Assessing the medical reasoning skills of GPT-4 in complex ophthalmology cases.

BACKGROUND/AIMS: This study assesses the proficiency of Generative Pre-trained Transformer (GPT)-4 in answering questions about complex clinical ophthalmology cases. METHODS: We tested GPT-4 on 422 Journal of the American Medical Association Ophthalmology Clinical Challenges, and prompted the model to determine the diagnosis (open-ended question) and identify the next-step (multiple-choice question). We generated responses using two zero-shot prompting strategies, including zero-shot plan-and-solve+ (PS+), to improve the reasoning of the model. We compared the best-performing model to human graders in a benchmarking effort. RESULTS: Using PS+ prompting, GPT-4 achieved mean accuracies of 48.0% (95% CI (43.1% to 52.9%)) and 63.0% (95% CI (58.2% to 67.6%)) in diagnosis and next step, respectively. Next-step accuracy did not significantly differ by subspecialty (p=0.44). However, diagnostic accuracy in pathology and tumours was significantly higher than in uveitis (p=0.027). When the diagnosis was accurate, 75.2% (95% CI (68.6% to 80.9%)) of the next steps were correct. Conversely, when the diagnosis was incorrect, 50.2% (95% CI (43.8% to 56.6%)) of the next steps were accurate. The next step was three times more likely to be accurate when the initial diagnosis was correct (p<0.001). No significant differences were observed in diagnostic accuracy and decision-making between board-certified ophthalmologists and GPT-4. Among trainees, senior residents outperformed GPT-4 in diagnostic accuracy (p≤0.001 and 0.049) and in accuracy of next step (p=0.002 and 0.020). CONCLUSION: Improved prompting enhances GPT-4's performance in complex clinical situations, although it does not surpass ophthalmology trainees in our context. Specialised large language models hold promise for future assistance in medical decision-making and diagnosis.

Yellow fever vaccine-associated neurologic and viscerotropic disease: a 10-year case series of the French National Reference Center for Arboviruses with clinical and immunological insights.

BACKGROUND: Immunization against the Yellow fever virus (YFV) with the 17D live-attenuated vaccine is the most effective way to prevent the disease. However, unexpected severe adverse events can occur. They consist in a neurological impairment - neurological disease (YEL-AND), a YF-like illness - viscerotropic disease (YEL-AVD) or anaphylaxis. In this article, we describe the epidemiology, clinical and biological features of YEL-AND and YEL-AVD cases reported to the French National Reference Center for Arboviruses (NRCA) in the past 10 years. METHODS: We conducted a national, retrospective study using the database of the NRCA from June 2012 to June 2022. All patients whose biological samples were sent to the NRCA for detection of YFV by serology and/or RT-qPCR for a suspected vaccine-associated adverse event were included. We collected data by reading medical records and conducted complementary neuro-immunological analysis, followed by a search for autoimmunity against type-1-interferon when samples were available at the NRCA. RESULTS: There were 10 cases of YEL-AND and 2 cases of YEL-AVD reported to the NRCA in the past 10 years, which represented an overall incidence of 0.6 for 100 000 doses. A total of 6/12 cases were previously healthy patients (50%, mean age 31 years), and 4/12 cases had cardiovascular co-morbidities (42%, mean age 56 years). The majority of YEL-AND had a favourable outcome at 6 months of follow up. One YEL-AVD patient passed. In secondary analyses, we evidenced a significant blood cerebrospinal fluid (CSF) barrier dysfunction, without intrathecal synthesis of immunoglobulin and without argument for a neuron damage. We further detected a significant rate of anti-type-1alpha interferon antibodies in 3/10 tested patients (2 YEL-AND and 1 YEL-AVD). CONCLUSION: YEL-AND and YEL-AVD are rare events that can underlie defect in the innate immunity of apparently healthy or mild co-morbid subjects. Outcome was generally favourable in the YEL-AND cases of our series, but still life-threatening or even fatal in the YEL-AVD cases.

Proximal Molecular Probe Transfer (PROMPT), a new approach for identifying sites of protein/nucleic acid interaction in cells by correlated light and electron microscopy.

The binding and interaction of proteins with nucleic acids such as DNA and RNA constitutes a fundamental biochemical and biophysical process in all living organisms. Identifying and visualizing such temporal interactions in cells is key to understanding their function. To image sites of these events in cells across scales, we developed a method, named PROMPT for PROximal Molecular Probe Transfer, which is applicable to both light and correlative electron microscopy. This method relies on the transfer of a bound photosensitizer from a protein known to associate with specific nucleic acid sequence, allowing the marking of the binding site on DNA or RNA in fixed cells. The method produces a fluorescent mark at the site of their interaction, that can be made electron dense and reimaged at high resolution in the electron microscope. As proof of principle, we labeled in situ the interaction sites between the histone H2B and nuclear DNA. As an example of application for specific RNA localizations we labeled different nuclear and nucleolar fractions of the protein Fibrillarin to mark and locate where it associates with RNAs, also using electron tomography. While the current PROMPT method is designed for microscopy, with minimal variations, it can be potentially expanded to analytical techniques.

Proximal Molecular Probe Transfer (PROMPT), a new approach for identifying sites of protein/nucleic acid interaction in cells by correlated light and electron microscopy.

The binding and interaction of proteins with nucleic acids such as DNA and RNA constitutes a fundamental biochemical and biophysical process in all living organisms. Identifying and visualizing such temporal interactions in cells is key to understanding their function. To image sites of these events in cells across scales, we developed a method, named PROMPT for PROximal Molecular Probe Transfer, which is applicable to both light and correlative electron microscopy. This method relies on the transfer of a bound photosensitizer from a protein known to associate with specific nucleic acid sequence, allowing the marking of the binding site on DNA or RNA in fixed cells. The method produces a fluorescent mark at the site of their interaction, that can be made electron dense and reimaged at high resolution in the electron microscope. As proof of principle, we labeled in situ the interaction sites between the histone H2B and nuclear DNA. As an example of application for specific RNA localizations we labeled different nuclear and nucleolar fractions of the protein Fibrillarin to mark and locate where it associates with RNAs, also using electron tomography. While the current PROMPT method is designed for microscopy, with minimal variations, it can be potentially expanded to analytical techniques.

Adjusting the Fitting of Fluorescence-Based Dose-Response Kinase Inhibition Assay to Account for Fluorescent Inhibitors.

Fluorescence is routinely used to monitor kinase inhibition in commercial assays. Occasionally fluorescent compounds can interfere with the fluorescent reading. To address this issue, the problematic data are usually truncated to improve the fit, however, this approach raises ethical and reproducibility concerns. Instead, it is suggested to adjust the fitting formula, to account for the autofluorescence of the compounds and improve the fit of the data compared with a naive approach. Finally, it was noticed that truncating the data can result in a small underestimation of the IC50 values and should therefore be used carefully.

Fe-TAMLs as a new class of small molecule peroxidase probes for correlated light and electron microscopy.

We introduce Fe-TAML, a small molecule-based peroxidase as a versatile new member of the correlated fluorescence and electron microscopy toolkit. The utility of the probe is demonstrated by high resolution imaging of newly synthesized DNA (through biorthogonal labeling), genetically tagged proteins (using HaloTag), and untagged endogenous proteins (via immunostaining). EM visualization in these applications is facilitated by exploiting Fe-TAML's catalytic activity for the deposition of localized osmiophilic precipitates based on polymerized 3,3'-diaminobenzidine. Optimized conditions for synthesizing and implementing Fe-TAML based probes are also described. Overall, Fe-TAML is a new chemical biology tool that can be used to visualize diverse biomolecular species along nanometer and micron scales within cells.

An Updated Guide to Robust Statistical Methods in Neuroscience.

There is a vast array of new and improved methods for comparing groups and studying associations that offer the potential for substantially increasing power, providing improved control over the probability of false positives, and yielding a deeper and more nuanced understanding of data. These new techniques effectively deal with four insights into when and why conventional methods can be unsatisfactory. But for the non-statistician, this vast array of techniques for comparing groups and studying associations can seem daunting. This article briefly reviews when and why conventional methods can have relatively low power and yield misleading results. The main goal is to suggest guidelines regarding the use of modern techniques that improve upon classic approaches such as Pearson's correlation, ordinary linear regression, ANOVA, and ANCOVA. This updated version includes recent advances dealing with effect sizes, including situations where there is a covariate. The R code, figures, and accompanying notebooks have been updated as well. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.

Rating norms should be calculated from cumulative link mixed effects models.

Studies which provide norms of Likert ratings typically report per-item summary statistics. Traditionally, these summary statistics comprise the mean and the standard deviation (SD) of the ratings, and the number of observations. Such summary statistics can preserve the rank order of items, but provide distorted estimates of the relative distances between items because of the ordinal nature of Likert ratings. Inter-item relations in such ordinal scales can be more appropriately modelled by cumulative link mixed effects models (CLMMs). In a series of simulations, and with a reanalysis of an existing rating norms dataset, we show that CLMMs can be used to more accurately norm items, and can provide summary statistics analogous to the traditionally reported means and SDs, but which are disentangled from participants' response biases. CLMMs can be applied to solve important statistical issues that exist for more traditional analyses of rating norms.

Prognostic and therapeutic interest of a new classification in inferior hip dislocation: a systematic review of the literature.

INTRODUCTION: Reported cases of inferior dislocation in the literature are found under several names (inferior, anteroinferior, obturator, or erecta), which may be source of confusion. The purpose of this comprehensive review of the literature is to collect as many cases of inferior dislocation as possible to determine better therapeutic strategies, outcome after reduction, complications, and prognostic factors. METHODS: In April 2020, a literature search was performed in Pubmed, Medline, Scopus, Cochrane, and Embase databases. The MeSH keywords were "OBTURATOR DISLOCATION HIP" or "ANTERIOR DISLOCATION HIP" or "INFERIOR DISLOCATION HIP." Authors independently selected articles that met the selection criteria, with no time limit. RESULTS: Out of the 97 articles selected, there were 119 cases of primary inferior hip dislocations. This review of the literature has allowed us to differentiate 3 radiographic subtypes of inferior dislocations, which correspond to 3 different anatomical positions of the femoral head: "obturator" dislocation, "proximal anterior-inferior" dislocation, and "distal anterior-inferior" dislocation. Our subtype classification yielded 39 obturator subtype inferior dislocations (32.8%), 66 proximal anteroinferior subtypes (55.4%), and 14 distal anteroinferior (11.8%). The obturator subtype is at risk of reduction failure and femoral neck fracture during the reduction manoeuver. CONCLUSIONS: Our study identified 3 subtypes with different prognosis, with obturator and distal anteroinferior dislocations having a poorer prognosis because of their pre- and post-reduction complications. We were unable to determine the correct manoeuver to reduce inferior dislocations without taking the risk of femoral neck fracture, but each of these subtypes may require a different manoeuver.

Identification of New Purpuroine Analogues from the Arctic Echinodermata Pteraster militaris That Inhibit FLT3-ITD+ AML Cell Lines.

Isolation of bioactive products from the marine environment is considered a very promising approach to identify new compounds that can be used for further drug development. In this work we have isolated three new compounds from the purpuroine family by mass-guided preparative HPLC; purpuroine K-M. These compounds where screened for antibacterial- and antifungal activity, antibiofilm formation and anti-cell proliferation activity. Additionally, apoptosis-, cell cycle-, kinase binding- and docking studies were performed to evaluate the mechanism-of-action. None of the compounds showed activity in antibacterial-, antibiofilm- or antifungal assays. However, one of the isolated compounds, purpuroine K, showed activity against two cell lines, MV-4-11 and MOLM-13, two AML cell lines both carrying the FTL3-ITD mutation. In MV-4-11 cells, purpuroine K was found to increase apoptosis and arrest cells cycle in G1/G0, which is a common feature of FLT3 inhibitors. Interactions between purpuroine K and the FLT3 wild type or FLT3 ITD mutant proteins could however not be elucidated in our kinase binding and docking studies. In conclusion, we have isolated three novel molecules, purpuroine K-M, one of which (purpuroine K) shows a potent activity against FLT3-ITD mutated AML cell lines, however, the molecular target(s) of purpuroine K still need to be further investigated.

Prevalence of Obesity and Anemia Among Early Adolescents in Junior Secondary Schools: A Cross-Sectional Study in Ouagadougou, Burkina Faso.

BACKGROUND: The school presents an ideal environment to positively impact the long-term health and nutrition outcomes of early adolescents, who are at risk of obesity and anemia. METHODS: In this cross-sectional survey, we described differences in weight and anemia by sociodemographic, diet and physical activity indicators among 1059 students aged 11 to 15 years from 22 junior secondary schools in Ouagadougou, Burkina Faso. Weight was based on body mass index (BMI) z-scores according to the WHO reference and anemia status was defined by standardized hemoglobin (Hb) measure cut-offs. We calculated dietary diversity scores (DDS) from a 24-hour dietary recall and a global diet quality score (GDQS) from a 7-day dietary recall. RESULTS: The prevalence of obesity (5%) and anemia (50%) was relatively high among the students, which differed significantly between gender, household wealth and school grade, but not age groups. Eighteen percent of the female adolescents were overweight or obese and 22% were moderately anemic compared to 13% and 16% of the male adolescents. Dietary diversity was significantly different between weight categories, but not anemia status. For physical activity, those taking transportation to school were significantly more likely to be overweight or obese. In adjusted multivariable Poisson regression analyses, only the DDS was significantly associated with thinness and both thinness and anemia, while taking transportation to school was significantly associated with overweight among adolescents. CONCLUSION: We encourage the promotion of school-based interventions and provision of a curriculum on health and healthy eating in order to reduce obesity, anemia, and its comorbidities.

Defining the ultrastructure of the hematopoietic stem cell niche by correlative light and electron microscopy.

The blood system is supported by hematopoietic stem and progenitor cells (HSPCs) found in a specialized microenvironment called the niche. Many different niche cell types support HSPCs, however how they interact and their ultrastructure has been difficult to define. Here, we show that single endogenous HSPCs can be tracked by light microscopy, then identified by serial block-face scanning electron microscopy (SBEM) at multiscale levels. Using the zebrafish larval kidney marrow (KM) niche as a model, we followed single fluorescently labeled HSPCs by light sheet microscopy, then confirmed their exact location in a 3D SBEM dataset. We found a variety of different configurations of HSPCs and surrounding niche cells, suggesting there could be functional heterogeneity in sites of HSPC lodgement. Our approach also allowed us to identify dopamine beta-hydroxylase (dbh) positive ganglion cells as a previously uncharacterized functional cell type in the HSPC niche. By integrating multiple imaging modalities, we could resolve the ultrastructure of single rare cells deep in live tissue and define all contacts between an HSPC and its surrounding niche cell types.

Metabolite Damage and Damage Control in a Minimal Genome.

Analysis of the genes retained in the minimized Mycoplasma JCVI-Syn3A genome established that systems that repair or preempt metabolite damage are essential to life. Several genes known to have such functions were identified and experimentally validated, including 5-formyltetrahydrofolate cycloligase, coenzyme A (CoA) disulfide reductase, and certain hydrolases. Furthermore, we discovered that an enigmatic YqeK hydrolase domain fused to NadD has a novel proofreading function in NAD synthesis and could double as a MutT-like sanitizing enzyme for the nucleotide pool. Finally, we combined metabolomics and cheminformatics approaches to extend the core metabolic map of JCVI-Syn3A to include promiscuous enzymatic reactions and spontaneous side reactions. This extension revealed that several key metabolite damage control systems remain to be identified in JCVI-Syn3A, such as that for methylglyoxal. IMPORTANCE Metabolite damage and repair mechanisms are being increasingly recognized. We present here compelling genetic and biochemical evidence for the universal importance of these mechanisms by demonstrating that stripping a genome down to its barest essentials leaves metabolite damage control systems in place. Furthermore, our metabolomic and cheminformatic results point to the existence of a network of metabolite damage and damage control reactions that extends far beyond the corners of it that have been characterized so far. In sum, there can be little room left to doubt that metabolite damage and the systems that counter it are mainstream metabolic processes that cannot be separated from life itself.

The suppressor of copper sensitivity protein C from Caulobacter crescentus is a trimeric disulfide isomerase that binds copper(I) with subpicomolar affinity.

The introduction of disulfide bonds into periplasmic proteins is a critical process in many Gram-negative bacteria. The formation and regulation of protein disulfide bonds have been linked to the production of virulence factors. Understanding the different pathways involved in this process is important in the development of strategies to disarm pathogenic bacteria. The well characterized disulfide bond-forming (DSB) proteins play a key role by introducing or isomerizing disulfide bonds between cysteines in substrate proteins. Curiously, the suppressor of copper sensitivity C proteins (ScsCs), which are part of the bacterial copper-resistance response, share structural and functional similarities with DSB oxidase and isomerase proteins, including the presence of a catalytic thioredoxin domain. However, the oxidoreductase activity of ScsC varies with its oligomerization state, which depends on a poorly conserved N-terminal domain. Here, the structure and function of Caulobacter crescentus ScsC (CcScsC) have been characterized. It is shown that CcScsC binds copper in the copper(I) form with subpicomolar affinity and that its isomerase activity is comparable to that of Escherichia coli DsbC, the prototypical dimeric bacterial isomerase. It is also reported that CcScsC functionally complements trimeric Proteus mirabilis ScsC (PmScsC) in vivo, enabling the swarming of P. mirabilis in the presence of copper. Using mass photometry and small-angle X-ray scattering (SAXS) the protein is demonstrated to be trimeric in solution, like PmScsC, and not dimeric like EcDsbC. The crystal structure of CcScsC was also determined at a resolution of 2.6 Å, confirming the trimeric state and indicating that the trimerization results from interactions between the N-terminal α-helical domains of three CcScsC protomers. The SAXS data analysis suggested that the protomers are dynamic, like those of PmScsC, and are able to sample different conformations in solution.

Expression, purification and characterization of the suppressor of copper sensitivity (Scs) B membrane protein from Proteus mirabilis.

Suppressor of copper sensitivity (Scs) proteins play a role in the bacterial response to copper stress in many Gram-negative bacteria, including in the human pathogen Proteus mirabilis. Recently, the ScsC protein from P. mirabilis (PmScsC) was characterized as a trimeric protein with isomerase activity that contributes to the ability of the bacterium to swarm in the presence of copper. The CXXC motif catalytic cysteines of PmScsC are maintained in their active reduced state by the action of its membrane-bound partner protein, the Proteus mirabilis ScsB (PmScsB). Thus, PmScsC and PmScsB form a redox relay in vivo. The predicted domain arrangement of PmScsB comprises a central transmembrane ß-domain and two soluble, periplasmic domains, the N-terminal α-domain and C-terminal γ-domain. Here, we provide a procedure for the recombinant expression and purification of the full-length PmScsB protein. Using Lemo21 (DE3) cells we expressed PmScsB and, after extraction and purification, we were able to achieve a yield of 3 mg of purified protein per 8 L of bacterial culture. Furthermore, using two orthogonal methods - AMS labelling of free thiols and a scrambled RNase A activity assay - PmScsB is shown to catalyze the reduction of PmScsC. Our results demonstrate that the PmScsC and PmScsB redox relay can be reconstituted in vitro using recombinant full-length PmScsB membrane protein. This finding provides a promising starting point for the in vitro biochemical and structural characterization of the P. mirabilis ScsC and ScsB interaction.

Identification and characterization of two drug-like fragments that bind to the same cryptic binding pocket of Burkholderia pseudomallei DsbA.

Disulfide-bond-forming proteins (Dsbs) play a crucial role in the pathogenicity of many Gram-negative bacteria. Disulfide-bond-forming protein A (DsbA) catalyzes the formation of the disulfide bonds necessary for the activity and stability of multiple substrate proteins, including many virulence factors. Hence, DsbA is an attractive target for the development of new drugs to combat bacterial infections. Here, two fragments, bromophenoxy propanamide (1) and 4-methoxy-N-phenylbenzenesulfonamide (2), were identified that bind to DsbA from the pathogenic bacterium Burkholderia pseudomallei, the causative agent of melioidosis. The crystal structures of oxidized B. pseudomallei DsbA (termed BpsDsbA) co-crystallized with 1 or 2 show that both fragments bind to a hydrophobic pocket that is formed by a change in the side-chain orientation of Tyr110. This conformational change opens a `cryptic' pocket that is not evident in the apoprotein structure. This binding location was supported by 2D-NMR studies, which identified a chemical shift perturbation of the Tyr110 backbone amide resonance of more than 0.05 p.p.m. upon the addition of 2 mM fragment 1 and of more than 0.04 p.p.m. upon the addition of 1 mM fragment 2. Although binding was detected by both X-ray crystallography and NMR, the binding affinity (Kd) for both fragments was low (above 2 mM), suggesting weak interactions with BpsDsbA. This conclusion is also supported by the crystal structure models, which ascribe partial occupancy to the ligands in the cryptic binding pocket. Small fragments such as 1 and 2 are not expected to have a high energetic binding affinity due to their relatively small surface area and the few functional groups that are available for intermolecular interactions. However, their simplicity makes them ideal for functionalization and optimization. The identification of the binding sites of 1 and 2 to BpsDsbA could provide a starting point for the development of more potent novel antimicrobial compounds that target DsbA and bacterial virulence.