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BACKGROUND: Cardiomyocyte differentiation involves a stepwise clearance of repressors and fate-restricting regulators through the modulation of BMP (bone morphogenic protein)/Wnt-signaling pathways. However, the mechanisms and how regulatory roadblocks are removed with specific developmental signaling pathways remain unclear. METHODS: We conducted a genome-wide CRISPR screen to uncover essential regulators of cardiomyocyte specification in human embryonic stem cells using a myosin heavy chain 6 (MYH6)-GFP (green fluorescence protein) reporter system. After an independent secondary single guide ribonucleic acid validation of 25 candidates, we identified NF2 (neurofibromin 2), a moesin-ezrin-radixin like (MERLIN) tumor suppressor, as an upstream driver of early cardiomyocyte lineage specification. Independent monoclonal NF2 knockouts were generated using CRISPR-Cas9, and cell states were inferred through bulk RNA sequencing and protein expression analysis across differentiation time points. Terminal lineage differentiation was assessed by using an in vitro 2-dimensional-micropatterned gastruloid model, trilineage differentiation, and cardiomyocyte differentiation. Protein interaction and post-translation modification of NF2 with its interacting partners were assessed using site-directed mutagenesis, coimmunoprecipitation, and proximity ligation assays. RESULTS: Transcriptional regulation and trajectory inference from NF2-null cells reveal the loss of cardiomyocyte identity and the acquisition of nonmesodermal identity. Sustained elevation of early mesoderm lineage repressor SOX2 and upregulation of late anticardiac regulators CDX2 and MSX1 in NF2 knockout cells reflect a necessary role for NF2 in removing regulatory roadblocks. Furthermore, we found that NF2 and AMOT (angiomotin) cooperatively bind to YAP (yes-associated protein) during mesendoderm formation, thereby preventing YAP activation, independent of canonical MST (mammalian sterile 20-like serine-threonine protein kinase)-LATS (large tumor suppressor serine-threonine protein kinase) signaling. Mechanistically, cardiomyocyte lineage identity was rescued by wild-type and NF2 serine-518 phosphomutants, but not NF2 FERM (ezrin-radixin-meosin homology protein) domain blue-box mutants, demonstrating that the critical FERM domain-dependent formation of the AMOT-NF2-YAP scaffold complex at the adherens junction is required for early cardiomyocyte lineage differentiation. CONCLUSIONS: These results provide mechanistic insight into the essential role of NF2 during early epithelial-mesenchymal transition by sequestering the repressive effect of YAP and relieving regulatory roadblocks en route to cardiomyocytes.
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Diferenciação Celular , Linhagem da Célula , Miócitos Cardíacos , Neurofibromina 2 , Humanos , Miócitos Cardíacos/metabolismo , Neurofibromina 2/genética , Neurofibromina 2/metabolismo , Sistemas CRISPR-Cas , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/citologiaRESUMO
BACKGROUND: Adult mammalian cardiomyocytes have limited proliferative capacity, but in specifically induced contexts they traverse through cell-cycle reentry, offering the potential for heart regeneration. Endogenous cardiomyocyte proliferation is preceded by cardiomyocyte dedifferentiation (CMDD), wherein adult cardiomyocytes revert to a less matured state that is distinct from the classical myocardial fetal stress gene response associated with heart failure. However, very little is known about CMDD as a defined cardiomyocyte cell state in transition. METHODS: Here, we leveraged 2 models of in vitro cultured adult mouse cardiomyocytes and in vivo adeno-associated virus serotype 9 cardiomyocyte-targeted delivery of reprogramming factors (Oct4, Sox2, Klf4, and Myc) in adult mice to study CMDD. We profiled their transcriptomes using RNA sequencing, in combination with multiple published data sets, with the aim of identifying a common denominator for tracking CMDD. RESULTS: RNA sequencing and integrated analysis identified Asparagine Synthetase (Asns) as a unique molecular marker gene well correlated with CMDD, required for increased asparagine and also for distinct fluxes in other amino acids. Although Asns overexpression in Oct4, Sox2, Klf4, and Myc cardiomyocytes augmented hallmarks of CMDD, Asns deficiency led to defective regeneration in the neonatal mouse myocardial infarction model, increased cell death of cultured adult cardiomyocytes, and reduced cell cycle in Oct4, Sox2, Klf4, and Myc cardiomyocytes, at least in part through disrupting the mammalian target of rapamycin complex 1 pathway. CONCLUSIONS: We discovered a novel gene Asns as both a molecular marker and an essential mediator, marking a distinct threshold that appears in common for at least 4 models of CMDD, and revealing an Asns/mammalian target of rapamycin complex 1 axis dependency for dedifferentiating cardiomyocytes. Further study will be needed to extrapolate and assess its relevance to other cell state transitions as well as in heart regeneration.
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Aspartato-Amônia Ligase , Desdiferenciação Celular , Fator 4 Semelhante a Kruppel , Miócitos Cardíacos , Animais , Camundongos , Aspartato-Amônia Ligase/genética , Aspartato-Amônia Ligase/metabolismo , Células Cultivadas , Miócitos Cardíacos/metabolismo , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/genética , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/metabolismoRESUMO
PURPOSE: Aspirin has been suggested to reduce the risk of cancer. However, previous studies have been inconsistent regarding the relationship between aspirin use and the risk of occurrence of hepatocellular carcinoma (HCC). The purpose of this study was to assess the effect of aspirin on clinical outcomes in patients with HCC in a meta-analysis and to explore the possible dose-response relationship. METHODS: A systematic literature search was conducted in 10 electronic databases and 4 registries. The combined hazard ratios (HRs) were calculated using a random-effects model with 95% confidence interval (CIs) to assess the effect of aspirin on the risk of HCC. Relevant subgroup analyses and sensitivity analyses were performed. RESULTS: The results show that aspirin use correlated with lower incidence of HCC (HR: 0.75, 95% CI: 0.71-0.80), decreased risk of HCC recurrence (HR: 0.79, 95% CI: 0.65-0.96), and reduced mortality (HR: 0.72, 95% CI: 0.60-0.87). The results of the subgroup analysis showed that aspirin use was consistently associated with reduced incidence of HCC across different regions, study designs, and populations. A linear relationship was found for both dosage and duration of aspirin use. An increased of bleeding with aspirin use among patients was also observed (HR 1.10, 95% CI: 1.02-1.20). CONCLUSIONS: This meta-analysis found that aspirin use was independently associated with a reduced risk of HCC incidence, recurrence, and death. Furthermore, aspirin use influenced HCC occurrence in a dose-dependent and duration-dependent manner. However, an increased risk of bleeding with aspirin use was noted.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Aspirina/uso terapêutico , Carcinoma Hepatocelular/epidemiologia , Carcinoma Hepatocelular/prevenção & controle , Incidência , Neoplasias Hepáticas/epidemiologia , Neoplasias Hepáticas/prevenção & controle , Modelos de Riscos ProporcionaisRESUMO
Local recurrence and distant metastasis of non-small cell lung cancer (NSCLC) caused by immune escape is one of the root causes of treatment difficulties. We aim to investigate the mechanism of immune escape in NSCLC. NSCLC tissues were collected. Cell proliferation was detected by CCK-8 assay. Cell migration and invasion ability was measured by Transwell assay. The expressions of E-cadherin, N-cadherin and PD-L1 were detected by Western blot. NSCLC cells were co-cultured with CD8+ T cells to simulate tumor microenvironment in vitro. The proportion of CD8+ T cells and apoptosis were detected by flow cytometry. Dual-luciferase reporter gene assay confirmed the targeting relationship of circDENND2D and STK11. The expressions of circDENND2D and STK1 were down-regulated, while miR-130b-3p expression was up-regulated in NSCLC tissues. Overexpression of circDENND2D or STK11 inhibited NSCLC cells proliferation, migration and invasion, and attenuated the immune escape of NSCLC cells. CircDENND2D targeted miR-130b-3p to competitively promote STK11 expression. STK11 knockdown or miR-130b-3p overexpression attenuated the function of circDENND2D overexpression on NSCLC cells. CircDENND2D inhibited metastasis and immune escape of NSCLC by regulating miR-130b-3p/STK11 axis.
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Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , MicroRNAs , Humanos , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Proteínas Serina-Treonina Quinases/genética , Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica , Microambiente Tumoral , Quinases Proteína-Quinases Ativadas por AMPRESUMO
Cardiomyocytes (CMs) lost during cardiac injury and heart failure (HF) cannot be replaced due to their limited proliferative capacity. Regenerating the failing heart by promoting CM cell-cycle re-entry is an ambitious solution, currently vigorously pursued. Some genes have been proven to promote endogenous CM proliferation, believed to be preceded by CM dedifferentiation, wherein terminally differentiated CMs are initially reversed back to the less mature state which precedes cell division. However, very little else is known about CM dedifferentiation which remains poorly defined. We lack robust molecular markers and proper understanding of the mechanisms driving dedifferentiation. Even the term dedifferentiation is debated because there is no objective evidence of pluripotency, and could rather reflect CM plasticity instead. Nonetheless, the significance of CM transition states on cardiac function, and whether they necessarily lead to CM proliferation, remains unclear. This review summarises the current state of knowledge of both natural and experimentally induced CM dedifferentiation in non-mammalian vertebrates (primarily the zebrafish) and mammals, as well as the phenotypes and molecular mechanisms involved. The significance and potential challenges of studying CM dedifferentiation are also discussed. In summary, CM dedifferentiation, essential for CM plasticity, may have an important role in heart regeneration, thereby contributing to the prevention and treatment of heart disease. More attention is needed in this field to overcome the technical limitations and knowledge gaps.
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Desdiferenciação Celular , Cardiopatias/terapia , Miócitos Cardíacos/citologia , Regeneração , Animais , Cardiopatias/patologia , HumanosRESUMO
Background Asthma is a chronic inflammatory heterogeneous respiratory disease. Previous studies showed that the lncRNA NEAT1 (nuclear paraspeckle assembly transcript 1) might play an important role in the pathogenesis of asthma, but its potential mechanism in airway smooth muscle cell (ASMC) inflammation remains largely unknown and needs further investigation.Methods We performed cellular immunofluorescence to identify the features of ASMCs and detected the expression levels of lncRNA NEAT1, miR-139, TNF-α, IL-6, IL-8 and IL-1ß by quantitative real-time PCR (Q-PCR) and ELISA. Western blotting (WB) was used to measure the protein expression of the related genes, and bioinformatics as well as dual luciferase assays were used to validate the interaction between lncRNA NEAT1 and miR-139 and the interaction between miR-139 and the 3'-UTR of JAK3.Results The expression of lncRNA NEAT1 was increased in the ASMCs of asthma patients, but miR-139 was decreased. Overexpression of lncRNA NEAT1 promoted the expression of the inflammatory cytokines such as TNF-α, IL-6, IL-8 and IL-1ß in ASMCs. LncRNA NEAT1 was able to target miR-139 to activate the JAK3/STAT5 signaling pathway and induced the expression of these inflammatory cytokines in ASMCs. Overexpression of miR-139 or suppression of the JAK3/STAT5 signaling pathway reversed the inflammatory effect of lncRNA NEAT1.Conclusion LncRNA NEAT1 played a pivotal role in ASMC inflammation and exerted its function through the miR-139/JAK3/STAT5 signaling network.
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MicroRNAs , Miócitos de Músculo Liso/patologia , RNA Longo não Codificante , Humanos , Inflamação/genética , Janus Quinase 3 , MicroRNAs/genética , RNA Longo não Codificante/genética , Fator de Transcrição STAT5Assuntos
Insuficiência Cardíaca , Linfócitos T , Coração , Insuficiência Cardíaca/terapia , Humanos , Recém-Nascido , RegeneraçãoRESUMO
Flexible pressure sensors have attracted great interest due to their bendable, stretchable, and lightweight characteristics compared to rigid pressure sensors. However, the contradictions among sensitivity, detection limit, thickness, and detection range restrict the performance of flexible pressure sensors and the scope of their applications, especially for scenarios requiring conformal fitting, such as rough surfaces such as the human skin. This paper proposes a novel flexible pressure sensor by combining the nanoengineering strategy and nanocomposite structures. The nanoengineering strategy utilizes the bending deformation of nanofilm instead of the compression of the active layer to achieve super high sensitivity and low detection limit; meanwhile, the nanocomposite structures introduce distributed microbumps that delay the adhesion of nanofilm to enlarge the detection range. As a result, this device not only ensures an ultrathin thickness of 1.6 µm and a high sensitivity of 84.29 kPa-1 but also offers a large detection range of 20 kPa and an ultralow detection limit of 0.07 Pa. Owing to the ultrathin thickness as well as high performance, this device promotes applications in detecting fingertip pressure, flexible mechanical gripping, and so on, and demonstrates significant potential in wearable electronics, human-machine interaction, health monitoring, and tactile perception. This device offers a strategy to resolve the conflicts among thickness, sensitivity, detection limit, and detection range; therefore, it will advance the development of flexible pressure sensors and contribute to the community and other related research fields.
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Nanocompostos , Pressão , Dispositivos Eletrônicos Vestíveis , Nanocompostos/química , Humanos , Nanotecnologia/instrumentação , Limite de DetecçãoRESUMO
Foodborne illnesses, caused by harmful microorganisms in food, are a significant global health issue. Current methods for identifying these pathogens are both labor-intensive and time-consuming. In this research, we devised a swift and precise detection technique using recombinase polymerase amplification combined with a lateral flow dipstick (RPA-LFD) for three foodborne pathogens found in meat. By employing a dedicated detection device, RPA-LFD allows for the rapid analysis of DNA from Escherichia coli O157 (E. coli O157), Salmonella, and Shigella-pathogens that are prohibited in food. The detection thresholds for E. coli O157, Salmonella, and Shigella are 0.168 fg/µl (1.04 CFU/ml), 0.72 fg/µl (27.49 CFU/ml), and 1.25 fg/µl (48.84 CFU/ml), respectively. This method provides a short detection window, operates at low temperatures, follows simple procedures, and exhibits high sensitivity. Our study establishes the RPA-LFD method for simultaneously identifying the nucleic acid of three foodborne pathogens, offering an efficient solution for quickly identifying multiple contaminants.
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Escherichia coli O157 , Contaminação de Alimentos , Microbiologia de Alimentos , Técnicas de Amplificação de Ácido Nucleico , Recombinases , Salmonella , Shigella , Escherichia coli O157/isolamento & purificação , Escherichia coli O157/genética , Salmonella/isolamento & purificação , Salmonella/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Microbiologia de Alimentos/métodos , Recombinases/metabolismo , Shigella/isolamento & purificação , Shigella/genética , Contaminação de Alimentos/análise , Carne/microbiologia , DNA Bacteriano/genética , Animais , Sensibilidade e Especificidade , Doenças Transmitidas por Alimentos/microbiologiaRESUMO
Background: Limited data are available regarding the current microbiological characteristics of extracorporeal membrane oxygenation (ECMO)-related infections in intensive care units (ICUs) in China. This retrospective study aimed to determine the epidemiology, risk factors and impact on the outcome of ECMO-related infections. Methods: A retrospective observational study from January 2014 to December 2019 was performed, and adult patients receiving ECMO support for more than 48 hours were included in this study. The primary outcome was the incidence rate of ECMO-related infection. Clinical data were recorded, and risk factors associated with an increased risk of ECMO-related infection were analyzed. Results: A total of 174 adult patients who received ECMO and underwent ECMO for 1,670 days were included in this study. Forty-six patients (26.4%) developed ECMO-related infections, corresponding to 27.5 first episodes/1,000 ECMO days. The most common ECMO-related infection observed was ventilator-associated pneumonia (VAP). Infected patients had longer durations of mechanical ventilation {20.2 [interquartile range (IQR), 12.6, 30.7] vs. 9.0 (IQR, 5.8, 14.7) days, P<0.001}, ECMO support [11.6 (IQR, 8.1, 17.3) vs. 7.6 (IQR, 5.6, 9.7) days, P<0.001] and hospital stays (28.2±20.7 vs. 22.0±15.6 days, P<0.001). The factors independently associated with ECMO-related infection were a dynamic decrease in lymphocyte count [adjusted odds ratio (OR) =3.578, 95% confidence interval (CI): 2.175-4.906, P<0.001] and ECMO duration (adjusted OR =1.207, 95% CI: 1.096-1.330, P<0.001). Compared to patients without infection, infected patients had greater hospital mortality (39.1% vs. 78.3%, P<0.001) and 90-day mortality (40.6% vs. 87.0%, P<0.001). ECMO-related infections were associated with worse outcomes (adjusted Kaplan-Meier curve, log rank test P<0.001). Conclusions: Patients supported by ECMO had a high risk of developing ECMO-related infection. The most common ECMO-related infection observed was VAP. A dynamic decrease in lymphocyte counts was significantly associated with an increased risk of ECMO-related infection.
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Background: Executive dysfunction in children with attention deficit hyperactivity disorder (ADHD) is thought to be closely related to the prefrontal cortex (PFC). However, there is controversy over the activation of the PFC in children with ADHD. Differences could be related to the subtype. Meanwhile, no study to date has used functional near-infrared spectroscopy (fNIRS) to explore the differences between subtypes. Thus, this study aimed to explore the activation of the PFC in children with different subtypes of ADHD during executive function task. Methods: Participants in this study include typically developing (TD) children (n = 28), ADHD-predominantly inattentive (ADHD-PI) (n = 39) and ADHD-combined (ADHD-C) (n = 24). To examine the executive function of ADHD, the Go/No-go task is chosen to assess the response inhibition function. The activation of PFC in all participants during the Go/No-go task was recorded by fNIRS. Meanwhile, behavioral data were also recorded. Results: Both TD and ADHD children activated the right PFC [middle frontal gyrus (MFG) and inferior frontal gyrus (IFG)] during response inhibition. However, the range and degree of activation differed among these groups. Compared with TD children, those with ADHD-PI had a smaller extent of activation in the right PFC, and those with ADHD-C only had a tendency to enhance activation. In addition, children with ADHD-PI and ADHD-C had impaired activation of the temporal gyrus. Besides, compared with ADHD-C and TD, those with ADHD-PI also had impaired activation of the right precentral gyrus (PG), and the supplementary motor area (SMA). Compared with ADHD-PI, ADHD-C showed decreased activation of the right MFG. The activation of Ch34 (BA44, rPFC) in children with ADHD-PI and ADHD-C was negatively correlated with their clinical symptoms. Conclusion: The activation of the PFC in children with different subtypes of ADHD has both commonalities and differences. The degree of activation of the right PFC Ch34 in children with ADHD is negatively correlated with clinical symptoms. fNIRS could be served as a candidate hemodynamic biomarker for the diagnosis of ADHD.
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In the clinical intensive care units (ICU), the traditional Chinese medicine (TCM) formulation of Xuebijing has been frequently used for treating sepsis. Nevertheless, the underlying pharmacological mechanisms of Xuebijing remain largely unclear. Caenorhabditis elegans is an important experimental host for bacterial infections. Using C. elegans as an animal model, we here examined the potential of Xuebijing treatment against bacterial infection and the underlying mechanisms. Xuebijing treatment could inhibit the reduction tendency of lifespan caused by Pseudomonas aeruginosa infection. For the cellular mechanisms of this antibacterial infection property, we found that Xuebijing treatment rescued C. elegans lifespan to be against P. aeruginosa infection by inhibiting Pseudomonas colonization in the intestinal lumen. Meanwhile, the increase in the expression of antimicrobial genes induced by Pseudomonas infection was also suppressed by Xuebijing treatment. Moreover, the beneficial effect of Xuebijing against Pseudomonas infection depended on insulin, p38 MAPK, Wnt, DBL-1/TGF-ß, ELT-2, and programmed cell death (PCD)-related signals. Although Xuebijing did not show obvious antibacterial activity, Xuebijing (100%) treatment could inhibit the Pseudomonas biofilm formation and decrease the expression of virulence genes (lasA, lasB, rhlA, rhlC, phzA, phzM, phzH, and phzS) and quorum sensing (QS)-related genes (lasI, lasR, rhlI, rhlR, pqsA, and pqsR). Our results support the potential role of Xuebijing treatment against bacterial infection in hosts.
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BACKGROUND: Breast cancer (BC) is a common malignant tumor. Apatinib in combination with other treatments has been used for BC; however, its safety and efficacy are not well-known. Therefore, this meta-analysis was performed to assess the efficacy and safety of apatinib in the treatment of BC. METHODS: Studies comparing the effects of apatinib-based therapy versus control among BC patients were included. On January 21, 2022, a systematic search was performed in 9 databases. The risk ratio (RR) with 95% confidence interval (CI) was used to estimate efficacy and safety. The I square value (I2) was used to assess heterogeneity. A leave-one-out sensitivity analysis was also conducted. Publication bias was assessed by funnel plots and Egger's and Begg's tests. RESULTS: A total of 31 studies including 2,258 BC patients were included. The results showed that apatinib group had a significant improvement in disease control rate (DCR, RR = 1.43, 95% CI = 1.35-1.52, I2 = 43.8%) and objective response rate (ORR, RR = 1.79, 95% CI = 1.51-2.13, I2 = 61.8%) compared to the control group. Except for hemorrhage, hypertension, and hand-foot syndrome, the adverse events were similar between apatinib group and control group. Subgroup analyses found statistically significant differences in DCR in all subgroups except for apatinib combined with radiation therapy and with paclitaxel liposome plus S1. For ORR, there were statistically significant differences in all subgroups except for the radiation therapy, and apatinib monotherapy subgroups. CONCLUSIONS: Our study shown apatinib showed good efficacy and acceptable safety in the treatment of BC patients. More high-quality randomized controlled trials from different regions and countries are needed to confirm our findings.
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Neoplasias da Mama , Neoplasias da Mama/tratamento farmacológico , Feminino , Humanos , Lipossomos , Paclitaxel , Piridinas , Resultado do TratamentoRESUMO
BACKGROUND: Epilepsy is one of the most common brain disorders worldwide. It is usually hard to be identified properly, and a third of patients are drug-resistant. Genes related to the progression and prognosis of epilepsy are particularly needed to be identified. METHODS: In our study, we downloaded the Gene Expression Omnibus (GEO) microarray expression profiling dataset GSE143272. Differentially expressed genes (DEGs) with a fold change (FC) >1.2 and a P-value <0.05 were identified by GEO2R and grouped in male, female and overlapping DEGs. Functional enrichment analysis and Protein-Protein Interaction (PPI) network analysis were performed. RESULTS: In total, 183 DEGs overlapped (77 ups and 106 downs), 302 DEGs (185 ups and 117 downs) in the male dataset, and 750 DEGs (464 ups and 286 downs) in the female dataset were obtained from the GSE143272 dataset. These DEGs were markedly enriched under various Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) terms. 16 following hub genes were identified based on PPI network analysis: ADCY7, C3AR1, DEGS1, CXCL1 in male-specific DEGs, TOLLIP, ORM1, ELANE, QPCT in female-specific DEGs and FCAR, CD3G, CLEC12A, MOSPD2, CD3D, ALDH3B1, GPR97, PLAUR in overlapping DEGs. CONCLUSION: This discovery-driven study may be useful to provide a novel insight into the diagnosis and treatment of epilepsy. However, more experiments are needed in the future to study the functional roles of these genes in epilepsy.
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Biomarcadores/metabolismo , Biologia Computacional/métodos , Epilepsia/genética , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Mapas de Interação de Proteínas , Biomarcadores/análise , Estudos de Casos e Controles , Epilepsia/patologia , Feminino , Perfilação da Expressão Gênica , Humanos , MasculinoRESUMO
HIV-1 transactivator of transcription (Tat) has a great impact on the development of HIV-1 associated neurocognitive disorders through disrupting dopamine transmission. This study determined the mutational effects of human dopamine transporter (hDAT) on basal and Tat-induced inhibition of dopamine transport. Compared to wild-type hDAT, the maximal velocity (Vmax) of [3H]dopamine uptake was decreased in D381L and Y88F/D206L/H547A, increased in D206L/H547A, and unaltered in D206L. Recombinant TatR1 - 86 inhibited dopamine uptake in wild-type hDAT, which was attenuated in either DAT mutants (D206L, D206L/H547A, and Y88F/D206L/H547A) or mutated TatR1 - 86 (K19A and C22G), demonstrating perturbed Tat-DAT interaction. Mutational effects of hDAT on the transporter conformation were evidenced by attenuation of zinc-induced increased [3H]WIN35,428 binding in D206L/H547A and Y88F/D206A/H547A and enhanced basal MPP+ efflux in D206L/H547A. H547A-induced outward-open transport conformational state was further validated by enhanced accessibility to MTSET ([2-(trimethylammonium)ethyl]-methanethiosulfonate) of an inserted cysteine (I159C) on a hDAT background.. Furthermore, H547A displayed an increase in palmitoylation inhibitor-induced inhibition of dopamine uptake relative to wide-type hDAT, indicating a change in basal palmitoylation in H547A. These results demonstrate that Y88F, D206L, and H547A attenuate Tat inhibition while preserving DA uptake, providing insights into identifying targets for improving DAT-mediated dopaminergic dysregulation. HIV-1 Tat inhibits dopamine uptake through human dopamine transporter (hDAT) on the presynaptic terminal through a direct allosteric interaction. Key hDAT residues D-H547, D-Y88, and D-D206 are predicted to be involved in the HIV-1 Tat-DAT binding. Mutating these residues attenuates this inhibitory effect by disrupting the Tat-hDAT interaction.
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HIV-1 , Dopamina , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , HIV-1/metabolismo , Humanos , Mutação , Transativadores , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Bronchial asthma poses a serious threat to human health. Previous studies have documented the role of long noncoding RNAs (lncRNAs) in asthma. However, the molecular mechanism underlying bronchial asthma remains unclear. The aim of the present study was to evaluate the role of the lncRNA Opainteracting protein 5 antisense RNA1 (OIP5AS1) in the house dust miteinduced inflammatory response in human bronchial epithelial cells. BEAS2B cells were treated with Dermatophagoides pteronyssinus peptidase 1 (Der p1) to establish an in vitro model of asthma. OIP5AS1 expression levels increased in BEAS2B cells following Der p1 treatment, while microRNA (miR)1433p was downregulated. Additionally, the levels of the proinflammatory factors tumor necrosis factorα, interleukin (IL)6 and IL8 were measured, and apoptosis was evaluated following OIP5 silencing. OIP5AS1 knockdown reduced the inflammatory response and apoptosis in BEAS2B cells. Furthermore, using dual luciferase reporter assays and cotransfection experiments, it was demonstrated that the function of OIP5AS1 was mediated by miR1433p. miR1433p overexpression attenuated the Der p1induced inflammatory response and apoptosis of BEAS2B cells by targeting high mobility group box 1 (HMGB1). In summary, OIP5AS1 exacerbated Der p1induced inflammation and apoptosis in BEAS2B cells by targeting miR1433p via HMGB1.
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Asma/genética , Brônquios/metabolismo , RNA Longo não Codificante/genética , Células Epiteliais Alveolares/metabolismo , Animais , Apoptose/genética , Asma/patologia , Brônquios/imunologia , Linhagem Celular , Células Epiteliais/metabolismo , Proteína HMGB1/metabolismo , Humanos , Inflamação/genética , MicroRNAs/genética , Pyroglyphidae/patogenicidade , RNA Longo não Codificante/metabolismo , Transdução de Sinais/genéticaRESUMO
AIMS: Cisplatin is a major therapeutic drug for solid tumors, but can cause severe nephrotoxicity. However, the role and therapeutic potential of hydrogen sulfide (H2S), an endogenous gasotransmitter, in cisplatin-induced nephrotoxicity remain to be defined. RESULTS: Cisplatin led to the impairment of H2S production in vitro and in vivo by downregulating the expression level of cystathionine γ-lyase (CSE), which may contribute to the subsequent renal proximal tubule (RPT) cell death and thereby renal toxicity. H2S donors NaHS and GYY4137, but not AP39, mitigated cisplatin-induced RPT cell death and nephrotoxicity. The mechanisms underlying the protective effect of H2S donors included the suppression of intracellular reactive oxygen species generation and downstream mitogen-activated protein kinases by inhibiting NADPH oxidase activity, which may be possibly through persulfidating the subunit p47phox. Importantly, GYY4137 not only ameliorated cisplatin-caused renal injury but also added on more anticancer effect to cisplatin in cancer cell lines. Innovation and Conclusion: Our study provides a comprehensive understanding of the role and therapeutic potential of H2S in cisplatin-induced nephrotoxicity. Our results indicate that H2S may be a novel and promising therapeutic target to prevent cisplatin-induced nephrotoxicity. Antioxid. Redox Signal. 29, 455-470.
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Antineoplásicos/efeitos adversos , Cisplatino/efeitos adversos , Sulfeto de Hidrogênio/farmacologia , Rim/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Linhagem Celular , Cistationina gama-Liase/genética , Cistationina gama-Liase/metabolismo , Ativação Enzimática , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Sulfeto de Hidrogênio/metabolismo , Rim/metabolismo , Rim/patologia , Córtex Renal/efeitos dos fármacos , Córtex Renal/metabolismo , Testes de Função Renal , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NADPH Oxidases/metabolismo , Espécies Reativas de OxigênioRESUMO
5-Florouracil (5-FU) is the basic agent used in the treatment of gastric cancer. Capecitabine, a prodrug of 5-FU, displays increased antitumor efficacy compared with 5-FU in the clinic. 5'-Deoxy-5-fluorouracil (5'-DFUR), the metabolite of capecitabine, is converted to 5-FU by the enzyme thymidine phosphorylase (TP), which is present at high concentrations in human tumors. In this study, we investigated the effect of interferon-α (IFN-α) on the sensitivity of gastric cancer cells to treatment with 5'-DFUR and its relationship with TP expression. Preincubation of gastric cancer cells with IFN-α enhanced 5'-DFUR-induced apoptosis via IFN-α-mediated upregulation of TP. The depletion of TP with small interfering RNA (siRNA) obviously inhibited IFN-α-induced upregulation of TP expression and thus prevented apoptosis induced by IFN-α and 5'-DFUR. Treatment with IFN-α and combined IFN-α and 5'-DFUR treatment were also associated with concomitant activation of ERK signaling. Treatment with the ERK inhibitor PD98059 or depletion of ERK with siRNA partially reversed IFN-α-induced upregulation of TP expression, thus partially preventing apoptosis induced by IFN-α and 5'-DFUR. Taken together, our study shows that IFN-α enhanced 5'-DFUR-induced apoptosis in gastric cancer cells by upregulation of TP expression, which is partially regulated by activation of ERK signaling.