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BACKGROUND: The COVID-19 pandemic is protracted and episodic surges from viral variants continue to place significant strain on healthcare systems. COVID-19 vaccines, antiviral therapy and monoclonal antibodies have significantly reduced COVID-19 associated morbidity and mortality. Concurrently, telemedicine has gained acceptance as a model of care and a tool for remote monitoring. These advances allow us to safely transit our inpatient-based care for COVID-19 infected kidney transplant recipients (KTRs) to a hospital-at-home (HaH) model of care. METHODS: KTRs with PCR-proven COVID-19 infection were triaged by teleconsult and laboratory tests. Suitable patients were enrolled into the HaH. Remote monitoring via teleconsults were conducted daily until patients were de-isolated based on a time-based criterion. Monoclonal antibodies were administered in a dedicated clinic where indicated. RESULTS: Eighty-one KTRs with COVID-19 were enrolled into the HaH between February and June 2022, 70 (86.4%) completed HaH recovery without complications. Eleven (13.6%) patients required inpatient hospitalization for medical issues (n = 8) and weekend monoclonal antibody infusion (n = 3). Patients requiring inpatient hospitalization had longer transplant vintage (15 years vs. 10 years, p = .03), anaemia (haemoglobin 11.6 g/dL vs. 13.1 g/dL, p = .01), lower eGFR (39.8 vs. 62.9 mL/min/1.73 m2 , p < .05) and lower RBD levels (<50 AU/mL vs. 1435 AU/mL, p = .02). HaH saved 753 inpatient patient-days with no deaths observed. Hospital admission rates from the HaH programme was 13.6%. Patients who required inpatient care had direct access admission without utilization of emergency department resources. CONCLUSION: Selected KTRs with COVID-19 infection can be safely managed in a HaH programme; alleviating strain on inpatient and emergency healthcare resources.
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COVID-19 , Transplante de Rim , Humanos , Anticorpos Monoclonais , COVID-19/epidemiologia , COVID-19/terapia , Hospitais , Pacientes AmbulatoriaisRESUMO
The purpose of this literature review was to explore the qualitative evidence on coping strategies used by patients with end stage kidney disease (ESKD) to manage the challenges and outcomes associated with the condition. A systematic review design following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines was used, and a thematic analysis was used to analyze the data. Four themes were identified from the 14 selected studies: external support, emotion management strategies, reliance on faith or spirituality, and self-care practices. Implications of these findings are discussed. Further primary qualitative studies using interviews and focus groups are needed to gain additional in-depth evidence of ESKD-related coping strategies.
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Adaptação Psicológica , Falência Renal Crônica , Diálise Renal , Humanos , Falência Renal Crônica/psicologia , Falência Renal Crônica/terapia , Pesquisa Qualitativa , EspiritualidadeRESUMO
This protocol describes how to prepare mouse brain tissue for quantification of multiple inflammatory mediators using a multiplex bead-based immunoassay. It is important to have methods that allow quantification of multiple analytes from small amounts of tissue. Bio-Plex is a Luminex xMAP-based multiplex bead-based immunoassay technology that permits simultaneous analysis of up to 100 analytes from a single tissue sample. This assay has been used extensively to investigate analytes in plasma and serum samples as well as cultured and primary cells. Here, we describe a method for simultaneous analysis of 33 different inflammatory cytokines and chemokines from mouse brain tissue using the Bio-Plex Pro Mouse Chemokine Panel 33-Plex.
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Bioensaio/métodos , Quimiocinas/análise , Citocinas/análise , Ensaios de Triagem em Larga Escala/métodos , Malária Cerebral/diagnóstico , Animais , Bioensaio/instrumentação , Biomarcadores/análise , Encéfalo/imunologia , Encéfalo/patologia , Quimiocinas/imunologia , Citocinas/imunologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática/instrumentação , Ensaio de Imunoadsorção Enzimática/métodos , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Malária Cerebral/imunologia , Malária Cerebral/parasitologia , Malária Cerebral/patologia , Camundongos , Microesferas , Plasmodium berghei/imunologiaRESUMO
BACKGROUND: Gemcitabine is used as a standard drug treatment for non-small cell lung cancer (NSCLC), but treatment responses vary among patients. Our previous studies demonstrated that POLA2 + 1747 GG/GA single nucleotide polymorphism (SNP) improves differential survivability and mortality in NSCLC patients. Here, we determined the association between POLA2 and gemcitabine treatment in human lung cancer cells. RESULTS: Human PC9, H1299 and H1650 lung cancer cell lines were treated with 0.01-100 µM gemcitabine for 72 h. Although all 3 cell lines showed decreased cell viability upon gemcitabine treatment, H1299 was found to be the most sensitive to gemcitabine treatment. Next, sequencing was performed to determine if POLA2 + 1747 SNP might be involved in gemcitabine sensitivity. Data revealed that all 3 cell lines harbored the wild-type POLA2 + 1747 GG SNP, indicating that the POLA2 + 1747 SNP might not be responsible for gemcitabine sensitivity in the cell lines studied. Silencing of POLA2 gene in H1299 was then carried out by siRNA transfection, followed by gemcitabine treatment to determine the effect of POLA2 knockdown on chemosensitivity to gemcitabine. Results showed that H1299 exhibited increased resistance to gemcitabine after POLA2 knockdown, suggesting that POLA2 does not act alone and may cooperate with other interacting partners to cause gemcitabine resistance. CONCLUSIONS: Collectively, our findings showed that knockdown of POLA2 increases gemcitabine resistance in human lung cancer cells. We propose that POLA2 may play a role in gemcitabine sensitivity and can be used as a prognostic biomarker of patient outcome in NSCLC pathogenesis.
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Antimetabólitos Antineoplásicos/farmacologia , DNA Polimerase I/genética , Desoxicitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pulmonares/genética , Linhagem Celular Tumoral , Mapeamento Cromossômico , Biologia Computacional/métodos , Desoxicitidina/farmacologia , Epistasia Genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Polimorfismo de Nucleotídeo Único , GencitabinaRESUMO
BACKGROUND: Platinum based therapy is commonly used in the treatment of advanced gastric cancer. However, resistance to chemotherapy is a major challenge that causes marked variation in individual response rate and survival rate. In this study, we aimed to identify the expression of GTSE1 and its correlation with cisplatin resistance in gastric cancer cells. METHODS: Methylation profiling was carried out in tissue samples from gastric cancer patients before undergoing neoadjuvent therapy using docetaxel, cisplatin and 5FU (DCX) and in gastric cancer cell lines. The correlation between GTSE1 expression and methylation in gastric cancer cells was determined by RT-PCR and MSP respectively. GTSE1 expression was knocked-down using shRNA's and its effects on cisplatin cytotoxicity and cell survival were detected by MTS, proliferation and clonogenic survival assays. Additionally, the effect of GTSE1 knock down in drug induced apoptosis was determined by western blotting and apoptosis assays. RESULTS: GTSE1 exhibited a differential methylation index in gastric cancer patients and in cell lines that correlated with DCX treatment response and cisplatin sensitivity, respectively. In-vitro, GTSE1 expression showed a direct correlation with hypomethylation. Interestingly, Cisplatin treatment induced a dose dependent up regulation as well as nuclear translocation of GTSE1 expression in gastric cancer cells. Knock down of GTSE1 enhanced cisplatin cytotoxity and led to a significant reduction in cell proliferation and clonogenic survival. Also, loss of GTSE1 expression caused a significant increase in P53 mediated apoptosis in cisplatin treated cells. CONCLUSION: Our study identifies GTSE1 as a biomarker for cisplatin resistance in gastric cancer cells. This study also suggests the repressive role of GTSE1 in cisplatin induced apoptosis and signifies its potential utility as a therapeutic target for better clinical management of gastric cancer patients.
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Antineoplásicos/administração & dosagem , Cisplatino/administração & dosagem , Resistencia a Medicamentos Antineoplásicos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Neoplasias Gástricas/tratamento farmacológico , Adulto , Idoso , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Metilação de DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Transdução de Sinais/efeitos dos fármacos , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologiaRESUMO
Dispersal is an important process that is widely studied across species, and it can be influenced by intrinsic and extrinsic factors. Intrinsic factors commonly assessed include the sex and age of individuals, while landscape features are frequently-tested extrinsic factors. Here, we investigated the effects of both sex and landscape composition and configuration on genetic distances among bare-nosed wombats (Vombatus ursinus)-one of the largest fossorial mammals in the world and subject to habitat fragmentation, threats from disease, and human persecution including culling as an agricultural pest. We analyzed a data set comprising 74 Tasmanian individuals (30 males and 44 females), genotyped for 9,064 single-nucleotide polymorphisms. We tested for sex-biased dispersal and the influence of landscape features on genetic distances including land use, water, vegetation, elevation, and topographic ruggedness. We detected significant female-biased dispersal, which may be related to females donating burrows to their offspring due to the energetic cost of excavation, given their large body sizes. Land use, waterbodies, and elevation appeared to be significant landscape predictors of genetic distance. Land use potentially reflects land clearing and persecution over the last 200 years. If our findings based on a limited sample size are valid, retention and restoration of nonanthropogenic landscapes in which wombats can move and burrow may be important for gene flow and maintenance of genetic diversity.
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This prospective study used antibody suspension bead arrays to identify biomarkers capable of predicting post-operative recurrence with distal metastasis in patients with colorectal cancer. One hundred colorectal cancer patients who underwent surgery were enrolled in this study. The median follow-up period was 3.9 years. The pre-operative plasma concentrations of 24 angiogenesis-related molecules were analyzed with regard to the TNM stage and the development of post-operative recurrence. The concentrations of half of the examined molecules (13/24) increased significantly according to the TNM stage (P < 0.05). Meanwhile, a multivariate logistic regression analysis revealed that the concentrations of vascular cell adhesion molecule 1 (VCAM-1) and plasminogen activator inhibitor-1 (PAI-1) were significantly higher in the post-operative recurrence group. The VCAM-1 and PAI-1 model discriminated post-operative recurrence with an area under the curve of 0.82, a sensitivity of 0.75, and a specificity of 0.73. A leave-one-out cross-validation was applied to the model to assess the prediction performance, and the result indicated that the cross-validated error rate was 12.5% (12/96). In conclusion, our results demonstrate that antibody suspension bead arrays are a powerful tool to screen biomarkers in the clinical setting, and the plasma levels of VCAM-1 and PAI-1 together may be a promising biomarker for predicting post-operative recurrence in patients with colorectal cancer.
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Biomarcadores Tumorais/sangue , Neoplasias Colorretais/sangue , Recidiva Local de Neoplasia/sangue , Inibidor 1 de Ativador de Plasminogênio/sangue , Molécula 1 de Adesão de Célula Vascular/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/patologia , Neoplasias Colorretais/cirurgia , Feminino , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Estadiamento de NeoplasiasRESUMO
Exportin-1 (XPO1) controls the nucleo-cytoplasmic trafficking of several key growth regulatory and tumor suppressor proteins. Nuclear export blockade through XPO1 inhibition is a target for therapeutic inhibition in many cancers. Studies have suggested XPO1 upregulation as an indicator of poor prognosis in gastric cancer. In the current study, we investigated the anti-tumor efficacy of selective inhibitors of nuclear export (SINE) compounds KPT-185, KTP-276 and clinical stage selinexor (KPT-330) in gastric cancer. XPO1 was found to be overexpressed in gastric cancer as compared to adjacent normal tissues and was correlated with poor survival outcomes. Among the 3 SINE compounds, in vitro targeting of XPO1 with selinexor resulted in greatest potency with significant anti-proliferative effects at nano molar concentrations. XPO1 inhibition by selinexor resulted in nuclear accumulation of p53, causing cell cycle arrest and apoptosis. Also, inhibition of XPO1 lead to the cytoplasmic retention of p21 and suppression of survivin. Orally administered selienxor caused significant inhibition of tumor growth in xenograft models of gastric cancer. Furthermore, combination of selinexor with irinotecan exhibited greater anti-tumor effect compared to individual treatment. Taken together, our study underscores the therapeutic utility of XPO1 targeting in gastric cancer and suggests the potential benefits of XPO1 inhibition in-combination with chemotherapy.
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Antineoplásicos/farmacologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Hidrazinas/farmacologia , Neoplasias Gástricas/patologia , Triazóis/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Adulto , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Masculino , Camundongos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The problem hindering the use of data-driven modelling methods for training controllers on-line is the lack of control over the amount by which the plant is excited. As the operating schedule determines the information available on-line, the knowledge of the process may degrade if the setpoint remains constant for an extended period. This paper proposes an identification algorithm that alleviates "learning interference" by incorporating fuzzy theory into the normalized least-mean-square update rule. The ability of the proposed methodology to achieve faster learning is examined by employing the algorithm to train a neurofuzzy feedforward controller for controlling a liquid level process. Since the proposed identification strategy has similarities with the normalized least-mean-square update rule and the recursive least-square estimator, the on-line learning rates of these algorithms are also compared.
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Algoritmos , Inteligência Artificial , Modelos Estatísticos , Redes Neurais de Computação , Reconhecimento Automatizado de Padrão/métodos , Simulação por Computador , Retroalimentação , Análise dos Mínimos Quadrados , Sistemas On-LineRESUMO
Increasingly, genetic algorithms (GAs) are used to optimize the parameters of fuzzy logic controllers (FLCs). Although GAs provide a systematic design approach, the optimization process is generally performed off-line using a plant model. Differences between the model and physical plant may result in unsatisfactory control performance when the FLCs are deployed in practice. Type-2 FLCs are an attractive alternative because they can better cope with modeling uncertainties. Unfortunately, type-2 FLCs are computationally intensive. This paper presents a simplified type-2 FLC that is suitable for real-time applications. The key idea is to only replace some critical type-1 fuzzy sets by type-2 sets. Experimental results indicate that the proposed simplified type-2 FLC is as robust as a conventional type-2 FLC, while lowering the computational cost.
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Tissue samples (plasma, saliva, serum or urine) from 169 patients classified as either normal or having one of seven possible diseases are analysed across three 96-well plates for the presences of 37 analytes using cytokine inflammation multiplexed immunoassay panels. Censoring for concentration data caused problems for analysis of the low abundant analytes. Using fluorescence analysis over concentration based analysis allowed analysis of these low abundant analytes. Mixed-effects analysis on the resulting fluorescence and concentration responses reveals a combination of censoring and mapping the fluorescence responses to concentration values, through a 5PL curve, changed observed analyte concentrations. Simulation verifies this, by showing a dependence on the mean florescence response and its distribution on the observed analyte concentration levels. Differences from normality, in the fluorescence responses, can lead to differences in concentration estimates and unreliable probabilities for treatment effects. It is seen that when fluorescence responses are normally distributed, probabilities of treatment effects for fluorescence based t-tests has greater statistical power than the same probabilities from concentration based t-tests. We add evidence that the fluorescence response, unlike concentration values, doesn't require censoring and we show with respect to differential analysis on the fluorescence responses that background correction is not required.
Assuntos
Citocinas/sangue , Técnicas Imunoenzimáticas/normas , Espectrometria de Fluorescência/normas , Análise de Variância , Artrite Reumatoide/sangue , Artrite Reumatoide/diagnóstico , Artrite Reumatoide/imunologia , Estudos de Casos e Controles , Citocinas/imunologia , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/imunologia , Humanos , Mononucleose Infecciosa/sangue , Mononucleose Infecciosa/diagnóstico , Mononucleose Infecciosa/imunologia , Mieloma Múltiplo/sangue , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/imunologia , Psoríase/sangue , Psoríase/diagnóstico , Psoríase/imunologia , Doença Pulmonar Obstrutiva Crônica/sangue , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Doença Pulmonar Obstrutiva Crônica/imunologia , Kit de Reagentes para Diagnóstico/normas , Sepse/sangue , Sepse/diagnóstico , Sepse/imunologiaRESUMO
BACKGROUND: Melanoma-associated antigen (MAGE)-A3 is a member of the family of cancer-testis antigens and has been found to be epigenetically regulated and aberrantly expressed in various cancer types. It has also been found that MAGE-A3 expression may correlate with an aggressive clinical course and with chemo-resistance. The objectives of this study were to assess the relationship between MAGE-A3 promoter methylation and expression and (1) gastric cancer patient survival and (2) its functional consequences in gastric cancer-derived cells. METHODS: Samples from two independent gastric cancer cohorts (including matched non-malignant gastric samples) were included in this study. MAGE-A3 methylation and mRNA expression levels were determined by methylation-specific PCR (MSP) and quantitative real-time PCR (qPCR), respectively. MAGE-A3 expression was knocked down in MKN1 gastric cancer-derived cells using miRNAs. In addition, in vitro cell proliferation, colony formation, apoptosis, cell cycle, drug treatment, immunohistochemistry and Western blot assays were performed. RESULTS: Clinical analysis of 223 primary patient-derived samples (ntumor = 161, nnormal = 62) showed a significant inverse correlation between MAGE-A3 promoter methylation and expression in the cancer samples (R = -0.63, p = 5.99e-19). A lower MAGE-A3 methylation level was found to be associated with a worse patient survival (HR: 1.5, 95 % CI: 1.02-2.37, p = 0.04). In addition, we found that miRNA-mediated knockdown of MAGE-A3 expression in MKN1 cells caused a reduction in its proliferation and colony forming capacities, respectively. Under stress conditions MAGE-A3 was found to regulate the expression of Bax and p21. MAGE-A3 knock down also led to an increase in Puma and Noxa expression, thus contributing to an enhanced docetaxel sensitivity in the gastric cancer-derived cells. CONCLUSIONS: From our results we conclude that MAGE-A3 expression is regulated epigenetically by promoter methylation, and that its expression contributes to gastric cell proliferation and drug sensitivity. This study underscores the potential implications of MAGE-A3 as a therapeutic target and prognostic marker in gastric cancer patients.
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Antígenos de Neoplasias/metabolismo , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Neoplasias/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Antígenos de Neoplasias/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Docetaxel , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Humanos , Proteínas de Neoplasias/genética , Regiões Promotoras Genéticas , Estresse Fisiológico/efeitos dos fármacos , Análise de Sobrevida , Taxoides/farmacologia , Ensaio Tumoral de Célula-TroncoRESUMO
In cancer biology, cells and molecules that form the fundamental components of the tumor microenvironment play a major role in tumor initiation, and progression as well as responses to therapy. Therapeutic approaches that would enable and harness the immune system to target tumor cells mark the future of anticancer therapy as it could induce an immunological memory specific to the tumor type and further enhance tumor regression and relapse-free survival in cancer patients. Gastric cancer is one of the leading causes of cancer-related mortalities that has a modest survival benefit from existing treatment options. The advent of immunotherapy presents us with new approaches in gastric cancer treatment where adaptive cell therapies, cancer vaccines, and antibody therapies have all been used with promising outcomes. In this paper, we review the current advances and prospects in the gastric cancer immunotherapy. Special focus is laid on new strategies and clinical trials that attempt to enhance the efficacy of various immunotherapeutic modalities in gastric cancer.
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Fenômenos do Sistema Imunitário , Imunoterapia/métodos , Neoplasias Gástricas/imunologia , Neoplasias Gástricas/terapia , Vacinas Anticâncer/imunologia , Ensaios Clínicos como Assunto , Humanos , Memória Imunológica , Monitorização Imunológica , Microambiente TumoralRESUMO
INTRODUCTION: Weight management programmes (WMPs) can help overweight individuals lose weight, and thus prevent complications associated with obesity. Herein, we describe the demographic profile, clinical characteristics, motivations and expectations, and outcomes of patients enrolled in a nonsurgical WMP. METHODS: This was a retrospective study of consecutive patients with a body mass index (BMI) of > 23 kg/m2 enrolled in the four-month WMP at the Health For Life Clinic, Alexandra Hospital, Singapore, between 1 and 31 August 2009. Demographic data, medical history and source of referral were recorded. Details on personal motivations and weight loss goals were obtained from the completed self-administered questionnaires of the WMP participants. Weight, waist circumference, fat percentage and BMI were measured at the start and end of the WMP. A weight loss of ≥ 5% was deemed as a successful outcome. RESULTS: A total of 58 patients (mean age 37.2 years) were included in our study. Of these 58 patients, 58.6% were of Chinese ethnicity and 55.2% were male. Many patients (32.8%) attributed their weight gain to work- or study-related stress, and a minority to poor eating habits (12.1%) or a lack of exercise (10.3%). Patients' motivations included a desire for better health (53.4%) and better fitness (15.5%). However, only 53.4% patients scored their motivation as high (i.e. a score of > 7). The mean expected weight loss was 9.9 kg at 4 months, and 14.1 kg at 12 months. Among the 40 patients (69.0%) who completed the programme, the mean percentage weight loss was 1.8 ± 4.3%. A weight loss of ≥ 5% was achieved by 8 (13.8%) patients. CONCLUSION: Although the patients in our study cohort were young and educated, only a portion of them appeared to be highly motivated to lose weight, despite joining the WMP. There is a need for patients to be guided on how to set realistic weight loss goals.
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Motivação , Redução de Peso , Programas de Redução de Peso/métodos , Adulto , Idoso , Índice de Massa Corporal , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Obesidade/psicologia , Obesidade/terapia , Sobrepeso/psicologia , Sobrepeso/terapia , Estudos Retrospectivos , Singapura , Inquéritos e Questionários , Resultado do Tratamento , Adulto JovemRESUMO
We report the demonstration of miniaturized capillary isoelectric focusing (CIEF) in plastic microfluidic devices. Conventional CIEF technique was adapted to the microfluidic devices to separate proteins and to detect protein-protein interactions. Both acidic and basic proteins with isoelectric points (pI) ranging from 5.4 to 11.0 were rapidly focused, mobilized, and detected in a 1.2 cm long channel (50 microm deep x 120 microm wide) with a total analysis time of 150 s. In a device with a focusing distance of 4.7 cm, the separation efficiency for a basic protein, lysozyme, was achieved as high as 1.5 x 10(5) plates, corresponding to 3.2 million plates per meter. We also experimentally confirmed that IEF resolution is essentially independent of focusing length when the applied voltage is kept the same and within a range that it does not cause Joule heating. Further, we demonstrated the use of miniaturized CIEF to study the interactions between two pairs of proteins, immunoglobulin G (IgG) with protein G and anti-six histidine (anti-6xHis) with 6xHis-tagged green fluorescent protein (GFP). Using this approach, protein-protein interactions can be detected for as little as 50 fmol of protein. We believe miniaturized CIEF is useful for studying protein-protein interactions when there is a difference in pI between a protein-protein complex and its constitutent proteins.
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Focalização Isoelétrica/métodos , Proteínas/isolamento & purificação , Eletroforese Capilar/instrumentação , Eletroforese Capilar/métodos , Proteínas de Fluorescência Verde , Imunoglobulina G/isolamento & purificação , Focalização Isoelétrica/instrumentação , Proteínas Luminescentes/isolamento & purificação , Miniaturização , Proteínas do Tecido Nervoso/isolamento & purificação , Ligação ProteicaRESUMO
An integrated plastic microfluidic device was designed and fabricated for bacterial detection and identification. The device, made from poly(cyclic olefin) with integrated graphite ink electrodes and photopatterned gel domains, accomplishes DNA amplification, microfluidic valving, sample injection, on-column labeling, and separation. Polymerase chain reaction (PCR) is conducted in a channel reactor containing a volume as small as 29 nL; thermal cycling utilizes screen-printed graphite ink resistors. In situ gel polymerization was employed to form local microfluidic valves that minimize convective flow of the PCR mixture into other regions. After PCR, amplicons (products) are electrokinetically injected through the gel valve, followed by on-chip electrophoretic separation. An intercalating dye is admixed to label the amplicons; they are detected using laser-induced fluorescence. Two model bacteria, Escherichia coli O157 and Salmonella typhimurium, were chosen to demonstrate bacterial detection and identification based on amplification of several of their unique DNA sequences. The limit of detection is about six copies of target DNA.
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Bactérias/genética , Bactérias/isolamento & purificação , Eletroforese/instrumentação , Eletroforese/métodos , Reação em Cadeia da Polimerase/métodos , DNA Bacteriano/análise , Escherichia coli O157/genética , Escherichia coli O157/isolamento & purificação , Fluorescência , Microfluídica/instrumentação , Microfluídica/métodos , Plásticos , Salmonella typhimurium/genética , Salmonella typhimurium/isolamento & purificação , Temperatura , Fatores de TempoRESUMO
Four oligonucleotides (fluorescently labeled and unlabeled 16- and 90-mer), each containing a single adduct of benzo[a]pyrene diol epoxide (BPDE), were synthesized and used to study the binding stoichiometry between the DNA adduct and its antibody. The free oligonucleotide and its complexes with mouse monoclonal antibody were separated using capillary electrophoresis and detected with laser-induced fluorescence (LIF). Two complexes, representing the 1:1 and 1:2 stoichiometry between the antibody and the DNA adduct, were clearly demonstrated. The stoichiometry depended upon the relative concentrations of the antibody and the DNA adducts. A new approach examining the binding of the antibody with a mixture of a tetramethylrhodamine (TMR)-labeled and unlabeled BPDE-16-mer revealed insights on ligand redistribution and exchange between the labeled and unlabeled BPDE-16-mer oligonucleotides in the complexes. The observation of this unique behavior has not been possible previously with other binding studies. A mixture of the antibody with the TMR-labeled BPDE- 16-mer and an unlabeled BPDE-90-mer further revealed the formation of three fluorescent complexes: antibody with one TMR-BPDE-16-mer molecule, antibody with two TMR-BPDE- 16-mer molecules, and antibody with one TMR-BPDE-16-mer and one BPDE-90-mer. The three complexes clearly demonstrated binding stoichiometry and ligand redistribution/exchange.
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Carcinógenos/análise , Adutos de DNA/imunologia , 7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido , Animais , Anticorpos Monoclonais/química , Ligação Competitiva , Adutos de DNA/análise , Corantes Fluorescentes , Humanos , Sondas de Oligonucleotídeos/síntese química , Sondas de Oligonucleotídeos/imunologiaRESUMO
We report an accurate and reproducible DNA quantitation method using the polymerase chain reaction (PCR). The amount of PCR product is monitored after each PCR cycle by capillary electrophoresis. To ensure accurate quantitation, a non-amplified internal standard is added to each PCR-amplified electrophoresis sample to correct for variations in injection volume. Quantitation of the sample is based on the number of cycles necessary to generate a predetermined amount of PCR product. Duck hepatitis B virus genome was used as a model in this study. The genome was quantified with a linear relationship between cycle number and logarithm of sample DNA for amounts of sample DNA between 30 and 3.1 x 10(8) copies ( r(2)>0.999). The relative standard deviation for the corrected capillary electrophoresis signal was 2.7%, while the relative standard deviation for the overall assay was 3.0%. Results from a single-blind study generated a relative error of 1.3%.