High-throughput RNA sequencing (RNA-seq) dramatically expands the potential for novel genomics discoveries, but the wide variety of platforms, protocols and performance has created the need for comprehensive reference data. Here we describe the Association of Biomolecular Resource Facilities next-generation sequencing (ABRF-NGS) study on RNA-seq. We tested replicate experiments across 15 laboratory sites using reference RNA standards to test four protocols (polyA-selected, ribo-depleted, size-selected and degraded) on five sequencing platforms (Illumina HiSeq, Life Technologies’ PGM and Proton, Pacific Biosciences RS and Roche’s 454). The results show high intra-platform and inter-platform concordance for expression measures across the deep-count platforms, but highly variable efficiency and cost for splice junction and variant detection between all platforms. These data also demonstrate that ribosomal RNA depletion can both enable effective analysis of degraded RNA samples and be readily compared to polyA-enriched fractions. This study provides a broad foundation for cross-platform standardization, evaluation and improvement of RNA-seq.
Hydrogen sulfide (H
2
S) plays important roles in metabolism and health. Its enzymatic generation from sulfur-containing amino acids (SAAs) is well characterized. However, the existence of non-enzymatic H
2
S production from SAAs, the chemical mechanism, and its biological implications remain unclear. Here we present non-enzymatic H
2
S production in vitro and in blood via a reaction specific for the SAA cysteine serving as substrate and requires coordinated catalysis by Vitamin B
6
, pyridoxal(phosphate), and iron under physiological conditions. An initial cysteine-aldimine is formed by nucleophilic attack of the cysteine amino group to the pyridoxal(phosphate) aldehyde group. Free or heme-bound iron drives the formation of a cysteine-quinonoid, thiol group elimination, and hydrolysis of the desulfurated aldimine back to pyridoxal(phosphate). The reaction ultimately produces pyruvate, NH
3
, and H
2
S. This work highlights enzymatic production is inducible and robust in select tissues, whereas iron-catalyzed production contributes underappreciated basal H
2
S systemically with pathophysiological implications in hemolytic, iron overload, and hemorrhagic disorders.
Background & Aims
Selected-ion flow-tube mass spectrometry (SIFT-MS) can precisely identify trace gases in the human breath, in the parts-per-billion range. We investigated whether concentrations of volatile compounds in breath samples correlate with the diagnosis of alcoholic hepatitis (AH) and the severity of liver disease in patients with AH.
Methods
We recruited patients with liver disease from a single tertiary care center. The study population was divided those with AH with cirrhosis (n=40) and those with cirrhosis with acute decompensation from etiologies other than alcohol (n=40); individuals without liver disease served as controls (n=43). We used SIFT-MS to identify and measure 14 volatile compounds in breath samples from fasted subjects. We used various statistical analyses to compare clinical characteristics and breath levels of compounds among groups, and test the correlation between levels of compounds and severity of liver disease. Logistic regression analysis was performed to build a predictive model for AH.
Results
We identified 6 compounds (2-propanol, acetaldehyde, acetone, ethanol, pentane and trimethylamine [TMA]) whose levels were increased in patients with liver disease compared with controls. Mean concentrations of TMA, acetone, and pentane were particularly high in breath samples from patients with AH, compared to those with acute decompensation or controls (for both, P<.001). Using receiver operating characteristic curve analysis, we developed a model for the diagnosis of AH based on breath levels of TMA, acetone, and pentane (TAP). TAP scores of 36 or higher identified the patients with AH (AUC=0.92), with 90% sensitivity and 80% specificity. The levels of exhaled TMA had a low level of correlation with the severity of AH based on model for end-stage liver disease score (r=0.38; 95% confidence interval, 0.07–0.69; P=.018].
Conclusion
Based on levels of volatile compounds in breath samples, we can identify patients with AH vs patients with acute decompensation or individuals without liver disease. Levels of exhaled TMA moderately correlate with the severity of AH. These findings might be used in diagnosis of AH or in determining patient prognosis.
Exhaled breath analysis is a promising noninvasive method to detect fatty liver in children. Isoprene, acetone, trimethylamine, acetaldehyde, and pentane are novel biomarkers that may help to gain insight into pathophysiological processes leading to the development of NAFLD.
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