Structural and functional characterization of ubiquitin variant inhibitors for the JAMM-family deubiquitinases STAMBP and STAMBPL1.

Ubiquitination is a crucial posttranslational protein modification involved in a myriad of biological pathways. This modification is reversed by deubiquitinases (DUBs) that deconjugate the single ubiquitin (Ub) moiety or poly-Ub chains from substrates. In the past decade, tremendous efforts have been focused on targeting DUBs for drug discovery. However, most chemical compounds with inhibitory activity for DUBs suffer from mild potency and low selectivity. To overcome these obstacles, we developed a phage display-based protein engineering strategy for generating Ub variant (UbV) inhibitors, which was previously successfully applied to the Ub-specific protease (USP) family of cysteine proteases. In this work, we leveraged the UbV platform to selectively target STAMBP, a member of the JAB1/MPN/MOV34 (JAMM) metalloprotease family of DUB enzymes. We identified two UbVs (UbVSP.1 and UbVSP.3) that bind to STAMBP with high affinity but differ in their selectivity for the closely related paralog STAMBPL1. We determined the STAMBPL1-UbVSP.1 complex structure by X-ray crystallography, revealing hotspots of the JAMM-UbV interaction. Finally, we show that UbVSP.1 and UbVSP.3 are potent inhibitors of STAMBP isopeptidase activity, far exceeding the reported small-molecule inhibitor BC-1471. This work demonstrates that UbV technology is suitable to develop molecules as tools to target metalloproteases, which can be used to further understand the cellular function of JAMM family DUBs.

Virtual Care Integration: Balancing Physician Well-Being.

Background and Objectives: According to self-determination theory (SDT), fulfillment of three basic psychological needs-autonomy, competence, and relatedness-positively impacts people's health and well-being. Amid the COVID-19 pandemic, an accelerated adoption of virtual care practices coincided with a decline in the well-being of physicians. Taking into account the frequency of virtual care use, we examined the relationship between workplace need fulfillment and physician well-being. Methods: Using online survey methodology, in March through June 2022, we collected data from 156 family physicians (FPs) in Alberta, Canada. The survey contained scales that measured workplace need satisfaction and frustration, subjective well-being (physical, psychological, and relational), and frequency of virtual care use. We performed correlational and regression analyses of the data. Results: More frequent use of virtual care was associated with lower relatedness satisfaction among FPs. Controlling for the frequency of virtual care use, frustration of autonomy and competence needs negatively related to FPs' physical well-being; frustration of competence and relatedness needs negatively related to their psychological and relational well-being. Conclusions: Findings from this study align with SDT and underscore the importance of supporting FPs' basic psychological needs, while we work to integrate virtual care into clinical practice. In their day-to-day work, we encourage physicians to reflect on their own sense of autonomy, competence, and relatedness, and consider how using virtual care aligns with these basic needs.

Teaching Opportunities for Postgraduate Trainees in Primary Care.

ABSTRACT: The purpose of this AM Last Page is to help faculty and postgraduate medical trainees (residents) identify resident teaching opportunities and foster teaching skill development. The Fundamental Teaching Activities (FTA) framework includes three domains in which physicians teach: Clinical Preceptor; Teacher Outside the Clinical Setting; and Educational Leader. Based on interviews with faculty and residents as well as our collective and diverse teaching experience, we adapted the FTA framework to be more applicable to resident teaching. The resultant domains are: Clinical Teacher; Teacher in Nonclinical Settings; and Educational Collaborator.

Comparative study of the mechanical properties of woven and unidirectional fibres in discontinuous long-fibre composites.

Discontinuous long-fibre (DLF) composites can be made with randomly oriented unidirectional pre-impregnated composite chips. High fibre volume fraction unidirectional fibre chips provide good mechanical properties to the DLF composite architecture, which enables this material to contribute to bridging the gap between continuous-fibre and randomly-oriented short-fibre composites. However, it is well known that unidirectional fibres have highly anisotropic in-plane behaviour, which causes weak points in the parts when chips are oriented at unfavourable angles. This can be problematic since chips are randomly oriented in DLF composites. To overcome this problem, this research utilizes woven fibre chips instead of unidirectional fibre chips to fabricate DLF composites. Woven fibres diminish the potential for weak points due to their more homogenized in-plane mechanical properties. For comparison purposes, compression moulded carbon/PEI samples were made from both unidirectional chips and 5HS woven chips. Bending and tensile tests following ASTM guidelines were performed to compare both types of fibre arrangement. The results show that woven fibre chips increase the mechanical properties of the DLF composites and reduce their variability.

A 3D finite element model to simulate progressive damage in unidirectional- and woven-fibre thermoplastic discontinuous-long-fibre composites.

Discontinuous-long-fibre (DLF) composites fabricated from pre-impregnated unidirectional (UD) fibre chips are susceptible to structural deficiency. The in-plane highly anisotropic mechanical properties of the chips combined with the random nature of fibre orientation causes local weaknesses within the material when fibres are perpendicular to the load. Recent experimental results have shown that using woven-fibre chips could improve the performance of DLF composites by increasing their average mechanical properties and reducing their variability. To better understand the underlying phenomenon giving an advantage to the woven chips, a finite element model was developed to predict the mechanical properties obtained from a standard tensile test. DLF chips were modelled based on a voxel method where random chip positions were generated by an algorithm developed in this work. ANSYS® software was utilized to model the non-linear response associated with progressive damage of the composite. The maximum stress and the Puck failure criteria were employed to define damage initiation for the woven and UD fibres, respectively. Tensile modulus predictions for both types of chips showed good results when compared to experimental data. Strength predictions for the UD fibres also showed good correlation with experimental results, but the model overestimated the strength of the woven-fibre DLF composite. It appeared that the failure of the UD-fibre composites was associated with matrix failure (transverse tension and in-plane shear). Woven-fibre composites, however, showed damage modes linked to both fibre failure (longitudinal tension) and matrix failure (transverse tension and in-plane shear).

Targeting the Ubiquitin-Proteasome System for Cancer Therapeutics by Small-Molecule Inhibitors.

The ubiquitin-proteasome system (UPS) is a critical regulator of cellular protein levels and activity. It is, therefore, not surprising that its dysregulation is implicated in numerous human diseases, including many types of cancer. Moreover, since cancer cells exhibit increased rates of protein turnover, their heightened dependence on the UPS makes it an attractive target for inhibition via targeted therapeutics. Indeed, the clinical application of proteasome inhibitors in treatment of multiple myeloma has been very successful, stimulating the development of small-molecule inhibitors targeting other UPS components. On the other hand, while the discovery of potent and selective chemical compounds can be both challenging and time consuming, the area of targeted protein degradation through utilization of the UPS machinery has seen promising developments in recent years. The repertoire of proteolysis-targeting chimeras (PROTACs), which employ E3 ligases for the degradation of cancer-related proteins via the proteasome, continues to grow. In this review, we will provide a thorough overview of small-molecule UPS inhibitors and highlight advancements in the development of targeted protein degradation strategies for cancer therapeutics.

Distortion-free single point imaging of multi-layered composite sandwich panel structures.

The results of a magnetic resonance imaging (MRI) investigation concerning the effects of an aluminum honeycomb sandwich panel on the B1 and B0 fields and on subsequent image quality are presented. Although the sandwich panel structure, representative of an aircraft composite material, distorts B0 and attenuates B1, distortion-free imaging is possible using single point (constant time) imaging techniques. A new expression is derived for the error caused by gradient field distortion due to the heterogeneous magnetic susceptibility within a sample and this error is shown not to cause geometric distortion in the image. The origin of the B0 distortion in the sample under investigation was also examined. The graphite-epoxy 'skin' of the panel is the principal source of the B0 distortion. Successful imaging of these structures sets the stage for the development of methods for detecting moisture ingress and degradation within composite sandwich structures.