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Plasma homocysteine (Hcy) has been identified as a potential risk factor for cerebral small vessel disease. Cerebral small vessel disease (CSVD) leads to cognitive impairment, depression, and other symptoms and is a common disease in middle-aged and elderly people. To investigate the relationship between 5,10-methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism and CSVD in elderly patients, plasma levels of homocysteine (Hcy) and MTHFR genotyping were assessed. MRI and MRA were performed at the same time to analyze the relationship between different genotypes and cerebrovascular lesions. We showed that Hcy plasma levels in the TT group were significantly higher than those in the CC and CT groups. Moreover, we observed that the severity of white matter lesions was associated with women and positively correlated with age, previous coronary heart disease, luminal infarction, and MTHFR polymorphism. The multivariate logistic regression analysis showed that age, TT genotype, and lacunar infarction were independent risk factors for white matter hyperintensity (WMH). Importantly, we showed that there was a significant correlation between Hcy plasma levels and MTHFR gene polymorphism, with the TT genotype constituting an independent risk factor for WMH. Therefore, we recommended early detection of MTHFR gene polymorphisms with concomitant early intervention concerning risk factors to delay the occurrence of cognitive impairment in CSVD elderly patients.
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Rapid screening of infectious pathogens at the point-of-care (POC) is ideally low-cost, portable, easy to use, and capable of multiplex detection with high sensitivity. However, satisfying all these features in a single device without compromise remains a challenging task. Here, we introduce an ultraportable, automated RNA amplification testing device that allows rapid screening of infectious pathogens from clinical samples. In this device, 3D-printed structural parts incorporated with off-the-shelf mechanic/electronic components are utilized to create an inexpensive and automated droplet manipulation platform. On this platform, a simple configuration that couples a linear displacement of the chip with a tunable magnet array allows parallel and versatile droplet operations, including mixing, splitting, transporting, and merging. By exploiting a multi-channel droplet array chip to preload necessary reagents in "water-in-oil" format, bacteria lysis, RNA extraction and amplification are seamlessly integrated and implemented by the combination of droplet operations. Furthermore, visual readout and geometrically-multiplexed quantitative detection are provided by an integrated wireless video camera-enabled wide-field fluorescence imaging. We demonstrated that this droplet-based device could have a shorter RNA extraction time (12 min) and lower detection limits for pathogenic RNA (approaching to 102 copies per reaction). We also verified its clinical applicability for the rapid screening of four sexually transmitted pathogens from urine specimens. Results show that the sample-to-answer assay could be completed in approximately 42 min, with 100% concordance with the laboratory-based molecular testing. The exhibiting features may render this microdevice an easily accessible POC molecular diagnostic platform for infectious disease, especially in resource-limited settings.
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Técnicas Biossensoriais , Doenças Transmissíveis , Humanos , Sistemas Automatizados de Assistência Junto ao Leito , Testes Imediatos , RNARESUMO
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.
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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
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.
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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
We introduce a novel and versatile microfluidic technology that allows parallel and multi-step bioanalytical procedures to be simply implemented by switching reagent-containing droplet arrays among alternative interaction zones for intended mass or energy transport in a programmable manner. This enables multiplexed complex bioassays for point-of-care testing.
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Bioensaio , Técnicas Analíticas Microfluídicas , Testes Imediatos , Transferência de Energia , Tamanho da PartículaRESUMO
BACKGROUND: A simple and reliable DNA extraction of hepatitis B virus (HBV) is critical in developing an ultrasensitive detection method for HBV infection. Current commercially available serum Hepatitis B Virus (HBV) DNA extraction methods are time-consuming, expensive and/or require specialized equipment, which hinders wide adoption of clinical laboratories. This study offers a report on an ultrasensitive HBV DNA detection method by coupling serum HBV DNA extraction by ultrafiltration (UF) with real-time PCR (qPCR) detection. METHODS: Serum proteins were precipitated by phenol to release HBV DNA in the supernatant which was then transferred to the UF devices. The resultant DNA concentrate was eluted and released into qPCR pre-mixture. The UF-qPCR assay performance, including recovery rate, linearity, detection sensitivity, precision and diagnostic accuracy that compared to the CAP-CTM V2.0 assay by analyzing batched low viral load clinical samples was evaluated. RESULTS: The recovery rate of the UF-based HBV DNA extraction method was above 80%. The assay linearity was demonstrated with a slope of 0.95 and R2 values of 0.99. Limit-of-detection (LOD) of the UF-qPCR assay was determined to be 12.1IU/ml. The coefficient of variation (CV) of HBV quantitation for high, low and limit titer samples was 2.28%, 5.77% and 25.59%, respectively. Accuracy of the UF-qPCR assay was confirmed with the reference panel, and there was a strong correlation between these two methods (R2 = 0.55, p < 0.01). CONCLUSIONS: The UF-qPCR assay is reliable, highly sensitive, affordable and time-saving, and the method can be used for ultrasensitive detection of serum HBV.
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DNA Viral , Vírus da Hepatite B/genética , Hepatite B , Reação em Cadeia da Polimerase em Tempo Real/métodos , DNA Viral/sangue , DNA Viral/genética , DNA Viral/isolamento & purificação , Feminino , Hepatite B/sangue , Hepatite B/diagnóstico , Hepatite B/genética , Humanos , Masculino , Sensibilidade e Especificidade , UltrafiltraçãoRESUMO
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.
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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
The current work presents the development of a capillary-based oscillation droplet approach to maximize the potential of a continuous-flow polymerase chain reaction (PCR). Through the full utilization of interfacial chemistry, a water-in-oil (w/o) droplet was generated by allowing an oil-water plug to flow along a polytetrafluoroethylene (PTFE) capillary. The w/o droplet functioned as the reactor for oscillating-flow PCR to provide a stable reaction environment, accelerate reagent mixing, and eliminate surface adsorption. The capillary PCR approach proposed in the current research offers high amplification efficiency, fast reaction speed, and easy system control attributable to the oscillation droplet reactor. Experimental results show that the droplet-based micro-PCR assay requires lower reaction volume (2 µL) and shorter reaction time (12 min) compared with conventional PCR methods. Taking the amplification of the New Delhi metallo-beta-lactamase (NDM-1) gene as an example, the present work demonstrates that the oscillation droplet PCR assay is capable of achieving high efficiency up to 89.5% and a detection limit of 10 DNA copies. The miniature PCR protocol developed in the current work is fast, robust, and low-cost, thus exhibiting the potential for expansion into various practical applications.
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Miniaturização/instrumentação , Reação em Cadeia da Polimerase/instrumentação , Desenho de Equipamento , Óleos/química , Reação em Cadeia da Polimerase/economia , Politetrafluoretileno/química , Água/químicaRESUMO
OBJECTIVE: To investigate NF-kappaB and IkappaBalpha activities in HL-60 induced by TNF-alpha in order to understand the molecular mechanism of GbE in asthma treatment. METHODS: The amount of IkappaBalpha in HL-60 cells stimulated by TNF-alpha and GbE was measured by western blotting. Plasmid pNF-kappaB-LuC was transfected and NF-kappaB activity was analyzed by measuring the expression level of luciferase. RESULTS: It showed in the luciferase assay that the activity of NF-kappaB could significantly be suppressed in HL-60 cells after the pretreatment with CGbE. However, the phosphorylation and subsequent degradation of IKBalpha induced by TNF-alpha can not be inhibited in HL-60 cells even we prolonged the treatment time or increased the concentration of GhE. CONCLUSION: GhE can suppress the NF-kappaB gene expression actively on independent of NIK/ IKK/ IkappaBalpha pathway in HL-60 cells.
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Ginkgo biloba/química , Proteínas I-kappa B/efeitos dos fármacos , NF-kappa B/efeitos dos fármacos , Extratos Vegetais/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Asma/tratamento farmacológico , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Células HL-60 , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Immunoblotting , NF-kappa B/genética , NF-kappa B/metabolismo , Folhas de Planta/química , Plasmídeos , TransfecçãoRESUMO
OBJECTIVE: To investigate the value of serum soluble CD40 ligand (sCD40L) detection in risk evaluation of acute coronary syndromes (ACS). METHODS: This study involved 200 patients with established diagnosis of ACS, with death or nonfatal myocardial infarction as the end point of observation during the 6-month-long follow-up. Blood samples were obtained from the patients within the initial 72 h of ACS onset, and the levels of sCD40L and C-reactive protein (CRP) were determined with enzyme-linked immunosorbent assay (ELISA). Cardiac troponin I (cTnI) measurement was performed using chemiluminescent immunoassay. RESULTS: Of the 200 patients, 108 had serum sCD40L levels higher than 5.0 microg/L, and the levels of sCD40L, CRP and cTnI were found to significantly correlate with ACS. CONCLUSION: Independent detection of serum sCD40L, CRP and cTnI can help predict the risks of ACS, and their combined measurement may increase the sensitivity of the risk prediction and provide new cardiac makers to replace the cardiac enzymes for laboratory diagnosis and risk evaluation of cardiovascular events.