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1.
Background
The tremendous output of massive parallel sequencing technologies requires automated robust and scalable sample preparation methods to fully exploit the new sequence capacity.Methodology
In this study, a method for automated library preparation of RNA prior to massively parallel sequencing is presented. The automated protocol uses precipitation onto carboxylic acid paramagnetic beads for purification and size selection of both RNA and DNA. The automated sample preparation was compared to the standard manual sample preparation.Conclusion/Significance
The automated procedure was used to generate libraries for gene expression profiling on the Illumina HiSeq 2000 platform with the capacity of 12 samples per preparation with a significantly improved throughput compared to the standard manual preparation. The data analysis shows consistent gene expression profiles in terms of sensitivity and quantification of gene expression between the two library preparation methods. 相似文献2.
Rationale
Previous in vitro research demonstrated that ascorbate enhances potency and duration of activity of agonists binding to alpha 1 adrenergic and histamine receptors.Objectives
Extending this work to beta 2 adrenergic systems in vitro and in vivo.Methods
Ultraviolet spectroscopy was used to study ascorbate binding to adrenergic receptor preparations and peptides. Force transduction studies on acetylcholine-contracted trachealis preparations from pigs and guinea pigs measured the effect of ascorbate on relaxation due to submaximal doses of beta adrenergic agonists. The effect of inhaled albuterol with and without ascorbate was tested on horses with heaves and sheep with carbachol-induced bronchoconstriction.Measurements
Binding constants for ascorbate binding to beta adrenergic receptor were derived from concentration-dependent spectral shifts. Dose- dependence curves were obtained for the relaxation of pre-contracted trachealis preparations due to beta agonists in the presence and absence of varied ascorbate. Tachyphylaxis and fade were also measured. Dose response curves were determined for the effect of albuterol plus-and-minus ascorbate on airway resistance in horses and sheep.Main Results
Ascorbate binds to the beta 2 adrenergic receptor at physiological concentrations. The receptor recycles dehydroascorbate. Physiological and supra-physiological concentrations of ascorbate enhance submaximal epinephrine and isoproterenol relaxation of trachealis, producing a 3–10-fold increase in sensitivity, preventing tachyphylaxis, and reversing fade. In vivo, ascorbate improves albuterol''s effect on heaves and produces a 10-fold enhancement of albuterol activity in “asthmatic” sheep.Conclusions
Ascorbate enhances beta-adrenergic activity via a novel receptor-mediated mechanism; increases potency and duration of beta adrenergic agonists effective in asthma and COPD; prevents tachyphylaxis; and reverses fade. These novel effects are probably caused by a novel mechanism involving phosphorylation of aminergic receptors and have clinical and drug-development applications. 相似文献3.
Kawa Amin Christer Janson Lahja Sevéus Kaoru Miyazaki Ismo Virtanen Per Venge 《Respiratory research》2005,6(1):110
Background
Laminins are a group of proteins largely responsible for the anchorage of cells to basement membranes. We hypothesized that altered Laminin chain production in the bronchial mucosa might explain the phenomenon of epithelial cell shedding in asthma. The aim was to characterize the presence of Laminin chains in the SEBM and epithelium in allergic and non-allergic asthmatics.Patients and methods
Biopsies were taken from the bronchi of 11 patients with allergic and 9 patients with non-allergic asthma and from 7 controls and stained with antibodies against the Laminin (ln) chains alpha1-alpha5, beta1-beta2 and gamma1-gamma2.Results
Lns-2,-5 and -10 were the main Laminins of SEBM. The layer of ln-10 was thicker in the two asthmatic groups while an increased thickness of lns-2 and -5 was only seen in allergic asthmatics. The ln gamma2-chain, which is only found in ln 5, was exclusively expressed in epithelial cells in association with epithelial injury and in the columnar epithelium of allergic asthmatics.Conclusion
The uncoordinated production of chains of ln-5 in allergic asthma could have a bearing on the poor epithelial cell anchorage in these patients. 相似文献4.
Pierre-Benoit Pagès Olivier Facy Pierre Mordant Sylvain Ladoire Guy Magnin Francois Lokiec Francois Ghiringhelli Alain Bernard 《PloS one》2013,8(3)
Background
The lung is a frequent site of colorectal cancer (CRC) metastases. After surgical resection, lung metastases recurrences have been related to the presence of micrometastases, potentially accessible to a high dose chemotherapy administered via adjuvant isolated lung perfusion (ILP). We sought to determine in vitro the most efficient drug when administered to CRC cell lines during a short exposure and in vivo its immediate and delayed tolerance when administered via ILP.Methods
First, efficacy of various cytotoxic molecules against a panel of human CRC cell lines was tested in vitro using cytotoxic assay after a 30-minute exposure. Then, early (operative) and delayed (1 month) tolerance of two concentrations of the molecule administered via ILP was tested on 19 adult pigs using hemodynamic, biological and histological criteria.Results
In vitro, gemcitabine (GEM) was the most efficient drug against selected CRC cell lines. In vivo, GEM was administered via ILP at regular (20 µg/ml) or high (100 µg/ml) concentrations. GEM administration was associated with transient and dose-dependant pulmonary vasoconstriction, leading to a voluntary decrease in pump inflow in order to maintain a stable pulmonary artery pressure. After this modulation, ILP using GEM was not associated with any systemic leak, systemic damage, and acute or delayed histological pulmonary toxicity. Pharmacokinetics studies revealed dose-dependant uptake associated with heterogenous distribution of the molecule into the lung parenchyma, and persistent cytotoxicity of venous effluent.Conclusions
GEM is effective against CRC cells even after a short exposure. ILP with GEM is a safe and reproducible technique. 相似文献5.
Background
Various fabrication methods are used to improve the stability and osseointegration of screws within the host bone. The aim of this study was to investigate whether roughened surface titanium screws fabricated by electron beam melting can provide better stability and osseointegration as compared with smooth titanium screws in sheep cervical vertebrae.Methods
Roughened surface titanium screws, fabricated by electron beam melting, and conventional smooth surface titanium screws were implanted into sheep for 6 or 12 weeks (groups A and B, respectively). Bone ingrowth and implant stability were assessed with three-dimensional imaging and reconstruction, as well as histological and biomechanical tests.Results
No screws in either group showed signs of loosening. Fibrous tissue formation could be seen around the screws at 6 weeks, which was replaced with bone at 12 weeks. Bone volume/total volume, bone surface area/bone volume, and the trabecular number were significantly higher for a define region of interest surrounding the roughened screws than that surrounding the smooth screws at 12 weeks. Indeed, for roughened screws, trabecular number was significantly higher at 12 weeks than at 6 weeks. On mechanical testing, the maximum pullout strength was significantly higher at 12 weeks than at 6 weeks, as expected; however, no significant differences were found between smooth and roughened screws at either time point. The maximum torque to extract the roughened screws was higher than that required for the smooth screws.Conclusions
Electron beam melting is a simple and effective method for producing a roughened surface on titanium screws. After 12 weeks, roughened titanium screws demonstrated a high degree of osseointegration and increased torsional resistance to extraction over smooth titanium screws. 相似文献6.
Gustavo A. Santa Cruz 《Reports of Practical Oncology and Radiotherapy》2016,21(2):135-139
Aim
to present the most important aspects of Microdosimetry, a research field in radiation biophysics.Background
microdosimetry is the branch of radiation biophysics that systematically studies the spatial, temporal and spectral aspects of the stochastic nature of the energy deposition processes in microscopic structures.Materials and Methods
we briefly review its history, the people, the formalism and the theories and devices that allowed researchers to begin to understand the true nature of radiation action on living matter.Results and Conclusions
we outline some of its applications, especially to Boron Neutron Capture Therapy, attempting to explain the biological effectiveness of the boron thermal neutron capture reaction. 相似文献7.
Ola B. Nilsson Theresa Neimert-Andersson Mattias Bronge Jeanette Grundstr?m Ranjana Sarma Hannes Uchtenhagen Alexey Kikhney Tatyana Sandalova Erik Holmgren Dmitri Svergun Adnane Achour Marianne van Hage Hans Gr?nlund 《PloS one》2014,9(10)
Background
Dog dander extract used for diagnosis and allergen-specific immunotherapy is often of variable and of poor quality.Objective
To assemble four well-established dog allergen components into one recombinant folded protein for improved diagnosis and vaccination of allergy to dog.Methods
A linked molecule, comprising the four dog lipocalin allergens Can f 1, Can f 2, Can f 4 and Can f 6 was constructed. The tetrameric protein was structurally characterized by small angle X-ray scattering, and compared with each single recombinant lipocalin allergen or an equimolar mix of the four allergens by analytical size exclusion chromatography, circular dichroism, allergen-specific IgE in serum by ELISA and allergen-dependent capacity to activate basophils. The immunogenicity of the fusion protein was evaluated in immunized mice by assessing splenocyte proliferation and antibody production.Results
The linked tetrameric construct was produced as a soluble fusion protein, with the specific folds of the four individual allergens conserved. This multi-allergen molecule was significantly more efficient (p<0.001) than each single recombinant allergen in binding to dog-specific IgE, and the epitope spectrum was unaffected compared to an equimolar mix of the four allergens. Basophil degranulation revealed that the biologic activity of the linked molecule was retained. Immunization of mice with the linked construct induced comparable allergen-specific IgG responses with blocking capacity towards all included allergens and generated comparably low T-cell responses.Conclusion
We provide the first evidence for a linked recombinant molecule covering the major dog allergens for potential use in diagnostics and allergy vaccination of dog allergic patients. 相似文献8.
Vitellogenin functions as a multivalent pattern recognition receptor with an opsonic activity 总被引:2,自引:0,他引:2
Background
Vitellogenin (Vg), a major reproductive protein, has been associated with infection-resistant response in fish. However, the underlying mechanisms by which Vg is involved in anti-infectious response are not understood.Methodology/Results
By both protein-microbe interaction analysis and enzyme-linked immunosorbent assay as well as phagocytosis test, we demonstrate for the first time that fish Vg acts as a pattern recognition molecule with multiple specificities that can recognize bacteria as well as fungus rather than self components from fish, and functions as an opsonin that can enhance macrophage phagocytosis.Conclusions
This study shows that fish Vg plays an integrative function in regulating immunity via its pleiotropic effects on both recognizing pathogen-associated molecular patterns and promoting macrophage phagocytosis. It also supports the notion that factors normally involved in control of female reproduction are associated with immunity in organisms that rely on Vg for oocyte development. 相似文献9.
Background
Perfluorododecanoic acid (PFDoA) is a perfluorinated carboxylic chemical (PFC) that has broad applications and distribution in the environment. While many studies have focused on hepatotoxicity, immunotoxicity, and reproductive toxicity of PFCAs, few have investigated renal toxicity.Methodology/Principal Findings
Here, we used comparative proteomic and metabonomic technologies to provide a global perspective on renal response to PFDoA. Male rats were exposed to 0, 0.05, 0.2, and 0.5 mg/kg/day of PFDoA for 110 days. After 2-D DIGE and MALDI TOF/TOF analysis, 79 differentially expressed proteins between the control and the PFDoA treated rats (0.2 and 0.5 mg-dosed groups) were successfully identified. These proteins were mainly involved in amino acid metabolism, the tricarboxylic acid cycle, gluconeogenesis, glycolysis, electron transport, and stress response. Nuclear magnetic resonance-based metabonomic analysis showed an increase in pyruvate, lactate, acetate, choline, and a variety of amino acids in the highest dose group. Furthermore, the profiles of free amino acids in the PFDoA treated groups were investigated quantitatively by high-coverage quantitative iTRAQ-LC MS/MS, which showed levels of sarcosine, asparagine, histidine, 1-methylhistidine, Ile, Leu, Val, Trp, Tyr, Phe, Cys, and Met increased markedly in the 0.5 mg dosed group, while homocitrulline, α-aminoadipic acid, β-alanine, and cystathionine decreased.Conclusion/Significance
These observations provide evidence that disorders in glucose and amino acid metabolism may contribute to PFDoA nephrotoxicity. Additionally, α2u globulin may play an important role in protecting the kidneys from PFDoA toxicity. 相似文献10.
Xiufen Zheng Motohiko Suzuki Xusheng Zhang Thomas E Ichim Fei Zhu Hong Ling Aminah Shunnar Michael H Wang Bertha Garcia Robert D Inman Wei-Ping Min 《Arthritis research & therapy》2010,12(1):R13
Introduction
We have previously demonstrated that ex vivo inhibition of costimulatory molecules on antigen-pulsed dendritic cells (DCs) can be useful for induction of antigen-specific immune deviation and suppression of autoimmune arthritis in the collagen induced arthritis (CIA) model. The current study evaluated a practical method of immune modulation through temporary systemic inhibition of the costimulatory molecule CD40.Methods
Mice with collagen II (CII)-induced arthritis (CIA) were administered siRNA targeting the CD40 molecule. Therapeutic effects were evaluated by clinical symptoms, histopathology, Ag-specific T cell and B cell immune responses.Results
Systemic administration of CD40-targeting siRNA can inhibit antigen-specific T cell response to collagen II, as well as prevent pathogenesis of disease in both a pre- and post-immunization manner in the CIA model. Disease amelioration was associated with suppression of Th1 cytokines, attenuation of antibody production, and upregulation of T regulatory cells.Conclusions
These studies support the feasibility of transient gene silencing at a systemic level as a mechanism of resetting autoreactive immunity. 相似文献11.
Purpose
To assess the capacity of methylxanthines (caffeine, theophylline, theobromine and paraxanthine) to inhibit uric acid crystallization, and to evaluate their potential application in the treatment of uric acid nephrolithiasis.Materials and Methods
The ability of methylxathines to inhibit uric acid nucleation was assayed turbidimetrically. Crystal morphology and its modification due to the effect of theobromine were evaluated by scanning electron microscopy (SEM). The ability of theobromine to inhibit uric acid crystal growth on calculi fragments resulting from extracorporeal shock wave lithotripsy (ESWL) was evaluated using a flow system.Results
The turbidimetric assay showed that among the studied methylxanthines, theobromine could markedly inhibit uric acid nucleation. SEM images showed that the presence of theobromine resulted in thinner uric acid crystals. Furthermore, in a flow system theobromine blocked the regrowth of post-ESWL uric acid calculi fragments.Conclusions
Theobromine, a natural dimethylxanthine present in high amounts in cocoa, acts as an inhibitor of nucleation and crystal growth of uric acid. Therefore, theobromine may be clinically useful in the treatment of uric acid nephrolithiasis. 相似文献12.
Aim
Review of recent advances and vision for future developments in clinical practice of Radiation Oncology.Background
There have been substantial research and technological developments in Radiation Oncology over the past 40 years.Materials and methods
The relevant literature was reviewed and the authors offer their perspective on future opportunities for advancement in Radiation Oncology.Conclusions
Significant innovative technological developments have been introduced in the practice of Radiation Oncology, with more precise target delineation and tracking and three dimensional treatment planning, optimal delivery of radiation therapy to the target and lower doses to surrounding Organs at Risk. This dose optimization and adaptive therapy have enhanced the role of Radiation Therapy to more effectively treat patients with cancer. Further creativity and refinements will continue to advance the field into new applications of ionizing radiations in cancer therapy. 相似文献13.
Steven S An Peter S Askovich Thomas I Zarembinski Kwangmi Ahn John M Peltier Moritz von Rechenberg Sudhir Sahasrabudhe Jeffrey J Fredberg 《Respiratory research》2011,12(1):8
Background
A newly identified mechanism of smooth muscle relaxation is the interaction between the small heat shock protein 20 (HSP20) and 14-3-3 proteins. Focusing upon this class of interactions, we describe here a novel drug target screening approach for treating airflow obstruction in asthma.Methods
Using a high-throughput fluorescence polarization (FP) assay, we screened a library of compounds that could act as small molecule modulators of HSP20 signals. We then applied two quantitative, cell-based biophysical methods to assess the functional efficacy of these molecules and rank-ordered their abilities to relax isolated human airway smooth muscle (ASM). Scaling up to the level of an intact tissue, we confirmed in a concentration-responsive manner the potency of the cell-based hit compounds.Results
Among 58,019 compound tested, 268 compounds caused 20% or more reduction of the polarized emission in the FP assay. A small subset of these primary screen hits, belonging to two scaffolds, caused relaxation of isolated ASM cell in vitro and attenuated active force development of intact tissue ex vivo.Conclusions
This staged biophysical screening paradigm provides proof-of-principle for high-throughput and cost-effective discovery of new small molecule therapeutic agents for obstructive lung diseases. 相似文献14.
Menglei Huan Bangle Zhang Zenghui Teng Han Cui Jieping Wang Xinyou Liu Hui Xia Siyuan Zhou Qibing Mei 《PloS one》2012,7(9)
Background
Conventional chemotherapy agent such as doxorubicin (DOX) is of limited clinical use because of its inherently low selectivity, which can lead to systemic toxicity in normal healthy tissue.Methods
A pH stimuli-sensitive conjugate based on polyethylene glycol (PEG) with covalently attachment doxorubicin via hydrazone bond (PEG-hyd-DOX) was prepared for tumor targeting delivery system. While PEG-DOX conjugates via amid bond (PEG-ami-DOX) was synthesized as control.Results
The synthetic conjugates were confirmed by proton nuclear magnetic resonance (NMR) spectroscopy, the release profile of DOX from PEG-hyd-DOX was acid-liable for the hydrazone linkage between DOX and PEG, led to different intracellular uptake route; intracellular accumulation of PEG-hyd-DOX was higher than PEG-ami-DOX due to its pH-triggered profile, and thereby more cytotoxicity against MCF-7, MDA-MB-231 (breast cancer models) and HepG2 (hepatocellular carcinoma model) cell lines. Following the in vitro results, we xenografted MDA-MB-231 cell onto SCID mice, PEG-hyd-DOX showed stronger antitumor efficacy than free DOX and was tumor-targeting.Conclusions
Results from these in vivo experiments were consistent with our in vitro results; suggested this pH-triggered PEG-hyd-DOX conjugate could target DOX to tumor tissues and release free drugs by acidic tumor environment, which would be potent in antitumor drug delivery. 相似文献15.
Background
The ErbB receptor tyrosine kinases and nucleolin are major contributors to malignant transformation. Recently we have found that cell-surface ErbB receptors interact with nucleolin via their cytoplasmic tail. Overexpression of ErbB1 and nucleolin leads to receptor phosphorylation, dimerization and anchorage independent growth.Methodology/Principal Findings
In the present study we explored the regions of nucleolin and ErbB responsible for their interaction. Using mutational analyses, we addressed the structure–function relationship of the interaction between ErbB1 and nucleolin. We identified the ErbB1 nuclear localization domain as nucleolin interacting region. This region is important for nucleolin-associated receptor activation. Notably, though the tyrosine kinase domain is important for nucleolin-associated receptor activation, it is not involved in nucleolin/ErbB interactions. In addition, we demonstrated that the 212 c-terminal portion of nucleolin is imperative for the interaction with ErbB1 and ErbB4. This region of nucleolin is sufficient to induce ErbB1 dimerization, phosphorylation and growth in soft agar.Conclusions/Significance
The oncogenic potential of ErbB depends on receptor levels and activation. Nucleolin affects ErbB dimerization and activation leading to enhanced cell growth. The C-terminal region of nucleolin and the ErbB1 NLS-domain mediate this interaction. Moreover, when the C-terminal 212 amino acids region of nucleolin is expressed with ErbB1, it can enhance anchorage independent cell growth. Taken together these results offer new insight into the role of ErbB1 and nucleolin interaction in malignant cells. 相似文献16.
17.
Background
Src homology 2 (SH2) domain is a conserved module involved in various biological processes. Tensin family member was reported to be involved in tumor suppression by interacting with DLC-1 (deleted-in-liver-cancer-1) via its SH2 domain. We explore here the important questions that what the structure of tensin2 SH2 domain is, and how it binds to DLC-1, which might reveal a novel binding mode.Principal Findings
Tensin2 SH2 domain adopts a conserved SH2 fold that mainly consists of five β-strands flanked by two α-helices. Most SH2 domains recognize phosphorylated ligands specifically. However, tensin2 SH2 domain was identified to interact with nonphosphorylated ligand (DLC-1) as well as phosphorylated ligand.Conclusions
We determined the solution structure of tensin2 SH2 domain using NMR spectroscopy, and revealed the interactions between tensin2 SH2 domain and its ligands in a phosphotyrosine-independent manner. 相似文献18.
Background
Nulliparity is a major risk factor of preeclampsia investigated in numerous trials of its prevention.Objective
We aimed to assess whether these trials considered nulliparity in subject selection or analysis of results.Search Strategy
01 April 2013 search of MEDLINE via PubMed, EMBASE and the Cochrane Library. 01 April 2013 search of trials registered in Clinicaltrials.gov.Selection Criteria
Randomised controlled trials and metaanalyses of preeclampsia prevention with no restriction to period of publication or language. Metaanalyses were selected to fully identify relevant trials.Data Collection and Analysis
One reader appraised each selected article/registered protocol using a pretested, standardized data abstraction form developed in a pilot test. For each article, he recorded whether both nulliparous and multiparous were included and, in case of mixed populations, whether randomisation was stratified, and whether subgroup analyses had been reported. For registered protocols, he only assessed whether it was planned to include mixed populations.Main Results
88 randomised controlled trials were identified, representing 83,396 included women. In 58 of the 88 articles identified (65.9%), preeclampsia was the primary outcome. In 31 of these (53.4%), the investigation combined nulliparous and multiparous women; only two reports in 31 (6.5%) stated that randomisation was stratified on parity and only four (12.9%) described a subgroup analysis by parity. Of the 30 registered trials, 20 (66.6%) planned to include both nulliparous and multiparous women.Conclusion
Parity is largely ignored in randomised controlled trials of preeclampsia prevention, which raises difficulties in interpreting the results. 相似文献19.
Ponce J Calvet X Gallach M Ponce M;Esophagitis Study Group of the Asociación Española de Gastroenterología 《PloS one》2011,6(10):e25051
Background
Few data are available on the prevalence of erosive and severe esophagitis in Western countries.Objective
To retrospectively determine the prevalence and the factors predicting erosive esophagitis and severe esophagitis in a large series of endoscopies in Spain.Design
Retrospective observational study. A multivariate analysis was performed to determine variables predicting severe esophagitis.Setting
Databases of 29 Spanish endoscopy units.Patients
Patients submitted to a diagnostic endoscopy during the year 2005.Interventions
Retrospective review of the databases.Main Outcome Measurements
Esophagitis severity (graded according to the Los Angeles classification) and associated endoscopic findings.Results
Esophagitis was observed in 8.7% of the 93,699 endoscopies reviewed. Severe esophagitis (LA grade C or D) accounted for 22.5% of cases of the disease and was found in 1.9% of all endoscopies. Incidences of esophagitis and those of severe esophagitis were 86.2 and 18.7 cases per 100,000 inhabitants per year respectively. Male sex (OR 1.89) and advanced age (OR 4.2 for patients in the fourth age quartile) were the only variables associated with severe esophagitis. Associated peptic ulcer was present in 8.8% of cases.Limitations
Retrospective study, no data on individual proton pump inhibitors use.Conclusions
Severe esophagitis is an infrequent finding in Spain. It occurs predominantly in males and in older individuals. Peptic ulcer disease is frequently associated with erosive esophagitis. 相似文献20.
Sébastien Rodrigue Arne C. Materna Sonia C. Timberlake Matthew C. Blackburn Rex R. Malmstrom Eric J. Alm Sallie W. Chisholm 《PloS one》2010,5(7)