Noninvasive proteomic biomarkers for alcohol-related liver disease.

Alcohol-related liver disease (ALD) is a major cause of liver-related death worldwide, yet understanding of the three key pathological features of the disease-fibrosis, inflammation and steatosis-remains incomplete. Here, we present a paired liver-plasma proteomics approach to infer molecular pathophysiology and to explore the diagnostic and prognostic capability of plasma proteomics in 596 individuals (137 controls and 459 individuals with ALD), 360 of whom had biopsy-based histological assessment. We analyzed all plasma samples and 79 liver biopsies using a mass spectrometry (MS)-based proteomics workflow with short gradient times and an enhanced, data-independent acquisition scheme in only 3 weeks of measurement time. In plasma and liver biopsy tissues, metabolic functions were downregulated whereas fibrosis-associated signaling and immune responses were upregulated. Machine learning models identified proteomics biomarker panels that detected significant fibrosis (receiver operating characteristic-area under the curve (ROC-AUC), 0.92, accuracy, 0.82) and mild inflammation (ROC-AUC, 0.87, accuracy, 0.79) more accurately than existing clinical assays (DeLong's test, P < 0.05). These biomarker panels were found to be accurate in prediction of future liver-related events and all-cause mortality, with a Harrell's C-index of 0.90 and 0.79, respectively. An independent validation cohort reproduced the diagnostic model performance, laying the foundation for routine MS-based liver disease testing.

A Previously Undescribed Highly Prevalent Phage Identified in a Danish Enteric Virome Catalog.

Gut viruses are important, yet often neglected, players in the complex human gut microbial ecosystem. Recently, the number of human gut virome studies has been increasing; however, we are still only scratching the surface of the immense viral diversity. In this study, 254 virus-enriched fecal metagenomes from 204 Danish subjects were used to generate the Danish Enteric Virome Catalog (DEVoC) containing 12,986 nonredundant viral scaffolds, of which the majority was previously undescribed, encoding 190,029 viral genes. The DEVoC was used to compare 91 healthy DEVoC gut viromes from children, adolescents, and adults that were used to create the DEVoC. Gut viromes of healthy Danish subjects were dominated by phages. While most phage genomes (PGs) only occurred in a single subject, indicating large virome individuality, 39 PGs were present in more than 10 healthy subjects. Among these 39 PGs, the prevalences of three PGs were associated with age. To further study the prevalence of these 39 prevalent PGs, 1,880 gut virome data sets of 27 studies from across the world were screened, revealing several age-, geography-, and disease-related prevalence patterns. Two PGs also showed a remarkably high prevalence worldwide-a crAss-like phage (20.6% prevalence), belonging to the tentative AlphacrAssvirinae subfamily, and a previously undescribed circular temperate phage infecting Bacteroides dorei (14.4% prevalence), called LoVEphage because it encodes lots of viral elements. Due to the LoVEphage's high prevalence and novelty, public data sets in which the LoVEphage was detected were de novo assembled, resulting in an additional 18 circular LoVEphage-like genomes (67.9 to 72.4 kb). IMPORTANCE Through generation of the DEVoC, we added numerous previously uncharacterized viral genomes and genes to the ever-increasing worldwide pool of human gut viromes. The DEVoC, the largest human gut virome catalog generated from consistently processed fecal samples, facilitated the analysis of the 91 healthy Danish gut viromes. Characterizing the biggest cohort of healthy gut viromes from children, adolescents, and adults to date confirmed the previously established high interindividual variation in human gut viromes and demonstrated that the effect of age on the gut virome composition was limited to the prevalence of specific phage (groups). The identification of a previously undescribed prevalent phage illustrates the usefulness of developing virome catalogs, and we foresee that the DEVoC will benefit future analysis of the roles of gut viruses in human health and disease.

Progressive alcohol-related liver fibrosis is characterised by imbalanced collagen formation and degradation.

BACKGROUND: Liver fibrosis accumulation is considered a turnover disease, with formation exceeding degradation, although this hypothesis has never been tested in humans. AIMS: To investigate extracellular matrix (ECM) remodelling in a biopsy-controlled study of alcohol-related liver disease (ALD) patients. METHODS: We evaluated the relationship between formation and degradation of four collagens as a function of histological fibrosis, inflammation and steatosis in 281 patients with ALD and 50 matched healthy controls. Post hoc, we tested the findings in a cohort of patients with alcohol-related cirrhosis and assessed the collagens' prognostic accuracy. We assessed the fibrillar collagens type III (PRO-C3/C3M) and V (PRO-C5/C5M), the basement membrane collagen IV (PRO-C4/C4M), and the microfilament interface collagen VI (PRO-C6/C6M). RESULTS: Mean age was 54 ± 6 years, 74% male, fibrosis stage F0/1/2/3/4 = 33/98/84/18/48. Compared to controls, patients with ALD had higher levels of type III collagen formation and degradation, with the highest concentrations in those with cirrhosis (PRO-C3 = 8.2 ± 1.7 ng/mL in controls, 14.6 ± 13.5 in ALD, 34.8 ± 23.1 in cirrhosis; C3M 7.4 ± 1.9 in controls, 9.3 ± 4.4 in ALD, 14.0 ± 5 in cirrhosis). ECM remodelling became increasingly imbalanced in higher stages of liver fibrosis, with formation progressively superseding degradation. This was particularly pronounced for type III collagen. We observed similar imbalance for inflammatory severity, but not steatosis. CONCLUSIONS: ALD is characterised by both elevated collagen formation and degradation, which becomes increasingly imbalanced with more severe disease. Net increase in fibrillar collagens contributes to fibrosis progression. This has important implications for monitoring and very early identification of patients at highest risk of progressing to cirrhosis.

Describing the fecal metabolome in cryogenically collected samples from healthy participants.

The chemical composition of feces plays an important role in human metabolism. Metabolomics and lipidomics are valuable tools for screening the metabolite composition in feces. Here we set out to describe fecal metabolite composition in healthy participants in frozen stools. Frozen stool samples were collected from 10 healthy volunteers and cryogenically drilled in four areas along the specimen. Polar metabolites were analyzed using derivatization followed by two-dimensional gas chromatography and time of flight mass spectrometry. Lipids were detected using ultra high-performance liquid chromatography coupled with quadruple time-of-flight mass spectrometry. 2326 metabolic features were detected. Out of a total of 298 metabolites that were annotated we report here 185 that showed a technical variation of x < 30%. These metabolites included amino acids, fatty acid derivatives, carboxylic acids and phenolic compounds. Lipids predominantly belonged to the groups of diacylglycerols, triacylglycerols and ceramides. Metabolites varied between sampling areas, some were broadly homogeneous, others varied 80%. A LASSO-computed network using metabolites present in all areas showed two main clusters describing the system, DAG lipids and phenyllactic acid. In feces from healthy participants, the main groups detected were phenolic compounds, ceramides, diacylglycerols and triacylglycerols.