Investigating the Crime Scene-Molecular Signatures in Inflammatory Bowel Disease.

Inflammatory bowel diseases (IBD) are without cure and troublesome to manage because of the considerable diversity between patients and the lack of reliable biomarkers. Several studies have demonstrated that diet, gut microbiota, genetics and other patient factors are essential for disease occurrence and progression. Understanding the link between these factors is crucial for identifying molecular signatures that identify biomarkers to advance the management of IBD. Recent technological breakthroughs and data integration have fuelled the intensity of this research. This research demonstrates that the effect of diet depends on patient factors and gut microbial activity. It also identifies a range of potential biomarkers for IBD management, including mucosa-derived cytokines, gasdermins and neutrophil extracellular traps, all of which need further evaluation before clinical translation. This review provides an update on cutting-edge research in IBD that aims to improve disease management and patient quality of life.

Sample Preparation for High-Throughput Urine Proteomics Using 96-Well Polyvinylidene Fluoride (PVDF) Membranes.

Proteomics analysis of urine samples allows for studying the impact of system perturbation. However, meaningful proteomics-based biomarker discovery projects often require the analysis of large patient cohorts with hundreds of samples to describe the biological variability. Thus, robust high-throughput sample processing methods are a prerequisite for clinical proteomics pipelines that minimize experimental bias due to individual sample processing methods. Herein we describe a high-throughput method for parallel 96-well plate-based processing of urine samples for subsequent LC/MS-based proteomic analyses. Protein digestion and subsequent sample processing steps are efficiently performed in 96-well polyvinylidene fluoride (PVDF) membrane plate allowing for the use of vacuum manifolds for rapid liquid transfer, and multichannel pipettes and/or liquid handing robots. In this chapter we make available a detailed step-by-step protocol for our 'MStern blotting' sample processing strategy applied to patient urine samples followed by mass spectrometry-based proteomics analysis. Subsequently, we provide an example application using minimal volume of urine samples (e.g. 150 µL) collected from children pre and post thoracotomy to identify the predominant sites of protein catabolism and aid in the design of therapies to ameliorate protein catabolism and breakdown during critical illness. Furthermore, we demonstrate how the systemic state is reflected in the urine as an easily obtainable, stable, and safe biofluid.

The Pig PeptideAtlas: A resource for systems biology in animal production and biomedicine.

Biological research of Sus scrofa, the domestic pig, is of immediate relevance for food production sciences, and for developing pig as a model organism for human biomedical research. Publicly available data repositories play a fundamental role for all biological sciences, and protein data repositories are in particular essential for the successful development of new proteomic methods. Cumulative proteome data repositories, including the PeptideAtlas, provide the means for targeted proteomics, system-wide observations, and cross-species observational studies, but pigs have so far been underrepresented in existing repositories. We here present a significantly improved build of the Pig PeptideAtlas, which includes pig proteome data from 25 tissues and three body fluid types mapped to 7139 canonical proteins. The content of the Pig PeptideAtlas reflects actively ongoing research within the veterinary proteomics domain, and this article demonstrates how the expression of isoform-unique peptides can be observed across distinct tissues and body fluids. The Pig PeptideAtlas is a unique resource for use in animal proteome research, particularly biomarker discovery and for preliminary design of SRM assays, which are equally important for progress in research that supports farm animal production and veterinary health, as for developing pig models with relevance to human health research.

Complement killing of clinical Klebsiella pneumoniae isolates is serum concentration dependent.

Klebsiella pneumoniae is an opportunistic gram-negative pathogen causing serious infections, including sepsis. In plasma, activation of the complement cascades is important for killing bacteria. Thirty clinical Klebsiella spp. blood isolates were analyzed for serum susceptibility in 75% normal human serum (NHS). Twenty-two were serum resistant and eight were serum sensitive, and subsequently tested in 5-75% NHS. Two isolates were killed in 5% and the remaining six in 50%-75% NHS. The two 5% sensitive isolates showed binding of complement (C)4 and C3 in 5% NHS with formation of membrane attack complex (MAC). Inhibition of the classical/lectin mediated pathways (CP/LP) using a C4 specific nanobody, hC4Nb8, led to survival of both isolates in 5% NHS. Using nanobody hC3Nb1, inhibiting the alternative pathway (AP), the isolates were killed in 5% NHS, and amplification of the CP/LP by AP was not necessary for killing. Sole AP killing of these isolates when inhibiting CP/LP with hC4Nb8 was observed in 50% NHS, stressing the concentration dependent functionality of AP. For the less sensitive isolates, killing required activation of CP/LP and AP demonstrated by inhibition with nanobodies. AP inhibition resulted in no C3 deposition on the serum resistant isolate, supporting that AP was the sole activation pathway.

Dynamic molecular changes during the first week of human life follow a robust developmental trajectory.

Systems biology can unravel complex biology but has not been extensively applied to human newborns, a group highly vulnerable to a wide range of diseases. We optimized methods to extract transcriptomic, proteomic, metabolomic, cytokine/chemokine, and single cell immune phenotyping data from <1 ml of blood, a volume readily obtained from newborns. Indexing to baseline and applying innovative integrative computational methods reveals dramatic changes along a remarkably stable developmental trajectory over the first week of life. This is most evident in changes of interferon and complement pathways, as well as neutrophil-associated signaling. Validated across two independent cohorts of newborns from West Africa and Australasia, a robust and common trajectory emerges, suggesting a purposeful rather than random developmental path. Systems biology and innovative data integration can provide fresh insights into the molecular ontogeny of the first week of life, a dynamic developmental phase that is key for health and disease.