RESUMO
Animals integrate sensory information from the environment and display various behaviors in response to external stimuli. In Caenorhabditis elegans hermaphrodites, 33 types of sensory neurons are responsible for chemosensation, olfaction, and mechanosensation. However, the functional roles of all sensory neurons have not been systematically studied due to the lack of facile genetic accessibility. A bipartite cGAL-UAS system has been previously developed to study tissue- or cell-specific functions in C. elegans. Here, we report a toolkit of new cGAL drivers that can facilitate the analysis of a vast majority of the 60 sensory neurons in C. elegans hermaphrodites. We generated 37 sensory neuronal cGAL drivers that drive cGAL expression by cell-specific regulatory sequences or intersection of two distinct regulatory regions with overlapping expression (split cGAL). Most cGAL-drivers exhibit expression in single types of cells. We also constructed 28 UAS effectors that allow expression of proteins to perturb or interrogate sensory neurons of choice. This cGAL-UAS sensory neuron toolkit provides a genetic platform to systematically study the functions of C. elegans sensory neurons.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Células Receptoras Sensoriais/metabolismoRESUMO
Pulmonary fibrosis is a progressive and debilitating lung disease characterized by the excessive accumulation of extracellular matrix (ECM) components within the lung parenchyma. However, the underlying mechanism remains largely elusive, and the treatment options available for pulmonary fibrosis are limited. Interleukin 5 receptor, alpha (IL5RA) is a well-established regulator of eosinophil activation, involved in eosinophil-mediated anti-parasitic activities and allergic reactions. Recent studies have indicated additional roles of IL5RA in lung epithelium and fibroblasts. Nevertheless, its involvement in pulmonary fibrosis remains unclear. In present study, we employed single-cell analyses alongside molecular and cellular assays to unveil the expression of IL5RA in lung epithelial cells. Moreover, using both in vitro and in vivo models, we demonstrated a notable upregulation of epithelial IL5RA during the progression of pulmonary fibrosis. This upregulated IL5RA expression subsequently promotes epithelial-mesenchymal transition (EMT), leading to the generation of mesenchymal phenotype with augmented capability for ECM production. Importantly, our findings uncovered that the pro-fibrotic function of IL5RA is mediated by Jak2/STAT3 signaling cascades. Inhibiting IL5RA has the potential to deactivate Jak2/STAT3 and suppress the downstream EMT process and ECM production, thereby offering a promising therapeutic strategy for pulmonary fibrosis.
Assuntos
Fibrose Pulmonar , Humanos , Transição Epitelial-Mesenquimal/fisiologia , Fibrose , Subunidade alfa de Receptor de Interleucina-5/metabolismo , Pulmão/metabolismo , Fibrose Pulmonar/metabolismo , Receptores de Interleucina-5/metabolismo , Fator de Transcrição STAT3/metabolismoRESUMO
Recent findings suggest that extracellular heat shock protein 90α (eHSP90α) promotes pulmonary fibrosis, but the underlying mechanisms are not well understood. Aging, especially cellular senescence, is a critical risk factor for idiopathic pulmonary fibrosis (IPF). Here, we aim to investigate the role of eHSP90α on cellular senescence in IPF. Our results found that eHSP90α was upregulated in bleomycin (BLM)-induced mice, which correlated with the expression of senescence markers. This increase in eHSP90α mediated fibroblast senescence and facilitated mitochondrial dysfunction. eHSP90α activated TGF-ß signaling through the phosphorylation of the SMAD complex. The SMAD complex binding to p53 and p21 promoters triggered their transcription. In vivo, the blockade of eHSP90α with 1G6-D7, a specific eHSP90α antibody, in old mice attenuated the BLM-induced lung fibrosis. Our findings elucidate a crucial mechanism underlying eHSP90α-induced cellular senescence, providing a framework for aging-related fibrosis interventions.
Assuntos
Bleomicina , Fibrose Pulmonar Idiopática , Animais , Bleomicina/toxicidade , Senescência Celular , Fibroblastos/metabolismo , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fator de Crescimento Transformador beta/metabolismoRESUMO
OBJECTIVE: This study aimed to explore the roles of duration and burden of atrial high-rate episode (AHRE) on ischemic stroke in patients with pacemaker implantation. METHODS: Patients with pacemaker implantation for bradycardia from 2013 to 2017 were consecutively enrolled. Data such as gender, age, combined diseases, type of AF, left atrial size, left ventricular size, left ventricular ejection fraction, CHA2 DS2 -VASc score, and anticoagulants were collected. The burden and duration of AHRE based on different interval partition were also recorded in detail to evaluate the impacts on ischemic stroke. Cox regression analysis with time-dependent covariates was conducted. RESULTS: A total of 220 patients with AHRE were enrolled. The average follow-up time was 48.42 ± 17.20 months. Univariate regression analysis showed that diabetes (p = .024), high CHA2 DS2 -VASc score (≥ 2) (p = .021), long mean AHRE burden (p = .011), long maximal AHRE burden (p = .015), long AHRE duration lasting≥48 h (p = .001) or 24 h (p = .001) or 12 h (p = .005) were prone to ischemic stroke. Further multivariate regression analysis showed that long duration of AHRE (≥48 h) (HR 10.77; 95% CI 3.22-55.12; p = .030) were significantly correlated with stroke in patients with paroxysmal AF. There was no significant correlation between the type of AF and stroke (p = .927). CONCLUSION: The longer duration of AHRE (≥48 h) was more favorable in predicting ischemic stroke than high CHA2 DS2 -VASc score (≥2).
Assuntos
Fibrilação Atrial , AVC Isquêmico , Humanos , Medição de Risco , Fatores de Risco , Volume Sistólico , Função Ventricular EsquerdaRESUMO
Lung squamous cell carcinoma (LSCC) is a prevalent and progressive subtype of lung cancer. This study aimed to substantiate the regulatory effect of the PAK2/SOX2/DEK axis on the LSCC development. LSCC tissues (n = 83) and adjacent normal tissues were collected and SOX2 expression was determined by qRT-PCR and Western blotting. Correlation between SOX2 expression and the prognosis of LSCC patients was then explored utilizing Kaplan-Meier analysis. Co-immunoprecipitation and glutathione-S-transferase pull-down assays were conducted to validate the binding of SOX2 to DEK. Gain- and loss- of function assays were then performed on LSCC cells, with CCK-8 and Transwell assays applied to detect the malignant behaviors of cells. A mouse xenograft model of LSCC was further established for in vivo validation. The expression levels of SOX2, PAK2 and DEK were up-regulated in LSCC tissues and cells. SOX2 overexpression was correlated with poor prognosis of LSCC patients. Knockdown of SOX2 weakened the viability and the migratory and invasive potential of LSCC cells. Further, PAK2 directly interacted with SOX2. PAK2 overexpression accelerated the malignant phenotypes of LSCC cells through interplay with SOX2. Moreover, SOX2 activated the expression of DEK, and silencing DEK attenuated the malignant behaviors of LSCC cells. In conclusion, PAK2 could bind to the transcription factor SOX2 and thus activate the expression of DEK, thereby driving the malignant phenotypes of LSCC cells both in vivo and in vitro.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Carcinoma de Células Escamosas , Neoplasias Laríngeas , Neoplasias Pulmonares , MicroRNAs , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Regulação Neoplásica da Expressão Gênica , Glutationa/metabolismo , Humanos , Neoplasias Laríngeas/genética , Neoplasias Laríngeas/patologia , Pulmão/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , MicroRNAs/genética , Proteínas Oncogênicas , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Fatores de Transcrição SOXB1 , Sincalida/genética , Sincalida/metabolismo , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/metabolismoRESUMO
We previously developed chicken interleukin-1ß (IL-1ß) mutants as single-dose adjuvants that induce protective immunity when co-administered with an avian vaccine. However, livestock such as pigs may require a vaccine adjuvant delivery system that provides long-lasting protection to reduce the need for successive booster doses. Therefore, we developed chitosan-coated alginate microparticles as a carrier for bovine serum albumin (BSA) or porcine IL-1ß (pIL-1ß) and assessed their physical, chemical, and biological properties. Electrospraying of the BSA-loaded alginate microparticles (BSA/ALG MPs) resulted in an encapsulation efficiency of 50%, and those MPs were then coated with chitosan (BSA/ALG/CHI MPs). Optical and scanning electron microscopy, zeta potential analysis, and Fourier transform infrared spectroscopy were used to characterize these MPs. The BSA encapsulation parameters were applied to ALG/CHI MPs loaded with pIL-1ß, which were not cytotoxic to porcine fibroblasts but had enhanced bio-activity over unencapsulated pIL-1ß. The chitosan layer of the BSA/ALG/CHI MPs prevented burst release and facilitated sustained release of pIL-1ß for at least 28 days. In conclusion, BSA/ALG/CHI MPs prepared as a carrier for pIL-1ß may be used as an adjuvant for the formulation of pig vaccines.
Assuntos
Quitosana , Vacinas , Alginatos/química , Animais , Quitosana/química , Ácido Glucurônico/química , Ácido Glucurônico/farmacologia , Ácidos Hexurônicos/química , Ácidos Hexurônicos/farmacologia , Interleucina-1beta , Soroalbumina Bovina/química , SuínosRESUMO
The COVID-19 pandemic is caused by SARS-CoV-2; the spike protein is a key structural protein that mediates infection of the host by SARS-CoV-2. In this study, we aimed to evaluate the effects of signal peptide on the secretion and release of SARS-CoV-2 spike protein. Therefore, we constructed a signal peptide deletion mutant and three signal peptide site-directed mutants. The (H) region and (C) region in the signal peptide of L5F-S13I mutant have changed significantly, compared with wild type, L5F and S13I. We demonstrated the effects of signal peptide on the secretion and synthesis of RBD protein, finding that mutation of S13 to I13 on the signal peptide is more conducive to the secretion of RBD protein, which was mainly due to the shift of the signal peptide cleavage site in the mutant S13I. Here, we not only investigated the structure of the N-terminal signal peptide of the SARS-CoV-2 spike protein but also considered possible secretory pathways. We suggest that the development of drugs that target the signal peptide of the SARS-CoV-2 spike protein may have potential to treat COVID-19 in the future.
Assuntos
COVID-19 , Glicoproteína da Espícula de Coronavírus , Humanos , Pandemias , Sinais Direcionadores de Proteínas/genética , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
Pheromones are critical cues for attracting mating partners for successful reproduction. Sexually mature Caenorhabditis remanei virgin females and self-sperm-depleted Caenorhabditis elegans hermaphrodites produce volatile sex pheromones to attract adult males of both species from afar. The chemoresponsive receptor in males has remained unknown. Here, we show that the male chemotactic behavior requires amphid sensory neurons (AWA neurons) and the G-protein-coupled receptor SRD-1. SRD-1 expression in AWA neurons is sexually dimorphic, with the levels being high in males but undetectable in hermaphrodites. Notably, srd-1 mutant males lack the chemotactic response and pheromone-induced excitation of AWA neurons, both of which can be restored in males and hermaphrodites by AWA-specific srd-1 expression, and ectopic expression of srd-1 in AWB neurons in srd-1 mutants results in a repulsive behavioral response in both sexes. Furthermore, we show that the C-terminal region of SRD-1 confers species-specific differences in the ability to perceive sex pheromones between C. elegans and C. remanei These findings offer an excellent model for dissecting how a single G-protein-coupled receptor expressed in a dimorphic neural system contributes to sex-specific behaviors in animals.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células Receptoras Sensoriais/metabolismo , Atrativos Sexuais/metabolismo , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Cálcio/metabolismo , Feminino , Regulação da Expressão Gênica , Masculino , Receptores de Superfície Celular/química , Receptores Acoplados a Proteínas G/química , Caracteres Sexuais , Transdução de Sinais , Especificidade da Espécie , VolatilizaçãoRESUMO
Idiopathic pulmonary fibrosis (IPF), a progressive and fatal lung disease, usually leads to an irreversible distortion of the pulmonary structure. The functional roles of bone marrow-derived mesenchymal stem cells (BMSC)-secreted extracellular vesicles (EVs) in fibroblasts have been implicated, yet their actions in the treatment of IPF are not fully understood. This study investigated the roles of BMSC-derived EVs expressing miR-29b-3p in fibroblasts in IPF treatment. EVs derived from BMSCs were successfully isolated and could be internalized by pulmonary fibroblasts, and Cell Counting Kit-8 (CCK-8) and Transwell assay results identified that EVs inhibited the activation of fibroblast in IPF. miR-29b-3p, frizzled 6 (FZD6), α-skeletal muscle actin (α-SMA), and Collagen I expressions were examined, which revealed that miR-29b-3p was poorly expressed and FZD6, α-SMA, and Collagen I were overexpressed in pulmonary tissues. Dual-luciferase reporter assay results demonstrated that miR-29b-3p could inversely target FZD6 expression. The gain- and loss-of-function assays were conducted to determine regulatory effects of FZD6 and miR-29b-3p on IPF. CCK-8 and Transwell assays results displayed that BMSCs-derived EVs overexpressing miR-29b-3p contributed to inhibited pulmonary interstitial fibroblast proliferation, migration, invasion, and differentiation. Furthermore, the effects of BMSCs-derived EVs overexpressing miR-29b-3p on IPF progression were assessed in vivo, which confirmed the repressive effects of BMSCs-derived EVs overexpressing miR-29b-3p on IPF progression. Collectively, BMSCs-derived EVs overexpressing miR-29b-3p relieve IPF through FZD6.
Assuntos
Fibroblastos/metabolismo , Receptores Frizzled/genética , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , Idoso , Animais , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Colágeno Tipo I/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/metabolismo , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Pessoa de Meia-IdadeRESUMO
Idiopathic pulmonary fibrosis (IPF) is a condition that results in the progressive deterioration of lung function with poor prognosis. The current study is aimed at exploring how microRNA-448 (miR-448) targeting ABCC3 affects fibroblast proliferation, apoptosis, and collagen synthesis of mice with IPF via the Jun N-terminal kinase (JNK) signaling pathway. Bioinformatics and dual-luciferase polymerase chain reaction were used to predict the relationship of miR-448 and ABCC3. The expression of miR-448 and ABCC3 was detected in IPF tissues. Using IPF mouse models, lung fibroblasts for the experiments were treated with miR-448 mimic, miR-448 inhibitor, si-ABCC3, or SP600125 (inhibitor of JNK) to evaluate the cell proliferation and apoptosis in response to miR-448. Reverse transcription quantitative polymerase chain reaction and western blot analysis were used to identify the expression of miR-448, ABCC3, and the activation of the JNK signaling pathway. ABCC3 was targeted and downregulated by miR-448 based on bioinformatics prediction and dual-luciferase reporter gene assay. Additionally, miR-448 was found to be highly expressed in IPF lung tissues with low expression levels of ABCC3. In response to the treatment of miR-448 mimic or si-ABCC3, lung fibroblasts exhibited decreased cell proliferation and increased apoptotic rates, whereas the miR-448 inhibitor reversed the conditions. Notably, we also found that miR-448 mimic inhibited the JNK signaling pathway. In conclusion, by using miR-448 to target and downregulate ABCC3 to block the JNK signaling pathway in mice with IPF, we found an increase in fibroblast apoptosis, inhibited cell proliferation, and decreased collagen synthesis of fibroblasts.
Assuntos
Colágeno/biossíntese , Fibroblastos/patologia , Fibrose Pulmonar Idiopática/patologia , Sistema de Sinalização das MAP Quinases/fisiologia , MicroRNAs/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Animais , Apoptose/genética , Proliferação de Células/genética , Colágeno/genética , Fibroblastos/metabolismo , Regulação da Expressão Gênica/genética , Fibrose Pulmonar Idiopática/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genéticaRESUMO
BACKGROUND: It is reported that medical students do not receive adequate opportunities to learn surgical skill and are at risk of being unable to perform simple surgical procedures safely. The usefulness of peer-assisted learning (PAL) as a tool to assist in delivering surgical skills training is worth exploring. METHODS: This is a randomised single blinded controlled trial. Fourth-year students from the university's Surgical Society were asked to volunteer as peer tutors and those in 3rd-year were asked to undertake surgical skills training. A cohort of 35 students were selected and randomised to receive basic surgical skills training conducted either by faculty members or peers. The students' performance of basic suturing skills was assessed using a checklist, through directly observed procedural skills (DOPS) technique. The assessment was conducted by faculty blinded to the training. Students' perception to surgical skills training was assessed using a questionnaire survey. RESULTS: The suturing and knotting skills of students learned from their peers was comparable to that acquired from faculty. The students' perceived that their peers could conduct surgical skills training similar to their faculty. CONCLUSION: PAL approach for basic surgical skills training is as effective as faculty-led training. PAL has the potential to optimise the delivery of surgical skills training in undergraduate medical education.
RESUMO
BACKGROUND: The dysfunction of airway epithelial barrier is closely related to the pathogenesis of asthma. Secreted Hsp90α participates in inflammation and Hsp90 inhibitor protects endothelial dysfunction. In the current study, we aimed to explore the role of secreted Hsp90α in asthmatic airway epithelial barrier function. METHODS: Male BALB/c mice were sensitized and challenged with HDM to generate asthma model. The 16HBE and Hsp90α-knockdown cells were cultured and treated according to the experiment requirements. Transepithelial Electric Resistance (TEER) and permeability of epithelial layer in vitro, distribution and expression of junction proteins both in vivo and in vitro were used to evaluate the epithelial barrier function. Western Blot was used to evaluate the expression of junction proteins and phosphorylated AKT in cells and lung tissues while ELISA were used to evaluate the Hsp90α expression and cytokines release in the lung homogenate. RESULTS: HDM resulted in a dysfunction of airway epithelial barrier both in vivo and in vitro, paralleled with the increased expression and release of Hsp90α. All of which were rescued in Hsp90α-knockdown cells or co-administration of 1G6-D7. Furthermore, either 1G6-D7 or PI3K inhibitor LY294002 suppressed the significant phosphorylation of AKT, which caused by secreted and recombinant Hsp90α, resulting in the restoration of epithelial barrier function. CONCLUSIONS: Secreted Hsp90α medicates HDM-induced asthmatic airway epithelial barrier dysfunction via PI3K/AKT pathway, indicating that anti-secreted Hsp90α therapy might be a potential treatment to asthma in future.
Assuntos
Asma/fisiopatologia , Brônquios/efeitos dos fármacos , Cromonas/farmacologia , Células Epiteliais/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/metabolismo , Morfolinas/farmacologia , Animais , Asma/tratamento farmacológico , Brônquios/enzimologia , Brônquios/imunologia , Caderinas/metabolismo , Linhagem Celular , Citocinas/metabolismo , Impedância Elétrica , Células Epiteliais/enzimologia , Células Epiteliais/imunologia , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico HSP90/genética , Humanos , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação , Pyroglyphidae/imunologia , Mucosa Respiratória/metabolismoRESUMO
How environmental cues regulate adult stem cell and cancer cell activity through surface receptors is poorly understood. Angiopoietin-like proteins (ANGPTLs), a family of seven secreted glycoproteins, are known to support the activity of haematopoietic stem cells (HSCs) in vitro and in vivo. ANGPTLs also have important roles in lipid metabolism, angiogenesis and inflammation, but were considered 'orphan ligands' because no receptors were identified. Here we show that the immune-inhibitory receptor human leukocyte immunoglobulin-like receptor B2 (LILRB2) and its mouse orthologue paired immunoglobulin-like receptor (PIRB) are receptors for several ANGPTLs. LILRB2 and PIRB are expressed on human and mouse HSCs, respectively, and the binding of ANGPTLs to these receptors supported ex vivo expansion of HSCs. In mouse transplantation acute myeloid leukaemia models, a deficiency in intracellular signalling of PIRB resulted in increased differentiation of leukaemia cells, revealing that PIRB supports leukaemia development. Our study indicates an unexpected functional significance of classical immune-inhibitory receptors in maintenance of stemness of normal adult stem cells and in support of cancer development.
Assuntos
Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Leucemia/metabolismo , Leucemia/patologia , Glicoproteínas de Membrana/metabolismo , Receptores Imunológicos/metabolismo , Animais , Diferenciação Celular , Divisão Celular , Células Cultivadas , Modelos Animais de Doenças , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Células HEK293 , Humanos , Glicoproteínas de Membrana/genética , Camundongos , Proteína de Leucina Linfoide-Mieloide , Receptores Imunológicos/genéticaRESUMO
Pulmonary fibrosis is characterized by lung fibroblast activation and ECM deposition and has a poor prognosis. Heat shock protein 90 (Hsp90) participates in organ fibrosis, and extracellular Hsp90α (eHsp90α) promotes fibroblast activation and migration. This study aimed to investigate whether a selective anti-Hsp90α monoclonal antibody, 1G6-D7, could attenuate lung fibrosis and whether 1G6-D7 presents a protective effect by inactivating the profibrotic pathway. Our results showed that eHsp90α was increased in mice with BLM-induced pulmonary fibrosis and that 1G6-D7 attenuated inflammation and collagen deposition in the lung. TGF-ß1 induced eHsp90α secretion, concomitantly promoting HFL-1 activation and ECM synthesis. 1G6-D7-mediated inhibition of eHsp90α significantly blocked these effects, meanwhile inhibiting downstream profibrotic pathways such as ERK, Akt, and P38. Human recombinant (hr)Hsp90α mimicked the effects of TGF-ß1, by activating profibrotic pathways and by upregulating LRP-1. Moreover, ERK inhibition effectively blocked the effect of (hr)Hsp90α. In conclusion, 1G6-D7 significantly protects against BLM-induced pulmonary fibrosis by ameliorating fibroblast activation and ECM production, which may be through blocking ERK signaling. Our results suggest a safer molecular therapy, 1G6-D7, in pulmonary fibrosis.
Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fibrose Pulmonar/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Bleomicina/efeitos adversos , Bleomicina/farmacologia , Linhagem Celular , Proteínas de Choque Térmico HSP90/imunologia , Humanos , Sistema de Sinalização das MAP Quinases/imunologia , Camundongos , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/patologia , Fator de Crescimento Transformador beta1/imunologiaRESUMO
BACKGROUND: The disruption and hyperpermeability of bronchial epithelial barrier are closely related to the pathogenesis of asthma. House dust mite (HDM), one of the most important allergens, could increase the airway epithelial permeability. Heat shock protein (Hsp) 90α is also implicated in the lung endothelial barrier dysfunction by disrupting RhoA signaling. However, the effect of extracellular Hsp90α (eHsp90α) on the bronchial epithelial barrier disruption induced by HDM has never been reported. METHODS: To investigate the involvement of eHsp90α in the bronchial epithelial barrier disruption induced by HDM, normal human bronchial epithelial cell line 16HBE14o- (16HBE) cells were treated by HDM, human recombinant (hr) Hsp90α and hrHsp90ß respectively and pretreated by1G6-D7, a specific anti-secreted Hsp90α monoclonal antibody (mAb). Hsp90α-silencing cells were also constructed. To further evaluate the role of RhoA signaling in this process, cells were pretreated by inhibitors of Rho kinase, GSK429286A and Y27632 2HCl. Transepithelial electrical resistance (TEER) and FITC-dextran flux (FITC-DX) were examined as the epithelial barrier function. Expression and localization of adherens junctional proteins E-cadherin and ß-catenin were evaluated by western blotting and immunofluorescence respectively. The level of eHsp90α was investigated by concentration and purification of condition media. RhoA activity was determined by using a Rho G-LISA® RhoA activation assay kitTM biochem kit, and the phosphorylation of myosin light chain (MLC), the downstream signal molecule of RhoA, was assessed by western blotting. RESULTS: The epithelial barrier disruption and the loss of adherens junctional proteins E-cadherin and ß-catenin in cytomembrane were observed in HDM-treated 16HBE cells, paralleled with the increase of eHsp90α secretion. All of which were rescued in Hsp90α-silencing cells or by pretreating 16HBE cells with 1G6-D7. Also, 1G6-D7 suppressed RhoA activity and MLC phosphorylation induced by HDM. Furthermore, inhibitors of Rho kinase prevented and restored the airway barrier disruption. Consistently, it was hrHsp90α instead of hrHsp90ß that promoted barrier dysfunction and activated RhoA/MLC signaling in 16HBE cells. CONCLUSIONS: The eHsp90α mediates HDM-induced human bronchial epithelial barrier dysfunction by activating RhoA/MLC signaling, suggesting that eHsp90α is a potential therapeutic target for treatment of asthma.
Assuntos
Antiasmáticos/farmacologia , Brônquios/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/farmacologia , Cadeias Leves de Miosina/metabolismo , Pyroglyphidae/imunologia , Transdução de Sinais/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Antígenos CD , Brônquios/enzimologia , Brônquios/imunologia , Caderinas/metabolismo , Linhagem Celular , Dextranos/metabolismo , Impedância Elétrica , Células Epiteliais/enzimologia , Células Epiteliais/imunologia , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Permeabilidade , Fosforilação , Interferência de RNA , Fatores de Tempo , Transfecção , beta Catenina/metabolismo , Quinases Associadas a rho/metabolismoRESUMO
A better understanding of the interaction between extrinsic factors and surface receptors on stem cells will greatly benefit stem cell research and applications. Recently, we showed that several angiopoietin-like proteins (Angptls) bind and activate the immune inhibitory receptor human leukocyte immunoglobulin (Ig)-like receptor B2 (LILRB2) to support ex vivo expansion of hematopoietic stem cells (HSCs) and leukemia development. However, the molecular basis for the interaction between Angptls and LILRB2 was unclear. Here, we demonstrate that Angptl2 expressed in mammalian cells forms high-molecular-weight species and that ligand multimerization is required for activation of LILRB2 for downstream signaling. A novel motif in the first and fourth Ig domains of LILRB2 was identified that is necessary for the receptor to be bound and activated by Angptl2. The binding of Angptl2 to LILRB2 is more potent than and not completely overlapped with the binding of another ligand, HLA-G. Immobilized anti-LILRB2 antibodies induce a more potent activation of LILRB2 than Angptl2, and we developed a serum-free culture containing defined cytokines and immobilized anti-LILRB2 that supports a net expansion of repopulating human cord blood HSCs. Our elucidation of the mode of Angptl binding to LILRB2 enabled the development of a new approach for ex vivo expansion of human HSCs.
Assuntos
Angiopoietinas/química , Angiopoietinas/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Receptores Imunológicos/química , Receptores Imunológicos/metabolismo , Proteína 2 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Angiopoietinas/genética , Animais , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Xenoenxertos , Humanos , Glicoproteínas de Membrana/genética , Camundongos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores Imunológicos/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de SinaisRESUMO
Excessive production of connective tissue growth factor (CTGF, CCN2) and increased motor ability of the activated fibroblast phenotype contribute to the pathogenesis of idiopathic pulmonary fibrosis (IPF). However, molecules and signal pathways regulating CCN2 expression and migration of lung fibroblasts are still elusive. We hypothesize that rapamycin, via binding and blocking mammalian target of rapamycin (mTOR) complex (mTORC), affects CCN2 expression and migration of lung fibroblasts in vitro. Primary normal and fibrotic human lung fibroblasts were isolated from lung tissues of three patients with primary spontaneous pneumothorax and three with IPF. Cells were incubated with regular medium, or medium containing rapamycin, human recombinant transforming growth factor (TGF)-ß1, or both. CCN2 and tissue inhibitor of metalloproteinase (TIMP)-1 expression in cells or supernatant was detected. Wound healing and migration assay was used to measure the migratory potential. TGF-ß type I receptor (TßRI)/Smad inhibitor, SB431542 and phosphoinositide 3-kinase (PI3K) inhibitor, LY294002 were used to determine rapamycin's mechanism of action. We demonstrated that rapamycin amplified basal or TGF-ß1-induced CCN2 mRNA and protein expression in normal or fibrotic fibroblasts by Smad-independent but PI3K-dependent pathway. Additionally, rapamycin also enhanced TIMP-1 expression as indicated by ELISA. However, wound healing and migrating assay showed rapamycin did not affect the mobility of fibroblasts. Collectively, this study implies a significant fibrogenic induction activity of rapamycin by activating AKT and inducing CCN2 expression in vitro and provides the possible mechanisms for the in vivo findings which previously showed no antifibrotic effect of rapamycin on lung fibrosis.
Assuntos
Fator de Crescimento do Tecido Conjuntivo/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Pulmão/citologia , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Benzamidas/farmacologia , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Fator de Crescimento do Tecido Conjuntivo/análise , Fator de Crescimento do Tecido Conjuntivo/genética , Dioxóis/farmacologia , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Fosfatidilinositol 3-Quinases/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína Smad2/metabolismo , Proteína Smad3/metabolismoRESUMO
Chemical communication is vital in organismal health, reproduction, and overall well-being. Understanding the molecular pathways, neural processes, and computations governing these signals remains an active area of research. The nematode Caenorhabditis elegans provides a powerful model for studying these processes as it produces a volatile sex pheromone. This pheromone is synthesized by virgin females or sperm-depleted hermaphrodites and serves as an attractant for males. This protocol describes a detailed method for isolating the volatile sex pheromone from several C. elegans strains (WT strain N2, daf-22, and fog-2) and C. remanei. We also provide a protocol for quantifying the male chemotaxis response to the volatile sex pheromone. Our analysis utilizes measurements such as chemotaxis index (C.I.), arrival time (A.T.), and a trajectory plot to compare male responses under various conditions accurately. This method allows for robust comparisons between males of different genetic backgrounds or developmental stages. Furthermore, the experimental setup outlined here is adaptable to investigating other chemoattraction chemicals.
Assuntos
Caenorhabditis elegans , Quimiotaxia , Atrativos Sexuais , Animais , Atrativos Sexuais/farmacologia , Atrativos Sexuais/química , Quimiotaxia/fisiologia , Quimiotaxia/efeitos dos fármacos , Masculino , FemininoRESUMO
To assess the blending effect of field snails with grass carp muscle, the effects of paramyosin (PM) and actomyosin (AM) with different mixture ratios on the gel properties of the binary blend system were investigated in our work. The purified PM from field snail muscle was about 95 kDa on SDS-PAGE. Its main secondary structure was α-helix, which reached to 97.97 %. When the amount of PM increased in the binary blend system, their rheological indices and gel strength were improved. The water holding capacity (WHC) increased to 86.30 % at a mixture ratio of 2:8. However, the WHC and the area of immobile water (P22) dramatically decreased, and the area of free water (P23) increased when the mixture ratio exceeded 4:6. The low level of PM in binary blend system promoted the formation of a homogenous and dense gel network through non-covalent interactions as observed results of SEM and FTIR. When there were redundant PM molecules, the development of heterostructure via hydrophobic interaction of tail-tail contributed to the reduced gel properties of the binary blend system. These findings provided new insight into the binary blend system of PM and AM with different ratios to change the gel properties of myofibrillar protein.