RESUMO
"Multi-stage" search strategies have become widely accepted for peptide identification and are implemented in a number of available software packages. We describe limitations of these strategies for validation and decoy-based statistical analyses and demonstrate these limitations using a set of control sample spectra. We propose a solution that corrects the statistical deficiencies and describe its implementation using the open-source software X!Tandem.
Assuntos
Proteômica , Cromatografia Líquida , Software , Espectrometria de Massas em TandemRESUMO
CONTEXT.: As electronic health records (EHRs) become more ubiquitous, physicians have come to expect that laboratory data from a variety of sources will be incorporated into the EHR in a structured format. The Clinical Laboratory Improvement Amendments have standards for data transmission traditionally met by pathologist review of their own hospital laboratory information system transmissions. However, with third-party laboratory data now being sent through external (nonhospital laboratory) interfaces, ownership of this review is less clear. Lack of an expert laboratory review process prior to changes being implemented can result in mapping and interfacing errors that could lead to misinterpretation and diagnostic errors. OBJECTIVE.: To determine the impact of retrospective and prospective laboratorian-assisted review on the volume of interface errors and new builds. DESIGN.: A seminal event led to a restructuring of the process for review of EHR laboratory builds, using laboratory expertise. RESULTS.: A review of 26 500 test result fields found 61 of 4282 (1.4%) unique codes that could have led to misinterpretation. These were corrected and a process for proactive review and maintenance by laboratory experts was implemented. This resulted in monthly decreases in outbound error message from 4270 to 1820 (57.4%), in new test builds from 586 to 274 (53.2%), and in new result builds from 1116 to 552 (50.5%). CONCLUSIONS.: Regular review and maintenance of external laboratory test builds in EHRs by a laboratory review team reduces interface error messages and reduces the number of new builds required for results to file into the EHR.
Assuntos
Registros Eletrônicos de Saúde/normas , Laboratórios , Garantia da Qualidade dos Cuidados de Saúde/métodos , Controle de Qualidade , HumanosRESUMO
OBJECTIVES: Thyroid and rheumatologic autoimmune testing are areas where evidence-based guidance from specialty organizations and Choosing Wisely support utilizing screening tests for autoimmune and thyroid disorders prior to more specialized testing. Adjustment of the orderable options in the electronic health record (EHR) can influence ordering patterns without requiring manual review or additional effort by the clinician. METHODS: The menu was adjusted to reflect recommendations from Choosing Wisely to favor screening tests that automatically reflex to specialized testing on primary care providers' preference lists. Effectiveness was evaluated by reviewing total orders for individual tests. RESULTS: Shifts in ordering from individual screening tests (antinuclear antibody and thyrotropin) to ones that reflexed to specialized testing were observed in parallel with significant reductions in the corresponding specialized testing. CONCLUSIONS: Optimization of the EHR laboratory ordering menu can be used to shift ordering patterns toward Choosing Wisely recommendations.
Assuntos
Registros Eletrônicos de Saúde , Sistemas de Registro de Ordens Médicas , Padrões de Prática Médica/estatística & dados numéricos , Design de Software , Algoritmos , Anticorpos Antinucleares/análise , Humanos , New Jersey , Reflexo , Centros de Atenção Terciária , Tireotropina/análiseRESUMO
OBJECTIVES: Traditional laboratory utilization measures are unable to detect the results of small-scale utilization improvement efforts in a background of rising patient volumes and acuity. However, accurate assessment is necessary to document effectiveness of these efforts. METHODS: Test menu changes, physician education, and laboratory utilization feedback were used to address costs and overused tests. Effectiveness was evaluated using cost/case mix index (CMI)-adjusted hospital day and inpatient tests/CMI-adjusted discharge. These metrics were compared with the more traditional measures of cost/test and inpatient tests/discharge. RESULTS: Cost/CMI-adjusted hospital day and inpatient tests/CMI-adjusted discharge better demonstrated the impact of utilization improvement efforts compared with more traditional measures because they account for changes in patient volume and acuity. CONCLUSIONS: Cost/CMI-adjusted hospital day and tests/CMI-adjusted discharge show the effectiveness of laboratory utilization efforts despite increasing patient volume and acuity.
Assuntos
Centros Médicos Acadêmicos/economia , Laboratórios Hospitalares/economia , Custos e Análise de Custo , Grupos Diagnósticos Relacionados , Humanos , Pacientes Internados/estatística & dados numéricos , Laboratórios Hospitalares/estatística & dados numéricos , Tempo de Internação/estatística & dados numéricos , Programas de Assistência Gerenciada , Pacientes Ambulatoriais/estatística & dados numéricosRESUMO
BACKGROUND AND PURPOSE: Plasma glutathione peroxidase (GPx-3) is a major antioxidant enzyme in plasma and the extracellular space that scavenges reactive oxygen species produced during normal metabolism or after oxidative insult. A deficiency of this enzyme increases extracellular oxidant stress, promotes platelet activation, and may promote oxidative posttranslational modification of fibrinogen. We recently identified a haplotype (H(2)) in the GPx-3 gene promoter that increases the risk of arterial ischemic stroke among children and young adults. METHODS: The aim of this study is to identify possible relationships between promoter haplotypes in the GPx-3 gene and cerebral venous thrombosis (CVT). We studied the GPx-3 gene promoter from 23 patients with CVT and 123 young controls (18 to 45 years) by single-stranded conformational polymorphism and sequencing analysis. RESULTS: Over half of CVT patients (52.1%) were heterozygous (H(1)H(2)) or homozygous (H(2)H(2)) carriers of the H(2) haplotype compared with 12.2% of controls, yielding a more than 10-fold independent increase in the risk of CVT (OR=10.7; 95% CI, 2.70 to 42.36; P<0.0001). Among women, the interaction of the H(2) haplotype with hormonal risk factors increased the OR of CVT to almost 70 (P<0.0001). CONCLUSIONS: These findings show that a novel GPx-3 promoter haplotype is a strong, independent risk factor for CVT. As we have previously shown that this haplotype is associated with a reduction in transcriptional activity, which compromises antioxidant activity and antithrombotic benefits of the enzyme, these results suggest that a deficiency of GPx-3 leads to a cerebral venous thrombophilic state.
Assuntos
Glutationa Peroxidase/genética , Trombose Intracraniana/epidemiologia , Trombose Intracraniana/genética , Trombose Venosa/epidemiologia , Trombose Venosa/genética , Adolescente , Adulto , Predisposição Genética para Doença/epidemiologia , Glutationa Peroxidase/sangue , Haplótipos , Hormônios , Humanos , Trombose Intracraniana/sangue , Modelos Logísticos , Pessoa de Meia-Idade , Polimorfismo Conformacional de Fita Simples , Regiões Promotoras Genéticas/genética , Fatores de Risco , Acidente Vascular Cerebral/sangue , Acidente Vascular Cerebral/epidemiologia , Acidente Vascular Cerebral/genética , Trombose Venosa/sangueRESUMO
BACKGROUND AND PURPOSE: Plasma glutathione peroxidase (GPx-3)-deficiency increases extracellular oxidant stress, decreases bioavailable nitric oxide, and promotes platelet activation. The aim of this study is to identify polymorphisms in the GPx-3 gene, examine their relationship to arterial ischemic stroke (AIS) in a large series of children and young adults, and determine their functional molecular consequences. METHODS: We studied the GPx-3 gene promoter from 123 young adults with idiopathic AIS and 123 age- and gender-matched controls by single-stranded conformational polymorphism and sequencing analysis. A second, independent population with childhood stroke was used for a replication study. We identified 8 novel, strongly linked polymorphisms in the GPx-3 gene promoter that formed 2 main haplotypes (H1 and H2). The transcriptional activity of the 2 most prevalent haplotypes was studied with luciferase reporter gene constructs. RESULTS: The H2 haplotype was over-represented in both patient populations and associated with an independent increase in the risk of AIS in young adults (odds ratio=2.07, 95% CI=1.03 to 4.47; P=0.034) and children (odds ratio=2.13, 95% CI=1.23 to 4.90; P=0.027). In adults simultaneously exposed to vascular risk factors, the risk of AIS approximately doubled (odds ratio=5.18, 95% CI=1.82 to 15.03; P<0.001). Transcriptional activity of the H2 haplotype was lower than that of the H1 haplotype, especially after upregulation by hypoxia (normalized relative luminescence: 3.54+/-0.32 versus 2.47+/-0.26; P=0.0083). CONCLUSIONS: These findings indicate that a novel GPx-3 promoter haplotype is an independent risk factor for AIS in children and young adults. This haplotype reduces the gene's transcriptional activity, thereby compromising gene expression and plasma antioxidant and antithrombotic activities.
Assuntos
Isquemia Encefálica/genética , Predisposição Genética para Doença/genética , Glutationa Peroxidase/genética , Polimorfismo Genético/genética , Regiões Promotoras Genéticas/genética , Acidente Vascular Cerebral/genética , Adulto , Antioxidantes/metabolismo , Coagulação Sanguínea/genética , Isquemia Encefálica/sangue , Isquemia Encefálica/enzimologia , Criança , Análise Mutacional de DNA , Ativação Enzimática/genética , Feminino , Regulação Enzimológica da Expressão Gênica/genética , Frequência do Gene , Marcadores Genéticos/genética , Testes Genéticos , Haplótipos , Humanos , Hipóxia/genética , Hipóxia/metabolismo , Hipóxia/fisiopatologia , Masculino , Mutação/genética , Fatores de Risco , Acidente Vascular Cerebral/sangue , Acidente Vascular Cerebral/enzimologia , Ativação Transcricional/genéticaRESUMO
Multiplexed protein analysis using planar microarrays or microbeads is growing in popularity for simultaneous assays of antibodies, cytokines, allergens, drugs and hormones. However, this new assay format presents several new operational issues for the clinical laboratory, such as the quality control of protein-microarray-based assays, the release of unrequested test data and the use of diagnostic algorithms to transform microarray data into diagnostic results.
Assuntos
Biomarcadores/análise , Técnicas de Diagnóstico Molecular , Análise Serial de Proteínas , Kit de Reagentes para Diagnóstico , Algoritmos , Animais , Pesquisa Biomédica/ética , Confidencialidade/ética , Interpretação Estatística de Dados , Humanos , Técnicas de Diagnóstico Molecular/ética , Valor Preditivo dos Testes , Análise Serial de Proteínas/ética , Análise Serial de Proteínas/métodos , Proteínas/análise , Controle de Qualidade , Kit de Reagentes para Diagnóstico/normas , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Previous in vitro experiments have shown that hyperhomocysteinemia leads to oxidative inactivation of nitric oxide, in part by inhibiting the expression of cellular glutathione peroxidase (GPx-1). To elucidate the role of intracellular redox status on homocysteine-induced endothelial dysfunction and oxidant stress, heterozygous cystathionine beta-synthase-deficient (CBS(-/+)) and wild-type (CBS(+/+)) mice were treated with the cysteine donor L-2-oxothiazolidine-4-carboxylic acid (OTC). CBS(-/+) mice had significantly lower GPx-1 activity compared with their CBS(+/+) littermates, and OTC treatment led to a modest increase in tissue GPx-1 activity and significant increases in total thiols and in reduced glutathione levels in both CBS(+/+) and CBS(-/+) mice. Superfusion of the mesentery with beta-methacholine or bradykinin produced dose-dependent vasodilation of mesenteric arterioles in CBS(+/+) mice and in CBS(+/+) mice treated with OTC. In contrast, mesenteric arterioles from CBS(-/+) mice manifested dose-dependent vasoconstriction in response to both agonists. OTC treatment of CBS(-/+) mice restored normal microvascular vasodilator reactivity to beta-methacholine and bradykinin. These findings demonstrate that mild hyperhomocysteinemia leads to endothelial dysfunction in association with decreased bioavailable nitric oxide. Increasing the cellular thiol and reduced glutathione pools and increasing GPx-1 activity restores endothelial function. These findings emphasize the importance of intracellular redox balance for nitric oxide bioactivity and endothelial function.
Assuntos
Glutationa Peroxidase/metabolismo , Hiper-Homocisteinemia/metabolismo , Óxido Nítrico/metabolismo , Estresse Oxidativo , Vasodilatação/fisiologia , Animais , Bradicinina/farmacologia , Cistationina beta-Sintase/deficiência , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiopatologia , Homocisteína/sangue , Hiper-Homocisteinemia/fisiopatologia , Fígado/metabolismo , Mesentério/irrigação sanguínea , Cloreto de Metacolina/farmacologia , Camundongos , Oxirredução , Ácido Pirrolidonocarboxílico , Compostos de Sulfidrila/sangue , Tiazóis/farmacologia , Tiazolidinas , Vasodilatação/efeitos dos fármacosRESUMO
OBJECTIVES: We examined the impact of weekly feedback reports on the test-ordering behavior of internal medicine residents. STUDY DESIGN: Retrospective analysis of a performance improvement effort. METHODS: In a large, urban, academic medical center, we extracted raw data on every inpatient laboratory test ordered by all internal medicine residents during two 26-week time periods. The pre-intervention phase established baseline ordering volume as each resident rotated through the various clinical services. The intervention consisted of a 1-hour educational seminar detailing the potential harm and costs of laboratory overutilization followed by the post intervention phase, which consisted of weekly feedback reports graphically illustrating individual versus group ordering patterns, where the identity of individual residents was protected. The total numbers of tests ordered during the 2 phases were compared using an independent t test. RESULTS: During the post intervention phase, we observed a net reduction of 21% in tests ordered-an average of 941 tests per week-with the greatest reduction in the chemistry section of the laboratory, followed by hematology, coagulation, and all others combined. This reduction in test volume corresponded to a $1.3 million reduction in charges. CONCLUSIONS: Providing physicians-in-training with a weekly feedback report detailing their test ordering volume in comparison with those of their peers is an effective method for reducing laboratory overutilization. Benefits to our approach include maintaining physician autonomy without alteration of existing infrastructure or disclosure of test fees.
Assuntos
Controle de Custos/métodos , Testes Diagnósticos de Rotina/estatística & dados numéricos , Feedback Formativo , Centros Médicos Acadêmicos , Controle de Custos/economia , Testes Diagnósticos de Rotina/economia , Humanos , Medicina Interna/educação , Medicina Interna/estatística & dados numéricos , Internato e Residência/estatística & dados numéricos , Padrões de Prática Médica/estatística & dados numéricos , Avaliação de Programas e Projetos de Saúde , Melhoria de Qualidade/organização & administração , Melhoria de Qualidade/estatística & dados numéricos , Estudos Retrospectivos , Procedimentos Desnecessários/economia , Procedimentos Desnecessários/estatística & dados numéricosRESUMO
Proteins involved in mitochondrial metabolic pathways engage in functionally relevant multi-enzyme complexes. We previously described an interaction between short-chain 3-hydroxyacyl-coenzyme A dehydrogenase (SCHAD) and glutamate dehydrogenase (GDH) explaining the clinical phenotype of hyperinsulinism in SCHAD-deficient patients and adding SCHAD to the list of mitochondrial proteins capable of forming functional, multi-pathway complexes. In this work, we provide evidence of SCHAD's involvement in additional interactions forming tissue-specific metabolic super complexes involving both membrane-associated and matrix-dwelling enzymes and spanning multiple metabolic pathways. As an example, in murine liver, we find SCHAD interaction with aspartate transaminase (AST) and GDH from amino acid metabolic pathways, carbamoyl phosphate synthase I (CPS-1) from ureagenesis, other fatty acid oxidation and ketogenesis enzymes and fructose-bisphosphate aldolase, an extra-mitochondrial enzyme of the glycolytic pathway. Most of the interactions appear to be independent of SCHAD's role in the penultimate step of fatty acid oxidation suggesting an organizational, structural or non-enzymatic role for the SCHAD protein.
Assuntos
3-Hidroxiacil-CoA Desidrogenases/metabolismo , Fígado/metabolismo , Redes e Vias Metabólicas , Complexos Multienzimáticos/metabolismo , 3-Hidroxiacil-CoA Desidrogenases/química , 3-Hidroxiacil-CoA Desidrogenases/genética , Animais , Aspartato Aminotransferases/química , Aspartato Aminotransferases/metabolismo , Carbamoil-Fosfato Sintase (Amônia)/química , Carbamoil-Fosfato Sintase (Amônia)/metabolismo , Frutose-Bifosfato Aldolase/química , Frutose-Bifosfato Aldolase/metabolismo , Glutamato Desidrogenase/química , Glutamato Desidrogenase/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Complexos Multienzimáticos/químicaRESUMO
A classic physiologic response to hypoxia in humans is the up-regulation of the ERYTHROPOIETIN (EPO) gene, which is the central regulator of red blood cell mass. The EPO gene, in turn, is activated by hypoxia inducible factor (HIF). HIF is a transcription factor consisting of an alpha subunit (HIF-alpha) and a beta subunit (HIF-beta). Under normoxic conditions, prolyl hydroxylase domain protein (PHD, also known as HIF prolyl hydroxylase and egg laying-defective nine protein) site specifically hydroxylates HIF-alpha in a conserved LXXLAP motif (where underlining indicates the hydroxylacceptor proline). This provides a recognition motif for the von Hippel Lindau protein, a component of an E3 ubiquitin ligase complex that targets hydroxylated HIF-alpha for degradation. Under hypoxic conditions, this inherently oxygen-dependent modification is arrested, thereby stabilizing HIF-alpha and allowing it to activate the EPO gene. We previously identified and characterized an erythrocytosis-associated HIF2A mutation, G537W. More recently, we reported two additional erythrocytosis-associated HIF2A mutations, G537R and M535V. Here, we describe the functional characterization of these two mutants as well as a third novel erythrocytosis-associated mutation, P534L. These mutations affect residues C-terminal to the LXXLAP motif. We find that all result in impaired degradation and thus aberrant stabilization of HIF-2alpha. However, each exhibits a distinct profile with respect to their effects on PHD2 binding and von Hippel Lindau interaction. These findings reinforce the importance of HIF-2alpha in human EPO regulation, demonstrate heterogeneity of functional defects arising from these mutations, and point to a critical role for residues C-terminal to the LXXLAP motif in HIF-alpha.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Policitemia/metabolismo , Sequência de Aminoácidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Biocatálise , Linhagem Celular , Humanos , Hidroxilação , Dados de Sequência Molecular , Mutação/genética , Policitemia/genética , Pró-Colágeno-Prolina Dioxigenase/genética , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Prolina/genética , Prolina/metabolismo , Ligação Proteica , Alinhamento de Sequência , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismoRESUMO
We present four patients with late AMR following cardiac transplantation, which was associated with de novo post-transplant anti-HLA class II antibody production. All patients had negative anti-HLA class I and class II antibodies prior to transplantation (as assessed by sensitive Flow PRA bead assays) and had a negative retrospective T- and B-cell flow cytometric cross-match. Upon presentation with late graft rejection due to AMR, all patients were treated with rituximab and serial plasmapheresis with IVIg plus triple-drug immunosuppression therapy. Despite initial responses to therapy, relapses occurred in all of the patients and necessitated prolonged or multiple hospital admissions and second transplants in two cases. Post-transplant serum antibody monitoring did not prove to be predictive of treatment success or failure. Serum anti-HLA antibodies should be monitored after heart transplantation. We recommend an assessment of anti-HLA antibodies following a decline in immunosuppressant drug levels or in the presence of heart failure symptoms. Anti-HLA antibody detection should be performed using very sensitive techniques such as microparticle-based assays.
Assuntos
Linfócitos B/imunologia , Rejeição de Enxerto/imunologia , Transplante de Coração/imunologia , Isoanticorpos/sangue , Depleção Linfocítica , Plasmaferese , Adulto , Humanos , Isoanticorpos/imunologia , Isoanticorpos/isolamento & purificação , Masculino , Pessoa de Meia-Idade , Resultado do TratamentoRESUMO
Plasma glutathione peroxidase (GPx-3) is a selenocysteine-containing protein with antioxidant properties. GPx-3 deficiency has been associated with cardiovascular disease and stroke. The regulation of GPx-3 expression remains largely uncharacterized, however, and we studied its transcriptional and translational determinants in a cultured cell system. In transient transfections of a renal cell line (Caki-2), the published sequence cloned upstream of a luciferase reporter gene produced minimal activity (relative luminescence (RL) = 0.6 +/- 0.4). Rapid amplification of cDNA ends was used to identify a novel transcription start site that is located 233 bp downstream (3') of the published site and that produced a >25-fold increase in transcriptional activity (RL = 16.8 +/- 1.9; p < 0.0001). Analysis of the novel GPx-3 promoter identified Sp-1- and hypoxia-inducible factor-1-binding sites, as well as the redox-sensitive metal response element and antioxidant response element. Hypoxia was identified as a strong transcriptional regulator of GPx-3 expression, in part through the presence of the hypoxia-inducible factor-1-binding site, leading to an almost 3-fold increase in expression levels after 24 h compared with normoxic conditions (normalized RL = 3.5 +/- 0.3 versus 1.2 +/- 0.1; p < 0.001). We also investigated the role of the translational cofactors tRNA(Sec), SECIS-binding protein-2, and SelD (selenophosphate synthetase D) in GPx-3 protein expression. tRNA(Sec) and SelD significantly enhanced GPx-3 expression, whereas SECIS-binding protein-2 showed a trend toward increased expression. These results demonstrate the presence of a novel functional transcription start site for the human GPx-3 gene with a promoter regulated by hypoxia, and identify unique translational determinants of GPx-3 expression.
Assuntos
Glutationa Peroxidase/biossíntese , Animais , Sequência de Bases , Sítios de Ligação , Hipóxia Celular/genética , DNA Complementar/análise , DNA Complementar/genética , Expressão Gênica , Genes Reporter/genética , Glutationa Peroxidase/sangue , Glutationa Peroxidase/genética , Humanos , Rim/citologia , Luciferases/genética , Dados de Sequência Molecular , Fosfotransferases/genética , Regiões Promotoras Genéticas/genética , RNA de Transferência/genética , Proteínas de Ligação a RNA/genética , Selenocisteína/genética , Compostos de Sulfidrila/farmacologia , Sítio de Iniciação de Transcrição , Transfecção , Xenopus laevis/genéticaRESUMO
Cellular glutathione peroxidase (GPx-1) is the most abundant intracellular isoform of the GPx antioxidant enzyme family. In this study, we hypothesized that GPx-1 deficiency directly induces an increase in vascular oxidant stress, with resulting endothelial dysfunction. We studied vascular function in a murine model of homozygous deficiency of GPx-1 (GPx-1(-/-)). Mesenteric arterioles of GPx-1(-/-) mice demonstrated paradoxical vasoconstriction to beta-methacholine and bradykinin, whereas wild-type (WT) mice showed dose-dependent vasodilation in response to both agonists. One week of treatment of GPx-1(-/-) mice with L-2-oxothiazolidine-4-carboxylic acid (OTC), which increases intracellular thiol pools, resulted in restoration of normal vascular reactivity in the mesenteric bed of GPx-1(-/-) mice. We observed an increase of the isoprostane iPF(2alpha)-III, a marker of oxidant stress, in the plasma and aortas of GPx-1(-/-) mice compared with WT mice, which returned toward normal after OTC treatment. Aortic sections from GPx-1(-/-) mice showed increased binding of an anti-3-nitrotyrosine antibody in the absence of frank vascular lesions. These findings demonstrate that homozygous deficiency of GPx-1 leads to impaired endothelium-dependent vasodilator function presumably due to a decrease in bioavailable nitric oxide and to increased vascular oxidant stress. These vascular abnormalities can be attenuated by increasing bioavailable intracellular thiol pools.