PepSeq: a fully in vitro platform for highly multiplexed serology using customizable DNA-barcoded peptide libraries.

PepSeq is an in vitro platform for building and conducting highly multiplexed proteomic assays against customizable targets by using DNA-barcoded peptides. Starting with a pool of DNA oligonucleotides encoding peptides of interest, this protocol outlines a fully in vitro and massively parallel procedure for synthesizing the encoded peptides and covalently linking each to a corresponding cDNA tag. The resulting libraries of peptide/DNA conjugates can be used for highly multiplexed assays that leverage high-throughput sequencing to profile the binding or enzymatic specificities of proteins of interest. Here, we describe the implementation of PepSeq for fast and cost-effective epitope-level analysis of antibody reactivity across hundreds of thousands of peptides from <1 µl of serum or plasma input. This protocol includes the design of the DNA oligonucleotide library, synthesis of DNA-barcoded peptide constructs, binding of constructs to sample, preparation for sequencing and data analysis. Implemented in this way, PepSeq can be used for a number of applications, including fine-scale mapping of antibody epitopes and determining a subject's pathogen exposure history. The protocol is divided into two main sections: (i) design and synthesis of DNA-barcoded peptide libraries and (ii) use of libraries for highly multiplexed serology. Once oligonucleotide templates are in hand, library synthesis takes 1-2 weeks and can provide enough material for hundreds to thousands of assays. Serological assays can be conducted in 96-well plates and generate sequencing data within a further ~4 d. A suite of software tools, including the PepSIRF package, are made available to facilitate the design of PepSeq libraries and analysis of assay data.

Impact of Intravenous Fluids and Enteral Nutrition on the Severity of Gastrointestinal Dysfunction: A Systematic Review and Meta-analysis.

INTRODUCTION: Gastrointestinal dysfunction (GDF) is one of the primary causes of morbidity and mortality in critically ill patients. Intensive care interventions, such as intravenous fluids and enteral feeding, can exacerbate GDF. There exists a paucity of high-quality literature on the interaction between these two modalities (intravenous fluids and enteral feeding) as a combined therapy on its impact on GDF. AIM: To review the impact of intravenous fluids and enteral nutrition individually on determinants of gut function and implications in clinical practice. METHODS: Randomized controlled trials on intravenous fluids and enteral feeding on GDF were identified by a comprehensive database search of MEDLINE and EMBASE. Extraction of data was conducted for study characteristics, provision of fluids or feeding in both groups and quality of studies was assessed using the Cochrane criteria. A random-effects model was applied to estimate the impact of these interventions across the spectrum of GDF severity. RESULTS: Restricted/ goal-directed intravenous fluid therapy is likely to reduce 'mild' GDF such as vomiting (p = 0.03) compared to a standard/ liberal intravenous fluid regime. Enterally fed patients experienced increased episodes of vomiting (p = <0.01) but were less likely to develop an anastomotic leak (p = 0.03) and peritonitis (p = 0.03) compared to parenterally fed patients. Vomiting (p = <0.01) and anastomotic leak (p = 0.04) were significantly lower in the early enteral feeding group. CONCLUSIONS: There is less emphasis on the combined approach of intravenous fluid resuscitation and enteral feeding in critically ill patients. Conservative fluid resuscitation and aggressive enteral feeding are presumably key factors contributing to severe life-threatening GDF. Future trials should evaluate the impact of cross-interaction between conservative and aggressive modes of these two interventions on the severity of GDF.

Gastrointestinal Dysfunction in Critical Illness: A Review of Scoring Tools.

Gastrointestinal dysfunction/failure (GDF) is a common feature of critical illness. There is no consensus on the best way to measure severity of GDF. The aim of this systematic review was to evaluate and compare all gastrointestinal dysfunction scoring tools (GDSTs) to determine aspects that might be useful to construct a new gut scoring tool for intensive care patients. A comprehensive database search of MEDLINE and EMBASE on GDSTs in acute, surgical, and critically ill patients was conducted. Extraction of data was conducted for study characteristics, GDST domains, feasibility of the scoring process, clinical outcomes, and quality of life attributes; independent authors conducted the search, applied the selection criteria, and extracted the data. Methodological quality of studies was assessed. A GDST matrix was created using 14 scoring tools and was evaluated for validity. The 14 GDSTs identified were used in different clinical settings, including critical illness, acute intestinal failure, gastrointestinal disorders, and postoperative patients. There was marked variation between these GDSTs. There is a lack of emphasis in the use of objective laboratory parameters and gut-specific biomarkers to measure GDF in majority of the studies. The overall quality of evidence was poor, and most tools lacked formal validation. This review has highlighted the lack of an agreed and validated approach to scoring GDF in critical illness. The identified aspects of GDF relevant to critical illness should now be incorporated in a new scoring tool and prospectively tested in intensive care patients.