Intestinal Protein Characterisation of SARS-CoV-2 Entry Molecules ACE2 and TMPRSS2 in Inflammatory Bowel Disease (IBD) and Fatal COVID-19 Infection

Abstract—The coronavirus SARS-CoV-2 contributes to morbidity and mortality mainly as a result of immune-pathology in the lungs. Recent data has shown multi-system involvement with widespread viral tropism. Here we present a detailed intestinal protein characterisation of SARS-Cov-2 entry molecules ACE2 and TMPRSS2 in patients with inflammatory bowel disease ([IBD]; ulcerative colitis [UC] and Crohn’s disease [CD]) with age- and sex-matched non-IBD controls, and in those with fatal COVID-19 infection. In our dataset, ACE2 and TMPRSS2 displayed a membrane enterocyte staining in the ileum (due to presence of brush border/microvilli) in contrast to a cytoplasmic pattern in the colon. We also showed a high ACE2/low TMPRSS2 expression pattern in the ileum with a reverse trend in the colon. In UC, colonic ACE2 and TMPRSS2 are cytoplasmic in nature, with significantly higher ACE2 staining intensity compared to non-IBD controls. In inflamed and unaffected IBD mucosa, ileal and colonic enterocyte ACE2 and TMPRSS2 expressions are not modified in the histologic presence of inflammation. We observed immune cells within the lamina propria that expressed ACE2 and TMPRSS2, at higher frequencies in IBD when compared to non-IBD controls. These were identified as plasma cells with multiple myeloma oncogene 1/interferon regulatory factor 4 (MUM1/IRF4) expression. We further analysed the gut histology of six fatal COVID-19 cases, with no difference in colonic and ileal ACE2/TMRPSS2 staining (compared to non-IBD controls) and identified ACE2 + lamina propria plasma cells. Of interest, in this COVID-19 cohort, there was no histologic evidence gut inflammation despite known evidence of viral tropism within the enterocytes. Our data provides evidence for tissue expression of entry molecules ACE2 and TMPRSS2 including a close apposition to plasma cells — both pointing towards a role of the gut in the antecedent immune response to SARS-CoV-2 infection.

Protein characterisation and antioxidant potential of fresh cheese obtained by kombucha inoculum

Protein profile, in vitro gastrointestinal digestion and antioxidant potential of two fresh cheese samples obtained by kombucha inoculum and traditional starter culture have been investigated in this study. There is a significant difference in protein profile and bioactive potential between fresh cheese samples. Kombucha fresh cheese contains higher proportion of protein, predominantly the casein fractions (α-casein and β-casein) compared to fresh cheese obtained by traditional culture. After gastrointestinal digestion (with pepsin and pancreatin), significantly higher amount of soluble protein and higher degree of hydrolyses were measured in kombucha fresh cheese sample than sample obtained by traditional culture. During gastrointestinal digestion, for fresh cheese with traditional starter both fractions of casein are degraded more rapidly by pepsin than in kombucha fresh cheese. The hydrolysates of cheese obtained by kombucha inoculum showed higher phenolic content and higher antiradical scavenging abilities than hydrolysates of fresh cheese produced with traditional starter. These results suggest that kombucha inoculum contributes to improvement of product’s bioactive potential.

Intestinal Protein Characterisation of SARS-CoV-2 Entry Molecules ACE2 and TMPRSS2 in Inflammatory Bowel Disease (IBD) and Fatal COVID-19 Infection

The coronavirus SARS-CoV-2 contributes to morbidity and mortality mainly as a result of immune-pathology in the lungs. Recent data has shown multi-system involvement with widespread viral tropism. Here we present a detailed intestinal protein characterisation of SARS-Cov-2 entry molecules ACE2 and TMPRSS2 in patients with Inflammatory Bowel Disease ([IBD]; Ulcerative Colitis [UC] and Crohn’s disease [CD]) with age- and sex-match non-IBD controls; and in those with fatal COVID19 infection. Our study showed that IBD gut inflammation did not influence ACE2 and TRPMSS2 expression. Of interest, colonic protein expression of ACE2 and TRPMSS2 are cytoplasmic distinct to the membranous pattern in the ileum. We observed a significant increase in immune cells within the lamina propria in UC and CD that expressed ACE2 and TRMPSS2 when compared to non-IBD controls. These were identified as plasma cells with multiple myeloma oncogene 1/interferon regulatory factor 4 (MUM1/IRF4) expression. In six fatal COVID19 cases, there was no gut inflammation despite evidence of viral tropism within the enterocytes. Our data provides evidence for tissue expression of entry molecules ACE2 and TMPRSS2 including a close apposition to plasma cells – both pointing towards a role of the gut in the immune response to SARS-CoV-2 infection.

Machine learning aided top-down proteomics on a microfluidic platform

The ability to determine the identity of specific proteins is a critical challenge in many areas of cellular and molecular biology, and in medical diagnostics. Here, we present a microfluidic protein characterisation strategy that within a few minutes generates a three-dimensional fingerprint of a protein sample indicative of its amino acid composition and size and, thereby, creates a unique signature for the protein. By acquiring such multidimensional fingerprints for a set of ten proteins and using machine learning approaches to classify the fingerprints, we demonstrate that this strategy allows proteins to be classified at a high accuracy, even though classification using a single dimension is not possible. Moreover, we show that the acquired fingerprints correlate with the amino acid content of the samples, which makes it is possible to identify proteins directly from their sequence without requiring any prior knowledge about the fingerprints. These findings suggest that such a multidimensional profiling strategy can lead to the development of novel method for protein identification in a microfluidic format.

Multidimensional protein characterisation using microfluidic post-column analysis

Direct combination of liquid chromatography with multidimensional post-column analysis through microfluidics for individual quantification of biophysical parameters.