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The conserved co-activator complex Mediator enables regulated transcription initiation by RNA polymerase (Pol) II. Here we reconstitute an active 15-subunit core Mediator (cMed) comprising all essential Mediator subunits from Saccharomyces cerevisiae. The cryo-electron microscopic structure of cMed bound to a core initiation complex was determined at 9.7 Å resolution. cMed binds Pol II around the Rpb4-Rpb7 stalk near the carboxy-terminal domain (CTD). The Mediator head module binds the Pol II dock and the TFIIB ribbon and stabilizes the initiation complex. The Mediator middle module extends to the Pol II foot with a 'plank' that may influence polymerase conformation. The Mediator subunit Med14 forms a 'beam' between the head and middle modules and connects to the tail module that is predicted to bind transcription activators located on upstream DNA. The Mediator 'arm' and 'hook' domains contribute to a 'cradle' that may position the CTD and TFIIH kinase to stimulate Pol II phosphorylation.
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Microscopia Crioeletrônica , Complexo Mediador/química , Complexo Mediador/ultraestrutura , RNA Polimerase II/química , RNA Polimerase II/ultraestrutura , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/ultraestrutura , Regulação Alostérica , Sítios de Ligação , DNA/química , DNA/metabolismo , Ativação Enzimática , Complexo Mediador/metabolismo , Modelos Moleculares , Fosforilação , Estabilidade Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Fator de Transcrição TFIIB/química , Fator de Transcrição TFIIB/metabolismo , Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/metabolismo , Iniciação da Transcrição GenéticaRESUMO
This Article was originally published under Nature Research's License to Publish, but has now been made available under a [CC BY 4.0] license. The PDF and HTML versions of the Article have been modified accordingly.
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Protein mass spectrometry (MS) is an indispensable tool to detect molecular signatures that can be associated with cellular dysregulation and disease. Despite its huge success in the life sciences, where it has led to novel insights into disease mechanisms and the identification of potential protein biomarkers, protein MS is rarely used for clinical protein assays. While conventional matrix-assisted laser desorption/ionization (MALDI) MS is not compatible with complex samples, liquid chromatography-MS (LC-MS)-based assays may be too complex and may lack the robustness and ease of automation required for routine use in the clinic. Therefore, clinical protein assays are dominated by immunohistochemistry and immunoassays which, however, often lack standardization and fully depend on antibody specificity. Immuno-MALDI (iMALDI) MS may overcome these hurdles by utilizing anti-peptide antibodies for the specific enrichment of targeted analytes and on-target detection of the captured analytes, thus combining the unique properties of MS for the unambiguous detection and quantitation of analytes with a workflow that can be fully automated. Here we discuss the requirements for clinical protein assays, the pitfalls of existing methods, how iMALDI has been successfully used to quantify endogenous peptides and proteins from clinical samples, as well as its potential as a powerful tool for companion diagnostics in the light of precision medicine.
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Técnicas e Procedimentos Diagnósticos , Proteínas/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Cromatografia Líquida , Humanos , Peptídeos , Espectrometria de Massas em TandemRESUMO
OBJECTIVES: In progressive glioblastoma, salvage treatment remains unstandardized, response is highly variable, and detailed analysis of individual approaches is mandatory. Re-irradiation is an established option in the therapy of progressive glioblastoma. Thus, we analysed outcome and prognostic parameters of patients with re-irradiated glioblastoma treated at our institution since 1998. MATERIALS AND METHODS: In a total of 51 patients, clinical and treatment parameters were collected and analysed retrospectively. Re-irradiation protocols included radiosurgery, hypofractionated radiotherapy or normofractionated radiotherapy. Outcome was analysed regarding prognostic factors in this highly selected cohort. RESULTS: Median overall survival after primary diagnosis was 28.8 months. Patients re-irradiated with single-dose stereotactic radiosurgery or hypofractionated regimes showed a superior overall survival after primary diagnosis compared to normofractionated treatment. Positive prognostic factors included a smaller gross tumour volume and younger age. A methylated MGMT promoter approached statistical significance as a positive factor regarding overall survival after re-irradiation. Further well-known prognostic factors as extension of the initial resection and the concomitance of temozolomide with the initial radiation treatment only appeared relevant in a subgroup of four long-term survivors. CONCLUSIONS: The favourable results regarding overall survival are probably due to patient selection for re-irradiation. If technically feasible, stereotactic radiosurgery or hypofractionated regimes should be preferred. In this highly selected re-irradiation cohort, only some of the well-known prognostic factors of the primary tumour setting were found to influence overall survival significantly. In contrast, also some patients presenting with unfavourable predictive parameters showed an encouraging course of disease and thus should not be excluded from re-irradiation.
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Neoplasias Encefálicas/radioterapia , Glioblastoma/radioterapia , Radiocirurgia , Reirradiação , Adulto , Idoso , Neoplasias Encefálicas/tratamento farmacológico , Dacarbazina/análogos & derivados , Dacarbazina/uso terapêutico , Feminino , Glioblastoma/tratamento farmacológico , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sobrevida , TemozolomidaRESUMO
This article provides a review of the routine methods currently utilized for total naphthenic acid analyses. There is a growing need to develop chemical methods that can selectively distinguish compounds found within industrially derived oil sands process affected waters (OSPW) from those derived from the natural weathering of oil sands deposits. Attention is thus given to the characterization of other OSPW components such as oil sands polar organic compounds, PAHs, and heavy metals along with characterization of chemical additives such as polyacrylamide polymers and trace levels of boron species. Environmental samples discussed cover the following matrices: OSPW containments, on-lease interceptor well systems, on- and off-lease groundwater, and river and lake surface waters. There are diverse ranges of methods available for analyses of total naphthenic acids. However, there is a need for inter-laboratory studies to compare their accuracy and precision for routine analyses. Recent advances in high- and medium-resolution mass spectrometry, concomitant with comprehensive mass spectrometry techniques following multi-dimensional chromatography or ion-mobility separations, have allowed for the speciation of monocarboxylic naphthenic acids along with a wide range of other species including humics. The distributions of oil sands polar organic compounds, particularly the sulphur containing species (i.e., OxS and OxS2) may allow for distinguishing sources of OSPW. The ratios of oxygen- (i.e., Ox) and nitrogen-containing species (i.e., NOx, and N2Ox) are useful for differentiating organic components derived from OSPW from natural components found within receiving waters. Synchronous fluorescence spectroscopy also provides a powerful screening technique capable of quickly detecting the presence of aromatic organic acids contained within oil sands naphthenic acid mixtures. Synchronous fluorescence spectroscopy provides diagnostic profiles for OSPW and potentially impacted groundwater that can be compared against reference groundwater and surface water samples. Novel applications of X-ray absorption near edge spectroscopy (XANES) are emerging for speciation of sulphur-containing species (both organic and inorganic components) as well as industrially derived boron-containing species. There is strong potential for an environmental forensics application of XANES for chemical fingerprinting of weathered sulphur-containing species and industrial additives in OSPW.
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Ácidos Carboxílicos/análise , Monitoramento Ambiental/métodos , Poluentes Químicos da Água/análise , Espectrometria de Massas , Hidrocarbonetos Policíclicos Aromáticos/análiseRESUMO
For many years, mRNA abundance has been used as the surrogate measure of gene expression in biological systems. However, recent genome-scale analyses in both bacteria and eukaryotes have revealed that mRNA levels correlate with steady-state protein abundance for only 50-70% of genes, indicating that translation and post-translation processes also play important roles in determining gene expression. What is not yet clear is whether dynamic processes such as cell cycle progression, differentiation, or response to environmental changes change the relationship between mRNA and protein abundance. Here, we describe a systems approach to interrogate promastigote-to-amastigote differentiation in the obligatory intracellular parasitic protozoan Leishmania donovani. Our results indicate that regulation of mRNA levels plays a major role early in the differentiation process, while translation and post-translational regulation are more important in the latter part. In addition, it appears that the differentiation signal causes a transient global increase in the rate of protein synthesis, which is subsequently down-regulated by phosphorylation of α-subunit of translation initiation factor 2. Thus, Leishmania dynamically changes the relationship between mRNA and protein abundance as it adapts to new environmental circumstances. It is likely that similar mechanisms play a more important role than previously recognized in regulation of gene expression in other organisms.
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Regulação da Expressão Gênica/fisiologia , Leishmania donovani/genética , Leishmania donovani/metabolismo , Animais , Diferenciação Celular , Análise de Sequência com Séries de Oligonucleotídeos , Dobramento de Proteína , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , Fatores de TempoRESUMO
The interest in the role of the gravitational factor during landing after long-term space flights (SF) leads to the search for various innovative approaches to assessing the compliance of external changes observed by clinicians. The results of special research methods such as Omics technologies that may reflect physiological responses to the conditions created during landing are of great interest. Our purpose is to compare the blood plasma proteome changes associated with the trauma and endothelial dysfunction processes prior to launch and on the day of landing, as well as the groups of cosmonauts with and without the secondary hemorrhagic purpura. In our study, the concentrations of 125 plasma proteins in 18 Russian cosmonauts, measured using targeted proteomic analysis based on liquid chromatography and tandem mass spectrometry were analyzed. The results reveal the trends of 12 proteins participating in the processes that trigger hemorrhagic purpura under the effect of re-entry g-forces. Exposure to intense g-forces and return to the gravity are the key factors for external manifestations of changes in the body systems induced by a long-term stay in space microgravity. Our results may be useful for further research to experts in gravitational physiology, aviation and space medicine.
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Astronautas , Púrpura , Humanos , Plasma/química , Proteoma/análise , ProteômicaRESUMO
Quantitative bronchoalveolar lavage (BAL) is used to diagnose ventilator-associated pneumonia (VAP). We prospectively compared semiquantitative (SQ) and quantitative (Qu) culture of BAL for VAP diagnosis. Ventilated patients suspected of VAP underwent bronchoscopic BAL. BAL fluid was examined by both Qu (colony-forming units [CFUs]/mL) and SQ culture (none, sparse, moderate, or heavy) and results were compared. VAP was defined as 105 CFU/mL or greater on Qu culture. Over 36 months, 319 BALs were performed. Sixty-three of 319 (20%) showed diagnostic growth by Qu culture identifying a total of 81 organisms causing VAP. All 63 specimens showed growth of some organism(s) on SQ culture with 79 of 81 causative organisms identified and two (Pseudomonas, one; Corynebacterium, one) not identified. The remaining 256 specimens did not meet the threshold for VAP by the Qu method. Among these, 79 did not show any growth on SQ culture. Among the 240 specimens showing some growth on SQ culture, a total of 384 organisms were identified. VAP rates in relation to strength of growth on SQ culture were: sparse, 10 of 140 (7%); moderate, 24 of 147 (16%); and heavy, 45 of 97 (46%). Sensitivity (Sn), specificity (Sp), positive (PPV), and negative (NPV) predictive values of SQ culture of BAL fluid for the diagnosis of VAP were 97, 21, 21, and 97 per cent, respectively. Nonquantitative culture of BAL fluid is fairly accurate in ruling out VAP (high Sn and NPV). It however has poor Sp and PPV and using this method will lead to unnecessary antimicrobial use with its attendant complications of toxicity, cost, and resistance.
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Infecções Bacterianas/diagnóstico , Líquido da Lavagem Broncoalveolar/microbiologia , Broncoscopia , Cuidados Críticos , Pneumonia Associada à Ventilação Mecânica/diagnóstico , Pneumonia Associada à Ventilação Mecânica/microbiologia , Adulto , Infecções Bacterianas/microbiologia , Infecções Bacterianas/terapia , Contagem de Colônia Microbiana , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pneumonia Associada à Ventilação Mecânica/terapia , Valor Preditivo dos TestesRESUMO
We evaluated the benefit of a central venous line (CVL) protocol on bloodstream infections (BSIs) and outcome in a trauma intensive care unit (ICU) population. We prospectively compared three groups: Group 1 (January 2003 to June 2004) preprotocol; Group 2 (July 2004 to June 2005) after the start of the protocol that included minimizing CVL use and strict universal precautions; and Group 3 (July 2005 to December 2006) after the addition of a line supply cart and nursing checklist. There were 1622 trauma patients admitted to the trauma ICU during the study period of whom 542 had a CVL. Group 3 had a higher Injury Severity Score (ISS) compared with both Groups 2 and 1 (28.3 +/- 13.0 vs 23.5 +/- 11.7 vs 22.8 +/- 12.0, P = 0.0002) but had a lower BSI rate/1000 line days (Group 1: 16.5; Group 2: 15.0; Group 3: 7.7). Adjusting for ISS group, three had shorter ICU length of stay (LOS) compared with Group 1 (12.11 +/- 1.46 vs 18.16 +/- 1.51, P = 0.01). Logistic regression showed ISS (P = 0.04; OR, 1.025; CI, 1.001-1.050) and a lack of CVL protocol (P = 0.01; OR, 0.31; CI, 0.13-0.76) to be independent predictors of BSI. CVL protocols decrease both BSI and LOS in trauma patients. Strict enforcement by a nurse preserves the integrity of the protocol.
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Bacteriemia/prevenção & controle , Cateterismo Venoso Central/normas , Protocolos Clínicos , Infecção Hospitalar/prevenção & controle , Tempo de Internação , Ferimentos e Lesões/terapia , Adulto , Bacteriemia/epidemiologia , Cateterismo Venoso Central/efeitos adversos , Infecção Hospitalar/epidemiologia , Humanos , Escala de Gravidade do Ferimento , Unidades de Terapia Intensiva/economia , Unidades de Terapia Intensiva/estatística & dados numéricos , Unidades de Terapia Intensiva/tendências , Modelos Logísticos , Estudos Prospectivos , Virginia , Ferimentos e Lesões/economiaRESUMO
It is well-known that pluripotent human embryonic stem cells (hPSC) can differentiate into any cell type. Recently, we reported that hPSC colonies enter stasis when immersed in an extremely soft hydrogel comprising hydroxyl-functional block copolymer worms (I. Canton, N. J. Warren, A. Chahal, K. Amps, A. Wood, R. Weightman, E. Wang, H. Moore and S. P. Armes, ACS Centr. Sci., 2016, 2, 65-74). The gel modulus and chemical structure of this synthetic hydrogel are similar to that of natural mucins, which are implicated in the mechanism of diapause for mammalian embryos. Does stasis induction occur merely because of the very soft nature of such hydrogels or does chemical functionality also play a role? Herein, we address this key question by designing a new hydrogel of comparable softness in which the PGMA stabilizer chains are replaced with non-hydroxylated poly(ethylene glycol) [PEG]. Immunolabeling studies confirm that hPSC colonies immersed in such PEG-based hydrogels do not enter stasis but instead proliferate (and differentiate if no adhesion substrate is present). However, pluripotency is retained if an appropriate adhesion substrate is provided. Thus, the chemical functionality of the hydrogel clearly plays a decisive role in the stasis induction mechanism.
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BACKGROUND: Quantitative broncho-alveolar lavage (qBAL) is increasingly being used for diagnosing ventilator-associated pneumonia (VAP). The current study prospectively evaluates the accuracy of broncho-alveolar lavage fluid Gram's stain (GS) in predicting both the presence of VAP and the class of causative microorganism in patients suspected of VAP. METHODS: Patients suspected of VAP in a trauma or surgical intensive care unit underwent bronchoscopic qBAL with GS. Presence and class of organisms seen on GS were correlated respectively with the presence of VAP, as diagnosed by qBAL, and class of causative microorganism. VAP was defined as qBAL >10(5) colony forming units/mL. All data were gathered prospectively. RESULTS: During a 28-month study period, 229 patients underwent 309 qBALs for suspected VAP. Seventy-one (23%) specimens were positive for VAP (qBAL>10(5) CFU/mL). Fifty-four specimens (77%) had one causative microorganism, 13 (18%) had two, 3 (4%) had three, and 1 (1%) demonstrated four microorganisms giving a total of 93 VAPs. Forty-one (62%) of 66 specimens showing moderate or many microorganisms on GS were positive for VAP. However, 7 (4%) of 167 specimens showing none and 23 (30%) of 76 showing few microorganisms on GS were also positive for VAP. Of the 64 qBAL specimens positive for VAP and where the GS showed microorganisms, 6 (23%) of 26 showing only G+ microorganisms on GS had G- VAP (G- alone, 4; G+ and G-, 2), and 1 (8%) of 12 showing G- microorganisms only had G+ and G- VAP. Of the seven qBAL specimens positive for VAP where the GS did not show microorganisms, one had G+ and six had G- VAP. With the threshold of positivity of GS at more than none, the sensitivity, specificity, positive, and negative predictive values of GS for the presence of VAP were 90%, 67%, 45%, and 96% respectively. CONCLUSIONS: Broncho-alveolar lavage fluid GS is poor in predicting the presence of VAP and predicting the class of causative microorganism. Using GS to determine necessity of and to select class of antimicrobial therapy will result in delayed or inappropriate VAP therapy or both.
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Líquido da Lavagem Broncoalveolar/microbiologia , Pneumonia Associada à Ventilação Mecânica/diagnóstico , Pneumonia Associada à Ventilação Mecânica/epidemiologia , Respiração Artificial/efeitos adversos , Adulto , Idoso , Estudos de Coortes , Cuidados Críticos/métodos , Contaminação de Equipamentos/estatística & dados numéricos , Feminino , Seguimentos , Bactérias Gram-Negativas/isolamento & purificação , Bactérias Gram-Positivas/isolamento & purificação , Humanos , Incidência , Unidades de Terapia Intensiva , Masculino , Pessoa de Meia-Idade , Pneumonia Associada à Ventilação Mecânica/microbiologia , Valor Preditivo dos Testes , Probabilidade , Estudos Prospectivos , Respiração Artificial/instrumentação , Medição de Risco , Sensibilidade e Especificidade , Taxa de SobrevidaRESUMO
A diagnostic blood test for stroke is desirable but will likely require multiple proteins rather than a single "troponin." Validating large protein panels requires large patient numbers. Mass spectrometry (MS) is a cost-effective tool for this task. We compared differences in the abundance of 147 protein markers to distinguish 20 acute cerebrovascular syndrome (ACVS) patients who presented to the Emergency Department of one urban hospital within < 24 h from onset) and from 20 control patients who were enrolled via an outpatient neurology clinic. We targeted proteins from the stroke literature plus cardiovascular markers previously studied in our lab. One hundred forty-one proteins were quantified using MS, 8 were quantified using antibody protein enrichment with MS, and 32 were measured using ELISA, with some proteins measured by multiple techniques. Thirty proteins (4 by ELISA and 26 by the MS techniques) were differentially abundant between mimic and stroke after adjusting for age in robust regression analyses (FDR < 0.20). A logistic regression model using the first two principal components of the proteins significantly improved discrimination between strokes and controls compared to a model based on age alone (p < 0.001, cross-validated AUC 0.93 vs. 0.78). Significant proteins included markers of inflammation (47%), coagulation (40%), atrial fibrillation (7%), neurovascular unit injury (3%), and other (3%). These results suggest the potential value of plasma proteins as biomarkers for ACVS diagnosis and the role of plasma-based MS in this area.
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Proteínas Sanguíneas/metabolismo , Isquemia Encefálica/complicações , Proteômica/métodos , Acidente Vascular Cerebral/etiologia , Acidente Vascular Cerebral/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Isquemia Encefálica/diagnóstico por imagem , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Projetos Piloto , Análise de Componente Principal , Curva ROC , Acidente Vascular Cerebral/diagnóstico por imagemRESUMO
Proteins can provide insights into biological processes at the functional level, so they are very promising biomarker candidates. The quantification of proteins in biological samples has been routinely used for the diagnosis of diseases and monitoring the treatment. Although large-scale protein quantification in complex samples is still a challenging task, a great amount of effort has been made to advance the technologies that enable quantitative proteomics. Seven years ago, in 2009, we wrote an article about the current trends in quantitative proteomics. In writing this current paper, we realized that, today, we have an even wider selection of potential tools for quantitative proteomics. These tools include new derivatization reagents, novel sampling formats, new types of analyzers and scanning techniques, and recently developed software to assist in assay development and data analysis. In this review article, we will discuss these innovative methods, and their current and potential applications in proteomics. Copyright © 2017 John Wiley & Sons, Ltd.
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Proteoma/análise , Proteômica/métodos , Animais , Biomarcadores/análise , Teste em Amostras de Sangue Seco , Humanos , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Espectrometria de Massas/normas , Proteômica/normas , Proteômica/tendências , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Vapor-liquid-solid is a well-established process in catalyst guided growth of 1-D nanostructures, i.e., nanobelts and nanowires. The catalyst particle is generally believed to be in the liquid state during growth, and is the site for impinging molecules. The crystalline structure of the catalyst may not have any influence on the structure of the grown nanostructures. In this work, using Au guided growth of ZnO, we show that the interfaces between the catalyst droplet and the nanostructure grow in well-defined mutual crystallographic relationships. The nanostructure defines the crystallographic orientation of the solidifying Au droplet. Possible alloy, intermetallic, or eutectic phase formation during catalysis are elucidated with the help of a proposed ternary Au-Zn-O phase diagram.
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Nanoestruturas/química , Óxido de Zinco/química , Catálise , Ouro/química , Microscopia Eletrônica de Varredura , Propriedades de Superfície , TermodinâmicaRESUMO
The effect of graphite addition on the mechanism of hydrogen uptake by titanium during mechanochemical activation in hydrogen flow was studied using kinetic, structural, microscopic, and spectroscopic techniques. As was found, already a small graphite admixture of about 0.5 wt % changed the kinetics of mechanically induced H2 sorption and significantly stimulated Ti-H2 interaction. Two new types of occupation sites available for hydrogen were observed, which are characterized by low H2 desorption temperatures: about 650 and 750 K instead of 1000 K.
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Little data exists regarding extubation protocols in critically injured trauma patients. The objective of the current study was to prospectively examine the impact of implementing an extubation protocol on the outcomes of ventilated trauma patients in a surgical intensive care unit (STICU). Trauma patients admitted to the STICU over a 15-month period at a Level 1 trauma center were prospectively evaluated. The total period was divided into an education and institution period (April 2002-November 2003) and an evaluation period (December 2003-July 2003). Patient demographics, hospital course, complications, and outcomes from period I were compared with those obtained during period II. From April 8, 2002 through July 5, 2003, 69 patients intubated for greater than 24 hours were included in our analysis. Thirty-three were treated during period I and 36 were treated during period II. Both groups were well matched in terms of age, sex, Injury Severity Score, and chest Abbreviated Injury Score. Ventilation days significantly decreased from a mean of 16.3 to 8.2 days (P = 0.04). ICU length of stay also decreased, nearly meeting significance. A rigorously enforced extubation protocol significantly decreased ventilator days in STICU patients. Continued education of health care providers is key to the success of the protocol.
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Protocolos Clínicos , Cuidados Críticos , Desmame do Respirador/métodos , Ferimentos e Lesões/terapia , Ferimentos por Arma de Fogo/terapia , Acidentes de Trânsito , Adulto , Feminino , Humanos , Escala de Gravidade do Ferimento , Unidades de Terapia Intensiva , Tempo de Internação , Masculino , Pessoa de Meia-Idade , Pneumonia/epidemiologia , Estudos Prospectivos , VirginiaRESUMO
The effect of graphite admixture on hydrogen sorption-desorption properties of mechanically activated electron microscopy, temperature-programmed reaction, and temperature-programmed desorption techniques. The major effect of graphite addition was found to be the formation of microporous carbon matrix particles containing randomly distributed titanium particles only several nanometers in diameter. This powder architecture enhances hydrogen transport to the titanium surface without hindrances and promotes titanium-hydrogen interaction by modifying the titanium surface and subsurface layers with interstitial carbon atoms.
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OBJECTIVES: To determine the feasibility of continuous measurement of limb movement via wrist and ankle actigraphy (an activity measure) in critically ill patients and to compare actigraphy measurements with observed activity, subjective scores on sedation-agitation scales, and heart rate and blood pressure of patients. METHODS: In a prospective, descriptive, correlational study, all activity of 20 adult patients in medical and coronary care units in a university medical center were observed for 2 hours and documented. Wrist and ankle actigraphy, heart rate, and systolic and diastolic blood pressure data were collected every minute. The Comfort Scale and the Richmond Agitation-Sedation Scale were completed at the beginning of the observation period and 1 and 2 hours later. RESULTS: Wrist actigraphy data correlated with scores on the Richmond Agitation-Sedation Scale (r = 0.58) and the Comfort Scale (r = 0.62) and with observed stimulation and activity events of patients (r = 0.45). Correlations with systolic, diastolic, and mean arterial pressures were weaker. Wrist and ankle actigraphy data were significantly correlated (r = 0.69; P < .001); however, their mean values (wrist, 418; ankle, 147) were significantly different (t = 5.77; P < .001). CONCLUSIONS: Actigraphy provides a continuous recording of patients' limb movement. Actigraphy measurements correlate well with patients' observed activity and with subjective scores on agitation and sedation scales. Actigraphy may become particularly important as a continuous measurement of activity for use in behavioral research and may enhance early recognition and management of the excessive activity that characterizes agitation.
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Sedação Consciente , Atividade Motora/fisiologia , Polissonografia/métodos , Agitação Psicomotora/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Tornozelo/fisiologia , Pressão Sanguínea/fisiologia , Estado Terminal , Eletrocardiografia/métodos , Estudos de Viabilidade , Feminino , Frequência Cardíaca/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Monitorização Fisiológica/métodos , Estudos Prospectivos , Agitação Psicomotora/etiologia , Fatores de Tempo , Punho/fisiologiaRESUMO
A combination of epitope excision, epitope extraction, and differential chemical modification followed by mass spectrometric peptide mapping was used for the characterization of a discontinuous epitope that is recognized by the mouse anti-HIV-p24 monoclonal antibody 5E2.A3. In epitope excision, the protein is first bound to an immobilized antibody and then digested with proteolytic enzymes. In epitope extraction, the protein is first digested and subsequently allowed to react with the antibody. After epitope excision of the p24-5E2.A3 complex with endoproteinase Lys-C, a large fragment remained affinity bound corresponding to amino acids 1-158 of HIV-p24 (fragment 1-158). Further digestion, however, resulted in loss of affinity. Moreover, no affinity-bound fragments were observed after an epitope extraction experiment. These data from the epitope excision and extraction experiments suggest that the epitope is discontinuous. For the further characterization of the epitope, amino groups in the epitope-containing fragment were acetylated in both the affinity bound and free states followed by mass spectrometric analysis. Two successive acetylation reactions were performed: (1) the first used a low molar excess of acetic anhydride, and (2) the second, after separation from the antibody, a high molar excess of its hexadeuteroderivative. This isotopic labeling procedure, in combination with high resolution mass spectrometry, allowed the precise determination of relative reactivities of amino groups. In this study, no differences were observed in the ranking of the relative reactivities of five lysine residues. However, the N-terminal amino group was found to be part of the discontinuous epitope.
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Epitopos , Proteína do Núcleo p24 do HIV/química , Fragmentos de Peptídeos/química , Acetilação , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Complexo Antígeno-Anticorpo/química , Cromatografia Líquida , Proteína do Núcleo p24 do HIV/imunologia , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mapeamento de Peptídeos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Cytosolic sulfotransferases sulfate steroids such as estrogens and hydroxysteroids. The enzymes, including human estrogen sulfotransferase (hEST) and hydroxysteroid sulfotransferase (hHST), are generally homodimers in solution with mouse estrogen sulfotransferase (mEST) being one of few exceptions. To identify the amino acid residues responsible for the dimerization, eight residues on the surface of hEST were mutated to their counterparts in mEST and mutated hESTs were then analyzed by gel filtration chromatography. A single mutation of Val(269) to Glu was sufficient to convert hEST to a monomer and the corresponding mutation of Val(260) also altered hHST to a monomer. The hHST crystal structure revealed a short stretch of peptide with the side-chains from two hHST monomers forming a hydrophobic zipper-like structure enforced by ion pairs at both ends. This peptide consisted of 10 residues near the C-terminus that, including the critical Val residue, is conserved as KXXXTVXXXE in nearly all cytosolic sulfotransferases. When mEST underwent the double mutations Pro269Thr/Glu270Val dimerization resulted. Thus, the KXXXTVXXXE sequence appears to be the common protein-protein interaction motif that mediates the homo- as well as heterodimerization of cytosolic sulfotransferases.