Development and Validation of an Automated High-Throughput System for Zebrafish In Vivo Screenings

The zebrafish is a vertebrate model compatible with the paradigms of drug discovery. The small size and transparency of zebrafish embryos make them amenable for the automation necessary in high-throughput screenings. We have developed an automated high-throughput platform for in vivo chemical screenings on zebrafish embryos that includes automated methods for embryo dispensation, compound delivery, incubation, imaging and analysis of the results. At present, two different assays to detect cardiotoxic compounds and angiogenesis inhibitors can be automatically run in the platform, showing the versatility of the system. A validation of these two assays with known positive and negative compounds, as well as a screening for the detection of unknown anti-angiogenic compounds, have been successfully carried out in the system developed. We present a totally automated platform that allows for high-throughput screenings in a vertebrate organism

The impact of patient skin colour on diagnostic ability and confidence of medical students.

Previous literature has explored unconscious racial biases in clinical education and medicine, finding that people with darker skin tones can be underrepresented in learning resources and managed differently in a clinical setting. This study aimed to examine whether patient skin colour can affect the diagnostic ability and confidence of medical students, and their cognitive reasoning processes. We presented students with 12 different clinical presentations on both white skin (WS) and non-white skin (NWS). A think aloud (TA) study was conducted to explore students' cognitive reasoning processes (n = 8). An online quiz was also conducted where students submitted a diagnosis and confidence level for each clinical presentation (n = 185). In the TA interviews, students used similar levels of information gathering and analytical reasoning for each skin type but appeared to display increased uncertainty and reduced non-analytical reasoning methods for the NWS images compared to the WS images. In the online quiz, students were significantly more likely to accurately diagnose five of the 12 clinical presentations (shingles, cellulitis, Lyme disease, eczema and meningococcal disease) on WS compared to NWS (p < 0.01). With regards to students' confidence, they were significantly more confident diagnosing eight of the 12 clinical presentations (shingles, cellulitis, Lyme disease, eczema, meningococcal disease, urticaria, chickenpox and Kawasaki disease) on WS when compared to NWS (p < 0.01). These findings highlight the need to improve teaching resources to include a greater diversity of skin colours exhibiting clinical signs, to improve students' knowledge and confidence, and ultimately, to avoid patients being misdiagnosed due to the colour of their skin

Knowledge attainment and engagement among medical students : a comparison of three forms of online learning

This study compared knowledge attainment and student enjoyment and engagement between clinical case vignette, patient-testimony videos and mixed reality (MR) teaching via the Microsoft HoloLens 2, all delivered remotely to third year medical students. The feasibility of conducting MR teaching on a large scale was also assessed. Medical students in Year 3 at Imperial College London participated in three online teaching sessions, one in each format. All students were expected to attend these scheduled teaching sessions and to complete the formative assessment. Inclusion of their data used as part of the research trial was optional. The primary outcome measure was performance on a formative assessment, which served to compare knowledge attainment between three forms of online learning. Moreover, we aimed to explore student engagement with each form of learning via a questionnaire, and also feasibility of applying MR as a teaching tool on a large scale. Comparisons between performances on the formative assessment between the three groups were investigated using a repeated measures two-way ANOVA. Engagement and enjoyment were also analysed in the same manner. A total of 252 students participated in the study. Knowledge attainment of students using MR was comparable with the other two methods. Participants reported higher enjoyment and engagement (p<0.001) for the case vignette method, compared with MR and video-based teaching. There was no difference in enjoyment or engagement ratings between MR and the video-based methods. This study demonstrated that the implementation of MR is an effective, acceptable, and feasible way of teaching clinical medicine to undergraduate students on a large scale. However, case-based tutorials were found to be favoured most by students. Future work could further explore the best uses for MR teaching within the medical curriculum. [Abstract copyright: © 2023 Stackhouse et al.

Structural and immunologic correlates of chemically stabilized HIV-1 envelope glycoproteins

<div><p>Inducing broad spectrum neutralizing antibodies against challenging pathogens such as HIV-1 is a major vaccine design goal, but may be hindered by conformational instability within viral envelope glycoproteins (Env). Chemical cross-linking is widely used for vaccine antigen stabilization, but how this process affects structure, antigenicity and immunogenicity is poorly understood and its use remains entirely empirical. We have solved the first cryo-EM structure of a cross-linked vaccine antigen. The 4.2 Å structure of HIV-1 BG505 SOSIP soluble recombinant Env in complex with a CD4 binding site-specific broadly neutralizing antibody (bNAb) Fab fragment reveals how cross-linking affects key properties of the trimer. We observed density corresponding to highly specific glutaraldehyde (GLA) cross-links between gp120 monomers at the trimer apex and between gp120 and gp41 at the trimer interface that had strikingly little impact on overall trimer conformation, but critically enhanced trimer stability and improved Env antigenicity. Cross-links were also observed within gp120 at sites associated with the N241/N289 glycan hole that locally modified trimer antigenicity. In immunogenicity studies, the neutralizing antibody response to cross-linked trimers showed modest but significantly greater breadth against a global panel of difficult-to-neutralize Tier-2 heterologous viruses. Moreover, the specificity of autologous Tier-2 neutralization was modified away from the N241/N289 glycan hole, implying a novel specificity. Finally, we have investigated for the first time T helper cell responses to next-generation soluble trimers, and report on vaccine-relevant immunodominant responses to epitopes within BG505 that are modified by cross-linking. Elucidation of the structural correlates of a cross-linked viral glycoprotein will allow more rational use of this methodology for vaccine design, and reveals a strategy with promise for eliciting neutralizing antibodies needed for an effective HIV-1 vaccine.</p></div

Rabbit antibody responses to SOSIP and GLA-SOSIP trimer.

<p>(<b>a</b>) ELISA endpoint titers of rabbit sera binding to SOSIP (top panel) and GLA-SOSIP trimer (bottom panel). Arrows represent immunizations at weeks 0, 4, 20 and 50, datum points are means of 5 animals/group and error bars indicate SD. *<i>p</i><0.05; ****<i>p</i><0.0001, One-way ANOVA with Sidak’s multiple-comparison correction. (<b>b</b>) Linear peptide array mapping of rabbit serum responses, pie charts represent proportion of response to individual epitopes with the region for each epitope listed with dominant percentage reactivity shown. Values plotted are magnitude of binding to any strain among the 13 strains in the array library for each epitope. Regions and sequences listed on left. Supporting data in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006986#ppat.1006986.s007" target="_blank">S5 Fig</a>. (<b>c, d</b>) Neutralization of (<b>c</b>) neutralization-sensitive Tier-1 PV and (<b>d</b>) autologous Tier-2 PV by sera from SOSIP and GLA-SOSIP trimer-immunized rabbits. Supporting data in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006986#ppat.1006986.s008" target="_blank">S6 Fig</a>. (<b>d</b>) ns = not significant, Mann-Whitney U test. (<b>e</b>) Loss of neutralization activity of SOSIP relative to GLA-SOSIP trimer sera on mutant PV. (<b>f</b>) Cross-competition ELISA of sera with the indicated glycan-hole specific mAbs is shown as maximum percent inhibition (MPI). *<i>p</i><0.05, One-way ANOVA with Sidak’s multiple-comparison correction. (<b>g</b>) Neutralization of heterologous Tier-2 PV, ns = not significant, *<i>p</i><0.05, Kruskal-Wallis test with Dunn’s multiple-comparison correction.</p

Murine antibody and CD4 Th cell responses to GLA-SOSIP trimer.

<p>(<b>a</b>) Endpoint titers of week 8 sera from SOSIP or GLA-SOSIP trimer-immunized mice. Datum points represent individual mouse sera (n = 10) from two independent experiments, and bars represent median ± SD. X-axis represents ELISA coating antigen. ***<i>p</i><0.001 Mann Whitney U. (<b>b</b>) CD4 T cells negatively isolated from splenocytes at week 8 post-prime were re-stimulated in vitro with SOSIP or GLA-SOSIP trimer and assayed for proliferation, IFN-γ or IL-4 production. Data represent readouts from pooled Th cells from each mouse group merged from two independent experiments, bars represent median ± SD. (<b>c</b>) CD4 T cells negatively isolated from splenocytes at week 8 post-prime were pooled and re-stimulated in vitro using an overlapping 15-mer BG505 peptide library (peptides 1–165), and assayed for Th cell proliferation (top panel), IFN-γ production (middle panel) and IL-4 production (lower panel). Data are averaged from two independent experiments. Horizontal bar represents secondary structure regions of BG505. Gold bars represent regions in which cross-links were identified in the GLA-SOSIP trimer structure, and dotted black lines represent assay cutoff. Supporting data in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006986#ppat.1006986.s009" target="_blank">S7</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006986#ppat.1006986.s010" target="_blank">S8</a> Figs.</p

Unobserved potential cross-linking targets in GLA-SOSIP trimer.

<p>Unobserved potential cross-linking targets in GLA-SOSIP trimer.</p

Visualization of cross-links in GLA-trimer.

<p>(<b>a</b>) Top view of the GLA-SOSIP trimer-PGV04 Fab complex with lysines (K)169 and arginines (R)166 displayed in green. Extra density corresponding to GLA crosslinks between K and R side chains is colored in yellow. (<b>b</b>) Distances between K169 and R166 side chains in the GLA-SOSIP trimer. (<b>c</b>) Distances between K169 and R166 side chains in the PGT145-bound SOSIP trimer structure (PDB ID 5V8L), with the PGT145 CDRH3 contact region shown in pink. (<b>d</b>) Tilted side view of GLA-SOSIP trimer revealing extra density (yellow) corresponding to an inter-gp120 protomer cross-link between K155 and R178. (<b>e</b>) Tilted side view of GLA-SOSIP trimer revealing cross-linking (yellow) between gp120 residues K227, K229 and K485, proximal to the BG505 N241 and N289 glycan holes. (<b>f</b>) Tilted side view of GLA-SOSIP trimer revealing extra density (yellow) corresponding to a cross-link between K490 (gp120) and R585 (gp41).</p

Cryo-EM model of GLA-SOSIP trimer at 4.2 Å.

<p>(<b>a</b>) Cryo-EM 3D reconstruction at 4.2 Å resolution of GLA-SOSIP trimer complexed with 3 PGV04 Fabs showing top, side and bottom views. Cryo-EM data analysis parameters are presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006986#ppat.1006986.s004" target="_blank">S2 Table</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006986#ppat.1006986.s006" target="_blank">S4 Fig</a>. (<b>b</b>) Superposition of SOSIP (PDB ID 5CEZ) and GLA-SOSIP trimer single gp120-gp41 protomers where the unmodified SOSIP structure is in grey and the GLA-SOSIP structure is in blue (gp120) and orange (gp41). The differential Cα RMSD for the gp120-gp41 protomer is 1.48 Å.</p