Coagulopathy of Dengue and COVID-19: Clinical Considerations.

Thrombocytopenia and platelet dysfunction commonly occur in both dengue and COVID-19 and are related to clinical outcomes. Coagulation and fibrinolytic pathways are activated during an acute dengue infection, and endothelial dysfunction is observed in severe dengue. On the other hand, COVID-19 is characterised by a high prevalence of thrombotic complications, where bleeding is rare and occurs only in advanced stages of critical illness; here thrombin is the central mediator that activates endothelial cells, and elicits a pro-inflammatory reaction followed by platelet aggregation. Serological cross-reactivity may occur between COVID-19 and dengue infection. An important management aspect of COVID-19-induced immunothrombosis associated with thrombocytopenia is anticoagulation with or without aspirin. In contrast, the use of aspirin, nonsteroidal anti-inflammatory drugs and anticoagulants is contraindicated in dengue. Mild to moderate dengue infections are treated with supportive therapy and paracetamol for fever. Severe infection such as dengue haemorrhagic fever and dengue shock syndrome often require escalation to higher levels of support in a critical care facility. The role of therapeutic platelet transfusion is equivocal and should not be routinely used in patients with dengue with thrombocytopaenia and mild bleeding. The use of prophylactic platelet transfusion in dengue fever has strained financial and healthcare systems in endemic areas, together with risks of transfusion-transmitted infections in low- and middle-income countries. There is a clear research gap in the management of dengue with significant bleeding.

Paired sensitivity analysis of four SARS-CoV-2 serological immunoassays in a longitudinal cohort of convalescent hospital staff.

BACKGROUND: SARS-CoV-2 serological testing has seen extensive academic and clinical use from investigating correlates of immunity to seroprevalence, convalescent plasma and vaccine trials. Interpretation of these studies will depend on robust validation of the longitudinal sensitivities of these assays, especially in the context of mild disease which makes up the majority of the Coronavirus Disease 2019 (COVID-19) caseload. METHODS: Hospital staff (n = 94) returning to work following polymerase chain reaction confirmed COVID-19 were offered antibody testing to assist with laboratory verification. Initial specimens were collected at median 29 days post-symptom onset and run on the Roche, Abbott, Siemens and DiaSorin platforms. Re-sampling occurred at median 142 days from a subset of the initial cohort (n = 62) that had volunteered to provide further serum samples to assist in longitudinal sensitivity analysis. Samples that were not run across all four platforms were excluded from analysis. RESULTS: Comparative sensitivity analysis was conducted on 89/94 of the initial specimens and 55/62 of the repeat specimens. Sensitivity at initial sampling ranged from 78 to 87% across platforms. At re-sampling, sensitivities were: 100% (Roche), 45% (Abbott), 100% (Siemens), and 80% (DiaSorin). Paired analysis using the longitudinal cohort (n = 55) demonstrated stable or increasing median assay values on three platforms, with a clear reduction seen only on the Abbott platform (4.78 to 1.34) with corresponding sensitivity drop-off (81.8% to 45.4%). CONCLUSION: The Abbott assay demonstrated sensitivity drop-off and decrease in median assay signal below detection threshold at four to five months. This has implications on the interpretation and design of future studies.

A functional Kv1.2-hERG chimaeric channel expressed in Pichia pastoris.

Members of the six-transmembrane segment family of ion channels share a common structural design. However, there are sequence differences between the members that confer distinct biophysical properties on individual channels. Currently, we do not have 3D structures for all members of the family to help explain the molecular basis for the differences in their biophysical properties and pharmacology. This is due to low-level expression of many members in native or heterologous systems. One exception is rat Kv1.2 which has been overexpressed in Pichia pastoris and crystallised. Here, we tested chimaeras of rat Kv1.2 with the hERG channel for function in Xenopus oocytes and for overexpression in Pichia. Chimaera containing the S1-S6 transmembrane region of HERG showed functional and pharmacological properties similar to hERG and could be overexpressed and purified from Pichia. Our results demonstrate that rat Kv1.2 could serve as a surrogate to express difficult-to-overexpress members of the six-transmembrane segment channel family.

Imaging and quantification of recycled KATP channels.

This chapter describes immunochemistry-based methods to investigate recycling of membrane proteins at the cell surface. Two methods are described, one qualitative and the other quantitative. Both methods consist of two rounds of extracellular antibody capture. Firstly, a primary antibody is captured by an extracellular epitope presented by the target membrane protein and is subsequently internalized. Secondly, the primary antibody-labelled protein is recycled back to the membrane where it is captured by a probe--conjugated secondary antibody. In the qualitative assay, the probe is a fluorophore, which can be imaged by fluorescence microscopy. In the quantitative assay, the probe is horse-radish peroxidase (HRP) and enzyme activity can be assayed by chemiluminescence.

Mapping the membrane-aqueous border for the voltage-sensing domain of a potassium channel.

Voltage-sensing domains (VSDs) play diverse roles in biology. As integral components, they can detect changes in the membrane potential of a cell and couple these changes to activity of ion channels and enzymes. As independent proteins, homologues of the VSD can function as voltage-dependent proton channels. To sense voltage changes, the positively charged fourth transmembrane segment, S4, must move across the energetically unfavorable hydrophobic core of the bilayer, which presents a barrier to movement of both charged species and protons. To reduce the barrier to S4 movement, it has been suggested that aqueous crevices may penetrate the protein, reducing the extent of total movement. To investigate this hypothesis in a system containing fully functional channels in a native environment with an intact membrane potential, we have determined the contour of the membrane-aqueous border of the VSD of KvAP in Escherichia coli by examining the chemical accessibility of introduced cysteines. The results revealed the contour of the membrane-aqueous border of the VSD in its activated conformation. The water-inaccessible regions of S1 and S2 correspond to the standard width of the membrane bilayer (~28 A), but those of S3 and S4 are considerably shorter (> or = 40%), consistent with aqueous crevices pervading both the extracellular and intracellular ends. One face of S3b and the entire S3a were water-accessible, reducing the water-inaccessible region of S3 to just 10 residues, significantly shorter than for S4. The results suggest a key role for S3 in reducing the distance S4 needs to move to elicit gating.