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Intramuscular fat (IMF) content significantly impacts meat quality. influenced by complex interactions between skeletal muscle cells and adipocytes. Adipogenesis plays a pivotal role in IMF formation. Exosomes, extracellular membranous nanovesicles, facilitate intercellular communication by transporting proteins, nucleic acids (DNA and RNA), and other biomolecules into target cells, thereby modulating cellular behaviors. Recent studies have linked exosome-derived microRNAs (miRNAs) and other cargo to adipogenic processes. Various cell types, including skeletal muscle cells, interact with adipocytes via exosome secretion and uptake. Exosomes entering adipocytes regulate adipogenesis by modulating key signaling pathways, thereby influencing the extent and distribution of IMF deposition. This review comprehensively explores the origin, formation, and mechanisms of exosome action, along with current research and their applications in adipogenesis. Emphasis is placed on exosome-mediated regulation of miRNAs, non-coding RNAs (ncRNAs), proteins, lipids, and other biomolecules during adipogenesis. Leveraging exosomal contents for genetic breeding and treating obesity-related disorders is discussed. Insights gathered contribute to advancing understanding and potential therapeutic applications of exosome-regulated adipogenesis mechanisms.
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Adipogenia , Exossomos , MicroRNAs , Adipogenia/genética , Exossomos/metabolismo , Exossomos/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Humanos , Animais , Adipócitos/metabolismoRESUMO
Technology for spatial multi-omics aids the discovery of new insights into cellular functions and disease mechanisms. Here we report the development and applicability of multi-omics in situ pairwise sequencing (MiP-seq), a method for the simultaneous detection of DNAs, RNAs, proteins and biomolecules at subcellular resolution. Compared with other in situ sequencing methods, MiP-seq enhances decoding capacity and reduces sequencing and imaging costs while maintaining the efficacy of detection of gene mutations, allele-specific expression and RNA modifications. MiP-seq can be integrated with in vivo calcium imaging and Raman imaging, which enabled us to generate a spatial multi-omics atlas of mouse brain tissues and to correlate gene expression with neuronal activity and cellular biochemical fingerprints. We also report a sequential dilution strategy for resolving optically crowded signals during in situ sequencing. High-throughput in situ pairwise sequencing may facilitate the multidimensional analysis of molecular and functional maps of tissues.
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Sequenciamento de Nucleotídeos em Larga Escala , Animais , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Camundongos , Encéfalo/metabolismo , Cálcio/metabolismo , Genômica/métodos , MultiômicaRESUMO
Beef and dairy products are rich in protein and amino acids, making them highly nutritious for human consumption. The increasing use of gene editing technology in agriculture has paved the way for genetic improvement in cattle breeding via the development of the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) system. Gene sequences are artificially altered and employed in the pursuit of improving bovine breeding research through targeted knockout, knock-in, substitution, and mutation methods. This review offers a comprehensive analysis of the advancements in gene editing technology and its diverse applications in enhancing both quantitative and qualitative traits across livestock. These applications encompass areas such as meat quality, milk quality, fertility, disease resistance, environmental adaptability, sex control, horn development, and coat colour. Furthermore, the review considers prospective ideas and insights that may be employed to refine breeding traits, enhance editing efficiency, and navigate the ethical considerations associated with these advancements. The review's focus on improving the quality of beef and milk is intended to enhance the economic viability of these products. Furthermore, it constitutes a valuable resource for scholars and researchers engaged in the fields of cattle genetic improvement and breeding.
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Cruzamento , Sistemas CRISPR-Cas , Edição de Genes , Bovinos/genética , Animais , Edição de Genes/métodos , Cruzamento/métodos , Carne , Leite/metabolismoRESUMO
The purpose of this study was to systematically evaluate the prognosis of patients with hepatocellular carcinoma (HCC) smaller than 5 cm using microwave ablation (MWA) versus radiofrequency ablation (RFA). PubMed, Cochrane Library and Embase databases were searched for studies reporting comparisons of two interventions (MWA versus RFA) for patients with early-stage HCC published up to 31 December, 2022. The analysis evaluated the recurrence-free survival (RFS), overall survival (OS) and complications. A total of 894 patients were enrolled in six studies (two randomised controlled trials and four propensity score cohort studies). There were 446 patients in the MWA group and 448 patients in the RFA group. Compared with RFA, MWA had a significant advantage in the post-operative 1-, 2-, 3- and 5-year RFS (odds ratios [OR] = 0.58, 95% confidence interval [CI]: 0.40, 0.84; OR = 0.60, 95% CI: 0.45, 0.80; OR = 0.56, 95% CI: 0.33, 0.93; and OR = 0.44, 95% CI: 0.30, 0.65). The OS of MWA was significantly higher than that of RFA in 5 years after ablation (OR = 0.48, 95% CI: 0.34, 0.68). Moreover, MWA had an advantage in the incidence of complications (OR = 2.23, 95% CI: 1.16, 4.29). In the comparison of percutaneous MWA and RFA in the treatment of HCC with a diameter smaller than 5 cm, MWA may have more advantages in improving the prognosis.
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Intramuscular fat content is closely related to the quality of beef, where the forkhead box protein O1 (FOXO1) is involved in adipocyte differentiation and lipid metabolism, but the specific mechanism of its involvement is still unclear. In this study, interfering with FOXO1 promoted the G1/S transformation of bovine adipocytes by enhancing the expression of proliferation marker genes PCNA, CDK1, CDK2, CCNA2, CCNB1, and CCNE2, thereby positively regulating the proliferation of bovine adipocytes. Additionally, interfering with FOXO1 negatively regulated the expression of adipogenic differentiation marker genes PPARG and CEBPA, as well as lipid anabolism marker genes ACC, FASN, SCD1, SREBP1, FABP4, ACSL1, LPL, and DGAT1, thus reducing triglyceride (TG) content and inhibiting the generation of lipid droplets in bovine adipocytes. A combination of transcriptomic and metabolomics analyses revealed that FOXO1 could regulate the lipogenesis of cattle by influencing the AMPK and PI3K/AKT pathways. Importantly, chromatin immunoprecipitation (ChIP) and site-directed mutagenesis revealed that FOXO1 could regulate bovine lipogenesis by binding to the promoter regions of the CD36 and STEAP4 genes and affecting their transcriptional activities. These results provide a foundation for studying the role and molecular mechanism of FOXO1 in the bovine adipogenesis.
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Adipócitos , Fosfatidilinositol 3-Quinases , Bovinos , Animais , Fosfatidilinositol 3-Quinases/metabolismo , Adipócitos/metabolismo , Metabolismo dos Lipídeos/genética , Adipogenia/genética , Perfilação da Expressão Gênica , Diferenciação CelularRESUMO
Circular RNAs (CircRNAs) are covalently closed-loop non-coding RNA (ncRNA) molecules present in eukaryotes. Numerous studies have demonstrated that circRNAs are important regulators of bovine fat deposition, but their precise mechanisms remain unclear. Previous transcriptome sequencing studies have indicated that circADAMTS16, a circRNA derived from the a disintegrin-like metalloproteinases with the thrombospondin motif 16 (ADAMTS16) gene, is high expressed in bovine adipose tissue. This gives a hint that the circRNA may be involved in the process of bovine lipid metabolism. In this study, the targeting relationship between circADAMTS16 and miR-10167-3p was verified using a dual-luciferase reporter assay. Then, the functions of circADAMTS16 and miR-10167-3p in bovine adipocytes were explored through gain-of-function and lose-of-function. The mRNA expression levels of genes were detected by real-time quantitative PCR (qPCR), and lipid droplet formation was phenotypically evaluated by Oil Red O staining. Cell proliferation and apoptosis were detected using CCK-8, EdU, and flow cytometry. We demonstrated that circADAMTS16 targeted binding to miR-10167-3p. The up-regulation of circADAMTS16 inhibited the differentiation of bovine preadipocytes, and the overexpression of miR-10167-3p promoted the differentiation of bovine preadipocytes. Meanwhile, CCK-8 and EdU results indicated that circADAMTS16 promoted adipocyte proliferation. Subsequently, flow cytometry analysis showed that circADAMTS16 promoted cell transition from G0/G1 phase to S phase, and inhibited cell apoptosis. However, up-regulation of miR-10167-3p inhibited cell proliferation and promoted apoptosis. Briefly, circADAMTS16 inhibited the differentiation and promotes the proliferation of bovine adipocytes by targeting miR-10167-3p during bovine fat deposition, which provides new insights into the mechanism of circRNAs regulation of beef quality.
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MicroRNAs , Bovinos , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Circular/genética , RNA Circular/metabolismo , Sincalida/metabolismo , Diferenciação Celular/genética , Proliferação de Células/genética , Adipócitos/metabolismoRESUMO
Fibroblast growth factor (FGF) family genes are a class of polypeptide factors with similar structures that play an important role in regulating cell proliferation and differentiation, nutritional metabolism, and neural activity. In previous studies, the FGF gene has been widely studied and analyzed in many species. However, the systematic study of the FGF gene in cattle has not been reported. In this study, 22 FGF genes distributed on 15 chromosomes were identified in the Bos taurus genome and clustered into seven subfamilies according to phylogenetic analysis and conservative domains. Collinear analysis showed that the bovine FGF gene family was homologous to Bos grunniens, Bos indicus, Hybrid-Bos taurus, Bubalus bubalis, and Hybrid-Bos indicus, and tandem replication and fragment replication were the key driving forces for the expansion of the gene family. Tissue expression profiling showed that bovine FGF genes were commonly expressed in different tissues, with FGF1, FGF5, FGF10, FGF12, FGF16, FGF17, and FGF20 being highly expressed in adipose tissue. In addition, real-time fluorescence quantitative PCR (qRT-PCR) detection showed that some FGF genes were differentially expressed before and after adipocyte differentiation, indicating their diverse role in the formation of lipid droplets. This study made a comprehensive exploration of the bovine FGF family and laid a foundation for further study on the potential function in the regulation of bovine adipogenic differentiation.
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Fatores de Crescimento de Fibroblastos , Genoma , Bovinos , Animais , Filogenia , Fatores de Crescimento de Fibroblastos/genética , Diferenciação Celular/genética , Búfalos , AdipócitosRESUMO
OBJECTIVE: The purpose of this study was to report the efficacy and safety of no-touch radiofrequency ablation (NT-RFA) in the treatment of small hepatocellular carcinoma (HCC). METHODS: We systematically searched for eligible studies in PubMed, Embase and Cochrane library until June 1, 2022. Random effect model was applied to synthesize the pooled proportions of local tumor progression-free survival (LTP), recurrence-free survival (RFS) and overall survival (OS) respectively, as well as adverse events, for small HCC treated by NT-RFA. RESULTS: Of the 10 included studies, 3 of them reported local tumor recurrence. One reported local tumor recurrence at 19 months (range, 12-24), and 2 studies had no tumor recurrence with 24-months of follow-up. The 1- and 2-year LTP pooled proportions were 99.3% (95%CI, 97.5-100) and 97.8% (95%CI, 94.6-99.6) respectively, and two studies reported a 3-year LTP rate of 96.4% (204/212, 36/37). The 1-yearRFS rates was 91.3% (95%CI, 84.1-98.4), 2-year was 86.4% (95%CI, 75.3-97.5). The 1-, 2- and 3- year OS rates were 92.4% (95%CI, 82.8-92.7), 84.1% (95%CI, 74.7-93.6) and 81.8% (116/181, 33/36) respectively, and only one study reported a 5-year OS rate of 47.0% (85/181). The ablative success rate of the HCC nodules was 96.6% (95%CI, 91.3-99.5) and the proportions of mild and severe adverse events following ablation were 18.3% (95%CI, 8.1-41.6) and 5.0%, respectively. CONCLUSION: NT-RFA provides safely very high rate of sustained local control for the treatment of HCC up to 5 cm.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Ablação por Radiofrequência , Humanos , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/cirurgia , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/cirurgia , Recidiva Local de Neoplasia/patologia , Ablação por Radiofrequência/efeitos adversos , Resultado do TratamentoAssuntos
Células Germinativas , Maturidade Sexual , Animais , Masculino , Maturidade Sexual/genética , Suínos/genética , TestículoRESUMO
OBJECTIVES: To systematically evaluate the clinical efficacy of rectal nonsteroidal anti-inflammatory drugs (NSAIDs) alone or in combination with other agents for preventing pancreatitis after endoscopic retrograde cholangiopanography. METHODS: We carried out a literature search of random controlled trials (RCTs) on preventing post-operative pancreatitis by administration of the anti-inflammatory drugs, indomethacin and diclofenac, following endoscopic retrograde cholangiopancreatography (ERCP). The databases searched for relevant publications up to July 7, 2021, included PubMed, Cochrane Library, and Embase. We screened the literature according to inclusion criteria and analyzed the extracted data. The overall population and high-risk patient groups were analyzed, with the main outcome being the incidence of PEP. RESULTS: The search identified 32 RCTs that included 15019 patients with post-ERCP pancreatitis and 9 different interventions. The results of the overall population network meta-analysis showed that NSAIDs alone, high-dose NSAIDs, and a combination of NSAIDs significantly reduced the incidence of PEP compared with placebo. However, compared with placebo, there was no statistically significant difference between the two interventions (NSAIDs + standard hydration and high-dose NSAIDs). In addition, NSAIDs + sublingual nitrates were associated with a lower incidence of PEP compared to that observed with NSAIDs alone. Probability ranking results showed that NSAIDs + sublingual nitrate had the best effect, followed by NSAIDs + standard hydration, NSAIDs + melatonin, NSAIDs + aggressive hydration, NSAIDs + somatostatin, NSAIDs alone, NSAIDs + epinephrine, high-dose NSAIDs, and placebo. In the high-risk subgroup, the results of the network meta-analysis showed that NSAIDs alone, high-dose NSAIDs, and a combination of NSAIDs showed no statistically significant difference in their ability to reduce the incidence of PEP compared with placebo. Probability ranking results showed that NSAIDs + hydration had the best effect, followed by NSAIDs + sublingual nitroglycerin and NSAIDs + aggressive hydration. CONCLUSION: Of the nine interventions, NSAIDs + sublingual nitrates had considerably better efficacy than the other drugs for reducing the incidence of PEP in the overall population. In high-risk patients, NSAIDs + standard hydration may be the best preventive treatment; however, more randomized, controlled trials are needed to validate our results. TRIAL REGISTRATION: Name of the registry: PROSPERO-International prospective register of systematic reviews. Unique identifying number or registration ID: CRD42021282205.
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Melatonina , Pancreatite , Administração Retal , Anti-Inflamatórios não Esteroides/uso terapêutico , Colangiopancreatografia Retrógrada Endoscópica/efeitos adversos , Diclofenaco/uso terapêutico , Epinefrina , Humanos , Indometacina , Metanálise em Rede , Nitratos , Nitroglicerina , Pancreatite/etiologia , Pancreatite/prevenção & controle , Somatostatina , Revisões Sistemáticas como AssuntoRESUMO
Spermatogenesis is the process by which diploid male germ cells propagate and differentiate into haploid flagellated spermatozoa. This highly complicated process is dependent on testicular somatic cells maturation. While the role of these somatic cells in spermatogenesis is relatively well established, knowledge about their development and maturation, particularly at the molecular level, is limited. In this study, we profiled the testicular single-cell transcriptomes of Guanzhong black pigs at the age of 7, 30, 60, 90, and 150 days. Five types of Sertoli cells, five types of Leydig cells, and four types of peritubular myoid cells were identified. Histological analysis revealed the changes in proliferation levels and marker gene expressions, and the prion-like protein gene (PRND) was identified as a novel marker for Sertoli cells. Additionally, integrated analyses of porcine and human datasets revealed similarities between human and pig testicular somatic cells. Overall, the data obtained in this study contribute to the understanding of testicular development in pigs as a model species.
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Diploide , Células de Sertoli , Masculino , Humanos , Animais , Suínos/genética , Células de Sertoli/metabolismo , Células de Sertoli/patologia , Espermatogênese/genética , Testículo/metabolismo , Análise de Sequência de RNARESUMO
Spermatogonial stem cells (SSCs) are able to undergo both self-renewal and differentiation. Unlike self-renewal, which replenishes the SSC and progenitor pool, differentiation is an irreversible process committing cells to meiosis. Although the preparations for meiotic events in differentiating spermatogonia (Di-SG) are likely to be accompanied by alterations in chromatin structure, the three-dimensional chromatin architectural differences between SSCs and Di-SG, and the higher-order chromatin dynamics during spermatogonial differentiation, have not been systematically investigated. Here, we performed in situ high-throughput chromosome conformation capture, RNA-seq, and chromatin immunoprecipitation-sequencing analyses on porcine undifferentiated spermatogonia (which consist of SSCs and progenitors) and Di-SG. We identified that Di-SG exhibited less compact chromatin structural organization, weakened compartmentalization, and diminished topologically associating domains in comparison with undifferentiated spermatogonia, suggesting that diminished higher-order chromatin architecture in meiotic cells, as shown by recent reports, might be preprogrammed in Di-SG. Our data also revealed that A/B compartments, representing open or closed chromatin regions respectively, and topologically associating domains were related to dynamic gene expression during spermatogonial differentiation. Furthermore, we unraveled the contribution of promoter-enhancer interactions to premeiotic transcriptional regulation, which has not been accomplished in previous studies due to limited cell input and resolution. Together, our study uncovered the three-dimensional chromatin structure of SSCs/progenitors and Di-SG, as well as the interplay between higher-order chromatin architecture and dynamic gene expression during spermatogonial differentiation. These findings provide novel insights into the mechanisms for SSC self-renewal and differentiation and have implications for diagnosis and treatment of male sub-/infertility.
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Células-Tronco Germinativas Adultas , Cromatina , Espermatogênese , Espermatogônias , Células-Tronco Germinativas Adultas/citologia , Células-Tronco Germinativas Adultas/metabolismo , Animais , Diferenciação Celular/fisiologia , Cromatina/metabolismo , Masculino , Espermatogênese/fisiologia , Espermatogônias/citologia , SuínosRESUMO
The macro- and micro-physical properties of cement-stabilized steel slag (CSS) base materials in a highway project were studied. A discrete element model of CSS with a real steel slag shape was constructed using Particle Flow Code 3D and 3D scanning technology. The sensitivity between the macro- and micro-parameters of the sample was explored, and a nonlinear regression equation was established to analyze the relationship between these parameters. Uniaxial compression simulation tests were conducted on CSS with steel slag contents of 0%, 10%, 30%, and 50%. By combining contact calculation, crack location, energy tracking, acoustic emission (AE) monitoring, and other program systems, the macro- and micro-mechanical properties and micro-crack evolution law of the samples in the failure process were analyzed in terms of strength, energy, and fracture damage. The damage mechanism of CSS was also revealed. Results showed that with the increase in steel slag content, the elastic modulus and peak stress of the samples increased, the Poisson's ratio decreased, and the post-peak stress curve steepened, indicating obvious brittle failure characteristics. With the increase in steel slag content, the crack initiation stress, thickness of the fracture surface, and number of internal micro-cracks in CSS increased exponentially. In the uniaxial compression test, AE intensity underwent five stages, in which the peak moment of AE intensity exhibited hysteresis compared with the moment of the peak stress. Absorption and release phenomena of strain energy were observed in the process of specimen failure. When the steel slag content increased, the total strain energy absorbed by the specimen increased. When the absorbed energy exceeded the bond strength, the bond ruptured with the release of energy. The main crack of the sample penetrated and stretched to the direction of strain energy release to form a macroscopic fracture surface.
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BACKGROUND: Spermatogenesis is the process by which male gametes are formed from spermatogonial stem cells and it is essential for the reliable transmission of genetic information between generations. To date, the dynamic transcriptional changes of defined populations of male germ cells in pigs have not been reported. RESULTS: To characterize the atlas of porcine spermatogenesis, we profiled the transcriptomes of ~ 16,966 testicular cells from a 150-day-old pig testis through single-cell RNA-sequencing (scRNA-seq). The scRNA-seq analysis identified spermatogonia, spermatocytes, spermatids and three somatic cell types in porcine testes. The functional enrichment analysis demonstrated that these cell types played diverse roles in porcine spermatogenesis. The accuracy of the defined porcine germ cell types was further validated by comparing the data from scRNA-seq with those from bulk RNA-seq. Since we delineated four distinct spermatogonial subsets, we further identified CD99 and PODXL2 as novel cell surface markers for undifferentiated and differentiating spermatogonia, respectively. CONCLUSIONS: The present study has for the first time analyzed the transcriptome of male germ cells and somatic cells in porcine testes through scRNA-seq. Four subsets of spermatogonia were identified and two novel cell surface markers were discovered, which would be helpful for studies on spermatogonial differentiation in pigs. The datasets offer valuable information on porcine spermatogenesis, and pave the way for identification of key molecular markers involved in development of male germ cells.
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Spermatogenesis is a continual process that occurs in the testes, in which diploid spermatogonial stem cells (SSCs) differentiate and generate haploid spermatozoa. This highly efficient and intricate process is orchestrated at multiple levels. N6-methyladenosine (m6A), an epigenetic modification prevalent in mRNAs, is implicated in transcriptional regulation during spermatogenesis. However, the dynamics of m6A modification in non-rodent mammalian species remains unclear. Here we systematically investigated the profile and role of m6A during spermatogenesis in pigs. By analyzing the transcriptomic distribution of m6A in spermatogonia, spermatocytes, and round spermatids, we identified a globally conserved m6A pattern between porcine and murine genes with spermatogenic function. We found that m6A was enriched in a group of genes that specifically encode the metabolic enzymes and regulators. In addition, transcriptomes in porcine male germ cells could be subjected to the m6A modification. Our data showed that m6A played the regulatory roles during spermatogenesis in pigs, which is similar to that in mice. Illustrations of this point were three genes (SETDB1, FOXO1, and FOXO3) that were crucial to the determination of the fate of SSCs. To the best of our knowledge, this study has for the first time uncovered the expression profile and role of m6A during spermatogenesis in large animals and contributes to insights into the intricate transcriptional regulation underlying the lifelong male fertility in non-rodent mammalian species.
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BACKGROUND: Spermatogonial stem cells (SSCs), as tissue-specific stem cells, are capable of both self-renewal and differentiation and supporting the continual and robust spermatogenesis for male fertility. As a rare sub-fraction of undifferentiated spermatogonia, SSCs share most molecular markers with the progenitor spermatogonia. Thus, the heterogeneity of the progenitor cells often obscures the characteristics of stem cells. Distinguishing SSCs from the progenitors is of paramount importance to understand the regulatory mechanisms governing their actions. OBJECTIVES: The present study was designed to reveal that SSEA4 can be a marker for putative porcine SSCs that distinguished it from the progenitors and to build a sorting program for efficient enrichment of porcine SSCs. METHODS: To explore expression of SSEA4 within the undifferentiated spermatogonial population, we performed co-immunofluorescent staining for SSEA4 and common molecular markers (VASA, DBA, PLZF, c-KIT, and SOX9) in the 7-, 90-, and 150-day-old porcine testicular tissues. SSEA4-positive cells were isolated from the 90-day-old porcine testes by flow cytometry. Immunofluorescent, RNA-sequencing, and transplantation analysis were used to reveal that SSEA4-positive fraction holds the stem cell capacity. RESULTS: We found that SSEA4 was expressed in a rare sub-fraction of porcine undifferentiated spermatogonia, and RNA-sequencing analysis revealed that the genes for stem cell maintenance and SSC-specific markers (ID4 and PAX7) were up-regulated in the SSEA4-sorted fraction, compared with undifferentiated spermatogonia. In addition, germ cell transplantation assay demonstrated that SSEA4-positive spermatogonia colonized in the recipient testicular tubules. Sorting of the undifferentiated spermatogonia with anti-SSEA4 antibody resulted in a 2.5-fold enrichment of SSCs compared with the germ cell gate group, and 21-fold enrichment of SSCs compared with the SSEA4-negative spermatogonia group. CONCLUSIONS: Our findings revealed that SSEA4 is a new surface marker for porcine undifferentiated spermatogonia. This finding helps to elucidate the characteristics of porcine SSCs and facilitates the culture and manipulation of SSCs.
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Células-Tronco Germinativas Adultas/metabolismo , Células-Tronco Germinativas Adultas/transplante , Espermatogênese/fisiologia , Espermatogônias/metabolismo , Antígenos Embrionários Estágio-Específicos/metabolismo , Células-Tronco Germinativas Adultas/citologia , Animais , Biomarcadores/metabolismo , Diferenciação Celular/fisiologia , Xenoenxertos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Espermatogônias/citologia , Espermatozoides/crescimento & desenvolvimento , Suínos , Testículo/metabolismo , Transplante HeterólogoRESUMO
In recent years, cell-free protein synthesis (CFPS) systems have been used to synthesize proteins, prototype genetic elements, manufacture chemicals, and diagnose diseases. These exciting, novel applications lead to a new wave of interest in the development of new CFPS systems that are derived from prokaryotic and eukaryotic organisms. The eukaryotic Pichia pastoris is emerging as a robust chassis host for recombinant protein production. To expand the current CFPS repertoire, we report here the development and optimization of a eukaryotic CFPS system, which is derived from a protease-deficient strain P. pastoris SMD1163. By developing a simple crude extract preparation protocol and optimizing CFPS reaction conditions, we were able to achieve superfolder green fluorescent protein (sfGFP) yields of 50.16 ± 7.49 µg/ml in 5 h batch reactions. Our newly developed P. pastoris CFPS system fits to the range of the productivity achieved by other eukaryotic CFPS platforms, normally ranging from several to tens of micrograms protein per milliliter in batch mode reactions. Looking forward, we believe that our P. pastoris CFPS system will not only expand the CFPS toolbox for synthetic biology applications, but also provide a novel platform for cost-effective, high-yielding production of complex proteins that need post-translational modification and functionalization.
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Amphoteric modification can simultaneously improve the adsorption of organic pollutants and heavy metals on clay minerals. Study of the adsorption and interaction of multiple heavy metals on amphoteric modified soils is therefore of practical significance. Here, bentonite-(CK) and 150BS-12-modified bentonites (150BS-12) were characterized both before and after metal ion adsorption using Fourier-transform infrared spectroscopy (FTIR). The equilibrium adsorption characteristics and differences of Cu2+ and Pb2+ in single and binary systems were studied by batch methods. The interaction mechanism of the metals on modified bentonites is also discussed. The results showed that the adsorption capacity of Cu2+ and Pb2+ in single and binary systems was ranked, in descending order, as 150BS-12 > 100BS-12 > 50BS-12 > CK, and that the adsorption isotherm could be described by the Langmuir and Freundlich equations. Although the modification of BS-12 was more beneficial for the adsorption of Cu2+, the adsorption capacity and selectivity coefficient of Pb2+ on BS-12-modified bentonites were larger than for Cu2+. Cu2+ and Pb2+ had a mutually antagonistic effect on each other; modification with BS-12 enhanced the effect of Cu2+ on Pb2+ and weakened the effect of Pb2+ on Cu2+, which was always stronger than the effect of Cu2+ on Pb2+. Increases in temperature and pH, and a reduction in ionic strength, can increase the adsorption of Cu2+ and Pb2+; however, the influence of these factors varied between the two metals, which also affected the interaction between the metals.
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DNA methylation and histone modifications critically regulate the expression of many genes and repeat regions during spermatogenesis. However, the molecular details of these processes in male germ cells remain to be addressed. Here, using isolated murine sperm cells, ultra-low-input native ChIP-Seq (ULI-NChIP-Seq), and whole genome bisulfite sequencing (WGBS), we investigated genome-wide DNA methylation patterns and histone 3 Lys-9 trimethylation (H3K9me3) modifications during mouse spermatogenesis. We found that DNA methylation and H3K9me3 have distinct sequence preferences and dynamics in promoters and repeat elements during spermatogenesis. H3K9me3 modifications in histones at gene promoters were highly enriched in round spermatids. H3K9me3 modification on long terminal repeats (LTRs) and long interspersed nuclear elements (LINEs) was involved in silencing active transcription from these regions in conjunction with reestablishment of DNA methylation. Furthermore, H3K9me3 remodeling on the X chromosome was involved in meiotic sex chromosome inactivation and in partial transcriptional reactivation of sex chromosomes in spermatids. Our findings also revealed the DNA methylation patterns and H3K9me3 modification profiles of paternal and maternal germline imprinting control regions (gICRs) during spermatogenesis. Taken together, our results provide a genome-wide map of H3K9me3 modifications during mouse spermatogenesis that may be helpful for understanding male reproductive disorders.