The apo-acyl coenzyme A binding protein of Leishmania major forms a unique 'AXXA' motif mediated dimer.

Acyl-Coenzyme A binding domain containing proteins (ACBDs) are ubiquitous in nearly all eukaryotes. They can exist as a free protein, or a domain of a large, multidomain, multifunctional protein. Besides modularity, ACBDs also display multiplicity. The same organism may have multiple ACBDs, differing in sequence and organization. By virtue of this diversity, ACBDs perform functions ranging from transport, synthesis, trafficking, signal transduction, transcription, and gene regulation. In plants and some microorganisms, these ACBDs are designated ACBPs (acyl-CoA binding proteins). The simplest ACBD/ACBP is a small, ∼10 kDa, soluble protein, comprising the acyl-CoA binding (ACB) domain. Most of these small ACBDs exist as monomers, while a few show a tendency to oligomerize. In sync with those studies, we report the crystal structure of two ACBDs from Leishmania major, named ACBP103, and ACBP96 based on the number of residues present. Interestingly, ACBP103 crystallized as a monomer and a dimer under different crystallization conditions. Careful examination of the dimer disclosed an exposed 'AXXA' motif in the helix I of the two ACBP103 monomers, aligned in a head-to-tail arrangement in the dimer. Glutaraldehyde cross-linking studies confirm that apo-ACBP103 can self-associate in solution. Isothermal titration calorimetry studies further show that ACBP103 can bind ligands ranging from C8 - to C20-CoA, and the data could be best fit to a 'two sets of sites'/sequential binding site model. Taken together, our studies show that Leishmania major ACBP103 can self-associate in the apo-form through a unique dimerization motif, an interaction that may play an important role in its function.

Drug repurposing screen identifies lonafarnib as respiratory syncytial virus fusion protein inhibitor.

Respiratory syncytial virus (RSV) is a common cause of acute lower respiratory tract infection in infants, older adults and the immunocompromised. Effective directly acting antivirals are not yet available for clinical use. To address this, we screen the ReFRAME drug-repurposing library consisting of 12,000 small molecules against RSV. We identify 21 primary candidates including RSV F and N protein inhibitors, five HSP90 and four IMPDH inhibitors. We select lonafarnib, a licensed farnesyltransferase inhibitor, and phase III candidate for hepatitis delta virus (HDV) therapy, for further follow-up. Dose-response analyses and plaque assays confirm the antiviral activity (IC50: 10-118 nM). Passaging of RSV with lonafarnib selects for phenotypic resistance and fixation of mutations in the RSV fusion protein (T335I and T400A). Lentiviral pseudotypes programmed with variant RSV fusion proteins confirm that lonafarnib inhibits RSV cell entry and that these mutations confer lonafarnib resistance. Surface plasmon resonance reveals RSV fusion protein binding of lonafarnib and co-crystallography identifies the lonafarnib binding site within RSV F. Oral administration of lonafarnib dose-dependently reduces RSV virus load in a murine infection model using female mice. Collectively, this work provides an overview of RSV drug repurposing candidates and establishes lonafarnib as a bona fide fusion protein inhibitor.

Wooden foreign body impalement through the right shoulder region - an unusual penetrating injury: A case report.

BACKGROUND: Impalement of the body is a rare injury and comes with varied presentation. There is no set classification or defined protocols for managing this injury. This case report aims to create awareness among trauma surgeons about unusual presentation and management of such case. CASE SUMMARY: A 45-year-old man presented to the emergency department with a sharp penetrating wooden plank at right clavicular region between the neck and shoulder following a road traffic accident. The vehicle had crashed into a roadside wooden hut, thus causing an impalement injury. He was meticulously worked up and taken to emergency theatre. The wooden plank was removed and the wound healed uneventfully. Postoperatively, he had fairly good shoulder function and was able to return back to work successfully. CONCLUSION: Each impalement injury brings in challenges in management as no two cases are the same. The varied presentation and risks involved should be known to medical professionals handling the emergency. Coordinated multidisciplinary team approach is needed for successful outcome.

Impact of COVID-19 first wave on the in-hospital length of stay of operated proximal femur fracture patients in an industrial hospital in Eastern India.

Objective: The objective of this article is to study the impact of coronavirus disease 2019 (COVID-19) pandemic first wave on the in-hospital length of stay of operated proximal femur fractures. Materials and Methods: A retrospective analysis of data collected through the electronic record system of the hospital, after applying inclusion and exclusion criteria, was done. The data were collected from the pre-pandemic, early part first wave and later part first wave of COVID-19 pandemic to calculate the average preoperative stay (POS) and total length of stay (LOS) of operated proximal femur fracture patients. Also, a sub-analysis of POS and LOS was done as per age (male/female), sex (<60/≥60 years) and fracture subtype (intertrochanteric, neck of femur and subtrochanteric fracture) of the patients to study if any of these had a significant direct impact on the POS and LOS. Results: The LOS and POS were found to be significantly increased during early part of first wave of COVID-19 pandemic in comparison to the pre-pandemic era (13.6 ± 7.7 days vs. 11.1 ± 5.7 days). The later part of the first wave of the pandemic however saw the LOS and POS to return to near pre-pandemic values, although still remaining higher. Conclusion: The study highlights that unpreparedness during the early part of the unprecedented pandemic event leads to a significant increase in LOS of operated patients with its associated implications; however, prompt action by the government, hospital administration and hospital staff the LOS could be reduced to near pre-pandemic values in the later part of the first wave of the pandemic. Analysis of the causes that lead to a significant increase in LOS can help for better future management of similar events in future.

Leishmania major biotin protein ligase forms a unique cross-handshake dimer.

Biotin protein ligase catalyses the post-translational modification of biotin carboxyl carrier protein (BCCP) domains, a modification that is crucial for the function of several carboxylases. It is a two-step process that results in the covalent attachment of biotin to the ϵ-amino group of a conserved lysine of the BCCP domain of a carboxylase in an ATP-dependent manner. In Leishmania, three mitochondrial enzymes, acetyl-CoA carboxylase, methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase, depend on biotinylation for activity. In view of the indispensable role of the biotinylating enzyme in the activation of these carboxylases, crystal structures of L. major biotin protein ligase complexed with biotin and with biotinyl-5'-AMP have been solved. L. major biotin protein ligase crystallizes as a unique dimer formed by cross-handshake interactions of the hinge region of the two monomers formed by partial unfolding of the C-terminal domain. Interestingly, the substrate (BCCP domain)-binding site of each monomer is occupied by its own C-terminal domain in the dimer structure. This was observed in all of the crystals that were obtained, suggesting a closed/inactive conformation of the enzyme. Size-exclusion chromatography studies carried out using high protein concentrations (0.5 mM) suggest the formation of a concentration-dependent dimer that exists in equilibrium with the monomer.

Chemical shift assignments of the biotin carboxyl carrier protein domain of L. major Methylcrotonyl-CoA carboxylase.

Methylcrotonyl-CoA carboxylase (MCCC) is a biotin dependent enzyme, that plays a crucial role in leucine metabolism. The enzyme comprises a biotin carboxylase (BC), a carboxyltransferase (CT), and a biotin carboxyl carrier protein (BCCP) domain. MCCC is synthesized as an apo-protein, and is posttranslationally modified at a lysine residue, conserved in the biotin carboxyl carrier protein (BCCP) domain. In order to understand the structure, function and interactions of L. major MCCC, we have expressed and characterized its domains. Here we report the complete chemical shift assignments of MCCC BCCP domain of L. major. Furthermore, we have used the assignments to generate a model of the same, using CS-Rosetta. We have also followed its chemical shift perturbations upon biotin modification. Changes were observed at the lysine 51 amide, that undergoes biotin modification, and a few others present in its immediate neighborhood.

Bipolar hemiarthroplasty for intracapsular femoral neck fractures in elderly patients.

PURPOSE: To report outcome of 30 patients who underwent bipolar hemiarthroplasty for intracapsular femoral neck fractures. METHODS: 18 women and 12 men aged 56 to 86 (mean, 70) years with Garden type III (n=7) or IV (n=23) intracapsular femoral neck fractures underwent cemented (n=5) or uncemented (n=25) bipolar hemiarthroplasty. Pain was evaluated using the visual analogue score. Mobility status was classified as ambulation with or without aid (a cane or walker). Stability of the prosthesis was classified as stable or unstable. Functional outcome was evaluated using the Harris Hip Score. RESULTS: The mean length of hospital stay was 15.3 (range, 4-29) days. At the 6-month follow-up, 21 patients had no pain, 6 had mild pain, and 3 had moderate pain. The mean Harris Hip Score was 83.1; functional outcome was excellent in 10 patients, good in 13, fair in 5, and poor in 2. The latter 2 were non-ambulatory; one of whom had sustained a periprosthetic fracture intra-operatively, which was managed by encerclage wiring, and the other had a neglected prosthetic dislocation at 3 months, which was converted to an excision arthroplasty. CONCLUSION: Bipolar hemiarthroplasty conferred good short-term outcome.