Ane Landajuela, Martha Braun, Christopher D.A. Rodrigues, Erdem Karatekin

Membrane fission is an essential process in all domains of life. The underlying mechanisms remain poorly understood in bacteria, partly because suitable assays are lacking. Here, we describe an assay to detect membrane fission during endospore formation in single Bacillus subtilis cells with a temporal resolution of ∼1 min. Other cellular processes can be quantified and temporally aligned to the membrane fission event in individual cells, revealing correlations and causal relationships.

STAR Protocols. March 2024

Kevin D. Whitley, James Grimshaw, David M. Roberts, Eleni Karinou, Phillip J. Stansfeld & Seamus Holden

Bacterial cell division requires septal peptidoglycan (sPG) synthesis by the divisome complex. Treadmilling of the essential tubulin homologue FtsZ has been implicated in septal constriction, though its precise role remains unclear. Here we used live-cell single-molecule imaging of the divisome transpeptidase PBP2B to investigate sPG synthesis dynamics in Bacillus subtilis. In contrast to previous models, we observed a single population of processively moving PBP2B molecules whose motion is driven by peptidoglycan synthesis and is not associated with FtsZ treadmilling. However, despite the asynchronous motions of PBP2B and FtsZ, a partial dependence of PBP2B processivity on FtsZ treadmilling was observed. Additionally, through single-molecule counting experiments we provide evidence that the divisome synthesis complex is multimeric. Our results support a model for B. subtilis division where a multimeric synthesis complex follows a single track dependent on sPG synthesis whose activity and dynamics are asynchronous with FtsZ treadmilling.

Nature Microbiology. March 2024

Talal Al-Yazeedi, Sally Adams, Sophie Tandonnet, Anisa Turner, Jun Kim, Junho Lee, Andre Pires-daSilva

Auanema freiburgense is a nematode with males, females, and selfing hermaphrodites. When XO males mate with XX females, they typically produce a low proportion of XO offspring because they eliminate nullo-X spermatids. This occurs because of an unequal distribution of essential cellular organelles during sperm formation. Intriguingly, the interbreeding of two A. freiburgense strains results in hybrid males capable of producing viable nullo-X sperm. Consequently, when these hybrid males mate with females, they yield a high percentage of male offspring. To uncover the genetic basis of nullo-spermatid elimination and X- chromosome drive, we generated a genome assembly for A. freiburgense, and genotyped the intercrossed lines. This analysis identified a Quantitative Trait Locus spanning several X chromosome genes linked to the non-Mendelian inheritance patterns observed in A. freiburgense. This finding provides valuable clues to the underlying factors involved in asymmetric organelle partitioning during male meiotic division and thus non-Mendelian transmission of the X chromosome and sex ratios.

Genetics. March 2024

Javier Sánchez Romano, Jaione Simón-Santamaria, Peter McCourt, Bård Smedsrød, Kim Erlend Mortensen, Antonia P. Sagona, Karen Kristine Sørensen, Anett Kristin Larsen

Blood-borne phages are believed to be captured by macrophages in the liver and spleen. Since liver sinusoids also consist of specialized scavenger liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs), this study investigated the contribution of both cell types in the elimination of Escherichia coli phage K1Fg10b::gfp (K1Fgfp) in mice. The results presented herein contribute to increased knowledge about the pharmacokinetics of the T7-like phage K1F in the mammalian system. The cell types of the liver that are responsible for rapid phage blood clearance are identified. Our results highlight the need for more research about appropriate dose regimens when phage therapy is delivered intravenously and advise essential knowledge about cell systems that should be investigated further for detailed phage pharmacodynamics.

mSphere. February 2024

Lan X.Y., Liang X.S., Cao M.X., Qin H.M., Chu C.Y., Boltze J., Li S.

The neural cell adhesion molecule (NCAM) promotes neural development and regeneration. Whether NCAM mimetic peptides could synergize with bone marrow mesenchymal stem cells (BMSCs) in stroke treatment deserves investigation. We found that the NCAM mimetic peptide P2 promoted BMSC proliferation, migration, and neurotrophic factor expression, protected neurons from oxygen-glucose deprivation through ERK and PI3K/AKT activation and anti-apoptotic mechanisms in vitro. Following middle cerebral artery occlusion (MCAO) in rats, P2 alone or in combination with BMSCs inhibited neuronal apoptosis and induced the phosphorylation of ERK and AKT. P2 combined with BMSCs enhanced neurotrophic factor expression and BMSC proliferation in the ischemic boundary zone. Moreover, combined P2 and BMSC therapy induced translocation of nuclear factor erythroid 2-related factor, upregulated heme oxygenase-1 expression, reduced infarct volume, and increased functional recovery as compared to monotreatments. Treatment with LY294002 (PI3K inhibitor) and PD98059 (ERK inhibitor) decreased the neuroprotective effects of combined P2 and BMSC therapy in MCAO rats. Collectively, P2 is neuroprotective while P2 and BMSCs work synergistically to improve functional outcomes after ischemic stroke, which may be attributed to mechanisms involving enhanced BMSC proliferation and neurotrophic factor release, anti-apoptosis, and PI3K/AKT and ERK pathways activation.

Journal of Cerebral Blood Flow & Metabolism. January 2024

Huba L Marton, Apoorva Bhatt, Antonia P Sagona, Peter Kilbride, Matthew I Gibson

Bacteriophages have many biotechnological and therapeutic applications, but as with other biologics, cryopreservation is essential for storage and distribution. Macromolecular cryoprotectants are emerging for a range of biologics, but the chemical space for polymer-mediated phage cryopreservation has not been explored. Here we screen the cryoprotective effect of a panel of polymers against five distinct phages, showing that nearly all the tested polymers provide a benefit. This work shows that phages are amenable to protection with hydrophilic polymers and opens up new opportunities for advanced formulations for future phage therapies and to take advantage of the additional functionality brought by the polymers.

Biomacromolecules. December 2023

Mononuclear cell therapy of neonatal hypoxic-ischemic encephalopathy in preclinical versus clinical studies: a systematic analysis of therapeutic efficacy and study design

Scrutton A. M., Ollis F., Boltze J

Hypoxic-ischemic encephalopathy (HIE) is a devastating condition affecting around 8.5 in 1000 newborns globally. Therapeutic hypothermia (TH) can reduce mortality and, to a limited extent, disability after HIE. Nevertheless, there is a need for new and effective treatment strategies. Here, we conducted a systematic review and meta-analysis. and analyzed overall MNC efficacy in preclinical trials, the methodological quality of preclinical trials, and relevant design features in preclinical versus clinical trials. Based on the analyzed data, it is unlikely that therapeutic effect size is massively overestimated in preclinical studies. It is more plausible that the many design differences between preclinical and clinical trials are responsible for the so far lacking proof of the efficacy of MNC treatments in HIE. Additional preclinical and clinical research is required to optimize the application of MNC for experimental HIE treatment.

Neuroprotection. December 2023

MCC950 reduces autophagy and improves cognitive function by inhibiting NLRP3-dependent neuroinflammation in a rat model of Alzheimer's disease

Abdul Naeem, Ravi Prakash, Neha Kumari, Mohsin Ali Khan, Abdul Quaiyoom Khan, Shahab Uddin, Sandeep Verma, Avril AB Robertson, Johannes Boltze, Syed Shadab Raza

In this study, we investigated the potential therapeutic effects of MCC950 on NLRP3-mediated inflammasome-driven inflammation and autophagy in Alzheimer's disease (AD). . MCC950 effectively suppressed STZ-induced cognitive impairment and anxiety by inhibiting NLRP3-dependent neuroinflammation. Moreover, our findings indicate that MCC950 exerts neuroprotective effects by attenuating autophagy in neuronal cells. The inhibiting effects of MCC950 on inflammasome activation and autophagy were reproduced in vitro, provding further mechansistic insights into MCC950 therapeutic action. Our findings suggest that MCC950 impedes the progression of AD and may also improve cognitive function through the mitigation of autophagy and NLRP3 inflammasome.

Brain, Behavior and Immunity. December 2023

María Florencia Mammarella, Leandro Lucero, Nosheen Hussain, Aitor Muñoz-Lopez, Ying Huang, Lucia Ferrero, Guadalupe L Fernandez-Milmanda, Pablo Manavella, Moussa Benhamed, Martin Crespi, Carlos L Ballare, Jose Gutierrez-Marcos, Pilar Cubas, Federico Ariel

The long noncoding RNA (lncRNA) AUXIN-REGULATED PROMOTER LOOP (APOLO) recognizes a subset of target loci across the Arabidopsis thaliana genome by forming RNA–DNA hybrids (R-loops) and modulating local three-dimensional chromatin conformation. Here, we show that APOLO regulates shade avoidance syndrome by dynamically modulating expression of key factors. We show that direct application of APOLO RNA to leaves results in a rapid increase in auxin signaling that is associated with changes in the plant response to far-red light. Collectively, our data support the view that lncRNAs coordinate shade avoidance syndrome in A. thaliana, and reveal their potential as exogenous bioactive molecules. Deploying exogenous RNAs that modulate plant–environment interactions may therefore become a new tool for sustainable agriculture.

EMBO Journal. December 2023

Chaweewan Sirakawin, Dongfa Lin, Ziyue Zhou, Xiaoxin Wang, Rhianne Kelleher, Shangyuan Huang, Weimiao Long, Andre Pires-da Silva, Yu Liu, Jingjing Wang, Ilya A. Vinnikov

Vitamin A (VA) is a micronutrient essential for the physiology of many organisms, but its role in longevity and age-related diseases remains unclear. In this work, we used Caenorhabditis elegans to study the impact of various bioactive compounds on lifespan. We demonstrate that VA extends lifespan and reduces lipofuscin and fat accumulation while increasing resistance to heat and oxidative stress. This resistance can be attributed to high levels of detoxifying enzymes called glutathione S-transferases, induced by the transcription factor skinhead-1 (SKN-1). Notably, VA upregulated the transcript levels of skn-1 or its mammalian ortholog NRF2 in both C. elegans, human cells, and liver tissues of mice. Moreover, the loss-of-function genetic models demonstrated a critical involvement of the SKN-1 pathway in longevity extension by VA. Our study thus provides novel insights into the molecular mechanism of anti-aging and anti-oxidative effects of VA, suggesting that this micronutrient could be used for the prevention and/or treatment of age-related disorders.

Aging Cell. December 2023

Taguchi A., Okinaka Y., Takeda A., Okamoto T., Boltze J., Claussen C., Gul S

It is proposed that age-related brain dysfunction may not necessarily result from the accumulation of uncontrollable disorders, but rather the natural deterioration of brain function following expiration of the limited innate program to preserve the brain in a healthy condition. We now have identified a means by which this process could potentially be mitigated or even partially reversed by applying stem cell therapy. Furthermore, we propose that the activation of neurogenesis in the hippocampus, also through stem cell therapy, is a promising therapeutic target in AD.

Neuroprotection. November 2023

T. Bertie Ansell, Wanling Song, Claire E. Coupland, Loic Carrique, Robin A. Corey, Anna L. Duncan, C. Keith Cassidy, Maxwell M. G. Geurts, Tim Rasmussen, Andrew B. Ward, Christian Siebold, Phillip J. Stansfeld, Mark S. P. Sansom

Cryo-electron microscopy (cryo-EM) enables the determination of membrane protein structures in native-like environments. Characterising how membrane proteins interact with the surrounding membrane lipid environment is assisted by resolution of lipid-like densities visible in cryo-EM maps. Nevertheless, establishing the molecular identity of putative lipid and/or detergent densities remains challenging. Here we present LipIDens, a pipeline for molecular dynamics (MD) simulation-assisted interpretation of lipid and lipid-like densities in cryo-EM structures.

Nature Communications. November 2023

Tang T., Li D., Fan T.P., Guo L.J., Lan X.Y., Bi C.J., Boltze J., Thomas A.M., Zhao X.S., Mo M., Zhao M.H., Ji X., Li S

Timely recognition of futile recanalization might enable a prompter response and thus improve outcomes in patients receiving successful thrombectomy. This study aims to evaluate whether postoperative fibrinogen-to-albumin ratio (FAR) could act as an indicator of futile recanalization.This is a single-center, retrospective analysis of patients with acute anterior circulation large-vessel occlusion and successful thrombectomy between May 2019 and June 2022. A total of 255 patients were enrolled, amongst which 34.1% had high postoperative FAR. Futile recanalization was more prevalent among patients with high FAR compared to those with low FAR. After adjusting for potential confounders, high postoperative FAR was found to independently correspond with the occurrence of futile. This association was consistently observed regardless of prior antithrombotic therapy, treatment of intravenous thrombolysis, occlusion site, time from symptom onset to groin puncture, and reperfusion status. Our findings support high postoperative FAR serving as an indicator of futile recanalization in patients with anterior circulation large-vessel occlusion and successful thrombectomy.

Brain & Behavior. November 2023

Patrick Becker, Fiona B. Naughton, Deborah H. Brotherton, Raul Pacheco-Gomez, Oliver Beckstein, Alexander D. Cameron

The bile acid sodium symporter (BASS) family transports a wide array of molecules across membranes, including bile acids in humans, and small metabolites in plants. These transporters, many of which are sodium-coupled, have been shown to use an elevator mechanism of transport, but exactly how substrate binding is coupled to sodium ion binding and transport is not clear. Here, we solve the crystal structure at 2.3 Å of a transporter from Neisseria meningitidis (ASBTNM) in complex with pantoate, a potential substrate of ASBTNM.. Comparison of structures in the presence and absence of pantoate demonstrates that pantoate elicits a conformational change in one of the cross-over helices. This modifies the interface between the two domains that move relative to one another to elicit the elevator mechanism. These results have implications, not only for ASBTNM but for the BASS family as a whole and indeed other transporters that work through the elevator mechanism.

eLife. November 2023

Rie Nygaard, Chris L. B. Graham, Meagan Belcher Dufrisne, Jonathan D. Colburn, Joseph Pepe, Molly A. Hydorn, Silvia Corradi, Chelsea M. Brown, Khuram U. Ashraf, Owen N. Vickery, Nicholas S. Briggs, John J. Deering, Brian Kloss, Bruno Botta, Oliver B. Clarke, Linda Columbus, Jonathan Dworkin, Phillip J. Stansfeld, David I. Roper & Filippo Mancia

Peptidoglycan (PG) assembly requires a glycosyltransferase (GT) to generate a glycan polymer using a Lipid II substrate, which is then crosslinked to the existing PG via a transpeptidase (TP) reaction. A Shape, Elongation, Division and Sporulation (SEDS) GT enzyme and a Class B Penicillin Binding Protein (PBP) form the core of the multi-protein complex required for PG assembly. Here we used single particle cryo-electron microscopy to determine the structure of a cell elongation-specific E. coli RodA-PBP2 complex. We combine this information with biochemical, genetic, spectroscopic, and computational analyses to identify the Lipid II binding sites and propose a mechanism for Lipid II polymerization. Our data suggest a hypothesis for the movement of the glycan strand from the Lipid II polymerization site of RodA towards the TP site of PBP2, functionally linking these two central enzymatic activities required for cell wall peptidoglycan biosynthesis.

Nature Communications. August 2023

Chatzichristofi, Agathangelos, Sagris, Vasileios, Pallaris, Aristos, Eftychiou, Marios, Kalvari, Ioanna, Price, Nicholas, Theodosiou, Theodosios, Iliopoulos, Ioannis, Nezis, Ioannis P. and Promponas, Vasilis J

Several selective macroautophagy receptor and adaptor proteins bind members of the Atg8 (autophagy related 8) family using short linear motifs (SLiMs), most often referred to as Atg8-family interacting motifs (AIMs) or LC3-interacting regions (LIRs). AIM/LIR motifs have been extensively studied during the last fifteen years, since they can uncover the underlying biological mechanisms and possible substrates for this key catabolic process of eukaryotic cells. Prompted by the fact that experimental information regarding LIR motifs can be found scattered across heterogeneous literature resources, we have developed LIRcentral (https://lircentral.euLink opens in a new window), a freely available online repository for user-friendly access to comprehensive, high-quality information regarding LIR motifs from manually curated publications. Herein, we describe the development of LIRcentral and showcase currently available data and features, along with our plans for the expansion of this resource.

Autophagy. August 2023

Björn Nitzsche, Sabine Schulze, Johannes Boltze, Martin J. Schmidt

Pathomorphological alterations of the central nervous system in dogs, such as syringomyelia and Chiari-like malformation, can cause cranial and cervical hyperesthesia and neuropathic pain. The long-term activity of the pain network can induce functional alteration and eventually even morphological changes in the pain network. This may happen especially in the prefrontal and cingulate cortex, where atrophy of the gray matter (GM) was observed in humans with chronic pain, irrespective of the nature of the pain syndrome. We tested the hypothesis that Cavalier King Charles Spaniels (CKCS) with Chiari-like malformation and associated syringomyelia (SM) and pain show cerebral morphological differences compared to animals without signs of syringomyelia and pain. We found that GM atrophy in the CG is associated with chronic pain and thus may serve as an objective readout parameter for the diagnosis or treatment of canine pain syndromes.

Frontiers in Neuroanatomy. July 2023

Kelsey Cremin, Gabriel N Meloni, Dimitrios Valavanis, Orkun S Soyer and Patrick R Unwin

Ultramicroelectrode (UME), or, equivalently, microelectrode, probes are increasingly used for single-cell measurements of cellular properties and processes, including physiological activity, such as metabolic fluxes and respiration rates. Major challenges for the sensitivity of such measurements include: (i) the relative magnitude of cellular and UME fluxes (manifested in the current); and (ii) issues around the stability of the UME response over time. To explore the extent to which these factors impact the precision of electrochemical cellular measurements, we undertake a systematic analysis of measurement conditions and experimental parameters for determining single cell respiration rates via the oxygen consumption rate (OCR) in single HeLa cells. We provide a set of model-based suggestions for improving these measurements in the future but highlight that extraordinary improvements in the stability and precision of SECM measurements will be required if single cell OCR measurements are to be realized.

ACS Measurement Science. July 2023

D Xu, W Jiang, . Wu, RG Gaudet, E-S Park, M Su, SK Cheppali, NR Cheemarla, P Kumar, PD Uchil, JR Grover, EF Foxman, CM Brown, PJ Stansfeld, J Bewersdorf, W Mothes, E Karatekin, CB Wilen, and JD MacMicking

Understanding protective immunity to COVID-19 facilitates preparedness for future pandemics and combats new SARS-CoV-2 variants emerging in the human population. Neutralizing antibodies have been widely studied; however, on the basis of large-scale exome sequencing of protected versus severely ill patients with COVID-19, local cell-autonomous defence is also crucial. Here we identify phospholipid scramblase 1 (PLSCR1) as a potent cell-autonomous restriction factor against live SARS-CoV-2 infection in parallel genome-wide CRISPR–Cas9 screens of human lung epithelia and hepatocytes before and after stimulation with interferon-γ (IFNγ). IFNγ-induced PLSCR1 not only restricted SARS-CoV-2 USA-WA1/2020, but was also effective against the Delta B.1.617.2 and Omicron BA.1 lineages. . Our mechanistic studies, together with reports that COVID-associated PLSCR1 mutations are found in some susceptible people, identify an anti-coronavirus protein that interferes at a late entry step before viral RNA is released into the host-cell cytosol.

Nature. July 2023

Fuss MF, Wieferig JP, Corey RA, Hellmich Y, Tascón I, Sousa JS, Stansfeld PJ, Vonck J, Hänelt I

Cyclic di-AMP is the only known essential second messenger in bacteria and archaea, regulating different proteins indispensable for numerous physiological processes. In particular, it controls various potassium and osmolyte transporters involved in osmoregulation. In this study, we reveal the molecular basis of how c-di-AMP binding inhibits KimA. We report cryo-EM structures of KimA with bound c-di-AMP in detergent solution and reconstituted in amphipols. By combining structural data with functional assays and molecular dynamics simulations we reveal how c-di-AMP modulates transport. We show that an intracellular loop in the transmembrane domain interacts with c-di-AMP bound to the adjacent cytosolic domain. This reduces the mobility of transmembrane helices at the cytosolic side of the K+ binding site and therefore traps KimA in an inward-occluded conformation.

Nature Communications. June 2023

Sutthirat Sitthisak, Suphattra Manrueang, Supat Khongfak, Udomluk Leungtongkam, Rapee Thummeepak, Aunchalee Thanwisai, Nathan Burton, Gurneet K Dhanoa, Panagiotis Tsapras, Antonia P Sagona

Phage lytic enzymes are promising antimicrobial agents. In this study, an endolysin derived from vB_AbaM_PhT2 (vPhT2), was identified. This endolysin represented the conserved lysozyme domain. Recombinant endolysin (lysAB- vT2) and hydrophobic fusion endolysin (lysAB-vT2-fusion) were expressed and purified. Both endolysins showed lytic activity against bacterial crude cell wall of Gram-negative bacteria. The MIC of lysAB-vT2-fusion was 2 mg/ml corresponding to 100 µM, while the MIC of lysAB-vT2 was more than 10 mg/ml (400 µM). Combination of lysAB-vT2-fusion with colistin, polymyxin B or copper was synergistic against A. baumannii (FICI value as 0.25). Antibacterial activity of lysAB-vT2-fusion plus colistin at the fractional inhibitory concentrations (FICs) revealed that it can inhibit Escherichia coli, Klebsiella pneumoniae and various strains of extremely drug-resistant A. baumannii (XDRAB) and phage resistant A. baumannii. The lysAB- vT2-fusion still retained its antibacterial activity after incubating the enzyme at 4, 20, 40 and 60 °C for 30 min. The lysAB-vT2-fusion could inhibit the mature biofilm, and incubation of lysAB-vT2-fusion with T24 human cells infected with A. baumannii led to a partial reduction of LDH release from T24 cells. In summary, our study highlights the antimicrobial ability of engineered lysAB-vT2-fusion endolysin, which can be applied for the control of A. baumannii infection.

Scientific Reports. May 2023

Marton, Huba L., Kilbride, Peter, Ahmad, Ashfaq, Sagona, Antonia P. and Gibson, Matthew

Bioprocessing and biotechnology exploit microorganisms (such as bacteria) for the production of chemicals, biologics, therapies, and food. A major unmet challenge is that bacteriophage (phage) contamination compromises products and necessitates shut-downs and extensive decontamination using nonspecific disinfectants. Here we demonstrate that poly(acrylic acid) prevents phage-induced killing of bacterial hosts, prevents phage replication, and that induction of recombinant protein expression is not affected by the presence of the polymer. Poly(acrylic acid) was more active than poly(methacrylic acid), and poly(styrenesulfonate) had no activity showing the importance of the carboxylic acids. Initial evidence supported a virustatic, not virucidal, mechanism of action. This simple, low-cost, mass-produced additive offers a practical, scalable, and easy to implement solution to reduce phage contamination.

Journal of the American Chemical Society. April 2023 Press Release