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UNLABELLED: Recurrent outbreaks of bacterial gastroenteritis linked to the consumption of fresh fruits and vegetables highlight the paucity of understanding of the ecology of Salmonella enterica under crop production and postharvest conditions. These gaps in knowledge are due, at least in part, to the lack of suitable surrogate organisms for studies for which biosafety level 2 is problematic. Therefore, we constructed and validated an avirulent strain of Salmonella enterica serovar Typhimurium. The strain lacks major Salmonella pathogenicity islands SPI-1, SPI-2, SPI-3, SPI-4, and SPI-5 as well as the virulence plasmid pSLT. Deletions and the absence of genomic rearrangements were confirmed by genomic sequencing, and the surrogate behaved like the parental wild-type strain on selective media. A loss-of-function (phoN) selective marker allowed the differentiation of this strain from wild-type strains on a medium containing a chromogenic substrate for alkaline phosphatase. Lack of virulence was confirmed by oral infection of female BALB/c mice. The strain persisted in tomatoes, cantaloupes, leafy greens, and soil with the same kinetics as the parental wild-type and selected outbreak strains, and it reached similar final population levels. The responses of this strain to heat treatment and disinfectants were similar to those of the wild type, supporting its potential as a surrogate for future studies on the ecology and survival of Salmonella in production and processing environments. IMPORTANCE: There is significant interest in understanding the ecology of human pathogens in environments outside of their animal hosts, including the crop production environment. However, manipulative field experiments with virulent human pathogens are unlikely to receive regulatory approval due to the obvious risks. Therefore, we constructed an avirulent strain of S. enterica serovar Typhimurium and characterized it extensively.
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Microbiologia de Alimentos/métodos , Frutas/microbiologia , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/patogenicidade , Verduras/microbiologia , Animais , Modelos Animais de Doenças , Ilhas Genômicas , Camundongos Endogâmicos BALB C , Infecções por Salmonella/microbiologia , Infecções por Salmonella/patologia , Salmonella typhimurium/genética , Salmonella typhimurium/isolamento & purificação , Deleção de Sequência , Microbiologia do Solo , VirulênciaRESUMO
We describe the construction and characterization of a novel membrane designed to allow proteins separated by gel electrophoresis (SDS-PAGE) to be detected as peptides by mass spectrometry in an efficient and comprehensive manner. The key attribute of the membrane is a bifunctional design that allows for the digestion of protein(s) and retention of the resulting peptides with minimal lateral diffusion. Silane chemistries are used to differentially treat the opposing surfaces of a glass filter paper to enable this unique capability.
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Eletroforese em Gel de Poliacrilamida/instrumentação , Vidro/química , Peptídeos/isolamento & purificação , Proteínas/isolamento & purificação , Animais , Desenho de Equipamento , Filtração/instrumentação , Humanos , Espectrometria de Massas , Membranas Artificiais , Peptídeos/análise , Proteínas/análise , Silanos/químicaRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is characterized by highly heterogeneous manifestations ranging from asymptomatic cases to death for still incompletely understood reasons. As part of the IMmunoPhenotyping Assessment in a COVID-19 Cohort study, we mapped the plasma proteomes of 1117 hospitalized patients with COVID-19 from 15 hospitals across the United States. Up to six samples were collected within ~28 days of hospitalization resulting in one of the largest COVID-19 plasma proteomics cohorts with 2934 samples. Using perchloric acid to deplete the most abundant plasma proteins allowed for detecting 2910 proteins. Our findings show that increased levels of neutrophil extracellular trap and heart damage markers are associated with fatal outcomes. Our analysis also identified prognostic biomarkers for worsening severity and death. Our comprehensive longitudinal plasma proteomics study, involving 1117 participants and 2934 samples, allowed for testing the generalizability of the findings of many previous COVID-19 plasma proteomics studies using much smaller cohorts.
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Biomarcadores , COVID-19 , Hospitalização , Proteoma , Proteômica , SARS-CoV-2 , Humanos , COVID-19/sangue , COVID-19/diagnóstico , COVID-19/virologia , Proteômica/métodos , Feminino , Masculino , SARS-CoV-2/isolamento & purificação , Pessoa de Meia-Idade , Estudos Longitudinais , Idoso , Biomarcadores/sangue , Proteoma/análise , Índice de Gravidade de Doença , Proteínas Sanguíneas/análise , Prognóstico , AdultoRESUMO
Characterizing perturbation of molecular pathways in congenital Zika virus (ZIKV) infection is critical for improved therapeutic approaches. Leveraging integrative systems biology, proteomics, and RNA-seq, we analyzed embryonic brain tissues from an immunocompetent, wild-type congenital ZIKV infection mouse model. ZIKV induced a robust immune response accompanied by the downregulation of critical neurodevelopmental gene programs. We identified a negative correlation between ZIKV polyprotein abundance and host cell cycle-inducing proteins. We further captured the downregulation of genes/proteins, many of which are known to be causative for human microcephaly, including Eomesodermin/T-box Brain Protein 2 (EOMES/TBR2) and Neuronal Differentiation 2 (NEUROD2). Disturbances of distinct molecular pathways in neural progenitors and post-mitotic neurons may contribute to complex brain phenotype of congenital ZIKV infection. Overall, this report on protein- and transcript-level dynamics enhances understanding of the ZIKV immunopathological landscape through characterization of fetal immune response in the developing brain.
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OBJECTIVE: We investigated interferon-stimulated gene 15 (ISG15), a poorly understood ubiquitin-like modifier, and its enzymatic pathway in dermatomyositis (DM), an autoimmune disease primarily involving muscle and skin. METHODS: We generated microarray data measuring transcript abundance for approximately 18,000 genes in each of 113 human muscle biopsy specimens, and studied biopsy specimens and cultured skeletal muscle using immunohistochemistry, immunoblotting proteomics, real-time quantitative polymerase chain reaction, and laser-capture microdissection. RESULTS: Transcripts encoding ISG15-conjugation pathway proteins were markedly upregulated in DM with perifascicular atrophy (DM-PFA) muscle (ISG15 339-fold, HERC5 62-fold, and USP18 68-fold) compared with 99 non-DM samples. Combined analysis with publicly available microarray datasets showed that >50-fold ISG15 transcript elevation had 100% sensitivity and specificity for 28 biopsies from adult DM-PFA and juvenile DM patients compared with 199 muscle samples from other muscle diseases. Free ISG15 and ISG15-conjugated proteins were only found on immunoblots from DM-PFA muscle. Cultured human skeletal muscle exposed to type 1 interferons produced similar transcripts and ISG15 protein and conjugates. Laser-capture microdissection followed by proteomic analysis showed deficiency of titin in DM perifascicular atrophic myofibers. INTERPRETATION: A large-scale microarray study of muscle samples demonstrated that among a diverse group of muscle diseases DM was uniquely associated with upregulation of the ISG15 conjugation pathway. Exposure of human skeletal muscle cell culture to type 1 interferons produced a molecular picture highly similar to that seen in human DM muscle. Perifascicular atrophic myofibers in DM were deficient in a number of skeletal muscle proteins including titin.
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Citocinas/metabolismo , Dermatomiosite/metabolismo , Músculo Esquelético/metabolismo , Ubiquitinas/metabolismo , Células Cultivadas , Conectina , Citocinas/genética , Bases de Dados Genéticas , Dermatomiosite/diagnóstico , Dermatomiosite/genética , Humanos , Immunoblotting , Imuno-Histoquímica , Interferon Tipo I/metabolismo , Lasers , Microdissecção/métodos , Proteínas Musculares/deficiência , Doenças Musculares/diagnóstico , Doenças Musculares/genética , Doenças Musculares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Proteínas Quinases/deficiência , Proteômica/métodos , Sensibilidade e Especificidade , Ubiquitinas/genética , Regulação para CimaRESUMO
The anaphase promoting complex (APC) controls the degradation of proteins during exit from mitosis and entry into S-phase. The activity of the APC is regulated by phosphorylation during mitosis. Because the phosphorylation pattern provides insights into the complexity of regulation of the APC, we studied in detail the phosphorylation patterns at a single mitotic state of arrest generated by various antimitotic drugs. We examined the phosphorylation patterns of the APC in HeLa S3 cells after they were arrested in prometaphase with taxol, nocodazole, vincristine, or monastrol. There were 71 phosphorylation sites on nine of the APC subunits. Despite the common state of arrest, the various antimitotic drug treatments resulted in differences in the phosphorylation patterns and phosphorylation stoichiometries. The relative phosphorylation stoichiometries were determined by using a method adapted from the isotope-free quantitation of the extent of modification (iQEM). We could show that during drug arrest the phosphorylation state of the APC changes, indicating that the mitotic arrest is not a static condition. We discuss these findings in terms of the variable efficacy of antimitotic drugs in cancer chemotherapy.
Assuntos
Antimitóticos/farmacologia , Proteômica/métodos , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Células HeLa , Humanos , Espectrometria de Massas , Nocodazol/farmacologia , Paclitaxel/farmacologia , Fosforilação , Prometáfase/efeitos dos fármacos , Subunidades Proteicas/metabolismo , Pirimidinas/farmacologia , Fuso Acromático/efeitos dos fármacos , Tionas/farmacologia , Vincristina/farmacologiaRESUMO
A method has been established to map a bacterial colony to the ever-expanding database of publicly available bacterial genomes by means of matrix-assisted laser desorption/ionization (MALDI) spectra. To accomplish this, spectra are mapped to the predicted masses of â¼65 families of mostly ribosomal proteins. Each of the â¼40â¯000 bacterial strains in the database receives scores, together with tables listing identified protein sequences and how the highest ranking strains are related to one another. The approach was first confirmed with 16 distinct species of bacteria from the Vibrionales whose genome had been sequenced. Identifications of a few species of bacteria from environmental samples from compost, lakes, and streams in Massachusetts are also reported. Most of these organisms map to known species in the Gammaproteobacteria and Firmicutes. The clades of bacteria deducible from shared ribosomal protein sequences do not always correspond well to named bacterial species. Instead, the identifications made by this methodology indicate groupings of organisms that can readily be distinguished by MALDI-TOF and indicate which polymorphisms in highly conserved proteins demarcate the groupings. Successful identifications highlight organism interrelationships that can be deduced from the available genomes, sorting together genomes into new proposed clades typically consistent with relationships deduced from DNA sequence analysis. In contrast, if for a high-quality spectrum from a fresh colony, no group of related organisms receives high scores, one might infer that no closely related genome has yet been deposited into the database.
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Proteínas de Bactérias/química , Bases de Dados de Ácidos Nucleicos , Genoma Bacteriano/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Aeromonas/química , Aeromonas/genética , Aeromonas/isolamento & purificação , Bactérias/genética , Bactérias/isolamento & purificação , Proteínas de Bactérias/genética , Técnicas Bacteriológicas/métodos , Água Doce/microbiologia , Massachusetts , Reprodutibilidade dos Testes , Software , Vibrio/química , Vibrio/genéticaRESUMO
Our goal in this work is to evaluate a new combination linear/reflector MALDI-TOF instrument toward satisfying all "7S criteria" for the ideal MSI mass spectrometer. The linear analyzer satisfies all of the 7 criteria except for Specificity. The new instrument described here adds a reflector to provide up to 50,000 mass resolving power with ppm mass accuracy and with no sacrifice in speed, spatial resolution, and sensitivity demonstrated earlier for the linear MALDI-TOF. This instrument employs new laser optics that produces a 5 kHz laser beam with 2.5-25 µm diameter under computer control. The most important advance is the patented combination of laser and ion optics that provides very high efficiency for production and detection of ions generated by laser desorption using small diameter laser beams. This provides spectra with a wide dynamic range summing a relatively small number of laser shots/pixels. Rat and mouse brain tissues have been used for these initial studies. Examples of negative ion images of lipids and positive ion images from tryptic digestion of proteins are presented. These results demonstrate a very high speed for MSI. This speed is derived from a combination of high laser rate (5 kHz), fast motion of sample relative to the laser beam (20 mm/s), very high ionization efficiency (up to 50%), and the ability to acquire, process, and save spectra at a very high rate (1000/s). As a result, the speed that is possible is imposed by other limits, including the mass range, concentration of samples on the surface, and the spatial resolution required.
RESUMO
A new high performance linear MALDI-TOF mass spectrometer provides both high spatial resolution and high speed. This instrument employs a new ion optics system with a grounded ion source and efficient transfer and detection of ions over a broad mass range. This provides very high sensitivity, precision, and an extended dynamic range for both positive and negative ion detection. Here we demonstrate the capabilities of this system by imaging pancreatic tissue samples from rats and mice.
Assuntos
Imagem Molecular/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Diabetes Mellitus Experimental , Camundongos , Obesidade , Pâncreas/diagnóstico por imagem , RatosRESUMO
Vibrio cholerae O1 can cause diarrheal disease that may be life-threatening without treatment. Natural infection results in long-lasting protective immunity, but the role of T cells in this immune response has not been well characterized. In contrast, robust B-cell responses to V. cholerae infection have been observed. In particular, memory B-cell responses to T-cell-dependent antigens persist for at least 1 year, whereas responses to lipopolysaccharide, a T-cell-independent antigen, wane more rapidly after infection. We hypothesize that protective immunity is mediated by anamnestic responses of memory B cells in the gut-associated lymphoid tissue, and T-cell responses may be required to generate and maintain durable memory B-cell responses. In this study, we examined B- and T-cell responses in patients with severe V. cholerae infection. Using the flow cytometric assay of the specific cell-mediated immune response in activated whole blood, we measured antigen-specific T-cell responses using V. cholerae antigens, including the toxin-coregulated pilus (TcpA), a V. cholerae membrane preparation, and the V. cholerae cytolysin/hemolysin (VCC) protein. Our results show that memory T-cell responses develop by day 7 after infection, a time prior to and concurrent with the development of B-cell responses. This suggests that T-cell responses to V. cholerae antigens may be important for the generation and stability of memory B-cell responses. The T-cell proliferative response to VCC was of a higher magnitude than responses observed to other V. cholerae antigens.
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Cólera/imunologia , Imunidade Celular/imunologia , Memória Imunológica/imunologia , Subpopulações de Linfócitos T/imunologia , Adolescente , Adulto , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Linfócitos B/imunologia , Cólera/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Vibrio cholerae O1/imunologia , Adulto JovemRESUMO
An effective vaccine for Vibrio cholerae is not yet available for use in the developing world, where the burden of cholera disease is highest. Characterizing the proteins that are expressed by V. cholerae in the human host environment may provide insight into the pathogenesis of cholera and assist with the development of an improved vaccine. We analyzed the V. cholerae proteins present in the stools of 32 patients with clinical cholera. The V. cholerae outer membrane porin, OmpU, was identified in all of the human stool samples, and many V. cholerae proteins were repeatedly identified in separate patient samples. The majority of V. cholerae proteins identified in human stool are involved in protein synthesis and energy metabolism. A number of proteins involved in the pathogenesis of cholera, including the A and B subunits of cholera toxin and the toxin-coregulated pilus, were identified in human stool. In a subset of stool specimens, we also assessed which in vivo expressed V. cholerae proteins were recognized uniquely by convalescent-phase as opposed to acute-phase serum from cholera patients. We identified a number of these in vivo expressed proteins as immunogenic during human infection. To our knowledge, this is the first characterization of the proteome of a pathogenic bacteria recovered from a natural host.
Assuntos
Proteínas de Bactérias/análise , Cólera/microbiologia , Fezes/microbiologia , Proteoma/análise , Vibrio cholerae/química , Adolescente , Adulto , Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/imunologia , Humanos , Vibrio cholerae/isolamento & purificaçãoRESUMO
Measurement of glycated hemoglobin is widely used for the diagnosis and monitoring of diabetes mellitus. Matrix assisted laser desorption/ionization (MALDI) time of flight (TOF) mass spectrometry (MS) analysis of patient samples is used to demonstrate a method for quantitation of total glycation on the ß-subunit of hemoglobin. The approach is accurate and calibrated with commercially available reference materials. Measurements were linear (R(2) > 0.99) across the clinically relevant range of 4% to 20% glycation with coefficients of variation of ≤ 2.5%. Additional and independent measurements of glycation of the α-subunit of hemoglobin are used to validate ß-subunit glycation measurements and distinguish hemoglobin variants. Results obtained by MALDI-TOF MS were compared with those obtained in a clinical laboratory using validated HPLC methodology. MALDI-TOF MS sample preparation was minimal and analysis times were rapid making the method an attractive alternative to methodologies currently in practice.
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Hemoglobinas Glicadas/análise , Subunidades de Hemoglobina/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Cromatografia Líquida de Alta Pressão/métodos , Diabetes Mellitus/diagnóstico , Humanos , Modelos LinearesRESUMO
Forensic investigators routinely deploy side-scan sonar for submerged body searches. This study adds to the limited body of literature by undertaking a controlled project to understand how variables affect detection of submerged bodies using side-scan sonar. Research consisted of two phases using small and medium-sized pig (Sus scrofa) carcasses as proxies for human bodies to investigate the effects of terrain, body size, frequency, swath width, and state of decomposition. Results demonstrated that a clear, flat, sandy pond floor terrain was optimal for detection of the target as irregular terrain and/or vegetation are major limitations that can obscure the target. A higher frequency towfish was preferred for small bodies, and a 20 m swath width allowed greater visibility and easier maneuverability of the boat in this environment. Also, the medium-sized carcasses were discernable throughout the 81-day study period, indicating that it is possible to detect bodies undergoing decomposition with side-scan sonar.
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Imersão , Som , Água , Animais , Diagnóstico , Ciências Forenses/métodos , Humanos , Modelos Animais , Lagoas , Sus scrofaRESUMO
No universally accepted score is currently available to determine when a matrix-assisted laser desorption ionization (MALDI) peptide mass fingerprint (PMF) experiment has been successfully carried out. We describe a software program (ChemApplex) based on a calculated parameter (Combined Protein Score) that takes into account (1) peak intensity, (2) the mass accuracy of the match, and (3) ChemScore, a theoretical intensity factor that estimates the probability of observing a particular peptide based on a combination of chemical considerations, in particular the amino acid composition of the peptide and the amino acid sequence of the amino acids that span the cleavage site. When these three factors are taken into account both at the level of individual peptides and at the protein level, protein components in mixtures whose peptides contribute less than 1% of the total intensity can often be correctly identified, as is demonstrated for mixtures of standard proteins. Moreover, it is possible to make robust database identifications that are nearly independent of the number of masses submitted and the mass error threshold used for matching. Protein scoring based on Combined Protein Score is orthogonal to many of the commonly used probability-based scoring schemes, and makes it possible to archive a more complete set of parameters that more thoroughly characterize the validity of the database match, which increases the confidence in the identifications.
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Mapeamento de Peptídeos/métodos , Software , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sequência de Aminoácidos , Bases de Dados de Proteínas , Dados de Sequência Molecular , Peso Molecular , Muramidase/química , Ovalbumina/química , Fragmentos de Peptídeos/química , Soroalbumina Bovina/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/instrumentaçãoRESUMO
Chronic obstructive pulmonary disease (COPD) is associated with increased cardiovascular morbidity and mortality, yet the exact pathophysiological links remain unclear. Whether the presence and severity of COPD are associated with atrial or ventricular arrhythmias recorded on continuous electrocardiographic monitoring is unknown. We identified consecutive adult patients who underwent clinically indicated pulmonary function testing as well as 24-hour Holter monitoring at the Mayo Clinic, Rochester, from 2000 to 2009. Demographic data and relevant co-morbidities were gathered from the electronic medical record; severity of COPD was classified according to the GOLD classification, and arrhythmias were classified in concordance with the current clinical guidelines. From 7,441 patients who were included (age 64±16 years, 49% woman, 92% Caucasian), COPD was diagnosed in 3,121 (41.9%). Compared with those without COPD, the presence and severity of COPD were associated with increased likelihood of atrial fibrillation/atrial flutter (AF/AFL; 23.3% vs 11.0%, respectively, p<0.0001), nonsustained ventricular tachycardia (NSVT; 13.0% vs 5.9%, respectively, p<0.0001), and sustained ventricular tachycardia (0.9% vs 1.6%, respectively, p<0.0001). COPD remained a significant predictor of AF/AFL and NSVT (p<0.0001 and p<0.0001, respectively) after adjusting for age, gender, tobacco use, obesity, hypertension, coronary artery disease, heart failure, diabetes, anemia, cancer, chronic kidney disease, and rate/rhythm control medications. In conclusion, the independent association between the presence and severity of COPD and arrhythmias (AF/AFL and NSVT) provides further insight into the markedly increased cardiovascular mortality of patients with COPD. Further studies should explore which anti-arrhythmic strategies would best apply to the patients with COPD.
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Fibrilação Atrial/etiologia , Doença Pulmonar Obstrutiva Crônica/complicações , Taquicardia Ventricular/etiologia , Idoso , Fibrilação Atrial/epidemiologia , Fibrilação Atrial/fisiopatologia , Eletrocardiografia Ambulatorial , Feminino , Seguimentos , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Minnesota/epidemiologia , Pletismografia , Doença Pulmonar Obstrutiva Crônica/epidemiologia , Estudos Retrospectivos , Taxa de Sobrevida/tendências , Taquicardia Ventricular/epidemiologia , Taquicardia Ventricular/fisiopatologiaRESUMO
Forensic personnel must deal with numerous challenges when searching for submerged objects. While traditional water search methods have generally involved using dive teams, remotely operated vehicles (ROVs), and water scent dogs for cases involving submerged objects and bodies, law enforcement is increasingly integrating multiple methods that include geophysical technologies. There are numerous advantages for integrating geophysical technologies, such as side scan sonar and ground penetrating radar (GPR), with more traditional search methods. Overall, these methods decrease the time involved searching, in addition to increasing area searched. However, as with other search methods, there are advantages and disadvantages when using each method. For example, in instances with excessive aquatic vegetation or irregular bottom terrain, it may not be possible to discern a submersed body with side scan sonar. As a result, forensic personnel will have the highest rate of success during searches for submerged objects when integrating multiple search methods, including deploying multiple geophysical technologies. The goal of this paper is to discuss the methodology of various search methods that are employed for submerged objects and how these various methods can be integrated as part of a comprehensive protocol for water searches depending upon the type of underwater terrain. In addition, two successful case studies involving the search and recovery of a submerged human body using side scan sonar are presented to illustrate the successful application of integrating a geophysical technology with divers when searching for a submerged object.
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Imersão , Som , Animais , Arqueologia , Mergulho , Cães , Ciências Forenses , Humanos , Radar , Robótica , Processamento de Sinais Assistido por ComputadorRESUMO
BACKGROUND: The nonspecific (NS) pulmonary function (PF) pattern refers to a PF test with a normal total lung capacity (TLC), normal FEV1/FVC ratio, and a low FEV1, a low FVC, or both. Currently, no information is available regarding the long-term stability of the NS pattern or variables that predict changes in subjects with an initial NS PF pattern. METHODS: From 1990 to 2005 we identified 1,284 subjects with an NS pattern on initial PF testing with one or more follow-up PF tests 6 months or more after the initial NS test result. Lung volumes, diffusing capacity, and spirometry data were analyzed. A multivariate, multinomial logistic regression model was used to study the association between different variables and the final PF pattern. RESULTS: Overall, 3,674 PF tests were performed in 1,284 subjects over a median follow-up period of 3 years. At last follow-up, 818/1,284 (64%) subjects continued to show the NS pattern, whereas 208/1,284 (16%) showed a restrictive pattern, 191/1,284 (15%) an obstructive pattern, 42/1,284 (3%) a normal pattern, and 25/1,284 (2%) a mixed pattern. The multinomial logistic regression analysis showed that increasing values for specific airway resistance and the difference between TLC and alveolar volume were predictors of a change to an obstructive pattern on follow-up. CONCLUSIONS: The NS pattern is a distinct and stable PF test pattern with roughly two-thirds of patients continuing to show this pattern on follow-up testing. Current interpretation guidelines erroneously label the NS pattern as representing obstruction and need to be changed to reflect these data.
Assuntos
Fluxo Expiratório Forçado/fisiologia , Fibrose Pulmonar/fisiopatologia , Testes de Função Respiratória/métodos , Capacidade Pulmonar Total/fisiologia , Progressão da Doença , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Fibrose Pulmonar/diagnóstico , Estudos Retrospectivos , Índice de Gravidade de Doença , Fatores de TempoRESUMO
Sarcoplasmic accumulation of phosphorylated-tau has been widely stated to occur in and contribute to the pathogenesis of muscle disease in inclusion body myositis. Twenty inflammatory myopathy and 10 normal muscle samples along with a range of other tissues were stained with anti-"tau" antibodies (tau-5, pS422, and SMI-31). Myonuclear and sarcoplasmic fractions were prepared using differential solubilization and laser-capture microdissection, and immunoblots were performed using pS422 and SMI-31 antibodies. All three antibodies demonstrated anti-tau immunoreactivity in myonuclei from normal and diseased muscle, but not in nuclei from other tissues. Western blots showed pS422 and SMI-31 immunoreactivity against nuclear proteins outside the region expected for phosphorylated-tau. Antibodies previously reported to indicate abnormal accumulation of phosphorylated-tau in IBM myofibers react to normal myonuclei and recognize proteins other than tau. Normal myonuclei contain neurofilament H or other unidentified 200 kDa proteins with similar phosphorylated motifs accounting for SMI-31 immunoreactivity.
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Núcleo Celular/metabolismo , Células Musculares/metabolismo , Miosite de Corpos de Inclusão/metabolismo , Proteínas tau/metabolismo , Anticorpos/química , Especificidade de Anticorpos , Western Blotting , Núcleo Celular/patologia , Reações Cruzadas , Imunofluorescência , Humanos , Imuno-Histoquímica , Microdissecção , Células Musculares/patologia , Miosite de Corpos de Inclusão/patologia , Fosforilação , Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/patologia , Proteínas tau/imunologiaRESUMO
We characterized the human muscle proteome by studying muscle biopsy specimens through four different workflows, using 1 or 2D peptide separation, SDS gels, or differential solubilization. By performing MS/MS analyses of 178 4-h LC separations derived from 31 patients, we identified more than 2000 proteins, and determined how 370 very abundant proteins behave upon differential solubilization. The resulting semiquantitative database should serve as a resource for muscle biochemistry.
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Músculo Esquelético/patologia , Proteômica/métodos , Biópsia , Cromatografia Líquida/métodos , Análise por Conglomerados , Bases de Dados de Proteínas , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Humanos , Espectrometria de Massas , Peptídeos/química , Análise Serial de Proteínas , Proteoma/análise , Proteoma/metabolismo , Sarcômeros/metabolismo , SolubilidadeRESUMO
Inclusion body myositis (IBM) is an inflammatory disease of skeletal muscle of unknown cause. To further understand the nature of the tissue injury in this disease, we developed methods for large-scale detection and quantitation of proteins in muscle biopsy samples and analyzed proteomic data produced by these methods together with histochemical, immunohistochemical, and microarray data. Twenty muscle biopsy samples from patients with inflammatory myopathies (n = 17) or elderly subjects without neuromuscular disease (n = 3) were profiled by proteomic studies using liquid chromatographic separation of peptides followed by mass spectrometry. Thirteen of the diseased samples additionally underwent microarray studies. Seventy muscle specimens from patients with a range of neuromuscular disorders were examined by ATPase histochemical methods. Smaller numbers of samples underwent immunohistochemical and immunoblot studies. Mass spectrometric studies identified and quantified approximately 300 total distinct proteins in each muscle sample. In IBM and to a lesser extent in polymyositis, proteomic studies confirmed by histochemical, immunohistochemical, and immunoblot studies showed loss of many fast-twitch specific structural proteins and glycolytic enzymes despite relative preservation of transcript levels. Increased abundance of a nuclear membrane protein, immunoglobulins, and two calpain-3 substrates were present. The atrophy present in IBM muscle is accompanied by preferential loss of fast-twitch structural proteins and glycolytic enzymes, particularly glycogen debranching enzyme, with relative preservation of the abundance of their respective transcripts. Although muscle atrophy has long been recognized in IBM, these studies are the first to report specific proteins which are reduced in quantity in IBM muscle.