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Pediatric-onset colitis and inflammatory bowel disease (IBD) have significant effects on the growth of infants and children, but the etiopathogenesis underlying disease subtypes remains incompletely understood. Here, we report single-cell clustering, immune phenotyping, and risk gene analysis for children with undifferentiated colitis, Crohn's disease, and ulcerative colitis. We demonstrate disease-specific characteristics, as well as common pathogenesis marked by impaired cyclic AMP (cAMP)-response signaling. Specifically, infiltration of PDE4B- and TNF-expressing macrophages, decreased abundance of CD39-expressing intraepithelial T cells, and platelet aggregation and release of 5-hydroxytryptamine at the colonic mucosae were common in colitis and IBD patients. Targeting these pathways by using the phosphodiesterase inhibitor dipyridamole restored immune homeostasis and improved colitis symptoms in a pilot study. In summary, comprehensive analysis of the colonic mucosae has uncovered common pathogenesis and therapeutic targets for children with colitis and IBD.
Assuntos
Doenças Inflamatórias Intestinais/patologia , Doenças Inflamatórias Intestinais/terapia , Mucosa Intestinal/patologia , Antígenos CD/metabolismo , Apirase/metabolismo , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Morte Celular/efeitos dos fármacos , Microambiente Celular/efeitos dos fármacos , Criança , Estudos de Coortes , Colo/patologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Dipiridamol/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Predisposição Genética para Doença , Homeostase/efeitos dos fármacos , Humanos , Imunoglobulina G/sangue , Memória Imunológica , Inflamação/patologia , Doenças Inflamatórias Intestinais/sangue , Doenças Inflamatórias Intestinais/genética , Interferon Tipo I/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Metilprednisolona/farmacologia , Células Mieloides/efeitos dos fármacos , Células Mieloides/metabolismoRESUMO
The digital nucleic acid detection assay features the capability of absolute quantitation without the need for calibration, thereby facilitating the rapid identification of pathogens. Although several integrated digital nucleic acid detection techniques have been developed, there are still constraints in terms of automation and analysis throughput. To tackle these challenges, this study presents a digital-to-droplet microfluidic device comprising a digital microfluidics (DMF) module at the bottom and a droplet microfluidics module at the top. Following sample introduction, the extraction of nucleic acid and the dispensation of nucleic acid elution for mixing with the multiple amplification reagents are carried out in the DMF module. Subsequently, the reaction droplets are transported to the sample inlet of the droplet microfluidic module via a liquid outlet, and then droplet generation in four parallel units within the droplet microfluidics module is actuated by negative pressure generated by a syringe vacuum. The digital-to-droplet microfluidic device was employed to execute an integrated multiplex digital droplet nucleic acid detection assay (imDDNA) incorporating loop-mediated isothermal amplification (LAMP). This assay was specifically designed to enable simultaneous detection of four uropathogens, namely, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Enterococcus faecalis. The entire process of the imDDNA is completed within 75 min, with a detection range spanning 5 orders of magnitude (9.43 × 10-2.86 × 104 copies µL-1). The imDDNA was employed for the detection of batched clinical specimens, showing a consistency of 91.1% when compared with that of the conventional method. The imDDNA exhibits simplicity in operation and accuracy in quantification, thus offering potential advantages in achieving rapid pathogen detection.
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Toll-like receptors (TLRs) play a critical role in early immune recognition of Aspergillus, which can regulate host defense during invasive pulmonary Aspergillosis (IPA). However, the role of TLR7 in the pathogenesis of IPA remains unknown. In this study, an in vivo model of IPA was established to investigate the contribution of TLR7 to host anti-Aspergillus immunity upon invasive pulmonary Aspergillus fumigatus infection. The effects of TLR7 on phagocytosis and killing capacities of A. fumigatus by macrophages and neutrophils were investigated in vitro We found that TLR7 knockout mice exhibited lower lung inflammatory response and tissue injury, higher fungal clearance, and greater survival in an in vivo model of IPA compared with wild-type mice. TLR7 activation by R837 ligand led to wild-type mice being more susceptible to invasive pulmonary Aspergillus fumigatus infection. Macrophages, but not neutrophils, were required for the protection against IPA observed in TLR7 knockout mice. Mechanistically, TLR7 impaired phagocytosis and killing of A. fumigatus by macrophages but not neutrophils. Together, these data identify TLR7 as an important negative regulator of anti-Aspergillus innate immunity in IPA, and we propose that targeting TLR7 will be beneficial in the treatment of IPA.
Assuntos
Expressão Gênica , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Aspergilose Pulmonar Invasiva/etiologia , Macrófagos/imunologia , Macrófagos/metabolismo , Receptor 7 Toll-Like/genética , Animais , Biópsia , Citocinas/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Regulação da Expressão Gênica , Imunofenotipagem , Aspergilose Pulmonar Invasiva/metabolismo , Aspergilose Pulmonar Invasiva/patologia , Camundongos , Camundongos Knockout , Fagocitose/genética , Fagocitose/imunologia , Prognóstico , Receptor 7 Toll-Like/agonistas , Receptor 7 Toll-Like/deficiência , Receptor 7 Toll-Like/metabolismoRESUMO
We report a case of melioidosis in China and offer a comparison of 5 commercial detection systems for Burkholderia pseudomallei. The organism was misidentified by the VITEK 2 Compact, Phoenix, VITEK mass spectrometry, and API 20NE systems but was eventually identified by the Bruker Biotyper system and 16S rRNA sequencing.
Assuntos
Burkholderia pseudomallei , Melioidose , China , Humanos , RNA Ribossômico 16S , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
BACKGROUND: The intestinal microbiota plays a crucial role in human health, which could affect host immunity and the susceptibility to infectious diseases. However, the role of intestinal microbiota in the immunopathology of invasive candidiasis remains unknown. METHODS: In this work, an antibiotic cocktail was used to eliminate the intestinal microbiota of conventional-housed (CNV) C57/BL6 mice, and then both antibiotic-treated (ABX) mice and CNV mice were intravenously infected with Candida albicans to investigate their differential responses to infection. Furthermore, fecal microbiota transplantation (FMT) was applied to ABX mice in order to assess its effects on host immunity against invasive candidiasis after restoring the intestinal microbiota, and 16S ribosomal RNA gene sequencing was conducted on fecal samples from both uninfected ABX and CNV group of mice to analyze their microbiomes. RESULTS: We found that ABX mice displayed significantly increased weight loss, mortality, and organ damage during invasive candidiasis when compared with CNV mice, which could be alleviated by FMT. In addition, the level of IL-17A in ABX mice was significantly lower than that in the CNV group during invasive candidiasis. Treatment with recombinant IL-17A could improve the survival of ABX mice during invasive candidiasis. Besides, the microbial diversity of ABX mice was significantly reduced, and the intestinal microbiota structure of ABX mice was significantly deviated from the CNV mice. CONCLUSIONS: Our data revealed that intestinal microbiota plays a protective role in invasive candidiasis by enhancing IL-17A production in our model system.
Assuntos
Candidíase/imunologia , Interleucina-17/farmacologia , Microbiota/fisiologia , Animais , Candidíase/tratamento farmacológico , Transplante de Microbiota Fecal/métodos , Humanos , Interleucina-17/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Microbiota/efeitos dos fármacosRESUMO
Triple-negative breast cancer (TNBC) has a relatively poor outcome. Acquired chemoresistance is a major clinical challenge for TNBC patients. Previously, we reported that kinase-dead Aurora kinase A (Aurora-A) could effectively transactivate the FOXM1 promoter. Here, we demonstrate an additional pathway through which Aurora-A stabilizes FOXM1 by attenuating its ubiquitin in TNBC. Specifically, Aurora-A stabilizes FOXM1 in late M phase and early G1 phase of the cell cycle, which promotes proliferation of TNBC cells. Knock-down of Aurora-A significantly suppresses cell proliferation in TNBC cell lines and can be rescued by FOXM1 overexpression. We observe that paclitaxel-resistant TNBC cells exhibit high expression of Aurora-A and FOXM1. Overexpression of Aurora-A offers TNBC cells an additional growth advantage and protection against paclitaxel. Moreover, Aurora-A and FOXM1 could be simultaneously targeted by thiostrepton. Combination of thiostrepton and paclitaxel treatment reverses paclitaxel resistance and significantly inhibits cell proliferation. In conclusion, our study reveals additional mechanism through which Aurora-A regulates FOXM1 and provides a new therapeutic strategy to treat paclitaxel-resistant triple-negative breast cancer.
Assuntos
Aurora Quinase A/genética , Resistencia a Medicamentos Antineoplásicos/genética , Proteína Forkhead Box M1/genética , Paclitaxel/farmacologia , Neoplasias de Mama Triplo Negativas/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Células MCF-7 , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Tioestreptona/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológicoRESUMO
Identification of new therapeutic targets for the treatment of sepsis is imperative. We report here that cytokine IL-28 (IFN-λ) levels were elevated in clinical and experimental sepsis. Neutralization of IL-28 protected mice from lethal sepsis induced by cecal ligation and puncture (CLP), which was associated with improved bacterial clearance and enhanced neutrophil infiltration. Conversely, administration of recombinant IL-28 aggravated mortality, facilitated bacterial dissimilation and limited neutrophil recruitment, in the model of sepsis induced by CLP. This study defines IL-28 as a detrimental mediator during sepsis and identifies a potential therapeutic target for the immune therapy in sepsis.
Assuntos
Anticorpos Neutralizantes/farmacologia , Citocinas/antagonistas & inibidores , Interleucinas/antagonistas & inibidores , Interleucinas/imunologia , Infiltração de Neutrófilos/efeitos dos fármacos , Sepse/imunologia , Adulto , Idoso , Animais , Ceco/cirurgia , Citocinas/farmacologia , Modelos Animais de Doenças , Feminino , Humanos , Interleucinas/farmacologia , Perfuração Intestinal , Masculino , Camundongos , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Mortalidade , Infiltração de Neutrófilos/imunologiaRESUMO
BACKGROUND: Hepatitis C virus (HCV) infection represents an important risk factor for hepatocellular carcinoma (HCC). Alpha-fetoprotein (AFP) is a widely used biomarker for HCC. However, elevated serum AFP levels in different statuses of chronic hepatitis C (CHC) is not well defined. This study aimed to evaluate the relationship between AFP levels and HCV viral load in CHC patients. We also analyzed the correlation between three liver func-tion enzyme levels (AST, ALT, GGT) and HCV RNA viral load in CHC patients. METHODS: A total of 279 patients infected with HCV were included in this study. Patients were divided into two groups: HCV RNA positive and HCV RNA negative group. Serum HCV RNA load was measured by Quantitative Real-time PCR. Electrochemiluminescence assay (ECLA) was used to determine the serum AFP levels. The differences between two groups in AFP levels and biochemical profile (AST, ALT, GGT) was evaluated. RESULTS: The HCV RNA-positive group had significantly higher serum AFP levels than the negative groups (12.61 vs. 4.72 ng/mL, p < 0.0001). There was no correlation between AFP levels and HCV RNA viral load in HCV infection patients (p = 0.92). A significant correlation was observed between serum ALT (r = 0.243, p = 0.005), GGT (r = 0.212, p = 0.016) levels and HCV RNA viral load. Poor correlation (r = 0.148, p = 0.093) was found between AST levels and HCV RNA viral load. Interestingly, there was a positive correlation (r = 0.337, p < 0.001) between ALT and AFP levels in the whole study population. CONCLUSIONS: We concluded that serum HCV RNA positive patients were candidates for therapeutic prevention of HCC and liver inflammation regardless of the HCV RNA viral load. Furthermore, higher burden of HCV viral load was associated with more severe liver damage.
Assuntos
Biomarcadores/sangue , Hepatite C Crônica/sangue , RNA Viral/sangue , alfa-Fetoproteínas/análise , Feminino , Hepacivirus/genética , Hepacivirus/fisiologia , Hepatite C Crônica/diagnóstico , Hepatite C Crônica/virologia , Humanos , Masculino , Pessoa de Meia-Idade , RNA Viral/genética , Carga Viral/genéticaRESUMO
BACKGROUND: The apolipoprotein E (APOE) ε4 allele is considered as a risk factor for Alzheimer's disease (AD). However, the association of APOE allele with MRI evidence of intracranial lesions has not been well understood. METHODS: Quantitative real-time PCR was performed to detect the APOE genotype; MRI was examined for intracranial lesions. Their association was evaluated in a cohort of 226 AD patients and 2607 healthy individuals in southern China. RESULTS: The frequencies of ε2, ε3, and ε4 alleles were 8.0%, 82.9%, and 9.1% in the whole study population. The frequency of APOE-ε4 allele was significantly higher in the AD subjects than that in the control group (14.4% vs 8.6%, P < 0.001). We found that brain atrophy occurred at a rate of 12.3% in ε4 allele group vs 8.5% in non-ε4 genotype group, with a significance of P = 0.008. Severe brain atrophy occurred at a rate of 1.0% in ε4 allele group vs 0.2% in non-ε4 genotype group (P = 0.011). The individuals carrying APOE ε4/ε4 had an odds ratio (OR) of 7.64 (P < 0.01) for developing AD, while the APOE ε3/ε4 gene carriers had an OR of 1.47 (P = 0.031) and the OR in APOE ε2/ε3 carriers is 0.81 (P = 0.372). Interestingly, we found that the risk of ε4/ε4 allele carrier developing AD was significantly higher in male (P < 0.001) than female (P = 0.478). CONCLUSION: Compared to ε2 and ε3 alleles, the presence of APOE-ε4 allele might increase the risk for AD in a dose-dependent manner in southern China. Moreover, the presence of APOE-ε4 allele results in a higher incidence of brain atrophy.
Assuntos
Doença de Alzheimer/genética , Apolipoproteínas E/genética , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Estudos de Associação Genética , Predisposição Genética para Doença , Imageamento por Ressonância Magnética , Polimorfismo Genético , Alelos , Estudos de Casos e Controles , China , Estudos de Coortes , Feminino , Frequência do Gene , Humanos , Masculino , Razão de Chances , Fatores de RiscoRESUMO
BACKGROUND: Gut microbial dysbiosis contributes to the development of colorectal cancer (CRC). We evaluated the utility of fecal bacterial biomarker candidates identified by our 16S rDNA sequencing analysis for CRC diagnosis. METHODS: We measured the relative abundance of Fusobacterium nucleatum (Fn), Faecalibacterium prausnitzii (Fp), Bifidobacterium (Bb), and Lactobacillus (Lb) by quantitative PCR in fecal samples from 2 cohorts of 903 individuals. We evaluated and validated the diagnostic performance of these microbial ratios and investigated the antagonistic effect of Fn against 3 different indicator stains. RESULTS: The microbial ratio of Fn to Bb (Fn/Bb) had a superior sensitivity of 84.6% and specificity of 92.3% in detecting CRC (area under the curve, AUC = 0.911). The combination of Fn/Bb and Fn/Fp improved the diagnostic value (AUC = 0.943). Moreover, the combination of Fn/Bb and Fn/Fp offered 60.0% specificity and 90.0% sensitivity in detecting stage I of CRC (AUC = 0.804). In particular, Fn was negatively correlated with Fp in the CRC group. The performance for CRC diagnosis was confirmed in the validation cohort II. The culture supernatant from Fn exhibited strong bactericidal activity against probiotics Fp and Bb strains. CONCLUSIONS: This study found that Fn could play a role in microbiota dysbiosis via the secreted antagonistic substances against probiotics. Moreover, the ratio of Fn to the important probiotics Fp and Bb was identified as a valuable biomarker for screening early CRC.
Assuntos
Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/microbiologia , Fezes/microbiologia , Fusobacterium nucleatum/isolamento & purificação , Probióticos/isolamento & purificação , Biomarcadores/análise , DNA Ribossômico/genética , Fusobacterium nucleatum/genética , Microbioma Gastrointestinal , Humanos , Reação em Cadeia da Polimerase/métodos , RNA Ribossômico 16S/genéticaRESUMO
Serum hepatitis B surface antigen (HBsAg) level has been developed as an important marker to predict treatment outcome recent years. The authors aimed to identify the correlation between quantitative HBsAg and hepatitis B virus (HBV) DNA level in chronic hepatitis B (CHB) patients and explore whether quantitative HBsAg can be used as a surrogate marker of serum HBV DNA for CHB patients. One hundred seventy-three patients were included in this study. Patients were divided into two groups: Hepatitis B e antigen (HBeAg) positive and negative patients. There was a positive correlation between quantitative HBsAg and HBV DNA level in HBeAg positive patients (r = 0.509, P < 0.001) and poor correlation in HBeAg negative patients (r = 0.176, P = 0.096). Interestingly, completely no correlation (r = -0.01, P = 0.994) was found in younger HBeAg negative patients (<40 years old), whereas in older HBeAg negative patients (>40 years old) there is a positive correlation (r = 0.448, P = 0.003). Mean HBsAg titer and Alanine aminotransferase (ALT) level were significantly higher in HBeAg positive group (3.81 log10 IU/mL; 105 IU/mL) than in negative group (2.85 log10 â IU/mL; 32 IU/mL) (P < â 0.001). We concluded that quantitative HBsAg could reflect HBV DNA level in HBeAg positive patients, but could not surrogate for HBV DNA level in HBeAg negative patients. Our study improves understanding of the relationship between HBsAg titers and HBV DNA levels in CHB patient and may have implications for future treatment algorithms evaluating the HBsAg titers in both HBeAg positive and negative patients.
Assuntos
DNA Viral/sangue , Antígenos de Superfície da Hepatite B/sangue , Hepatite B Crônica/virologia , Adulto , Alanina Transaminase/sangue , Monitoramento de Medicamentos/métodos , Feminino , Antígenos E da Hepatite B/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Screening for potential drug combinations presents significant challenges to the current microfluidic cell culture systems, due to the requirement of flexibility in liquid handling. To overcome this limitation, we present here an open-access microfluidic tissue array system specifically designed for drug combination screening. The microfluidic chip features a key structure in which a nanoporous membrane is sandwiched by a cell culture chamber array layer and a corresponding media reservoir array layer. The microfluidic approach takes advantage of the characteristics of the nanoporous membrane: on one side, this membrane permits the flow of air but not liquid, thus acting as a flow-stop valve to enable automatic cell distribution; on the other side, it allows diffusion-based media exchange and thus mimics the endothelial layer. In synergy with a liquid-transferring platform, the open-access microfluidic system enables complex multistep operations involving long-term cell culture, medium exchange, multistep drug treatment, and cell-viability testing. By using the microfluidic protocol, a 10 × 10 tissue array was constructed in 90 s, followed by schedule-dependent drug testing. Morphological and immunohistochemical assays indicated that the resultant tumor tissue was faithful to that in vivo. Drug-testing assays showed that the incorporation of the nanoporous membrane further decreased killing efficacy of the anticancer agents, indicating its function as an endothelial layer. Robustness of the microfluidic system was demonstrated by implementing a three-factor, three-level orthogonal screening of anticancer drug combinations, with which 67% of the testing (9 vs. 27) was saved. Experimental results demonstrated that the microfluidic tissue system presented herein is flexible and easy-to-use, thus providing an ideal tool for performing complex multistep cell assays with high efficiencies.
Assuntos
Antineoplásicos/análise , Cisplatino/análise , Doxorrubicina/análise , Técnicas Analíticas Microfluídicas , Paclitaxel/análise , Análise Serial de Tecidos , Antineoplásicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cisplatino/farmacologia , Relação Dose-Resposta a Droga , Doxorrubicina/farmacologia , Avaliação Pré-Clínica de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Paclitaxel/farmacologia , Relação Estrutura-AtividadeRESUMO
A microfluidic chip was developed for one-step identification and antimicrobial susceptibility testing (AST) of multiple uropathogens. The polydimethylsiloxane (PDMS) microchip used had features of cell culture chamber arrays connected through a sample introduction channel. At the bottom of each chamber, a paper substrate preloaded with chromogenic media and antimicrobial agents was embedded. By integrating a hydrophobic membrane valve on the microchip, the urine sample can be equally distributed into and confined in individual chambers. The identification and AST assays on multiple uropathogens were performed by combining the spatial resolution of the cell culture arrays and the color resolution from the chromogenic reaction. The composite microbial testing assay was based on dynamic changes in color in a serial of chambers. The bacterial antimicrobial susceptibility was determined by the lowest concentration of an antimicrobial agent that is capable of inhibiting the chromogenic reaction. Using three common uropathogenic bacteria as test models, the developed microfluidic approach was demonstrated to be able to complete the multiple colorimetric assays in 15 h. The accuracy of the microchip method, in comparison with that of the conventional approach, showed a coincidence of 94.1%. Our data suggest this microfluidic approach will be a promising tool for simple and fast uropathogen testing in resource-limited settings.
Assuntos
Antibacterianos/análise , Técnicas de Cultura de Células , Técnicas Analíticas Microfluídicas , Papel , Antibacterianos/farmacologia , Dimetilpolisiloxanos , Enterococcus faecalis/citologia , Enterococcus faecalis/efeitos dos fármacos , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Staphylococcus aureus/citologia , Staphylococcus aureus/efeitos dos fármacosRESUMO
BACKGROUND/AIM: Triple-negative breast cancer (TNBC) represents a particular clinical challenge because these cancers do not respond to endocrine therapy or other available targeted agents. The lack of effective agents and obvious targets are major challenges in treating TNBC. In this study we explored the cytostatic effect of thiazole ring containing antibiotic drug thiostrepton on TNBC cell lines and investigated the molecular mechanism. METHODS: Cell viability was measured by MTT assay. Cell surface marker was monitored by FCM. Western blot was applied to assess the protein expression levels of target genes. RESULTS: We found that thiostrepton remarkably suppressed the CD44+/CD24- stem-like population and sphere forming capacity of TNBC cell lines. Notably, we showed for the first time that thiostrepton exerted its pharmacological action by targeting sonic hedgehog (SHH) signaling pathway. Thiostrepton repressed SHH ligand expression and reduced Gli-1 nuclear localization in TNBC cell line. Furthermore, the downstream target of SHH signaling undergone dose-dependent, rapid, and sustained loss of mRNA transcript level after thiostrepton treatment. Finally, we showed that SHH ligand was essential for maintaining CD44+/CD24- stem-like population in TNBC cell line. CONCLUSION: We conclude that thiostrepton suppresses the CD44+/CD24- stem-like population through inhibition of SHH signaling pathway. Our results give a new insight into the mechanism of thiostrepton anti-tumor activity and suggest thiostrepton as a promising agent that targets hedgehog signaling pathway in TNBC.
Assuntos
Antineoplásicos/farmacologia , Proteínas Hedgehog/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Esferoides Celulares/efeitos dos fármacos , Tioestreptona/farmacologia , Neoplasias de Mama Triplo Negativas/metabolismo , Antibacterianos/farmacologia , Antígeno CD24/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas Hedgehog/genética , Humanos , Receptores de Hialuronatos/metabolismo , Células MCF-7 , Células-Tronco Neoplásicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esferoides Celulares/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Early and rapid diagnostic of acute myocardial infarction (AMI) during its developing stage is crucial due to its high fatality rate. Heart-type fatty acid binding protein (h-FABP) is an ideal biomarker for the quantitative diagnosis of AMI, surpassing traditional markers such as myoglobin, creatine phosphokinase-MB, and troponin in terms of sensitivity, specificity, and prognostic value. To obtain diagnostic and prognostic information, a precise and fully quantitative measurement of h-FABP is essential, typically achieved through an immunosorbent assay like the enzyme-linked immunosorbent assay. Nevertheless, this method has several limitations, including extended detection time, complex assay procedures, the necessity for skilled technicians, and challenges in implementing automated detection. This research introduces a novel biosensor, utilizing aggregation-induced emission nanoparticles (AIENPs) and integrated with a digital microfluidic (DMF) workstation, designed for the sensitive, rapid, and automated detection of h-FABP in low-volume serum samples. AIENPs and magnetic beads in nanoscale were served as the capture particles and the fluorescent probe, which were linked covalently to anti-h-FABP antibodies respectively. The approach was based on a sandwich immunoassay and performed on a fully automated DMF workstation with assay time by 15 min. We demonstrated the determination of h-FABP in serum samples with detection limit of 0.14 ng/mL using this biosensor under optimal condition. Furthermore, excellent correlations (R2 = 0.9536, n = 50) were obtained between utilizing this biosensor and commercialized ELISA kits in clinical serum detecting. These results demonstrate that our flexible and reliable biosensor is suitable for direct integration into clinical diagnostics, and it is expected to be promising diagnostic tool for early detection and screening tests as well as prognosis evaluation for AMI patients.
Assuntos
Técnicas Biossensoriais , Proteína 3 Ligante de Ácido Graxo , Infarto do Miocárdio , Nanopartículas , Técnicas Biossensoriais/instrumentação , Humanos , Proteína 3 Ligante de Ácido Graxo/sangue , Infarto do Miocárdio/diagnóstico , Infarto do Miocárdio/sangue , Nanopartículas/química , Limite de Detecção , Biomarcadores/sangue , Proteínas de Ligação a Ácido Graxo/sangue , Proteínas de Ligação a Ácido Graxo/análise , Imunoensaio/métodos , Imunoensaio/instrumentação , Microfluídica/métodos , Desenho de Equipamento , Anticorpos Imobilizados/química , Ensaio de Imunoadsorção EnzimáticaRESUMO
Candidemia is a severe disease with high mortality in both intensive care unit (ICU) and non-ICU settings. Considering that progranulin (PGRN) is a potential therapeutic target for the candidemia caused by Candida albicans, we determined the serum level of PGRN after candidemia and evaluated its association with mortality. A retrospective discovery cohort (62 patients) and a validation cohort (70 patients) were enrolled. Blood was collected on day of first blood culture positivity for C. albicans, and serum PGRN levels were then measured. In the discovery cohort, all serum PGRN studied were expressed at higher levels in candidemia patients than in bacteremia patients and healthy volunteers, non-survivors presented with significantly higher serum PGRN concentrations when compared with survivors. Serum PGRN concentration was associated with 30-day mortality and patients at a higher risk of death showed higher serum PGRN levels. These results were confirmed in the independent validation cohort. Interestingly, in vitro study demonstrated that macrophages, neutrophils and lymphocytes may be the major source of PGRN production after C. albicans infection instead of epithelial cells. Our findings highlight that serum PGRN appears as a biomarker in candidemia patients and as a promising tool for mortality risk stratification in managing candidemia.
Assuntos
Biomarcadores , Candidemia , Estado Terminal , Progranulinas , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Biomarcadores/sangue , Candida albicans , Candidemia/mortalidade , Candidemia/sangue , Candidemia/microbiologia , Estado Terminal/mortalidade , Unidades de Terapia Intensiva , Progranulinas/sangue , Estudos RetrospectivosRESUMO
Loss-of-function mutations in CREBBP, which encodes for a histone acetyltransferase, occur frequently in B-cell malignancies, highlighting CREBBP deficiency as an attractive therapeutic target. Using established isogenic cell models, we demonstrated that CREBBP-deficient cells are selectively vulnerable to AURKA inhibition. Mechanistically, we found that co-targeting CREBBP and AURKA suppressed MYC transcriptionally and post-translationally to induce replication stress and apoptosis. Inhibition of AURKA dramatically decreased MYC protein level in CREBBP-deficient cells, implying a dependency on AURKA to sustain MYC stability. Furthermore, in vivo studies showed that pharmacological inhibition of AURKA was efficacious in delaying tumor progression in CREBBP-deficient cells and was synergistic with CREBBP inhibitors in CREBBP-proficient cells. Our study sheds light on a novel synthetic lethal interaction between CREBBP and AURKA, indicating that targeting AURKA represents a potential therapeutic strategy for high-risk B-cell malignancies harboring CREBBP inactivating mutations.
Assuntos
Aurora Quinase A , Proteína de Ligação a CREB , Proteínas Proto-Oncogênicas c-myc , Mutações Sintéticas Letais , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Aurora Quinase A/genética , Aurora Quinase A/metabolismo , Aurora Quinase A/antagonistas & inibidores , Humanos , Animais , Camundongos , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Apoptose/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: The aminoglycoside-resistance genes encoding aminoglycoside modifying enzymes and 16S rRNA methyltransferases are main factors contributing to increasing resistance to aminoglycosides. Characterization and distribution of antimicrobial resistance gene profiles provide important information on the potential difficulty of treatment of bacteria. Several molecular methods have been developed to investigate the prevalence of aminoglycoside-resistance genes. These existing methods are time-consuming, labor-intensive, expensive or limited sensitivity in the epidemiological investigation. Therefore, it is necessary to develop a rapid, less-costly and high throughput and sensitive method to investigate the distribution of antimicrobial resistance gene in clinical isolates. RESULTS: In this study, we developed a GeXP analyzer-based multiplex PCR assay to simultaneously detect seven aminoglycoside-resistance genes, including aac(3)-II, aac(6')-Ib, aac(6')-II, ant(3â³)-I,aph(3')-VI,armA and rmtB, and to analyze the distribution of these genes in clinical Enterobacteriaceae isolates. Under optimized conditions, this assay achieved a limit-of-detection as low as 10 copies of each of the seven genes. The presented method was applied to analyze the distribution of aminoglycoside-resistance genes in 56 clinical Enterobacteriaceae isolates, and the results were compared with that of the conventional single PCR assay. Kappa values of the two methods for detecting each of the seven resistance genes were 0.831, 0.846, 0.810, 0.909, 0.887, 0.810 and 0.825, respectively. CONCLUSION: This GeXP assay is demonstrated to be a rapid, cost-effective and high throughput method with high sensitivity and specificity for simultaneously detecting seven common aminoglycoside-resistance genes.
Assuntos
Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Enterobacteriaceae/efeitos dos fármacos , Genes Bacterianos , Ensaios de Triagem em Larga Escala/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Enterobacteriaceae/genética , Humanos , Testes de Sensibilidade Microbiana/métodos , Sensibilidade e EspecificidadeRESUMO
Since COVID-19 might have a lasting impact on global public health, it is crucial to analyze its effect on drug-resistant bacterial infections in the respiratory system for the prevention and control of hospital infections. This work aimed to investigate the impact of the COVID-19 outbreak on the clinical distribution and antibiotic resistance of bacterial infection among hospitalized patients in the respiratory unit in order to establish strategies to control antibiotic-resistant infections. Electronic clinical data registry records from 2018 to 2022 were retrospectively analyzed. A total of 36,829 clinical specimens, including sputum, bronchoalveolar lavage fluid, blood, and urine, were collected from 16,073 patients admitted to the Guangzhou First People's Hospital from January 2018 to December 2022. Among them, 2209 samples were culture-positive. The bacterial isolation rates of different types of samples showed a similar trend from 2019 to 2022, with an increase in 2020 and 2022 and a decrease in 2021. Different bacterial species were separated from different types of samples. The most reported pathogens were identified in sputum samples. Gram-positive isolates were prevalent in urine samples, while Gram-negative bacilli were the predominant pathogenic bacteria isolated from respiratory tract and blood samples. Pseudomonas aeruginosa (P. aeruginosa), Acinetobacter baumannii (A. baumannii) complex, and Klebsiella pneumoniae (K. pneumoniae) were the most abundant Gram-negative bacteria in sputum samples, of which A. baumannii complex had the highest resistance to all tested antibiotics except colistin. Notably, there has been a substantial prevalence of carbapenem-resistant P. aeruginosa, A. baumannii, and K. pneumoniae in the past five years. This alarming situation calls for greater attention and precaution with prescribed antibiotics to limit the generation and spread of new multidrug-resistant bacteria and improve therapeutic management.