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BACKGROUND: Delivering acute hospital care to patients at home might reduce costs and improve patient experience. Mayo Clinic's Advanced Care at Home (ACH) program is a novel virtual hybrid model of "Hospital at Home." This pragmatic randomized controlled non-inferiority trial aims to compare two acute care delivery models: ACH vs. traditional brick-and-mortar hospital care in acutely ill patients. METHODS: We aim to enroll 360 acutely ill adult patients (≥18 years) who are admitted to three hospitals in Arizona, Florida, and Wisconsin, two of which are academic medical centers and one is a community-based practice. The eligibility criteria will follow what is used in routine practice determined by local clinical teams, including clinical stability, social stability, health insurance plans, and zip codes. Patients will be randomized 1:1 to ACH or traditional inpatient care, stratified by site. The primary outcome is a composite outcome of all-cause mortality and 30-day readmission. Secondary outcomes include individual outcomes in the composite endpoint, fall with injury, medication errors, emergency room visit, transfer to intensive care unit (ICU), cost, the number of days alive out of hospital, and patient-reported quality of life. A mixed-methods study will be conducted with patients, clinicians, and other staff to investigate their experience. DISCUSSION: The pragmatic trial will examine a novel virtual hybrid model for delivering high-acuity medical care at home. The findings will inform patient selection and future large-scale implementation. TRIAL REGISTRATION: ClinicalTrials.gov NCT05212077. Registered on 27 January 2022.
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Hospitales , Calidad de Vida , Adulto , Servicios de Salud Comunitaria , Hospitalización , Humanos , Readmisión del Paciente , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
Human identification from biological material is largely dependent on the ability to characterize genetic polymorphisms in DNA. Unfortunately, DNA can degrade in the environment, sometimes below the level at which it can be amplified by PCR. Protein however is chemically more robust than DNA and can persist for longer periods. Protein also contains genetic variation in the form of single amino acid polymorphisms. These can be used to infer the status of non-synonymous single nucleotide polymorphism alleles. To demonstrate this, we used mass spectrometry-based shotgun proteomics to characterize hair shaft proteins in 66 European-American subjects. A total of 596 single nucleotide polymorphism alleles were correctly imputed in 32 loci from 22 genes of subjects' DNA and directly validated using Sanger sequencing. Estimates of the probability of resulting individual non-synonymous single nucleotide polymorphism allelic profiles in the European population, using the product rule, resulted in a maximum power of discrimination of 1 in 12,500. Imputed non-synonymous single nucleotide polymorphism profiles from European-American subjects were considerably less frequent in the African population (maximum likelihood ratio = 11,000). The converse was true for hair shafts collected from an additional 10 subjects with African ancestry, where some profiles were more frequent in the African population. Genetically variant peptides were also identified in hair shaft datasets from six archaeological skeletal remains (up to 260 years old). This study demonstrates that quantifiable measures of identity discrimination and biogeographic background can be obtained from detecting genetically variant peptides in hair shaft protein, including hair from bioarchaeological contexts.
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Antropología Forense/métodos , Cabello/química , Reacción en Cadena de la Polimerasa , Proteómica , Alelos , Población Negra/genética , Genotipo , Humanos , Polimorfismo de Nucleótido Simple/genética , Población Blanca/genéticaRESUMEN
Air-cathode microbial fuel cells (MFCs) are widely tested to recover electrical energy from waste streams containing organic matter. When high-strength wastewater, such as liquid animal manure, is used as a medium, inhibition on anode and cathode catalysts potentially impairs the effectiveness of MFC performance in power generation and pollutant removal. This study evaluated possible inhibitive effects of liquid swine manure components on MFC power generation, improved liquid manure-fed MFCs performance by pretreatment (dilution and selective adsorption), and modeled the kinetics of organic matter and nutrients removal kinetics. Parameters monitored included pH, conductivity, chemical oxygen demand (COD), volatile fatty acids (VFAs), total ammoniacal nitrogen (TAN), nitrite, nitrate, and phosphate concentrations. The removals of VFA and TAN were efficient, indicated by the short half-life times of 4.99 and 7.84 d, respectively. The mechanism for phosphate decrease was principally the salt precipitation on cathode, but the removal was incomplete after 42-d operation. MFC with an external resistor of 2.2 kΩ and fed with swine wastewater generated relatively small power (28.2 µW), energy efficiency (0.37%) and Coulombic efficiency (1.5%). Dilution of swine wastewater dramatically improved the power generation as the inhibitory effect was decreased. Zeolite and granular activated carbon were effective in the selective adsorption of ammonia or organic matter in swine wastewater, and so substantially improved the power generation, energy efficiency, and Coulombic efficiency. A smaller external resistor in the circuit was also observed to promote the organic matter degradation and thus to shorten the treatment time. Overall, air-cathode MFCs are promising for generating electrical power from livestock wastewater and meanwhile reducing the level of organic matter and nutrients.
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Fuentes de Energía Bioeléctrica/microbiología , Estiércol/microbiología , Amoníaco/análisis , Amoníaco/metabolismo , Animales , Reactores Biológicos/microbiología , Electricidad , Estiércol/análisis , Aguas del Alcantarillado/química , Porcinos , Eliminación de Residuos Líquidos , Aguas Residuales/análisis , Aguas Residuales/microbiologíaRESUMEN
TRIM5α is an antiviral, cytoplasmic, E3 ubiquitin (Ub) ligase that assembles on incoming retroviral capsids and induces their premature dissociation. It inhibits reverse transcription of the viral genome and can also synthesize unanchored polyubiquitin (polyUb) chains to stimulate innate immune responses. Here, we show that TRIM5α employs the E2 Ub-conjugating enzyme Ube2W to anchor the Lys63-linked polyUb chains in a process of TRIM5α auto-ubiquitination. Chain anchoring is initiated, in cells and in vitro, through Ube2W-catalyzed monoubiquitination of TRIM5α. This modification serves as a substrate for the elongation of anchored Lys63-linked polyUb chains, catalyzed by the heterodimeric E2 enzyme Ube2N/Ube2V2. Ube2W targets multiple TRIM5α internal lysines with Ub especially lysines 45 and 50, rather than modifying the N-terminal amino group, which is instead αN-acetylated in cells. E2 depletion or Ub mutation inhibits TRIM5α ubiquitination in cells and restores restricted viral reverse transcription, but not infection. Our data indicate that the stepwise formation of anchored Lys63-linked polyUb is a critical early step in the TRIM5α restriction mechanism and identify the E2 Ub-conjugating cofactors involved.
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Proteínas Portadoras/metabolismo , Modelos Biológicos , Transcripción Reversa/fisiología , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina/metabolismo , Factores de Restricción Antivirales , Células HEK293 , Células HeLa , Humanos , Mutagénesis Sitio-Dirigida , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína LigasasRESUMEN
BACKGROUND: Glycogen is a branched polysaccharide of glucose residues, consisting of α-1-4 glycosidic linkages with α-1-6 branches that together form multi-layered particles ranging in size from 30 nm to 300 nm. Glycogen spatial conformation and intracellular organization are highly regulated processes. Glycogen particles interact with their metabolizing enzymes and are associated with a variety of proteins that intervene in its biology, controlling its structure, particle size and sub-cellular distribution. The function of glycogen in adipose tissue is not well understood but appears to have a pivotal role as a regulatory mechanism informing the cells on substrate availability for triacylglycerol synthesis. To provide new molecular insights into the role of adipocyte glycogen we analyzed the glycogen-associated proteome from differentiated 3T3-L1-adipocytes. RESULTS: Glycogen particles from 3T3-L1-adipocytes were purified using a series of centrifugation steps followed by specific elution of glycogen bound proteins using α-1,4 glucose oligosaccharides, or maltodextrins, and tandem mass spectrometry. We identified regulatory proteins, 14-3-3 proteins, RACK1 and protein phosphatase 1 glycogen targeting subunit 3D. Evidence was also obtained for a regulated subcellular distribution of the glycogen particle: metabolic and mitochondrial proteins were abundant. Unlike the recently analyzed hepatic glycogen proteome, no endoplasmic proteins were detected, along with the recently described starch-binding domain protein 1. Other regulatory proteins which have previously been described as glycogen-associated proteins were not detected, including laforin, the AMPK beta-subunit and protein targeting to glycogen (PTG). CONCLUSIONS: These data provide new molecular insights into the regulation of glycogen-bound proteins that are associated with the maintenance, organization and localization of the adipocyte glycogen particle.
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Because HER-2 has been demonstrated in the nuclei of cancer cells, we hypothesized that it might interact with transcription factors that activate ERBB2 transcription. Macrohistone 2A1 (H2AFY; mH2A1) was found to interact with HER-2 in cancer cells that overexpress HER-2. Of the two human mH2A1 isoforms, mH2A1.2, but not mH2A1.1, interacted with HER-2 in human cancer cell lines. Overexpression of mH2A1.2, but not mH2A1.1, in cancer cells significantly increased HER-2 expression and tumorigenicity. Inhibition of HER-2 kinase activity diminished mH2A1 expression and mH2A1.2-induced ERBB2 transcription in cancer cells. Chromatin immunoprecipitation of mH2A1.2 in cancer cells stably transfected with mH2A1.2 showed enrichment of mH2A1.2 at the HER-2 promoter, suggesting a role for mH2A1.2 in driving HER-2 overexpression. The evolutionarily conserved macro domain of mH2A1.2 was sufficient for the interaction between HER-2 and mH2A1.2 and for mH2A1.2-induced ERBB2 transcription. Within the macro domain of mH2A1.2, a trinucleotide insertion (-EIS-) sequence not found in mH2A1.1 was essential for the interaction between HER-2 and mH2A1.2 as well as mH2A1.2-induced HER-2 expression and cell proliferation.
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Regulación Neoplásica de la Expresión Génica/fisiología , Histonas , Neoplasias/genética , Neoplasias/metabolismo , Receptor ErbB-2 , Animales , Neoplasias de la Mama/metabolismo , Células CACO-2 , División Celular/fisiología , Núcleo Celular/metabolismo , Neoplasias del Colon/metabolismo , Femenino , Células HEK293 , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias Ováricas/metabolismo , Regiones Promotoras Genéticas/fisiología , Dominios y Motivos de Interacción de Proteínas/fisiología , Receptor ErbB-2/química , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismoRESUMEN
The US Food and Drug Administration's Office of Food Additive Safety in the Center for Food Safety and Applied Nutrition conducts safety assessments of food additives, including food-contact substances such as polymeric and oligomeric materials that have the potential to migrate to food. Traditionally, little toxicity testing has been conducted on the low-molecular weight oligomeric fraction (< 1000 Da) of these food-contact substances. At lower exposures (≤ 150 µg/person/day), safety has been assessed based on the use of toxicity data on the monomeric components of these polymers as a sufficiently conservative approach for addressing the concern for genetic toxicity and carcinogenicity of the low-molecular weight oligomers (LMWOs). This paper discusses this assumption relative to the available data on these substances and their monomeric components in the context of exposures of ≤ 150 µg/person/day with emphasis on the evaluation of the potential genetic toxicity of these compounds. In most instances, data are available on either the monomers or the monomers' structural class to conservatively address the potential genetic toxicity of the LMWOs. Caveats to this generalization are also discussed. The assessment of LMWOs is important because they can be one of the primary migrants to food from a polymeric food-contact substance.
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Alimentos , Polímeros/toxicidad , Propiedades de Superficie , Estados Unidos , United States Food and Drug AdministrationRESUMEN
The dopamine (DA) transporter (DAT) is a key regulator of dopaminergic signaling as it mediates the reuptake of extrasynaptic DA and thereby terminates dopaminergic signaling. Emerging evidence indicates that DAT function is influenced through interactions with other proteins. The current report describes a method to identify such interactions following DAT immunoprecipitation from a rat striatal synaptosomal preparation. This subcellular fraction was selected since DAT function is often determined ex vivo by measuring DA uptake in this preparation and few reports investigating DAT-protein interactions have utilized this preparation. Following SDS-PAGE and colloidal Coomassie staining, selected protein bands from a DAT-immunoprecipitate were excised, digested with trypsin, extracted, and analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS). From the analysis of the tryptic peptides, several proteins were identified including DAT, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) ß, CaMKII δ, protein kinase C (PKC) ß, and PKC γ. Co-immunoprecipitation of PKC, CaMKII, and protein interacting with C kinase-1 with DAT was confirmed by Western blotting. Thus, the present study highlights a method to immunoprecipitate DAT and to identify co-immunoprecipitating proteins using LC/MS/MS and Western blotting. This method can be utilized to evaluate DAT protein-protein interactions but also to assess interactions involving other synaptic proteins. Ex vivo identification of protein-protein interactions will provide new insight into the function and regulation of a variety of synaptic, membrane-associated proteins, including DAT.
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Química Encefálica/fisiología , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Neuroquímica/métodos , Terminales Presinápticos/metabolismo , Mapeo de Interacción de Proteínas/métodos , Proteómica/métodos , Sinaptosomas/metabolismo , Animales , Masculino , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/fisiología , Neuroquímica/instrumentación , Terminales Presinápticos/química , Proteómica/instrumentación , Ratas , Ratas Sprague-Dawley , Sinaptosomas/químicaRESUMEN
Glycogen particles are associated with a population of proteins that mediate its biological functions, including: management of glucose flux into and out of the glycogen particle, maintenance of glycogen structure and regulation of particle size, number, and cellular location. A survey of the glycogen-associated proteome would be predicted to identify the relative representation of known members of this population, and associations with unexpected proteins that have the potential to mediate other functions of the glycogen particle. We therefore purified glycogen particles from both mouse and rat liver, using different techniques, and analyzed the resulting tryptic peptides by MS. We also specifically eluted glycogen-binding proteins from the pellet using malto-oligosaccharides. Comparison of the rat and mouse populations, and analysis of specifically eluted proteins allow some conclusions to be made about the hepatic glycogen sub-proteome. With the exception of glycogen branching enzyme all glycogen metabolic proteins were detected. Novel associations were identified, including ferritin and starch-binding domain protein 1, a protein that contains both a transmembrane endoplasmic reticulum signal peptide and a carbohydrate-binding module. This study therefore provides insight into the organization of the glycogen proteome, identifies other associated proteins and provides a starting point to explore the dynamic nature and cellular distribution of this metabolically important protein population.
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Glucógeno Hepático/metabolismo , Hígado/metabolismo , Proteínas/metabolismo , Animales , Ratones , Ratas , Espectrometría de Masas en TándemRESUMEN
Half the ribosomes translating the mRNA for phage T4 gene 60 topoisomerase subunit bypass a 50 nucleotide coding gap between codons 46 and 47. The pairing of codon 46 with its cognate peptidyl-tRNA anticodon dissociates, and following mRNA slippage, peptidyl-tRNA re-pairs to mRNA at a matched triplet 5' adjacent to codon 47, where translation resumes. Here, in studies with gene 60 cassettes, it is shown that the peptidyl-tRNA anticodon does not scan the intervening sequence for potential complementarity. However, certain coding gap mutants allow peptidyl-tRNA to scan sequences in the bypassed segment. A model is proposed in which the coding gap mRNA enters the ribosomal A-site and forms a structure that precludes peptidyl-tRNA scanning of its sequence. Dissipation of this RNA structure, together with the contribution of 16S rRNA anti-Shine-Dalgarno sequence pairing with GAG, facilitates peptidyl-tRNA re-pairing to mRNA.
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Anticodón/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , Aminoacil-ARN de Transferencia/genética , Secuencia de Aminoácidos , Animales , Bacteriófago T4/enzimología , Bacteriófago T4/genética , Secuencia de Bases , Codón/genética , ADN-Topoisomerasas/genética , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Mensajero/química , ARN Mensajero/metabolismo , Aminoacil-ARN de Transferencia/química , Aminoacil-ARN de Transferencia/metabolismo , Ribosomas/metabolismoRESUMEN
Near-infrared (NIR) fluorescence has the potential to provide surgeons with real-time intraoperative image-guidance. Increasing the signal-to-background ratio of fluorescent agents involves delivering a controllable excitation fluence rate of proper wavelength and/or using complementary imaging techniques such as FLIM. In this study we describe a low-cost linear driver circuit capable of driving Light Emitting Diodes (LEDs) from DC to 35 MHz, at high power, and which permit fluorescence CW and lifetime measurements. The electronic circuit Gerber files described in this article and the list of components are available online at www.frangionilab.org.
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When a eukaryotic mRNA sequence specifying an amino acid motif known as 2A is directly followed by a proline codon, two nonoverlapping proteins are synthesized. From earlier work, the second protein is known to start with this proline codon and is not created by proteolysis. Here we identify the C-terminal amino acid of an upstream 2A-encoded product from Perina nuda picorna-like virus that is glycine specified by the last codon of the 2A-encoding sequence. This is an example of recoding where 2A promotes unconventional termination after decoding of the glycine codon and continued translation beginning with the 3' adjacent proline codon.
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Codón de Terminación/genética , Biosíntesis de Proteínas , Secuencia de Aminoácidos , Modelos Genéticos , Datos de Secuencia Molecular , Picornaviridae/genética , ARN Mensajero/genética , ARN Viral/genética , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en Tándem , Proteínas Virales/biosíntesis , Proteínas Virales/genéticaRESUMEN
Eosinophil granule major basic protein 2 (MBP2 or major basic protein homolog) is a paralog of major basic protein (MBP1) and, similar to MBP1, is cytotoxic and cytostimulatory in vitro. MBP2, a small protein of 13,433 Da molecular weight, contains 10 cysteine residues. Mass spectrometry shows two cystine disulfide linkages (Cys20-Cys115 and Cys92-Cys107) and 6 cysteine residues with free sulfhydryl groups (Cys2, Cys23, Cys42, Cys43, Cys68, and Cys96). MBP2, similar to MBP1, has conserved motifs in common with C-type lectins. The disulfide bond locations are conserved among human MBP1, MBP2 and C-type lectins.
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Proteínas Sanguíneas/química , Proteínas Sanguíneas/aislamiento & purificación , Cistina/análisis , Cistina/química , Mapeo Peptídico , Proteoglicanos/química , Proteoglicanos/aislamiento & purificación , Secuencia de Aminoácidos , Proteínas Sanguíneas/metabolismo , Cisteína/química , Proteína Mayor Básica del Eosinófilo , Etilmaleimida/química , Humanos , Proteoglicanos/metabolismo , Alineación de Secuencia , Espectrometría de Masa por Ionización de Electrospray , Tripsina/metabolismoRESUMEN
Previous studies demonstrate that the covalent modification of thioredoxin reductase (TrxR) by both endogenous and exogenous electrophiles results in disruption of the conformation of the tumor suppressor protein p53. Here we report that the loss of normal cellular TrxR enzymatic activity by electrophilic modification or deletion of the C-terminal catalytic selenocysteine residue has functional consequences that are distinct from those resulting from depletion of TrxR protein in human RKO colon cancer cells. A thorough kinetic analysis was performed on purified TrxR in order to characterize the mechanism of its inhibition by electrophiles. Furthermore, electrospray mass spectrometry confirmed the alkylation of TrxR by lipid electrophiles and liquid chromatography-mass spectrometry/mass spectrometry identified the C-terminus as one target for alkylation. Then the consequences of TrxR modification by electrophiles on p53 conformation, transactivation and apoptosis were compared and contrasted with the effects of depletion of TrxR protein by treatment of cells with small interfering RNA directed against TrxR1. We found that cells depleted of TrxR were actually less sensitive to electrophile-induced disruption of p53 conformation and apoptosis than were cells expressing normal levels of TrxR. When RKO cells depleted of wild-type TrxR were transfected with C-terminal mutants of TrxR lacking the catalytic selenocysteine, p53 was found to be conformationally deranged, similar to cells treated with electrophiles. These results lead us to conclude that C-terminal modification of TrxR is both necessary and sufficient for the disruption of p53 and for the induction of apoptosis. Endogenous lipid electrophiles have been our primary focus; however, metabolic activation of hormones can generate endogenous mutagens, and we demonstrate that estrone-quinone attenuates p53 function in human MCF7 cells.
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Neoplasias del Colon/genética , Neoplasias del Colon/patología , Genes p53 , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Apoptosis , Línea Celular Tumoral , Cartilla de ADN , Inhibidores Enzimáticos/farmacología , Regulación Neoplásica de la Expresión Génica , Humanos , Cinética , Lípidos/fisiología , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reductasa de Tiorredoxina-Disulfuro/antagonistas & inhibidores , Reductasa de Tiorredoxina-Disulfuro/genéticaRESUMEN
The concept of a dichotomous versus a continuous aggression model continues to be debated within the research literature. The Impulsive/Premeditated Aggression Scale (IPAS; M. S. Stanford, R. J. Houston, C. W. Mathias, et al., 2003) is a newly developed self-report instrument designed to classify an individual's aggressive behavior as predominantly premeditated or predominantly impulsive. The IPAS consists of 30-items that are scored on a 5-point Likert scale. This study used a nonrandom sample of convenience (N = 85) from a forensic state hospital. Principal-components analysis of the 30 items revealed 2 distinct factors (Impulsive and Premeditated Aggression), which accounted for 33% of the variance. The results of this study further validate the bimodal classification of aggression through its application to a forensic sample. The implications for general assessment, diagnosis, and treatment are discussed.
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Agresión/psicología , Psiquiatría Forense , Trastornos Mentales/epidemiología , Trastornos Mentales/terapia , Adulto , Anciano , Anciano de 80 o más Años , Trastornos Disruptivos, del Control de Impulso y de la Conducta/epidemiología , Femenino , Humanos , Masculino , Trastornos Mentales/psicología , Persona de Mediana Edad , Prevalencia , Encuestas y CuestionariosRESUMEN
Bacterial analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry has been demonstrated in numerous laboratories, and a few attempts have been made to compare results from different laboratories on the same organism. It has been difficult to understand the causes behind the observed differences between laboratories when different instruments, matrices, solvents, etc. are used. In order to establish this technique as a useful tool for bacterial identification, additional efforts in standardizing the methods by which MALDI mass spectra are obtained and comparisons of spectra from different instruments with different operators are needed. Presented here is an extension of our previous single-laboratory reproducibility study with three different laboratories in a controlled experiment with aliquots of the same bacterial culture, matrix stock solution, and calibrant standards. Using automated spectral collection of whole-cell bacteria and automated data processing and analysis algorithms, fingerprints from three different laboratories were constructed and compared. Nine of the ions appeared reproducibly within all three laboratories, with additional unique ions observed within each of the laboratories. An initial evaluation of the ability to use a fingerprint generated within one laboratory for bacterial identification of a sample from another laboratory is presented, and strategies for improving identification rates between laboratories is discussed.
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Proteínas Bacterianas/análisis , Escherichia coli/química , Escherichia coli/metabolismo , Mapeo Peptídico/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Laboratorios , Proteoma , Reproducibilidad de los ResultadosRESUMEN
The measurement of 5-methyltetrahydrofolic acid (5 MT) blood levels is one of several factors used to diagnose folate deficiency in humans. 5 can be selectively purified from either human plasma or human serum via solid-phase extraction procedures and specifically detected and quantified in the extracts with liquid chromatography/isotope-dilution electrospray-ionization mass spectrometry. Two different, yet complementary, solid-phase extraction-liquid chromatography/mass spectrometry methods have been developed and applied to the quantification of 5 MT from such extracts. One method utilizes the high-affinity folate-binding protein from cow's milk coupled with multiple-reaction-monitoring-mode tandem mass spectrometry while the other method utilizes reversed-phase C(18) extraction followed by selected-ion-monitoring-mode mass spectrometry. The accuracy of each method is assessed through a comparative determination of 5 MT levels in homogenous plasma and serum pools. Additionally, each method is compared and evaluated against the "total folate" results provided by routine radioassay and microbiological assay determinations. On the basis of the experimental data presented in this report, it is suggested that both methods have the capacity to serve as potential reference methods for the quantification of circulating 5MT in plasma or serum.
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Ácido Fólico/sangre , Espectrometría de Masa por Ionización de Electrospray , Tetrahidrofolatos/sangre , Cromatografía Liquida , Humanos , Plasma/química , Sensibilidad y Especificidad , Suero/química , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
This study investigates bypassing initiated from codons immediately 5' of a stop codon. The mRNA slips and is scanned by the peptidyl-tRNA for a suitable landing site, and standard decoding resumes at the next 3' codon. This work shows that landing sites with potentially strong base pairing between the peptidyl-tRNA anticodon and mRNA are preferred, but sites with little or no potential for Watson-Crick or wobble base pairing can also be utilized. These results have implications for re-pairing in ribosomal frameshifting. Shine-Dalgarno sequences in the mRNA can alter the distribution of landing sites observed. The bacteriophage T4 gene 60 nascent peptide, known to influence take-off in its native context, imposes stringent P-site pairing requirements, thereby limiting the number of suitable landing sites.
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Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Aminoacil-ARN de Transferencia/metabolismo , Secuencia de Bases , Sistema de Lectura Ribosómico , Espectrometría de Masas , ARN Mensajero/química , Aminoacil-ARN de Transferencia/químicaRESUMEN
The predominant circulating folate monoglutamate in human plasma (>90%), and thus the most significant folate for accurately diagnosing folate deficiency, is 5-methyltetrahydrofolic acid (5 MT). Folate deficiency is typically indicated when circulating folate levels are < or = 3 ng/mL. The quantitative determination of plasma folates in general, and of 5 MT in particular, is complicated by their naturally low levels (pg/mL to ng/mL), their instability, and their tendency to interconvert. Highly specific and sensitive analytical methods are needed to accurately quantify endogenous 5 MT in human plasma. A method that utilizes the specific high-affinity binding sites of bovine folate binding protein (FBP) and the selectivity and sensitivity of selected ion monitoring mode isotope-dilution liquid chromatography/mass spectrometry (LC/MS) to quantify plasma 5 MT has been developed. The method is based on the solid-phase affinity extraction (SPAE) of 5 MT and its stable isotopically labeled analogue ([13C(5)]5 MT) from plasma (1 mL) using FBP immobilized to polymeric beads. The excess high-affinity binding sites on the affinity columns enable quantitative extraction of 5 MT from plasma under optimized sample pH conditions. Additionally, it is demonstrated that plasma proteins do not hinder the determination of 5 MT; therefore, protein precipitation is not required before the affinity extraction step. Detection and quantification of the extracted 5 MT is provided by positive-ion mode LC/MS in which the protonated molecular ions [M+H](+) of the analyte and the internal standard are monitored. The method shows linearity over three orders of magnitude (0.04-40 ng/mL) and has limits of detection and quantification of 0.04 and 0.4 ng/mL, respectively. Calibration curves obtained by spiking 5 MT into plasma exhibited good linearity between 0 and 25 ng/mL and both the plasma calibration standards and the plasma samples were stable for at least 48 h at room temperature. The recovery (average +/- % RSD) of 5 MT spiked into plasma from 5 to 25 ng/mL was 98.0% +/- 1.6% (n = 15). 5 MT levels determined by SPAE-LC/MS compared to "total folate" levels determined by radioassay and microbiological assay were discordant. Reasons for the discordancy are theorized, but it is clear that there exists an urgent need for clinical reference materials containing certified folate levels.