Small open reading frames (smORFs), which encode proteins under 100 amino acids, represent an underexplored dimension of the human gut microbiome, despite growing evidence of their essential biological roles. Due to small size and poor annotation, smORFs are typically excluded from metagenomic/metaproteomic analyses. Here, we present a high-resolution multi-omic workflow that integrates smORF prediction into metaproteome searches and enables ultra-deep detection of smORF-encoded proteins (SEPs), without experimental size-based enrichment, utilizing state-of-the-art mass spectrometry instrumentation. Applied to human gut microbiomes, this approach resulted in the largest number of detected SEPs to date, allowing identification of over 25,000 SEPs in the metaproteome, alongside the measurements of the larger proteins. Our multi-omics integrative strategy is critical for advancing human metaproteome research. It also provides a generalizable strategy for comprehensive SEP discovery across diverse microbial ecosystems greatly expanding the previously hidden proteomic landscape.

This study aimed to analyse COVID-19-related mortality during the pandemic, stratified by groups at risk of severe COVID-19. Patients with COVID-19 between March 2020 and February 2023 were enrolled using the international multicentric Lean European Open Survey on SARS-CoV-2-Infected Patients (LEOSS). The COVID-19 in-hospital mortality was calculated using a multivariable logistic regression model adjusted for age and sex. A total of 11,765 patients were included, with an overall mortality rate of 13.1

ObjectiveA subset of Covid-19 survivors will develop persisting health sequelae (i.e. Long Covid/LC or Post Covid/PC) similar to Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). In the absence of a reliable biomarker to diagnose LC/PC and ME/CFS, their classification based on symptoms becomes indispensable. Hence, we translated and validated the DePaul Symptom Questionnaire-2 (DSQ-2), to offer a screening tool for the German-speaking population.MethodsA sample of healthy adults, and adults with ME/CFS and LC/PC (N = 502) completed a reduced-item version of the DSQ-2 and SF-36 questionnaire online. We performed an exploratory factor analysis, assessed construct validity, diagnostic accuracy and compared the symptom profiles of individuals with ME/CFS versus LC/PC versus healthy adults.ResultsExploratory factor analysis revealed a 10-factor solution with excellent internal consistencies. The sensitivity of the DSQ-2 was excellent. The specificity was moderate with moderate inter-rater reliability. Construct validity of the DSQ-2 was supported by strong negative correlations with physical health subscales of the SF-36. A visual comparison of the symptom profiles of individuals with ME/CFS versus LC/PC revealed a comparable pattern.ConclusionDespite lower symptom severity, individuals with LC/PC reported significantly stronger limitations in general health and physical functioning and were more likely to meet ME/CFS diagnostic criteria with ongoing sickness duration, suggesting that ME/CFS can be considered a long-term sequela of LC/PC. This study offers a translated and validated version of the reduced-item DSQ-2 that can guide medical evaluation and aid physicians in identifying a ME/CFS-like subtype of LC/PC.

PURPOSE:This study assessed the clinical and immunological outcomes of SARS-CoV-2-infected patients with risk factors for severe disease depending on their immunological status.METHODS:In this retrospective study with single follow-up visit, clinical outcome and humoral immunity was monitored in SARS-CoV-2 infected patients at risk. The results were compared based on the patients' initial immunological status: unvaccinated (UV), patients who did not develop neutralizing antibodies after vaccination (vaccine non-responders, VNR), and patients who expressed neutralizing antibodies after vaccination (vaccine responders, VR). Patients who lacked neutralizing antibodies (VNR and UV) were treated with nMABs.RESULTS:In total, 113 patients at risk of severe COVID-19 consented to participate in the study. VR and UV were not admitted to the hospital. During the observation period, UVs had the highest rate of SARS-CoV-2 re-infections. Three of 41 VNRs (7.3%) were hospitalized due to severe COVID-19, with two of them having undergone iatrogenic B-cell depletion. The humoral immune response after infection was significantly lower in the VNR group than in the VR group in terms of anti-N, anti-receptor-binding domain (RBD), anti-S antibody titers, and anti-S antibody avidity. In a sub-analysis of VNR, B cell-deficient non-responders had significantly lower levels of anti-N antibodies and anti-S avidity after infection than other VNRs.CONCLUSION:VNR, particularly B-cell-depleted VNR, remained at risk of hospitalization due to COVID-19. In the VR group, however, no clinical complications or severe disease were observed, despite not receiving nMAbs. Tailoring the administration of nMABs according to patient vaccination and immunological status may be advisable.

Abstract Background To assess the long-term survival of patients with enterococcal bloodstream infections (BSI), encompassing various species and resistance patterns, in comparison to Escherichia coli (E. coli) BSI. Recruitment process Graphical Abstract Methods Between 2010 and 2019, 3,290 enterococcal and 3,415 E. coli BSI were retrospectively screened in seven hospitals in Munich, Germany. All vancomycin (VRE), vancomycin/linezolid (LVRE), and linezolid (LRE) resistant Enterococcus faecium (ECFM) BSI were included. Enterococcus faecalis (ECFA), vancomycin/Linezolid-susceptible ECFM, and E. coli BSI were randomly assigned. Cox-regression models were used to assess survival as the primary endpoint and were adjusted for limiting prognostic factors, which were measured for their importance using a random forest model (RFM). Results 952 patients with 916 episodes of enterococcal BSI and 193 E. coli BSI episodes were analysed. RFM identified multimorbidity and markers for disease severity as most indicative of low survival in enterococcal BSI. The 5-year survival was significantly lower for enterococcal BSI than for E. coli BSI (23.9% vs. 42.3%; p< 0.001). This difference remained significant in the Cox-regression analysis after adjusting for 17 prognostic factors and excluding patients with limited life expectancy (metastatic tumour disease, Charlson comorbidity index ≥5). Adjusted 5-year survival between E. coli and ECFA was similar but significantly different between ECFA and ECFM BSI (29.2% vs. 21.7%; p=0.002). The analysis conducted on monomicrobial ECFM and VRE BSI indicated that their respective 5-year survival was similar (19.6% vs. 21.2%; p=0.753). Conclusion ECFM BSI seems to be an independent risk factor for poor long-term survival. However, additional vancomycin resistance does not appear to be a contributing factor. Disclosures Jochen Schneider, MD, JS reports grants, personal fees, and nonfinancial support from AbbVie, Gilead Sciences, Janssen-Cilag, GSK/ViiV Healthcare, and MSD, Dr. Falk Pharma: Grant/Research Support Karl Dichtl, MD, KD reports grants from Euroimmun Medizinische Labordiagnostika and Fujifilm Wako Chemicals Europe outside of this study.: Advisor/Consultant Ulrich Seybold, MD, reports personal fees and nonfinancial support from Gilead Sciences, ViiV Healthcare, Janssen-Cilag, Falk Foundation, Sanofi-Aventis.: Advisor/Consultant Christoph Spinner, MD, AbbVie, Gilead Sciences, Janssen-C,MSD, Cepheid, GSK, ViiV Healthcare, AstraZeneca, Apeiron, Braun, Pfizer, Novartis, Lilly, Biontech…: Advisor/Consultant|AbbVie, Gilead Sciences, Janssen-C,MSD, Cepheid, GSK, ViiV Healthcare, AstraZeneca, Apeiron, Braun, Pfizer, Novartis, Lilly, Biontech…: Board Member|AbbVie, Gilead Sciences, Janssen-C,MSD, Cepheid, GSK, ViiV Healthcare, AstraZeneca, Apeiron, Braun, Pfizer, Novartis, Lilly, Biontech…: Grant/Research Support|CDS reports grants and personal fees from AbbVie, grants, fees and non-financial support from Gilead Sciences, grants and personal fees from Janssen-C: Advisor/Consultant|CDS reports grants and personal fees from AbbVie, grants, fees and non-financial support from Gilead Sciences, grants and personal fees from Janssen-C: Board Member|CDS reports grants and personal fees from AbbVie, grants, fees and non-financial support from Gilead Sciences, grants and personal fees from Janssen-C: Grant/Research Support

OBJECTIVE:Visceral hypersensitivity is considered a key symptom in inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS), both of which seriously affect health-related quality of life (HrQoL). Previous findings are mostly based on invasive procedures that may interfere with the assessment of visceral perception. The current study, therefore, investigates whether IBD and IBS are characterized by altered perception of 'natural' gastric distensions ('interoception').METHODS:Twenty IBD patients in remission (13 Crohn's disease, 7 ulcerative colitis), 12 IBS patients, and 20/12 matched healthy control (HC) individuals, respectively, underwent the water load test, in which they could drink ad libitum until the subjective thresholds of satiation (stage 1) and fullness (stage 2) were reached. Gastric motility was assessed using electrogastrography.RESULTS:IBD patients drank significantly more water until satiation than IBS patients, whereas no differences between patients and HC groups were observed. Electrogastrographic patterns were comparable between groups, suggesting no pathologies in gastric motility in IBD or IBS. The amount of water consumed until satiation negatively correlated with HrQoL related to bowel symptoms in IBD patients, but was positively associated with emotional well-being in IBS patients.CONCLUSION:Our findings implicate relative gastric hypersensitivity in IBS, and relative hyposensitivity in IBD patients, which are both related to specific HrQoL aspects.

Abstract As part of the revisions of our original manuscript, we performed 13C-labelling experiments with cultures of the microorganism most strongly correlated with 2-hydroxypyridine (2-HP), i.e. the archaeal species Methanobrevibacter smithii. Although unlabelled 2-HP was detected in the cultures, the measurements by gas chromatography-mass spectrometry (GC-MS) indicated that M. smithii was not the direct source of 2-HP as labelled 2-HP was not measured. Further experiments involving a labelled solvent (deuterated pyridine) and faecal samples from our original study alongside the use of additional analytical platforms and measurements on human blood plasma and mouse brain tissues demonstrate that 2-HP is an artefact of the measurements by GC-MS. It is produced in a sample-specific manner during the derivatisation process for GC-MS by a so far unknown chemical reaction. Our correlative links between archaea (M. smithii) and 2-HP remain but, based on these most recent results, cannot be directly mechanistically linked. Apart from this central limitation of our original study, we have so far not uncovered any reasons which would draw into question the validity of our in vitro and in vivo results linking 2-HP to the observed molecular, behavioural and pathological hallmarks of Parkinson’s disease.

BACKGROUND:An effective testing strategy is essential for pandemic control of the novel Coronavirus disease 2019 (COVID-19) caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Breath gas analysis can expand the available toolbox for diagnostic tests by using a rapid, cost-beneficial, high-throughput point-of-care test. We conducted a bi-center clinical pilot study in Germany to evaluate breath gas analysis using multi-capillary column ion mobility spectrometry (MCC-IMS) to detect SARS-CoV-2 infection.METHODS:Between September 23, 2020, and June 11, 2021, breath gas measurements were performed on 380 patients (SARS-CoV-2 real-time polymerase chain reaction (PCR) positive: 186; PCR negative: 194) presenting to the emergency department (ED) with respiratory symptoms.RESULTS:Breath gas analysis using MCC-IMS identified 110 peaks; 54 showed statistically significant differences in peak intensity between the SARS-CoV-2 PCR-negative and PCR-positive groups. A decision tree analysis classification resulted in a sensitivity of 83% and specificity of 86%, but limited robustness to dataset changes. Modest values for the sensitivity (74%) and specificity (52%) were obtained using linear discriminant analysis. A systematic search for peaks led to a sensitivity of 77% and specificity of 67%; however, validation by transferability to other data is questionable.CONCLUSIONS:Despite identifying several peaks by MCC-IMS with significant differences in peak intensity between PCR-negative and PCR-positive samples, finding a classification system that allows reliable differentiation between the two groups proved to be difficult. However, with some modifications to the setup, breath gas analysis using MCC-IMS may be a useful diagnostic toolbox for SARS-CoV-2 infection.TRIAL REGISTRATION:This study was registered at ClinicalTrials.gov on September 21, 2020 (NCT04556318; Study-ID: HC-N-H-2004).

Hepatobiliary involvement is a hallmark in cystic fibrosis (CF), as the causative CF Transmembrane Conductance Regulator (CFTR) defect is expressed in the biliary tree. However, bile acid (BA) compositions in regard to pancreatic insufficiency, which is present at an early stage in about 85% of CF patients, have not been satisfactorily understood. We assess the pattern of serum BAs in people with CF (pwCF) without CFTR modulator therapy in regard to pancreatic insufficiency and the CFTR genotype. In 47 pwCF, 10 free and 12 taurine- and glycine-conjugated BAs in serum were prospectively assessed. Findings were related to genotype, pancreatic insufficiency prevalence (PIP)-score, and hepatic involvement indicated by serum liver enzymes, as well as clinical and ultrasound criteria for CF-related liver disease. Serum concentrations of total primary BAs and free cholic acid (CA) were significantly higher in pwCF with higher PIP-scores (p = 0.025, p = 0.009, respectively). Higher total BAs were seen in pwCF with PIP-scores ≥0.88 (p = 0.033) and with pancreatic insufficiency (p = 0.034). Free CA was higher in patients with CF-related liver involvement without cirrhosis, compared to pwCF without liver disease (2.3-fold, p = 0.036). pwCF with severe CFTR genotypes, as assessed by the PIP-score, reveals more toxic BA compositions in serum. Subsequent studies assessing changes in BA homeostasis during new highly effective CFTR-modulating therapies are of high interest.

Abstract Patients with Parkinson’s disease (PD) exhibit differences in their gut microbiomes compared to healthy individuals. Although differences have most commonly been described in the abundances of bacterial taxa, changes to viral and archaeal populations have also been observed. Mechanistic links between gut microbes and PD pathogenesis remain elusive but could involve molecules that promote α-synuclein aggregation. Here, we show that 2-hydroxypyridine (2-HP) represents a key molecule for the pathogenesis of PD. We observe significantly elevated 2-HP levels in faecal samples from patients with PD or its prodrome, idiopathic REM sleep behaviour disorder (iRBD), compared to healthy controls. 2-HP is correlated with the archaeal species Methanobrevibacter smithii and with genes involved in methane metabolism, and it is detectable in isolate cultures of M. smithii. We demonstrate that 2-HP is selectively toxic to transgenic α-synuclein overexpressing yeast and increases α-synuclein aggregation in a yeast model as well as in human induced pluripotent stem cell derived enteric neurons. It also exacerbates PD-related motor symptoms, α-synuclein aggregation, and striatal degeneration when injected intrastriatally in transgenic mice overexpressing human α-synuclein. Our results highlight the effect of an archaeal molecule in relation to the gut-brain axis, which is critical for the diagnosis, prognosis, and treatment of PD.