Structure of the MlaC-MlaD complex reveals molecular basis of periplasmic phospholipid transport.

The Maintenance of Lipid Asymmetry (Mla) pathway is a multicomponent system found in all gram-negative bacteria that contributes to virulence, vesicle blebbing and preservation of the outer membrane barrier function. It acts by removing ectopic lipids from the outer leaflet of the outer membrane and returning them to the inner membrane through three proteinaceous assemblies: the MlaA-OmpC complex, situated within the outer membrane; the periplasmic phospholipid shuttle protein, MlaC; and the inner membrane ABC transporter complex, MlaFEDB, proposed to be the founding member of a structurally distinct ABC superfamily. While the function of each component is well established, how phospholipids are exchanged between components remains unknown. This stands as a major roadblock in our understanding of the function of the pathway, and in particular, the role of ATPase activity of MlaFEDB is not clear. Here, we report the structure of E. coli MlaC in complex with the MlaD hexamer in two distinct stoichiometries. Utilising in vivo complementation assays, an in vitro fluorescence-based transport assay, and molecular dynamics simulations, we confirm key residues, identifying the MlaD ß6-ß7 loop as essential for MlaCD function. We also provide evidence that phospholipids pass between the C-terminal helices of the MlaD hexamer to reach the central pore, providing insight into the trajectory of GPL transfer between MlaC and MlaD.

Serotonin reuptake inhibitors improve muscle stem cell function and muscle regeneration in male mice.

Serotonin reuptake inhibitor antidepressants such as fluoxetine are widely used to treat mood disorders. The mechanisms of action include an increase in extracellular level of serotonin, neurogenesis, and growth of vessels in the brain. We investigated whether fluoxetine could have broader peripheral regenerative properties. Following prolonged administration of fluoxetine in male mice, we showed that fluoxetine increases the number of muscle stem cells and muscle angiogenesis, associated with positive changes in skeletal muscle function. Fluoxetine also improved skeletal muscle regeneration after single and multiples injuries with an increased muscle stem cells pool and vessel density associated with reduced fibrotic lesions and inflammation. Mice devoid of peripheral serotonin treated with fluoxetine did not exhibit beneficial effects during muscle regeneration. Specifically, pharmacological, and genetic inactivation of the 5-HT1B subtype serotonin receptor also abolished the enhanced regenerative process induced by fluoxetine. We highlight here a regenerative property of serotonin on skeletal muscle.

Distance tuneable integral membrane protein containing floating bilayers via in situ directed self-assembly.

Model membranes allow for structural and biophysical studies on membrane biochemistry at the molecular level, albeit on systems of reduced complexity which can limit biological accuracy. Floating supported bilayers offer a means of producing planar lipid membrane models not adhered to a surface, which allows for improved accuracy compared to other model membranes. Here we communicate the incorporation of an integral membrane protein complex, the multidomain ß-barrel assembly machinery (Bam), into our recently developed in situ self-assembled floating supported bilayers. Using neutron reflectometry and quartz crystal microbalance measurements we show this sample system can be fabricated using a two-step self-assembly process. We then demonstrate the complexity of the model membrane and tuneability of the membrane-to-surface distance using changes in the salt concentration of the bulk solution. Results demonstrate an easily fabricated, biologically accurate and tuneable membrane assay system which can be utilized for studies on integral membrane proteins within their native lipid matrix.

A measles-vectored vaccine candidate expressing prefusion-stabilized SARS-CoV-2 spike protein brought to phase I/II clinical trials: candidate selection in a preclinical murine model.

In the early COVID-19 pandemic with urgent need for countermeasures, we aimed at developing a replicating viral vaccine using the highly efficacious measles vaccine as vector, a promising technology with prior clinical proof of concept. Building on our successful pre-clinical development of a measles virus (MV)-based vaccine candidate against the related SARS-CoV, we evaluated several recombinant MV expressing codon-optimized SARS-CoV-2 spike glycoprotein. Candidate V591 expressing a prefusion-stabilized spike through introduction of two proline residues in HR1 hinge loop, together with deleted S1/S2 furin cleavage site and additional inactivation of the endoplasmic reticulum retrieval signal, was the most potent in eliciting neutralizing antibodies in mice. After single immunization, V591 induced similar neutralization titers as observed in sera of convalescent patients. The cellular immune response was confirmed to be Th1 skewed. V591 conferred long-lasting protection against SARS-CoV-2 challenge in a murine model with marked decrease in viral RNA load, absence of detectable infectious virus loads, and reduced lesions in the lungs. V591 was furthermore efficacious in an established non-human primate model of disease (see companion article [S. Nambulli, N. Escriou, L. J. Rennick, M. J. Demers, N. L. Tilston-Lunel et al., J Virol 98:e01762-23, 2024, https://doi.org/10.1128/jvi.01762-23]). Thus, V591 was taken forward into phase I/II clinical trials in August 2020. Unexpected low immunogenicity in humans (O. Launay, C. Artaud, M. Lachâtre, M. Ait-Ahmed, J. Klein et al., eBioMedicine 75:103810, 2022, https://doi.org/10.1016/j.ebiom.2021.103810) revealed that the underlying mechanisms for resistance or sensitivity to pre-existing anti-measles immunity are not yet understood. Different hypotheses are discussed here, which will be important to investigate for further development of the measles-vectored vaccine platform.IMPORTANCESARS-CoV-2 emerged at the end of 2019 and rapidly spread worldwide causing the COVID-19 pandemic that urgently called for vaccines. We developed a vaccine candidate using the highly efficacious measles vaccine as vector, a technology which has proved highly promising in clinical trials for other pathogens. We report here and in the companion article by Nambulli et al. (J Virol 98:e01762-23, 2024, https://doi.org/10.1128/jvi.01762-23) the design, selection, and preclinical efficacy of the V591 vaccine candidate that was moved into clinical development in August 2020, 7 months after the identification of SARS-CoV-2 in Wuhan. These unique in-human trials of a measles vector-based COVID-19 vaccine revealed insufficient immunogenicity, which may be the consequence of previous exposure to the pediatric measles vaccine. The three studies together in mice, primates, and humans provide a unique insight into the measles-vectored vaccine platform, raising potential limitations of surrogate preclinical models and calling for further refinement of the platform.

A host-directed oxadiazole compound potentiates antituberculosis treatment via zinc poisoning in human macrophages and in a mouse model of infection.

Antituberculosis drugs, mostly developed over 60 years ago, combined with a poorly effective vaccine, have failed to eradicate tuberculosis. More worryingly, multiresistant strains of Mycobacterium tuberculosis (MTB) are constantly emerging. Innovative strategies are thus urgently needed to improve tuberculosis treatment. Recently, host-directed therapy has emerged as a promising strategy to be used in adjunct with existing or future antibiotics, by improving innate immunity or limiting immunopathology. Here, using high-content imaging, we identified novel 1,2,4-oxadiazole-based compounds, which allow human macrophages to control MTB replication. Genome-wide gene expression analysis revealed that these molecules induced zinc remobilization inside cells, resulting in bacterial zinc intoxication. More importantly, we also demonstrated that, upon treatment with these novel compounds, MTB became even more sensitive to antituberculosis drugs, in vitro and in vivo, in a mouse model of tuberculosis. Manipulation of heavy metal homeostasis holds thus great promise to be exploited to develop host-directed therapeutic interventions.

Study protocol for a cross-sectional online survey investigating patient preferences and experiences of waiting for elective cardiac surgery.

INTRODUCTION: Being on a waiting list for elective (planned) cardiac surgery can be physically and psychologically challenging for patients. Research suggests that stress associated with waiting for surgery is dependent on different individual and contextual factors. However, most data on patients' experiences of waiting for surgery and preferences for waiting list management derives from non-cardiac clinical populations. The aim of the current study is to explore patients' experiences of being on a waiting list for elective cardiac surgery, and their views on how the waiting experience could be improved in the future. This work will inform the patient management strategy during the waiting period for surgery across the four major hospitals in London directly involved in this study, and potentially beyond by transferring learning to other services. METHODS AND ANALYSIS: This is a mixed-methods study that will collect quantitative and qualitative data using a cross-sectional online survey. Patients who are on waiting lists for elective surgery across four major cardiac surgery departments in London hospitals, and are at least 18 years old, will be invited by their healthcare team via text message or letter to complete the survey. The target sample size of non-randomly selected participants will be 268. Bivariable and multivariable regression models will be used to assess associations between survey items measuring the impact of the cardiac condition on specific life domains (eg, daily activities, social and family relationships, hobbies, sexual life), anxiety and depression symptoms as measured by the Patient Health Questionnaire-4 and survey items evaluating experiences of health services. Data on experience and preferences for improvements to the waiting experience will be analysed with qualitative content analysis using an inductive approach. ETHICS AND DISSEMINATION: This study was reviewed and granted ethical approval by the East of England-East Cambridge Research Ethics Committee. Findings from this study will be disseminated through peer-reviewed journals, a research website and social media and with an online event engaging patients, members of the public, healthcare professionals and other relevant stakeholders. TRIAL REGISTRATION NUMB: NCT05996640.

An octameric PqiC toroid stabilises the outer-membrane interaction of the PqiABC transport system.

The E. coli Paraquat Inducible (Pqi) Pathway is a putative Gram-negative phospholipid transport system. The pathway comprises three components: an integral inner membrane protein (PqiA), a periplasmic spanning MCE family protein (PqiB) and an outer membrane lipoprotein (PqiC). Interactions between all complex components, including stoichiometry, remain uncharacterised; nevertheless, once assembled into their quaternary complex, the trio of Pqi proteins are anticipated to provide a continuous channel between the inner and outer membranes of diderms. Here, we present X-ray structures of both the native and a truncated, soluble construct of the PqiC lipoprotein, providing insight into its biological assembly, and utilise neutron reflectometry to characterise the nature of the PqiB-PqiC-membrane interaction. Finally, we employ phenotypic complementation assays to probe specific PqiC residues, which imply the interaction between PqiB and PqiC is less intimate than previously anticipated.

Retention of uninfected red blood cells causing congestive splenomegaly is the major mechanism of anemia in malaria.

Splenomegaly frequently occurs in patients with Plasmodium falciparum (Pf) or P. vivax (Pv) malarial anemia, but mechanisms underlying this co-occurrence are unclear. In malaria-endemic Papua, Indonesia, we prospectively analyzed red blood cell (RBC) concentrations in the spleen and spleen-mimetic retention in 37 subjects splenectomized for trauma or hyperreactive splenomegaly, most of whom were infected with Plasmodium. Splenomegaly (median 357 g [range: 80-1918 g]) was correlated positively with the proportion of red-pulp on histological sections (median 88.1% [range: 74%-99.4%]; r = .59, p = .0003) and correlated negatively with the proportion of white-pulp (median 8.3% [range: 0.4%-22.9%]; r = -.50, p = .002). The number of RBC per microscopic field (>95% uninfected) was correlated positively with spleen weight in both Pf-infected (r = .73; p = .017) and Pv-infected spleens (r = .94; p = .006). The median estimated proportion of total-body RBCs retained in Pf-infected spleens was 8.2% (range: 1.0%-33.6%), significantly higher than in Pv-infected (2.6% [range: 0.6%-23.8%]; p = .015) and PCR-negative subjects (2.5% [range: 1.0%-3.3%]; p = .006). Retained RBCs accounted for over half of circulating RBC loss seen in Pf infections. The proportion of total-body RBC retained in Pf- and Pv-infected spleens correlated negatively with hemoglobin concentrations (r = -.56, p = .0003), hematocrit (r = -.58, p = .0002), and circulating RBC counts (r = -.56, p = .0003). Splenic CD71-positive reticulocyte concentrations correlated with spleen weight in Pf (r = 1.0; p = .003). Retention rates of peripheral and splenic RBCs were correlated negatively with circulating RBC counts (r = -.69, p = .07 and r = -.83, p = .008, respectively). In conclusion, retention of mostly uninfected RBC in the spleen, leading to marked congestion of the red-pulp, was associated with splenomegaly and is the major mechanism of anemia in subjects infected with Plasmodium, particularly Pf.

Quantum Chemical Insight into 1,2-Shift Rearrangement in Bromination of Allylaryls.

In this work, we have provided mechanistic insight into the addition of bromine to an allylic double bond of allylaryl derivatives using experimental and DFT-based electronic structure methods. The experimental yields indicate the influence of the functional group on the aryl ring on the ratio of 1,2-dibromo and 1,3-dibromo adducts formed in the reaction. The optimized geometry and the electron density maps of the allylaryls and their cationic intermediates from DFT simulations revealed that electron-rich aryl rings promoted formation of cationic spiro[2.5] intermediate II, whereas electron-poor aryl rings resulted in formation of bromonium intermediate I. It was observed that electron-rich allylaryls promoted the 1,2-shift of the aryl ring that resulted in bond formation between the carbon atom (C1) on the aryl ring and the central carbon atom (C3) in the allylic double bond and formed spiro[2.5] intermediate II, a trend which was confirmed by harmonic oscillator model of aromaticity index. Also, Wiberg bond order analysis is in good agreement with the experimental work. Thermochemical analysis indicates that smaller C1···C3 distance resulted in favorable values for the difference in free energy change (ΔΔG). The favorable ΔΔG values are a result of higher electron density on the aryl ring, making it more nucleophilic toward C3 carbon and promoting 1,2-shift that led to formation of the spiro[2.5] intermediate. Thus, the underlying mechanism indicates that the electron-rich allylaryls promote the formation of 1,3-dibromo compounds through formation and stabilization of the spiro[2.5] intermediate II.

Revealing invisible cell phenotypes with conditional generative modeling.

Biological sciences, drug discovery and medicine rely heavily on cell phenotype perturbation and microscope observation. However, most cellular phenotypic changes are subtle and thus hidden from us by natural cell variability: two cells in the same condition already look different. In this study, we show that conditional generative models can be used to transform an image of cells from any one condition to another, thus canceling cell variability. We visually and quantitatively validate that the principle of synthetic cell perturbation works on discernible cases. We then illustrate its effectiveness in displaying otherwise invisible cell phenotypes triggered by blood cells under parasite infection, or by the presence of a disease-causing pathological mutation in differentiated neurons derived from iPSCs, or by low concentration drug treatments. The proposed approach, easy to use and robust, opens the door to more accessible discovery of biological and disease biomarkers.

Full eradication of pre-clinical human papilloma virus-induced tumors by a lentiviral vaccine.

Human papillomavirus (HPV) infections are the cause of all cervical and numerous oropharyngeal and anogenital cancers. The currently available HPV vaccines, which induce neutralizing antibodies, have no therapeutic effect on established tumors. Here, we developed an immuno-oncotherapy against HPV-induced tumors based on a non-integrative lentiviral vector encoding detoxified forms of the Early E6 and E7 oncoproteins of HPV16 and 18 genotypes, namely, "Lenti-HPV-07". A single intramuscular injection of Lenti-HPV-07 into mice bearing established HPV-induced tumors resulted in complete tumor eradication in 100% of the animals and was also effective against lung metastases. This effect correlated with CD8+ T-cell induction and profound remodeling of the tumor microenvironment. In the intra-tumoral infiltrates of vaccinated mice, the presence of large amounts of activated effector, resident memory, and transcription factor T cell factor-1 (TCF-1)+ "stem-like" CD8+ T cells was associated with full tumor eradication. The Lenti-HPV-07-induced immunity was long-lasting and prevented tumor growth after a late re-challenge, mimicking tumor relapse. Lenti-HPV-07 therapy synergizes with an anti-checkpoint inhibitory treatment and therefore shows promise as an immuno-oncotherapy against established HPV-mediated malignancies.

Prevalence of blood and skin trypanosomes in domestic and wild fauna from two sleeping sickness foci in Southern Cameroon.

Although studies on African Trypanosomiases revealed a variety of trypanosome species in the blood of various animal taxa, animal reservoirs of Trypanosoma brucei gambiense and anatomical niches such as skin have been overlooked in most epidemiological settings. This study aims to update epidemiological data on trypanosome infections in animals from human African trypanosomiasis (HAT) foci of Cameroon. Blood and skin snips were collected from 291 domestic and wild animals. DNA was extracted from blood and skin snips and molecular approaches were used to identify different trypanosomes species. Immunohistochemical analyses were used to confirm trypanosome infections in skin snips. PCR revealed 137 animals (47.1%) with at least one trypanosome species in the blood and/or in the skin. Of these 137 animals, 90 (65.7%) and 32 (23.4%) had trypanosome infections respectively in the blood and skin. Fifteen (10.9%) animals had trypanosome infections in both blood and skin snip. Animals from the Campo HAT focus (55.0%) were significantly (X2 = 17.6; P< 0.0001) more infected than those (29.7%) from Bipindi. Trypanosomes of the subgenus Trypanozoon were present in 27.8% of animals while T. vivax, T. congolense forest type and savannah type were detected in 16.5%, 10.3% and 1.4% of animals respectively. Trypanosoma b. gambiense infections were detected in the blood of 7.6% (22/291) of animals. No T. b. gambiense infection was detected in skin. This study highlights the presence of several trypanosome species in the blood and skin of various wild and domestic animals. Skin appeared as an anatomical reservoir for trypanosomes in animals. Despite methodological limitations, pigs, sheep, goats and wild animals were confirmed as potential reservoirs of T. b. gambiense. These animal reservoirs must be considered for the designing of control strategies that will lead to sustainable elimination of HAT.

Neuroinvasion and anosmia are independent phenomena upon infection with SARS-CoV-2 and its variants.

Anosmia was identified as a hallmark of COVID-19 early in the pandemic, however, with the emergence of variants of concern, the clinical profile induced by SARS-CoV-2 infection has changed, with anosmia being less frequent. Here, we assessed the clinical, olfactory and neuroinflammatory conditions of golden hamsters infected with the original Wuhan SARS-CoV-2 strain, its isogenic ORF7-deletion mutant and three variants: Gamma, Delta, and Omicron/BA.1. We show that infected animals develop a variant-dependent clinical disease including anosmia, and that the ORF7 of SARS-CoV-2 contributes to the induction of olfactory dysfunction. Conversely, all SARS-CoV-2 variants are neuroinvasive, regardless of the clinical presentation they induce. Taken together, this confirms that neuroinvasion and anosmia are independent phenomena upon SARS-CoV-2 infection. Using newly generated nanoluciferase-expressing SARS-CoV-2, we validate the olfactory pathway as a major entry point into the brain in vivo and demonstrate in vitro that SARS-CoV-2 travels retrogradely and anterogradely along axons in microfluidic neuron-epithelial networks.

Mice humanized for MHC and hACE2 with high permissiveness to SARS-CoV-2 omicron replication.

Human Angiotensin-Converting Enzyme 2 (hACE2) is the major receptor enabling host cell invasion by SARS-CoV-2 via interaction with Spike. The murine ACE2 does not interact efficiently with SARS-CoV-2 Spike and therefore the laboratory mouse strains are not permissive to SARS-CoV-2 replication. Here, we generated new hACE2 transgenic mice, which harbor the hACE2 gene under the human keratin 18 promoter, in "HHD-DR1" background. HHD-DR1 mice are fully devoid of murine Major Histocompatibility Complex (MHC) molecules of class-I and -II and express only MHC molecules from Human Leukocyte Antigen (HLA) HLA 02.01, DRA01.01, DRB1.01.01 alleles, widely expressed in human populations. We selected three transgenic strains, with various hACE2 mRNA expression levels and distinctive profiles of lung and/or brain permissiveness to SARS-CoV-2 replication. These new hACE2 transgenic strains display high permissiveness to the replication of SARS-CoV-2 Omicron sub-variants, while the previously available B6.K18-ACE22Prlmn/JAX mice have been reported to be poorly susceptible to infection with Omicron. As a first application, one of these MHC- and ACE2-humanized strains was successfully used to show the efficacy of a lentiviral-based COVID-19 vaccine.

Anticancer Agents as Design Archetypes: Insights into the Structure-Property Relationships of Ionic Liquids with a Triarylmethyl Moiety.

A fundamental challenge underlying the design principles of ionic liquids (ILs) entails a lack of understanding into how tailored properties arise from the molecular framework of the constituent ions. Herein, we present detailed analyses of novel functional ILs containing a triarylmethyl (trityl) motif. Combining an empirically driven molecular design, thermophysical analysis, X-ray crystallography, and computational modeling, we achieved an in-depth understanding of structure-property relationships, establishing a coherent correlation with distinct trends between the thermophysical properties and functional diversity of the compound library. We observe a coherent relationship between melting (T m) and glass transition (T g) temperatures and the location and type of chemical modification of the cation. Furthermore, there is an inverse correlation between the simulated dipole moment and the T m/T g of the salts. Specifically, chlorination of the ILs both reduces and reorients the dipole moment, a key property controlling intermolecular interactions, thus allowing for control over T m/T g values. The observed trends are particularly apparent when comparing the phase transitions and dipole moments, allowing for the development of predictive models. Ultimately, trends in structural features and characterized properties align with established studies in physicochemical relationships for ILs, underpinning the formation and stability of these new lipophilic, low-melting salts.

#COVIDisAirborne: AI-enabled multiscale computational microscopy of delta SARS-CoV-2 in a respiratory aerosol.

We seek to completely revise current models of airborne transmission of respiratory viruses by providing never-before-seen atomic-level views of the SARS-CoV-2 virus within a respiratory aerosol. Our work dramatically extends the capabilities of multiscale computational microscopy to address the significant gaps that exist in current experimental methods, which are limited in their ability to interrogate aerosols at the atomic/molecular level and thus obscure our understanding of airborne transmission. We demonstrate how our integrated data-driven platform provides a new way of exploring the composition, structure, and dynamics of aerosols and aerosolized viruses, while driving simulation method development along several important axes. We present a series of initial scientific discoveries for the SARS-CoV-2 Delta variant, noting that the full scientific impact of this work has yet to be realized.

Application Experiences on a GPU-Accelerated Arm-based HPC Testbed.

This paper assesses and reports the experience of ten teams working to port, validate, and benchmark several High Performance Computing applications on a novel GPU-accelerated Arm testbed system. The testbed consists of eight NVIDIA Arm HPC Developer Kit systems, each one equipped with a server-class Arm CPU from Ampere Computing and two data center GPUs from NVIDIA Corp. The systems are connected together using InfiniBand interconnect. The selected applications and mini-apps are written using several programming languages and use multiple accelerator-based programming models for GPUs such as CUDA, OpenACC, and OpenMP offloading. Working on application porting requires a robust and easy-to-access programming environment, including a variety of compilers and optimized scientific libraries. The goal of this work is to evaluate platform readiness and assess the effort required from developers to deploy well-established scientific workloads on current and future generation Arm-based GPU-accelerated HPC systems. The reported case studies demonstrate that the current level of maturity and diversity of software and tools is already adequate for large-scale production deployments.

A lentiviral vector expressing a dendritic cell-targeting multimer induces mucosal anti-mycobacterial CD4+ T-cell immunity.

Most viral vectors, including the potently immunogenic lentiviral vectors (LVs), only poorly direct antigens to the MHC-II endosomal pathway and elicit CD4+ T cells. We developed a new generation of LVs encoding antigen-bearing monomers of collectins substituted at their C-terminal domain with the CD40 ligand ectodomain to target and activate antigen-presenting cells. Host cells transduced with such optimized LVs secreted soluble collectin-antigen polymers with the potential to be endocytosed in vivo and reach the MHC-II pathway. In the murine tuberculosis model, such LVs induced efficient MHC-II antigenic presentation and triggered both CD8+ and CD4+ T cells at the systemic and mucosal levels. They also conferred a significant booster effect, consistent with the importance of CD4+ T cells for protection against Mycobacterium tuberculosis. Given the pivotal role of CD4+ T cells in orchestrating innate and adaptive immunity, this strategy could have a broad range of applications in the vaccinology field.