Evaluation of the interaction between phosphohistidine analogues and phosphotyrosine binding domains.

We have investigated the interaction of peptides containing phosphohistidine analogues and their homologues with the prototypical phosphotyrosine binding SH2 domain from the eukaryotic cell signalling protein Grb2 by using a combination of isothermal titration calorimetry and a fluorescence anisotropy competition assay. These investigations demonstrated that the triazole class of phosphohistidine analogues are capable of binding too, suggesting that phosphohistidine could potentially be detected by this class of proteins in vivo

A Protein‐Based Pentavalent Inhibitor of the Cholera Toxin B‐Subunit

Protein toxins produced by bacteria are the cause of many life-threatening diarrheal diseases. Many of these toxins, including cholera toxin (CT), enter the cell by first binding to glycolipids in the cell membrane. Inhibiting these multivalent protein/carbohydrate interactions would prevent the toxin from entering cells and causing diarrhea. Here we demonstrate that the site-specific modification of a protein scaffold, which is perfectly matched in both size and valency to the target toxin, provides a convenient route to an effective multivalent inhibitor. The resulting pentavalent neoglycoprotein displays an inhibition potency (IC50) of 104 pM for the CT B-subunit (CTB), which is the most potent pentavalent inhibitor for this target reported thus far. Complexation of the inhibitor and CTB resulted in a protein heterodimer. This inhibition strategy can potentially be applied to many multivalent receptors and also opens up new possibilities for protein assembly strategies

Selective targeting of human TET1 by cyclic peptide inhibitors: Insights from biochemical profiling

Ten-Eleven Translocation (TET) enzymes are Fe(II)/2OG-dependent oxygenases that play important roles in epigenetic regulation, but selective inhibition of the TETs is an unmet challenge. We describe the profiling of previously identified TET1-binding macrocyclic peptides. TiP1 is established as a potent TET1 inhibitor (IC50 = 0.26 \ub5M) with excellent selectivity over other TETs and 2OG oxygenases. TiP1 alanine scanning reveals the critical roles of Trp10 and Glu11 residues for inhibition of TET isoenzymes. The results highlight the utility of the RaPID method to identify potent enzyme inhibitors with selectivity over closely related paralogues. The structure–activity relationship data generated herein may find utility in the development of chemical probes for the TETs

Predicting COVID-19 outcomes from clinical and laboratory parameters in an intensive care facility during the second wave of the pandemic in South Africa

Background: The second wave of coronavirus disease 2019 (COVID-19) in South Africa was caused by the Beta variant of severe acute respiratory syndrome coronavirurus-2. This study aimed to explore clinical and biochemical parameters that could predict outcome in patients with COVID-19. Methods: A prospective study was conducted between 5 November 2020 and 30 April 2021 among patients with confirmed COVID-19 admitted to the intensive care unit (ICU) of a tertiary hospital. The Cox proportional hazards model in Stata 16 was used to assess risk factors associated with survival or death. Factors with P<0.05 were considered significant. Results: Patients who died were found to have significantly lower median pH (P<0.001), higher median arterial partial pressure of carbon dioxide (P<0.001), higher D-dimer levels (P=0.001), higher troponin T levels (P=0.001), higher N-terminal-prohormone B-type natriuretic peptide levels (P=0.007) and higher C-reactive protein levels (P=0.010) compared with patients who survived. Increased standard bicarbonate (HCO3std) was associated with lower risk of death (hazard ratio 0.96, 95% confidence interval 0.93–0.99). Conclusions: The mortality of patients with COVID-19 admitted to the ICU was associated with elevated D-dimer and a low HCO3std level. Large studies are warranted to increase the identification of patients at risk of poor prognosis, and to improve the clinical approach

Comparison of patients with severe COVID-19 admitted to an intensive care unit in South Africa during the first and second wave of the COVID-19 pandemic

BACKGROUND: The second wave of coronavirus disease 2019 (COVID‑19), dominated by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Beta variant, has been reported to be associated with increased severity in South Africa (SA). OBJECTIVES: To describe and compare clinical characteristics, management and outcomes of COVID‑19 patients admitted to an intensive care unit (ICU) in SA during the first and second waves. METHODS: In a prospective, single-centre, descriptive study, we compared all patients with severe COVID‑19 admitted to ICU during the first and second waves. The primary outcomes assessed were ICU mortality and ICU length of stay (LOS). RESULTS: In 490 patients with comparable ages and comorbidities, no difference in mortality was demonstrated during the second compared with the first wave (65.9% v. 62.5%, p=0.57). ICU LOS was longer in the second wave (10 v. 6 days, p<0.001). More female admissions (67.1% v. 44.6%, p<0.001) and a greater proportion of patients were managed with invasive mechanical ventilation than with non-invasive respiratory support (39.0% v. 14%, p<0.001) in the second wave. CONCLUSIONS: While clinical characteristics were comparable between the two waves, a higher proportion of patients was invasively ventilated and ICU stay was longer in the second. ICU mortality was unchanged

Haematological predictors of poor outcome among COVID-19 patients admitted to an intensive care unit of a tertiary hospital in South Africa

BACKGROUND: Studies from Asia, Europe and the USA indicate that widely available haematological parameters could be used to determine the clinical severity of Coronavirus disease 2019 (COVID-19) and predict management outcome. There is limited data from Africa on their usefulness in patients admitted to Intensive Care Units (ICUs). We performed an evaluation of baseline haematological parameters as prognostic biomarkers in ICU COVID-19 patients. METHODS: Demographic, clinical and laboratory data were collected prospectively on patients with confirmed COVID-19, admitted to the adult ICU in a tertiary hospital in Cape Town, South Africa, between March 2020 and February 2021. Robust Poisson regression methods and receiver operating characteristic (ROC) curves were used to explore the association of haematological parameters with COVID-19 severity and mortality. RESULTS: A total of 490 patients (median age 54.1 years) were included, of whom 237 (48%) were female. The median duration of ICU stay was 6 days and 309/490 (63%) patients died. Raised neutrophil count and neutrophil/lymphocyte ratio (NLR) were associated with worse outcome. Independent risk factors associated with mortality were age (ARR 1.01, 95%CI 1.0–1.02; p = 0.002); female sex (ARR 1.23, 95%CI 1.05–1.42; p = 0.008) and D-dimer levels (ARR 1.01, 95%CI 1.002–1.03; p = 0.016). CONCLUSIONS: Our study showed that raised neutrophil count, NLR and D-dimer at the time of ICU admission were associated with higher mortality. Contrary to what has previously been reported, our study revealed females admitted to the ICU had a higher risk of mortality

Effects of deletion of the Streptococcus pneumoniae lipoprotein diacylglyceryl transferase gene lgt on ABC transporter function and on growth in vivo

Lipoproteins are an important class of surface associated proteins that have diverse roles and frequently are involved in the virulence of bacterial pathogens. As prolipoproteins are attached to the cell membrane by a single enzyme, prolipoprotein diacylglyceryl transferase (Lgt), deletion of the corresponding gene potentially allows the characterisation of the overall importance of lipoproteins for specific bacterial functions. We have used a Δlgt mutant strain of Streptococcus pneumoniae to investigate the effects of loss of lipoprotein attachment on cation acquisition, growth in media containing specific carbon sources, and virulence in different infection models. Immunoblots of triton X-114 extracts, flow cytometry and immuno-fluorescence microscopy confirmed the Δlgt mutant had markedly reduced lipoprotein expression on the cell surface. The Δlgt mutant had reduced growth in cation depleted medium, increased sensitivity to oxidative stress, reduced zinc uptake, and reduced intracellular levels of several cations. Doubling time of the Δlgt mutant was also increased slightly when grown in medium with glucose, raffinose and maltotriose as sole carbon sources. These multiple defects in cation and sugar ABC transporter function for the Δlgt mutant were associated with only slightly delayed growth in complete medium. However the Δlgt mutant had significantly reduced growth in blood or bronchoalveolar lavage fluid and a marked impairment in virulence in mouse models of nasopharyngeal colonisation, sepsis and pneumonia. These data suggest that for S. pneumoniae loss of surface localisation of lipoproteins has widespread effects on ABC transporter functions that collectively prevent the Δlgt mutant from establishing invasive infection