WPK5, a Novel Kunitz-Type Peptide from the Leech Whitmania pigra Inhibiting Factor XIa, and Its Loop-Replaced Mutant to Improve Potency

Kunitz-type proteins or peptides have been found in many blood-sucking animals, but the identity of them in leeches remained elusive. In the present study, five Kunitz-type peptides named WPK1-WPK5 were identified from the leech Whitmania pigra. Recombinant WPK1-WPK5 were expressed in Pichia pastoris GS115, and their inhibitory activity against Factor XIa (FXIa) was tested. WPK5 showed inhibitory activity against FXIa with an IC50 value of 978.20 nM. To improve its potency, the loop replacement strategy was used. The loop 1 (TGPCRSNLER) and loop 2 (QYGGC) in WPK5 were replaced by loop 1 (TGPCRAMISR) and loop 2 (FYGGC) in PN2KPI, respectively, and the resulting peptide named WPK5-Mut showed an IC50 value of 8.34 nM to FXIa, which is about 100-fold the potency of FXIa compared to that of WPK5. WPK5-Mut was further evaluated for its extensive bioactivity in vitro and in vivo. It dose-dependently prolonged APTT on both murine plasma and human plasma, and potently inhibited FeCl3-induced carotid artery thrombosis in mice at a dose of 1.5 mg/kg. Additionally, WPK5-Mut did not show significant bleeding risk at a dose of 6 mg/kg. Together, these results showed that WPK5-Mut is a promising candidate for the development of an antithrombotic drug.

Progress on 2—loop Amplitude Reduction

We collect results on 2-loop 2 → 3 amplitude reduction computations and discuss the progress done for the upgrade of the HELAC framework, in order to be able to compute 2—loop scattering amplitudes.

Extracellular loop 2 of G protein-coupled olfactory receptors is critical for odorant recognition

G protein-coupled olfactory receptors (ORs) enable us to detect innumerous odorants. They are also ectopically expressed, emerging as attractive drug targets. ORs can be promiscuous or highly specific, which is part of Nature′s strategy for odor discrimination. This work demonstrates that the extracellular loop 2 (ECL2) plays critical roles in OR promiscuity and specificity. Using site-directed mutagenesis and molecular modeling, we constructed 3D OR models in which ECL2 forms a lid of the orthosteric pocket. ECL2 controls the shape and the volume of the odorant-binding pocket, maintains the pocket hydrophobicity and acts as a gatekeeper of odorant binding. The interplay between the specific orthosteric pocket and the variable, less specific ECL2 controls OR specificity and promiscuity. The 3D models enabled virtual screening of new OR agonists and antagonists, exhibiting 78% hit rate in cell assays. This approach can be generalized to structure-based ligand screening for other GPCRs that lack high-resolution 3D structures.

Structural and Biochemical Characterization of PEDV Papain-like Protease 2

Coronaviral papain-like proteases (PLpros) are essential enzymes that mediate not only the proteolytic processes of viral polyproteins during virus replication, but also the deubiquitination and deISGylation of cellular proteins that attenuate host innate immune responses. Therefore, PLpros are attractive targets for antiviral drug development. Here we report the crystal structure of the papain-like protease 2 (PLP2) of porcine epidemic diarrhea virus (PEDV) in complex with ubiquitin (Ub). The X-ray structural analyses reveal that PEDV PLP2 interacts with Ub substrate mainly through the Ub core region and C-terminal tail. Mutations of Ub-interacting residues resulted in moderately or completely abolished deubiquitinylating function of PEDV PLP2. In addition, our analyses also indicate that the two residues-extended blocking loop 2 at the S4 subsite contributes to the substrate selectivity and binding affinity of PEDV PLP2. Furthermore, the PEDV PLP2 Glu99 residue, conserved in alpha-CoV PLpros, was found to govern the preference of a positively charged P4 residue of peptidyl substrates. Collectively, our data provided structure-based information for substrate binding and selectivity of PEDV PLP2. These findings may help us gain insights into the deubiquitinating and proteolytic functions of PEDV PLP2 from a structural perspective. Importance Current challenges in CoVs include a comprehensive understanding of mechanistic effects of associated enzymes, including the 3C-like and papain-like proteases. We have previously reported that the PEDV PLP2 exhibits a broader substrate preference, superior DUB function, and inferior peptidase activity. However, the structure basis for these functions remains largely unclear. Here, we show the high-resolution X-ray crystal structure of PEDV PLP2 in complex with Ub. Integrated structural and biochemical analyses revealed: (i) three Ub-core interacting residues are essential for DUB function, (ii) two-residue-elongated blocking loop 2 regulates substrate selectivity, and (iii) a conserved glutamate residue governs the substrate specificity of PEDV PLP2. Collectively, our findings provide not only the structural insights to the catalytic mechanism of PEDV PLP2 but also a model for developing antiviral strategies.

1126 Improving Hand Trauma Clerking Using an Electronic Proforma: A Full Cycle Clinical Audit

Aim The BSSH standards of care in hand trauma emphasise the importance of accurate and comprehensive documentation. This quality improvement project aimed to evaluate whether an electronic proforma could improve documentation rates in hand trauma. Method This prospective, closed-loop audit involved two cycles of seven consecutive days. Outcomes were evaluated against BSSH standards of care using 15 data points. Pre-audit, a bespoke proforma was piloted in the hand trauma clinic and revised following feedback. In the first cycle (‘Loop 1’), documentation in the hand trauma clinic was compared with that of the acute take, where clinicians were still using free text. The second cycle (‘Loop 2’) compared documentation pre- and post-introduction of the proforma within the acute take setting. Categorical data were interrogated using the χ2 test with significance set at p < 0.05. Results 122 patients were included in the Loop 1 dataset. Free text clerking quality varied between clinician groups (e.g., surgical trainees significantly outperformed advanced nurse practitioners in 5/15 data points); however, some data points were universally poorly documented. 99 patients were included in the Loop 2 dataset. There was excellent uptake of the proforma in both acute take (95%) and hand trauma clinic (100%) settings. Statistically significant improvements were seen in 11/15 data points following the introduction of the proforma. Conclusions This project demonstrates that the introduction of a hand trauma clerking proforma led to significant improvements in documentation quality. The proforma has now been developed into a Trust-wide ClinDoc which brings additional benefits in terms of research, audit and service evaluation.

Targeting N-Terminal Human Maltase-Glucoamylase to Unravel Possible Inhibitors Using Molecular Docking, Molecular Dynamics Simulations, and Adaptive Steered Molecular Dynamics Simulations

There are multiple drugs for the treatment of type 2 diabetes, including traditional sulfonylureas biguanides, glinides, thiazolidinediones, α-glucosidase inhibitors, glucagon-like peptide-1 (GLP-1) receptor agonists, dipeptidyl peptidase IV (DPP-4) inhibitors, and sodium-glucose cotransporter 2 (SGLT2) inhibitors. α-Glucosidase inhibitors have been used to control postprandial glucose levels caused by type 2 diabetes since 1990. α-Glucosidases are rather crucial in the human metabolic system and are principally found in families 13 and 31. Maltase-glucoamylase (MGAM) belongs to glycoside hydrolase family 31. The main function of MGAM is to digest terminal starch products left after the enzymatic action of α-amylase; hence, MGAM becomes an efficient drug target for insulin resistance. In order to explore the conformational changes in the active pocket and unbinding pathway for NtMGAM, molecular dynamics (MD) simulations and adaptive steered molecular dynamics (ASMD) simulations were performed for two NtMGAM-inhibitor [de-O-sulfonated kotalanol (DSK) and acarbose] complexes. MD simulations indicated that DSK bound to NtMGAM may influence two domains (inserted loop 1 and inserted loop 2) by interfering with the spiralization of residue 497–499. The flexibility of inserted loop 1 and inserted loop 2 can influence the volume of the active pocket of NtMGAM, which can affect the binding progress for DSK to NtMGAM. ASMD simulations showed that compared to acarbose, DSK escaped from NtMGAM easily with lower energy. Asp542 is an important residue on the bottleneck of the active pocket of NtMGAM and could generate hydrogen bonds with DSK continuously. Our theoretical results may provide some useful clues for designing new α-glucosidase inhibitors to treat type 2 diabetes.

The Changes in the p53 Protein across the Animal Kingdom Point to Its Involvement in Longevity

Recently, the quest for the mythical fountain of youth has produced extensive research programs that aim to extend the healthy lifespan of humans. Despite advances in our understanding of the aging process, the surprisingly extended lifespan and cancer resistance of some animal species remain unexplained. The p53 protein plays a crucial role in tumor suppression, tissue homeostasis, and aging. Long-lived, cancer-free African elephants have 20 copies of the TP53 gene, including 19 retrogenes (38 alleles), which are partially active, whereas humans possess only one copy of TP53 and have an estimated cancer mortality rate of 11–25%. The mechanism through which p53 contributes to the resolution of the Peto’s paradox in Animalia remains vague. Thus, in this work, we took advantage of the available datasets and inspected the p53 amino acid sequence of phylogenetically related organisms that show variations in their lifespans. We discovered new correlations between specific amino acid deviations in p53 and the lifespans across different animal species. We found that species with extended lifespans have certain characteristic amino acid substitutions in the p53 DNA-binding domain that alter its function, as depicted from the Phenotypic Annotation of p53 Mutations, using the PROVEAN tool or SWISS-MODEL workflow. In addition, the loop 2 region of the human p53 DNA-binding domain was identified as the longest region that was associated with longevity. The 3D model revealed variations in the loop 2 structure in long-lived species when compared with human p53. Our findings show a direct association between specific amino acid residues in p53 protein, changes in p53 functionality, and the extended animal lifespan, and further highlight the importance of p53 protein in aging.

Iterative Text-Based Editing of Talking-Heads Using Neural Retargeting

We present a text-based tool for editing talking-head video that enables an iterative editing workflow. On each iteration users can edit the wording of the speech, further refine mouth motions if necessary to reduce artifacts, and manipulate non-verbal aspects of the performance by inserting mouth gestures (e.g., a smile) or changing the overall performance style (e.g., energetic, mumble). Our tool requires only 2 to 3 minutes of the target actor video and it synthesizes the video for each iteration in about 40 seconds, allowing users to quickly explore many editing possibilities as they iterate. Our approach is based on two key ideas. (1) We develop a fast phoneme search algorithm that can quickly identify phoneme-level subsequences of the source repository video that best match a desired edit. This enables our fast iteration loop. (2) We leverage a large repository of video of a source actor and develop a new self-supervised neural retargeting technique for transferring the mouth motions of the source actor to the target actor. This allows us to work with relatively short target actor videos, making our approach applicable in many real-world editing scenarios. Finally, our, refinement and performance controls give users the ability to further fine-tune the synthesized results.