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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 β6-β7 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., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

New Findings: What is the topic of this review? We review barosensory vessel mechanics and their role in blood pressure regulation across the lifespan. What advances does it highlight? In young normotensive men, aortic unloading mechanics contribute to the resting operating point of the vascular sympathetic baroreflex; however, with advancing age, this contribution is removed. This suggests that barosensory vessel unloading mechanics are not driving the well-documented age-related increase in resting muscle sympathetic nerve activity., Abstract: An age-associated increase in arterial blood pressure is evident for apparently healthy humans. This is frequently attributed to stiffening of the central arteries and a concurrent increase in sympathetic outflow, potentially mediated by a reduced ability of the baroreceptive vessels to distend. This is supported, in part, by a reduced mechanical component of the vascular sympathetic baroreflex (i.e., a reduction in distension for a given pressure). Previous characterization of the mechanical component has assessed only carotid artery distension; however, evidence suggests that both the aortic and carotid baroreflexes are integral to blood pressure regulation. In addition, given that baroreceptors are located in the vessel wall, the change in wall tension, comprising diameter, pressure and vessel wall thickness, and the mechanics of this change might provide a better index of the baroreceptor stimulus than the previous method used to characterize the mechanical component that relies on diameter alone. This brief review summarizes the data using this new method of assessing barosensory vessel mechanics and their influence on the vascular sympathetic baroreflex across the lifespan., (© 2023 The Authors. Experimental Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Autophagy is a homeostatic process critical for cellular survival, and its malfunction is implicated in human diseases including neurodegeneration. Loss of autophagy contributes to cytotoxicity and tissue degeneration, but the mechanistic understanding of this phenomenon remains elusive. Here, we generated autophagy-deficient (ATG5 -/- ) human embryonic stem cells (hESCs), from which we established a human neuronal platform to investigate how loss of autophagy affects neuronal survival. ATG5 -/- neurons exhibit basal cytotoxicity accompanied by metabolic defects. Depletion of nicotinamide adenine dinucleotide (NAD) due to hyperactivation of NAD-consuming enzymes is found to trigger cell death via mitochondrial depolarization in ATG5 -/- neurons. Boosting intracellular NAD levels improves cell viability by restoring mitochondrial bioenergetics and proteostasis in ATG5 -/- neurons. Our findings elucidate a mechanistic link between autophagy deficiency and neuronal cell death that can be targeted for therapeutic interventions in neurodegenerative and lysosomal storage diseases associated with autophagic defect., Competing Interests: Declaration of interests R.J. is cofounder of Fate Therapeutics, Fulcrum Therapeutics, and Omega Therapeutics and advisor to Dewpoint Therapeutics. E.S. is founder of NMN Bio Ltd. V.I.K. is a scientific advisor for Longaevus Technologies., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mitochondrial dysfunction involving mitochondria-associated ER membrane (MAM) dysregulation is implicated in the pathogenesis of late-onset neurodegenerative diseases, but understanding is limited for rare early-onset conditions. Loss of the MAM-resident protein WFS1 causes Wolfram syndrome (WS), a rare early-onset neurodegenerative disease that has been linked to mitochondrial abnormalities. Here we demonstrate mitochondrial dysfunction in human induced pluripotent stem cell-derived neuronal cells of WS patients. VDAC1 is identified to interact with WFS1, whereas loss of this interaction in WS cells could compromise mitochondrial function. Restoring WFS1 levels in WS cells reinstates WFS1-VDAC1 interaction, which correlates with an increase in MAMs and mitochondrial network that could positively affect mitochondrial function. Genetic rescue by WFS1 overexpression or pharmacological agents modulating mitochondrial function improves the viability and bioenergetics of WS neurons. Our data implicate a role of WFS1 in regulating mitochondrial functionality and highlight a therapeutic intervention for WS and related rare diseases with mitochondrial defects., Competing Interests: Conflict of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mitochondria are responsible for many vital pathways governing cellular homeostasis, including cellular energy management, heme biosynthesis, lipid metabolism, cellular proliferation and differentiation, cell cycle regulation, and cellular viability. Electron transport and ADP phosphorylation coupled with proton pumping through the mitochondrial complexes contribute to the preservation of mitochondrial membrane potential (ΔΨ m ). Importantly, mitochondrial polarization is essential for reactive oxygen species (ROS) production and cytosolic calcium (Ca 2+ ) handling. Thus, changes in mitochondrial oxidative phosphorylation (OXPHOS), ΔΨ m , and ATP/ADP may occur in parallel or stimulate each other. Brain cells like neurons are heavily reliant on mitochondrial OXPHOS for its high-energy demands, and hence improper mitochondrial function is detrimental for neuronal survival. Indeed, several neurodegenerative disorders are associated with mitochondrial dysfunction. Modeling this disease-relevant phenotype in neuronal cells differentiated from patient-derived human induced pluripotent stem cells (hiPSCs) provide an appropriate cellular platform for studying the disease pathology and drug discovery. In this review, we describe high-throughput analysis of crucial parameters related to mitochondrial function in hiPSC-derived neurons. These methodologies include measurement of ΔΨ m , intracellular Ca 2+ , oxidative stress, and ATP/ADP levels using fluorescence probes via a microplate reader. Benefits of such an approach include analysis of mitochondrial parameters on a large population of cells, simultaneous analysis of different cell lines and experimental conditions, and for drug screening to identify compounds restoring mitochondrial function., (© 2022. Springer Science+Business Media, LLC.)

The outer membrane of Gram-negative bacteria presents a robust physicochemical barrier protecting the cell from both the natural environment and acting as the first line of defense against antimicrobial materials. The proteins situated within the outer membrane are responsible for a range of biological functions including controlling influx and efflux. These outer membrane proteins (OMPs) are ultimately inserted and folded within the membrane by the β-barrel assembly machine (Bam) complex. The precise mechanism by which the Bam complex folds and inserts OMPs remains unclear. Here, we have developed a platform for investigating Bam-mediated OMP insertion. By derivatizing a gold surface with a copper-chelating self-assembled monolayer, we were able to assemble a planar system containing the complete Bam complex reconstituted within a phospholipid bilayer. Structural characterization of this interfacial protein-tethered bilayer by polarized neutron reflectometry revealed distinct regions consistent with known high-resolution models of the Bam complex. Additionally, by monitoring changes of mass associated with OMP insertion by quartz crystal microbalance with dissipation monitoring, we were able to demonstrate the functionality of this system by inserting two diverse OMPs within the membrane, pertactin, and OmpT. This platform has promising application in investigating the mechanism of Bam-mediated OMP insertion, in addition to OMP function and activity within a phospholipid bilayer environment., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The polymeric mucin MUC5B provides the structural and functional framework of respiratory mucus, conferring both viscoelastic and antimicrobial properties onto this vital protective barrier. Whilst it is established that MUC5B forms disulfide-linked linear polymers, how this relates to their packaging in secretory granules, and their molecular form in mucus remain to be fully elucidated. Moreover, the role of the central heavily O-glycosylated mucin domains in MUC5B conformation is incompletely described. Here we have completed a detailed structural analysis on native MUC5B polymers purified from saliva and subsequently investigated how MUC5B conformation is affected by changes in calcium concentration and pH, factors important for mucin intragranular packaging and post-secretory expansion. The results identify that MUC5B has a beaded structure repeating along the polymer axis and suggest that these repeating motifs arise from distinct glycosylation patterns. Moreover, we demonstrate that the conformation of these highly entangled linear polymers is sensitive to calcium concentration and changes in pH. In the presence of calcium (Ca 2+ , 10 mM) at pH 5.0, MUC5B adopted a compact conformation which was lost either upon removal of calcium with EGTA, or by increasing the pH to 7.4. These results suggest a pathway of mucin collapse to enable intracellular packaging and mechanisms driving mucin expansion following secretion. They also point to the importance of the tight control of calcium and pH during different stages of mucin biosynthesis and secretion, and in the generation of correct mucus barrier properties.

The Mla pathway is believed to be involved in maintaining the asymmetrical Gram-negative outer membrane via retrograde phospholipid transport. The pathway is composed of three components: the outer membrane MlaA-OmpC/F complex, a soluble periplasmic protein, MlaC, and the inner membrane ATPase, MlaFEDB complex. Here, we solve the crystal structure of MlaC in its phospholipid-free closed apo conformation, revealing a pivoting β-sheet mechanism that functions to open and close the phospholipid-binding pocket. Using the apo form of MlaC, we provide evidence that the inner-membrane MlaFEDB machinery exports phospholipids to MlaC in the periplasm. Furthermore, we confirm that the phospholipid export process occurs through the MlaD component of the MlaFEDB complex and that this process is independent of ATP. Our data provide evidence of an apparatus for lipid export away from the inner membrane and suggest that the Mla pathway may have a role in anterograde phospholipid transport.

In bacteria, the translocation of proteins across the cytoplasmic membrane by the Sec machinery requires the ATPase SecA. SecA binds ribosomes and recognises nascent substrate proteins, but the molecular mechanism of nascent substrate recognition is unknown. We investigated the role of the C-terminal tail (CTT) of SecA in nascent polypeptide recognition. The CTT consists of a flexible linker (FLD) and a small metal-binding domain (MBD). Phylogenetic analysis and ribosome binding experiments indicated that the MBD interacts with 70S ribosomes. Disruption of the MBD only or the entire CTT had opposing effects on ribosome binding, substrate-protein binding, ATPase activity and in vivo function, suggesting that the CTT influences the conformation of SecA. Site-specific crosslinking indicated that F399 in SecA contacts ribosomal protein uL29, and binding to nascent chains disrupts this interaction. Structural studies provided insight into the CTT-mediated conformational changes in SecA. Our results suggest a mechanism for nascent substrate protein recognition., Competing Interests: MJ, TK, SW, DW, FM, KR, MW, GH, GK, BB, DH No competing interests declared, (© 2019, Jamshad et al.)

Cystic fibrosis (CF) is characterized by increased mucus viscosity and delayed mucociliary clearance that contributes to progressive decline of lung function. Mucus in the respiratory and GI tract is excessively adhesive in the presence of airway dehydration and excess extracellular Ca2+ upon mucin release, promoting hyperviscous, densely packed mucins characteristic of CF. Therapies that target mucins directly through ionic interactions remain unexploited. Here we show that poly (acetyl, arginyl) glucosamine (PAAG), a polycationic biopolymer suitable for human use, interacts directly with mucins in a Ca2+-sensitive manner to reduce CF mucus viscoelasticity and improve its transport. Notably, PAAG induced a linear structure of purified MUC5B and altered its sedimentation profile and viscosity, indicative of proper mucin expansion. In vivo, PAAG nebulization improved mucociliary transport in CF rats with delayed mucus clearance, and cleared mucus plugging in CF ferrets. This study demonstrates the potential use of a synthetic glycopolymer PAAG as a molecular agent that could benefit patients with a broad array of mucus diseases.

Aim: There is a requirement of an expansive and up to date review of surgical management of inflammatory bowel disease (IBD) that can dovetail with the medical guidelines produced by the British Society of Gastroenterology., Methods: Surgeons who are members of the ACPGBI with a recognised interest in IBD were invited to contribute various sections of the guidelines. They were directed to produce a procedure based document using literature searches that were systematic, comprehensible, transparent and reproducible. Levels of evidence were graded. An editorial board was convened to ensure consistency of style, presentation and quality. Each author was asked to provide a set of recommendations which were evidence based and unambiguous. These recommendations were submitted to the whole guideline group and scored. They were then refined and submitted to a second vote. Only those that achieved >80% consensus at level 5 (strongly agree) or level 4 (agree) after 2 votes were included in the guidelines., Results: All aspects of surgical care for IBD have been included along with 157 recommendations for management., Conclusion: These guidelines provide an up to date and evidence based summary of the current surgical knowledge in the management of IBD and will serve as a useful practical text for clinicians performing this type of surgery., (© 2018 Authors. Colorectal Disease published by John Wiley & Sons Ltd on behalf of Association of Coloproctology of Great Britain and Ireland.)

Gastrointestinal nematodes, such as Trichuris trichiura (human whipworm), are a major source of morbidity in humans and their livestock. There is a paucity of commercially available vaccines against these parasites, and vaccine development for T. trichiura has been impeded by a lack of known host protective antigens. Experimental vaccinations with T. muris (murine whipworm) soluble Excretory/Secretory (ES) material have demonstrated that it is possible to induce protective immunity in mice; however, the potential for extracellular vesicles (EVs) as a source of antigenic material has remained relatively unexplored. Here, we demonstrate that EVs isolated from T. muris ES can induce protective immunity in mice when administered as a vaccine without adjuvant and show that the protective properties of these EVs are dependent on intact vesicles. We also identified several proteins within EV preparations that are targeted by the host antibodies following vaccination and subsequent infection with T. muris. Many of these proteins, including VWD and vitellogenin N and DUF1943-domain-containing protein, vacuolar protein sorting-associated protein 52 and TSP-1 domain-containing protein, were detected in both soluble ES and EV samples and have homologues in other parasites of medical and veterinary importance, and as such are possible protective antigens., (© 2018 The Authors. Parasite Immunology Published by John Wiley & Sons Ltd.)

Mucosal surfaces represent critical routes for entry and exit of pathogens. As such, animals have evolved strategies to combat infection at these sites, in particular the production of mucus to prevent attachment and to promote subsequent movement of the mucus/microbe away from the underlying epithelial surface. Using biochemical, biophysical, and infection studies, we have investigated the host protective properties of the skin mucus barrier of the Xenopus tropicalis tadpole. Specifically, we have characterized the major structural component of the barrier and shown that it is a mucin glycoprotein (Otogelin-like or Otogl) with similar sequence, domain organization, and structural properties to human gel-forming mucins. This mucin forms the structural basis of a surface barrier (∼6 μm thick), which is depleted through knockdown of Otogl. Crucially, Otogl knockdown leads to susceptibility to infection by the opportunistic pathogen Aeromonas hydrophila To more accurately reflect its structure, tissue localization, and function, we have renamed Otogl as Xenopus Skin Mucin, or MucXS. Our findings characterize an accessible and tractable model system to define mucus barrier function and host-microbe interactions., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)