On pandemics and pivots: a COVID-19 reflection on envisioning the future of medical education.

The required adjustments precipitated by the coronavirus disease 2019 crisis have been challenging, but also represent a critical opportunity for the evolution and potential disruptive and constructive change of medical education. Given that the format of medical education is not fixed, but malleable and in fact must be adaptable to societal needs through ongoing reflexivity, we find ourselves in a potentially transformative learning phase for the field. An Association for Medical Education in Europe ASPIRE Academy group of 18 medical educators from seven countries was formed to consider this opportunity, and identified critical questions for collective reflection on current medical education practices and assumptions, with the attendant challenge to envision the future of medical education. This was achieved through online discussion as well as asynchronous collective reflections by group members. Four major themes and related conclusions arose from this conversation: Why we teach: the humanitarian mission of medicine should be reinforced; what we teach: disaster management, social accountability and embracing an environment of complexity and uncertainty should be the core; how we teach: open pathways to lean medical education and learning by developing learners embedded in a community context; and whom we teach: those willing to take professional responsibility. These collective reflections provide neither fully matured digests of the challenges of our field, nor comprehensive solutions; rather they are offered as a starting point for medical schools to consider as we seek to harness the learning opportunities stimulated by the pandemic.

Twelve tips for promoting consistent, good quality medical education across diverse clinical settings through faculty development approaches.

When students attend clinical attachments in diverse locations, a key challenge is in ensuring consistently good teaching over all areas. To meet this challenge, a faculty development intervention called TiMEtoTeach was created with the aim of reaching all involved in teaching medical students. The programme takes a holistic view of workplace (professional clinical attachments) learning with the recognition of all who are part of the student learning journey, including staff in clinical environments, charitable organisations, fellow students and the patients and carers. Empowering and upskilling this diverse group, we create a Universal Faculty. We engage this group with a comprehensive and accessible faculty development programme, enabling a consistent, authentic, and realistic learning experience for students. This supports graduate preparedness for their roles as junior doctors. The twelve tips described in this article relate to simple, achievable processes that faculty developers within medical education can apply to help improve consistency and quality in clinical workplace experience for students, recognising the challenges of engaging the large and diverse group of people who support education within the clinical arena.

Tissue non-specific alkaline phosphatase production by human dental pulp stromal cells is enhanced by high density cell culture.

The cell surface hydrolase tissue non-specific alkaline phosphatase (TNAP) (also known as MSCA-1) is used to identify a sub-population of bone marrow stromal cells (BMSCs) with high mineralising potential and is found on subsets of cells within the dental pulp. We aim to determine whether TNAP is co-expressed by human dental pulp stromal cells (hDPSCs) alongside a range of BMSC markers, whether this is an active form of the enzyme and the effects of culture duration and cell density on its expression. Cells from primary dental pulp and culture expanded hDPSCs expressed TNAP. Subsequent analyses revealed persistent TNAP expression and co-expression with BMSC markers such as CD73 and CD90. Flow cytometry and biochemical assays showed that increased culture durations and cell densities enhanced TNAP expression by hDPSCs. Arresting the hDPSC cell cycle also increased TNAP expression. These data confirm that TNAP is co-expressed by hDPSCs together with other BMSC markers and show that cell density affects TNAP expression levels. We conclude that TNAP is a potentially useful marker for hDPSC selection especially for uses in mineralised tissue regenerative therapies.

Hydrophobic and charged residues in the C-terminal arm of hepatitis C virus RNA-dependent RNA polymerase regulate initiation and elongation.

UNLABELLED: The RNA-dependent RNA polymerase (RdRp) of hepatitis C virus (HCV) is essential for viral genome replication. Crystal structures of the HCV RdRp reveal two C-terminal features, a ß-loop and a C-terminal arm, suitably located for involvement in positioning components of the initiation complex. Here we show that these two elements intimately regulate template and nucleotide binding, initiation, and elongation. We constructed a series of ß-loop and C-terminal arm mutants, which were used for in vitro analysis of RdRp de novo initiation and primer extension activities. All mutants showed a substantial decrease in initiation activities but a marked increase in primer extension activities, indicating an ability to form more stable elongation complexes with long primer-template RNAs. Structural studies of the mutants indicated that these enzyme properties might be attributed to an increased flexibility in the C-terminal features resulting in a more open polymerase cleft, which likely favors the elongation process but hampers the initiation steps. A UTP cocrystal structure of one mutant shows, in contrast to the wild-type protein, several alternate conformations of the substrate, confirming that even subtle changes in the C-terminal arm result in a more loosely organized active site and flexible binding modes of the nucleotide. We used a subgenomic replicon system to assess the effects of the same mutations on viral replication in cells. Even the subtlest mutations either severely impaired or completely abolished the ability of the replicon to replicate, further supporting the concept that the correct positioning of both the ß-loop and C-terminal arm plays an essential role during initiation and in HCV replication in general. IMPORTANCE: HCV RNA polymerase is a key target for the development of directly acting agents to cure HCV infections, which necessitates a thorough understanding of the functional roles of the various structural features of the RdRp. Here we show that even highly conservative changes, e.g., Tyr→Phe or Asp→Glu, in these seemingly peripheral structural features have profound effects on the initiation and elongation properties of the HCV polymerase.

Construction and crystal structure of recombinant STNV capsids.

A codon-optimised gene has been expressed in Escherichia coli to produce the coat protein (CP) of the Satellite Tobacco Necrosis Virus. This protein assembles in vivo into capsids closely resembling those of the T=1 wild-type virus. These virus-like particles (VLPs) package the recombinant mRNA transcript and can be disassembled and reassembled using different buffer conditions. The X-ray crystal structure of the VLP has been solved and refined at 1.4 Å resolution and shown to be very similar to that of wild-type Satellite Tobacco Necrosis Virus, except that icosahedral symmetry constraints could be removed to reveal differences between subunits, presumably owing to crystal packing. An additional low-resolution X-ray crystal structure determination revealed well-ordered RNA fragments lodged near the inside surface of the capsid, close to basic clusters formed by the N-terminal helices that project into the interior of the particle. The RNA consists of multiple copies of a 3-bp helical stem, with a single unpaired base at the 3' end, and probably consists of a number of short stem-loops where the loop region is disordered. The arrangement of the RNA is different from that observed in other satellite viruses.

Structural insights into substrate specificity in variants of N-acetylneuraminic Acid lyase produced by directed evolution.

The substrate specificity of Escherichia coli N-acetylneuraminic acid lyase was previously switched from the natural condensation of pyruvate with N-acetylmannosamine, yielding N-acetylneuraminic acid, to the aldol condensation generating N-alkylcarboxamide analogues of N-acetylneuraminic acid. This was achieved by a single mutation of Glu192 to Asn. In order to analyze the structural changes involved and to more fully understand the basis of this switch in specificity, we have isolated all 20 variants of the enzyme at position 192 and determined the activities with a range of substrates. We have also determined five high-resolution crystal structures: the structures of wild-type E. coli N-acetylneuraminic acid lyase in the presence and in the absence of pyruvate, the structures of the E192N variant in the presence and in the absence of pyruvate, and the structure of the E192N variant in the presence of pyruvate and a competitive inhibitor (2R,3R)-2,3,4-trihydroxy-N,N-dipropylbutanamide. All structures were solved in space group P2(1) at resolutions ranging from 1.65 Å to 2.2 Å. A comparison of these structures, in combination with the specificity profiles of the variants, reveals subtle differences that explain the details of the specificity changes. This work demonstrates the subtleties of enzyme-substrate interactions and the importance of determining the structures of enzymes produced by directed evolution, where the specificity determinants may change from one substrate to another.

Comparison of entropic contributions to binding in a "hydrophilic" versus "hydrophobic" ligand-protein interaction.

In the present study we characterize the thermodynamics of binding of histamine to recombinant histamine-binding protein (rRaHBP2), a member of the lipocalin family isolated from the brown-ear tick Rhipicephalus appendiculatus. The binding pocket of this protein contains a number of charged residues, consistent with histamine binding, and is thus a typical example of a "hydrophilic" binder. In contrast, a second member of the lipocalin family, the recombinant major urinary protein (rMUP), binds small hydrophobic ligands, with a similar overall entropy of binding in comparison with rRaHBP2. Having extensively studied ligand binding thermodynamics for rMUP previously, the data we obtained in the present study for HBP enables a comparison of the driving forces for binding between these classically distinct binding processes in terms of entropic contributions from ligand, protein, and solvent. In the case of rRaHBP2, we find favorable entropic contributions to binding from desolvation of the ligand; however, the overall entropy of binding is unfavorable due to a dominant unfavorable contribution arising from the loss of ligand degrees of freedom, together with the sequestration of solvent water molecules into the binding pocket in the complex. This contrasts with binding in rMUP where desolvation of the protein binding pocket makes a minor contribution to the overall entropy of binding given that the pocket is substantially desolvated prior to binding.

Co-translational myristoylation alters the quaternary structure of HIV-1 Nef in solution.

We have studied the solution properties of Nef, a 24-kDa cotranslationally myristoylated protein produced by HIV-1 and other primate lentiviruses. Nef is found in the cytosol and also in association with cytoplasmic membranes, the latter, mediated in part by the myristoyl group attached to the N-terminal glycine. Recombinant Nef was coexpressed in Escherichia coli in tandem with N-myristoyl-transferase and is fully myristoylated. Analysis by circular dichroism showed the myristoylated form to contain a greater alpha-helical content than the nonmyristoylated form. Analysis of modified and unmodified Nef in solution using small angle X-ray scattering, dynamic laser light scattering and analytical ultracentrifugation consistently showed differences in the oligomeric states of the two forms of Nef. Myristoylated Nef is predominantly monomeric and small oligomers which are also present, can be converted to the monomeric form under reducing conditions. By contrast, the nonmyristoylated form exists as a stable hexadecamer in solution which disassociates into tetramers upon addition of reducing agents. Shape reconstructions from small angle scattering curves of nonmyristoylated Nef are compatible with a large disc-like structure in the hexadecameric oligomer consisting of four Nef tetramers. From these findings, we hypothesize that Nef undergoes a substantial conformational change from an "open" into a "closed" form whereby the myristate group is sequestered in a hydrophobic pocket. The myristoylated protein can switch to the open conformation by association of the N-terminal region of molecule with membranes. These changes would allow Nef to carry out various functions depending on the conformational and oligomeric states.

HIV-1 Nef induces the release of inflammatory factors from human monocyte/macrophages: involvement of Nef endocytotic signals and NF-kappa B activation.

It has been recently reported that the endogenous expression of HIV-1 Nef in human monocyte/macrophages induces the release of chemokines and other as yet unidentified soluble factors leading to multiple effects of pathogenic significance, such as the recruitment and activation of quiescent lymphocytes. However, the description of underlying molecular mechanisms remained elusive. We recently demonstrated that human monocyte-derived macrophages (MDM) efficiently internalize soluble rNef, thereby inducing effects largely resembling those observed in cells endogenously expressing Nef. By exploiting the rNef/MDM model, we sought to gain more insights on the molecular mechanisms underlying the response of MDM to Nef. Array analysis for the detection of transcripts from a large number of monokines, chemokines, cytokines, and receptors thereof showed that MDM promptly responded to rNef treatment by increasing the transcription of genes for several inflammatory factors. Analysis of supernatants revealed that rNef treatment induced the release of macrophage inflammatory proteins 1alpha and 1beta, IL-1beta, IL-6, and TNF-alpha. Conversely, rNefs mutated in domains critical for the interaction with the endocytotic machinery (i.e., EE155-156QQ, and DD174-175AA) were ineffective. Interestingly, we found that the Nef-dependent release of inflammatory factors correlated with the activation of the NF-kappaB transcription factor, mainly in its p50/p50 homodimeric form, and in a de novo protein synthesis-independent manner. Our data add new hints supporting the idea that the presence of Nef is per se heavily detrimental for monocyte/macrophages and relative cross-talking cell types.