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Autophagy machinery as exploited by viruses.
Viruses adapt and modulate cellular pathways to allow their replication in host cells. The catabolic pathway of macroautophagy, for simplicity referred to as autophagy, is no exception. In this review, we discuss anti-viral functions of both autophagy and select components of the autophagy machinery, and how viruses have evaded them. Some viruses use the membrane remodeling ability of the autophagy machinery to build their replication compartments in the cytosol or efficiently egress from cells in a non-lytic fashion. Some of the autophagy machinery components and their remodeled membranes can even be found in viral particles as envelopes or single membranes around virus packages that protect them during spreading and transmission. Therefore, studies on autophagy regulation by viral infections can reveal functions of the autophagy machinery beyond lysosomal degradation of cytosolic constituents. Furthermore, they can also pinpoint molecular interactions with which the autophagy machinery can most efficiently be manipulated, and this may be relevant to develop effective disease treatments based on autophagy modulation.
Nanotechnology for immuno-oncology.
Although the first generation of cancer immunotherapeutics produced unprecedented improvements in clinical outcomes for individuals with cancer, novel strategies to increase treatment specificity, delivery efficiency and pharmacokinetics are still needed. In this Review, we describe the potential advantages and current limitations of nanomaterials for cancer immunotherapy and highlight rational uses of nanosystems to generate potent and durable antitumor immune responses. We close with a review of the current state of clinical development of nanomedicine for cancer immunotherapy.
Distinct immunity protein families mediate compartment-specific neutralisation of a bacterial toxin.
Staphylococcus aureus utilises a type VII secretion system (T7SS) to secrete antibacterial toxins that target competitor bacteria. EsxX is a T7SS substrate protein secreted by ST398 strains that has an LXG domain at its N-terminus. Here we show that the EsxX C-terminus is a membrane-depolarising toxin with a glycine zipper motif. EsxX is profoundly toxic to bacteria, displaying toxicity from both cytoplasmic and extracellular compartments. A pair of polytopic membrane proteins, ExiCD, protect cells from intoxication by extracellular EsxX. By contrast, a distinct soluble heterodimer, ExiAB, neutralises cytoplasmic EsxX by sequestration of its glycine zipper motif in a binding groove on ExiB. The exiA-exiB gene pair co-occur in staphylococcal genomes with esxX, invoking a model whereby ExiAB protects EsxX-producing cells from self-toxicity prior to EsxX secretion. By contrast ExiCD is encoded by both EsxX producers and in antitoxin islands of competitor strains that do not encode EsxX, consistent with providing immunity against the secreted form of the toxin. This work defines a new class of antibacterial toxin requiring two distinct types of immunity protein which follow different phylogenetic distributions.
Rabies glycoprotein engineering for improved stability and expression.
Current rabies vaccines require multiple doses and are relatively expensive, limiting their accessibility. Novel low-cost vaccines capable of inducing a protective antibody response against the rabies virus glycoprotein (RVG) are therefore desirable. Structure-guided engineering of the antigen may enhance its qualitative or quantitative immunogenicity, as may transgene cassette optimisation in the case of vectored vaccines. We investigated the potential of these approaches for the design of improved rabies vaccines. We evaluated twelve candidate cassette designs. While codon optimisation enhanced expression in vitro, it did not translate into improved immunogenicity. Co-expression or RVG with rabies matrix protein (RVM) did not detectably affect expression or immunogenicity. Inserting a C-terminal trimerisation domain was detrimental to expression in vitro and did not improve immunogenicity compared to the wild-type comparator. We screened 72 mutant constructs for in vitro expression and pre-fusion stabilisation. Several mutants enhanced expression and/or pre-fusion stability at low pH. Combination of the previously reported H270P mutation with the H419L substitution achieved enhanced stability. An L271Q + H419L double mutant achieved the greatest positive effect upon expression. Neither of double mutants improved immunogenicity compared to wild-type RVG when tested using an mRNA vaccine platform. These mutant constructs may be of value for protein subunit vaccines, but full length wild-type RVG may be sufficiently conformationally stable and well-expressed for optimal immunogenicity of adenovirus and mRNA vaccines in mice.
A Data-Driven Perspective on Bioisostere Evaluation: Mapping the Benzene Bioisostere Landscape with BioSTAR.
The bioisostere landscape is continually expanding, with new scaffolds emerging as alternatives in drug design. Increasingly, medicinal chemists face the challenge of selecting and prioritising these bioisosteres, often relying on personal experience and anecdotal evidence. In this Perspective, we lay out a data-driven approach to analyze the bioisostere landscape, using benzene bioisosteres as a representative example, and quantitatively compare replacements based on their impact on bioactivity, solubility, and metabolic stability. To support the findings of the analysis, we highlight recent and particularly elegant examples of benzene bioisostere applications while identifying areas where further development could significantly benefit the community. By providing this Perspective and associated data-mining workflow (BioSTAR), we aim to support more informed decision-making in bioisosteric replacement selection in drug design and inspire future innovations in bioisostere design.
Steric control of signaling bias in the immunometabolic receptor GPR84.
Biased signaling in G protein-coupled receptors offers therapeutic promise, yet rational design of biased ligands remains challenging due to limited mechanistic understanding. Here, we report a molecular framework for controlling signaling bias at the immunometabolic receptor GPR84. We identified three structurally-matched ligands (OX04529, OX04954, and OX04539) with varying steric profiles that exhibit comparable Gi protein activation but dramatically different β-arrestin recruitment capacities. A high-resolution cryo-EM structure of GPR84-Gi in complex with OX04529, complemented by molecular dynamics simulations and targeted mutagenesis, revealed that steric interactions between ligand substituents and Leu3366.52 and Phe1875.47 indirectly disrupt a critical polar network involving Tyr3326.48, Asn1043.36 and Asn3627.45 essential for β-arrestin recruitment. Based on these insights, we developed a steric-dependent model that enabled rational design of G protein-biased agonists with predictable β-arrestin recruitment profiles. This mechanistic framework provides a blueprint for designing biased agonists with customized signaling profiles at GPR84 and potentially other class A GPCRs.
Selecting outcome measures to validate prognostic biomarkers of paediatric mild traumatic brain injury: challenges and priorities.
Outcomes following paediatric mild traumatic brain injury (mTBI) are extremely heterogenous. While emerging biomarkers promise enhanced prognostic accuracy, a critical question remains unanswered-which outcome measures provide the most accurate assessment of injury impact? In this article, we highlight barriers to selecting appropriate outcome measures, including variability in how outcomes are defined and the wide range of assessment tools used. With reference to the most recent literature, we summarise current evidence of adverse outcomes following paediatric mTBI and highlight emerging candidate biomarkers of these outcomes. We emphasise the unique challenges associated with interpreting outcome measures in younger patients, from the impact of developmental stage and assessment timing to the influence of injury-independent factors. We assert the need to consider these obstacles when designing and interpreting mTBI biomarker studies. To realise the potential of prognostic biomarkers, future research should prioritise establishing consensus definitions, compiling a set of accessible and comprehensive outcome measures, and capturing injury-independent factors through longitudinal study designs.
Prevention and management of venous thromboembolism
Venous thromboembolism (VTE), which comprises deep vein thrombosis (DVT) and pulmonary embolism (PE), has an incidence of about 1 per 1000 per annum, with two-thirds presenting as DVT and one-third as PE. Approximately half of VTEs are associated with hospital admissions, and hospital VTE prevention programmes are important to help reduce this risk. The case fatality of VTE is approximately 5%, but there is also major morbidity caused by post-thrombotic syndrome (PTS) of the leg and chronic thromboembolic pulmonary hypertension (CTPH). Anticoagulants are highly effective in secondary prevention, but leave the patient at risk of bleeding. The risk of recurrence is considerable and patients need to be assessed to identify those who require long-term anticoagulation as secondary prevention. This chapter covers primary VTE prevention (risk assessment and thromboprophylaxis), VTE diagnosis and acute treatment and secondary VTE prevention.
Temporo-spatial cellular atlas of the regenerating alveolar niche in idiopathic pulmonary fibrosis.
Healthy alveolar repair relies on the ability of alveolar stem cells to differentiate into specialized epithelial cells for gas exchange. In chronic fibrotic lung diseases such as idiopathic pulmonary fibrosis (IPF), this regenerative process is abnormal but the underlying mechanisms remain unclear. Here, using human lung tissue that represents different stages of disease and a 33-plex single-cell imaging mass cytometry (IMC), we present a high-resolution, temporo-spatial cell atlas of the regenerating alveolar niche. With unbiased mathematical methods which quantify statistically enriched interactions, CD206himacrophage subtype and an alveolar basal intermediate epithelial cell emerge as the most statistically robust spatial association in the epithelial and immune cell interactome, found across all stages of disease. Spatially resolved receptor-ligand analysis further offers an in silico mechanism by which these macrophages may influence epithelial regeneration. These findings provide a foundational step toward understanding immune-epithelial dynamics in aberrant alveolar regeneration in IPF.
Neuro-Cells Mitigate Amyloid Plaque Formation and Behavioral Deficits in the APPswe/PS1dE9 Model of Alzheimer Disease While Also Reducing IL-6 Production in Human Monocytes
Neuroinflammation is a key feature of Alzheimer’s disease (AD), and stem cell therapies have emerged as promising candidates due to their immunomodulatory properties. Neuro-Cells (NC), a combination of unmodified mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs), have demonstrated therapeutic potential in models of central nervous system (CNS) injury and neurodegeneration. Here, we studied the effects of NC in APPswe/PS1dE9 mice, an AD mouse model. Twelve-month-old APPswe/PS1dE9 mice or their wild-type littermates were injected with NC or vehicle into the cisterna magna. Five to six weeks post-injection, cognitive, locomotor, and emotional behaviors were assessed. The brain was stained for amyloid plaque density using Congo red, and for astrogliosis using DAPI and GFAP staining. Gene expression of immune activation markers (Il-1β, Il-6, Cd45, Tnf) and plasticity markers (Tubβ3, Bace1, Trem2, Stat3) was examined in the prefrontal cortex. IL-6 secretion was measured in cultured human monocytes following endotoxin challenge and NC treatment. Untreated APPswe/PS1dE9 mice displayed impaired learning in the conditioned taste aversion test, reduced object exploration, and anxiety-like behavior, which were improved in the NC-treated mutants. NC treatment normalized the expression of several immune and plasticity markers and reduced the density of GFAP-positive cells in the hippocampus and thalamus. NC treatment decreased amyloid plaque density in the hippocampus and thalamus, targeting plaques of <100 μm2. Additionally, NC treatment suppressed IL-6 secretion by human monocytes. Thus, NC treatment alleviated behavioral deficits and reduced amyloid plaque formation in APPswe/PS1dE9 mice, likely via anti-inflammatory mechanisms. The reduction in IL-6 production in human monocytes further supports the potential of NC therapy for the treatment of AD.
Comparative performance of the InBios SCoV-2 DetectTM IgG ELISA and the in-house KWTRP ELISA in detecting SARS-CoV-2 spike IgG antibodies in Kenyan populations
Background The InBios SCoV-2 Detect™ IgG ELISA (InBios) and the in-house KWTRP ELISA (KWTRP) have both been used in the estimation of SARS-CoV-2 seroprevalence in Kenya. Whereas the latter has been validated extensively using local samples, the former has not. Such validation is important for informing the comparability of data across the sites and populations where seroprevalence has been reported. Methods We compared the assays directly using pre-pandemic serum/plasma collected in 2018 from 454 blood donors and 173 malaria cross-sectional survey participants, designated gold standard negatives. As gold standard SARS-CoV-2 positive samples: we assayed serum/plasma from 159 SARS-CoV-2 PCR-positive patients and 166 vaccination-confirmed participants. Results The overall agreement on correctly classified samples was >0.87 for both assays. The overall specificity was 0.89 (95% CI, 0.87–0.91) for InBios and 0.99 (95% CI, 0.97–0.99) for KWTRP among the gold standard negative samples while the overall sensitivity was 0.97 (95% CI, 0.94–0.98) and 0.93 (95% CI, 0.90– 0.95) for InBios and KWTRP ELISAs respectively, among the gold standard positive samples. In all, the positive predictive value for InBios was 0.83 (95% CI, 0.79-0.87) and 0.98 (95% CI, 0.96-0.99) for KWTRP while the negative predictive value was 0.98 (95% CI, 0.97- 0.99) and 0.97 (95% CI, 0.95-0.98) for InBios and KWTRP respectively. Conclusions Overall, both assays showed sufficient sensitivity and specificity to estimate SARS-CoV-2 antibodies in different populations in Kenya.