RESUMO
The prevalence of hepatitis B virus (HBV) is a global healthcare threat, particularly chronic hepatitis B (CHB) that might lead to hepatocellular carcinoma (HCC) should not be neglected. Although many types of HBV diagnosis detection methods are available, some technical challenges, such as the high cost or lack of practical feasibility, need to be overcome. In this study, the polycrystalline silicon nanowire field-effect transistors (pSiNWFETs) were fabricated through commercial process technology and then chemically functionalized for sensing hepatitis B virus surface antigen (HBsAg) and hepatitis B virus X protein (HBx) at the femto-molar level. These two proteins have been suggested to be related to the HCC development, while the former is also the hallmark for HBV diagnosis, and the latter is an RNA-binding protein. Interestingly, these two proteins carried opposite net charges, which could serve as complementary candidates for evaluating the charge-based sensing mechanism in the pSiNWFET. The measurements on the threshold voltage shifts of pSiNWFETs showed a consistent correspondence to the polarity of the charges on the proteins studied. We believe that this report can pave the way towards developing an approachable tool for biomedical applications.
Assuntos
Antígenos de Superfície da Hepatite B/análise , Hepatite B/diagnóstico , Nanofios , Transativadores/análise , Proteínas Virais Reguladoras e Acessórias/análise , Carcinoma Hepatocelular , Atenção à Saúde , Vírus da Hepatite B , Humanos , Neoplasias Hepáticas , SilícioRESUMO
Detecting proteins at low concentrations in high-ionic-strength conditions by silicon nanowire field-effect transistors (SiNWFETs) is severely hindered due to the weakened signal, primarily caused by screening effects. In this study, aptamer as a signal amplifier, which has already been reported by our group, is integrated into SiNWFET immunosensors employing antigen-binding fragments (Fab) as the receptors to improve its detection limit for the first time. The Fab-SiNWFET immunosensors were developed by immobilizing Fab onto Si surfaces modified with either 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde (GA) (Fab/APTES-SiNWFETs), or mixed self-assembled monolayers (mSAMs) of polyethylene glycol (PEG) and GA (Fab/PEG-SiNWFETs), to detect the rabbit IgG at different concentrations in a high-ionic-strength environment (150 mM Bis-Tris Propane) followed by incubation with R18, an aptamer which can specifically target rabbit IgG, for signal enhancement. Empirical results revealed that the signal produced by the sensors with Fab probes was greatly enhanced compared to the ones with whole antibody (Wab) after detecting similar concentrations of rabbit IgG. The Fab/PEG-SiNWFET immunosensors exhibited an especially improved limit of detection to determine the IgG level down to 1 pg/mL, which has not been achieved by the Wab/PEG-SiNWFET immunosensors.
Assuntos
Técnicas Biossensoriais , Nanofios , Animais , Imunoensaio , Limite de Detecção , Proteínas/análise , Coelhos , SilícioRESUMO
Although in situ atomic force microscopy (AFM) allows single-molecule detection of antibody-antigen binding, the practical applications of in situ AFM for disease diagnosis are greatly limited, due to its operational complexity and long operational times, including the execution time for the surface chemical/biological treatments in the equipped glass liquid cell. Herein, a method of graphically superimposed alignment that enables ex situ AFM analysis of an immobilized antibody at the same location on a semiconductor chip surface before and after incubation with its antigen is presented. All of the required chemical/biological treatments are executed feasibly using standard laboratory containers, allowing single-molecule ex situ AFM detection to be conducted with great practicality, flexibility, and versatility. As an example, the analysis of hepatitis B virus X protein (HBx) and its IgG antibody is described. Using ex situ AFM, individual information on the topographical characteristics of the immobilized single and aggregated IgG antibodies on the chip surface is extracted and the data are analyzed statistically. Furthermore, in a statistical manner, the changes in AFM-measured heights of the individual and aggregated IgG antibodies that occur as a result of changes in conformation upon formation of IgG-HBx complexes are investigated.
RESUMO
Protein tyrosine sulfation (PTS), a vital post-translational modification, facilitates protein-protein interactions and regulates many physiological and pathological responses. Monitoring PTS has been difficult owing to the instability of sulfated proteins and the lack of a suitable method for detecting the protein sulfate ester. In this study, we combined an in situ PTS system with a high-sensitivity polysilicon nanowire field-effect transistor (pSNWFET)-based sensor to directly monitor PTS formation. A peptide containing the tyrosine sulfation site of P-selectin glycoprotein ligand (PSGL)-1 was immobilized onto the surface of the pSNWFET by using 3-aminopropyltriethoxysilane and glutaraldehyde as linker molecules. A coupled enzyme sulfation system consisting of tyrosylprotein sulfotransferase and phenol sulfotransferase was used to catalyze PTS of the immobilized PSGL-1 peptide. Enzyme-catalyzed sulfation of the immobilized peptide was readily observed through the shift of the drain current-gate voltage curves of the pSNWFET before and after PTS. We expect that this approach can be developed as a next generation biochip for biomedical research and industries.
Assuntos
Técnicas Biossensoriais , Nanofios , Processamento de Proteína Pós-Traducional , Glicoproteínas de Membrana , Peptídeos , Silício , Tirosina/análogos & derivadosRESUMO
Debulking surgery followed by systemic chemotherapy-including three-weekly intravenous paclitaxel and carboplatin (GOG-158)-is the cornerstone for advanced epithelial ovarian, fallopian tubal, and peritoneal cancer (EOC) treatment. In this scenario, Federation of Gynecology and Obstetrics (FIGO) stage, cell types, completeness of surgery, lymph nodes (LN) status, adjuvant chemotherapy regimens, survival status, progression-free survival (PFS), and overall survival (OS) of 192 patients diagnosed as having stage IIIA1-IVB EOC over January 2008-December 2017 were analyzed retrospectively. Of them, 100 (52.1%) patients had been debulked optimally. Of all cases, 64.1% and 10.9% demonstrated serous and clear-cell carcinoma. Moreover, the FIGO stage, surgery completeness, and LN status affected recurrence/persistence and mortality (all p < 0.001). Clear cell carcinoma led to shorter survival than serous carcinoma (p = 0.002). Adjuvant chemotherapy regimens were divided into five main groups according to previous clinical trials. However, choice of chemotherapy failed to demonstrate significant differences in patient outcomes. Similar results were found in the sub-analysis of optimally debulked cases, except that intraperitoneal chemotherapy could reduce mortality risk when compared with GOG-158 (p = 0.042). Notably, retroperitoneal LN dissection in all cases or optimally debulked cases reduced risks of recurrence/persistence and mortality, and prolonged PFS and OS significantly (all p < 0.05). Without optimal debulking, LN dissection led to little improvement in outcomes. Various modified chemotherapy regimens did not prolong PFS and OS or reduce recurrence/persistence and mortality risks. LN dissection is strongly recommended to improve the completeness of surgery and patient outcome. Clear cell type has a poorer outcome than serous type, which requires more aggressive treatment and follow-up.
Assuntos
Quimioterapia Adjuvante , Neoplasias das Tubas Uterinas , Neoplasias Ovarianas , Neoplasias Peritoneais , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias das Tubas Uterinas/tratamento farmacológico , Neoplasias das Tubas Uterinas/cirurgia , Feminino , Humanos , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Estadiamento de Neoplasias , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/cirurgia , Neoplasias Peritoneais/tratamento farmacológico , Neoplasias Peritoneais/cirurgia , Estudos Retrospectivos , Resultado do TratamentoRESUMO
The pandemic outbreaks of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread all over the world in a short period of time. Efficient identification of the infection by SARS-CoV-2 has been one of the most important tasks to facilitate all the following counter measurements in dealing with the infectious disease. In Taiwan, a COVID-19 Open Science Platform adheres to the spirit of open science: sharing sources, data, and methods to promote progress in academic research while corroborating findings from various disciplines has established in mid-February 2020, for collaborative research in support of the development of detection methods, therapeutics, and a vaccine for COVID-19. Research priorities include infection control, epidemiology, clinical characterization and management, detection methods (including viral RNA detection, viral antigen detection, and serum antibody detection), therapeutics (neutralizing antibody and small molecule drugs), vaccines, and SARS-CoV-2 pathogenesis. In addition, research on social ethics and the law are included to take full account of the impact of the COVID-19 virus.
Assuntos
Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , COVID-19 , Teste para COVID-19 , Infecções por Coronavirus/virologia , Proteínas do Nucleocapsídeo de Coronavírus , Humanos , Proteínas do Nucleocapsídeo/isolamento & purificação , Pandemias , Fosfoproteínas , Pneumonia Viral/virologia , RNA Viral/isolamento & purificação , SARS-CoV-2 , Sensibilidade e Especificidade , Glicoproteína da Espícula de Coronavírus/isolamento & purificaçãoRESUMO
Ovarian clear cell carcinoma (OCCC) is the second common histology of epithelial ovarian cancer in Taiwan. Stage IC is common, especially during minimally invasive surgery. Adjuvant chemotherapy in stage IC OCCC is unavoidable, and paclitaxel-based chemotherapy in Taiwan is self-paid. However, surgical spillage from minimally invasive surgery as a cause of unfavorable prognosis is still uncertain. The information of patients with stage IC OCCC, corresponding to a period of January 1995 to December 2016, was retrospectively collected following a chart and pathology review. Data regarding surgical methods, cytology status, regimens of adjuvant chemotherapy, survivorship, progression-free survival (PFS), and overall survival (OS) period were analyzed. In total, 88 patients were analyzed, and 64 and 24 patients were treated with paclitaxel- and nonpaclitaxel-based chemotherapy, respectively. Recurrence was identical between the two groups: PFS (47.5 ± 41.36 versus 54.0 ± 53.9 months, p = 0.157) and OS (53.5 ± 38.14 versus 79.0 ± 49.42 months, p = 0.070). Of the 88 patients, 12 had undergone laparoscopy for histological confirmation before complete open staging surgery; however, their PFS (49.5 ± 46.84 versus 49.0 ± 35.55 months, p = 0.719) and OS (56.5 ± 43.4 versus 51.0 ± 32.77 months, p = 0.600) were still comparable. Cytology results were only available for 51 patients, and positive washing cytology results seemed to worsen PFS (p = 0.026) but not OS (p = 0.446). In conclusion, adjuvant nonpaclitaxel chemotherapy and laparoscopic tumor spillage before the staging operation did not worsen the outcome in stage IC OCCC. Positive washing cytology has a negative effect on PFS but not on OS.
Assuntos
Adenocarcinoma de Células Claras , Antineoplásicos/uso terapêutico , Neoplasias Ovarianas , Adenocarcinoma de Células Claras/tratamento farmacológico , Adenocarcinoma de Células Claras/mortalidade , Adenocarcinoma de Células Claras/patologia , Adenocarcinoma de Células Claras/cirurgia , Adulto , Quimioterapia Adjuvante , Feminino , Humanos , Laparoscopia , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/cirurgia , Paclitaxel/uso terapêutico , Análise de SobrevidaRESUMO
BACKGROUND: Sarcopenia is commonly observed in patients with advanced-stage epithelial ovarian cancer (EOC). However, the effect of body composition changes-during primary debulking surgery (PDS) and adjuvant platinum-based chemotherapy-on outcomes of patients with advanced-stage EOC is unknown. This study aimed to evaluate the association between body composition changes and outcomes of patients with stage III EOC treated with PDS and adjuvant platinum-based chemotherapy. METHODS: Pre-treatment and post-treatment computed tomography (CT) images of 139 patients with stage III EOC were analysed. All CT images were contrast-enhanced scans and were acquired according to a standardized protocol. The skeletal muscle index (SMI), skeletal muscle radiodensity (SMD), and total adipose tissue index were measured using CT images obtained at the L3 vertebral level. Predictors of overall survival were identified using Cox regression models. RESULTS: The median follow-up was 37.9 months. The median duration between pre-treatment and post-treatment CT was 182 days (interquartile range: 161-225 days). Patients experienced an average SMI loss of 1.8%/180 days (95% confidence interval: -3.1 to -0.4; P = 0.01) and SMD loss of 1.7%/180 days (95% confidence interval: -3.3 to -0.03; P = 0.046). SMI and SMD changes were weakly correlated with body mass index changes (Spearman ρ for SMI, 0.15, P = 0.07; ρ for SMD, 0.02, P = 0.82). The modified Glasgow prognostic score was associated with SMI loss (odds ratio: 2.42, 95% confidence interval: 1.03-5.69; P = 0.04). The median time to disease recurrence was significantly shorter in patients with SMI loss ≥5% after treatment than in those with SMI loss <5% or gain (5.4 vs. 11.2 months, P = 0.01). Pre-treatment SMI (1 cm2 /m2 decrease; hazard ratio: 1.08, 95% confidence interval: 1.03-1.11; P = 0.002) and SMI change (1%/180 days decrease; hazard ratio: 1.04, 95% confidence interval: 1.01-1.08; P = 0.002) were independently associated with poorer overall survival. SMD, body mass index, and total adipose tissue index at baseline and changes were not associated with overall survival. CONCLUSIONS: Skeletal muscle index decreased significantly during treatment and was independently associated with poor overall survival in patients with stage III EOC treated with PDS and adjuvant platinum-based chemotherapy. The modified Glasgow prognostic score might be a predictor of SMI loss during treatment.
Assuntos
Procedimentos Cirúrgicos de Citorredução/métodos , Músculo Esquelético/patologia , Neoplasias Ovarianas/complicações , Neoplasias Ovarianas/tratamento farmacológico , Feminino , Humanos , Pessoa de Meia-Idade , Neoplasias Ovarianas/mortalidade , PrognósticoRESUMO
Silicon nanowire field-effect transistors (SiNW-FETs) have been demonstrated as a highly sensitive platform for label-free detection of a variety of biological and chemical entities. However, detecting signal from immunoassays by nano-FETs is severely hindered by the distribution of different charged groups of targeted entities, their binding orientation, and distances to the surface of the FET. Aptamers have been widely applied as a recognition element for plentiful biosensors because of small molecular sizes and moderate to high specific binding affinity with different types of molecules. In this study, we propose an effective approach to enhance the electrical responses of both direct (6×-histidine) and sandwich (amyloid ß 1-42) immunoassays in SiNW-FETs with R18, a highly negative charged RNA aptamer against rabbit immunoglobulin G (IgG). Empirical results presented that the immunosensors targeted with R18 expressed a significantly stabilized and amplified signal compared to the ones without this aptamer. The research outcome provides applicability of the highly negative charged aptamer as a bioamplifier for immunoassays by FETs.
RESUMO
Silicon nanowire (SiNW) field-effect transistors (FETs) is a powerful tool in genetic molecule analysis because of their high sensitivity, short detection time, and label-free detection. In nucleic acid detection, GC-rich nucleic acid sequences form self- and cross-dimers and stem-loop structures, which can easily obtain data containing signals from nonspecific DNA binding. The features of GC-rich nucleic acid sequences cause inaccuracies in nucleic acid detection and hinder the development of precision medicine. To improve the inaccurate detection results, we used phosphate-methylated (neutral) nucleotides to synthesize the neutralized chimeric DNA oligomer probe. The probe fragment originated from a primer for the detection of hepatitis C virus (HCV) genotype 3b, and single-mismatched and perfect-matched targets were designed for single nucleotide polymorphisms (SNP) detection on the SiNW FET device. Experimental results revealed that the HCV-3b chimeric neutralized DNA (nDNA) probe exhibited better performance for SNP discrimination in 10 mM bis-tris propane buffer at 25 °C than a regular DNA probe. The SNP discrimination of the nDNA probe could be further improved at 40 °C on the FET device. Consequently, the neutralized chimeric DNA probe could successfully distinguish SNP in the detection of GC-rich target sequences under optimal operating conditions on the SiNW FET device.
Assuntos
Técnicas Biossensoriais/métodos , Sondas de DNA/genética , Técnicas de Genotipagem/métodos , Nanofios/química , Transistores Eletrônicos , Polimorfismo de Nucleotídeo Único , Sensibilidade e Especificidade , Silício/químicaRESUMO
Genetic epidemiological studies show that genetic factors contribute significantly to cervical cancer carcinogenesis. Several genome-wide association studies (GWAS) have revealed novel genetic variants associated with cervical cancer susceptibility. We aim to replicate 4 GWAS-identified single nucleotide polymorphisms (SNPs), which were associated with invasive cervical cancer in Chinese women, in a Taiwanese population. The rs13117307 C/T, rs8067378 A/G, rs4282438 G/T, and rs9277952 A/G SNPs were genotyped in 507 women with cervical squamous cell carcinoma (CSCC) and 432 age/sex matched healthy controls by using TaqMan PCR Assay. Human papillomavirus (HPV) DNA test and typing were performed in CSCC patients. Only the rs4282438 SNP was found to be significantly associated (G allele, odds ratio [OR] = 0.67, P = 1.5 × 10-5). This protective association remained in HPV-16 positive CSCC subgroup (G allele, OR = 0.60, P = 1.2 × 10-5). In conclusion, our study confirms the association of rs4282438 SNP with CSCC in a Taiwanese population. However, larger sample sets of other ethnic groups are required to confirm these findings.
Assuntos
Carcinoma de Células Escamosas/genética , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único , Neoplasias do Colo do Útero/genética , Adulto , Povo Asiático/genética , Estudos de Casos e Controles , Feminino , Estudos de Associação Genética , Humanos , Pessoa de Meia-Idade , TaiwanRESUMO
Protein tyrosine sulfation (PTS), catalyzed by membrane-anchored tyrosylprotein sulfotransferase (TPST), is one of the most common post-translational modifications of secretory and transmembrane proteins. PTS, a key modulator of extracellular protein-protein interactions, accounts for various important biological activities, namely, virus entry, inflammation, coagulation, and sterility. The preparation and characterization of TPST is fundamental for understanding the synthesis of tyrosine-sulfated proteins and for studying PTS in biology. A sulfated protein was prepared using a TPST-coupled protein sulfation system that involves the generation of the active sulfate 3'-phosphoadenosine-5'-phosphosulfate (PAPS) through either PAPS synthetase (PAPSS) or phenol sulfotransferase. The preparation of sulfated proteins was confirmed through radiometric or immunochemical assays. In this study, enzymatically active Drosophila melanogaster TPST (DmTPST) and human TPSTs (hTPST1 and hTPST2) were expressed in Escherichia coli BL21(DE3) host cells and purified to homogeneity in high yield. Our results revealed that recombinant DmTPST was particularly useful considering its catalytic efficiency and ease of preparation in large quantities. This study provides tools for high-efficiency, one-step synthesis of sulfated proteins and peptides that are useful for further deciphering the mechanisms, functions, and future applications of PTS.
RESUMO
Neutral DNA analogs as probes for the detection of target oligomers on the biosensors based on the field-effect transistor (FET) configuration feature advantages in the enhancement of sensitivity and signal-to-noise ratio. Herein, we used phosphate-methylated nucleotides to synthesize two partially neutralized chimeric DNA products and a fully neutralized DNA sequence and adopted a regular DNA oligomer as probes on the polycrystalline silicon nanowire (NW) FET devices. The sequences of two neutralized chimeric DNAs close to the 5' end were alternately modified with the phosphate-methylated nucleotides, and all probes were immobilized via their 5' end on the NW surface. The non-specific-to-specific binding ratio indicated that the two 5'-end partially neutralized chimeric DNAs featured better performance than the regular and fully neutralized DNA oligomers. The partially neutralized probe design reduces the ionic strength needed for hybridization and increases the Debye length of detection, thus promoting the detection sensitivity of FET and achieving the limit of detection of 0.1 fM. By using an appropriate probe design, applying DNA oligomers with embedded phosphate-methylated nucleotides in the FET biosensors is a promising way for gene detection with high sensitivity and specificity.
Assuntos
Aptâmeros de Nucleotídeos/síntese química , Sondas de DNA/síntese química , Nanofios/química , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Técnicas Biossensoriais/instrumentação , DNA/genética , Desenho de Equipamento/métodos , Hibridização de Ácido Nucleico , Nucleotídeos/síntese química , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Sensibilidade e Especificidade , Silício/química , Transistores EletrônicosRESUMO
An implementation of neutralized chimeric DNA oligomer as a probe for sensitive detection of single nucleotide polymorphisms (SNPs) on a surface plasmon resonance imaging sensor is investigated. The chimeric DNA oligomer was synthesized in a conventional DNA synthesizer, containing neutral nucleotides with a methylated phosphate group. The secondary structures and melting points of the chimeric DNA fragment and its complexes with perfect-matched and single-mismatched complementary DNA molecules were examined by using circular dichroism and UV-vis spectroscopy in comparison with the native probe DNA counterpart. The results indicate that the chimeric DNA complexes can form a B-form structure and exhibit high thermostability. Moreover, the hybridization and discrimination efficiency of the chimeric probe DNA for the SNP genotyping were verified by using the SPRi sensor under different experimental conditions. The data reveal the effects of the ionic strength and operation temperature on the selectivity of the chimeric probe DNA for the SNP detection. The hybridization condition with a low ionic strength and high temperature allows the chimeric probe DNA distinguishing perfect-matched and single-mismatched target DNA molecules to the best extent, likely due to the reduced electrostatic repulsive force and presence of the additional methyl group on the backbone. Consequently, the direct and label-free detection with the SPR technique and neutralized chimeric probe DNA can be realized for the SNP genotyping by optimizing the operation condition and sequence design.
Assuntos
Técnicas Biossensoriais , Polimorfismo de Nucleotídeo Único/genética , Ressonância de Plasmônio de Superfície , Dicroísmo Circular , Sondas de DNA , Genótipo , Hibridização de Ácido NucleicoRESUMO
Protein tyrosine sulfation (PTS) is a key modulator of extracellular protein-protein interaction (PPI), which regulates principal biological processes. For example, the capsid protein VP1 of enterovirus 71 (EV71) specifically interacts with sulfated P-selectin glycoprotein ligand-1 (PSGL-1) to facilitate virus invasion. Currently available methods cannot be used to directly observe PTS-induced PPI. In this study, atomic force microscopy was used to measure the interaction between sulfated or mutated PSGL-1 and VP1. We found that the binding strength increased by 6.7-fold following PTS treatment on PSGL-1 with a specific antisulfotyrosine antibody. Similar results were obtained when the antisulfotyrosine antibody was replaced with the VP1 protein of EV71; however, the interaction forces of VP1 were only approximately one-third of those of the antisulfotyrosine antibody. We also found that PTS on the tyrosine-51 residue of glutathione S-transferases fusion-PSGL-1 was mainly responsible for the PTS-induced PPI. Our results contribute to the fundamental understanding of PPI regulated through PTS.
Assuntos
Proteínas do Capsídeo/fisiologia , Glicoproteínas de Membrana/fisiologia , Microscopia de Força Atômica , Proteínas Virais/fisiologia , Glutationa Transferase/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Glicoproteínas de Membrana/genética , Mutação , Tirosina/metabolismo , Ligação ViralRESUMO
Interlocking nailing is a common surgical operation to stabilize fractures in long bones. One of the difficult parts of the surgery is how to locate the position and direction of a screw hole on the interlocking nail, which is invisible to the naked eye after insertion of the nail into the medullary canal. Here, we propose a novel two-stage targeting process using two passive magnetic devices to locate the position and direction of the screw hole without radiation for the locking screw procedure. This involves a ring-shape positioning magnet inside the nail to generate a magnetic field for targeting. From the accuracy test results of these two-stage targeting devices, the search region can be identified in less than 20 seconds by the 1st-stage targeting device, while the total targeting time to locate the drilling position and direction takes less than 4 minutes, with 100% successful rate in 50 attempts. The drilling test further combines the two-stage targeting process and drilling process on the swine tibia, and it is shown that a 100% successful rate is achieved in all 10 attempts, where the total time needed is less than 5 minutes.
Assuntos
Pinos Ortopédicos , Fixação Intramedular de Fraturas/instrumentação , Magnetismo , Fraturas da Tíbia/cirurgia , Animais , Humanos , SuínosRESUMO
B-cell-activating factor (BAFF) belongs to the tumor necrosis factor family that not only stimulates B and T cells but also counteracts immune tolerance. BAFF is also a type II membrane protein, which is secreted through the endoplasmic reticulum (ER)-Golgi apparatus pathway. Fusing an antigen to BAFF might enhance the presentation of major histocompatibility complex class I molecules. These characteristics represent an opportunity to enhance the antitumor effects of DNA vaccines. Therefore, we fused BAFF to human papillomavirus type 16 E7 as a DNA vaccine and evaluated its antitumor effects. We found that this vaccine increased E7-specific CD8+ T-cell immune responses, engendered major antitumor effects against E7-expressing tumors, and prolonged the survival of the immunized mice. Interestingly, vaccinating B-cell-deficient mice with BAFF-E7 revealed considerable E7-specific CD8+ T-cell immune responses, suggesting that B cells do not contribute to this immune response. Image analysis through confocal fluorescence microscopy revealed that fusing BAFF to E7 targeted the protein to the ER, but not BAFF lacking 128 N-terminal residues that generated a lower number of E7-specific CD8+ T cells in the vaccinated mice. Our data indicated that the ER-targeting characteristic of BAFF is the main factor improving the potency of DNA vaccines.
Assuntos
Fator Ativador de Células B/genética , Vacinas Anticâncer/administração & dosagem , Carcinoma Pulmonar de Células não Pequenas/virologia , Neoplasias Pulmonares/virologia , Proteínas E7 de Papillomavirus/genética , Infecções por Papillomavirus/prevenção & controle , Vacinas de DNA/administração & dosagem , Animais , Fator Ativador de Células B/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Vacinas Anticâncer/imunologia , Carcinoma Pulmonar de Células não Pequenas/prevenção & controle , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Pulmonares/prevenção & controle , Camundongos , Papillomaviridae/genética , Papillomaviridae/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Vacinas de DNA/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Protein tyrosine sulfation (PTS) is a widespread posttranslational modification that induces intercellular and extracellular responses by regulating protein-protein interactions and enzymatic activity. Although PTS affects numerous physiological and pathological processes, only a small fraction of the total predicted sulfated proteins has been identified to date. Here, we localized the potential sulfation sites of Escherichia coli proteins on a proteome microarray by using a 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthase-coupled tyrosylprotein sulfotransferase (TPST) catalysis system that involves in situ PAPS generation and TPST catalysis. Among the 4256 E. coli K12 proteins, 875 sulfated proteins were identified using antisulfotyrosine primary and Cy3-labeled antimouse secondary antibodies. Our findings add considerably to the list of potential proteins subjected to tyrosine sulfation. Similar procedures can be applied to identify sulfated proteins in yeast and human proteome microarrays, and we expect such approaches to contribute substantially to the understanding of important human diseases.
Assuntos
Proteínas de Escherichia coli/análise , Proteínas de Escherichia coli/química , Ensaios de Triagem em Larga Escala , Análise Serial de Proteínas , Proteoma , Tirosina/análogos & derivados , Animais , Drosophila melanogaster/enzimologia , Escherichia coli K12 , Proteínas de Escherichia coli/genética , Humanos , Complexos Multienzimáticos/isolamento & purificação , Complexos Multienzimáticos/metabolismo , Sulfato Adenililtransferase/isolamento & purificação , Sulfato Adenililtransferase/metabolismo , Sulfotransferases/isolamento & purificação , Sulfotransferases/metabolismo , Tirosina/químicaRESUMO
Host immunogenetic background plays an important role in human papillomavirus (HPV) infection and cervical cancer development. Inositol 1,4,5-triphosphate receptor type 3 (ITPR3) is essential for both immune activation and cancer pathogenesis. We aim to investigate if ITPR3 genetic polymorphisms are associated with the risk of cervical cancer in Taiwanese women. ITPR3 rs3748079 A/G and rs2229634 C/T polymorphisms were genotyped in a hospital-based study of 462 women with cervical squamous cell carcinoma (CSCC) and 921 age-matched healthy control women. The presence and genotypes of HPV in CSCC was determined. No significant association of individual ITPR3 variants were found among controls, CSCC, and HPV-16 positive CSCC. However, we found a significant association of haplotype AT between CSCC and controls (OR = 2.28, 95% CI 1.31-3.97, P = 2.83 × 10-3) and the OR increased further in CSCC patients infected with HPV-16 (OR = 2.89, 95% CI 1.55-5.37, P = 4.54 × 10-4). The linkage disequilibrium analysis demonstrated that ITPR3 association with CSCC was independent of HLA-DRB1 alleles. In conclusion, these findings suggest that AT haplotype in the ITPR3 gene may serve as a potential marker for genetic susceptibility to CSCC.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Haplótipos , Receptores de Inositol 1,4,5-Trifosfato/genética , Polimorfismo de Nucleotídeo Único , Neoplasias do Colo do Útero/genética , Adulto , Idoso , Povo Asiático/genética , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/etnologia , Carcinoma de Células Escamosas/virologia , Estudos de Casos e Controles , Distribuição de Qui-Quadrado , Feminino , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Cadeias HLA-DRB1/genética , Heterozigoto , Homozigoto , Papillomavirus Humano 16/patogenicidade , Humanos , Desequilíbrio de Ligação , Pessoa de Meia-Idade , Razão de Chances , Infecções por Papillomavirus/virologia , Fenótipo , Medição de Risco , Fatores de Risco , Taiwan , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/etnologia , Neoplasias do Colo do Útero/virologiaRESUMO
Human papillomavirus (HPV) infection and the fate of HPV infected cervical epithelial cells are strictly associated with cervical cancer development. P2X7 receptor has been implicated in both the regulation of immune responses and apoptosis of cervical cancer cells. The study aims to investigate if polymorphisms in the P2RX7 gene are associated with the risk of cervical cancer in Taiwanese women. P2RX7 253 T/C, 835 G/A, and 1513 A/C loss-of-function polymorphisms were genotyped in a hospital-based study of 507 women with cervical squamous cell carcinoma (CSCC) and 1619 age-matched healthy control women. The presence and genotypes of HPV in CSCC was determined. The frequency of 253 C/C genotype was found to increase significantly in patients with HPV-16 positive CSCC compared with controls (odds ratio = 10.2, 95% confidence interval 1.39-87.8, Pc = 0.03). No significant associations were found for other 2 polymorphisms. Analysis of haplotypes also revealed no significant differences among women with CSCC, those with HPV-16 positive CSCC and controls. In conclusion, inheritance of the C/C genotype at position 253 in the P2RX7 gene may contribute to the risk of HPV-16 associated CSCC in Taiwanese women.