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OBJECTIVES: Autoimmune hepatitis (AIH) is an aberrant autoimmune condition mediated by T cell abnormality, which may cause fulminant liver failure and persistent liver injury. This study aimed to disclose the histopathological and functional engagement of interleukin (IL)-26, a potent inflammation mediator, in AIH disease progression. METHODS: We conducted immunohistochemical staining on liver biopsy samples to evaluate intrahepatic expression of IL-26. Cellular sources of hepatic IL-26 were detected by confocal microscopy. Flow cytometry was employed to determine the immunological alterations of CD4+ and CD8+ T cells following in vitro IL-26 treatment on primary peripheral blood mononuclear cells from healthy controls. RESULTS: Statistically significant increase in IL-26 level was observed in AIH (n = 48) liver samples in comparison with patients having chronic hepatitis B (n = 25), nonalcoholic fatty liver disease (n = 18), and healthy donors for living donor liver transplantation (n = 10). The number of intrahepatic IL-26+ cells was positively correlated with histological and serological severity. An immunofluorescence staining indicated that liver-infiltrating CD4+ T cells, CD8+ T cells, and CD68+ macrophages orchestrated IL-26 secretion in AIH. Both CD4+ and CD8+ T cells demonstrated effective activation, lytic, and proinflammatory functions upon IL-26 stimulation. CONCLUSION: We observed elevated IL-26 in AIH liver which promoted T cell activation and cytotoxic capacity, indicating a therapeutic potential of IL-26 intervention in AIH.
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Hepatite Autoimune , Transplante de Fígado , Humanos , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Fígado/patologia , Doadores VivosRESUMO
Quantitative polymerase chain reaction as a powerful tool for DNA detection has been pivotal to a vast range of applications, including disease screening, food safety assessment, environmental monitoring, and many others. However, the essential target amplification step in combination with fluorescence readout poses a significant challenge to rapid and streamlined analysis. The discovery and engineering of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) technology have recently paved the way for a novel approach to nucleic acid detection, but the majority of current CRISPR-mediated DNA detection platforms are limited by insufficient sensitivity and still require target preamplification. Herein, we report a CRISPR-Cas12a-mediated graphene field-effect transistor (gFET) array, named CRISPR Cas12a-gFET, for amplification-free, ultrasensitive, and reliable detection of both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) targets. CRISPR Cas12a-gFET leverages the multiturnover trans-cleavage activity of CRISPR Cas12a for intrinsic signal amplification and ultrasensitivity of gFET. As demonstrated, CRISPR Cas12a-gFET achieves a limit of detection of 1 aM for the ssDNA human papillomavirus 16 synthetic target and 10 aM for the dsDNA Escherichia coli plasmid target without target preamplification. In addition, an array of 48 sensors on a single 1.5 cm × 1.5 cm chip is employed to improve data reliability. Finally, Cas12a-gFET demonstrates the capability to discriminate single-nucleotide polymorphisms. Together, the CRISPR Cas12a-gFET biosensor array provides a detection tool for amplification-free, ultrasensitive, reliable, and highly specific DNA detections.
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Sistemas CRISPR-Cas , Grafite , Humanos , Sistemas CRISPR-Cas/genética , Polimorfismo de Nucleotídeo Único , Reprodutibilidade dos Testes , DNA/genética , DNA de Cadeia Simples/genética , Escherichia coli/genética , NucleotídeosRESUMO
Breast cancer is the most fatal disease among female cancers yet its detection still relies on needle biopsy. The unique physical and immune characteristics of breast cancer cells different from blood cells make them suitable to be employed as excellent biomarkers in liquid biopsy, through which breast cancer cells are collected from peripheral blood for further cancer diagnosis, medical treatment monitoring, and drug screening. Although the separation and enrichment of breast cancer cells from peripheral blood have been studied for years, there are still two problems to be solved in these methods: the low efficiency of on-chip immunologic capture in the flow state and the influence of the conjugated antibodies for the following analyses during cell release. In this paper, a vein-shaped microchip with self-assembled surface was developed for the specific and robust capture (91.2%) of breast cancer cells in the flow state. A protein-recovery process was proposed, in which trypsin served as a mild release reagent, releasing 92% of cells with high viability (96%), normal adherent proliferation, and complete proteins on the cell membrane, avoiding disturbance of the conjugated chemical molecules in the following clinical study. The excellent performance demonstrated in isolating free breast cancer cells from real peripheral blood sample, originating from the orthotopic 4T1 breast cancer metastatic models, suggest the microchip could be utilized as a multiple circulating tumor cell capture and release platform that could allow providing more reliable information in liquid biopsies.
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Neoplasias da Mama , Células Neoplásicas Circulantes , Neoplasias da Mama/diagnóstico , Linhagem Celular Tumoral , Separação Celular , Feminino , Humanos , Análise em MicrossériesRESUMO
Serving as an effective biomarker in liquid biopsy, circulating tumor cells (CTCs) can provide an accessible source for cancer biology study. For the in-depth evaluation of CTCs in cancer analysis, their efficient enrichment is essential, owing to their low abundance in peripheral blood. In this paper, self-assembled immunomagnetic beads were developed to isolate CTCs from the ordered bundles of cells under the assistance of the spiral inertial effect. Parametric numerical simulations were performed to explore the velocity distribution in the cross section. Based on this chip, rare CTCs could be recovered under the throughput of 500 µL/min, making this device a valuable supplement in cancer analysis, diagnostics, and therapeutics.
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Contagem de Células , Células Neoplásicas Circulantes , Linhagem Celular Tumoral , Separação Celular , Humanos , Separação Imunomagnética , Neoplasias PulmonaresRESUMO
Three-dimensional (3D) liver tumor segmentation from Computed Tomography (CT) images is a prerequisite for computer-aided diagnosis, treatment planning, and monitoring of liver cancer. Despite many years of research, 3D liver tumor segmentation remains a challenging task. In this paper, we propose an effective and efficient method for tumor segmentation in liver CT images using encoder-decoder based octave convolution networks. Compared with other convolution networks utilizing standard convolution for feature extraction, the proposed method utilizes octave convolutions for learning multiple-spatial-frequency features, thus can better capture tumors with varying sizes and shapes. The proposed network takes advantage of a fully convolutional architecture which performs efficient end-to-end learning and inference. More importantly, we introduce a deep supervision mechanism during the learning process to combat potential optimization difficulties, and thus the model can acquire a much faster convergence rate and more powerful discrimination capability. Finally, we integrate octave convolutions into the encoder-decoder architecture of UNet, which can generate high resolution tumor segmentation in one single forward feeding without post-processing steps. Both architectures are trained on a subset of the LiTS (Liver Tumor Segmentation) Challenge. The proposed approach is shown to significantly outperform other networks in terms of various accuracy measures and processing speed.
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Automatic segmentation of brain tumors from multi-modalities magnetic resonance image data has the potential to enable preoperative planning and intraoperative volume measurement. Recent advances in deep convolutional neural network technology have opened up an opportunity to achieve end-to-end segmenting the brain tumor areas. However, the medical image data used in brain tumor segmentation are relatively scarce and the appearance of brain tumors is varied, so that it is difficult to find a learnable pattern to directly describe tumor regions. In this paper, we propose a novel cross-modalities interactive feature learning framework to segment brain tumors from the multi-modalities data. The core idea is that the multi-modality MR data contain rich patterns of the normal brain regions, which can be easily captured and can be potentially used to detect the non-normal brain regions, i.e., brain tumor regions. The proposed multi-modalities interactive feature learning framework consists of two modules: cross-modality feature extracting module and attention guided feature fusing module, which aim at exploring the rich patterns cross multi-modalities and guiding the interacting and the fusing process for the rich features from different modalities. Comprehensive experiments are conducted on the BraTS 2018 benchmark, which show that the proposed cross-modality feature learning framework can effectively improve the brain tumor segmentation performance when compared with the baseline methods and state-of-the-art methods.
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BACKGROUND: Adoptive cell therapy based on the infusion of chimeric antigen receptor (CAR) T cells has shown remarkable efficacy for the treatment of hematologic malignancies. The primary mechanism of action of these infused T cells is the direct killing of tumor cells expressing the cognate antigen. However, understanding why only some T cells are capable of killing, and identifying mechanisms that can improve killing has remained elusive. METHODS: To identify molecular and cellular mechanisms that can improve T-cell killing, we utilized integrated high-throughput single-cell functional profiling by microscopy, followed by robotic retrieval and transcriptional profiling. RESULTS: With the aid of mathematical modeling we demonstrate that non-killer CAR T cells comprise a heterogeneous population that arise from failure in each of the discrete steps leading to the killing. Differential transcriptional single-cell profiling of killers and non-killers identified CD137 as an inducible costimulatory molecule upregulated on killer T cells. Our single-cell profiling results directly demonstrate that inducible CD137 is feature of killer (and serial killer) T cells and this marks a different subset compared with the CD107apos (degranulating) subset of CAR T cells. Ligation of the induced CD137 with CD137 ligand (CD137L) leads to younger CD19 CAR T cells with sustained killing and lower exhaustion. We genetically modified CAR T cells to co-express CD137L, in trans, and this lead to a profound improvement in anti-tumor efficacy in leukemia and refractory ovarian cancer models in mice. CONCLUSIONS: Broadly, our results illustrate that while non-killer T cells are reflective of population heterogeneity, integrated single-cell profiling can enable identification of mechanisms that can enhance the function/proliferation of killer T cells leading to direct anti-tumor benefit.
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Ligante 4-1BB/genética , Perfilação da Expressão Gênica , Imunoterapia Adotiva , Leucemia/terapia , Neoplasias Ovarianas/terapia , Receptores de Antígenos Quiméricos/genética , Análise de Célula Única , Linfócitos T/transplante , Transcriptoma , Ligante 4-1BB/metabolismo , Animais , Citotoxicidade Imunológica/genética , Feminino , Receptor Celular 2 do Vírus da Hepatite A/genética , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Humanos , Imunofenotipagem , Células K562 , Leucemia/genética , Leucemia/imunologia , Leucemia/metabolismo , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/metabolismo , Fenótipo , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Efficient and reliable manipulation of biological particles is crucial in medical diagnosis and chemical synthesis. Inertial microfluidic devices utilizing passive hydrodynamic forces in the secondary flow have drawn considerable attention for their high throughputs, low costs, and harmless particle manipulation. However, as the dominant mechanism, the inertial lift force is difficult to quantitatively analyze because of the uncertainties of its magnitude and direction. The equilibrium position of particles varies along the migration process, thus inducing the instabilities of particle separation. Herein, we present a designable inertial microfluidic chip combining a spiral channel with periodic expansion structures for the sheathless separation of particles with different sizes. The stable vortex-induced lift force arising from the periodic expansion and the Dean drag force significantly enhanced the focusing process and determined the final equilibrium position. The experimental results showed that over 99% of target particles could be isolated with the high target sample purity of 86.12%. In the biological experiment, 93.5% of the MCF-7, 89.5% of the Hela, and 88.6% of the A549 cells were steadily recovered with excellent viabilities to verify the potential of the device in dealing with biological particles over a broad range of throughputs. The device presented in this study can further serve as a lab-on-chip platform for liquid biopsy and diagnostic analysis.
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Separação Celular/métodos , Dispositivos Lab-On-A-Chip , Microfluídica/métodos , Linhagem Celular Tumoral , Separação Celular/instrumentação , Desenho de Equipamento , Humanos , Microfluídica/instrumentação , Microesferas , Tamanho da Partícula , Poliestirenos/química , Poliestirenos/isolamento & purificaçãoRESUMO
MicroRNA (miRNA) is a type of noncoding RNA and plays a crucial role in gene expression regulation. Sensitive identification and detection of miRNA can offer vast information for transcriptome analysis and cancer studies. Conventional PCR and molecular hybridization techniques suffer from low specificity when used for miRNA detection due to the short nucleotide length of miRNA. The extremely low abundance of miRNA in real biological samples also sets higher demands for the sensitivity of detection methods. A novel method based on target-assisted fluorescence resonance energy transfer (FRET) signal amplification was proposed for the simple and ultrasensitive detection of miRNA. A hairpin fluorescence DNA probe could hybridize with target miRNA to expose the hybridization site for the primer. After being duplexed with the nanogold-labeled primer, the fluorescent dye in the DNA probe was quenched via the FRET mechanism. In the presence of a DNA polymerase, primer-activated strand displacement released the miRNA, and then the miRNA strand could recognize and function with another DNA probe. The recycling of target miRNA and a high quenching efficiency of nanogold greatly improved the sensitivity. The detection limit of the method (1.5 fM) was lower than that of the reported strategies using a target cycling reaction, allowing trace detection of miRNA in real samples. By making full use of miRNA sequences, the method could also recognize single-base mismatches and distinguish homologous miRNAs.
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Transferência Ressonante de Energia de Fluorescência , Limite de Detecção , MicroRNAs/análise , Polimorfismo de Nucleotídeo Único , Sequência de Bases , Sondas de DNA/genética , Sondas de DNA/metabolismo , Estudos de Viabilidade , Células HeLa , Humanos , Cinética , MicroRNAs/química , MicroRNAs/metabolismo , Técnicas de Amplificação de Ácido NucleicoRESUMO
Single-cell capture plays an important role in single-cell manipulation and analysis. This paper presents a microfluidic device for deterministic single-cell trapping based on the hydrodynamic trapping mechanism. The device is composed of an S-shaped loop channel and thousands of aligned trap units. This arrayed structure enables each row of the device to be treated equally and independently, as it has row periodicity. A theoretical model was established and a simulation was conducted to optimize the key geometric parameters, and the performance was evaluated by conducting experiments on MCF-7 and Jurkat cells. The results showed improvements in single-cell trapping ability, including loading efficiency, capture speed, and the density of the patterned cells. The optimized device can achieve a capture efficiency of up to 100% and single-cell capture efficiency of up to 95%. This device offers 200 trap units in an area of 1 mm², which enables 100 single cells to be observed simultaneously using a microscope with a 20× objective lens. One thousand cells can be trapped sequentially within 2 min; this is faster than the values obtained with previously reported devices. Furthermore, the cells can also be recovered by reversely infusing solutions. The structure can be easily extended to a large scale, and a patterned array with 32,000 trap sites was accomplished on a single chip. This device can be a powerful tool for high-throughput single-cell analysis, cell heterogeneity investigation, and drug screening.
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Análise de Célula Única/métodos , Simulação por Computador , Humanos , Hidrodinâmica , Células Jurkat , Dispositivos Lab-On-A-Chip , Células MCF-7 , Pressão , Fatores de TempoRESUMO
Quantum dot (QD) multicolor analysis was achieved by labeling the epidermal growth factor and labeling the antibody against the epidermal growth factor receptor (anti-EGFR) using different QDs. On the basis of the fluorescence intensity of the two types of QDs, the amount of epidermal growth factors (EGFs) in the cells and anti-EGFRs bound to EGFRs were analyzed. The functions of anti-EGFR in preventing HeLa cells from engulfing EGF and inhibiting overproliferation of the HeLa cells were also studied. Meanwhile, parallel analysis was conducted to analyze the heterogeneity of cells at the single-cell level using a single-cell array. This provides a novel approach for the multiplexed analysis for the anti-EGFR functions in cells together with the cell heterogeneity. It also lays a foundation for parallel analysis and detection, using various fluorescence probes, simultaneous tracking, and detection of multiple targets at an overall level or individual level, and multichannel analysis of drug effects.
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Compostos de Cádmio/farmacologia , Receptores ErbB/antagonistas & inibidores , Corantes Fluorescentes/farmacologia , Pontos Quânticos , Compostos de Selênio/farmacologia , Compostos de Cádmio/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Receptores ErbB/metabolismo , Corantes Fluorescentes/química , Células HeLa , Humanos , Compostos de Selênio/química , Relação Estrutura-AtividadeRESUMO
Fluorescent probes based on the principle of resonance energy transfer (RET) or the principle of fluorescence polarization (FP) are already used to detect biomolecules independently. However, there were no in-depth studies about the impact of RET on FP. Also, very few studies gave a comprehensive analysis on how to effectively design such a fluorescent probe. Based on the principle of resonance energy transfer (RET), we constructed fluorescent probes (SA-488-sub-nanogold) using streptavidin labeled Alexa488 (SA-488), nanogold and biotinylated substrate peptide (biotin-subpeptide). The influence of the structure and the ingredients of the substrate peptide were discussed. After SA-488 was combined with the biotin-subpeptide and the nanogold, its fluorescence intensity (FI) would be suppressed due to the energy transfer, leading to an increase in its volume and mass. The suppression of the FI led to a decrease in SA-488's effective concentration, and the increase in the volume or mass prolonged the SA-488's rotational relaxation time. Both changes increased SA-488's polarization in the solution. Therefore, the FP performance of the probe is enhanced by the RET. Using the probe, trypsin and biotin were detected by the change in both fluorescence intensity and fluorescence polarization, showing higher reliability, higher sensitivity, and a lower detection limit.
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Biotina/análise , Polarização de Fluorescência/métodos , Corantes Fluorescentes/química , Tripsina/análise , Sequência de Aminoácidos , Técnicas Biossensoriais/métodos , Biotinilação , Ouro/química , Dados de Sequência Molecular , Peptídeos/química , Estreptavidina/químicaRESUMO
The gas sensors based on polymer-coated resonant microcantilevers for volatile organic compounds (VOCs) detection are investigated. A method to characterize the gas sensors through sensor calibration is proposed. The expressions for the estimation of the characteristic parameters are derived. The effect of the polymer coating location on the sensor's sensitivity is investigated and the formula to calculate the polymer-analyte partition coefficient without knowing the polymer coating features is presented for the first time. Three polymers: polyethyleneoxide (PEO), polyethylenevinylacetate (PEVA) and polyvinylalcohol (PVA) are used to perform the experiments. Six organic solvents: toluene, benzene, ethanol, acetone, hexane and octane are used as analytes. The response time, reversibility, hydrophilicity, sensitivity and selectivity of the polymer layers are discussed. According to the results, highly sensitive sensors for each of the analytes are proposed. Based on the characterization method, a convenient and flexible way to the construction of electric nose system by the polymer-coated resonant microcantilevers can be achieved.
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In the course of studies on human copper-zinc superoxide dismutase (SOD1), we observed a modified form of the protein whose mass was increased by 158 mass units. The covalent modification was characterized, and we established that it is a novel heptasulfane bridge connecting the two Cys111 residues in the SOD1 homodimer. The heptasulfane bridge was visualized directly in the crystal structure of a recombinant human mutant SOD1, H46R/H48Q, produced in yeast. The modification is reversible, with the bridge being cleaved by thiols, by cyanide, and by unfolding of the protein to expose the polysulfane. The polysulfane bridge can be introduced in vitro by incubation of purified SOD1 with elemental sulfur, even under anaerobic conditions and in the presence of a metal chelator. Because polysulfanes and polysulfides can catalyze the generation of reactive oxygen and sulfur species, the modification may endow SOD1 with a toxic gain of function.
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Cisteína/química , Compostos de Enxofre/química , Superóxido Dismutase/química , Cátions Bivalentes/química , Cobre/química , Cristalografia por Raios X , Dimerização , Estabilidade Enzimática , Humanos , Concentração de Íons de Hidrogênio , Peso Molecular , Proteínas Recombinantes/química , Sulfetos/química , Superóxido Dismutase-1 , Zinco/químicaRESUMO
Micro-cantilever sensors for mass detection using resonance frequency have attracted considerable attention over the last decade in the field of gas sensing. For such a sensing system, an oscillator circuit loop is conventionally used to actuate the micro-cantilever, and trace the frequency shifts. In this paper, gas experiments are introduced to investigate the mechanical resonance frequency shifts of the micro-cantilever within the circuit loop(mechanical resonance frequency, MRF) and resonating frequency shifts of the electric signal in the oscillator circuit (system working frequency, SWF). A silicon beam with a piezoelectric zinc oxide layer is employed in the experiment, and a Self-Actuating-Detecting (SAD) circuit loop is built to drive the micro-cantilever and to follow the frequency shifts. The differences between the two resonating frequencies and their shifts are discussed and analyzed, and a coefficient α related to the two frequency shifts is confirmed.
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Gases/análise , EletrônicaRESUMO
OBJECTIVE: To investigate the chest CT manifestations of paragonimiasis at different clinical phases. METHODS: Forty-eight cases of paragonimiasis confirmed by laboratory studies were retrospectively analyzed. There were 30 males and e18 females ageing 9 - 66 years (average, 31.4 years). Conventional chest CT was performed in all cases and contrast enhanced CT in some cases. RESULTS: The chest CT findings of paragonimiasis included: (1) peribronchial inflammation (n = 9); (2) infiltration (n = 13), manifested as unilateral or bilateral patchy infiltrates with hazy borders. The location and shape were variable. The typical "canal gas-path" sign was seen in a few patients; (3) nodular and masses (n = 11); (4) cysts and cavity (n = 6); (5) pleural effusion (n = 9). The CT appearances of paragonimiasis were different at different clinical phases. Peribronchial inflammation, infiltration and pleural effusion were early presentations, not distinguishable from other common infections. The appearance of nodules, masses, cysts and cavities was more common in the clinical course. At the stabilization phase, nodular cavity or pleura thickening and calcification were revealed. CONCLUSION: There are various abnormalities on chest CT at different phases of paragonimiasis. Combination of clinical manifestations with chest CT characteristics is important in the diagnosis of paragonimiasis.
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Paragonimíase/diagnóstico por imagem , Tomografia Computadorizada por Raios X , Adolescente , Adulto , Idoso , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Radiografia Torácica , Adulto JovemRESUMO
OBJECTIVE: To investigate the CT features of pulmonary sarcoidosis and to follow the changes after glucocorticoid therapy. METHODS: CT scans and clinical data of 90 patients with histologically confirmed pulmonary sarcoidosis were retrospectively reviewed. CT follow-up was carried out 5-30 d following therapy in 43 cases. The follow-up lasted 3-48 months. RESULTS: The main CT finding of pulmonary sarcoidosis was nodules which were present in 69 cases (77%), mostly distributed around bronchovascular bundles (n = 37, 41%). Other abnormalities included consolidation (n = 31, 34%), ground-grass ( n = 39, 43%), thickening of bronchovascular bundles (n = 30, 33%) interlobular septal lines (n = 58, 64%), fibrosis (n = 17, 19%), air-trapping (n = 3, 3%) bronchial narrowing (n = 8, 9%), pleural thickening (n = 42, 47%), hilar and mediastinal adenopathy (n =76, 84%). Two or more radiological patterns were present in 83 cases. Twenty-five cases of nodules (25/30), 9 cases of consolidation (9/15), 11 cases of ground-grass (11/16), 10 cases of thickening of bronchovascular bundles (10/12) were improved after therapy. Ten cases of interlobular septa (10/22), 1 case of diffuse linear changes (1/3), but no bronchial distortion (0/4) and honeycombing (0/2), were improved. CONCLUSIONS: The CT manifestations of pulmonary sarcoidosis are varied, with some specific radiographic features. The radiological diagnosis and the effect of glucocorticoid therapy can be evaluated by repeated CT scanning. Nodules, consolidation, ground-grass, and thickening of bronchovascular bundles can be improved markedly after glucocorticoid therapy, but bronchial distortion, linear changes and honeycombing can not.
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Radiografia Torácica , Sarcoidose Pulmonar/diagnóstico por imagem , Sarcoidose Pulmonar/terapia , Tomografia Computadorizada por Raios X , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
OBJECTIVE: To explore the mechanism of Chaiqin Chengqi Decoction (CQCQD) in treatment of rats with acute necrotizing pancreatitis (ANP). METHODS: Thirty SD rats were randomly divided into 3 groups: sham-operated (SO) group, ANP group and CQCQD-treated group. ANP was induced by retro-pumping 3.5% sodium cholate to common bile duct. Blood sample was collected from abdominal vein for examination and the pancreatic tissue samples were taken for making pathology section 6 hours later. The pancreatic tissue (HE staining) was observed by light microscope. The content of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) was detected with the method of enzyme-linked immunosorbent assay, and the activation of nuclear factor-kappaB (NF-kappaB) in pancreas was detected by immunohistochemical method. RESULTS: Compared with the SO group, there was dramatic increase in the white blood cell (WBC) counts and AMY level in the ANP group (P<0.05, P<0.01). Compared with the ANP group, the WBC counts and AMY level in CQCQD-treated group were significantly reduced (P<0.05). The edema, inflammatory infiltration, haemorrhage and necrosis scores and total pathological score in the ANP group were obviously higher than those in the SO group (P<0.01). The edema, haemorrhage and inflammatory infiltration scores and the total pathological score in CQCQD-treated group were decreased (P<0.05). The integral optical density of NF-kappaB p65 positive cells of pancreas in CQCQD-treated group was lower than that in the ANP group (P<0.05). CONCLUSION: CQCQD can reduce the content of serum TNF-alpha and IL-6, depress the activation of NF-kappaB, and lessen the pancreatic lesions.
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Medicamentos de Ervas Chinesas/uso terapêutico , NF-kappa B/metabolismo , Pâncreas/metabolismo , Pancreatite Necrosante Aguda/tratamento farmacológico , Fitoterapia , Animais , Feminino , Interleucina-6/sangue , Masculino , Pâncreas/patologia , Pancreatite Necrosante Aguda/patologia , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/sangueRESUMO
The non-heme iron dioxygenase PtlH from the soil organism Streptomyces avermitilis is a member of the iron(II)/alpha-ketoglutarate-dependent dioxygenase superfamily and catalyzes an essential reaction in the biosynthesis of the sesquiterpenoid antibiotic pentalenolactone. To investigate the structural basis for substrate recognition and catalysis, we have determined the x-ray crystal structure of PtlH in several complexes with the cofactors iron, alpha-ketoglutarate, and the non-reactive enantiomer of the substrate, ent-1-deoxypentalenic acid, in four different crystal forms to up to 1.31 A resolution. The overall structure of PtlH forms a double-stranded barrel helix fold, and the cofactor-binding site for iron and alpha-ketoglutarate is similar to other double-stranded barrel helix fold enzymes. Additional secondary structure elements that contribute to the substrate-binding site in PtlH are not conserved in other double-stranded barrel helix fold enzymes. Binding of the substrate enantiomer induces a reorganization of the monoclinic crystal lattice leading to a disorder-order transition of a C-terminal alpha-helix. The newly formed helix blocks the major access to the active site and effectively traps the bound substrate. Kinetic analysis of wild type and site-directed mutant proteins confirms a critical function of two arginine residues in substrate binding, while simulated docking of the enzymatic reaction product reveals the likely orientation of bound substrate.
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Coenzimas/química , Dioxigenases/química , Ferro/química , Dobramento de Proteína , Streptomyces/enzimologia , Sítios de Ligação/genética , Catálise , Coenzimas/metabolismo , Cristalografia por Raios X , Dioxigenases/genética , Ferro/metabolismo , Ácidos Cetoglutáricos/química , Ácidos Cetoglutáricos/metabolismo , Mutagênese Sítio-Dirigida , Estrutura Secundária de Proteína/genética , Estrutura Terciária de Proteína/genética , Sesquiterpenos/química , Sesquiterpenos/metabolismo , Streptomyces/genética , Especificidade por Substrato/genéticaRESUMO
OBJECTIVE: To study the CT manifestations of cryptogenic organizing pneumonia (COP) after glucocorticoid treatment. METHODS: The diagnosis of COP was made based on clinical and radiological features and confirmed by lung biopsy and pathological examination in 21 cases from 2001 to 2005. CT follow-up was carried out 5 - 75 days following therapy. The follow-up lasted 3 - 43 months. All data were analyzed and relevant literatures were reviewed. RESULTS: There were 2 male an 19 female patients with ages of 39 - 74 years. CT scans revealed multiple patchy shadows or patchy air-space consolidations, often by a predominantly subpleural and/or basal distribution. Ground-glass opacities and bronchiectasis were common findings. Migratory lesions were found in some patients. Glucocorticoid treatment resulted in significant improvement in most cases. Cure was achieved in 4 cases, significant improvement in 16, and failure in 1 case. Recurrence was found in 2 cases. CONCLUSIONS: The diagnosis of COP requires combination of clinical, CT and pathological manifestations. The effect of glucocorticoid therapy can be evaluated by repeated CT scanning.