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1.
Anal Bioanal Chem ; 415(5): 809-821, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36507958

RESUMO

The US National Institute of Standards and Technology (NIST) developed a Standard Reference Material® (SRM®) 3949 Folate Vitamers in Frozen Human Serum to replace SRM 1955 Homocysteine and Folate in Human Serum. The presence of increased endogenous levels of folic acid and 5-methyltetrahydrofolate (5mTHF) in SRM 3949, enhanced folate stability via addition of ascorbic acid, and inclusion of values for additional minor folates are improvements over SRM 1955 that should better serve the clinical folate measurement community. The new SRM contains folates at three levels. To produce SRM 3949, pilot sera were collected from 15 individual donors, 5 of whom were given a 400-µg folic acid supplement 1 h prior to blood draw to increase serum levels of 5mTHF and folic acid for the high-level material. To stabilize the folates, 0.5% (mass concentration) ascorbic acid was added as soon as possible after preparation of serum. These pilot sera were screened for five folates plus the pyrazino-s-triazine derivative of 4-α-hydroxy-5-methyltetrahydrofolate (MeFox) at the US Centers for Disease Control and Prevention (CDC) by isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS). Based on these results, a blending protocol was specified to obtain the three desired folate concentrations for SRM 3949. ID-LC-MS/MS analysis at the CDC and NIST was utilized to assign values for folic acid and 5mTHF, as well as several minor folates.


Assuntos
Ácido Fólico , Espectrometria de Massas em Tandem , Humanos , Ácido Fólico/análise , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Padrões de Referência , Ácido Ascórbico
2.
Clin Chem Lab Med ; 61(1): 78-85, 2023 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-36279170

RESUMO

OBJECTIVES: Vitamin D-binding protein (VDBP), a serum transport protein for 25-hydroxyvitamin D [25(OH)D], has three common proteoforms which have co-localized amino acid variations and glycosylation. A monoclonal immunoassay was found to differentially detect VDBP proteoforms and methods using liquid chromatography-tandem mass spectrometry (LC-MS/MS) might be able to overcome this limitation. Previously developed multiple reaction monitoring LC-MS/MS methods for total VDBP quantification represent an opportunity to probe the potential effects of proteoforms on proteolysis, instrument response and quantification accuracy. METHODS: VDBP was purified from homozygous human donors and quantified using proteolysis or acid hydrolysis and LC-MS/MS. An interlaboratory comparison was performed using pooled human plasma [Standard Reference Material® 1950 (SRM 1950) Metabolites in Frozen Human Plasma] and analyses with different LC-MS/MS methods in two laboratories. RESULTS: Several shared peptides from purified proteoforms were found to give reproducible concentrations [≤2.7% coefficient of variation (CV)] and linear instrument responses (R2≥0.9971) when added to human serum. Total VDBP concentrations from proteolysis or amino acid analysis (AAA) of purified proteoforms had ≤1.92% CV. SRM 1950, containing multiple proteoforms, quantified in two laboratories resulted in total VDBP concentrations with 7.05% CV. CONCLUSIONS: VDBP proteoforms were not found to cause bias during quantification by LC-MS/MS, thus demonstrating that a family of proteins can be accurately quantified using shared peptides. A reference value was assigned for total VDBP in SRM 1950, which may be used to standardize methods and improve the accuracy of VDBP quantification in research and clinical samples.


Assuntos
Espectrometria de Massas em Tandem , Proteína de Ligação a Vitamina D , Humanos , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Proteólise , Vitamina D , Proteínas Sanguíneas/metabolismo , Aminoácidos/metabolismo
3.
Nat Biotechnol ; 40(6): 938-945, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35145312

RESUMO

Recruiting endogenous adenosine deaminases using exogenous guide RNAs to edit cellular RNAs is a promising therapeutic strategy, but editing efficiency and durability remain low using current guide RNA designs. In this study, we engineered circular ADAR-recruiting guide RNAs (cadRNAs) to enable more efficient programmable adenosine-to-inosine RNA editing without requiring co-delivery of any exogenous proteins. Using these cadRNAs, we observed robust and durable RNA editing across multiple sites and cell lines, in both untranslated and coding regions of RNAs, and high transcriptome-wide specificity. Additionally, we increased transcript-level specificity for the target adenosine by incorporating interspersed loops in the antisense domains, reducing bystander editing. In vivo delivery of cadRNAs via adeno-associated viruses enabled 53% RNA editing of the mPCSK9 transcript in C57BL/6J mice livers and 12% UAG-to-UGG RNA correction of the amber nonsense mutation in the IDUA-W392X mouse model of mucopolysaccharidosis type I-Hurler syndrome. cadRNAs enable efficient programmable RNA editing in vivo with diverse protein modulation and gene therapeutic applications.


Assuntos
Edição de RNA , RNA Guia de Cinetoplastídeos , Adenosina/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL , RNA/genética , RNA/metabolismo , Edição de RNA/genética , RNA Circular , RNA Guia de Cinetoplastídeos/genética , Proteínas de Ligação a RNA/metabolismo
4.
J AOAC Int ; 105(4): 1162-1174, 2022 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-35188206

RESUMO

BACKGROUND: In collaboration with the Office of Dietary Supplements at the National Institutes of Health, the National Institute of Standards and Technology issued a suite of botanical matrix reference materials (RMs) and Standard Reference Material® (SRM) for determination of isoflavones and toxic elements in kudzu dietary supplement ingredients. OBJECTIVE: RM 8650 Pueraria montana var. lobata (Kudzu) Rhizome, SRM 3268 Pueraria montana var. lobata (Kudzu) Extract, and RM 8652 Kudzu-Containing Solid Oral Dosage Form were issued with values assigned for isoflavones (puerarin, daidzin, and daidzein), toxic elements (arsenic, cadmium, and lead), and selenium. METHODS: Isoflavone values were assigned using liquid chromatography with UV absorbance or mass spectrometry detection. Element values were assigned using inductively coupled plasma mass spectrometry and results from an interlaboratory comparison exercise. RESULTS: Mass fractions for puerarin were 32.2 ± 3.2 mg/g, 128 ± 13 mg/g, and 68.2 ± 6.9 mg/g in RM 8650, SRM 3268, and RM 8652, respectively. Arsenic increases from 156 ± 14 ng/g to 849 ± 83 ng/g and cadmium decreases from 348 ± 14 ng/g to 82.1 ± 4.9 ng/g from rhizome to extract. CONCLUSION: The kudzu RM/SRM suite complements previously issued soy-related SRMs with values assigned for isoflavones, which have been studied for their potential health benefits, and expands the analytical resource by providing values for puerarin, an isoflavone not found in soy. HIGHLIGHTS: The three new kudzurmaterials are for use in the determination of isoflavones, toxic elements, and selenium. For the isoflavones, these new kudzu materials provide higher levels of daidzin and daidzein than existing soy-related SRMs, and they provide a value for an isoflavone not in existing SRMs (puerarin). Toxic elements in RM 8650 and SRM 3268 provide new botanical matrixes for use by dietary supplement manufacturers for the verification of the safety of their raw materials.


Assuntos
Arsênio , Isoflavonas , Pueraria , Selênio , Cádmio , Isoflavonas/análise , Pueraria/química
5.
Proc Natl Acad Sci U S A ; 117(35): 21319-21327, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817489

RESUMO

Organisms can adapt to a broad spectrum of sudden and dramatic changes in their environment. These abrupt changes are often perceived as stress and trigger responses that facilitate survival and eventual adaptation. The ubiquitin-proteasome system (UPS) is involved in most cellular processes. Unsurprisingly, components of the UPS also play crucial roles during various stress response programs. The budding yeast SCFMet30 complex is an essential cullin-RING ubiquitin ligase that connects metabolic and heavy metal stress to cell cycle regulation. Cadmium exposure results in the active dissociation of the F-box protein Met30 from the core ligase, leading to SCFMet30 inactivation. Consequently, SCFMet30 substrate ubiquitylation is blocked and triggers a downstream cascade to activate a specific transcriptional stress response program. Signal-induced dissociation is initiated by autoubiquitylation of Met30 and serves as a recruitment signal for the AAA-ATPase Cdc48/p97, which actively disassembles the complex. Here we show that the UBX cofactor Shp1/p47 is an additional key element for SCFMet30 disassembly during heavy metal stress. Although the cofactor can directly interact with the ATPase, Cdc48 and Shp1 are recruited independently to SCFMet30 during cadmium stress. An intact UBX domain is crucial for effective SCFMet30 disassembly, and a concentration threshold of Shp1 recruited to SCFMet30 needs to be exceeded to initiate Met30 dissociation. The latter is likely related to Shp1-mediated control of Cdc48 ATPase activity. This study identifies Shp1 as the crucial Cdc48 cofactor for signal-induced selective disassembly of a multisubunit protein complex to modulate activity.


Assuntos
Proteínas F-Box/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Proteína com Valosina/metabolismo , Cádmio , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mutação , Domínios Proteicos , Multimerização Proteica , Proteínas de Saccharomyces cerevisiae/genética , Saccharomycetales , Estresse Fisiológico
6.
Clin Mass Spectrom ; 16: 11-17, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-34820515

RESUMO

Vitamin D plays a vital role in successful pregnancy outcomes for both the mother and fetus. Vitamin D is bound to vitamin D binding protein (VDBP) in blood and is carried to the liver, kidneys and other target tissues. Accurate measurements of the clinically measured metabolite of vitamin D, 25-hydroxyvitamin D [25(OH)D], depend on complete removal from the binding protein. It has been found that VDBP concentrations increase in maternal serum during pregnancy, obfuscating the accuracy of 25(OH)D concentration measurements in pregnant women. Additionally, measurements of VDBP concentrations during pregnancy have been performed using immunoassays, which suffer from variations due to differences in antibody epitopes, making clinical comparisons difficult. Quantification of VDBP is also of interest because changes in VDBP expression levels may indicate negative outcomes during pregnancy, such as preterm delivery and restricted fetal growth. To address the need for accurate measurement of VDBP during pregnancy, a method using liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) was developed to quantify VDBP using isotopically labeled peptides as internal standards. This method was used to quantify VDBP in Standard Reference Material® (SRM) 1949 Frozen Human Prenatal Serum, which was prepared from separate serum pools of women who were not pregnant and women during each trimester of pregnancy. VDBP concentrations were found to be lowest in the serum pool from non-pregnant women and increased in each trimester. These data had good repeatability and were found to be suitable for reference value assignment of VDBP in SRM 1949.

7.
Anal Bioanal Chem ; 410(9): 2321-2329, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29435636

RESUMO

Dietary fatty acids can be both beneficial and detrimental to human health depending on the degree and type of saturation. Healthcare providers and research scientists monitor the fatty acid content of human plasma and serum as an indicator of health status and diet. In addition, both the Centers for Disease Control & Prevention (CDC) and the National Institutes of Health - Office of Dietary Supplements are interested in circulating fatty acids (FAs) because they may be predictive of coronary heart disease. The National Institute of Standards and Technology (NIST) provides a wide variety of reference materials (RMs) and Standard Reference Materials® (SRM®s) including blood, serum, plasma, and urine with values assigned for analytes of clinical interest. NIST SRM 2378 Fatty Acids in Frozen Human Serum was introduced in 2015 to help validate methods used for the analysis of FAs in serum, and consists of three different pools of serum acquired from (1) healthy donors who had taken fish oil dietary supplements (at least 1000 mg per day) for at least one month (level 1 material), (2) healthy donors who had taken flaxseed oil dietary supplements (at least 1000 mg per day) for at least one month (level 2 material), and (3) healthy donors eating "normal" diets who had not taken dietary supplements containing fish or plant oils (level 3 material). The use of dietary supplements by donors provided SRMs with natural endogenous ranges of FAs at concentrations observed in human populations. Results from analyses using two methods at NIST, including one involving a novel microwave-assisted acid hydrolysis procedure, and one at the CDC are presented here. These results and their respective uncertainties were combined to yield certified values with expanded uncertainties for 12 FAs and reference values with expanded uncertainties for an additional 18 FAs.


Assuntos
Cromatografia Gasosa/métodos , Suplementos Nutricionais , Ácidos Graxos/sangue , Ionização de Chama/métodos , Cromatografia Gasosa-Espectrometria de Massas/métodos , Preservação de Sangue , Cromatografia Gasosa/normas , Criopreservação , Suplementos Nutricionais/análise , Ácidos Graxos/normas , Ácidos Graxos Insaturados/sangue , Ácidos Graxos Insaturados/normas , Óleos de Peixe/administração & dosagem , Óleos de Peixe/sangue , Ionização de Chama/normas , Congelamento , Cromatografia Gasosa-Espectrometria de Massas/normas , Humanos , Óleos de Plantas/administração & dosagem , Óleos de Plantas/análise , Padrões de Referência
8.
Forensic Sci Int ; 284: 15-32, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29331680

RESUMO

Estimating error rates for firearm evidence identification is a fundamental challenge in forensic science. This paper describes the recently developed congruent matching cells (CMC) method for image comparisons, its application to firearm evidence identification, and its usage and initial tests for error rate estimation. The CMC method divides compared topography images into correlation cells. Four identification parameters are defined for quantifying both the topography similarity of the correlated cell pairs and the pattern congruency of the registered cell locations. A declared match requires a significant number of CMCs, i.e., cell pairs that meet all similarity and congruency requirements. Initial testing on breech face impressions of a set of 40 cartridge cases fired with consecutively manufactured pistol slides showed wide separation between the distributions of CMC numbers observed for known matching and known non-matching image pairs. Another test on 95 cartridge cases from a different set of slides manufactured by the same process also yielded widely separated distributions. The test results were used to develop two statistical models for the probability mass function of CMC correlation scores. The models were applied to develop a framework for estimating cumulative false positive and false negative error rates and individual error rates of declared matches and non-matches for this population of breech face impressions. The prospect for applying the models to large populations and realistic case work is also discussed. The CMC method can provide a statistical foundation for estimating error rates in firearm evidence identifications, thus emulating methods used for forensic identification of DNA evidence.

9.
Anal Bioanal Chem ; 410(4): 1265-1278, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29222652

RESUMO

A Standard Reference Material (SRM) of seaweed, SRM 3232 Kelp Powder (Thallus laminariae) has been developed to support food and dietary supplement measurements in compliance with the Food Safety Modernization Act (FSMA) and the Dietary Supplement Health and Education Act of 1994 (DSHEA). The material was characterized for nutritional minerals, arsenic species, isomers of vitamin K1, proximates, and toxic elements. Kelp is a rich source of vitamins and minerals, and it is an excellent source of dietary iodine. Kelp also contains a large amount of arsenic, which is toxic as inorganic species but much less so as organic species. To capture the dietary profile of kelp, certified values were issued for As, Ca, Cd, Cr, Cu, Fe, Hg, I, K, Mg, Mn, Mo, Na, Pb, and Zn. Reference values for proximates were assigned. For the first time, a certified value for iodine, reference values for isomers of vitamin K1, and reference values for arsenic species including arsenosugars were assigned in a seaweed. SRM 3232 fills a gap in Certified Reference Materials (CRMs) needed for quality assurance and method validation in the compositional measurements of kelp and similar seaweeds used as food and as dietary supplements. Graphical Absract Arsenic species and isomers of vitamin K1 were determined in the development of SRM 3232 Kelp Powder (Thallus laminariae).


Assuntos
Kelp/química , Pós , Cromatografia Líquida , Padrões de Referência , Espectrometria de Massas em Tandem
10.
Anal Chem ; 89(19): 10461-10467, 2017 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-28930436

RESUMO

A new tobacco filler Standard Reference Material (SRM) has been issued by the National Institute of Standards and Technology (NIST) in September 2016 with certified and reference mass fraction values for nicotine, N-nitrosonornicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and volatiles. The constituents have been determined by multiple analytical methods with measurements at NIST and at the Centers for Disease Control and Prevention, and with confirmatory measurements by commercial laboratories. This effort highlights the development of the first SRM for reduced nicotine and reduced tobacco-specific nitrosamines with certified values for composition.


Assuntos
Cromatografia Gasosa-Espectrometria de Massas/métodos , Espectrometria de Massas em Tandem/métodos , Produtos do Tabaco/análise , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida de Alta Pressão/normas , Congelamento , Cromatografia Gasosa-Espectrometria de Massas/normas , Nicotina/análise , Nicotina/normas , Nitrosaminas/análise , Nitrosaminas/normas , Transição de Fase , Padrões de Referência , Espectrometria de Massas em Tandem/normas , Produtos do Tabaco/normas , Compostos Orgânicos Voláteis/análise , Compostos Orgânicos Voláteis/normas
11.
J AOAC Int ; 100(5): 1294-1303, 2017 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-28917261

RESUMO

The National Institute of Standards and Technology (NIST), in collaboration with the National Institutes of Health Office of Dietary Supplements and the Vitamin D Standardization Program, has recently issued a new serum-matrix Standard Reference Material (SRM): 2973 Vitamin D Metabolites in Frozen Human Serum (High Level). SRM 2973 was designed to provide a serum material with a total 25-hydroxyvitamin D [25(OH)D] concentration near 100 nmol/L to complement the existing serum-based SRMs with values assigned for total 25(OH)D between 20 and 80 nmol/L. Values were assigned for 25-hydroxyvitamin D2 [25(OH)D2], 25-hydroxyvitamin D3 [25(OH)D3], 3-epi-25(OH)D3, and total 25(OH)D [the sum of 25(OH)D2 + 25(OH)D3] using the NIST isotope dilution LC with tandem MS (MS/MS) reference measurement procedure (RMP) and related methods. SRM 2973 has a certified value of 98.4 ± 2.1 nmol/L for 25(OH)D3 and reference values of 1.59 ± 0.05 nmol/L for 25(OH)D2 and 5.23 ± 0.20 nmol/L for 3-epi-25(OH)D3. In addition, a candidate RMP for 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] based on LC-MS/MS was used to assign values to SRM 2973 and the existing SRM 972a Vitamin D Metabolites in Frozen Human Serum. Reference values for 24R,25(OH)2D3 were assigned to SRM 2973 (7.51 ± 0.26 nmol/L) and the four levels of SRM 972a: Level 1 (6.38 ± 0.23 nmol/L), Level 2 (3.39 ± 0.12 nmol/L), Level 3 (3.88 ± 0.013 nmol/L), and Level 4 (6.32 ± 0.22 nmol/L). The development of SRM 2973 [with a higher concentration of 25(OH)D3] and the addition of values for 24R,25(OH)2D3 assigned to both SRM 972a and SRM 2973 provide laboratories involved in vitamin D measurements with improved QA tools.


Assuntos
25-Hidroxivitamina D 2/sangue , Análise Química do Sangue/normas , Calcifediol/sangue , Humanos , Espectrometria de Massas em Tandem/normas , Estados Unidos , Vitamina D
12.
Anal Chem ; 89(9): 4907-4913, 2017 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-28375002

RESUMO

The National Institute of Standards and Technology (NIST) has developed Standard Reference Material (SRM) 972a Vitamin D Metabolites in Frozen Human Serum as a replacement for SRM 972, which is no longer available. SRM 972a was developed in collaboration with the National Institutes of Health's Office of Dietary Supplements. In contrast to the previous reference material, three of the four levels of SRM 972a are composed of unmodified human serum. This SRM has certified and reference values for the following 25-hydroxyvitamin D [25(OH)D] species: 25(OH)D2, 25(OH)D3, and 3-epi-25(OH)D3. The value assignment and certification process included three isotope-dilution mass spectrometry approaches, with measurements performed at NIST and at the Centers for Disease Control and Prevention (CDC). The value assignment methods employed have been modified from those utilized for the previous SRM, and all three approaches now incorporate chromatographic resolution of the stereoisomers, 25(OH)D3 and 3-epi-25(OH)D3.


Assuntos
25-Hidroxivitamina D 2/sangue , Calcifediol/sangue , Cromatografia Líquida/normas , Espectrometria de Massas/normas , 25-Hidroxivitamina D 2/normas , Calcifediol/química , Calcifediol/normas , Humanos , Padrões de Referência , Valores de Referência , Estereoisomerismo , Estados Unidos , United States Government Agencies
13.
Anal Bioanal Chem ; 409(4): 949-960, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27832301

RESUMO

Two independent analytical approaches, based on liquid chromatography with absorbance detection and liquid chromatography with mass spectrometric detection, have been developed for determination of isoflavones in soy materials. These two methods yield comparable results for a variety of soy-based foods and dietary supplements. Four Standard Reference Materials (SRMs) have been produced by the National Institute of Standards and Technology to assist the food and dietary supplement community in method validation and have been assigned values for isoflavone content using both methods. These SRMs include SRM 3234 Soy Flour, SRM 3236 Soy Protein Isolate, SRM 3237 Soy Protein Concentrate, and SRM 3238 Soy-Containing Solid Oral Dosage Form. A fifth material, SRM 3235 Soy Milk, was evaluated using the methods and found to be inhomogeneous for isoflavones and unsuitable for value assignment. Graphical Abstract Separation of six isoflavone aglycones and glycosides found in Standard Reference Material (SRM) 3236 Soy Protein Isolate.


Assuntos
Cromatografia Líquida/métodos , Isoflavonas/análise , Alimentos de Soja/análise , Espectrofotometria Ultravioleta/métodos , Isótopos , Espectrometria de Massas , Espectroscopia de Prótons por Ressonância Magnética , Padrões de Referência
14.
Bioenergy Res ; 9(1): 303-314, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27019676

RESUMO

Biomass compositional methods are used to compare different lignocellulosic feedstocks, to measure component balances around unit operations and to determine process yields and therefore the economic viability of biomass-to-biofuel processes. Four biomass reference materials (RMs NIST 8491-8494) were prepared and characterized, via an interlaboratory comparison exercise in the early 1990s to evaluate biomass summative compositional methods, analysts, and laboratories. Having common, uniform, and stable biomass reference materials gives the opportunity to assess compositional data compared to other analysts, to other labs, and to a known compositional value. The expiration date for the original characterization of these RMs was reached and an effort to assess their stability and recharacterize the reference values for the remaining material using more current methods of analysis was initiated. We sent samples of the four biomass RMs to 11 academic, industrial, and government laboratories, familiar with sulfuric acid compositional methods, for recharacterization of the component reference values. In this work, we have used an expanded suite of analytical methods that are more appropriate for herbaceous feedstocks, to recharacterize the RMs' compositions. We report the median values and the expanded uncertainty values for the four RMs on a dry-mass, whole-biomass basis. The original characterization data has been recalculated using median statistics to facilitate comparisons with this data. We found improved total component closures for three out of the four RMs compared to the original characterization, and the total component closures were near 100 %, which suggests that most components were accurately measured and little double counting occurred. The major components were not statistically different in the recharacterization which suggests that the biomass materials are stable during storage and that additional components, not seen in the original characterization, were quantified here.

15.
PLoS Genet ; 11(12): e1005727, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26656496

RESUMO

Abundance of substrate receptor subunits of Cullin-RING ubiquitin ligases (CRLs) is tightly controlled to maintain the full repertoire of CRLs. Unbalanced levels can lead to sequestration of CRL core components by a few overabundant substrate receptors. Numerous diseases, including cancer, have been associated with misregulation of substrate receptor components, particularly for the largest class of CRLs, the SCF ligases. One relevant mechanism that controls abundance of their substrate receptors, the F-box proteins, is autocatalytic ubiquitylation by intact SCF complex followed by proteasome-mediated degradation. Here we describe an additional pathway for regulation of F-box proteins on the example of yeast Met30. This ubiquitylation and degradation pathway acts on Met30 that is dissociated from Skp1. Unexpectedly, this pathway required the cullin component Cdc53/Cul1 but was independent of the other central SCF component Skp1. We demonstrated that this non-canonical degradation pathway is critical for chromosome stability and effective defense against heavy metal stress. More importantly, our results assign important biological functions to a sub-complex of cullin-RING ligases that comprises Cdc53/Rbx1/Cdc34, but is independent of Skp1.


Assuntos
Proteínas Culina/genética , Proteínas F-Box/genética , Proteínas Ligases SKP Culina F-Box/genética , Proteínas de Saccharomyces cerevisiae/genética , Complexos Ubiquitina-Proteína Ligase/genética , Ubiquitinação/genética , Sequência de Aminoácidos , Cádmio/toxicidade , Ciclo Celular/genética , Divisão Celular/genética , Homeostase/efeitos dos fármacos , Redes e Vias Metabólicas/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Saccharomyces cerevisiae , Ubiquitinas/genética
16.
Dermatol Surg ; 41(9): 1020-3, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26230329

RESUMO

BACKGROUND: It is a common practice to biopsy clinically atypical nevi, which may signify an individual's increased risk of developing melanoma. There is no consensus in the current literature, however, as to what the best method is to manage biopsy-proven atypical nevi. OBJECTIVE: The objective was to compare margin clearance rates between reshave excision and full-thickness surgical excision performed to manage biopsy-proven atypical nevi. MATERIALS AND METHODS: In this retrospective observational study, histopathology specimens from 201 patients whose atypical nevi were surgically removed were analyzed. RESULTS: For the majority (76%-79%) of the atypical nevi studied, all atypical melanocytes were removed by the initial shave biopsy. Of those atypical nevi with positive margins, shave re-excision was shown to have a lower clearance rate (76.2%) when compared with surgical excision (87.5%). CONCLUSION: This study shows that in most cases, no residual atypical melanocytes are left after the initial shave biopsy. However, of the lesions where margins are not clear, full-thickness surgical excision may have a higher rate of success at eventual clearance than reshave excision.


Assuntos
Procedimentos Cirúrgicos Dermatológicos/métodos , Nevo Pigmentado/patologia , Nevo Pigmentado/cirurgia , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/cirurgia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos
17.
Anal Chem ; 85(24): 11732-8, 2013 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-24187941

RESUMO

The National Institute of Standards and Technology (NIST), in collaboration with the National Institutes of Health (NIH), has developed a Standard Reference Material (SRM) to support technology development in metabolomics research. SRM 1950 Metabolites in Human Plasma is intended to have metabolite concentrations that are representative of those found in adult human plasma. The plasma used in the preparation of SRM 1950 was collected from both male and female donors, and donor ethnicity targets were selected based upon the ethnic makeup of the U.S. population. Metabolomics research is diverse in terms of both instrumentation and scientific goals. This SRM was designed to apply broadly to the field, not toward specific applications. Therefore, concentrations of approximately 100 analytes, including amino acids, fatty acids, trace elements, vitamins, hormones, selenoproteins, clinical markers, and perfluorinated compounds (PFCs), were determined. Value assignment measurements were performed by NIST and the Centers for Disease Control and Prevention (CDC). SRM 1950 is the first reference material developed specifically for metabolomics research.


Assuntos
Análise Química do Sangue/normas , Metabolômica/normas , Adulto , Aminoácidos/sangue , Biomarcadores/sangue , Carotenoides/sangue , Ácidos Graxos/sangue , Feminino , Humanos , Masculino , National Institutes of Health (U.S.) , Padrões de Referência , Estados Unidos , Vitaminas/sangue
18.
Anal Bioanal Chem ; 405(13): 4539-48, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23529413

RESUMO

The vitamin C concentrations in three food-matrix Standard Reference Materials (SRMs) from the National Institute of Standards and Technology (NIST) have been determined by liquid chromatography (LC) with absorbance detection. These materials (SRM 1549a Whole Milk Powder, SRM 1849a Infant/Adult Nutritional Formula, and SRM 3233 Fortified Breakfast Cereal) have been characterized to support analytical measurements made by food processors that are required to provide information about their products' vitamin C content on the labels of products distributed in the United States. The SRMs are primarily intended for use in validating analytical methods for the determination of selected vitamins, elements, fatty acids, and other nutrients in these materials and in similar matrixes. They can also be used for quality assurance in the characterization of test samples or in-house control materials, and for establishing measurement traceability. Within-day precision of the LC method used to measure vitamin C in the food-matrix SRMs characterized in this study ranged from 2.7% to 6.5%.


Assuntos
Ácido Ascórbico/normas , Cromatografia Líquida/normas , Suplementos Nutricionais/normas , Alimentos Formulados/normas , Ácido Ascórbico/análise , Suplementos Nutricionais/análise , Alimentos Formulados/análise , Humanos , Lactente , Controle de Qualidade , Padrões de Referência , Valores de Referência , Reprodutibilidade dos Testes
19.
Anal Bioanal Chem ; 405(13): 4531-8, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23371533

RESUMO

As part of a collaboration with the National Institutes of Health's Office of Dietary Supplements and the Food and Drug Administration's Center for Drug Evaluation and Research, the National Institute of Standards and Technology has developed Standard Reference Material (SRM) 3274 Botanical Oils Containing Omega-3 and Omega-6 Fatty Acids and SRM 3275 Omega-3 and Omega-6 Fatty Acids in Fish Oil. SRM 3274 consists of one ampoule of each of four seed oils (3274-1 Borage (Borago officinalis), 3274-2 Evening Primrose (Oenothera biennis), 3274-3 Flax (Linium usitatissimum), and 3274-4 Perilla (Perilla frutescens)), and SRM 3275 consists of two ampoules of each of three fish oils (3275-1 a concentrate high in docosahexaenoic acid, 3275-2 an anchovy oil high in docosahexaenoic acid and eicosapentaenoic acid, and 3275-3 a concentrate containing 60% long-chain omega-3 fatty acids). Each oil has certified and reference mass fraction values for up to 20 fatty acids. The fatty acid mass fraction values are based on results from analyses using gas chromatography with flame ionization detection (GC-FID) and mass spectrometry (GC/MS). These SRMs will complement other reference materials currently available with mass fractions for similar analytes and are part of a series of SRMs being developed for dietary supplements.


Assuntos
Suplementos Nutricionais/normas , Ácidos Docosa-Hexaenoicos/normas , Ácido Eicosapentaenoico/normas , Óleos de Peixe/normas , Óleos de Plantas/normas , Cromatografia Gasosa , Suplementos Nutricionais/análise , Ácidos Docosa-Hexaenoicos/isolamento & purificação , Ácido Eicosapentaenoico/isolamento & purificação , Óleos de Peixe/química , Ionização de Chama , Humanos , Óleos de Plantas/química , Padrões de Referência , Valores de Referência
20.
J AOAC Int ; 96(6): 1281-7, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24645505

RESUMO

Standard Reference Material 3280 Multivitamin/ Multielement Tablets was issued by the National Institute of Standards and Technology in 2009, and has certified and reference mass fraction values for 13 vitamins, 26 elements, and two carotenoids. Elements were measured using two or more analytical methods at NIST with additional data contributed by collaborating laboratories. This reference material is expected to serve a dual purpose: to provide quality assurance in support of a database of dietary supplement products and to provide a means for analysts, dietary supplement manufacturers, and researchers to assess the appropriateness and validity of their analytical methods and the accuracy of their results.


Assuntos
Química Farmacêutica/normas , Suplementos Nutricionais/análise , Suplementos Nutricionais/normas , Vitaminas/análise , Carotenoides/análise , Química Farmacêutica/métodos , Controle de Qualidade , Padrões de Referência , Reprodutibilidade dos Testes , Comprimidos , Estados Unidos , Vitaminas/química
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