Sofosbuvir and its tri-phosphate metabolite inhibit the RNA-dependent RNA polymerase activity of non-structural protein 5 from the Kyasanur forest disease virus.

Kyasanur forest disease is a neglected zoonotic disease caused by a single-stranded RNA-based flavivirus, the incidence of which was first recorded in 1957 in the Southern part of India. Kyasanur forest disease virus is transmitted to monkeys and humans through the infected tick bite of Haemophysalis spinigera. Kyasanur forest disease is a febrile illness, which in severe cases, results in neurological complications leading to mortality. The current treatment regimens are symptomatic and supportive, and no targeted therapies are available for this disease. In this study, we evaluated the ability of FDA-approved drugs sofosbuvir (and its active metabolite) and Dasabuvir to inhibit the RNA-dependent RNA polymerase activity of NS5 protein from the Kyasanur forest disease virus. NS5 protein containing the N-terminal methyl transferase domain and C-terminal RNA-dependent RNA polymerase domain was expressed in Escherichia coli, and RNA-dependent RNA polymerase activity was demonstrated with the purified protein. The RNA-dependent RNA polymerase assay conditions were optimized, followed by the determination of apparent Km,ATP to validate the enzyme preparation. Half maximal-inhibitory concentrations against RNA-dependent RNA polymerase activity were determined for Sofosbuvir and its active metabolite. Dasabuvir did not show detectable inhibition with the tested conditions. This is the first demonstration of the inhibition of RNA-dependent RNA polymerase activity of NS5 protein from the Kyasanur forest disease virus with small molecule inhibitors. These initial findings can potentially facilitate the discovery and development of targeted therapies for treating Kyasanur forest disease.

Refolding and characterization of a diabody against Pfs25, a vaccine candidate of Plasmodium falciparum.

Pfs25, a vaccine candidate, expressed on the surface of the malarial parasite, plays an important role in the development of Plasmodium falciparum. 1269, a monoclonal antibody targeting the epidermal growth factor-like domain 1 and epidermal growth factor-like domain 3 of Pfs25, blocks the transmission of parasites in mosquitoes. In this study, we refolded 1269-Db, a dimeric antibody fragment referred as diabody, designed from 1269, with a yield of 3 mg/litre of bacterial culture. Structural integrity of the protein was validated with thermal stability, disulphide bond analysis and glutaraldehyde crosslinking experiments. To evaluate the functionality of 1269-Db, recombinant monomeric MBP-Pfs25 was produced from bacteria. Qualitative binding assays demonstrated that 1269-Db recognized the epitopes on Pfs25 in its native, but not the denatured state. An apparent KD of 2.6 nM was determined for 1269-Db with monomeric MBP-Pfs25, using isothermal titration calorimetry. 1269-Db recognized the periphery of zygotes/ookinetes, demonstrating recognition of Pfs25, expressed on the surface of the parasite. As the established refolding method resulted in a functional diabody, the optimized method pipeline for 1269-Db can potentially facilitate engineering of antibody fragments with desired properties.

Triple endoscopy and recurrent croup in children: A single aerodigestive center experience.

OBJECTIVE: To determine the utility of triple endoscopy (combined direct laryngoscopy, bronchoscopy (DLB), flexible bronchoscopy with bronchoalveolar lavage (FB + BAL), and esophagogastroduodenoscopy (EGD)) in the diagnosis and management of patients with recurrent croup (RC), and to identify predictors of endoscopic findings METHODS: A retrospective chart review was performed of pediatric patients (age <18 years) with RC evaluated by triple endoscopy at a tertiary care pediatric hospital from 2010 to 2021. Data including presenting symptoms, airway findings, BAL and EGD with biopsy findings were collected. RESULTS: 42 patients with RC underwent triple endoscopy were included. The mean age was 4.55±2.84 years old. The most common symptom was chronic cough among 19 (45%) patients, while 23 (55%) patients had gastrointestinal (GI) symptoms. Airway findings included tracheomalacia in 19, laryngeal cleft in 17, and subglottic stenosis in 11 patients. On EGD with biopsy, abnormal gross findings were present in 6 and abnormal microscopic findings in 18 patients, including 6 with histologic findings suggestive of gastroesophageal reflux and 5 with eosinophilic esophagitis. Seventeen (40%) patients had positive culture on BAL. No findings in patient histories significantly predicted presence of lower airway malacia, subglottic stenosis, or abnormal EGD findings. CONCLUSIONS: Children with recurrent croup presenting to aerodigestive centers may not have any pertinent presenting symptoms that correlate with significant findings on triple endoscopy. Further work is needed to determine which children with recurrent croup may benefit from aerodigestive evaluation. LEVEL OF EVIDENCE: Level 3.