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1.
Cell Death Discov ; 6(1): 133, 2020 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-33298889

RESUMO

In mammalian early embryos, the transition from maternal to embryonic control of gene expression requires timely degradation of a subset of maternal mRNAs (MRD). Recently, zygotic genome activation (ZGA)-dependent MRD has been characterized in mouse 2-cell embryo. However, in early embryos, the dynamics of MRD is still poorly understood, and the maternal factor-mediated MRD before and along with ZGA has not been investigated. Argonaute 2 (Ago2) is highly expressed in mouse oocyte and early embryos. In this study, we showed that Ago2-dependent degradation involving RNA interference (RNAi) and RNA activation (RNAa) pathways contributes to the decay of over half of the maternal mRNAs in mouse early embryos. We demonstrated that AGO2 guided by endogenous small interfering RNAs (endosiRNAs), generated from double-stranded RNAs (dsRNAs) formed by maternal mRNAs with their complementary long noncoding RNAs (CMR-lncRNAs), could target maternal mRNAs and cooperate with P-bodies to promote MRD. In addition, we also showed that AGO2 may interact with small activating RNAs (saRNAs) to activate Yap1 and Tead4, triggering ZGA-dependent MRD. Thus, Ago2-dependent degradation is required for timely elimination of subgroups of maternal mRNAs and facilitates the transition between developmental states.

2.
Reprod Domest Anim ; 2020 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-33176019

RESUMO

Seminal plasma is a complex biological fluid containing many metabolites including amino acids, fructose, carbohydrates and lipids Metabolites play important roles in multiple biological processes, but details and significance of the seminal plasma metabolome related to boar fertility are unknown. The aim of the present study was to compare the comprehensive metabolome of seminal plasma from boars with different conception rate after artificial insemination and to identify the potential biomarkers. Semen samples were collected from boars which divided into two groups according to the conception rates in the offspring. Seminal plasma metabolites were isolated, purified, and then subjected to Ultra-high Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry (UHPLC-qTOF-MS) procession. A total of 576 (Positive ion mode) and 377 (Negative ion mode) metabolites were identified in seminal plasma. Metabolites were identified and categorized according to their major chemical classes, including carboxylic acids and derivatives, organooxygen compounds, amino acids, peptides, and alogues, fatty amides, fatty acyls, benzene and substituted derivatives, purine nucleotides, pyrimidine nucleotides, glycosyl compounds, fatty acids and conjugates. The results showed that 4-Aminobenzoate, Pro-Asn, Ile-Tyr, Homoveratric acid and D-Biotin were higher in semen of boar with higher conception rate (HG) versus lower conception rate (LG) (p < .05), whereas L-Serine, Butoxyacetic acid, S-Methyl-5'-thioadenosine, Capsaicin and 1-O-(cis-9-Octadecenyl)-2-O-acetyl-sn-glycero-3-phosphocholine (PAF) were lower in HG than in LG (p < .05). These metabolites may be considered as candidate biomarkers for different fertility in boars.

3.
Chemosphere ; : 128670, 2020 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-33109355

RESUMO

Neonicotinoid insecticides are neurotoxicants that cause serious environmental pollution and ecosystem risks. In the present study, a nitenpyram-degrading bacterium, Rhodococcus ruber CGMCC 17550, was isolated from a nitenpyram production sewage treatment tank. Liquid chromatography-mass spectrometry analysis revealed R. ruber degraded nitenpyram via a novel hydroxylation pathway to form three different metabolites, one of which was confirmed to hydroxylate nitenpyram at the C3 site of the 6-chlorpyridine cycle by nuclear magnetic resonance analysis. The nitenpyram degradation rate increased as the biomass of resting R. ruber CGMCC 17550 cells increased, reaching 98.37% at an OD600 of 9 in transformation broth containing 100 mg L-1 nitenpyram after 72 h of incubation. Nitenpyram degradation by R. ruber CGMCC 17550 was insensitive to dissolved oxygen levels. Use of glucose, fructose and pyruvate as co-substrates slightly increased nitenpyram degradation. The cytochrome P450 inhibitor 1-aminobenzotriazole strongly inhibited nitenpyram degradation, indicating that P450 enzymes may mediate nitenpyram hydroxylation. Inoculation of R. ruber CGMCC 17550 enhanced nitenpyram degradation in surface water. Additionally, R. ruber cells immobilized by calcium-alginate remediated 87.11% of 100 mg L-1 NIT in 8 d. Genome sequencing analysis confirmed that R. ruber CGMCC 17550 has metabolic diversity and abundant KEGG genes involved in xenobiotics biodegradation and metabolism. These findings demonstrate that R. ruber CGMCC 17550 is capable of unique biodegradation of nitenpyram via the hydroxylation pathway and is a promising bacterium for bioremediation of contaminants.

4.
Mol Cancer Res ; 18(12): 1789-1802, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32878967

RESUMO

Lung cancer, especially lung adenocarcinoma, is one of the most common neoplasms worldwide. However, the mechanisms underlying its initiation, development, and metastasis are still poorly understood. Destrin (DSTN) is a member of ADF/cofilin family. Its detailed biological function remains unknown, although it is reported that DSTN is involved in cytoskeleton remodeling and regulation of actin filament turnover. Recent evidence has shown that high expression of cofilin-1 is associated with invasion and poor prognosis of several types of human tumors, but the detailed mechanism is still entirely unclear, particularly in lung cancer tumorigenesis and malignancy. Here, we report that DSTN was highly expressed in a mouse lung cancer model induced by urethane and in clinical lung adenocarcinoma tissue samples. Its expression level was positively correlated with cancer development, as well as metastasis to the liver and lymph nodes. Consistently, it was directly associated with the poor prognosis of lung adenocarcinoma patients. Furthermore, we also found that DSTN promotes cell proliferation, invasion, and migration in vitro, and facilitates subcutaneous tumor formation and lung metastasis via intravenous injection in vivo. Mechanically, DSTN associates with and facilitates nuclear translocation of ß-catenin, which promotes epithelial-to-mesenchymal transition (EMT). Taken together, our results indicated that DSTN enhances lung cancer malignancy through facilitating ß-catenin nuclear translocation and inducing EMT. Combined with multivariate analyses, DSTN might potentially serve as a therapeutic target and an independent prognostic marker of lung adenocarcinoma. IMPLICATIONS: This finding indicates that DSTN facilitates ß-catenin nuclear translocation and promotes malignancy in lung adenocarcinoma.

5.
Biomed Pharmacother ; 130: 110514, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32707438

RESUMO

Processing of dark tea varieties, such as Fu brick tea, Liupao tea, Qianliang tea, and Qing brick tea, includes solid-state fermentation involving microorganisms. In this study, we analyzed the major chemical constituents of dark tea extracts and evaluated their modulatory effect on the gastrointestinal function in normal mice, including the improvement of gastrointestinal transit and intestinal microbial, as well as the attenuation of intestinal microbial dysbiosis and intestinal pathological damage, and the adjustment of immune function in antibiotic-treated mice. Substantial differences in major chemical constituents, including total polyphenols, total organic acids, water extract content, 18 free amino acids, gallic acid, and six tea catechins, were observed among Fu brick tea, Qianliang tea, Qing brick tea, and Liupao tea extracts. Extracts from the four dark tea varieties significantly promoted gastrointestinal transit and colonization of beneficial Bifidobacterium and Lactobacillus, and inhibited the growth of harmful Escherichia coli and Enterococcus in normal mice. In addition, Qianliang tea, Qing brick tea, and Liupao tea extracts significantly accelerated the reversal of the ampicillin sodium-induced pathological damage in the ileum, intestinal bacterial dysbiosis (Bifidobacterium, Lactobacillus, E. coli, and Enterococcus), and low immunity.

6.
PeerJ ; 8: e9397, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32587808

RESUMO

Background: Previous studies recruited unrepresentative samples of Chinese patients with cataract and reported a wide range of prevalence of depressive symptoms in this patient population (18.0-89.7%). The present study determined the prevalence and correlates of depressive symptoms among a consecutive sample of Chinese patients with cataract treated in tertiary general hospitals. Methods: A total of 339 patients with cataract were consecutively selected from ophthalmology departments of two large general hospitals in Wuhan, China. Depressive symptoms were assessed with the Chinese Hospital Anxiety and Depression Scale. Logistic regression was used to identify factors that were associated with depression. Results: The prevalence of depressive symptoms was 23.9% (95% CI [19.4-28.4]%) among patients with cataract. Correlates for depressive symptoms include an education level of primary school and below (OR = 1.93, P = 0.038), marital status of "others" (OR =3.15, P < 0.001), poor family economic status (OR = 2.26, P = 0.010), nuclear cataract (OR =4.32, P < 0.001), and mixed cataract (OR = 2.76, P = 0.017). Conclusions: Depressive symptoms are common among Chinese patients with cataract treated in large general hospitals. Patients who are poorly educated, have a marital status other than "married", have poor family economic status, and suffer from nuclear and mixed cataracts are at greater risk for depressive symptoms.

8.
Chin J Nat Med ; 18(3): 219-225, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32245592

RESUMO

The rapid detection of pathogenic bacteria is vital for the prevention of outbreaks of infectious diseases, including infections by the common foodborne bacteria E.coli and Salmonella Carbohydrate microarrays have been developed as a powerful method to investigate carbohydrate-protein interaction with only very small amounts of glycans, which show great potential for detect the carbohydrate mediated interaction with pathogens. Here, different mannose-coated microarrays were constructed and tested with E.coli (K-12 and BL-21) and Salmonella enterica strains (ATCC9184 and ATCC31685) exhibiting different mannose binding affinities. The optimized carbohydrate microarray was then applied to test the binding of 12 Salmonella enterica and 9 E.coli isolates from local patients for the first time and showed strong binding with certain serovars or subtypes. The results showed that microarray probed with the single mannose structure is not enough for the detection of bacteria with various serovars or subtypes, which contain a high degree of allelic variation in adhesin. We suggest that a complex carbohydrate microarray containing different glycan conformation may be needed for detection of different bacteria isolates.


Assuntos
Carboidratos/química , Escherichia coli/isolamento & purificação , Análise em Microsséries/métodos , Salmonella enterica/isolamento & purificação , Adesinas Bacterianas/química , Contaminação de Alimentos , Microbiologia de Alimentos , Humanos , Manose/química , Polissacarídeos/química
9.
Eur J Nutr ; 59(8): 3603-3615, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32078065

RESUMO

PURPOSE: Data from in vitro and animal studies support the preventive effect of tea (Camellia sinensis) against colorectal cancer. Further, many epidemiologic studies evaluated the association between tea consumption and colorectal cancer risk, but the results were inconsistent. We conducted a meta-analysis of prospective cohort studies to systematically assess the association between tea consumption and colorectal cancer risk. METHODS: A comprehensive literature review was conducted to identify the related articles by searching PubMed and Embase up to June, 2019. Summary relative risks (RRs) and 95% confidence intervals (CIs) were calculated using a fixed effect model. RESULTS: Twenty cohort articles were included in the present meta-analysis involving 2,068,137 participants and 21,437 cases. The combined RR of colorectal cancer for the highest vs. lowest tea consumption was determined to 0.97 (95% CI 0.94-1.01) with marginal heterogeneity (I2 = 24.0%, P = 0.093) among all studies. This indicated that tea consumption had no significant association with colorectal cancer risk. Stratified analysis showed that no significant differences were found in all subgroups. We further conducted the gender-specific meta-analysis for deriving a more precise estimation. No significant association was observed between tea consumption and colorectal cancer risk in male (combined RR = 0.97; 95% CI 0.90-1.04). However, tea consumption had a marginal significant inverse impact on colorectal cancer risk in female (combined RR = 0.93; 95% CI 0.86-1.00). Further, we found a stronger inverse association between tea consumption and risk of colorectal cancer among the female studies with no adjustment of coffee intake (RR: 0.90; 95% CI 0.82-1.00, P < 0.05) compared to the female studies that adjusted for coffee intake (RR = 0.97; 95% CI 0.87-1.09, P > 0.05). CONCLUSIONS: Our finding indicates that tea consumption has no significant impact on the colorectal cancer risk in both genders combined, but gender-specific meta-analysis shows that tea consumption has a marginal significant inverse impact on colorectal cancer risk in female.

10.
Food Chem ; 312: 126043, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31896450

RESUMO

Dark tea is a unique fermented tea produced by solid-state fermentation of tea leaves (Camellia sinensis). It includes ripe Pu-erh tea, Fu brick tea, Liupao tea, and other teas. Microbial fermentation is considered to be the key factor controlling the quality of dark tea. It involves a series of reactions that modify the chemical constituents of tea leaves. These chemical conversions during microbial fermentation of dark tea are associated with a variety of functional core microorganisms. Further, Multi-omics approaches have been used to reveal the microbial impact on the conversion of the chemical components in dark tea. In the present review, we provide an overview of the most recent advances in the knowledge of the microbial bioconversion of the chemical components in dark tea, including the chemical composition of dark tea, microbial community composition and dynamics during the fermentation process, and the role of microorganisms in biotransformation of chemical constituents.


Assuntos
Camellia sinensis/química , Chá/química , Camellia sinensis/metabolismo , Fermentação , Humanos , Microbiota , Folhas de Planta/química , Folhas de Planta/metabolismo , Chá/metabolismo
11.
Theriogenology ; 146: 162-170, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31791612

RESUMO

Aberrant epigenetic reprogramming is a major cause of the developmental failure of embryos after somatic cell nuclear transfer (SCNT). Histone H3 lysine 9 trimethylation (H3K9me3), a histone marker of transcriptional repression, is considered a key barrier to the development of cloned embryos. In the present study, H3K9me3 levels were much higher in SCNT embryos than IVF embryos at the 4-cell and 2-cell stages. The microinjection of the kdm4a mRNA encoding an H3K9me3 demethylase significantly increased the developmental efficiency of cloned porcine embryos. Moreover, we evaluated the effect of chaetocin, an inhibitor of histone methyltransferases suv39h1/2, on SCNT embryo development. Chaetocin did not suppress the H3K9me3 modification in porcine embryonic fibroblast (PEF) but downregulated the expression of suv39h1, suv39h2, and kdm4d. However, 10 nM chaetocin treatment efficiently decreased the H3K9me3 level in cloned embryos. Importantly, a chaetocin treatment at the 4-cell stage for 6 h significantly increased the blastocyst rate and total cell numbers. Furthermore, the inhibitor treatment upregulated the expression of related developmental genes. In summary, both overexpression of kdm4a and treatment with a suv39h1/2 inhibitor improve the epigenetic reprogramming of cloned embryos and further improve the developmental competence in vitro.

12.
Yi Chuan ; 41(10): 950-961, 2019 Oct 20.
Artigo em Chinês | MEDLINE | ID: mdl-31624057

RESUMO

SOX2 (sex determining region Y-box2) is one of the critical pluripotent factors that play a crucial role in the first lineage differentiation and maintenance of pluripotency in inner cell mass during early embryonic development. However, there are few researches about the regulation of the SOX2 promoter, especially in Sus scrofa. To analyzed the activity of SOX2 promoter in early porcine embryos, we determined the control system and established the microinjection system for assessing SOX2 promoter activity by analyzing the embryonic development and the expression of enhanced green fluorescence protein (EGFP) after micro-injected different EGFP plasmids at different times after activation of the oocytes. Then, we analyzed the structure of 5000 bp upstream of the SOX2 translation initiation site and found there were four transcription factor binding site clusters. Next, we designed and constructed promoter-containing plasmids to analyze the function of each cluster. To detect the activity of different promoters, we assessed the mCherry expression in protein levels and mRNA levels by analyzing the mCherry fluorescence intensity and qRT-PCR after injecting plasmids into embryos. These results showed that the activity of the shorted promoter, with the region from 2254 bp to 2442 bp upstream of translation initiation site deleted, decreased to 17.8% at 4-cell and 8-cell stages compared with the full-length promoter. This region included two NF-AT transcription factor binding sites, which indicated that the NF-AT binding site is a key region to regulate the activity of the SOX2 promoter. The results provide important data for determination the mechanism of porcine SOX2 regulation.


Assuntos
Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Regiões Promotoras Genéticas , Fatores de Transcrição SOXB1/genética , Animais , Sítios de Ligação , Diferenciação Celular , Embrião de Mamíferos , Proteínas de Fluorescência Verde , Fatores de Transcrição NFATC , Plasmídeos , Suínos
13.
Theriogenology ; 135: 19-24, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31189122

RESUMO

Approximately 40% of mammalian genome is made of transposable elements (TEs), and during specific biological processes, such as gametogenesis, they may be activated by global demethylation, so strict silencing mechanism is indispensable for genomic stability. Here, we performed small RNA-seq on Dicer1 knockdown (KD) oocytes in pig, and observed short interspersed nuclear elements 1B (SINE1B) derived endogenous small interfering RNAs (endo-siRNAs), termed SINE1B-siRNAs, were significantly decreased and their biogenesis was dependent on Dicer1 and transcript of SINE1B. Furthermore, by injection of mimics and inhibitors of the SINE1B-siRNAs into germinal vesicle-stage (GV-stage) oocytes, we found the maturation rate was significantly decreased by SINE1B-siRNAs, indicating the SINE1B-siRNAs are indispensible for in vitro maturation (IVM) of porcine oocyte. To figure out the mechanism, we checked the expression pattern and DNA methylation status of SINE1B during IVM of porcine oocytes, and demonstrated the SINE1B-siRNAs could repress SINE1B expression induced by hypomethylation at a post-transcriptional level. Our results suggest that during gametogenesis when the erasure of DNA methylation occurs, endo-siRNAs act as a chronic response to limit retrotransposon activation.


Assuntos
Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/fisiologia , Elementos Nucleotídeos Curtos e Dispersos/fisiologia , Animais , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Metilação de DNA , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , RNA Interferente Pequeno , Retroelementos , Elementos Nucleotídeos Curtos e Dispersos/genética , Suínos
14.
Gene ; 699: 8-15, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-30851424

RESUMO

Epigallocatechin-3-O-(3-O-methyl) gallate (EGCG3"Me) in tea (Camellia sinensis (L.) O. Kuntze) is a major source of O-methylated catechin and renowned for a wide range of health effects. However, the transcriptional regulation mechanisms of EGCG3"Me biosynthesis remain unclear. In the present work, the basic Helix-Loop-Helix (bHLH) transcription factor, designated as CsbHLH62, belonging to GBOF group of bHLH families, was isolated and characterized from Camellia sinensis. CsbHLH62 contains an Open Reading Frame of 1662 bp and encodes a polypeptide of 553 amino acids. Subcellular location and transcriptional activity analysis showed it as a nucleus protein and possessed transcriptional inhibition activity. Furthermore, the expression of CsbHLH62 was decreased during EGCG3"Me accumulation. More importantly, E-box motifs (5'-CANNTG-3') were found in the promoters of CCoAOMT, CsLAR, and CsDFR, and further transient expression assays showed that CsbHLH62 repressed the transcription of CCoAOMT, CsLAR, and CsDFR. Collectively, these results suggest that CsbHLH62 acts as a transcriptional repressor that might be negatively affecting the accumulation of EGCG3"Me. These findings provide novel insights into the regulatory mechanism of EGCG3"Me biosynthesis, which might help to breed high EGCG3"Me-content tea plants.


Assuntos
Camellia sinensis/genética , Ácido Gálico/análogos & derivados , Proteínas de Plantas/genética , Transcrição Genética/genética , Catequina/genética , Ácido Gálico/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Metiltransferases/metabolismo , Fases de Leitura Aberta/genética , Folhas de Planta/genética , Folhas de Planta/metabolismo , Regiões Promotoras Genéticas/genética , Chá/genética , Chá/metabolismo
15.
Cell Prolif ; 52(3): e12591, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30896067

RESUMO

OBJECTIVES: To date, many efforts have been made to establish porcine embryonic stem (pES) cells without success. Extraembryonic endoderm (XEN) cells can self-renew and differentiate into the visceral endoderm and parietal endoderm. XEN cells are derived from the primitive endoderm of the inner cell mass of blastocysts and may be an intermediate state in cell reprogramming. MATERIALS AND METHODS: Porcine XEN cells (pXENCs) were generated from porcine pluripotent stem cells (pPSCs) and were characterized by RNA sequencing and immunofluorescence analyses. The developmental potential of pXENCs was investigated in chimeric mouse embryos. RESULTS: Porcine XEN cells derived from porcine pPSCs were successfully expanded in N2B27 medium supplemented with bFGF for least 30 passages. RNA sequencing and immunofluorescence analyses showed that pXENCs expressed the murine and canine XEN markers Gata6, Gata4, Sox17 and Pdgfra but not the pluripotent markers Oct4, Sox2 and TE marker Cdx2. Moreover, these cells contributed to the XEN when injected into four-cell stage mouse embryos. Supplementation with Chir99021 and SB431542 promoted the pluripotency of the pXENCs. CONCLUSIONS: We successfully derived pXENCs and showed that supplementation with Chir99021 and SB431542 confer them with pluripotency. Our results provide a new resource for investigating the reprogramming mechanism of porcine-induced pluripotent stem cells.


Assuntos
Endoderma/citologia , Endoderma/embriologia , Suínos/embriologia , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem Celular , Técnicas de Cocultura , Cães , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Endoderma/metabolismo , Expressão Gênica , Camundongos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Análise de Sequência de RNA , Transdução de Sinais , Suínos/genética , Suínos/metabolismo , Quimeras de Transplante
16.
Phytother Res ; 33(4): 1019-1026, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30746789

RESUMO

Epigallocatechin-3-gallate (EGCG) and caffeine in tea exert anti-obesity effects and induces nonalcoholic fatty liver disease (NAFLD) amelioration. However, previous studies usually performed a high-dose EGCG administration, whereas the insecurity was arisen in recent researches. In this study, we treated obese rats with an elaborate dose-40 mg/kg EGCG, 20 mg/kg caffeine, and the coadministration of them as low dose, which were similar to the daily intake; 160 mg/kg EGCG as high dose, which was the maximum safe dose had touched the contentious edge. The results suggested that the coadministration of EGCG and caffeine exerted more remarkable function on suppressing body weight gain, reducing white adipose tissue weight and decreasing the energy intake than single use. This may be due to the variation in serum lipid profile, oxidative stress, and adipose-derived and inflammatory cytokines. The pathological micrographs showed long-term high-fat diets caused severe NAFLD, but it was ameliorated at different levels by all of the administrations. In summary, low dose of EGCG or caffeine only showed a mild effect of anti-obesity and NAFLD amelioration. The coadministration of them could exert a superior curative effect as well as high dose EGCG but no anxiety regarding safety.


Assuntos
Cafeína/administração & dosagem , Catequina/análogos & derivados , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Obesidade/tratamento farmacológico , Animais , Peso Corporal/efeitos dos fármacos , Catequina/administração & dosagem , Dieta Hiperlipídica , Relação Dose-Resposta a Droga , Quimioterapia Combinada , Masculino , Hepatopatia Gordurosa não Alcoólica/complicações , Hepatopatia Gordurosa não Alcoólica/patologia , Obesidade/complicações , Obesidade/patologia , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Chá/química
17.
Theriogenology ; 126: 75-80, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30537656

RESUMO

In vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) are important breeding techniques for livestock. High-quality MII oocytes produced from in vitro maturation (IVM) are required for the two techniques listed above. The ovaries used for IVM operations are primarily acquired from commercial abattoirs, and the pathogen status of slaughtered animals becomes an unavoidable issue. Our previous monitoring data showed that porcine circovirus type 2 (PCV-2) is the main pathogen present in ovaries from abattoirs. However, the characteristics and effects of PCV-2 infection in oocyte maturation and in vitro production (IVP) of embryos are unclear, and currently there are no relevant studies. Therefore, the aim of this study was to determine the PCV-2 infection pattern and determine whether it affects oocyte in vitro maturation and IVP embryo development. More than five hundred ovaries and five thousand oocytes were utilized in the present study. Polymerase chain reaction (PCR) was used to detect PCV-2 DNA in ovaries, follicular fluid (FF), oocytes, cumulus cells and IVP embryos. The effects of viral infections on the rate of oocyte maturation and IVP embryo development were evaluated. We also analyzed the number of copies of the virus in the IVM and IVP process by absolute quantitative fluorescence PCR. Our study showed that the prevalent virus subgenotype in ovaries was PCV-2a. PCV-2a infection did not significantly affect ovarian/oocyte morphology and maturation. Moreover, virus infection did not have a significant effect on the development of the IVP embryos except for a reduction in IVF blastocyst cell numbers. Further tests showed that the viral copy numbers fluctuated at different stages between the IVP embryos and culture medium. For the first time, this study identified the infection pattern of naturally sourced PCV-2 in the course of oocyte maturation and embryo development.


Assuntos
Infecções por Circoviridae/veterinária , Circovirus , Fertilização In Vitro/veterinária , Oócitos/virologia , Doenças dos Suínos/embriologia , Suínos/embriologia , Animais , Infecções por Circoviridae/embriologia , Meios de Cultura , DNA Viral/isolamento & purificação , Desenvolvimento Embrionário , Fertilização In Vitro/métodos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/crescimento & desenvolvimento , Suínos/virologia
18.
Acta Pharmacol Sin ; 40(7): 859-866, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30382183

RESUMO

Human genetic and pharmacological studies have demonstrated that voltage-gated sodium channels (VGSCs) are promising therapeutic targets for the treatment of pain. Spider venom contains many toxins that modulate the activity of VGSCs. To date, only 0.01% of such spider toxins has been explored, and thus there is a great potential for discovery of novel VGSC modulators as useful pharmacological tools or potential therapeutics. In the current study, we identified a novel peptide, µ-TRTX-Ca1a (Ca1a), in the venom of the tarantula Cyriopagopus albostriatus. This peptide consisted of 38 residues, including 6 cysteines, i.e. IFECSISCEIEKEGNGKKCKPKKCKGGWKCKFNICVKV. In HEK293T or ND7/23 cells expressing mammalian VGSCs, this peptide exhibited the strongest inhibitory activity on Nav1.7 (IC50 378 nM), followed by Nav1.6 (IC50 547 nM), Nav1.2 (IC50 728 nM), Nav1.3 (IC50 2.2 µM) and Nav1.4 (IC50 3.2 µM), and produced negligible inhibitory effect on Nav1.5, Nav1.8, and Nav1.9, even at high concentrations of up to 10 µM. Furthermore, this peptide did not significantly affect the activation and inactivation of Nav1.7. Using site-directed mutagenesis of Nav1.7 and Nav1.4, we revealed that its binding site was localized to the DIIS3-S4 linker region involving the D816 and E818 residues. In three different mouse models of pain, pretreatment with Cala (100, 200, 500 µg/kg) dose-dependently suppressed the nociceptive responses induced by formalin, acetic acid or heat. These results suggest that Ca1a is a novel neurotoxin against VGSCs and has a potential to be developed as a novel analgesic.


Assuntos
Analgésicos/farmacologia , Proteínas de Artrópodes/farmacologia , Neurotoxinas/farmacologia , Venenos de Aranha/farmacologia , Aranhas/química , Sequência de Aminoácidos , Analgésicos/isolamento & purificação , Analgésicos/metabolismo , Animais , Proteínas de Artrópodes/isolamento & purificação , Proteínas de Artrópodes/metabolismo , Linhagem Celular Tumoral , Gânglios Espinais/efeitos dos fármacos , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Neurônios/efeitos dos fármacos , Neurotoxinas/isolamento & purificação , Neurotoxinas/metabolismo , Periplaneta , Ligação Proteica , Venenos de Aranha/isolamento & purificação , Venenos de Aranha/metabolismo , Bloqueadores do Canal de Sódio Disparado por Voltagem/isolamento & purificação , Bloqueadores do Canal de Sódio Disparado por Voltagem/metabolismo , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia
19.
Poult Sci ; 98(1): 430-439, 2019 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-30085302

RESUMO

The generation of transgenic chickens is of both biomedical and agricultural significance, and recently chicken transgenesis technology has been greatly advanced. However, major issues still exist in the efficient production of transgenic chickens. This study was designed to optimize the production of enhanced green fluorescence protein (EGFP)-transgenic broilers, including egg windowing at the blunt end (air cell) of egg, and the direct transfection of circulating primordial germ cells by microinjection of the Tol2 plasmid-liposome complex into the early embryonic dorsal aorta. For egg windowing, we discovered that proper manipulation of the inner shell membrane at the blunt end could improve the rate of producing G0 transgenic roosters. From 27 G0 roosters, we successfully collected semen with EGFP-positive sperms from 16 and 19 roosters after direct fluorescence observation and fluorescence-activated cell sorting analyses (13 detected by both methods), respectively. After artificial insemination using the G0 rooster with the highest number of EGFP fluorescent sperm, one G1 EGFP transgenic broiler (1/81, 1.23%) was generated. Our results indicate that appropriate egg windowing and screening of potentially transgene-positive roosters can improve the production of germline-transmitted transgenic birds.


Assuntos
Animais Geneticamente Modificados , Galinhas/genética , Técnicas de Transferência de Genes/veterinária , Transfecção/veterinária , Animais , Embrião não Mamífero , Feminino , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Inseminação Artificial/veterinária , Masculino , Transfecção/métodos , Transgenes
20.
Yi Chuan ; 40(12): 1120-1128, 2018 Dec 20.
Artigo em Chinês | MEDLINE | ID: mdl-30559101

RESUMO

With the rapid development of stem cell research, the demands for high quality stem cells in cell differentiation studies, cell therapy and clinical applications increase significantly. Human umbilical cord mesenchymal stem cells (hUCMSCs) hold great potentials in these applications due to its wide availability, self-renewal capacity, good pluripotency, and in particular, self-immunomodulation ability. However, the conventional adherent culture system remains challenging in mass production of high-quality stem cells. In this study, we set up a 3D suspension culture system by using spinner flasks added with microcarrier. By optimizing the seeding density and rotation speed, we achieved a cell density as higher as 7×10 8cells/L per vessel. The cultured MSCs had good viability, and the expression levels of the MSC markers were similar to those of 2D-cultured MSCs. After being transferred back into a 2D culture system, the MSCs still maintained normal morphology and proliferation ability. These results indicated that the 3D suspension culture system established in this study provides a fundamental basis for mass production of high-quality MSCs for future research and clinical applications.


Assuntos
Técnicas de Cultura de Células , Células-Tronco Mesenquimais/citologia , Cordão Umbilical/citologia , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Humanos
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