Design and Escherichia coli Expression of a Natively Folded Multi-Disulfide Bonded Influenza H1N1-PR8 Receptor-Binding Domain (RBD).

Refolding multi-disulfide bonded proteins expressed in E. coli into their native structure is challenging. Nevertheless, because of its cost-effectiveness, handiness, and versatility, the E. coli expression of viral envelope proteins, such as the RBD (Receptor-Binding Domain) of the influenza Hemagglutinin protein, could significantly advance research on viral infections. Here, we show that H1N1-PR8-RBD (27 kDa, containing four cysteines forming two disulfide bonds) expressed in E. coli and was purified with nickel affinity chromatography, and reversed-phase HPLC was successfully refolded into its native structure, as assessed with several biophysical and biochemical techniques. Analytical ultracentrifugation indicated that H1N1-PR8-RBD was monomeric with a hydrodynamic radius of 2.5 nm. Thermal denaturation, monitored with DSC and CD at a wavelength of 222 nm, was cooperative with a midpoint temperature around 55 °C, strongly indicating a natively folded protein. In addition, the 15N-HSQC NMR spectrum exhibited several 1H-15N resonances indicative of a beta-sheeted protein. Our results indicate that a significant amount (40 mg/L) of pure and native H1N1-PR8-RBD can be produced using an E. coli expression system with our refolding procedure, offering potential insights into the molecular characterization of influenza virus infection.

Treatment-related cardiovascular events in patients with non-small cell lung cancer: Evidence from real-world data with a competing risks approach.

BACKGROUND: Understanding cancer treatment-related cardiovascular (CV) events is important for cancer care; however, comprehensive evaluation of CV events in patients with lung cancer is limited. This study aimed to assess the cumulative incidence and associated risks of various CV event types in patients with non-small cell lung cancer (NSCLC). METHODS: A total of 7868 individuals aged 40 years and older, recently diagnosed with NSCLC (2007-2018), were assessed with data obtained from the National Cancer Center, Korea. This study included nine types of CV events. A 2-year cumulative incidence function (CIF) of CV events was estimated, with death as a competing event. The associated risks were assessed by subdistribution hazard ratio (sHR) in the Fine-Gray competing risks model. RESULTS: CV events were observed in 7.8% of patients with NSCLC, with the most frequently observed types being atrial fibrillation and flutter (AF) (2.7%), venous thromboembolic disease (2.0%), and cerebrovascular disease (CeVD) (1.5%). Overall, all CV events were highest in the group treated with systemic therapy (CIF, 10.6%; 95% confidence interval [CI], 9.5%-11.8%), followed by those treated with surgery (CIF, 10.0%; 95% CI, 8.6%-11.6%); the incidence of AF (CIF, 5.7%; 95% CI, 4.6%-7.0%) was highest in patients treated with surgery. Individuals treated with systemic therapy were found to exhibit a higher CeVD risk than those treated with surgery (sHR, 4.12; 95% CI, 1.66-10.23). Among the patients who underwent surgery, those with lobectomy and pneumonectomy had a higher AF risk (vs. wedge resection/segmentectomy; sHR, 7.79; 95% CI, 1.87-32.42; sHR, 8.10; 95% CI, 1.60-40.89). CONCLUSIONS: These findings revealed treatment-related CV event risks in patients with NSCLC, which suggests that the risk of AF in surgery and CeVD in systemic therapy should be paid more attention to achieve a better prognosis and improve cancer survivorship outcomes. PLAIN LANGUAGE SUMMARY: Atrial fibrillation and flutter (AF) is the most common cardiovascular event, particularly at a high risk in patients with non-small cell lung cancer (NSCLC) undergoing surgery. Patients receiving surgery with poor performance status, diagnosed with regional stage, and undergoing lobectomy or pneumonectomy are at a high risk of AF. Systemic/radiotherapy is associated with cerebrovascular and ischemic heart disease in patients with NSCLC.