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AIMS: The relationship between the p38MAPK signaling pathway and osterix in osteogenic differentiation of BMMSCs subjected to intermittent stretching was investigated. METHODS: BMMSCs derived from C57BL/6J mice were divided into the following groups: 1) control, 2) stretch, and 3) SB203580+stretch (SB203580 is a p38MAPK signal pathway inhibitor). BMMSCs were exposed to an intermittent mechanical strain of 0.8% (8000µ strain) at 0.5 Hz, twice a day for 30 min each application. BMMSCs were harvested on days 1, 3, and 5 post-treatment. The expression of ALP, COL I, OCN, and osterix mRNA was assessed utilizing RT-PCR while the expression of P-p38MAPK and osterix protein was assessed by Western blot analysis. The osterix gene in mouse BMMSCs was knocked down using RNAi technology and its protein expression was also assessed by Western blot. RT-PCR was used to detect ALP, COL I, and OCN mRNA expression. RESULTS: Intermittent stretching was found to promote expression of ALP, COL I, OCN, and osterix mRNA. Silencing the osterix gene was found to reduce levels of ALP, COL I, and OCN mRNA. Western blot analysis demonstrated that the levels of osterix and P-p38MAPK proteins in the stretch group were significantly higher than in the control group (P<0.05). There was less expression of ALP, COL I, OCN, and osterix mRNA in the SB203580+stretch group than in the control and stretch groups. CONCLUSIONS: Data demonstrate that intermittent stretching promotes osteogenic differentiation of BMMSCs, and the p38MAPK-osterix pathway has an important role in the control of osteogenesis-related gene expression.
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Regulação da Expressão Gênica , Mecanotransdução Celular/genética , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Fatores de Transcrição/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Adesão Celular , Diferenciação Celular , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Masculino , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/citologia , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogênese/genética , Cultura Primária de Células , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fator de Transcrição Sp7 , Estresse Mecânico , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
BACKGROUND: Eukaryotic mitogen-activated protein kinase (MAPK/MPK) signaling cascades transduce and amplify environmental signals via three types of reversibly phosphorylated kinases to activate defense gene expression. Canola (oilseed rape, Brassica napus) is a major crop in temperate regions. Identification and characterization of MAPK and MAPK kinases (MAPKK/MKK) of canola will help to elucidate their role in responses to abiotic and biotic stresses. RESULTS: We describe the identification and analysis of seven MKK (BnaMKK) and 12 MPK (BnaMPK) members from canola. Sequence alignments and phylogenetic analyses of the predicted amino acid sequences of BnaMKKs and BnaMPKs classified them into four different groups. We also examined the subcellular localization of four and two members of BnaMKK and BnaMPK gene families, respectively, using green fluorescent protein (GFP) and, found GFP signals in both nuclei and cytoplasm. Furthermore, we identified several interesting interaction pairs through yeast two-hybrid (Y2H) analysis of interactions between BnaMKKs and BnaMPKs, as well as BnaMPK and BnaWRKYs. We defined contiguous signaling modules including BnaMKK9-BnaMPK1/2-BnaWRKY53, BnaMKK2/4/5-BnaMPK3/6-BnaWRKY20/26 and BnaMKK9-BnaMPK5/9/19/20. Of these, several interactions had not been previously described in any species. Selected interactions were validated in vivo by a bimolecular fluorescence complementation (BiFC) assay. Transcriptional responses of a subset of canola MKK and MPK genes to stimuli including fungal pathogens, hormones and abiotic stress treatments were analyzed through real-time RT-PCR and we identified a few of BnaMKKs and BnaMPKs responding to salicylic acid (SA), oxalic acid (OA), Sclerotinia sclerotiorum or other stress conditions. Comparisons of expression patterns of putative orthologs in canola and Arabidopsis showed that transcript expression patterns were generally conserved, with some differences suggestive of sub-functionalization. CONCLUSIONS: We identified seven MKK and 12 MPK genes from canola and examined their phylogenetic relationships, transcript expression patterns, subcellular localization, and protein-protein interactions. Not all expression patterns and interactions were conserved between canola and Arabidopsis, highlighting the limitations of drawing inferences about crops from model species. The data presented here provide the first systematic description of MKK-MPK-WRKY signaling modules in canola and will further improve our understanding of defense responses in general and provide a basis for future crop improvement.
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Brassica napus/enzimologia , Brassica napus/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Sequência de Aminoácidos , Ascomicetos/fisiologia , Brassica napus/microbiologia , Brassica napus/fisiologia , Clonagem Molecular , Espaço Intracelular/metabolismo , Proteínas Quinases Ativadas por Mitógeno/química , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Filogenia , Estrutura Terciária de Proteína , Transporte Proteico , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Estresse Fisiológico/genética , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic has become an immense public health burden, first in China and subsequently worldwide. Developing effective control measures for COVID-19, especially measures that can halt the worsening of severe cases to a critical status is of urgent importance. CASE SUMMARY: A 52-year-old woman presented with a high fever (38.8 °C), chills, dizziness, and weakness. Epidemiologically, she had not been to Wuhan where COVID-19 emerged and did not have a family history of a disease cluster. A blood test yielded a white blood cell count of 4.41 × 109/L (60.6 ± 2.67% neutrophils and 30.4 ± 1.34% lymphocytes). Chest imaging revealed bilateral ground-glass lung changes. Based on a positive nasopharyngeal swab nucleic acid test result and clinical characteristics, the patient was diagnosed with COVID-19. Following treatment with early non-invasive ventilation and a bundle pharmacotherapy, she recovered with a good outcome. CONCLUSION: Early non-invasive ventilation with a bundle pharmacotherapy may be an effective treatment regimen for the broader population of patients with COVID-19.
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BACKGROUND: Early detection of suspected critical patients infected with coronavirus disease 2019 (COVID-19) is very important for the treatment of patients. This study aimed to investigate the role of COVID-19 associated coagulopathy (CAC) to preview and triage. METHODS AND RESULTS: A cohort study was designed from government designated COVID-19 treatment center. CAC was defined as International Society on Thrombosis and Haemostasis (ISTH) score ≥2. Data from 117 patients COVID-19 were reviewed on admission. The primary and secondary outcomes were admission to Intensive Care Unit (ICU), the use of mechanical ventilation, vital organ dysfunction, discharges of days 14, 21 and 28 from admission and hospital mortality. Among them, admission to ICU was increased progressively from 16.1% in patients with non-CAC to 42.6% in patients with CAC (P < 0.01). Likely, invasive ventilation and noninvasive ventilation were increased from 1.8%, 21.4% in patients with non-CAC to 21.3%, 52.5% in patients with CAC, respectively (P < 0.01). The incidences of acute hepatic injury and acute respiratory distress syndrome in non-CAC and CAC were 28.6% vs. 62.3%, 8.9% vs. 27.9%, respectively (P < 0.01). The discharges of days 14, 21 and 28 from admission were more in non-CAC than those of CAC (P < 0.05). Multiple logistic regression results showed that ISTH score ≥2 was obviously associated with the admission to ICU (OR 4.07, 95% CI 1.47-11.25 P = 0.007) and the use of mechanical ventilation (OR 5.54, 95% CI 2.01-15.28 P = 0.001) in patients with COVID-19. CONCLUSION: All results show ISTH score ≥2 is an important indicator to preview and triage for COVID-19 patients.
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Infecções por Coronavirus/complicações , Coagulação Intravascular Disseminada/etiologia , Pneumonia Viral/complicações , Adulto , Idoso , Idoso de 80 Anos ou mais , Betacoronavirus/isolamento & purificação , COVID-19 , China/epidemiologia , Infecções por Coronavirus/sangue , Infecções por Coronavirus/terapia , Coagulação Intravascular Disseminada/sangue , Coagulação Intravascular Disseminada/terapia , Feminino , Hospitalização , Humanos , Unidades de Terapia Intensiva , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/sangue , Pneumonia Viral/terapia , Respiração Artificial , Estudos Retrospectivos , SARS-CoV-2 , Resultado do TratamentoRESUMO
BACKGROUND: Patients with critical coronavirus disease 2019 (COVID-19), characterized by respiratory failure requiring mechanical ventilation (MV), are at high risk of mortality. An effective and practical MV weaning protocol is needed for these fragile cases. CASE SUMMARY: Here, we present two critical COVID-19 patients who presented with fever, cough and fatigue. COVID-19 diagnosis was confirmed based on blood cell counts, chest computed tomography (CT) imaging, and nuclei acid test results. To address the patients' respiratory failure, they first received noninvasive ventilation (NIV). When their condition did not improve after 2 h of NIV, each patient was advanced to MV [tidal volume (Vt), 6 mL/kg ideal body weight (IBW); 8-10 cmH2O of positive end-expiratory pressure; respiratory rate, 20 breaths/min; and 40%-80% FiO2] with prone positioning for 12 h/day for the first 5 d of MV. Extensive infection control measures were conducted to minimize morbidity, and pharmacotherapy consisting of an antiviral, immune-enhancer, and thrombosis prophylactic was administered in both cases. Upon resolution of lung changes evidenced by CT, the patients were sequentially weaned using a weaning screening test, spontaneous breathing test, and airbag leak test. After withdrawal of MV, the patients were transitioned through NIV and high-flow nasal cannula oxygen support. Both patients recovered well. CONCLUSION: A MV protocol attentive to intubation/extubation timing, prone positioning early in MV, infection control, and sequential withdrawal of respiratory support, may be an effective regimen for patients with critical COVID-19.
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The ventricular septal defect (VSD) is the most common congenital heart defect and no candidate susceptibility gene has been identified. Endocardial cushion and outflow septal morphogenesis, malalignment of which induces VSD, have been suggested to be mediated by the vascular endothelial growth factor (VEGF). Three single-nucleotide polymorphism (SNP) variants in promoter and 5'-UTR region of the VEGF gene, C-2578A (rs699947), G-1154A (rs1570360) and G-634C (rs2010963), were reported to alter its expression. We assessed the association in a Chinese population between these SNPs and VSD using a double approach: case-control and TDT designs. Among the three SNPs, only -634C allele was less frequently present in 222 patients compared to 352 controls (odds ratio: 0.76, 95% CI: 0.59-0.97, X(2)=5.06, P=0.024, not significant after a Bonferroni correction). This was significantly less transmitted to VSD patients (trios: 142) (odds ratio: 0.39, 95% CI: 0.25-0.62, X(2)=8.11, df=1, P=0.004, corrected P=0.024). A similar result was observed for haplotype -2578C/-1154G/-634C allele in both studies (in TDT: X(2)=7.51, df=1, P=0.006, corrected P=0.048). All these associations for the first time demonstrated that -634C allele was in a significant protective association against VSD, suggesting that VEGF dysregulation was involved in the pathological processes of VSD.
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Citosina , Guanina , Comunicação Interventricular/genética , Comunicação Interventricular/prevenção & controle , Desequilíbrio de Ligação/genética , Polimorfismo de Nucleotídeo Único/genética , Fator A de Crescimento do Endotélio Vascular/genética , Adolescente , Alelos , Estudos de Casos e Controles , Criança , Pré-Escolar , Família , Feminino , Haplótipos , Humanos , Lactente , Masculino , Fator A de Crescimento do Endotélio Vascular/sangueRESUMO
OBJECTIVE: To investigate the association between the single nucleotide polymorphisms (SNPs) of cluster of differentiation 14 (CD14) and susceptibility in patients with sepsis. METHODS: One hundred and twelve patients with sepsis admitted to the intensive care unit (ICU) from December 2004 to March 2006 were enrolled for study. Another 100 healthy volunteer donors were chosen serving as the normal control group. Genotypes and alleles of CD14-159 C/T, CD14-260 C/T were determined in patients and healthy controls by means of restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction (PCR) products. Distributional differences of genotypes and alleles between patients and healthy controls were analyzed. The sepsis patients were divided into survivor group and non-survivor group depending on survival for 28 days. Distributional differences of genotypes and alleles between survivor group and non-survivor group were determined. RESULTS: The rates of CD14-159 C/T genotypes and T-alleles in patients with sepsis were significantly higher than those of healthy volunteers (P=0.005 and P=0.000). There were no significant differences in the rates of CD14-260 C/T genotypes and T-alleles in two groups (P=0.912 and P=0.706). The rates of CD14 -159 C/T genotypes and T-alleles in non-survivors were significantly higher than those of survivors (both P=0.000). There were no significant differences in the rates of CD14-260 C/T genotypes and T-alleles in two groups (P=0.643 and P=0.890). CONCLUSION: The differences of CD14-159 C/T genotypes of SNPs may be associated with the higher risk of susceptibility and mortality of sepsis.
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Predisposição Genética para Doença , Receptores de Lipopolissacarídeos/genética , Polimorfismo de Nucleotídeo Único , Sepse/genética , Adolescente , Adulto , Idoso , Alelos , Estudos de Casos e Controles , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Fragmento de Restrição , Adulto JovemRESUMO
Side population (SP) cells, characterized by their ability to efflux the fluorescent dye Hoechst 33342, were isolated from the small intestine of mice. In the abdominal irradiation model, small intestinal organoid-derived SP (sioSP) cells from ROSA 26 mice were submucosally injected into the small intestinal of the irradiated C57BL/6 mice. In contrast to the control mice, mice receiving sioSP cell transplantation demonstrated far less skin injury. Most importantly, hairs in the irradiated body part of the transplanted mice almost remained black, whereas the counterpart in the control mice almost turned white. Histochemistry studies showed the donor cells gave rise to skin cells in the irradiated skin. Thus, our study demonstrated for the first time that stem cells from the small intestine can differentiate into skin cells under local cues and thus supports the theory of stem cell plasticity.
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Transplante de Células/métodos , Intestino Delgado/citologia , Lesões por Radiação/terapia , Pele/lesões , Transplante Homólogo/fisiologia , Animais , Animais Recém-Nascidos , Antígenos de Diferenciação/análise , Modelos Animais de Doenças , Intestino Delgado/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos EndogâmicosRESUMO
AIM: To investigate IL-1beta+3 594 in the 5th intron, IL-10-1 082 and CD14-159 polymorphisms in patients with acute pancreatitis (AP) and septic shock. METHODS: The study included 215 patients (109 with acute severe pancreatitis (SAP), 106 with acute mild pancreatitis (MAP)) and 116 healthy volunteers. Genomic DNA was prepared from peripheral blood leukocytes. Genotypes and allele frequencies were determined in patients and healthy controls using restriction fragment length polymorphism analysis of PCR products. RESULTS: The frequencies of IL-1beta+3 594T, IL-10-1082G and CD14-159T allele were similar in patients with mild or severe pancreatitis and in controls. Within SAP patients, no significant differences were found in the allele distribution examined when etiology was studied again. Patients with septic shock showed a significantly higher prevalence of IL-10-1082G allele than those without shock (chi2= 5.921, P = 0.015). CONCLUSION: IL-10-1082G plays an important role in the susceptibility of SAP patients to septic shock. Genetic factors are not important in determination of disease severity or susceptibility to AP.
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Interleucina-10/genética , Interleucina-1/genética , Receptores de Lipopolissacarídeos/genética , Pancreatite/genética , Choque Séptico/genética , Doença Aguda , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Polimorfismo Genético , Índice de Gravidade de DoençaRESUMO
OBJECTIVE: To explore the change in the protein content and procoagulant activity of platelet-associated tissue factor (TF) in thrombotic diseases. METHODS: Platelets were isolated with Sepharose 2B gel column. ELISA was employed to detect the TF protein content in the lysates of washed platelets, procoagulant activity of platelet-associated TF was measured with one stage clotting time assay. RESULTS: A certain amount of TF antigen (16.37 +/- 6.39) ng/L was detected in resting platelets in normal controls. The procoagulant activity of resting platelets and platelets activated by collagen were (0.89 +/- 0.26), (2.27 +/- 0.24) U/ml respectively. While specific TF McAb was added, the procoagulant activity of activated platelets can be markedly inhibited (1.39 +/- 0.28) U/ml (P < 0.001). It is thus proved that the procoagulant activity of activated platelets mainly comes from TF. TF protein content of the lysates of washed platelets in coronary heart disease group, brain infarction group and diabetes mellitus group was (20.71 +/- 8.78), (23.83 +/- 8.03), (22.12 +/- 8.24) ng/L respectively. The procoagulant activity of platelets without collagen treatment in these thrombotic diseases was (1.71 +/- 0.24), (1.69 +/- 0.25), (1.75 +/- 0.31) U/ml respectively. When specific TF McAb was added specific TF McAb, the procoagulant activity was (1.43 +/- 0.25), (1.39 +/- 0.25), (1.40 +/- 0.29) U/ml respectively. The TF protein content of lysates of washed platelets and the procoagulant activity of platelets without collagen treatment in patients with thrombotic diseases were significantly higher than those in normal controls. This procoagulant activity can be inhibited by TF McAb (P < 0.01). CONCLUSION: The change in platelet-associated TF in thrombotic diseases may contribute to the maintenance of hypercoagulability.
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Plaquetas/metabolismo , Tromboplastina/metabolismo , Trombose/sangue , Adolescente , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ativação Plaquetária , Trombose/fisiopatologiaRESUMO
OBJECTIVE: To investigate the influence and its significance of expression of Toll-like receptor mRNA in lung tissue of mice with hemorrhagic shock without resuscitation. METHODS: Forty-five C57BL/6 mice were randomly divided into hemorrhagic shock group, lipopolysaccharide (LPS) group (positive control, 5 mg/kg LPS was injected into caudal vein), and sham-operation group (negative control), with 15 mice in each group. The hemorrhagic shock model was reproduced by heart puncture. The total RNA from lung tissue of mice in each group was extracted by Tripure reagent and the expression of Toll-like receptor 2 (TLR2) and TLR4 mRNA were measured by semi-quantitative reverse transcription-polymerase chain reaction. RESULTS: Hemorrhagic shock and LPS challenge increased lung neutrophil infiltration and erythrocyte diapedesis. The expression of TLR2 mRNA and TLR4 mRNA could be found in normal lung tissues, but they were increased at 0, 1, 2, 4, and 6 hours after hemorrhagic shock and LPS challenge. There was no change in the sham-operation group. CONCLUSION: The increased expression of TLR2 and TLR4 gene in lung tissue after hemorrhagic shock is closely correlated with the onset of acute lung injury. Though the innate immunity is enhanced, the animals are more susceptible to subsequent irritations. Over-expression of TLR2 and TLR4 may induce structural and functional damage to organs.
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Pulmão/metabolismo , Choque Hemorrágico/metabolismo , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , Distribuição Aleatória , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/genéticaRESUMO
AIM: To investigate TNF-alpha-308 and TNFB polymorphisms in acute biliary pancreatitis (ABP) and to related them to the plasma TNF-alpha levels. METHODS: Genomic DNA was prepared from peripheral blood leukocytes. Genotypes and allele frequencies were determined in patients (n=127) and healthy controls (n=102) using restriction fragment length polymorphism analysis of polymerase chain reaction (PCR) products. Reading the size of digested bands from polyacrylamide gel demonstrated the two alleles TNF1 and TNF2, or the two alleles TNFB1 and TNFB2. RESULTS: The frequencies of TNF2 polymorphism and TNFB2 polymorphism were both similar in patients with mild or severe pancreatitis, so were in pancreatitis patients and in controls. Patients with septic shock showed a significantly higher prevalence of the TNF2 than those without. No significant differences were found in the genotype distribution of TNF-alpha-308 and TNFB among different groups. Plasma TNF-alpha levels did not differ significantly in ASBP patients displaying different alleles of the TNF gene studied. CONCLUSION: Results indicate that TNF gene polymorphisms studied play no part in determination of disease severity or susceptibility to acute biliary pancreatitis; however, TNF2 polymorphism is associated with septic shock from ASBP. Genetic factors are not important in determining plasma TNF-alpha levels in ASBP.
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Linfotoxina-alfa/genética , Pancreatite/genética , Polimorfismo Genético , Fator de Necrose Tumoral alfa/genética , APACHE , Doença Aguda , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Pancreatite/fisiopatologia , Mapeamento por RestriçãoRESUMO
OBJECTIVE: To investigate the association of Single nucleotide polymorphism (SNPs) tumor necrosis factor (TNF) and CD14 promoter with the systematic inflammatory response syndromec (SIRS) and sepsis in surgical patients. METHODS: The DNA and RNA sample of PBMC from 113 patients, 40 of them being complicated with sepsis, and 100 healthy volunteers were extracted. The SNP genotypes of TNF-alpha -308 G/A, -863 C/A, CD14-159C/T and TNFB1/B2 were examined by restriction fragment length polymorphism PCR (PCR-RFLP). The expressions of TNF-alpha mRNA of PBMC in parts of the patients who have at least one genotype of SNP were detected by RT-PCR. The risks for sepsis associated with polymorphisms in the TNF-alpha or CD14 promoter were determined by multivariate analysis. RESULTS: The rates of TNF2, -863A, CD14-159T alleles were 15%, 32.5%, and 40% respectively in patients with sepsis, significantly higher than those in the patients with SIRS (8.9%, 22%, and 23.3%), and those in the healthy volunteers (5%, 16% and 26%). The expression of TNF-alpha mRNA was much higher in those patients with at least one kind of SNP than those without SNP. CONCLUSION: The A-allele at the -308 and -863 position in the TNF-alpha promoter and the T-allele at the -159 position in the CD14 promoter increase the risk for sepsis. The effect of SNP genotypes on TNF-alpha expression can modulate inflammatory response.