RESUMO
BACKGROUND: Abdominal aortic aneurysm (AAA) is a catastrophic disease with little effective therapy, likely due to the limited understanding of the mechanisms underlying AAA development and progression. ATF3 (activating transcription factor 3) has been increasingly recognized as a key regulator of cardiovascular diseases. However, the role of ATF3 in AAA development and progression remains elusive. METHODS: Genome-wide RNA sequencing analysis was performed on the aorta isolated from saline or Ang II (angiotensin II)-induced AAA mice, and ATF3 was identified as the potential key gene for AAA development. To examine the role of ATF3 in AAA development, vascular smooth muscle cell-specific ATF3 knockdown or overexpressed mice by recombinant adeno-associated virus serotype 9 vectors carrying ATF3, or shRNA-ATF3 with SM22α (smooth muscle protein 22-α) promoter were used in Ang II-induced AAA mice. In human and murine vascular smooth muscle cells, gain or loss of function experiments were performed to investigate the role of ATF3 in vascular smooth muscle cell proliferation and apoptosis. RESULTS: In both Ang II-induced AAA mice and patients with AAA, the expression of ATF3 was reduced in aneurysm tissues but increased in aortic lesion tissues. The deficiency of ATF3 in vascular smooth muscle cell promoted AAA formation in Ang II-induced AAA mice. PDGFRB (platelet-derived growth factor receptor ß) was identified as the target of ATF3, which mediated vascular smooth muscle cell proliferation in response to TNF-alpha (tumor necrosis factor-α) at the early stage of AAA. ATF3 suppressed the mitochondria-dependent apoptosis at the advanced stage by upregulating its direct target BCL2. Our chromatin immunoprecipitation results also demonstrated that the recruitment of NFκB1 and P300/BAF/H3K27ac complex to the ATF3 promoter induces ATF3 transcription via enhancer activation. NFKB1 inhibitor (andrographolide) inhibits the expression of ATF3 by blocking the recruiters NFKB1 and ATF3-enhancer to the ATF3-promoter region, ultimately leading to AAA development. CONCLUSIONS: Our results demonstrate a previously unrecognized role of ATF3 in AAA development and progression, and ATF3 may serve as a novel therapeutic and prognostic marker for AAA.
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Fator 3 Ativador da Transcrição , Aneurisma da Aorta Abdominal , Músculo Liso Vascular , Miócitos de Músculo Liso , Fator 3 Ativador da Transcrição/genética , Fator 3 Ativador da Transcrição/metabolismo , Animais , Aneurisma da Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/induzido quimicamente , Humanos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Camundongos , Masculino , Camundongos Endogâmicos C57BL , Apoptose , Células Cultivadas , Angiotensina II , Proliferação de Células , Aorta Abdominal/patologia , Aorta Abdominal/metabolismo , Modelos Animais de DoençasRESUMO
BACKGROUND: Aberrant expression of circular RNA contributes to human diseases. Circular RNAs regulate gene expression by sequestering specific microRNAs. In this study, we investigated whether circMAP3K5 (circular mitogen-activated protein kinase 5) could act as a competing endogenous microRNA-22-3p (miR-22-3p) sponge and regulate neointimal hyperplasia. METHODS: Circular RNA profiling from genome-wide RNA sequencing data was compared between human coronary artery smooth muscle cells (SMCs) treated with or without platelet-derived growth factor. Expression levels of circMAP3K5 were assessed in human coronary arteries from autopsies on patients with dilated cardiomyopathy or coronary heart disease. The role of circMAP3K5 in intimal hyperplasia was further investigated in mice with adeno-associated virus 9-mediated circMAP3K5 transfection. SMC-specific Tet2 (ten-eleven translocation-2) knockout mice and global miR-22-3p knockout mice were used to delineate the mechanism by which circMAP3K5 attenuated neointimal hyperplasia using the femoral arterial wire injury model. RESULTS: RNA sequencing demonstrated that treatment with platelet-derived growth factor-BB significantly reduced expression of circMAP3K5 in human coronary artery SMCs. Wire-injured mouse femoral arteries and diseased arteries from patients with coronary heart disease (where platelet-derived growth factor-BB is increased) confirmed in vivo downregulation of circMAP3K5 associated with injury and disease. Lentivirus-mediated overexpression of circMAP3K5 inhibited the proliferation of human coronary artery SMCs. In vivo adeno-associated virus 9-mediated transfection of circMap3k5 (mouse circular Map3k5) specifically inhibited SMC proliferation in the wire-injured mouse arteries, resulting in reduced neointima formation. Using a luciferase reporter assay and RNA pull-down, circMAP3K5 (human circular MAP3K5) was found to sequester miR-22-3p, which, in turn, inhibited the expression of TET2. Both in vitro and in vivo results demonstrate that the loss of miR-22-3p recapitulated the antiproliferative effect of circMap3k5 on vascular SMCs. In SMC-specific Tet2 knockout mice, loss of Tet2 abolished the circMap3k5-mediated antiproliferative effect on vascular SMCs. CONCLUSIONS: We identify circMAP3K5 as a master regulator of TET2-mediated vascular SMC differentiation. Targeting the circMAP3K5/miR-22-3p/TET2 axis may provide a potential therapeutic strategy for diseases associated with intimal hyperplasia, including restenosis and atherosclerosis.
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Proteínas de Ligação a DNA/metabolismo , Dioxigenases/metabolismo , MicroRNAs/metabolismo , Miócitos de Músculo Liso/patologia , RNA Circular/metabolismo , Túnica Íntima/metabolismo , Animais , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Diferenciação Celular/fisiologia , Modelos Animais de Doenças , Feminino , Humanos , Hiperplasia/metabolismo , Hiperplasia/patologia , Masculino , Camundongos , Camundongos Knockout , MicroRNAs/genética , Miócitos de Músculo Liso/metabolismo , RNA Circular/genética , Túnica Íntima/patologiaRESUMO
RATIONALE: Kawasaki disease (KD) is an acute vasculitis of early childhood that can result in permanent coronary artery structural damage. The cause for this arterial vulnerability in up to 15% of patients with KD is unknown. Vascular smooth muscle cell dedifferentiation play a key role in the pathophysiology of medial damage and aneurysm formation, recognized arterial pathology in KD. Platelet hyperreactivity is also a hallmark of KD. We recently demonstrated that uptake of platelets and platelet-derived miRNAs influences vascular smooth muscle cell phenotype in vivo. OBJECTIVE: We set out to explore whether platelet/vascular smooth muscle cell (VSMC) interactions contribute to coronary pathology in KD. METHODS AND RESULTS: We prospectively recruited and studied 242 patients with KD, 75 of whom had documented coronary artery pathology. Genome-wide miRNA sequencing and droplet digital PCR demonstrated that patient with KD platelets have significant induction of miR-223 compared with healthy controls (HCs). Platelet-derived miR-223 has recently been shown to promote vascular smooth muscle quiescence and resolution of wound healing after vessel injury. Paradoxically, patients with KD with the most severe coronary pathology (giant coronary artery aneurysms) exhibited a lack of miR-223 induction. Hyperactive platelets isolated from patients with KD are readily taken up by VSMCs, delivering functional miR-223 into the VSMCs promoting VSMC differentiation via downregulation of PDGFRß (platelet-derived growth factor receptor ß). The lack of miR-223 induction in patients with severe coronary pathology leads to persistent VSMC dedifferentiation. In a mouse model of KD (Lactobacillus casei cell wall extract injection), miR-223 knockout mice exhibited increased medial thickening, loss of contractile VSMCs in the media, and fragmentation of medial elastic fibers compared with WT mice, which demonstrated significant miR-223 induction upon Lactobacillus casei cell wall extract challenge. The excessive arterial damage in the miR-223 knockout could be rescued by adoptive transfer of platelet, administration of miR-223 mimics, or the PDGFRß inhibitor imatinib mesylate. Interestingly, miR-223 levels progressively increase with age, with the lowest levels found in <5-year-old. This provides a basis for coronary pathology susceptibility in this very young cohort. CONCLUSIONS: Platelet-derived miR-223 (through PDGFRß inhibition) promotes VSMC differentiation and resolution of KD induced vascular injury. Lack of miR-223 induction leads to severe coronary pathology characterized by VSMC dedifferentiation and medial damage. Detection of platelet-derived miR-223 in patients with KD (at the time of diagnosis) may identify patients at greatest risk of coronary artery pathology. Moreover, targeting platelet miR-223 or VSMC PDGFRß represents potential therapeutic strategies to alleviate coronary pathology in KD. Graphic Abstract: A graphic abstract is available for this article.
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Plaquetas/metabolismo , Doença da Artéria Coronariana/etiologia , MicroRNAs/sangue , MicroRNAs/metabolismo , Síndrome de Linfonodos Mucocutâneos/complicações , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Adulto , Fatores Etários , Animais , Estudos de Casos e Controles , Células Cultivadas , Criança , Pré-Escolar , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/metabolismo , Doença da Artéria Coronariana/patologia , Vasos Coronários/metabolismo , Vasos Coronários/patologia , Modelos Animais de Doenças , Feminino , Humanos , Lactente , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , Síndrome de Linfonodos Mucocutâneos/sangue , Síndrome de Linfonodos Mucocutâneos/diagnóstico , Síndrome de Linfonodos Mucocutâneos/genética , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Ativação Plaquetária , Estudos Prospectivos , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Índice de Gravidade de Doença , Transdução de Sinais , Adulto JovemRESUMO
BACKGROUND: The insulin-like growth factor (IGF) pathway was involved in the occurrence of spontaneous preterm birth (SPTB), but little is known regarding the relationship between genetic variations in IGF pathway and the risk of SPTB. We aimed to investigate the associations of IGF1 rs972936 and IGF1 receptor (IGF1R) rs2229765 polymorphisms with SPTB risk in a Chinese population. METHOD: A total of 114 cases of SPTB and 250 controls of term delivery were included from Guangzhou Women and Children's Medical Center, China. The odds ratios (ORs) and the corresponding 95% confidence intervals (CIs) were calculated using multivariate logistic regression. RESULTS: We found that the GA and GA/AA genotypes of IGF1 rs972936 were associated with an increased risk of SPTB, and the adjusted ORs (95% CI) were 1.74 (1.01-3.02) and 1.75 (1.04-2.93) respectively. Women carrying GA and GA/AA genotypes of IGF1R rs2229765 had a reduced risk compared to those with the GG genotype (0.60 [0.37-0.98] and 0.64 [0.40-1.00] respectively). There were significant interactions between IGF1 rs972936 and GDM status (P for interaction=.02), as well as between IGF1R rs2229765 and pre-pregnancy BMI (P for interaction <.001) on the risk of SPTB. CONCLUSION: Our findings suggest that polymorphisms of IGF1 rs972936 and IGF1R rs2229765 were associated with the risk of SPTB in Chinese pregnant women and these effects depend on the maternal metabolic status.
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Fator de Crescimento Insulin-Like I/genética , Polimorfismo de Nucleotídeo Único/genética , Nascimento Prematuro/epidemiologia , Nascimento Prematuro/genética , Receptor IGF Tipo 1/genética , Adulto , Estudos de Casos e Controles , China , Feminino , Humanos , Gravidez , Resultado da Gravidez/epidemiologiaRESUMO
Extracellular matrix (ECM) hydrogels are used as scaffolds to facilitate the repair and reconstruction of tissues. This study aimed to optimize the decellularization process of porcine skeletal muscle ECM and to formulate a matrix hydrogel scaffold. Five multi-step methods (methods A-E) were used to generate acellular ECM from porcine skeletal muscle [rinsing in SDS, trypsin, ethylenediaminetetraacetic acid (EDTA), Triton X-100 and/or sodium deoxycholate at 4-37°C]. The resulting ECM was evaluated using haematoxylin and eosin, 4-6-diamidino-2-phenylindole (DAPI) staining, and DNA quantification. Acellular matrix was dissolved in pepsin and gelled at 37°C. Hydrogel response to temperature was observed in vivo and in vitro. ECM components were assessed by Masson, Sirius red, and alcian blue staining, and total protein content. Acellular porcine skeletal muscle exhibited a uniform translucent white appearance. No intact nuclear residue was detected by haematoxylin and eosin staining, while DAPI staining showed a few nuclei in the matrixes produced by methods B, C, and D. Method A generated a gel that was too thin for gelation. However, the matrix obtained by rinsing in 0.2% trypsin/0.1% EDTA, 0.5% Triton X-100, and 1% Triton X-100/0.2% sodium deoxycholate was nuclei-free and produced a viscous solution that formed a structurally stable white jelly-like hydrogel. The residual DNA content of this solution was 49.37 ± 0.72 ng/mg, significantly less than in fresh skeletal muscle, and decreased to 19.22 ± 0.85 ng/mg after gelation (P < 0.05). The acellular matrix was rich in collagen and glycosaminoglycan, with a total protein concentration of 64.8 ± 6.9%. An acellular ECM hydrogel from porcine skeletal muscle was efficiently produced.
Assuntos
Matriz Extracelular/química , Hidrogéis/química , Músculo Esquelético/química , Alicerces Teciduais , Animais , Sobrevivência Celular/fisiologia , Colágeno/química , DNA/química , Ácido Desoxicólico/química , Ácido Edético/química , Glicosaminoglicanos/química , Indóis , Camundongos , Células NIH 3T3 , Octoxinol/química , Transição de Fase , Dodecilsulfato de Sódio/química , Suínos , Temperatura , Engenharia Tecidual , Tripsina/químicaRESUMO
OBJECTIVE: To study the high risk factors of the third level of lymphatic metastasis in breast cancer patients to guide clinical practice. METHODS: The clinical data of 746 breast cancer patients (all female, aged from 33 to 80 years with a median of 46 years) received radical or modified mastectomy between 2001 and 2011 was analyzed retrospectively. Eleven individual variables were selected to investigate high risk factors of the third level of lymphatic metastasis in different conditions. RESULTS: Axillary nodes metastasis status (OR = 4.541, 95%CI:3.569-5.776), tumor site (OR = 1.437, 95%CI:1.029-2.007), external nodes involved (OR = 3.809, 95%CI:1.683-8.618) and estrogen receptor (OR = 0.740, 95%CI:0.569-0.964) were high risk factors of the third level of lymphatic metastasis. Further analysis found that it is prone to happen a metastasis, especially when the tumor with a size over 5 cm and located at the lateral quadrant. Negative estrogen receptor was a risk factor of the third level lymphatic metastasis along with the tumor stage. CONCLUSION: For preoperative tumor biopsy shows Negative estrogen receptor of tumor stage T3 and over stage T3 when considering suspicious lymph node metastasis or external tissues metastasis intraoperatively should take in account into third level axillary lymph node dissection actively.
Assuntos
Neoplasias da Mama/patologia , Neoplasias da Mama/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Axila/patologia , Feminino , Humanos , Excisão de Linfonodo , Linfonodos/patologia , Metástase Linfática , Mastectomia/métodos , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Fatores de RiscoRESUMO
OBJECTIVE: To study the relationship between SIRT1 and glaucoma trabecular meshwork cell (GTM) cell on DNA double-strand breaks (DSBs) repair capability and resist cellular senescence. METHODS: The expressions of SIRT1 in GTM and normal trabecular meshwork (HTM) cell detected by RT-RCR and Western blot; HTM and GTM cells divided into four groups separately: Res group (treat cells with 0.5 micromol/L Resveratrol for 24 h), SIRT1-ShRNA group (cells infected with recombinant SIRT1-ShRNA), microRNA34a group (cells infected with recombinant microRNA34a) and control group. The expression level of SIRT1 in groups was detected by Western blot. SA-beta-Gal staining was applied to each group of cells at 10 h, 32 h, 3 d and 6 d to evaluate the senescence of the cells. DSBs and the expression of gamma-H2AX after treated with 1.33 mol/L H2O2 at 0 h, 1 h, 2 h were detected by comet electrophoresis and Western blot. RESULTS: The expression of SIRT1 were observed in both HTM and GTM cells, but the expression level in HTM was higher than that of GTM cells have the ability to express SIRT1, however the expression of SIRT1 was lower than HTM. Expression levels of SIRT1 presented following treads: Res > Control > microRNA34a > SIRT1-ShRNA. The dgree of senescence in GTM was higher than that in HTM cells when detected at the same time point with SA-beta-Gal staining. In the same cell line, the signs of senescence were appeared firstly and seriously in the cells treated with SIRT1-ShRNA in a time-dependent manner. Differently, after 24 h treatment with Res, the degree of senescence was decreased. The DSBs in GTM group was more than that of HTM group after treatment with oxidant when detected with Comet Electrophoresis and the the trends of the change was SIRT1-ShRNA > microRNA34a > Control > Res. The similar results also observed in the expression of gamma-H2AX. CONCLUSION: SIRT1 may be useful in predicting the development and prognosis of glaucoma; Res promotes the expression of SIRT1 significantly, and the SIRT1 may protects GTM from oxidative stress-induced DSBs, aging even apoptosis, and promotes cell cycle arrest, which may provide a new target to treat glaucoma.
Assuntos
Senescência Celular , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Sirtuína 1/metabolismo , Malha Trabecular/citologia , Células Cultivadas , Glaucoma , Humanos , Peróxido de Hidrogênio , Estresse Oxidativo , RNA Interferente Pequeno , Resveratrol , Estilbenos/farmacologiaRESUMO
OBJECTIVE: To determine miRNA-34a regulated cell senescence indirectly through targeting silent mating-type information regulation 2 homologue 1 (SIRT1) in vitro experiment. METHODS: A constructed pre-miRNA -34a expression vector and a miRNA-1792 expression vector (not directly against any gene) were transfected into HEK293 and HUVEC cell lines respectively. The expression levels of SIRT1 in each cell groups were detected by RT-PCR and Western blot. The HUVEC cells were divided into different group: transfected with pre-miRNA-34a expression vector (HUVEC-pre-miRNA-34a), transfected with miRNA-1792 expression vector (HUVEC-pre-miRNA-1792), treated HUVEC cell with SIRT1 activator resveratrol (final concentration 1 micromol/L, treatment for 2 h)(HUVEC-Res), and HUVEC cells without any treatment as the control. Comet assay was applied to detect the oxidative damage of above-mentioned cells after H2O2 treatment for 2 h, and beta-galactosidase (SA-beta-gal) staining was used to detect the senescence of them in different time points after doxorubicin treatment for 2 h. RESULTS: Pre-miRNA-34a expression vector was constructed successfully by sequencing confirmation. RT-PCR and Western blot indicated that the overexpression of miRNA-34a down regulated mRNA and protein level of SIRT1 in HEK293-miRNA-34a and HUVEC-miRNA-34a cell groups (P < 0.001). Comet assay revealed that HUVEC-miRNA-34a cell group was the most sensitive to H2O2 treatment, and the DNA damage of HUVEC-Res cell group was the most minor. HUVEC-miRNA-34a cell group displayed higher frequency of SA-beta-gal staining than that of other cell groups. CONCLUSION: miRNA-34a regulated cell senescence indirectly through targeting SIRT1.
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Senescência Celular , MicroRNAs/metabolismo , Sirtuína 1/metabolismo , Regulação para Baixo , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , RNA Mensageiro , Resveratrol , Estilbenos , TransfecçãoRESUMO
OBJECTIVE: Investigate the association between genetic polymorphism of DSBs repair gene XRCC4, RAD51 and susceptibility to esophageal cancer (EC). METHODS: A hospital based case-control study with 123 EC cases and 61 controls in a Chinese population was conducted. PCR-RFLP was applied to investigate the genotype of XRCC4 promoter G-1394T (rs6869366) and RAD51-G135C and then statistical analysis was conducted by calculating the adjusted odds ratios (OR) and 95% confidence intervals (95% CI). RESULTS: A significant difference of XRCC4-1394 polymorphism was observed between EC cases and controls (P < 0.05). Carriers of the XRCC4 rs6869366 G allele (GC+GG) were at a higher risk of developing EC with the TT genotype as reference (OR = 3.022, 95% CI = 1.487-6.142, P = 0.002). When GG served as the reference group of RAD51-G135C allele, variant genotype (GC and CC) had a significant increased risk of lung cancer (OR = 3.643, 95% CI = 1.501-8. 842, P < 0.05). CONCLUSION: Our findings indicated that genetic variants in DNA repair pathways may be involved in esophageal tumorigenesis. XRCC4 G-1394T and RAD51-G135C conferred risk for the process of developing EC.
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Proteínas de Ligação a DNA/genética , Neoplasias Esofágicas/genética , Predisposição Genética para Doença/genética , Polimorfismo de Nucleotídeo Único , Rad51 Recombinase/genética , Idoso , Estudos de Casos e Controles , Reparo do DNA/genética , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Objectives: The association between biomarkers and the risk of gestational diabetes mellitus (GDM) or preeclampsia (PE) has been extensively studied. However, there is still a lack of convenient, specific, and sensitive indicators for early identification of GMD and PE. Therefore, we conducted a meta-analysis of published articles to investigate the value of afamin circulating levels in the early diagnosis of GDM and PE. Methods: We searched the PubMed, Embase, Cochrane Library, and Web of Science databases for English studies published before November 16, 2022, that examined the association between afamin and GDM or PE. In addition, we searched Clinicaltrials.gov for the relevant completed and ongoing clinical trials. Pooled standard mean differences (SMDs) and weighted mean differences (WMDs) with 95% confidence intervals (CIs) were used to compare the levels of afamin in different groups. Results: Eleven studies were included in our analysis (N = 3047 participants: 1195 GDM, 1407 non-GDM, 195 PE, and 250 non-PE). Subgroup analysis based on different blood collection periods found that the plasma afamin levels in pregnant women with GDM in the first trimester were significantly higher than those in healthy pregnant women (SMD = 0.481, 95% CI: 0.280-0.682), but the analysis showed the opposite results in the second and late stages (SMD = 0.292, 95% CI: -0.092-0.676). The plasma afamin levels of pregnant women with PE in the first trimester (SMD = 0.808, 95% CI: 0.558-1.059) and second/third trimesters (SMD = 0.904, 95% CI: 0.570-1.239) were significantly higher than those in healthy pregnant women. Conclusion: The plasma afamin levels in pregnant women with GDM in the first trimester were significantly higher than those in healthy pregnant women, but the analysis showed the opposite results in the second and third trimesters. The plasma afamin levels in pregnant women with PE in the first, second, and third trimesters were significantly higher than those in healthy pregnant women. Additional large-scale prospective studies are desired to verify these findings, and it is recommended that afamin should be included as a routine diagnostic test for women with GDM and PE. Systematic review registration: https://www.crd.york.ac.uk/PROSPERO/display_record.php?RecordID=339171, identifier CRD42022339171.
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Diabetes Gestacional , Pré-Eclâmpsia , Gravidez , Feminino , Humanos , Diabetes Gestacional/diagnóstico , Pré-Eclâmpsia/diagnóstico , Estudos Prospectivos , Terceiro Trimestre da Gravidez , Primeiro Trimestre da GravidezRESUMO
Background: Traditional drug susceptibility testing cannot be performed in clinical laboratories due to the slow-growing characteristics of Mycoplasma genitalium when cultured in vitro. Sanger sequencing is the standard method for detecting drug resistance-associated mutations. It has been used in some laboratories to guide the choice of macrolide antibiotics for Mycoplasma genitalium infected patients. Furthermore, resistance to fluoroquinolone has become another emerging clinical challenge. Objective: Sequencing analysis can detect unknown mutations, but it is time-consuming, requires professional analytical skills and the appropriate testing equipment. The main objective of this study was to establish a nested real-time PCR method for the simultaneous detection of 23S rRNA and parC genotypes in relation to the macrolide and fluoroquinolone resistance. Results: 105 MG-positive samples and 27 samples containing other pathogens were used for validation. The limit of the nested real-time PCR detection was 500 copies/reaction and there was no cross-reaction with Ureaplasma urealyticum, Mycoplasma hominis, Chlamydia trachomatis, Neisseria gonorrhoeae, Human papillomavirus, Herpes simplex virus, Candida albicans and Ureaplasma parvum, but the 23S rRNA assay cross-reacted with Mycoplasma pneumoniae. Compared with sequencing results, the sensitivity of 23S rRNA was 100% (95% CI; 93.3 -100), the specificity was 94.3% (95% CI; 79.4 - 99.0), the overall consistency was 98% (95% CI; 92.5 - 99.7) and kappa value was 0.96 (P < 0.001); the sensitivity of parC was 100% (95% CI; 93.4 - 100), the specificity was 89.7% (95% CI; 71.5 - 97.3) and the overall consistency was 96.9% (95% CI; 90.7 - 99.2) with a kappa value of 0.92 (P < 0.001). Conclusions: The results of this sensitive and rapid alternative for identifying resistant genotypes of Mycoplasma genitalium are intuitive and easy to interpret, especially for mixed MG populations. Although the relevant 23S rRNA primers need further adjustment, this reliable method would provide an effective diagnostic tool for the selection of antibiotics in clinical practice.
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Mycobacterium tuberculosis , Infecções por Mycoplasma , Mycoplasma genitalium , Humanos , Fluoroquinolonas/farmacologia , Mycoplasma genitalium/genética , RNA Ribossômico 23S/genética , Reação em Cadeia da Polimerase em Tempo Real , Macrolídeos/farmacologia , Testes de Sensibilidade Microbiana , Farmacorresistência Bacteriana/genética , Infecções por Mycoplasma/diagnóstico , Mycobacterium tuberculosis/genética , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , MutaçãoRESUMO
Platelets have emerged as key inflammatory cells implicated in the pathology of sepsis, but their contributions to rapid clinical deterioration and dysregulated inflammation have not been defined. Here, we show that the incidence of thrombocytopathy and inflammatory cytokine release was significantly increased in patients with severe sepsis. Platelet proteomic analysis revealed significant upregulation of gasdermin D (GSDMD). Using platelet-specific Gsdmd-deficient mice, we demonstrated a requirement for GSDMD in triggering platelet pyroptosis in cecal ligation and puncture (CLP)-induced sepsis. GSDMD-dependent platelet pyroptosis was induced by high levels of S100A8/A9 targeting toll-like receptor 4 (TLR4). Pyroptotic platelet-derived oxidized mitochondrial DNA (ox-mtDNA) potentially promoted neutrophil extracellular trap (NET) formation, which contributed to platelet pyroptosis by releasing S100A8/A9, forming a positive feedback loop that led to the excessive release of inflammatory cytokines. Both pharmacological inhibition using Paquinimod and genetic ablation of the S100A8/A9-TLR4 signaling axis improved survival in mice with CLP-induced sepsis by suppressing platelet pyroptosis.
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OBJECTIVE: To study the flavanoids extracted from onion on the blood-brain barrier (BBB) permeation, and their effects on primary cultured neuron cell proliferation and apoptosis of SD rats using ethanol reflux method. METHODS: The brain microvascular endothelial cells (BMVECs) were first successfully primary cultured. Then rats BMVECs and astrocytes (ACs) were co-cultured to establish the in vitro BBB model. The flavanoids were extracted from onion using ethanol reflux method. The model was verified by transmission electron microscopy (TEM) and trans-epithelial electric resistance (TEER). The flavanoids permeability was tested using high performance liquid chromatography (HPLC). Meanwhile, rat neuron cells were cultured and exposed to H2O2 and flavanoids. Their effects on the cell proliferation and apoptosis were observed using MTT assay. The injury of neuron DNA was analyzed using single-cell gel electrophoresis (SCGE) and immunofluorescent assay. RESULTS: The in vitro BBB model was successfully established by TEM and TEER. Results of HPLC proved flavanoids extracts could effectively permeate the BBB with the permeability of 60.58%. The extractive at 10 - 20 microg/mL showed obvious inhibition on the apoptosis of neuron cells induced by H2O2, and attenuated the injury of neuron DNA. CONCLUSIONS: The flavanoids extracted from onion ethanol reflux method could effectively penetrate the BBB. They also showed obvious inhibition on the H2O2 induced neuron cell apoptosis and DNA injury.
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Barreira Hematoencefálica/efeitos dos fármacos , Flavonoides/farmacologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Cebolas/química , Animais , Animais Recém-Nascidos , Apoptose , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Células Cultivadas , Dano ao DNA , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Peróxido de Hidrogênio , Neurônios/citologia , RatosRESUMO
OBJECTIVE: To study the DNA damage and recovery induced by hydrogen peroxide in normal aging and premature aging human cells. METHODS: The immunofluorescent assay and comet assay were used to estimate basal DNA damage in normal aging BJ cells and premature aging Hutchinson-Gilford progeria syndrome (HGPS) cells, which were divided into three and two distinct population doubling (PD) number groups (BJ 14, 30, 45 and HGPS 8, 17) respectively. The DNA damage induced by hydrogen peroxide of these cell populations, as well as their repair activity, was also studied. Finally, the recovery capability before and after the xeroderma pigmentosum group A (XPA) knocked down in these groups was measured. RESULTS: Our results indicated that the normal BJ cells of older PD number showed higher basal levels of DNA damage and were more sensitive to the effects of the DNA-damaging agents than the adult one, who, in turn, was more sensitive than the younger ones. The basal damage level of HGPS cells was same or higher than the older BJ cells. HGPS cells also had the same consistent damage change as BJ cells after treatment. The decline of the repair efficiency with age to the DNA damage induced by the agent was also observed. CONCLUSION: The DNA repair of HGPS is repressed by dysfunction of XPA.
Assuntos
Senescência Celular/fisiologia , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Senilidade Prematura/patologia , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Fibroblastos/citologia , HumanosRESUMO
Lung adenocarcinoma (LUAD) is the most common subtype of lung cancer, but the prognosis of LUAD patients remains unsatisfactory. Here, we retrieved the RNA-seq data of LUAD cohort from The Cancer Genome Atlas (TCGA) database and then identified differentially expressed immune-related lncRNAs (DEirlncRNAs) between LUAD and normal controls. Based on a new method of cyclically single pairing along with a 0-or-1 matrix, we constructed a novel prognostic signature of 8 DEirlncRNA pairs in LUAD with no dependence upon specific expression levels of lncRNAs. This prognostic model exhibited significant power in distinguishing good or poor prognosis of LUAD patients and the values of the area under the curve (AUC) were all over 0.70 in 1, 3, 5 years receiver operating characteristic (ROC) curves. Moreover, the risk score of the model could serve as an independent prognostic factor for patients with LUAD. In addition, the risk model was significantly associated with clinicopathological characteristics, tumor-infiltrating immune cells, immune-related molecules and sensitivity of anti-tumor drugs. This novel signature of DEirlncRNA pairs in LUAD, which did not require specific expression levels of lncRNAs, might be used to guide the administration of patients with LUAD in clinical practice.
Assuntos
Adenocarcinoma de Pulmão/genética , Biomarcadores Tumorais/genética , RNA Longo não Codificante/genética , Transcriptoma/genética , Idoso , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Prognóstico , RNA Longo não Codificante/classificação , RNA-SeqRESUMO
OBJECTIVE: To investigate whether the polymorphism of DNA repair genes XPC (Ala499Val and Lys939Gln) and XPG (His1104Asp) is associated with the susceptibility to hepatocellular carcinoma (HCC). METHODS: A hospital-based case-control study was conducted in 500 cases with HCC and 507 controls. Genotypes of XPC and XPG were determined by real-time polymerase chain reaction with the TaqMan MGB probe. RESULTS: Compared to the CC genotype, the CT genotype and the TT genotype of XPC Ala499Val were not associated with the susceptibility to HCC (adjusted OR = 1.34, 95% CI: 0.85-2.12; adjusted OR = 1.30, 95% CI: 0.68-2.51, respectively). Compared to the AA genotype, the AC genotype and the CC genotype of Lys939Gln were not associated with the susceptibility to HCC (adjusted OR = 1.20, 95% CI: 0.78-1.85; adjusted OR = 1.81, 95% CI: 0.88-3.73, respectively). Compared to the CC genotype, the CG genotype and the GG genotype of XPG His1104Asp were not associated with the susceptibility to HCC (adjusted OR = 0.85, 95% CI: 0.56-1.27; adjusted OR = 1.12, 95% CI: 0.67-1.87, respectively) However, the stratified analysis revealed that the females with the AC+CC genotype of XPC Lys939Gln had increased risk of HCC compared to those with AA genotype (OR = 2.17, 95% CI: 1.01-4.64). CONCLUSION: Our results suggest that XPC and XPG polymorphisms do not independently affect on the susceptibility to HCC, but the joint effect of C allele of XPC Lys939Gln and female may modify the risk of HCC.
Assuntos
Carcinoma Hepatocelular/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Neoplasias Hepáticas/genética , Proteínas Nucleares/genética , Polimorfismo de Nucleotídeo Único , Fatores de Transcrição/genética , Estudos de Casos e Controles , Reparo do DNA , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Platelet hyperactivity is the hallmark of diabetes, and platelet activation plays a crucial role in diabetic vascular complications. Recent studies have shown that upon activation, platelet-derived miRNAs are incorporated into vascular smooth muscle cells (VSMCs), regulating the phenotypic switch of VSMC. Under diabetes, miRNA deficiency in platelets fails to regulate the VSMC phenotypic switch. Therefore, manipulation of platelet-derived miRNAs expression may provide therapeutic option for diabetic vascular complications. We seek to investigate the effect of calpeptin (calpain inhibitor) on the expression of miRNAs in diabetic platelets, and elucidate the downstream signaling pathway involved in protecting from neointimal formation in diabetic mice with femoral wire injury model. Using human cell and platelet coculture, we demonstrate that diabetic platelet deficient of miR-223 fails to suppress VSMC proliferation, while overexpression of miR-223 in diabetic platelets suppressed the proliferation of VSMC to protect intimal hyperplasia. Mechanistically, miR-223 directly targets the insulin-like growth factor-1 receptor (IGF-1R), which inhibits the phosphorylation of GSK3ß and activates the phosphorylation of AMPK, resulting in reduced VSMC dedifferentiation and proliferation. Using a murine model of vascular injury, we show that calpeptin restores the platelet expression of miR-223 in diabetes, and the horizontal transfer of platelet miR-223 into VSMCs inhibits VSMC proliferation in the injured artery by targeting the expression of IGF-1R. Our data present that the platelet-derived miR-223 suppressed VSMC proliferation via the regulation miR-223/IGF-1R/AMPK signaling pathways, and inhibition of calpain alleviates neointimal formation by restoring the expression of miR-223 in diabetic platelet.
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Previous studies have demonstrated that inhibition of canonical Wnt signaling promotes zebrafish heart regeneration and that treatment of injured heart tissue with the Wnt activator 6-bromo-indirubin-3-oxime (BIO) can impede cardiomyocyte proliferation. However, the mechanism by which Wnt signaling regulates downstream gene expression following heart injury remains unknown. In this study, we have demonstrated that inhibition of injury-induced myocardial wnt2bb and jnk1/creb1/c-jun signaling impedes heart repair following apex resection. The expression of jnk1, creb1, and c-jun were inhibited in wnt2bb dominant negative (dn) mutant hearts and elevated in wnt2bb-overexpresssing hearts following ventricular amputation. The overexpression of creb1 sufficiently rescued the dn-wnt2bb-induced phenotype of reduced nkx2.5 expression and attenuated heart regeneration. In addition, wnt2bb/jnk1/c-jun/creb1 signaling was increased in Tg(hsp70l:dkk1) transgenic fish, whereas it was inhibited in Tg(hsp70l:wnt8) transgenic fish, indicating that canonical Wnt and non-canonical Wnt antagonize each other to regulate heart regeneration. Overall, the results of our study demonstrate that the wnt2bb-mediated jnk1/c-jun/creb1 non-canonical Wnt pathway regulates cardiomyocyte proliferation.
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Ethidium bromide (EtBr) is widely used as DNA-staining dyes for the detection of nucleic acids in laboratories and known to be powerful mutagens and carcinogens. In the present paper, the removal of EtBr from aqueous solutions in a batch system using DNA-loaded Fe3O4 nanoparticles as a simple and efficient method was investigated. DNA was covalently loaded on the surface of Fe3O4 magnetic nanoparticles, which was confirmed by FT-IR analysis and zeta potential measurements. The morphology and crystal structure were characterized by SEM, TEM, and XRD. The influence factors on the removal efficiency such as initial EtBr concentration, contact time, adsorbent dose, pH, and temperature were also studied. The removal process of EtBr can be completed quickly within 1 min. The removal efficiency was more than 99% while the EtBr concentration was routinely used (0.5 mg L-1) in biology laboratories and the dosages of nanoparticles were 1 g L-1. For the different EtBr concentrations from 0.5 to 10 mg L-1 in aqueous solution, the goal of optimized removal was achieved by adjusting the dosage of DNA-loaded Fe3O4 nanoparticles. The optimum pH was around 7 and the operational temperature from 4 to 35 °C was appropriate. Kinetic studies confirmed that the adsorption followed second-order reaction kinetics. Thermodynamic data revealed that the process was spontaneous and exothermic. The adsorption of EtBr on DNA-loaded Fe3O4 nanoparticles fitted well with the Freundlich isotherm model. These results indicated that DNA-loaded Fe3O4 nanoparticles are a promising adsorbent for highly efficient removal of EtBr from aqueous solution in practice.
Assuntos
DNA/química , Etídio/isolamento & purificação , Nanopartículas de Magnetita/química , Poluentes Químicos da Água/isolamento & purificação , Adsorção , Corantes/isolamento & purificação , Etídio/química , Cinética , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Soluções , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura , Termodinâmica , Eliminação de Resíduos Líquidos/métodos , Água/química , Poluentes Químicos da Água/química , Purificação da Água/métodos , Difração de Raios XRESUMO
Insulin-like growth factor I (IGF-I) is an anabolic growth hormone indispensable for cell growth, proliferation, differentiation, and other metabolic processes. Three missense mutations in IGF-I have been identified to be disease-related, while more mutations are waiting for phenotype annotation. However, there is no previous work regarding effective and accurate identification of pathological mutations of IGF-I, neither regarding the effects of mutations on the protein structure and dynamics. In this study, we first predicted potential deleterious mutations present in IGF-I using 16 in silico tools. Then, these mutations were further evaluated through multiple bioinformatics methods including conservation analysis, physicochemical characterization, and molecular dynamics simulation. After rigorous screening, five mutations (T4M, V17M, V44M, R50W, and M59R) were finally selected, of which two have been previously reported to be deleterious. These mutations locate at conserved regions and change the residue size locally. In the conventional simulations, the mutations destabilized the overall IGF-I structure by destroying two important hydrogen bonds within the key region of "C-neck." This finding was further confirmed by the thermal unfolding simulations and the free-energy calculations, where the mutants were associated with faster and greater loss of helix and lower energy barriers in comparison with the wild-type protein. The rigorous phenotype prediction and comprehensive structural analysis of missense mutations will not only pave the way of screening for harmful mutations in IGF-I but also provide new prospects for the rational design of IGF-I analogues and tailored medicine.