Immobilization of azide-functionalized proteins to micro- and nanoparticles directly from cell lysate.

Immobilization of proteins and enzymes on solid supports has been utilized in a variety of applications, from improved protein stability on supported catalysts in industrial processes to fabrication of biosensors, biochips, and microdevices. A critical requirement for these applications is facile yet stable covalent conjugation between the immobilized and fully active protein and the solid support to produce stable, highly bio-active conjugates. Here, we report functionalization of solid surfaces (gold nanoparticles and magnetic beads) with bio-active proteins using site-specific and biorthogonal labeling and azide-alkyne cycloaddition, a click chemistry. Specifically, we recombinantly express and selectively label calcium-dependent proteins, calmodulin and calcineurin, and cAMP-dependent protein kinase A (PKA) with N-terminal azide-tags for efficient conjugation to nanoparticles and magnetic beads. We successfully immobilized the proteins on to the solid supports directly from the cell lysate with click chemistry, forgoing the step of purification. This approach is optimized to yield low particle aggregation and high levels of protein activity post-conjugation. The entire process enables streamlined workflows for bioconjugation and highly active conjugated proteins.

Thromboelastography-Guided Anticoagulant Therapy for the Double Hazard of Thrombohemorrhagic Events in COVID-19: A Report of 3 Cases.

BACKGROUND The novel coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), often manifests a coagulopathy in severely ill patients, which may cause hemorrhage and/or thrombosis of varying severity. This report comprises the cases of 3 patients with COVID-19-associated coagulopathy who were evaluated with thromboelastography (TEG) and activated partial thromboplastin time (aPTT) to enable personalized anticoagulant therapy. CASE REPORT Three patients presented with COVID-19 pneumonia, confirmed by reverse transcription-polymerase chain reaction, who developed thrombohemorrhagic coagulopathy.Case 1: A 72-year-old woman on long-term warfarin therapy for a history of venous thromboembolism developed a right upper lobe pulmonary embolus, despite an international normalized ratio of 6.4 and aPTT of 120.7 s. TEG enabled successful anticoagulation with heparin, and her pulmonary infarct was no longer present 2 weeks later.Case 2: A 55-year-old woman developed a rectus sheath hematoma while on heparin, and TEG demonstrated increased fibrinolysis despite COVID-19 patients more commonly undergoing fibrinolytic shutdown.Case 3: A 43-year-old woman had significant thrombus burden while severely hypocoagulable according to laboratory testing. As the venous thrombi enlarged in a disseminated intravascular coagulopathic-like state, the heparin dose was escalated to achieve a target aPTT of 70 to 80 s, resulting in a flat line TEG tracing. CONCLUSIONS These 3 cases of COVID-19 pneumonia with complex and varied clinical histories demonstrated the clinical value of TEG combined with the measurement of aPTT to facilitate personalized anticoagulation, resulting in good clinical outcomes.

A Case Series of Thromboelastography-Guided Anticoagulation in COVID-19 Patients with Inherited and Acquired Hypercoagulable States.

One of the complications of the novel coronavirus disease 2019 (COVID-19) is hypercoagulability. For this reason, patients presenting with COVID-19 are often put on therapeutic or intermediate anticoagulation upon hospitalization. A common issue of this anticoagulation is the progression to hypocoagulability resulting in hemorrhage. Therefore, monitoring the hemostatic integrity of critically ill COVID-19 patients is of utmost importance. In this case series, we present the cases of three coagulopathic COVID-19 patients whose anticoagulation was guided by thromboelastography (TEG). In each case, TEG permitted the clinical team to simultaneously prevent thrombotic and hemorrhagic events, a difficult task for COVID-19 patients admitted to the intensive care unit. The first two cases illustrate the utility of TEG to guide anticoagulant dosing for COVID-19 patients when the activated partial thromboplastin time (aPTT) is inaccurate. The first case was a severely ill COVID-19 patient with end-stage renal disease and a falsely elevated aPTT secondary to hypertriglyceridemia. The second case was a severely ill COVID-19 patient with chronic pulmonary disease who demonstrated a falsely elevated aPTT due to polycythemia and hemoconcentration. In both cases, TEG was sensitive to the hypercoagulability caused by the metabolic derangements which enabled the goal-directed titration of anticoagulants. The last case depicts a severely ill COVID-19 patient with an inherited factor V Leiden mutation who required abnormally high dosing to achieve therapeutic anticoagulation, guided by TEG. Hypercoagulopathic COVID-19 patients are difficult to anticoagulate without development of hypocoagulopathy. Treatment of these patients demands goal-directed therapy by diligent laboratory monitoring. This can be accomplished by the use of TEG coupled with aPTT to guide anticoagulation. This case series illustrates the necessity for active hemostatic monitoring of critically ill COVID-19 patients.

Tension pneumomediastinum and diffuse subcutaneous emphysema with severe acute respiratory syndrome coronavirus 2 infection requiring operative management for impending airway collapse: A case report.

Tension pneumomediastinum is a rare complication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection that has increased in incidence with the novel coronavirus disease 2019 pandemic. Although traditionally managed with conservative measures, we present the indications and methods for the first operative management of tension pneumomediastinum with concomitant SARS-CoV-2 infection.