•Validation of a simple LC-MS/MS protocol to analyze underivatized acylcarnitines.•Separation of clinically important isomeric acylcarnitine species.•Expanded coverage of free carnitine, carnitine ...metabolic precursors, and TMAO.•Novel application of mixed mode chromatography in a clinical setting.•Detection of peroxisome biomarkers long/very long chain dicarboxylic acylcarnitines.
Acylcarnitines are intermediate metabolites of the mitochondria that serve as biomarkers for inherited disorders of fatty acid oxidation and amino acid metabolism. The prevailing clinical method used to quantify acylcarnitines involves flow-injection tandem mass spectrometry, an approach with a number of limitations; foremost the inability to separate and therefore distinguish key isobaric acylcarnitine species. To address these issues, we report a clinically validated liquid chromatography tandem mass spectrometry method to quantify acylcarnitines, free carnitine, and carnitine metabolic intermediates in human plasma. Importantly, this method resolves clinically relevant isobaric and isomeric acylcarnitine species in a single 22 min analysis without the use of ion pairing or derivatization reagents. This unique combination of features is not achievable by existing acylcarnitine methods and is made possible by the use of a novel mixed-mode chromatographic separation. Further clinical validation studies demonstrate excellent limits of quantification, linearity, accuracy, and inter-assay precision for analyses of 38 different calibrated analytes. An additional 28 analytes are semi-quantitatively analyzed using surrogate calibrators. The study of residual patient specimens confirms the clinical utility of this method and suggests expanded applicability to the diagnosis of peroxisomal disorders. In summary, we report a clinically validated acylcarnitine method that utilizes a novel mixed-mode chromatographic separation to provide a number of advantages in terms of specificity, accuracy, sample preparation time, and clinical utility.
Acylcarnitine analysis is a useful test for identifying patients with inborn errors of mitochondrial fatty acid β-oxidation and certain organic acidemias. Plasma is routinely used in the diagnostic ...workup of symptomatic patients. Urine analysis of targeted acylcarnitine species may be helpful in the diagnosis of glutaric acidemia type I and other disorders in which polar acylcarnitine species accumulate. For newborn screening applications, dried blood spot acylcarnitine analysis can be performed as a multiplex assay with other analytes, including amino acids, succinylacetone, guanidinoacetate, creatine, and lysophosphatidylcholines. Tandem mass spectrometric methodology, established more than 30 years ago, remains a valid approach for acylcarnitine analysis. The method involves flow-injection analysis of esterified or underivatized acylcarnitines species and detection using a precursor-ion scan. Alternative methods utilize liquid chromatographic separation of isomeric and isobaric species and/or detection by selected reaction monitoring. These technical standards were developed as a resource for diagnostic laboratory practices in acylcarnitine analysis, interpretation, and reporting.
Existing mouse models of lethal Ebola virus infection do not reproduce hallmark symptoms of Ebola hemorrhagic fever, neither delayed blood coagulation and disseminated intravascular coagulation nor ...death from shock, thus restricting pathogenesis studies to nonhuman primates. Here we show that mice from the Collaborative Cross panel of recombinant inbred mice exhibit distinct disease phenotypes after mouse-adapted Ebola virus infection. Phenotypes range from complete resistance to lethal disease to severe hemorrhagic fever characterized by prolonged coagulation times and 100% mortality. Inflammatory signaling was associated with vascular permeability and endothelial activation, and resistance to lethal infection arose by induction of lymphocyte differentiation and cellular adhesion, probably mediated by the susceptibility allele Tek. These data indicate that genetic background determines susceptibility to Ebola hemorrhagic fever.
The covalent attachment of SUMO (small ubiquitin-like modifier) to other intracellular proteins affects a broad range of nuclear processes in yeast and animals, including chromatin maintenance, ...transcription, and transport across the nuclear envelope, as well as protects proteins from ubiquitin addition. Substantial increases in SUMOylated proteins upon various stresses have also implicated this modification in the general stress response. To help understand the role(s) of SUMOylation in plants, we developed a stringent method to isolate SUMO-protein conjugates from Arabidopsis thaliana that exploits a tagged SUMO1 variant that faithfully replaces the wild-type protein. Following purification under denaturing conditions, SUMOylated proteins were identified by tandem mass spectrometry from both nonstressed plants and those exposed to heat and oxidative stress. The list of targets is enriched for factors that direct SUMOylation and for nuclear proteins involved in chromatin remodeling/repair, transcription, RNA metabolism, and protein trafficking. Targets of particular interest include histone H2B, components in the LEUNIG/TOPLESS corepressor complexes, and proteins that control histone acetylation and DNA methylation, which affect genome-wide transcription. SUMO attachment site(s) were identified in a subset of targets, including SUMO1 itself to confirm the assembly of poly-SUMO chains. SUMO1 also becomes conjugated with ubiquitin during heat stress, thus connecting these two posttranslational modifications in plants. Taken together, we propose that SUMOylation represents a rapid and global mechanism for reversibly manipulating plant chromosomal functions, especially during environmental stress.
METHANESULFONATE-SENSITIVE21 (MMS21) ligases having critical roles in stress protection and DNA endoreduplication/repair, respectively. To help identify their corresponding targets in Arabidopsis ...thaliana, we used siz1 and mms21 mutants for proteomic analyses of SUMOylated proteins enriched via an engineered SUMO1 isoform suitable for mass spectrometric studies. Through multiple data sets from seedlings grown at normal temperatures or exposed to heat stress, we identified over 1000 SUMO targets, most of which are nuclear localized. Whereas no targets could be assigned to MMS21, suggesting that it modifies only a few low abundance proteins, numerous targets could be assigned to SIZ1, including major transcription factors, coactivators/repressors, and chromatin modifiers connected to abiotic and biotic stress defense, some of which associate into multisubunit regulatory complexes. SIZ1 itself is also a target, but studies with mutants protected from SUMOylation failed to uncover a regulatory role. The catalog of SIZ1 substrates indicates that SUMOylation by this ligase provides stress protection by modifying a large array of key nuclear regulators.
This Protocol Extension describes the adaptation of an existing Protocol detailing the use of targetable reactive electrophiles and oxidants, an on-demand redox targeting toolset in cultured cells. ...The adaptation described here is for use of reactive electrophiles and oxidants technologies in live zebrafish embryos (Z-REX). Zebrafish embryos expressing a Halo-tagged protein of interest (POI)-either ubiquitously or tissue specifically-are treated with a HaloTag-specific small-molecule probe housing a photocaged reactive electrophile (either natural electrophiles or synthetic electrophilic drug-like fragments). The reactive electrophile is then photouncaged at a user-defined time, enabling proximity-assisted electrophile-modification of the POI. Functional and phenotypic ramifications of POI-specific modification can then be monitored, by coupling to standard downstream assays, such as click chemistry-based POI-labeling and target-occupancy quantification; immunofluorescence or live imaging; RNA-sequencing and real-time quantitative polymerase chain reaction analyses of downstream-transcript modulations. Transient expression of requisite Halo-POI in zebrafish embryos is achieved by messenger RNA injection. Procedures associated with generation of transgenic zebrafish expressing a tissue-specific Halo-POI are also described. The Z-REX experiments can be completed in <1 week using standard techniques. To successfully execute Z-REX, researchers should have basic skills in fish husbandry, imaging and pathway analysis. Experience with protein or proteome manipulation is useful. This Protocol Extension is aimed at helping chemical biologists study precision redox events in a model organism and fish biologists perform redox chemical biology.
In late December 2019, an outbreak caused by a novel severe acute respiratory syndrome coronavirus 2 emerged in Wuhan, China. Data on the clinical characteristics and outcomes of infected patients in ...urban communities in the US are limited.
To describe the clinical characteristics and outcomes of patients with coronavirus disease 2019 (COVID-19) and to perform a comparative analysis of hospitalized and ambulatory patient populations.
This study is a case series of 463 consecutive patients with COVID-19 evaluated at Henry Ford Health System in metropolitan Detroit, Michigan, from March 9 to March 27, 2020. Data analysis was performed from March to April 2020.
Laboratory-confirmed severe acute respiratory syndrome coronavirus 2 infection.
Demographic data, underlying comorbidities, clinical presentation, complications, treatment, and outcomes were collected.
Of 463 patients with COVID-19 (mean SD age, 57.5 16.8 years), 259 (55.9%) were female, and 334 (72.1%) were African American. Most patients (435 94.0%) had at least 1 comorbidity, including hypertension (295 patients 63.7%), chronic kidney disease (182 patients 39.3%), and diabetes (178 patients 38.4%). Common symptoms at presentation were cough (347 patients 74.9%), fever (315 patients 68.0%), and dyspnea (282 patients 60.9%). Three hundred fifty-five patients (76.7%) were hospitalized; 141 (39.7%) required intensive care unit management and 114 (80.8%) of those patients required invasive mechanical ventilation. Male sex (odds ratio OR, 2.0; 95% CI, 1.3-3.2; P = .001), severe obesity (OR, 2.0; 95% CI, 1.4-3.6; P = .02), and chronic kidney disease (OR, 2.0; 95% CI, 1.3-3.3; P = .006) were independently associated with intensive care unit admission. Patients admitted to the intensive care unit had longer length of stay and higher incidence of respiratory failure and acute respiratory distress syndrome requiring invasive mechanical ventilation, acute kidney injury requiring dialysis, shock, and mortality (57 patients 40.4% vs 15 patients 7.0%) compared with patients in the general practice unit. Twenty-nine (11.2%) of those discharged from the hospital were readmitted and, overall, 20.0% died within 30 days. Male sex (OR, 1.8; 95% CI, 1.1-3.1; P = .03) and age older than 60 years (OR, 5.3; 95% CI, 2.9-9.7; P < .001) were significantly associated with mortality, whereas African American race was not (OR, 0.98; 95% CI, 0.54-1.8; P = .86).
In this review of urban metropolitan patients with COVID-19, most were African American with a high prevalence of comorbid conditions and high rates of hospitalization, intensive care unit admission, complications, and mortality due to COVID-19.
Global metabolic profiling currently achievable by untargeted mass spectrometry-based metabolomic platforms has great potential to advance our understanding of human disease states, including ...potential utility in the detection of novel and known inborn errors of metabolism (IEMs). There are few studies of the technical reproducibility, data analysis methods, and overall diagnostic capabilities when this technology is applied to clinical specimens for the diagnosis of IEMs. We explored the clinical utility of a metabolomic workflow capable of routinely generating semi-quantitative z-score values for ~900 unique compounds, including ~500 named human analytes, in a single analysis of human plasma. We tested the technical reproducibility of this platform and applied it to the retrospective diagnosis of 190 individual plasma samples, 120 of which were collected from patients with a confirmed IEM. Our results demonstrate high intra-assay precision and linear detection for the majority compounds tested. Individual metabolomic profiles provided excellent sensitivity and specificity for the detection of a wide range of metabolic disorders and identified novel biomarkers for some diseases. With this platform, it is possible to use one test to screen for dozens of IEMs that might otherwise require ordering multiple unique biochemical tests. However, this test may yield false negative results for certain disorders that would be detected by a more well-established quantitative test and in its current state should be considered a supplementary test. Our findings describe a novel approach to metabolomic analysis of clinical specimens and demonstrate the clinical utility of this technology for prospective screening of IEMs.
A molecular test to distinguish between sepsis and systemic inflammation of noninfectious etiology could potentially have clinical utility.
This study evaluated the diagnostic performance of a ...molecular host response assay (SeptiCyte LAB) designed to distinguish between sepsis and noninfectious systemic inflammation in critically ill adults.
The study employed a prospective, observational, noninterventional design and recruited a heterogeneous cohort of adult critical care patients from seven sites in the United States (n = 249). An additional group of 198 patients, recruited in the large MARS (Molecular Diagnosis and Risk Stratification of Sepsis) consortium trial in the Netherlands ( www.clinicaltrials.gov identifier NCT01905033), was also tested and analyzed, making a grand total of 447 patients in our study. The performance of SeptiCyte LAB was compared with retrospective physician diagnosis by a panel of three experts.
In receiver operating characteristic curve analysis, SeptiCyte LAB had an estimated area under the curve of 0.82-0.89 for discriminating sepsis from noninfectious systemic inflammation. The relative likelihood of sepsis versus noninfectious systemic inflammation was found to increase with increasing test score (range, 0-10). In a forward logistic regression analysis, the diagnostic performance of the assay was improved only marginally when used in combination with other clinical and laboratory variables, including procalcitonin. The performance of the assay was not significantly affected by demographic variables, including age, sex, or race/ethnicity.
SeptiCyte LAB appears to be a promising diagnostic tool to complement physician assessment of infection likelihood in critically ill adult patients with systemic inflammation. Clinical trial registered with www.clinicaltrials.gov (NCT01905033 and NCT02127502).
The stress-induced attachment of small ubiquitin-like modifier (SUMO) to a diverse collection of nuclear proteins regulating chromatin architecture, transcription, and RNA biology has been implicated ...in protecting plants and animals against numerous environmental challenges. In order to better understand stress-induced SUMOylation, we combined stringent purification of SUMO conjugates with isobaric tag for relative and absolute quantification mass spectrometry and an advanced method to adjust for sample-to-sample variation so as to study quantitatively the SUMOylation dynamics of intact Arabidopsis seedlings subjected to stress. Inspection of 172 SUMO substrates during and after heat shock (37 °C) revealed that stress mostly increases the abundance of existing conjugates, as opposed to modifying new targets. Some of the most robustly up-regulated targets participate in RNA processing and turnover and RNA-directed DNA modification, thus implicating SUMO as a regulator of the transcriptome during stress. Many of these targets were also strongly SUMOylated during ethanol and oxidative stress, suggesting that their modification is crucial for general stress tolerance. Collectively, our quantitative data emphasize the importance of SUMO to RNA-related processes protecting plants from adverse environments.
PDF
