RESUMO
In both cancer and infections, diseased cells are presented to human Vγ9Vδ2 T cells through an 'inside out' signalling process whereby structurally diverse phosphoantigen (pAg) molecules are sensed by the intracellular domain of butyrophilin BTN3A11-4. Here we show how-in both humans and alpaca-multiple pAgs function as 'molecular glues' to promote heteromeric association between the intracellular domains of BTN3A1 and the structurally similar butyrophilin BTN2A1. X-ray crystallography studies visualized that engagement of BTN3A1 with pAgs forms a composite interface for direct binding to BTN2A1, with various pAg molecules each positioned at the centre of the interface and gluing the butyrophilins with distinct affinities. Our structural insights guided mutagenesis experiments that led to disruption of the intracellular BTN3A1-BTN2A1 association, abolishing pAg-mediated Vγ9Vδ2 T cell activation. Analyses using structure-based molecular-dynamics simulations, 19F-NMR investigations, chimeric receptor engineering and direct measurement of intercellular binding force revealed how pAg-mediated BTN2A1 association drives BTN3A1 intracellular fluctuations outwards in a thermodynamically favourable manner, thereby enabling BTN3A1 to push off from the BTN2A1 ectodomain to initiate T cell receptor-mediated γδ T cell activation. Practically, we harnessed the molecular-glue model for immunotherapeutics design, demonstrating chemical principles for developing both small-molecule activators and inhibitors of human γδ T cell function.
Assuntos
Butirofilinas , Ativação Linfocitária , Fosfoproteínas , Receptores de Antígenos de Linfócitos T gama-delta , Linfócitos T , Animais , Humanos , Antígenos CD/imunologia , Antígenos CD/metabolismo , Butirofilinas/imunologia , Butirofilinas/metabolismo , Camelídeos Americanos/imunologia , Simulação de Dinâmica Molecular , Fosfoproteínas/imunologia , Fosfoproteínas/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Cristalografia por Raios X , Ressonância Magnética Nuclear Biomolecular , TermodinâmicaRESUMO
The direct ligands recognized by γδ T cell receptors (γδ TCRs) remain uncertain and controversial. In a study appearing in this issue, Willcox et al. use surface plasmon resonance and isothermal titration calorimetry to demonstrate that B7-like molecule BTNL3 makes physical contact with V4γ-bearing TCRs on γδ T cells.
Assuntos
Receptores de Antígenos de Linfócitos T gama-delta , Linfócitos T , Butirofilinas , Membrana Celular , Humanos , LigantesRESUMO
Human Vγ9Vδ2 T cells respond to microbial infections and malignancy by sensing diphosphate-containing metabolites called phosphoantigens, which bind to the intracellular domain of butyrophilin 3A1, triggering extracellular interactions with the Vγ9Vδ2 T cell receptor (TCR). Here, we examined the molecular basis of this "inside-out" triggering mechanism. Crystal structures of intracellular butyrophilin 3A proteins alone or in complex with the potent microbial phosphoantigen HMBPP or a synthetic analog revealed key features of phosphoantigens and butyrophilins required for γδ T cell activation. Analyses with chemical probes and molecular dynamic simulations demonstrated that dimerized intracellular proteins cooperate in sensing HMBPP to enhance the efficiency of γδ T cell activation. HMBPP binding to butyrophilin doubled the binding force between a γδ T cell and a target cell during "outside" signaling, as measured by single-cell force microscopy. Our findings provide insight into the "inside-out" triggering of Vγ9Vδ2 T cell activation by phosphoantigen-bound butyrophilin, facilitating immunotherapeutic drug design.
Assuntos
Antígenos CD/química , Butirofilinas/química , Ativação Linfocitária , Organofosfatos/metabolismo , Subpopulações de Linfócitos T/imunologia , Antígenos CD/metabolismo , Sítios de Ligação , Butirofilinas/metabolismo , Cristalografia por Raios X , Dimerização , Desenho de Fármacos , Humanos , Ligação de Hidrogênio , Imunoterapia , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Domínios Proteicos , Isoformas de Proteínas/química , Processamento de Proteína Pós-Traducional , Receptores de Antígenos de Linfócitos T gama-delta , Análise de Célula Única , Relação Estrutura-Atividade , Subpopulações de Linfócitos T/metabolismoRESUMO
Parkinson's disease (PD) is associated with olfactory defects in addition to dopaminergic degeneration. Dopaminergic signalling is necessary for subventricular zone (SVZ) proliferation and olfactory bulb (OB) neurogenesis. Alpha-synuclein (α-syn or Snca) modulates dopaminergic neurotransmission, and SNCA mutations cause familial PD, but how α-syn and its mutations affect adult neurogenesis is unclear. To address this, we studied a bacterial artificial chromosome transgenic mouse expressing the A30P SNCA familial PD point mutation on an Snca-/- background. We confirmed that the SNCA-A30P transgene recapitulates endogenous α-syn expression patterns and levels by immunohistochemical detection of endogenous α-syn in a wild-type mouse and transgenic SNCA-A30P α-syn protein in the forebrain. The number of SVZ stem cells (BrdU+GFAP+) was decreased in SNCA-A30P mice, whereas proliferating (phospho-histone 3+) cells were decreased in Snca-/- and even more so in SNCA-A30P mice. Similarly, SNCA-A30P mice had fewer Mash1+ transit-amplifying SVZ progenitor cells but Snca-/- mice did not. These data suggest the A30P mutation aggravates the effect of Snca loss in the SVZ. Interestingly, calbindin+ and calretinin (CalR)+ periglomerular neurons were decreased in both Snca-/-, and SNCA-A30P mice but tyrosine hydroxylase+ periglomerular OB neurons were only decreased in Snca-/- mice. Cell death decreased in the OB granule layer of Snca-/- and SNCA-A30P mice. In the same region, CalR+ numbers increased in Snca-/- and SNCA-A30P mice. Thus, α-syn loss and human A30P SNCA decrease SVZ proliferation, cell death in the OB and differentially alter interneuron numbers. Similar disruptions in human neurogenesis may contribute to the olfactory deficits, which are observed in PD.
Assuntos
Interneurônios/citologia , Ventrículos Laterais/citologia , Bulbo Olfatório/citologia , Doença de Parkinson/genética , alfa-Sinucleína/genética , Animais , Calbindina 2/metabolismo , Morte Celular , Proliferação de Células , Modelos Animais de Doenças , Dopamina/metabolismo , Humanos , Interneurônios/metabolismo , Ventrículos Laterais/metabolismo , Ventrículos Laterais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurogênese/genética , Doença de Parkinson/metabolismo , Mutação Puntual , Tirosina 3-Mono-Oxigenase/metabolismo , alfa-Sinucleína/metabolismoRESUMO
Germplasm preservation of livestock or endangered animals and expansion of germline stem cells are important. The purpose of this study is to investigate whether supplementation of trehalose to the freezing medium (FM) reduces tissular damage and improves the quality of testicular cells in the cryopreserved bovine testicular tissues. We herein established an optimized protocol for the cryopreservation of bovine testicular tissues, and the isolation as well as culture of bovine germ cells containing spermatogonial stem cells (SSCs) from these tissues. The results showed that FM containing 10% dimethyl sulfoxide (Me2SO/DMSO), 10% knockout serum replacement (KSR) and 20% trehalose (FM5) combined with the uncontrolled slow freezing (USF) procedures has the optimized cryoprotective effect on bovine testicular tissues. The FM5 + USF protocol reduced the cell apoptosis, maintained high cell viability, supported the structural integrity and seminiferous epithelial cohesion similar to that in the fresh tissues. Viable germ cells containing SSCs were effectively isolated from these tissues and they maintained germline marker expressions in the co-testicular cells and co-mouse embryonic fibroblasts (MEF) feeder culture systems respectively, during the short-term culture. Additionally, upregulated transcriptions of spermatogenic differentiation marker C-KIT and meiotic marker SYCP3 were detected in these cells after retinoic acid-induced differentiation. Together, FM5 + USF is suitable for the cryopreservation of bovine testicular tissues, with benefits of reducing the apoptosis, maintaining the cell viability, supporting the testicular structure integrity, and sustaining the survival and differentiation potential of bovine germ cells containing SSCs.
Assuntos
Criopreservação , Trealose , Animais , Bovinos , Sobrevivência Celular , Criopreservação/métodos , Crioprotetores/farmacologia , Dimetil Sulfóxido , Fibroblastos , Masculino , Camundongos , Espermatogônias , Testículo , Trealose/farmacologiaRESUMO
Germplasm cryopreservation and expansion of gonocytes/prospermatogonia or spermatogonial stem cells (SSCs) are important; however, it's difficult in cattle. Since inhibitors of Mek1/2 and Gsk3ß (2i) can enhance pluripotency maintenance, effects of 2i-based medium on the cultivation of bovine prospermatogonia from the cryopreserved tissues were examined. The testicular tissues of newborn bulls were well cryopreserved. High mRNA levels of prospermatogonium/SSC markers (PLZF, GFRα-1) and pluripotency markers (Oct4/Pouf5, Sox2, Nanog) were detected and the PLZF+ /GFRα-1+ prospermatogonia were consistently identified immunohistochemically in the seminiferous cords. Using differential plating and Percoll-based centrifugation, 41.59% prospermatogonia were enriched and they proliferated robustly in 2i medium. The 2i medium boosted mRNA abundances of Pouf5, Sox2, Nanog, GFRα-1, PLZF, anti-apoptosis gene Bcl2, LIF receptor gene LIFR and enhanced PLZF protein expression, but suppressed mRNA expressions of spermatogonial differentiation marker c-kit and pro-apoptotic gene Bax, in the cultured prospermatogonia. It also alleviated H2 O2 -induced apoptosis of the enriched cells and decreased histone H3 lysine (K9) trimethylation (H3K9me3) and its methylase Suv39h1/2 mRNA level in the cultured seminiferous cords. Overall, 2i medium improves the cultivation of bovine prospermatogonia isolated from the cryopreserved testes, by inhibiting Suv39h1/2-mediated H3K9me3 through Mek1/2 and Gsk3ß signalling, evidencing successful cryopreservation and expansion of bovine germplasm.
Assuntos
Células-Tronco Germinativas Adultas , Espermatogônias , Animais , Bovinos , Criopreservação , Meios de Cultura , Masculino , TestículoRESUMO
To enrich bovine gonocytes from cryopreserved testicular tissues, the cryoprotection effects of the freezing media containing knockout serum replacement (KSR) were examined. Using Minimum essential medium (MEM) + 10% dimethyl sulfoxide (Me2SO) as the basic medium, calf testicular tissues were cryopreserved in media containing 0, 5, 10, 20, 40, 90% KSR and 5% fetal bovine serum (FBS) respectively. Morphologically, the seminiferous cords and interstitium were well preserved in all groups. The gonocytes were all glial cell line-derived neurotrophic factor (GDNF) family receptor α-1 (GFRα-1) positive. The recovery rates in all KSR groups were higher than that of the 10% Me2SO group, while comparable to the 5% FBS group. The enriched gonocytes expressed gonocyte marker GFRα-1 typically. Collectively, supplementation of 5-10% KSR can achieve comparable cryoprotective effects with using 5% FBS, which is useful in future study due to its defined formulation that is more consistent in quality and stable in supply.
Assuntos
Criopreservação/métodos , Crioprotetores/farmacologia , Soro/metabolismo , Testículo/citologia , Animais , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Dimetil Sulfóxido/farmacologia , Congelamento , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Masculino , Soro/químicaRESUMO
Bovine bone marrow mesenchymal stem cells (bBMSC) are potential stem cell source which can be used for multipurpose. However, their application is limited because the in vitro maintenance of these cells is usually accompanied by aging and multipotency losing. Considering transforming growth factor-ß (TGF-ß) pathway inhibitor Repsox is beneficial for cell reprogramming, here we investigated its impacts on the maintenance and differentiation of bBMSC. The bBMSC were enriched and characterized by morphology, immunofluorescent staining, flow cytometry, and multilineage differentiation. The impacts of Repsox on their proliferation, apoptosis, cell cycle, multipotency, and differentiation were examined by Cell Counting Kit-8 (CCK-8), real-time polymerase chain reaction, induced differentiation and specific staining. The results showed that highly purified cluster of diffrentiation 73+ (CD73 + )/CD90 + /CD105 + /CD34 - /CD45 - bBMSC with adipogenic, osteogenic, and chondrogenic differentiation capacities were enriched. Repsox treatments (5 µM, 48 hr) enhanced the messenger RNA mRNA levels of the proliferation gene (telomerase reverse transcriptase [ TERT]; basic fibroblast growth factor [ bFGF]), apoptosis-related gene ( bax and Bcl2), antiapoptosis ratio ( Bcl2/bax), and pluripotency marker gene ( Oct4, Sox2, and Nanog), instead of changing the cell cycle, in bBMSC. Repsox treatments also enhanced the osteogenic differentiation but attenuated the chondrogenic differentiation of bBMSC, concomitant with decreased Smad2 and increased Smad3/4 expressions in TGF-ß pathway. Collectively, inhibiting TGF-ß/Smad signaling by Repsox regulates the in vitro maintenance and differentiation of bBMSC.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Mesenquimais/fisiologia , Pirazóis/farmacologia , Piridinas/farmacologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Células da Medula Óssea , Bovinos , Diferenciação Celular/efeitos dos fármacos , Condrogênese/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Osteogênese/fisiologia , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad3/genética , Proteína Smad3/metabolismo , Proteína Smad4/genética , Proteína Smad4/metabolismoRESUMO
Found recently in stignomatales, the Stig cyclases catalyze the Cope rearrangement and intramolecular cyclization to produce complex indole alkaloids. Five crystal structures were solved of subfamily 1 and 2 Stig cyclases, which adopt a ß-sandwich fold like the non-catalytic carbohydrate-binding motif. Several complex structures were also determined of indole-based compounds, which are bound to the hydrophobic terminal cavity, where a conserved Asp residue makes an H-bond to the indole N and triggers the acid-catalyzed Cope rearrangement. Through analyzing the enzyme-ligand interactions and mutagenesis experiments, several aromatic residues were found important in catalysis. Apart from a common substrate binding mode and catalytic mechanism, potential subfamily variations that may attribute to the different product specificity are implicated. These results shall expand our scope of enzymology, in particular for further investigation of the biosynthetic Cope rearrangement.
Assuntos
Proteínas de Bactérias/química , Cianobactérias/enzimologia , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Cianobactérias/química , Cianobactérias/metabolismo , Ciclização , Alcaloides Indólicos/química , Alcaloides Indólicos/metabolismo , Modelos Moleculares , Conformação Proteica , Especificidade por SubstratoRESUMO
INTRODUCTION: Lung cancer is the primary cause of cancer-related deaths worldwide, with high rates of morbidity and mortality. The most effective treatment for early stage (I-II) non-small cell lung cancer (NSCLC) is surgical resection. However, the extent of mediastinal lymph nodes removal required and the impact of their removal remains controversial. This systematic review and meta-analysis aimed to evaluate the postoperative complications in patients with stage I-II NSCLC who received mediastinal lymph node dissection (MLND) or mediastinal lymph node sampling (MLNS). METHODS AND ANALYSIS: According to the predefined inclusion criteria, we will conduct a comprehensive search for randomized controlled trials (RCTs) and observational studies examining the postoperative complications of MLND compared to MLNS in patients with stage I-II NSCLC. The search will be performed across multiple databases including PubMed, Embase, the Cochrane Library, CNKI, WanFang, Sinomed, VIP, Duxiu, and Web of Science from inception to February 2024. Additionally, relevant literature references will be retrieved and hand searching of pertinent journals will be conducted. Screening, data extraction, and quality assessment will be performed by two independent reviewers. Review Manager 5.4 will be applied in analyzing and synthesizing. The Grading of Recommendations Assessment, Development and Evaluation (GRADE) will be used to assess the quality of evidence for the whole RCTs and used Newcastle-Ottawa scale to assess the methodologic quality of observational studies. ETHICS AND DISSEMINATION: This study did not include personal information. Ethical approval was not required for this study. This study is based on a secondary analysis of the literature, so ethical review approval is not required. The final report will be published in a peer-reviewed journal. CONCLUSION: This systematic review will contribute to compare the safety and survival benefits of these two surgical techniques for the treatment of early stage NSCLC, to further guide the selection of surgical approaches. TRIAL REGISTRATION: The protocol of the systematic review has been registered on Open Science Framework, with a registration number of DOI https://doi.org/10.17605/OSF.IO/N2Y5D.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Excisão de Linfonodo , Mediastino , Metanálise como Assunto , Complicações Pós-Operatórias , Revisões Sistemáticas como Assunto , Humanos , Carcinoma Pulmonar de Células não Pequenas/cirurgia , Carcinoma Pulmonar de Células não Pequenas/patologia , Excisão de Linfonodo/efeitos adversos , Excisão de Linfonodo/métodos , Neoplasias Pulmonares/cirurgia , Neoplasias Pulmonares/patologia , Complicações Pós-Operatórias/etiologia , Mediastino/patologia , Mediastino/cirurgia , Linfonodos/patologia , Linfonodos/cirurgia , Estadiamento de NeoplasiasRESUMO
BACKGROUND: This systematic review and meta-analysis aimed to evaluate the effects of Taohong Siwu Decoction (THSWD) combined with low molecular weight heparin (LMWH), as well as THSWD alone, on the incidence of Deep vein thrombosis (DVT), D-dimer levels, prothrombin time (PT), activated partial thromboplastin time (APTT), visual analogue scale (VAS) pain score, and calf swelling in patients undergoing hip fracture or replacement surgery, compared to LMWH. METHODS: According to the predefined inclusion criteria, we conducted a comprehensive search for randomized controlled trials (RCTs) examining the efficacy of THSWD combined with LMWH or THSWD compared to LMWH in patients with hip fractures or undergoing replacement surgery. The search was performed across multiple databases including China National Knowledge Internet, WanFang, Sinomed, Duxiu, PubMed, Embase, Google Scholar, Cochrane, and Web of Science from their inception until December 2023. Additionally, relevant literature references were retrieved and hand searching of pertinent journals was conducted. The methodological quality assessment of the included trials was carried out following the guidelines outlined in the Cochrane Handbook. Review Manager 5.4 was applied in analyzing and synthesizing. RESULTS: A total of 18 RCTs with 1353 patients were included. The results of meta-analysis showed that compared with the control group, the combined group had a better effect on the incidence of DVT [RRâ =â 0.32, 95% CI(0.17, 0.58; Pâ =â .0002], D-dimer [SMDâ =â -5.88, 95% CI(-7.66, -4.11); Pâ <â .00001], VAS [MDâ =â -1.16, 95% CI(-1.81, -0.50); Pâ =â .0005], Calf circumference difference [MDâ =â -0.56, 95% CI(-1.05, -0.08); Pâ =â .02]. There was no significant difference in PT and APTT between the combined group and the control group. Meta-analysis results show that the D-dimer, incidence of DVT, PT, and APTT did not significantly differ between the THSWD and the LMWH groups. CONCLUSION: This meta-analysis shows that compared with LMWH, THSWD combined with LMWH has a better efficacy in the treatment of DVT after hip surgery, without a significant increase in the incidence of adverse events. Additionally, the combined therapy can also reduce D-dimer, VAS, and swelling. However, due to the limitations of the included studies (such as small sample size and low-quality evidence), the results need to be further verified in more rigorous multicenter clinical trials with a large sample size.
Assuntos
Anticoagulantes , Medicamentos de Ervas Chinesas , Trombose Venosa , Humanos , Anticoagulantes/uso terapêutico , Ensaios Clínicos Controlados Aleatórios como Assunto , Heparina de Baixo Peso Molecular , Trombose Venosa/tratamento farmacológico , Estudos Multicêntricos como AssuntoRESUMO
BACKGROUND: Pulmonary fibrosis (PF) leads to excessive deposition of fibrous connective tissue in the lungs, increasing the risk of lung cancer due to the enhanced activity of fibroblasts (FBs). Fibroblast-mediated collagen fiber deposition creates a tumor-like microenvironment, laying the foundation for tumorigenesis. Clinically, numerous cases of lung cancer induced by pulmonary fibrosis have been observed. In recent years, the study of nucleotide point mutations, which provide more detailed insights than gene expression, has made significant advancements, offering new perspectives for clinical research. METHODS: We initially employed Mendelian randomization to ascertain that the initial stage of lung cancer induced by PF belongs to small cell lung cancer (SCLC). Subsequently, pulmonary neuroendocrine cells (PNECs) were identified by using pseudo-time series analysis as cell clusters with carcinogenic potential. We categorized FBs into four groups according to their cellular metabolism, and then analyzed the cellular communication between FBs and PNECs, as well as changes in intracellular pathways of PNECs. Additionally, we examined the characteristic genome of FBs which is significantly associated with PF and investigated the impact of FBs on immune cells in the PF microenvironment. Finally, we explored strategies for preventing the progression from PF to lung cancer. RESULTS: The genetic features of cells with carcinogenic potential in PF tissues were revealed, characterized by upregulation of Achaete-Scute Family BHLH Transcription Factor 1 (ASCL1), Homeobox B2 (HOXB2), Teashirt Zinc Finger Homeobox 2 (TSHZ2), Insulinoma-associated 1 (INSM1), and reduced activity of RE1 Silencing Transcription Factor (REST). FBs characterized by high glycolysis and low tricarboxylic acid (TCA) cycling played a key role in the progression of PF. The microenvironment of PF resembles the tumor microenvironment, providing a conducive immunosuppressive environment for the occurrence of cancer cells. In dendritic cells, rs9265808 is a susceptibility locus for progression from pulmonary fibrosis to lung cancer, mutations at this locus increase the expression of Complement Factor B (CFB), and excessive activation of the complement pathway is a crucial factor leading to lung cancer development in patients with pulmonary fibrosis. Ensuring adequate nutritional supply and physical function is one of the effective measures to prevent progression from pulmonary fibrosis to lung cancer. CONCLUSION: CFB promotes lung cancer occurrence by inducing the accumulation and polarization of a large number of monocytes/macrophages in the lungs, driving disease progression by reducing the physical fitness of patients with pulmonary fibrosis.
Assuntos
Progressão da Doença , Neoplasias Pulmonares , Fibrose Pulmonar , Análise de Célula Única , Neoplasias Pulmonares/genética , Humanos , Fibrose Pulmonar/genética , Microambiente Tumoral/imunologia , Microambiente Tumoral/genética , Transcriptoma , Fibroblastos , Células Neuroendócrinas/patologia , Células Neuroendócrinas/metabolismo , Pulmão/patologia , Pulmão/imunologia , Carcinoma de Pequenas Células do Pulmão/genéticaRESUMO
With the improvement of global dietary conditions, non-alcoholic fatty liver disease (NAFLD) has gradually become prevalent. As the number of NAFLD patients increases, the coexistence of diseases associated with it has come into focus. In this study, based on immune phenotypes, intercellular communication activities, and clinical manifestations of NAFLD patients, IL1RN was identified as a central pro-inflammatory factor. Subsequently, potential downstream biological pathways of IL1RN in liver tissues and various cell types were enriched to describe its functions. Transcription factors Nfkb1, Jun, and Sp1, significantly associated with these functions, were also enriched. Functional studies of IL1RN suggest its potential to trigger autoimmune diseases. Given this, Mendelian randomization analysis was used to explore the causal relationship between NAFLD and various autoimmune diseases, with IL1RN considered as an intermediary introduced into Mendelian randomization studies. The results indicate that IL1RN and its partially related proteins play a certain mediating role in the process of NAFLD inducing rheumatoid arthritis (RA). Finally, additional research results suggest that intrahepatic ALT levels may influence IL1RN levels, possibly through amino acid metabolism.
Assuntos
Artrite Reumatoide , Doenças Autoimunes , Hepatopatia Gordurosa não Alcoólica , Humanos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Artrite Reumatoide/genética , Fenótipo , Estudo de Associação Genômica Ampla , Proteína Antagonista do Receptor de Interleucina 1/genéticaRESUMO
Feeder cells are essential to derive pluripotent stem cells (PSCs). Mouse embryonic fibroblasts (MEF) are widely used as feeder to generate and culture embryonic stem cells (ESCs) and induced PSCs (iPSCs) in many species. However it may not be suitable for livestock ESCs/iPSCs due to interspecies difference. Previously we derived bovine iPSCs from bovine Sertoli cells using MEF feeder. Here we compared the effects of MEF feeder and bovine embryonic fibroblasts (BEF) feeder on the maintenance of bovine iPSC pluripotency and morphology as well their contributions to the naïve-like conversion, based on a naïve medium (NM). The results showed successful conversion of the primed bovine iPSCs to naïve-like state within 3-4 days both on MEF feeder and BEF feeder in NM (termed as MNM and BNM respectively). These naïve-like iPSCs showed normal karyotype. There were more iPSC colonies under BNM condition than MNM condition. Epigenetically, histone modification H3K4 was upregulated, while H3K27 was downregulated in the naïve-like iPSCs. We further analyzed the naïve markers and differentiation potential both in vitro and in vivo of these cells, which were all reserved throughout the maintenance. Together, bovine naïve-like iPSCs can be generated both on MEF and BEF feeder in NM condition. The BNM condition is able to sustain the pluripotency and differentiation potential of the naïve-like bovine iPSCs, and improve the conversion efficiency.
Assuntos
Células-Tronco Pluripotentes Induzidas , Camundongos , Animais , Bovinos , Masculino , Células de Sertoli , FibroblastosRESUMO
Ozone (O3) has become the most critical air pollutant in the Yangtze River Delta (YRD) region of China. Research on the O3 formation mechanism and its precursor sources (including nitrogen oxides (NOX) and volatile organic compounds (VOCs)) could provide a theoretical basis for mitigating O3 pollution in this region. In this study, simultaneous field experiments were conducted for air pollutants in a typical urban area (Suzhou) in the YRD region in 2022. The capacity of in-situ O3 formation, O3-NOX-VOCs sensitivities and sources of O3 precursors were analyzed. The results showed that in-situ formation contributed 20.8 % of the O3 concentration in the warm season (April to October) of the Suzhou urban area. Compared with the warm season average, the concentrations of various O3 precursors increased on pollution days. The O3-NOX-VOCs sensitivity was the VOCs-limited regime based on the average concentrations during the warm season. O3 formation was most sensitive to anthropogenic VOCs, of which oxygenated VOCs, alkenes and aromatics were the key species. There was a VOCs-limited regime in spring and autumn, while a transitional regime in summer due to the changes in NOX concentrations. This study considered NOX emission from VOCs sources and calculated the contribution of various sources to O3 formation. The results of VOCs source apportionment showed that diesel engine exhaust and fossil fuel combustion had a dominant proportion, but O3 formation presented significant negative sensitivities to the above two sources because of their high NOX emissions. There were significant sensitivities of O3 formation to gasoline vehicle exhaust and VOCs evaporative emissions (gasoline evaporation and solvent usage). The contribution of VOCs evaporative emissions during the O3 pollution episode was significantly higher than the average; therefore, controlling VOCs evaporative emissions during the O3 pollution episode is critical. These results provide feasible strategies to mitigate O3 pollution.
Assuntos
Poluentes Atmosféricos , Ozônio , Compostos Orgânicos Voláteis , Ozônio/análise , Gasolina , Monitoramento Ambiental/métodos , China , Poluentes Atmosféricos/análise , Compostos Orgânicos Voláteis/análise , Emissões de Veículos/análiseRESUMO
Schisandrin A and B (Sch A and B) are the important components of Asian dietary supplement and phytomedicine Schisandra chinensis (S. chinensis). They can enhance adult neurogenesis in vivo; however, these effects still need to be verified. Here NE-4 C neural stem cells (NSCs) were employed as the in vitro model and treated with Sch A and B at 0.1 µg/mL. EdU (5-Ethynyl-2'-deoxyuridine) labeling showed that both Sch A and B treatments enhanced NSC proliferation. Real-time PCR analysis showed the mRNA abundances of telomerase gene Tert and cell cycle gene Cyclin D1 were significantly up-regulated after the treatments. During the neurosphere induction, Sch B enhanced the neurosphere formation and neuronal differentiation, and increased the neurosphere semidiameters. Detection of the neuron differentiation marker Mapt indicates that both Sch A and B, especially Sch B, benefits the induced neuronal differentiation. Sch B treatment also enhanced mRNA expressions of the neurosphere-specific adhesion molecule Cdh2 and Wnt pathway-related genes including Mmp9, Cyclin D1 and ß-catenin. Together, Sch A especially Sch B, promotes the proliferation, affects the survival, differentiation and neurogenesis of NSCs, which is consistent with their in vivo effects. This study provides further clue on the potential neuropharmacological effects of S. chinensis.
Assuntos
Células-Tronco Neurais , Diferenciação Celular , Proliferação de Células , Ciclo-Octanos , Lignanas , Neurogênese , Compostos PolicíclicosRESUMO
To study the carbon components in indoor and outdoor PM2.5, the samples of PM2.5 were collected from Nankai University in December 2015. The contents of eight carbon components were analyzed to use the thermo-optical reflection method. The results indicated that organic carbon (OC) mass concentration was 17.01, 19.48 and 18.92 µg/m3 in outdoor, dormitory and laboratory; elemental carbon (EC) mass concentration was 7.97, 3.56 and 3.53 µg/m3 in outdoor, dormitory and laboratory; and the total carbon aerosol was the proportion of more than 23% of PM2.5 samples. Lower wind speed and higher relative humidity were helpful to the accumulation of PM2.5. The ratio of OC/EC was > 2, and the SOC/OC ratio was > 30%, indicating that SOC was a crucial component indoors and outdoors. About 72% and 85% of the outdoor OC entering dormitory and laboratory environment, and about 59% and 71% of the outdoor EC entering dormitory and laboratory environment. Factor analysis of the eight carbon fractions indicated that the sources of OC and EC in outdoor, dormitory and laboratory is different.
RESUMO
OBJECTIVE: Sertoli cells (SCs) are important sustentacular cells in the seminiferous tubules of the testis. Isolation and identification of SCs are the premise for studying their functions. Since New Zealand rabbit is a stable strain which is widely used for biomedical research and animal farming, this study aimed to develop a simple and effective protocol for SC isolation in New Zealand rabbits. MATERIALS AND METHODS: The SCs of three 30-day-old New Zealand rabbits were isolated by incubation with enzymatic digestion I (Dulbecco's modified Eagle medium supplemented with 1 mg/ml collagenase IV and 50 µg/ml DNase I) and digestion II (digestion I + 1 mg/ml hyaluronidase + 1 mg/ml trypsin), as well as differential plating. The cells were enriched and identified by using immunocytochemical staining and reverse transcription polymerase chain reaction analysis. RESULTS: Homogeneous cells were obtained. They presented the typical large cell body and an irregular pyramidal shape after differential plating and passaging. These cells expressed mRNA of the SC marker sex-determining region Y-box 9 (SOX9) instead of the Leydig cell marker StAR. Immunocytochemically, they are positive of SOX9, GATA binding protein 4, and androgen-binding protein. CONCLUSION: The SCs were enriched from the testicular tissues of prepubertal New Zealand rabbits by a simple and effective protocol, which provides a basis for further theoretical researches and practical applications.
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Silicon carbide (SiC) ceramic is an ideal material for mechanical seal because of its super hardness, high strength, low friction coefficient, good thermal conductivity, and resistance to friction and wear. However, due to relatively high resistivity of SiC ceramic, the triboelectric charge caused by rubbing of mechanical seal end-faces could not be released. It is terrible that the accumulation of triboelectric charge could cause electrochemical corrosion, which would accelerate wear. To decrease the resistivity of SiC ceramic is a desire for improving the performance of mechanical seal. In this research, decreasing resistivity of pressureless sintered SiC ceramic was investigated by conductive pathways through semiconductive grains in a body by incorporation of graphene, which has an extremely low resistivity. With the increasing of graphene from 0 to 2 wt.%, the volume resistivity of SiC ceramics sintered with graphene decreased logarithmically from >106 to around 200 Ω·cm, and the bulk density decreased gradually, from 3.132 to 3.039 g/cm3. In order to meet the requirements of mechanical seal, SiC ceramic sintered with 1 wt.% of graphene, for which the volume resistivity is of 397 Ω·cm, the bulk density is of 3.076 g/cm3, and the flexural strength is of 364 MPa, was optimized when all properties were taken into consideration. It is possible to fabricate low-resistivity SiC ceramic as a useful friction pair material for mechanical seal in a special condition, without excessive loss of their excellent mechanical properties by the introduction of partially connected graphene as conductive pathway into semiconducting ceramic.
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Schisandrin A and B (Sch A and B) are the main effective components of Schisandra chinensis (S. chinensis), which is traditionally used to enhance mental and intellectual functions in eastern Asia. Previously, we reported Sch A and B remarkably affect adult neurogenesis in the subventricular zone of mouse lateral ventricle. Since the neurogenesis in the hippocampal dentate gyrus (DG) is more important to learning, memory and cognition, here we further examined their effects on the adult DG neurogenesis. Phosphohistone H3 (PHH3) immunostaining showed that Sch B significantly enhanced the cell proliferation in the DG. Glial fibrillary acidic protein (GFAP, mostly labels astrocytes and some stem cells) staining was used to further identify the proliferating cell type. Dramatically, increases of GFAP+ cells in both Sch A and B treated groups were observed. What's more, the total numbers of the mature neurons labeled by neuron-specific nuclear protein (NeuN) were also increased in both Sch A and B treated groups compared with the controls. Together, Sch A and B enhance the adult DG neurogenesis by increasing astrocytes/stem cells and improving the survival and maturation of DG neurons. Our study shed a new light on the neuropharmacological functions of the herbal medicine S. chinensis.