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Amphotericin B (AmpB) is an effective but toxic antifungal drug. Thus, improving its activity/toxicity relationship is of interest. AmpB disrupts fungal membranes by two proposed mechanisms: ergosterol sequestration from the membrane and pore formation. Whether these two mechanisms operate in conjunction and how they could be potentiated remains to be fully understood. Here, we report that gladiolin, a polyketide antibiotic produced by Burkholderia gladioli , is a strong potentiator of AmpB and acts synergistically against Cryptococcus and Candida species, including drug-resistant C. auris . Gladiolin also synergizes with AmpB against drug-resistant fungal biofilms, while exerting no mammalian cytotoxicity. To explain the mechanism of synergy, we show that gladiolin interacts with membranes via a previously unreported binding mode for polyketides. Moreover, gladiolin modulates lipid binding by AmpB and, in combination, causes faster and more pronounced lipid rearrangements relative to AmpB alone which include membrane thinning consistent with ergosterol extraction, areas of thickening, pore formation, and increased membrane destruction. These biophysical data provide evidence of a functional interaction between gladiolin and AmpB at the membrane interface. The data further indicate that the two proposed AmpB mechanisms (ergosterol sequestration and pore formation) act in conjunction to disrupt membranes, and that gladiolin synergizes by enhancing both mechanisms. Collectively, our findings shed light on AmpB's mechanism of action and characterize gladiolin as an AmpB potentiator, showing an antifungal mechanism distinct from its proposed antibiotic activity. We shed light on the synergistic mechanism at the membrane, and provide insights into potentiation strategies to improve AmpB's activity/toxicity relationship., Importance: Amphotericin B (AmpB) is one of the oldest antifungal drugs in clinical use. It is an effective therapeutic, but it comes with toxicity issues due to the similarities between its fungal target (the membrane lipid ergosterol) and its mammalian counterpart (cholesterol). One strategy to improve its activity/toxicity relationship is by combinatorial therapy with potentiators, which would enable a lower therapeutic dose of AmpB. Here, we report on the discovery of the antibiotic gladiolin as a potentiator of AmpB against several priority human fungal pathogens and fungal biofilms, with no increased toxicity against mammalian cells. We show that gladiolin potentiates AmpB by increasing and accelerating membrane damage. Our findings also provide insights into the on-going debate about the mechanism of action of AmpB by indicating that both proposed mechanisms, extraction of ergosterol from membranes and pore formation, are potentiated by gladiolin., Competing Interests: G.L.C. is a shareholder, non-executive director, and consultant of Erebagen Ltd. The other authors declare no competing interests.

Histone deacetylase 7 (HDAC7) is a member of the class IIa family of classical HDACs with important roles in cell development, differentiation, and activation, including in macrophages and other innate immune cells. HDAC7 and other class IIa HDACs act as transcriptional repressors in the nucleus but, in some cell types, they can also act in the cytoplasm to modify non-nuclear proteins and/or scaffold signalling complexes. In macrophages, HDAC7 is a cytoplasmic protein with both pro- and anti-inflammatory functions, with the latter activity involving activation of the pentose phosphate pathway (PPP) enzyme 6-phosphogluconate dehydrogenase (6PGD) and the generation of anti-inflammatory metabolite ribulose-5-phosphate. Here, we used ectopic expression systems and biochemical approaches to investigate the mechanism by which HDAC7 promotes 6PGD enzyme activity. We reveal that HDAC7 enzyme activity is not required for its activation of 6PGD and that the N-terminal protein-protein interaction domain of HDAC7 is sufficient to initiate this response. Mechanistically, the N-terminus of HDAC7 increases the affinity of 6PGD for NADP+, promotes the generation of a shorter form of 6PGD, and enhances the formation of higher order protein complexes, implicating its scaffolding function in engagement of the PPP. This contrasts with the pro-inflammatory function of HDAC7 in macrophages, in which it promotes deacetylation of the glycolytic enzyme pyruvate kinase M2 for inflammatory cytokine production., (© 2024 The Author(s).)

The urgent need for effective treatments for acute and chronic lung diseases underscores the significance of developing innovative preclinical human research tools. The 2023 ATS Workshop on Precision Cut Lung Slices (PCLS) brought together 35 experts to discuss and address the role of human tissue-derived PCLS as a unique tool for target and drug discovery and validation in pulmonary medicine. With increasing interest and usage, along with advancements in methods and technology, there is a growing need for consensus on PCLS methodology and readouts. The current document recommends standard reporting criteria and emphasizes the requirement for careful collection and integration of clinical metadata. We further discuss current clinically relevant readouts that can be applied to PCLS and highlight recent developments and future steps for implementing novel technologies for PCLS modeling and analysis. The collection and correlation of clinical metadata and multiomic analysis will further advent the integration of this preclinical platform into patient endotyping and the development of tailored therapies for lung disease patients.

The rapid and accurate identification of pathogenic bacteria is crucial for combating the growing threat of antibiotic resistance, nosocomial infections, and food safety concerns. This study presents a novel and comprehensive comparison of two vibrational spectroscopic techniques - attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and a low-cost miniature near-infrared (NIR) spectrometer - for distinguishing Gram-positive and Gram-negative bacterial samples grown using the same stock media solution. This is the first report of NIR spectroscopy being applied to differentiate Gram-positive and Gram-negative bacteria, as well as the first direct comparison of ATR-FTIR and NIR for the combined multimodal analysis of clinical bacterial isolates. Using a data set of five Gram-positive and seven Gram-negative species and recording spectra in triplicate, the study employed advanced data fusion and multivariate analysis techniques to classify the spectra and facilitate NIR band assignment. 2D correlation analysis revealed strong positive correlations between key spectral markers identified in both modalities. Partial least-squares- and support vector machine discriminant analysis models were validated using a methodology based on 100 repeated random sampling of calibration and test sets. Models demonstrated that both the standalone ATR-FTIR and the combined ATR-FTIR/NIR approach achieved exceptional classification accuracy (>98%) in differentiating the two bacterial groups. Differences observed in the spectra were attributed to the distinct cell wall compositions of Gram-Positive and Gram-negative bacteria. Notably, the low-cost NIR technique also showed promising performance, with classification accuracy values above 90%. The findings highlight the potential of these rapid, noninvasive, and cost-effective vibrational spectroscopic techniques, particularly the NIR method, for point-of-care applications in clinical microbiology and food safety monitoring. The combination of ATR-FTIR and NIR data further enhances the robustness and reliability of bacterial identification, paving the way for broader adoption of these advanced analytical tools in various healthcare and food safety settings.

Transcranial direct current stimulation (tDCS) has garnered significant interest for its potential to enhance cognitive functions and as a therapeutic intervention in various cognitive disorders. However, the clinical application of tDCS has been hampered by significant variability in its cognitive outcomes. Furthermore, the widespread use of tDCS has raised concerns regarding its safety and efficacy, particularly in light of our limited understanding of its underlying neural mechanisms at the cellular level. We still do not know 'where', 'when' and 'how' tDCS modulates information encoding by neurons, in order to lead to the observed changes in cognitive functions. Without elucidating these fundamental unknowns, the root causes of its outcome variability and long-term safety remain elusive, challenging the effective application of tDCS in clinical settings. Addressing this gap, our study investigates the effects of tDCS, applied over the dorsolateral prefrontal cortex, on cognitive abilities and individual neuron activity in macaque monkeys performing cognitive tasks. Like humans performing a delayed match-to-sample task, monkeys exhibited practice-related slowing in their responses (within-session behavioural adaptation). Concurrently, there were practice-related changes in simultaneously recorded activity of prefrontal neurons (within-session neuronal adaptation). Anodal tDCS attenuated both these behavioural and neuronal adaptations when compared with sham stimulation. Furthermore, tDCS abolished the correlation between response time of monkeys and neuronal firing rate. At a single-cell level, we also found that following tDCS, neuronal firing rate was more likely to exhibit task-specific modulation than after sham stimulation. These tDCS-induced changes in both behaviour and neuronal activity persisted even after the end of tDCS stimulation. Importantly, multiple applications of tDCS did not alter burst-like firing rates of individual neurons when compared with sham stimulation. This suggests that tDCS modulates neural activity without enhancing susceptibility to epileptiform activity, confirming a potential for safe use in clinical settings. Our research contributes unprecedented insights into the 'where', 'when' and 'how' of tDCS effects on neuronal activity and cognitive functions by showing that modulation of the behaviour of monkeys by the tDCS of the prefrontal cortex is accompanied by alterations in prefrontal cortical cell activity ('where') during distinct trial phases ('when'). Importantly, tDCS led to task-specific and state-dependent alterations in prefrontal cell activities ('how'). Our findings suggest a significant shift from the view that the effects of tDCS are merely attributable to polarity-specific shifts in cortical excitability and instead propose a more complex mechanism of action for tDCS that encompasses various aspects of cortical neuronal activity without increasing burst-like epileptiform susceptibility., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Maternal asthma is associated with increased rates of neonatal lung disease, and fetuses from asthmatic ewes have fewer surfactant-producing cells and lower surfactant-protein B gene (SFTPB) expression than controls. Antenatal betamethasone increases lung surfactant production in preterm babies, and we therefore tested this therapy in experimental maternal asthma. Ewes were sensitised to house dust mite allergen, and an asthmatic phenotype induced by fortnightly allergen lung challenges; controls received saline. Pregnant asthmatic ewes were randomised to receive antenatal saline (asthma) or 12 mg intramuscular betamethasone (asthma+beta) at 138 and 139 days of gestation (term = 150 days). Lambs were delivered by Caesarean section at 140 days of gestation and ventilated for 45 min before tissue collection. Lung function and structure were similar in control lambs (n = 16, 11 ewes) and lambs from asthma ewes (n = 14, 9 ewes). Dynamic lung compliance was higher in lambs from asthma+beta ewes (n = 12, 8 ewes) compared to those from controls (P = 0.003) or asthma ewes (P = 0.008). Lung expression of surfactant protein genes SFTPA (P = 0.048) and SFTPB (P < 0.001), but not SFTPC (P = 0.177) or SFTPD (P = 0.285), was higher in lambs from asthma+beta than those from asthma ewes. Female lambs had higher tidal volume (P = 0.007), dynamic lung compliance (P < 0.001), and SFTPA (P = 0.037) and SFTPB gene expression (P = 0.030) than males. These data suggest that betamethasone stimulates lung maturation and function of near-term neonates, even in the absence of impairment by maternal asthma., (© 2024 The Author(s). Experimental Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.)

Acute kidney injury (AKI) and chronic kidney disease (CKD) are increasingly recognized as interconnected conditions with overlapping pathophysiological mechanisms. This review examines the transition from AKI to CKD, focusing on the molecular mechanisms, animal models, and biomarkers essential for understanding and managing this progression. AKI often progresses to CKD due to maladaptive repair processes, persistent inflammation, and fibrosis, with both conditions sharing common pathways involving cell death, inflammation, and extracellular matrix (ECM) deposition. Current animal models, including ischemia-reperfusion injury (IRI) and nephrotoxic damage, help elucidate these mechanisms but have limitations in replicating the complexity of human disease. Emerging biomarkers such as kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), and soluble tumor necrosis factor receptors (TNFRs) show promise in early detection and monitoring of disease progression. This review highlights the need for improved animal models and biomarker validation to better mimic human disease and enhance clinical translation. Advancing our understanding of the AKI-to-CKD transition through targeted therapies and refined research approaches holds the potential to significantly improve patient outcomes.

Objective: Although the metabolic state of an organism affects olfactory function, the precise mechanisms and their impact on behavior and metabolism remain unknown. Here, we assess whether ghrelin receptors (GHSRs) in the olfactory bulb (OB) increase olfactory function and influence foraging behaviors and metabolism., Methods: We performed a detailed behavioural and metabolic analysis in mice lacking GHSRs in the OB (OB GHSR deletion). We also analsyed OB scRNA-seq and spatial transcriptomic datasets to assess GHSR+ cells in the main and accessory olfactory bulbs, as well as the anterior olfactory nucleus., Results: OB GHSR deletion affected olfactory discrimination and habituation to both food and non-food odors. Anxiety-like and depression-like behaviors were significantly greater after OB GHSR deletion, whereas exploratory behavior was reduced, with the greatest effect under fasted conditions. OB GHSR deletion impacted feeding behavior as evidenced by altered bout number and duration, as well as buried food-seeking. OB GHSR deletion increased body weight and fat mass, spared fat utilisation on a chow diet and impaired glucose metabolism indicating metabolic dysfunction. Cross referenced analysis of OB scRNA-seq and spatial transcriptomic datasets revealed GHSR+ glutamate neurons in the main and accessory olfactory bulbs, as well as the anterior olfactory nucleus. Ablation of glutamate neurons in the OB reduced ghrelin-induced food finding and phenocopied results seen after OB GHSR deletion., Conclusions: OB GHSRs help to maintain olfactory function, particularly during hunger, and facilitate behavioral adaptations that optimise food-seeking in anxiogenic environments, priming metabolic pathways in preparation for food consumption., Competing Interests: Declaration of competing interest J.M.Z. receives research funding from Novo Nordisk for another project and consulted for Helsinn Healthcare S.A. and Dexcel Pharma Technologies Ltd. during the time these studies were performed. The other authors have nothing to disclose. All other authors report no conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

The escalating global burden of antimicrobial resistance (AMR) represents a critical public health challenge. This rise in antibiotic resistance is concomitant with heightened antibiotic consumption, with an estimated annual usage of 100,000 to 200,000 tons. A recent systematic review, which analysed data from 204 countries, reported that AMR was responsible for 4.95 million deaths in 2019 (Murray et al., 2022). The growing threat of AMR is imposing a significant financial burden on the global economy, with the CDC reporting an additional annual cost of $20 billion in the U.S. and €9 billion in Europe. The emerging field of bacteriophage therapy offers promising potential as a game-changer in the era of AMR. However, existing literature reveals numerous research gaps and technological challenges, including insufficient information on phage pharmacology, genomics, and a lack of preclinical and clinical data. In addition to conducting further research to address existing knowledge gaps, establishing phage banks in clinical facilities could be a transformative advancement in the fight against AMR., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Immunomodulatory imide drugs (IMiDs) are central components of therapy for multiple myeloma (MM). IMiDs bind cereblon (CRBN), an adaptor for the CUL4-DDB1-RBX1 E3 ligase to change its substrate specificity and induce degradation of 'neosubstrate' transcription factors that are essential to MM cells. Mechanistic studies to date have largely focussed on mediators of therapeutic activity and insight into clinical IMiD toxicities is less developed. We adopted BioID2-dependent proximity labelling (BioID2-CRBN) to characterise the CRBN interactome in the presence and absence of various IMiDs and the proteasome inhibitor, bortezomib. We aimed to leverage this technology to further map CRBN interactions beyond what has been achieved by conventional proteomic techniques. In support of this approach, analysis of cells expressing BioID2-CRBN following IMiD treatment displayed biotinylation of known CRBN interactors and neosubstrates. We observed that bortezomib alone significantly modifies the CRBN interactome. Proximity labelling also suggested that IMiDs augment the interaction between CRBN and proteins that are not degraded, thus designating 'neointeractors' distinct from previously disclosed 'neosubstrates'. Here we identify Non-Muscle Myosin Heavy Chain IIA (MYH9) as a putative CRBN neointeractor that may contribute to the haematological toxicity of IMiDs. These studies provide proof of concept for proximity labelling technologies in the mechanistic profiling of IMiDs and related E3-ligase-modulating drugs., (© 2024 The Author(s). The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Millions of livestock animals worldwide are infected with the haematophagous barber's pole worm, Haemonchus contortus, the aetiological agent of haemonchosis. Despite the major significance of this parasite worldwide and its widespread resistance to current treatments, the lack of a high-quality genome for the well-defined strain of this parasite from Australia, called Haecon-5, has constrained research in a number of areas including host-parasite interactions, drug discovery and population genetics. To enable research in these areas, we report here a chromosome-contiguous genome (∼280 Mb) for Haecon-5 with high-quality models for 19,234 protein-coding genes. Comparative genomic analyses show significant genomic similarity (synteny) with a UK strain of H. contortus, called MHco3(ISE).N1 (abbreviated as "ISE"), but we also discover marked differences in genomic structure/gene arrangements, distribution of nucleotide variability (single nucleotide polymorphisms (SNPs) and indels) and orthology between Haecon-5 and ISE. We used the genome and extensive transcriptomic resources for Haecon-5 to predict a subset of essential single-copy genes employing a "cross-species" machine learning (ML) approach using a range of features from nucleotide/protein sequences, protein orthology, subcellular localisation, single-cell RNA-seq and/or histone methylation data available for the model organisms Caenorhabditis elegans and Drosophila melanogaster. From a set of 1,464 conserved single copy genes, transcribed in key life-cycle stages of H. contortus, we identified 232 genes whose homologs have critical functions in C. elegans and/or D. melanogaster, and prioritised 10 of them for further characterisation; nine of the 10 genes likely play roles in neurophysiological processes, germline, hypodermis and/or respiration, and one is an unknown (orphan) gene for which no detailed functional information exists. Future studies of these genes/gene products are warranted to elucidate their roles in parasite biology, host-parasite interplay and/or disease. Clearly, the present Haecon-5 reference genome and associated resources now underpin a broad range of fundamental investigations of H. contortus and could assist in accelerating the discovery of novel intervention targets and drug candidates to combat haemonchosis., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The human gastrointestinal microbiome encompasses bacteria, fungi, and viruses forming complex bionetworks which, for organismal health, must be in a state of homeostasis. An important homeostatic mechanism derives from microbial competition, which maintains the relative abundance of microbial species in a healthy balance. Microbes compete for nutrients and secrete metabolites that inhibit other microbes. Short-chain fatty acids (SCFAs) are one such class of metabolites made by gut bacteria to very high levels. SCFAs are metabolised by microbes and host cells and have multiple roles in regulating cell physiology. Here, we review the mechanisms by which SCFAs regulate the fungal gut commensal Candida albicans. We discuss SCFA's ability to inhibit fungal growth, limit invasive behaviours and modulate cell surface antigens recognised by immune cells. We review the mechanisms underlying these roles: regulation of gene expression, metabolism, signalling and SCFA-driven post-translational protein modifications by acylation, which contribute to changes in acylome dynamics of C. albicans with potentially large consequences for cell physiology. Given that the gut mycobiome is a reservoir for systemic disease and has also been implicated in inflammatory bowel disease, understanding the mechanisms by which bacterial metabolites, such as SCFAs, control the mycobiome might provide therapeutic avenues., Competing Interests: Declaration of interests The authors have no interests to declare., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In a survey of a waterway on Wurundjeri land, two sub-populations of the bacteriophage Merri-merri-uth nyilam marra-natj (phage MMNM) were isolated on a permissive host, Klebsiella B5055 of capsule-type K2, but were distinguished by minor phenotypic differences. The variant phage MMNM(Ala 134 ) showed an inhibited activity against Klebsiella AJ174-2, and this was used as a basis to select for further variation through experimental evolution. Over the course of an evolution experiment, 20 phages that evolved distinct phenotypes in terms of the morphologies of plaques formed when they infected host Klebsiella were subject to whole-genome sequencing. The evolved phages had mutations in a small set of proteins that contribute to the baseplate portion of the phage virion. Phages MMNM and MMNM(Ala 134 ) are minimalist phages, with baseplates formed from only five predicted subunits, akin to other minimalist phages Pam3 and XM1. The homology between all three minimalist phages provided a structural framework to interpret the two classes of mutations derived through evolution in the presence of the semi-permissive host: those that affect the interfacial surfaces between baseplate subunits, and those in a base-plate associated tail-fiber. This study evidences that multiple small mutations can be fixed into a sub-population of phage to provide a basis for phenotypic variation that we suggest could ultimately provide for a shift of virus properties, as an alternative evolutionary scenario to the major genetic events that result in more well-studied evolutionary mechanism of phage mosaicism., Importance: Bacteriophages (phages) are viruses that prey on bacteria. This study sampled natural phage populations to test the hypothesis that untapped genetic variation within a population can be the basis for the selection of phages to diversify their host-range. Sampling of a freshwater site revealed two populations of the phage Merri-merri-uth nyilam marra-natj (phage MMNM), differing by a variant residue (Val134Ala) in the baseplate protein MMNM&#95;26. This sequence variation modulated bacterial killing in plaques, and further evolution of the phages on a semi-permissive bacterial host led to a new generation of phages with more diverse phenotypes in killing the bacterium Klebsiella pneumoniae .

Genetic variation in host immunity impacts the disproportionate burden of infectious diseases that can be experienced by First Nations peoples. Polymorphic human leukocyte antigen (HLA) class I and killer cell immunoglobulin-like receptors (KIRs) are key regulators of natural killer (NK) cells, which mediate early infection control. How this variation impacts their responses across populations is unclear. We show that HLA-A ∗ 24:02 became the dominant ligand for inhibitory KIR3DL1 in First Nations peoples across Oceania, through positive natural selection. We identify KIR3DL1 ∗ 114, widespread across and unique to Oceania, as an allele lineage derived from archaic humans. KIR3DL1 ∗ 114 + NK cells from First Nations Australian donors are inhibited through binding HLA-A ∗ 24:02. The KIR3DL1 ∗ 114 lineage is defined by phenylalanine at residue 166. Structural and binding studies show phenylalanine 166 forms multiple unique contacts with HLA-peptide complexes, increasing both affinity and specificity. Accordingly, assessing immunogenetic variation and the functional implications for immunity are fundamental toward understanding population-based disease associations., Competing Interests: Declaration of interests A.G.I. has shares in Galatea Bio, Inc. G.F.H. is currently an employee of Tempus AI. S.C.W. is currently an employee of Miltenyi Biotec Asia Pacific Pte Ltd. L.H. is affiliated with the Department of Internal Medicine II, University Hospital Tübingen, Tübingen, Germany. J.W. is affiliated with The Malignant Hematology, Transplantation, and Cellular Therapy Services, Alfred Health, Melbourne, VIC, Australia. S.Z. is affiliated with the Protein Production Facility (PPF) of the Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia. G.M.-M. is affiliated with the HLA Histocompatibility and Immunogenetics Laboratory, Vitalant, Phoenix, AZ, USA. J.P.V. is affiliated with St. Vincent’s Institute of Medical Research, Fitzroy, VIC, Australia., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Staphylococcus aureus (S. aureus) is a significant Gram-positive opportunistic pathogen behind many debilitating infections. β-lactam antibiotics are conventionally prescribed for treating S. aureus infections. However, the adaptability of S. aureus in evolving resistance to multiple β-lactams contributed to the persistence and spread of infections, exemplified in the emergence of methicillin-resistant S. aureus (MRSA). In the present study, we investigated the efficacies of the synthetic teixobactin analogue, Leu 10 -teixobactin, combined with the penicillinase-resistant cephalosporin cefepime against MRSA strains. The Leu 10 -teixobactin and cefepime combination exerted synergism against most strains tested in broth microdilution assay. Time-kill profiles showed that both Leu 10 -teixobactin and cefepime predominantly exhibited synergistic activity, with > 2.0-log 10 CFU decrease compared to monotherapy at 24 h. Moreover, biofilm assays revealed a significant inhibition of biofilm production in ATCC™43300 cells treated with sub-MICs of Leu 10 -teixobactin and cefepime. Subsequent electron microscopy studies showed more extensive damage with the combination therapy compared to monotherapies, including aberrant bacterial morphology, vesicle formation and substantial lysis, indicating combined damage to the cell wall. Quantitative real-time PCR revealed marked perturbation of genes mecA, sarA, atlA, and icaA, substantiating the apparent mode of combined antibacterial action of both antibiotics against peptidoglycan synthesis and initial biofilm production. Hence, the study highlights the prospective utility of the Leu 10 -teixobactin-cefepime combination in treating MRSA infections via β-lactam potentiation., (© 2024. The Author(s).)

Asgard archaea are of great interest as the progenitors of Eukaryotes, but little is known about the mobile genetic elements (MGEs) that may shape their ongoing evolution. Here, we describe MGEs that replicate in Atabeyarchaeia, a wetland Asgard archaea lineage represented by two complete genomes. We used soil depth-resolved population metagenomic data sets to track 18 MGEs for which genome structures were defined and precise chromosome integration sites could be identified for confident host linkage. Additionally, we identified a complete 20.67 kbp circular plasmid and two family-level groups of viruses linked to Atabeyarchaeia, via CRISPR spacer targeting. Closely related 40 kbp viruses possess a hypervariable genomic region encoding combinations of specific genes for small cysteine-rich proteins structurally similar to restriction-homing endonucleases. One 10.9 kbp integrative conjugative element (ICE) integrates genomically into the Atabeyarchaeum deiterrae-1 chromosome and has a 2.5 kbp circularizable element integrated within it. The 10.9 kbp ICE encodes an expressed Type IIG restriction-modification system with a sequence specificity matching an active methylation motif identified by Pacific Biosciences (PacBio) high-accuracy long-read (HiFi) metagenomic sequencing. Restriction-modification of Atabeyarchaeia differs from that of another coexisting Asgard archaea, Freyarchaeia, which has few identified MGEs but possesses diverse defense mechanisms, including DISARM and Hachiman, not found in Atabeyarchaeia. Overall, defense systems and methylation mechanisms of Asgard archaea likely modulate their interactions with MGEs, and integration/excision and copy number variation of MGEs in turn enable host genetic versatility., (© 2024 Valentin-Alvarado et al.; Published by Cold Spring Harbor Laboratory Press.)

MicroRNAs (miRNAs) are short non-coding RNAs involved in various cellular processes, playing a crucial role in gene regulation. Identifying miRNA targets remains a central challenge and is pivotal for elucidating the complex gene regulatory networks. Traditional computational approaches have predominantly focused on identifying miRNA targets through perfect Watson-Crick base pairings within the seed region, referred to as canonical sites. However, emerging evidence suggests that perfect seed matches are not a prerequisite for miRNA-mediated regulation, underscoring the importance of also recognizing imperfect, or non-canonical, sites. To address this challenge, we propose Mimosa, a new computational approach that employs the Transformer framework to enhance the prediction of miRNA targets. Mimosa distinguishes itself by integrating contextual, positional and base-pairing information to capture in-depth attributes, thereby improving its predictive capabilities. Its unique ability to identify non-canonical base-pairing patterns makes Mimosa a standout model, reducing the reliance on pre-selecting candidate targets. Mimosa achieves superior performance in gene-level predictions and also shows impressive performance in site-level predictions across various non-human species through extensive benchmarking tests. To facilitate research efforts in miRNA targeting, we have developed an easy-to-use web server for comprehensive end-to-end predictions, which is publicly available at http://monash.bioweb.cloud.edu.au/Mimosa., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Diets that are high in fat cause over-eating and weight gain in multiple species of animals, suggesting that high dietary fat is sufficient to cause obesity. However, high-fat diets are typically provided freely to animals in obesity experiments, so it remains unclear if high-fat diets would still cause obesity if they required more effort to obtain. We hypothesized that unrestricted and easy access is necessary for high-fat diet induced over-eating, and the corollary that requiring mice to perform small amounts of work to obtain high-fat diet would reduce high-fat diet intake and associated weight gain. To test this hypothesis, we developed a novel home-cage based feeding device that either provided high-fat diet freely, or after mice poked their noses into a port one time - a simple action that is easy for them to do. We tested the effect of this intervention for six weeks, with mice receiving all daily calories from high-fat diet, modifying only how they accessed it. Requiring mice to nose-poke to access high-fat diet reduced intake and nearly completely prevented the development of obesity. In follow up experiments, we observed a similar phenomenon in mice responding for low-fat grain-based pellets that do not induce obesity, suggesting a general mechanism whereby animals engage with and consume more food when it is freely available vs. when it requires a simple action to obtain. We conclude that unrestricted access to food promotes overeating, and that a simple action such as a nose-poke can reduce over-eating and weight gain in mice. This may have implications for why over-eating and obesity are common in modern food environments, which are often characterized by easy access to low-cost unhealthy foods., Competing Interests: Declaration of Interests The authors declare no competing interests

Wildfire-specific particulate matter with diameters ≤ 2.5 µm (PM 2.5 ) is the key component of wildfire smoke, with potentially higher toxicity than PM 2.5 from other sources. In this nationwide population-based cohort study, we included 22,163,195 births from Brazil during 20102019. Daily wildfire-specific PM 2.5 was estimated through the chemical transport model. Time-varying Cox proportional hazards models were used to characterize the exposure-time-response (E-T-R) relationship between weekly wildfire-specific PM 2.5 exposure and preterm birth (PTB) risks, followed by subgroup analyses. A 10 µg/m 3 increment in wildfire-specific PM 2.5 was associated with a hazard ratio of 1.047 (95 % confidence interval [CI]: 1.032-1.063) for PTB. Stronger associations between wildfire-specific PM 2.5 and PTB were observed during earlier pregnancy, among female infants, and pregnant women < 18 years old, in ethnic minorities, with a length of education ≥ 11 years, from low-income or high-temperature municipalities, and residing in North/Northeast regions. An estimated 1.47 % (95 % CI: 1.01 %1.94 %) of PTBs were attributable to wildfire-specific PM 2.5 in Brazil, increasing from 2010 to 2019. The PTBs attributable to wildfire-specific PM 2.5 surpassed those attributed to non-wildfire PM 2.5 (0.31 %, 95 % CI: 0.09 %0.57 %). Wildfire emerged as a critical source contributing to the PM 2.5 -linked PTBs. Prioritized fire management and emission control strategies are warranted for PTB prevention., Competing Interests: Declaration of Competing Interest All other authors declare they have no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Kidney fibrosis is a hallmark of chronic kidney disease (CKD) and compromises the viability of transplanted human bone marrow-derived mesenchymal stromal cells (BM-MSCs). Hence, BM-MSCs were genetically-engineered to express the anti-fibrotic and renoprotective hormone, human relaxin-2 (RLX) and green fluorescent protein (BM-MSCs-eRLX + GFP), which enabled BM-MSCs-eRLX + GFP delivery via a single intravenous injection., Methods: BM-MSCs were lentiviral-transduced with human relaxin-2 cDNA and GFP, under a eukaryotic translation elongation factor-1α promoter (BM-MSCs-eRLX + GFP) or GFP alone (BM-MSCs-eGFP). The ability of BM-MSCs-eRLX + GFP to differentiate, proliferate, migrate, produce RLX and cytokines was evaluated in vitro, whilst BM-MSC-eRLX + GFP vs BM-MSCs-eGFP homing to the injured kidney and renoprotective effects were evaluated in preclinical models of ischemia reperfusion injury (IRI) and high salt (HS)-induced hypertensive CKD in vivo. The long-term safety of BM-MSCs-RLX + GFP was also determined 9-months after treatment cessation in vivo., Results: When cultured for 3- or 7-days in vitro, 1 × 10 6 BM-MSCs-eRLX + GFP produced therapeutic RLX levels, and secreted an enhanced but finely-tuned cytokine profile without compromising their proliferation or differentiation capacity compared to naïve BM-MSCs. BM-MSCs-eRLX + GFP were identified in the kidney 2-weeks post-administration and retained the therapeutic effects of RLX in vivo. 1-2 × 10 6 BM-MSCs-eRLX + GFP attenuated the IRI- or therapeutically abrogated the HS-induced tubular epithelial damage and interstitial fibrosis, and significantly reduced the HS-induced hypertension, glomerulosclerosis and proteinuria. This was to an equivalent extent as RLX and BM-MSCs administered separately but to a broader extent than BM-MSCs-eGFP or the angiotensin-converting enzyme inhibitor, perindopril. Additionally, these renoprotective effects of BM-MSCs-eRLX + GFP were maintained in the presence of perindopril co-treatment, highlighting their suitability as adjunct therapies to ACE inhibition. Importantly, no major long-term adverse effects of BM-MSCs-eRLX + GFP were observed., Conclusions: BM-MSCs-eRLX + GFP produced greater renoprotective and therapeutic efficacy over that of BM-MSCs-eGFP or ACE inhibition, and may represent a novel and safe treatment option for acute kidney injury and hypertensive CKD., (© 2024. The Author(s).)

Background: Checkpoint immunotherapy unleashes tumor control by T cells, but it is undermined in non-immunogenic tumors, e.g. with low MHC class I expression and low neoantigen burden, such as neuroblastoma (NB). Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an enzyme that trims peptides before loading on MHC class I molecules. Inhibition of ERAP1 results in the generation of new antigens able of inducing potent anti-tumor immune responses. Here, we identify a novel non-toxic combinatorial strategy based on genetic inhibition of ERAP1 and administration of the HDAC inhibitor (HDACi) entinostat that increase the immunogenicity of NB, making it responsive to PD-1 therapy., Methods: CRISPR/Cas9-mediated gene editing was used to knockout (KO) the ERAP1 gene in 9464D NB cells derived from spontaneous tumors of TH-MYCN transgenic mice. The expression of MHC class I and PD-L1 was evaluated by flow cytometry (FC). The immunopeptidome of these cells was studied by mass spectrometry. Cocultures of splenocytes derived from 9464D bearing mice and tumor cells allowed the assessment of the effect of ERAP1 inhibition on the secretion of inflammatory cytokines and activation and migration of immune cells towards ERAP1 KO cells by FC. Tumor cell killing was evaluated by Caspase 3/7 assay and flow cytometry analysis. The effect of ERAP1 inhibition on the immune content of tumors was analyzed by FC, immunohistochemistry and multiple immunofluorescence., Results: We found that inhibition of ERAP1 makes 9464D cells more susceptible to immune cell-mediated killing by increasing both the recall and activation of CD4 + and CD8 + T cells and NK cells. Treatment with entinostat induces the expression of MHC class I and PD-L1 molecules in 9464D both in vitro and in vivo. This results in pronounced changes in the immunopeptidome induced by ERAP1 inhibition, but also restrains the growth of ERAP1 KO tumors in vivo by remodelling the tumor-infiltrating T-cell compartment. Interestingly, the absence of ERAP1 in combination with entinostat and PD-1 blockade overcomes resistance to PD-1 immunotherapy and increases host survival., Conclusions: These findings demonstrate that ERAP1 inhibition combined with HDACi entinostat treatment and PD-1 blockade remodels the immune landscape of a non-immunogenic tumor such as NB, making it responsive to checkpoint immunotherapy., (© 2024. The Author(s).)

T cell surface CTLA4 sequesters the costimulatory ligands CD80 and CD86 on antigen-presenting cells (APCs) to prevent autoimmunity. Therapeutic immunosuppression by recombinant CTLA4-immunoglobulin (Ig) fusion proteins, including abatacept, is also attributed to CD80/CD86 blockade. Recent studies show that CTLA4-Ig binding to APC surface cis-CD80:PD-L1 complexes can release the inhibitory ligand PD-L1, but whether this contributes to T cell inhibition remains unclear. Here, we show that PD-L1 liberation by CTLA4-Ig is strictly limited, both in extent and context, relative to PD-L1-competing anti-CD80 antibodies. At APC surface CD80:PD-L1 ratios exceeding 2:1, CTLA4-Ig therapies fail to release PD-L1 regardless of their CD80 affinity. Additionally, introducing flexibility into CTLA4-Ig by modifying its rigid homodimer interface produces biologics that retain bivalent CD80 binding without dissociating cis-bound PD-L1. These findings demonstrate that CTLA4-Ig therapies liberate PD-L1 through a CD80 reorientation mechanism that imposes a strict context dependence to their PD-1 checkpoint agonism and resultant T cell inhibition., Competing Interests: Declaration of interests E.P.O., N.J.K., K.J.G., M.M.G., and R.A.D. are inventors on International Patent Application PCT/AU2022/051296 (filed October 28, 2022) related to this work. E.P.O., N.J.K., and R.A.D. are co-founders of FLEX Immunotherapeutics. N.D.H. is CSO of oNKo-Innate Pty Ltd and declares ownership and funding not related to this work., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background and Aims: Myeloperoxidase (MPO) plays a critical role in the innate immune response and has been suggested to be a surrogate marker of oxidative stress and inflammation, with elevated levels implicated in cardiovascular diseases, such as atherosclerosis and heart failure, as well as in conditions like rheumatoid arthritis and cancer. While MPO is well-known in leukocytes, its expression and function in human endothelial cells remain unclear. This study investigates MPO expression in patient-derived endothelial colony-forming cells (ECFCs) and its potential association with CAD and mitochondrial function., Methods: ECFCs were cultured from the peripheral blood of 93 BioHEART-CT patients. MPO expression and associated functions were examined using qRT-PCR, immunochemistry, flow cytometry, and MPO activity assays. CAD presence was defined using CT coronary angiography (CACS > 0)., Results: We report MPO presence in patient-derived ECFCs for the first time. MPO protein expression occurred in 70.7% of samples ( n = 41) which had nuclear co-localisation, an atypical observation given its conventional localisation in the granules of neutrophils and monocytes. This suggests potential alternative roles for MPO in nuclear processes. MPO mRNA expression was detected in 66.23% of samples ( n = 77). CAD patients had a lower proportion of MPO-positive ECFCs compared to non-CAD controls (57.45% vs. 80%, p = 0.04), a difference that persisted in the statin-naïve sub-cohort (53.85% vs. 84.62%, p = 0.02). Non-CAD patients with MPO expression showed upregulated mitochondrial-antioxidant genes ( AIFM2 , TXNRD1 , CAT , PRDX3 , PRDX6 ). In contrast, CAD patients with MPO gene expression had heightened mROS production and mitochondrial mass and decreased mitochondrial function compared to that of CAD patients without MPO gene expression., Conclusions: MPO is present in the nucleus of ECFCs. In non-CAD ECFCs, MPO expression is linked to upregulated mitochondrial-antioxidant genes, whereas in CAD ECFCs, it is associated with greater mitochondrial dysfunction.

Background: Caloric vestibular stimulation (CVS) is a well-established neurological diagnostic technique that also induces many phenomenological modulations, including reductions in phantom limb pain (PLP), spinal cord injury pain (SCIP), and central post-stroke pain., Objective: We aimed to assess in a variety of persistent pain (PP) conditions (i) short-term pain modulation by CVS relative to a forehead ice pack cold-arousal control procedure and (ii) the duration and repeatability of CVS modulations. The tolerability of CVS was also assessed and has been reported separately., Methods: We conducted a convenience-based non-randomised single-blinded placebo-controlled study. Thirty-eight PP patients were assessed (PLP, n = 8; SCIP, n = 12; complex regional pain syndrome, CRPS, n = 14; non-specific PP, n = 4). Patients underwent 1-3 separate-day sessions of iced-water right-ear CVS. All but four also underwent the ice pack procedure. Analyses used patient-reported numerical rating scale pain intensity (NRS-PI) scores for pain and allodynia., Results: Across all groups, NRS-PI for pain was significantly lower within 30 min post-CVS than post-ice pack ( p < 0.01). Average reductions were 24.8% (CVS) and 6.4% (ice pack). CRPS appeared most responsive to CVS, while PLP and SCIP responses were less than expected from previous reports. The strongest CVS pain reductions lasted hours to over three weeks. CVS also induced substantial reductions in allodynia in three of nine allodynic CRPS patients, lasting 24 h to 1 month. As reported elsewhere, only one patient experienced emesis and CVS was widely rated by patients as a tolerable PP management intervention., Conclusions: Although these results require interpretative caution, CVS was found to modulate pain relative to an ice pack control. CVS also modulated allodynia in some cases. CVS should be examined for pain management efficacy using randomised controlled trials.

Wildfires have devastating effects on society and public health. However, little evidence from population-based cohort has been performed to analyze the relationship of wildfire-related PM 2.5 , an important component of wildfire smoke, with cancer-specific mortality. We aimed to explore this relationship and identify vulnerable populations in UK with lower levels of wildfire-related PM 2.5 exposure. The study consisted of 492394 participants (age: 38-73 years) recruited by UK Biobank during 2004-2010. The cumulative wildfire-related PM 2.5 within 10 kilometers of residence over three years was used as exposure, which was assessed by chemical transport and machine learning models. A time-varying Cox regression was utilized to explore the relationship of exposure with diverse cancer-specific mortality outcomes. Subgroup analyses of a range of potential modifiers were performed. Each 10 μg/m 3 increment of 3-year cumulative exposure was related to a 0.4 % greater risk of total cancer (95 %CI: 1.001-1.007), a 1.1 % greater risk of lung cancer (95 %CI: 1.004-1.018), and a 2.7 % greater risk of lip, oral cavity and pharynx (LOP) cancer (95 %CI: 1.005-1.049). Higher vulnerability in the wildfire-related PM 2.5 -lung cancer relationship was found among participants being retired than those with other employment status. Even lower levels of exposure to PM 2.5 from wildfires were related to elevated mortality risks for cancer from total, lung, LOP, highlighting the importance of wildfire prevention and control. Further investigations are warranted to enrich and extend existing knowledge in this field., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Drug-drug interactions (DDIs) can result in unexpected pharmacological outcomes, including adverse drug events, which are crucial for drug discovery. Graph neural networks have substantially advanced our ability to model molecular representations; however, the precise identification of key local structures and the capture of long-distance structural correlations for better DDI prediction and interpretation remain significant challenges., Results: Here, we present DrugDAGT, a dual-attention graph transformer framework with contrastive learning for predicting multiple DDI types. The dual-attention graph transformer incorporates attention mechanisms at both the bond and atomic levels, thereby enabling the integration of short and long-range dependencies within drug molecules to pinpoint key local structures essential for DDI discovery. Moreover, DrugDAGT further implements graph contrastive learning to maximize the similarity of representations across different views for better discrimination of molecular structures. Experiments in both warm-start and cold-start scenarios demonstrate that DrugDAGT outperforms state-of-the-art baseline models, achieving superior overall performance. Furthermore, visualization of the learned representations of drug pairs and the attention map provides interpretable insights instead of black-box results., Conclusions: DrugDAGT provides an effective tool for accurately predicting multiple DDI types by identifying key local chemical structures, offering valuable insights for prescribing medications, and guiding drug development. All data and code of our DrugDAGT can be found at https://github.com/codejiajia/DrugDAGT ., (© 2024. The Author(s).)

Our understanding of heterochromatin nanostructure and its capacity to mediate gene silencing in a living cell has been prevented by the diffraction limit of optical microscopy. Thus, here to overcome this technical hurdle, and directly measure the nucleosome arrangement that underpins this dense chromatin state, we coupled fluorescence lifetime imaging microscopy (FLIM) of Förster resonance energy transfer (FRET) between histones core to the nucleosome, with molecular editing of heterochromatin protein 1 alpha (HP1α). Intriguingly, this super-resolved readout of nanoscale chromatin structure, alongside fluorescence fluctuation spectroscopy (FFS) and FLIM-FRET analysis of HP1α protein-protein interaction, revealed nucleosome arrangement to be differentially regulated by HP1α oligomeric state. Specifically, we found HP1α monomers to impart a previously undescribed global nucleosome spacing throughout genome architecture that is mediated by trimethylation on lysine 9 of histone H3 (H3K9me3) and locally reduced upon HP1α dimerisation. Collectively, these results demonstrate HP1α to impart a dual action on chromatin that increases the dynamic range of nucleosome proximity. We anticipate that this finding will have important implications for our understanding of how live cell heterochromatin structure regulates genome function., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Tissue-resident memory T (T RM ) cells are integral to tissue immunity, persisting in diverse anatomical sites where they adhere to a common transcriptional framework. How these cells integrate distinct local cues to adopt the common T RM cell fate remains poorly understood. Here, we show that whereas skin T RM cells strictly require transforming growth factor β (TGF-β) for tissue residency, those in other locations utilize the metabolite retinoic acid (RA) to drive an alternative differentiation pathway, directing a TGF-β-independent tissue residency program in the liver and synergizing with TGF-β to drive T RM cells in the small intestine. We found that RA was required for the long-term maintenance of intestinal T RM populations, in part by impeding their retrograde migration. Moreover, enhanced RA signaling modulated T RM cell phenotype and function, a phenomenon mirrored in mice with increased microbial diversity. Together, our findings reveal RA as a fundamental component of the host-environment interaction that directs immunosurveillance in tissues., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

αβ T cell receptors (αβTCRs) co-recognise antigens when bound to Major Histocompatibility Complex (MHC) or MHC class I-like molecules. Additionally, some αβTCRs can bind non-MHC molecules, but how much intact antigen reactivities are achieved remains unknown. Here, we identify an αβ T cell clone that directly recognises the intact foreign protein, R-phycoerythrin (PE), a multimeric (αβ) 6 γ protein complex. This direct αβTCR-PE interaction occurs in an MHC-independent manner, yet triggers T cell activation and bound PE with an affinity comparable to αβTCR-peptide-MHC interactions. The crystal structure reveals how six αβTCR molecules simultaneously engage the PE hexamer, mediated by the complementarity-determining regions (CDRs) of the αβTCR. Here, the αβTCR mainly binds to two α-helices of the globin fold in the PE α-subunit, which is analogous to the antigen-binding platform of the MHC molecule. Using retrogenic mice expressing this TCR, we show that it supports intrathymic T cell development, maturation, and exit into the periphery as mature CD4/CD8 double negative (DN) T cells with TCR-mediated functional capacity. Accordingly, we show how an αβTCR can recognise an intact foreign protein in an antibody-like manner., (© 2024. The Author(s).)

The emergence of multi-drug resistant Gram-negative bacteria has led to renewed interest in the antimicrobial activity of polymyxins and novel polymyxin analogues (e.g. nonapeptides and octapeptin). In some individuals, clinically used polymyxins can cause acute hypersensitivity reactions through mast cell activation, with a recent study attributing this effect to activation of the MAS-related G protein-coupled receptor X2 (MRGPRX2). In the present study, HEK293 cells expressing human MRGPRX2 and the human mast cell line LAD2 were used to characterize the activity of the broader family of polymyxins. Octapeptin C4, polymyxin B and colistin produced concentration-dependent calcium mobilization, degranulation, and CCL-2 (MCP-1) release in LAD2 mast cells, with the former being highly potent. CRISPR-Cas9 knockdown of MRGPRX2 in LAD2 cells and a MRGPRX2 inverse agonist caused a significant reduction in calcium mobilization, degranulation, and CCL-2 release, demonstrating dependency on MRGPRX2 expression. In contrast, polymyxin nonapeptides were far less potent calcium mobilisers and failed to induce functional degranulation in LAD2 cells. Our results confirm that activation of mast cells induced by polymyxin-related antibiotics is MRGPRX2-dependent and reveal that octapeptin C4 might be more liable, whilst nonapeptides are less liable, to trigger immediate hypersensitivity reactions clinically. The mechanism underpinning the difference in MRGPRX2 activation between polymyxin-related antibiotics is important to better understand as it may help design new, safer polymyxins and guide the optimal clinical use of existing polymyxin drugs., Competing Interests: Declaration of competing interest JL and TV have licensed their new-generation lipopeptide antibiotics to Qpex Biopharma and Brii Biosciences. JL is Chief Executive Officer of Sliabx Pharmaceuticals and Cinfinno Biotech and holds equity in the companies. JL received grants, seminar honoraria, and consultation fees from Northern Antibiotics, Qpex Biopharma, Genentech, Healcare Pharmaceuticals, CTTQ, Aosaikang, Jiayou Medicine, MedCom, DanDi Bioscience, Xellia ApS and Brii Biosciences. All other authors declare no conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

The type VI secretion system (T6SS) is a molecular machine utilised by many Gram-negative bacteria to deliver antibacterial toxins into adjacent cells. Here we present the structure of Tse15, a T6SS Rhs effector from the nosocomial pathogen Acinetobacter baumannii. Tse15 forms a triple layered β-cocoon Rhs domain with an N-terminal α-helical clade domain and an unfolded C-terminal toxin domain inside the Rhs cage. Tse15 is cleaved into three domains, through independent auto-cleavage events involving aspartyl protease activity for toxin self-cleavage and a nucleophilic glutamic acid for N-terminal clade cleavage. Proteomic analyses identified that significantly more peptides from the N-terminal clade and toxin domains were secreted than from the Rhs cage, suggesting toxin delivery often occurs without the cage. We propose the clade domain acts as an internal chaperone to mediate toxin tethering to the T6SS machinery. Conservation of the clade domain in other Gram-negative bacteria suggests this may be a common mechanism for delivery., (© 2024. The Author(s).)

Spermatogonial stem cells (SSCs) are essential for sustained sperm production, but SSC regulatory mechanisms and markers remain poorly defined. Studies have suggested that the Id family transcriptional regulator Id4 is expressed in SSCs and involved in SSC maintenance. Here, we used reporter and knockout models to define the expression and function of Id4 in the adult male germline. Within the spermatogonial pool, Id4 reporter expression and inhibitor of DNA-binding 4 (ID4) protein are found throughout the GFRα1+ fraction, comprising the self-renewing population. However, Id4 deletion is tolerated by adult SSCs while revealing roles in meiotic spermatocytes. Cultures of undifferentiated spermatogonia could be established following Id4 deletion. Importantly, ID4 loss in undifferentiated spermatogonia triggers ID3 upregulation, and both ID proteins associate with transcription factor partner TCF3 in wild-type cells. Combined inhibition of IDs in cultured spermatogonia disrupts the stem cell state and blocks proliferation. Our data therefore demonstrate critical but functionally redundant roles of IDs in SSC function., Competing Interests: Declaration of interests F.J.R. receives institutional support as a co-investigator and is subcontracted by Peter MacCallum Cancer Centre for an investigator-initiated trial, which receives funding from Sanofi/Regeneron Pharmaceuticals., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Major risk factors of cardiovascular disease (CVD) include hypertension, obesity, diabetes mellitus and metabolic syndrome; all of which are considered inflammatory conditions. Women are disproportionately affected by inflammatory conditions, with sex differences emerging as early as adolescence. Hormonal fluctuations associated with reproductive events such as menarche, pregnancy and menopause, are hypothesized to promote a pro-inflammatory state in women. Moreover, women who have experienced inflammatory-type conditions such as polycystic ovarian syndrome (PCOS), gestational diabetes or pre-eclampsia, have a cardiometabolic phenotype that pre-disposes to increased risk of myocardial infarction, stroke and coronary heart disease. Women with no notable CVD risk factors are often relatively protected from CVD pre-menopause; but overtake men in risk of major cardiovascular events when the cardiovascular protective effects of oestrogen begin to wane. Sex differences and female-specific factors have long been considered challenging to study and this has led to an underrepresentation of females in clinical trials and lack of female-specific data from pre-clinical studies. However, there is now a clear prerogative to include females at all stages of research, despite inherent complexities and potential variability in data. This review explores recent advancements in our understanding of CVD in women. We summarise the underlying factors unique to women that can promote CVD risk factors, ultimately contributing to CVD burden and the emerging therapies aimed to combat this., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)