONCLUSIONS: Compared to cobas, Alinity performed equally well for detecting HPV in cervical specimens obtained with ThinPrep and SurePath. The Cervi-Collect device compared well to the other collection methods. Alinity is a reliable assay for simultaneous detection of HPV-16/18 and other high-risk genotypes in cervical specimens.