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This work was supported by the the University of Tours (to IDP and FDG), the University of Montpellier (to SD and EC), the Deutsche Forschungsgemeinschaft (to RTS), the Wellcome Trust project grant 093228 (to TKS) and the Campus France/DAAD PHC PROCOPE 24931RE (to RTS and EC). The funding source has no involvement in the conduct of the research and preparation of the article. Glycosylphosphatidylinositols (GPIs) are glycolipids described as toxins of protozoan parasites due to their inflammatory properties in mammalian hosts characterized by the production of interleukin (IL)-1, IL-12 and tumor necrosis factor (TNF)-α. In the present work, we studied the cytokines produced by antigen presenting cells in response to ten different GPI species extracted from Babesia divergens, responsible for babesiosis. Interestingly, B. divergens GPIs induced the production of anti-inflammatory cytokines (IL-2, IL-5) and of the regulatory cytokine IL-10 by macrophages and dendritic cells. In contrast to all protozoan GPIs studied until now, GPIs from B. divergens did not stimulate the production of TNF-α and IL-12, leading to a unique Th1/Th2 profile. Analysis of the carbohydrate composition of the B. divergens GPIs indicated that the di-mannose structure was different from the evolutionary conserved tri-mannose structure, which might explain the particular cytokine profile they induce. Expression of major histocompatibility complex (MHC) molecules on dendritic cells and apoptosis of mouse peritoneal cells were also analysed. B. divergens GPIs did not change expression of MHC class I, but decreased expression of MHC class II at the cell surface, while GPIs slightly increased the percentages of apoptotic cells. During pathogenesis of babesiosis, the inflammation-coagulation auto-amplification loop can lead to thrombosis and the effect of GPIs on coagulation parameters was investigated. Incubation of B. divergens GPIs with rat plasma ex vivo led to increase of fibrinogen levels and to prolonged activated partial thromboplastin time, suggesting a direct modulation of the extrinsic coagulation pathway by GPIs. Publisher PDF

Funding: This study was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) (Grant BB/M010996/1) via EASTBIO Doctoral Training Partnership studentships to G.F. and B.J.R., and by the University of St Andrews via a StARIS summer research bursary to S.K.L. The bifunctional enzyme phosphoribosyl-ATP pyrophosphohydrolase/phosphoribosyl-AMP cyclohydrolase (HisIE) catalyzes the second and third steps of histidine biosynthesis: pyrophosphohydrolysis of N1-(5-phospho-β-D-ribosyl)-ATP (PRATP) to N1-(5-phospho-β-D-ribosyl)-AMP (PRAMP) and pyrophosphate in the C-terminal HisE-like domain, and cyclohydrolysis of PRAMP to N-(5′-phospho-D-ribosylformimino)-5-amino-1-(5″-phospho-D-ribosyl)-4-imidazolecarboxamide (ProFAR) in the N-terminal HisI-like domain. Here we use UV–VIS spectroscopy and LC–MS to show Acinetobacter baumannii putative HisIE produces ProFAR from PRATP. Employing an assay to detect pyrophosphate and another to detect ProFAR, we established the pyrophosphohydrolase reaction rate is higher than the overall reaction rate. We produced a truncated version of the enzyme-containing only the C-terminal (HisE) domain. This truncated HisIE was catalytically active, which allowed the synthesis of PRAMP, the substrate for the cyclohydrolysis reaction. PRAMP was kinetically competent for HisIE-catalyzed ProFAR production, demonstrating PRAMP can bind the HisI-like domain from bulk water, and suggesting that the cyclohydrolase reaction is rate-limiting for the overall bifunctional enzyme. The overall kcat increased with increasing pH, while the solvent deuterium kinetic isotope effect decreased at more basic pH but was still large at pH 7.5. The lack of solvent viscosity effects on kcat and kcat/KM ruled out diffusional steps limiting the rates of substrate binding and product release. Rapid kinetics with excess PRATP demonstrated a lag time followed by a burst in ProFAR formation. These observations are consistent with a rate-limiting unimolecular step involving a proton transfer following adenine ring opening. We synthesized N1-(5-phospho-β-D-ribosyl)-ADP (PRADP), which could not be processed by HisIE. PRADP inhibited HisIE-catalyzed ProFAR formation from PRATP but not from PRAMP, suggesting that it binds to the phosphohydrolase active site while still permitting unobstructed access of PRAMP to the cyclohydrolase active site. The kinetics data are incompatible with a build-up of PRAMP in bulk solvent, indicating HisIE catalysis involves preferential channeling of PRAMP, albeit not via a protein tunnel. Publisher PDF

Introduction Fluoropyrimidines, principally 5-fluorouracil (5-FU), remain a key component of chemotherapy regimens for multiple cancer types, in particular colorectal and other gastrointestinal malignancies. To overcome key limitations and pharmacologic challenges that hinder the clinical utility of 5-FU, NUC-3373, a phosphoramidate transformation of 5-fluorodeoxyuridine, was designed to improve the efficacy and safety profile as well as the administration challenges associated with 5-FU. Methods Human colorectal cancer cell lines HCT116 and SW480 were treated with sub-IC50 doses of NUC-3373 or 5-FU. Intracellular activation was measured by LC–MS. Western blot was performed to determine binding of the active anti-cancer metabolite FdUMP to thymidylate synthase (TS) and DNA damage. Results We demonstrated that NUC-3373 generates more FdUMP than 5-FU, resulting in a more potent inhibition of TS, DNA misincorporation and subsequent cell cycle arrest and DNA damage in vitro. Unlike 5-FU, the thymineless death induced by NUC-3373 was rescued by the concurrent addition of exogenous thymidine. 5-FU cytotoxicity, however, was only reversed by supplementation with uridine, a treatment used to reduce 5-FU-induced toxicities in the clinic. This is in line with our findings that 5-FU generates FUTP which is incorporated into RNA, a mechanism known to underlie the myelosuppression and gastrointestinal inflammation associated with 5-FU. Conclusion Taken together, these results highlight key differences between NUC-3373 and 5-FU that are driven by the anti-cancer metabolites generated. NUC-3373 is a potent inhibitor of TS that also causes DNA-directed damage. These data support the preliminary clinical evidence that suggest NUC-3373 has a favorable safety profile in patients.

This work was supported by grants RTI2018-097099-B-I00 and PID2021-125010OB-I00 from Ministerio de Ciencia e Innovación (Spain). EM-L was supported by grant BES-2013-064540 (Ministerio de Ciencia e Innovación; Spain). Work in JT laboratory was supported by the Scottish Government’s Rural and Environment Science and Analytical Services Division (RESAS). Pepino mosaic virus (PepMV) is pandemic in tomato crops, causing important economic losses worldwide. No PepMV resistant varieties have been developed yet. Identification of host factors interacting with PepMV proteins is a promising source of genetic targets to develop PepMV resistant varieties. The interaction between the PepMV coat protein (CP) and the tomato glutathione S-transferase SlGSTU38 was identified in a yeast-two hybrid (Y2H) screening and validated by directed Y2H and co-immunoprecipitation assays. SlGSTU38-knocked out Micro-Tom plants (gstu38) generated by the CRISPR/Cas9 technology together with live-cell imaging were used to understand the role of SlGSTU38 during infection. The transcriptomes of healthy and PepMV-infected wild type (WT) and gstu38 plants were profiled by RNA-seq analysis. SlGSTU38 functions as a PepMV-specific susceptibility factor in a cell autonomous manner and re-localizes to the virus replication complexes during infection. Besides, knocking out SlGSTU38 triggers ROS accumulation in leaves and the deregulation of stress-responsive genes. SlGSTU38 may play a dual role: on the one hand, SlGSTU38 may exert a proviral function depending on its specific interaction with the PepMV CP; and on the other hand, SlGSTU38 may delay sensing PepMV infection by participating in the redox intra-cellular homeostasis in a non-specific manner. Publisher PDF

Funding: AG acknowledges the UK-MRBT-CDT funded by EPSRC (EP/J500045/1), BEB acknowledges the Wellcome Trust Institutional Strategic Support fund (204821/Z/16/Z), JET acknowledges the Leverhulme Trust (Early Career Fellowship; ECF-2014-005). Nitroxides are a unique class of persistent radicals finding a wide range of applications, from spin probes to polarizing agents, and recently bis-nitroxides have been used as proof-of-concept molecules for quantum information processing. Here we present the syntheses of pyrroline-based nitroxide (NO) radicals and give a comparision of two possible synthetic routes to form two key intermediates, namely 2,2,5,5-tetramethylpyrroline-1-oxyl-3-acetylene (TPA) and 1-oxyl-2,2,5,5-tetramethylpyrroline-3-carboxylic acid (TPC). TPC and TPA were then used as precursors for the synthesis of three model compounds featuring two distant NO groups with a variable degree of conjugation and thus electronic communication between them. Using relatively facile synthetic routes, we produced a number of mono- and bis-nitroxides with the structures of multiple compounds unambiguously characterized by X-ray crystallography, while Continuous Wave Electron Paramagnetic Resonance (CW-EPR) allowed us quantify the electronic communication in the bis-nitroxides. Our study expands the repertoire of mono- and bis-nitroxides with possibilities of exploiting them for studying quantum coherence effects and as polarizing agents. Publisher PDF

Funding: British Heart Foundation (BHF): Research Excellence Award RE/13/3/30183; Kidney Research UK: Innovation Grant IN_001_201703 Postdoctoral Fellowship PDF_008_20151127; Scottish Funding Council (SFC): St Andrews Restarting Research Funding Scheme; Society for Endocrinology (SFE): Early Career Grant. We report the transcriptomes associated with acute corticosteroid regulation of ENaC activity in polarised mCCDcl1 collecting duct cells. 9 genes were regulated by aldosterone (ALDO), 0 with corticosterone alone and 151 with corticosterone when 11βHSD2 activity was inhibited. We validated 3 novel ALDO-induced genes: Rasd1, Sult1d1 and Gm43305 in primary cells isolated from a novel collecting duct reporter mouse. Background Corticosteroids regulate distal nephron and collecting duct Na+ reabsorption, contributing to fluid-volume and blood pressure homeostasis. The transcriptional landscape underpinning the acute stimulation of the epithelial sodium channel (ENaC) by physiological concentrations of corticosteroids remains unclear. Methods Transcriptomic profiles underlying corticosteroid-stimulated ENaC activity in polarised mCCDcl1 cells were generated by coupling electrophysiological measurements of amiloride-sensitive currents with RNAseq. Generation of a collecting-duct specific reporter mouse line, mT/mG-Aqp2Cre, enabled isolation of primary collecting duct cells by FACS and ENaC activity was measured in cultured primary cells following acute application of corticosteroids. Expression of target genes was assessed by qRT-PCR in cultured cells or freshly isolated cells following acute elevation of steroid hormones in mT/mG-Aqp2Cre mice. Results Physiological relevance of the mCCDcl1 model was confirmed with aldosterone-specific stimulation of SGK1 and ENaC activity. Corticosterone only modulated these responses at supraphysiological concentrations or when 11βHSD2 was inhibited. When 11βHSD2 protection was intact, corticosterone caused no significant change in transcripts. We identified a small number of aldosterone-induced transcripts associated with stimulated ENaC activity in mCCDcl1 cells and a much larger number with corticosterone in the absence of 11βHSD2 activity. Cells isolated from mT/mG-Aqp2Cre mice were validated as collecting duct-specific and assessment of identified aldosterone-induced genes revealed that Sgk1, Zbtbt16, Sult1d1, Rasd1 and Gm43305 are acutely upregulated by corticosteroids both in vitro and in vivo. Conclusions This study reports the transcriptome of mCCDcl1 collecting duct cells and identifies a small number of aldosterone-induced genes associated with acute stimulation of ENaC, including 3 previously undescribed genes. Postprint

International audience; In 2017, the order Mononegavirales was expanded by the inclusion of a total of 69 novel species. Five new rhabdovirus genera and one new nyamivirus genus were established to harbor 41 of these species, whereas the remaining new species were assigned to already established genera. Furthermore, non-Latinized binomial species names replaced all paramyxovirus and pneumovirus species names, thereby accomplishing application of binomial species names throughout the entire order. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).

International audience; In 2016, the order Mononegavirales was emended through the addition of two new families (Mymonaviridae and Sunviridae), the elevation of the paramyxoviral subfamily Pneumovirinae to family status (Pneumoviridae), the addition of five free-floating genera (Anphevirus, Arlivirus, Chengtivirus, Crustavirus, and Wastrivirus), and several other changes at the genus and species levels. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).

Bluetongue virus (BTV) is the causative agent of bluetongue, a major infectious disease of ruminants with serious consequences to both animal health and the economy. The clinical outcome of BTV infection is highly variable and dependent on a variety of factors related to both the virus and the host. In this study, we show that the BTV nonstructural protein NS4 favors viral replication in sheep, the animal species most affected by bluetongue. In addition, NS4 confers a replication advantage on the virus in interferon (IFN)-competent primary sheep endothelial cells and immortalized cell lines. We determined that in cells infected with an NS4 deletion mutant (BTV8ΔNS4), there is increased synthesis of type I IFN compared to cells infected with wild-type BTV-8. In addition, using RNA sequencing (RNA-seq), we show that NS4 modulates the host IFN response and downregulates mRNA levels of type I IFN and interferon-stimulated genes. Moreover, using reporter assays and protein synthesis assays, we show that NS4 downregulates the activities of a variety of promoters, such as the cytomegalovirus immediate-early promoter, the IFN-β promoter, and a promoter containing interferon-stimulated response elements (ISRE). We also show that the NS4 inhibitory activity on gene expression is related to its nucleolar localization. Furthermore, NS4 does not affect mRNA splicing or cellular translation. The data obtained in this study strongly suggest that BTV NS4 is an IFN antagonist and a key determinant of viral virulence. IMPORTANCE Bluetongue is one of the main infectious diseases of ruminants and is caused by bluetongue virus (BTV), an arthropod-borne virus transmitted from infected to susceptible animals by Culicoides biting midges. Bluetongue has a variable clinical outcome that can be related to both virus and host factors. It is therefore critical to understand the interplay between BTV and the host immune responses. In this study, we show that a nonstructural protein of BTV (NS4) is critical to counteract the innate immune response of the host. Infection of cells with a BTV mutant lacking NS4 results in increased synthesis of IFN-β and upregulation of interferon-stimulated genes. In addition, we show that NS4 is a virulence factor for BTV by favoring viral replication in sheep, the animal species most susceptible to bluetongue.

Funding: This work was supported by the Biotechnology and Biological Sciences Research Council (REF: BB/S000313/1 to MFW), Medical Research Council (REF: MR/S021647/1 to RS) and the China Scholarship Council (REF: 202008060345 to QS). Type I CRISPR systems are the most common CRISPR type found in bacteria. They use a multisubunit effector, guided by crRNA, to detect and bind dsDNA targets, forming an R-loop and recruiting the Cas3 enzyme to facilitate target DNA destruction, thus providing immunity against mobile genetic elements. Subtypes have been classified into families A-G, with type I-G being the least well understood. Here, we report the composition, structure and function of the type I-G Cascade CRISPR effector from Thioalkalivibrio sulfidiphilus, revealing key new molecular details. The unique Csb2 subunit processes pre-crRNA, remaining bound to the 3′ end of the mature crRNA, and seven Cas7 subunits form the backbone of the effector. Cas3 associates stably with the effector complex via the Cas8g subunit and is important for target DNA recognition. Structural analysis by cryo-Electron Microscopy reveals a strikingly curved backbone conformation with Cas8g spanning the belly of the structure. These biochemical and structural insights shed new light on the diversity of type I systems and open the way to applications in genome engineering. Publisher PDF

Funding: This work was supported by The "Holistic Approach to Unravel Antibacterial Resistance in East Africa” project which was a 3-year Global Context Consortia Award (MR/S004785/1) funded by the National Institute for Health Research, Medical Research Council and the Department of Health and Social Care, UK. Background The accumulation of resistance genes in Escherichia coli (E. coli) strains imposes limitations in the therapeutic options available for the treatment of infections caused by E.coli. Production of Klebsiella pneumoniae carbapenemase (KPC) by E. coli renders it resistant to broad-spectrum β-lactam antibiotics. Globally there is existing evidence of spread of carbapenem-resistant E. coli in both humans and livestock driven by acquisition of the several other carbapenemase genes. Overall, there is little information regarding the extent of KPC gene distribution in E. coli. We set out to determine the prevalence, and evaluate the phenotypic and genotypic patterns of KPC in E. coli isolated from humans and their livestock in rural south western Uganda. Methods A laboratory-based, descriptive cross-sectional study was conducted involving 96 human and 96 livestock isolates collected from agro-pastoralist communities in Mbarara district in south western Uganda. Phenotypic and molecular methods (PCR) were used for presence and identification of KPC genes in the E. coli isolates. A chi-square test of independence was used to evaluate the differences in resistant patterns between carbapenems and isolates. Results The overall prevalence of carbapenem resistance by disk diffusion susceptibility testing (DST) for both humans and livestock isolates were 41.7% (80/192). DST-based resistance was identical in both human and livestock isolates (41.7%). The prevalence of carbapenem resistance based on Modified Hodge Test (MHT) was 5% (2/40) and 10% (4/40) for humans and livestock isolates respectively. Both human and livestock isolates, 48.7% (95/192) had the KPC gene, higher than phenotypic expression; 41.7% (80/192). blaKPC gene prevalence was overall similar in human isolates (51%; 49/96) vs livestock isolates (47.9%; 46/96). Approximately, 19% (15/80) of the isolates were phenotypically resistant to carbapenems and over 70% (79/112) of the phenotypically sensitive strains harbored the blaKPC gene. Conclusion Our results suggest that both human and livestock isolates of E. coli in our setting carry the blaKPC gene with a high percentage of strains not actively expressing the blaKPC gene. The finding of fewer isolates carrying the KPC gene than those phenotypically resistant to carbapenems suggests that other mechanisms are playing a role in this phenomenon, calling for further researcher into this phenomenon. Publisher PDF

Funding: Horizon 2020 FETOPEN, GRANT #(s) 863203; European Union FAMOS, GRANT #(s) 317744; UK Engineering and Physical Engineering and Physical Sciences Research Council (EPSRC) EP/M000869/1 and EP/P030017/1; Australian Research Council, FUNDREF 10.13039/501100000923, GRANT #(s) FL210100099; Horizon 2020 Framework Programme, FUNDREF 10.13039/100010661, GRANT #(s) 871212. The penetration depth of optical coherence tomography (OCT) reaches well beyond conventional microscopy; however, signal reduction with depth leads to rapid degradation of the signal below the noise level. The pursuit of imaging at depth has been largely approached by extinguishing multiple scattering. However, in OCT, multiple scattering substantially contributes to image formation at depth. Here, we investigate the role of multiple scattering in OCT image contrast and postulate that, in OCT, multiple scattering can enhance image contrast at depth. We introduce an original geometry that completely decouples the incident and collection fields by introducing a spatial offset between them, leading to preferential collection of multiply scattered light. A wave optics–based theoretical framework supports our experimentally demonstrated improvement in contrast. The effective signal attenuation can be reduced by more than 24 decibels. Notably, a ninefold enhancement in image contrast at depth is observed in scattering biological samples. This geometry enables a powerful capacity to dynamically tune for contrast at depth. Publisher PDF

Funding: CMC and CJH are funded by the Wellcome Trust (210486/Z/18/Z), additional funding was provided by the University of St Andrews Impact Innovation Fund. MB (Bergkessel) is funded by the University of Dundee/Wellcome Trust Institutional Strategic Support Fund [204816/Z/16/Z] and UKRI Future Leaders Fellowship (funded by the Medical Research Council) [MR/T041811/1]. Pseudomonas aeruginosa is an opportunistic pathogen that causes serious illness, especially in immunocompromised individuals. P. aeruginosa forms biofilms that contribute to growth and persistence in a wide range of environments. Here we investigated the aminopeptidase, P. aeruginosa aminopeptidase (PaAP) from P. aeruginosa, which is highly abundant in the biofilm matrix. PaAP is associated with biofilm development and contributes to nutrient recycling. We confirmed that post-translational processing was required for activation and PaAP is a promiscuous aminopeptidase acting on unstructured regions of peptides and proteins. Crystal structures of wild-type enzymes and variants revealed the mechanism of autoinhibition, whereby the C-terminal propeptide locks the protease-associated domain and the catalytic peptidase domain into a self-inhibited conformation. Inspired by this, we designed a highly potent small cyclic-peptide inhibitor that recapitulates the deleterious phenotype observed with a PaAP deletion variant in biofilm assays and present a path toward targeting secreted proteins in a biofilm context. Publisher PDF

Funding: Matthias Waibel was funded by the University of Galway College of Science and the Irish Research Council under GOIPG/2016/1215. The James Hutton Institute receives funding support from the Rural and Environment Science and Analytical Services Division of the Scottish Government. Open access funding provided by IReL. Soil organic matter (SOM) is biologically, chemically, and physically complex. As a major store of nutrients within soil, it plays an important role in nutrient provision to plants. An enhanced understanding of SOM utilisation processes could underpin better fertiliser management for plant growth, with reduced environmental losses. Metaproteomics can allow the characterisation of protein profiles and could help gaining insights into SOM microbial decomposition mechanisms. Here, we applied three different extraction methods to two soil types to recover SOM with different characteristics. Specifically, water extractable organic matter, mineral associated organic matter and protein-bound organic matter were targeted with the aim to investigate the metaproteome enriched in those extractions. As a proof-of-concept replicated extracts from one soil were further analysed for peptide identification using liquid chromatography followed by tandem mass spectrometry. We employ a framework for mining mass spectra for both peptide assignment and fragmentation pattern characterisation. Different extracts were found to exhibit contrasting total protein and humic substance content for the two soils investigated. Overall, water extracts displayed the lowest humic substance content (in both soils) and the highest number of peptide identifications (in the soil investigated) with most frequent peptide hits associated with diverse substrate/ligand binding proteins of Proteobacteria and derived taxa. Our framework also highlighted a strong peptidic signal in unassigned and unmatched spectra, information that is currently not captured by the pipelines employed in this study. Taken together, this work points to specific areas for optimisation in chromatography and mass spectrometry to adequately characterise SOM associated metaproteomes. Publisher PDF

Funding: The Holistic Approach to Unravel Antibacterial Resistance in East Africa is a Global Context Consortia Award (MR/S004785/1) funded by the National Institute for Health Research, Medical Research Council, and the Department of Health and Social Care. The award is also part of the EDCTP2 programme supported by the European Union. This work is supported in part by the Makerere University-Uganda Virus Research Institute Centre of Excellence for Infection and Immunity Research and Training (MUII). MUII is supported through the DELTAS Africa Initiative (grant number 107743). The DELTAS Africa Initiative is an independent funding scheme of the African Academy of Sciences and Alliance for Accelerating Excellence in Science in Africa and is supported by the New Partnership for Africa’s Development Planning and Coordinating Agency with funding from the Wellcome Trust (grant number 107743) and the UK Government. This paper was funded in part by a grant from the National Institutes of Health (grant number U01CA207167), and a Scottish Funding Council GCRF Consolidator Award. BACKGROUND A key factor driving the development and maintenance of antibacterial resistance (ABR) is individuals' use of antibiotics (ABs) to treat illness. To better understand motivations and context for antibiotic use we use the concept of a patient treatment-seeking pathway: a treatment journey encompassing where patients go when they are unwell, what motivates their choices, and how they obtain antibiotics. This paper investigates patterns and determinants of patient treatment-seeking pathways, and how they intersect with AB use in East Africa, a region where ABR-attributable deaths are exceptionally high. METHODS The Holistic Approach to Unravelling Antibacterial Resistance (HATUA) Consortium collected quantitative data from 6,827 adult outpatients presenting with urinary tract infection (UTI) symptoms in Kenya, Tanzania, and Uganda between February 2019- September 2020, and conducted qualitative in-depth patient interviews with a subset (n = 116). We described patterns of treatment-seeking visually using Sankey plots and explored explanations and motivations using mixed-methods. Using Bayesian hierarchical regression modelling, we investigated the associations between socio-demographic, economic, healthcare, and attitudinal factors and three factors related to ABR: self-treatment as a first step, having a multi-step treatment pathway, and consuming ABs. RESULTS Although most patients (86%) sought help from medical facilities in the first instance, many (56%) described multi-step, repetitive treatment-seeking pathways, which further increased the likelihood of consuming ABs. Higher socio-economic status patients were more likely to consume ABs and have multi-step pathways. Reasons for choosing providers (e.g., cost, location, time) were conditioned by wider structural factors such as hybrid healthcare systems and AB availability. CONCLUSION There is likely to be a reinforcing cycle between complex, repetitive treatment pathways, AB consumption and ABR. A focus on individual antibiotic use as the key intervention point in this cycle ignores the contextual challenges patients face when treatment seeking, which include inadequate access to diagnostics, perceived inefficient public healthcare and ease of purchasing antibiotics without prescription. Pluralistic healthcare landscapes may promote more complex treatment seeking and therefore inappropriate AB use. We recommend further attention to healthcare system factors, focussing on medical facilities (e.g., accessible diagnostics, patient-doctor interactions, information flows), and community AB access points (e.g., drug sellers). Publisher PDF

Funding: This work was supported by the British Heart Foundation (grant code: PG/21/10468, FS/19/69/34639) and the Biotechnological and Biological Sciences Research Council (grant code: BB/V014684/1). Increasing evidence suggests that Zn2+ acts as a second messenger capable of transducing extracellular stimuli into intracellular signalling events. The importance of Zn2+ as a signalling molecule in cardiovascular functioning is gaining traction. In the heart, Zn2+ plays important roles in excitation-contraction (EC) coupling, excitation-transcription coupling, and cardiac ventricular morphogenesis. Zn2+ homeostasis in cardiac tissue is tightly regulated through the action of a combination of transporters, buffers and sensors. Zn2+-mishandling is a common feature of various cardiovascular diseases. However, the precise mechanisms controlling the intracellular distribution of Zn2+ and its variations during normal cardiac function and during pathological conditions are not fully understood. In this review we consider the major pathways by which the concentration of intracellular Zn2+ is regulated in the heart, the role of Zn2+ in EC coupling and discuss how Zn2+-dyshomeostasis resulting from altered expression levels and efficacy of Zn2+ regulatory proteins are key drivers in the progression of cardiac dysfunction. Publisher PDF

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Funding: This work is a subset of the large HATUA (Holistic approach to unravel antibacterial resistance) consortium funded by the UK Medical Research Council (MR/S004785/1). Introduction Culture is the gold-standard diagnosis for urinary tract infections (UTIs). However, most hospitals in low-resource countries lack adequately equipped laboratories and relevant expertise to perform culture and, therefore, rely heavily on dipstick tests for UTI diagnosis. Research gap In many Kenyan hospitals, routine evaluations are rarely done to assess the accuracy of popular screening tests such as the dipstick test. As such, there is a substantial risk of misdiagnosis emanating from inaccuracy in proxy screening tests. This may result in misuse, under-use or over-use of antimicrobials. Aim The present study aimed to assess the accuracy of the urine dipstick test as a proxy for the diagnosis of UTIs in selected Kenyan hospitals. Methods A hospital-based cross-sectional method was used. The utility of dipstick in the diagnosis of UTIs was assessed using midstream urine against culture as the gold standard. Results The dipstick test predicted 1416 positive UTIs, but only 1027 were confirmed positive by culture, translating to a prevalence of 54.1 %. The sensitivity of the dipstick test was better when leucocytes and nitrite tests were combined (63.1 %) than when the two tests were separate (62.6 and 50.7 %, respectively). Similarly, the two tests combined had a better positive predictive value (87.0 %) than either test alone. The nitrite test had the best specificity (89.8 %) and negative predictive value (97.4 %) than leucocytes esterase (L.E) or both tests combined. In addition, sensitivity in samples from inpatients (69.2 %) was higher than from outpatients (62.7 %). Furthermore, the dipstick test had a better sensitivity and positive predictive value among female (66.0 and 88.6 %) than male patients (44.3 and 73.9 %). Among the various patient age groups, the dipstick test’s sensitivity and positive predictive value were exceptionally high in patients ≥75 years old (87.5 and 93.3 %). Conclusion Discrepancies in prevalence from the urine dipstick test and culture, the gold standard, indicate dipstick test inadequacy for accurate UTI diagnosis. The finding also demonstrates the need for urine culture for accurate UTI diagnosis. However, considering it is not always possible to perform a culture, especially in low-resource settings, future studies are needed to combine specific UTI symptoms and dipstick results to assess possible increases in the test’s sensitivity. There is also a need to develop readily available and affordable algorithms that can detect UTIs where culture is not available. Publisher PDF

Funding: This study was funded by the Holistic Approach to Unravel Antibacterial Resistance in East Africa (HATUA) project funded by the National Institute for Health Research, Medical Research Council, and the Department of Health and Social Care, Award (MR/S004785/1). Background: Additional antimicrobial resistance to extended spectrum β-lactamases (ESBL)-producing E. coli exhausts treatment options. We investigated allele distribution and resistance to ciprofloxacin and gentamicin among ESBL-producing E. coli isolates from urine, stool, animals and environments of presumptive urinary tract infection (UTI) patients, in order to gain a crucial insight towards devising prevention and control measures, and treatment guidelines. Methods: Archived ESBL-producing E. coli isolates from urine, stool, animals and surrounding environments of presumptive UTI patients were retrieved. Antimicrobial susceptibility profiles to ciprofloxacin and gentamicin were done followed by multi-plex PCR for blaCTX-M, blaTEM and blaSHV, to determine ESBL alleles distribution. Data were analysed using STATA version 17. Results: A total of 472 confirmed ESBL-producing E. coli isolates from Mwanza 243 (51.5%), Kilimanjaro 143 (30.3%) and Mbeya 86 (18.2%) were analyzed. Of these, 75 (15.9%) were from urine, 199 (42.2%) from stool, 58 (12.3%) from rectal/cloaca swabs of animals and 140 (29.7%) from surrounding environments. Out of 472 ESBL producing E. coli, 98.9% (467) had at least one ESBL allele. The most frequent allele was blaCTX-M detected in 88.1% (416/472) isolates, followed by blaTEM allele detected in 51.5% (243/472) isolates. There were 40.7% (192/472) isolates harboring dual blaCTX-M + blaTEM alleles, and only 0.2% (1/472) isolate had dual blaCTX-M + blaSHV alleles whereas 2.3% (11/472) isolates had a combination of all three alleles (blaCTX-M + blaTEM + blaSHV). None of the isolates harbored a combination of blaTEM + blaSHV only. Resistance to ciprofloxacin and gentamicin was observed in 70.8% (334/472) and 46.0% (217/472) isolates, respectively. There was a significant difference in distribution of resistance to ciprofloxacin as well as to gentamicin among ESBL-producing E. coli isolated from various sources (p-value < 0.001 and 0.002, respectively. Conclusion: Almost all ESBL-producing E. coli isolates carry blaCTX-M, blaTEM and blaSHV either alone or in combination with the most common alleles being blaCTX-M. The resistance to cipropfloxacin and gentamicin which are front-line antibiotics for UTIs among ESBL producing E. coli is high. This implies the need to continuously revise the local guidelines used for optimal empirical therapy for UTI and continual surveillance using one health approach. Publisher PDF

Funding: Engineering and Physical Sciences Research Council - EP/P030017/1, EP/R004854/1; Australian Research Council - FL210100099; National Health and Medical Research Council - APP2003786; Future Making Fellowship (University of Adelaide); Hospital Research Foundation (Mid-career fellowship C-MCF58-2019). Embryo quality is a crucial factor affecting live birth outcomes. However, an accurate diagnostic for embryo quality remains elusive in the in vitro fertilization clinic. Determining physical parameters of the embryo may offer key information for this purpose. Here, we demonstrate that digital holographic microscopy (DHM) can rapidly and non-invasively assess the refractive index of mouse embryos. Murine embryos were cultured in either low- or high-lipid containing media and digital holograms recorded at various stages of development. The phase of the recorded hologram was numerically retrieved, from which the refractive index of the embryo was calculated. We showed that DHM can detect spatio-temporal changes in refractive index during embryo development that are reflective of its lipid content. As accumulation of intracellular lipid is known to compromise embryo health, DHM may prove beneficial in developing an accurate, non-invasive, multimodal diagnostic. Publisher PDF

Funding: The Holistic Approach to Unravel Antibacterial Resistance in East Africa (HATUA) is a 3-year Global Context Consortia Award (MR/S004785/1) funded by the National Institute for Health Research, Medical Research Council and the Department of Health and Social Care. The award is also part of the EDCTP2 program supported by the European Union. Background Evidence-based empirical antibiotic prescribing requires knowledge of local antimicrobial resistance patterns. The spectrum of pathogens and their susceptibility strongly influences guidelines for empirical therapies for urinary tract infections (UTI) management. Objective This study aimed to determine the prevalence of UTI causative bacteria and their corresponding antibiotic resistance profiles in three counties of Kenya. Such data could be used to determine the optimal empirical therapy. Methods In this cross-sectional study, urine samples were collected from patients who presented with symptoms suggestive of UTI in the following healthcare facilities; Kenyatta National Hospital, Kiambu Hospital, Mbagathi, Makueni, Nanyuki, Centre for Microbiology Research, and Mukuru Health Centres. Urine cultures were done on Cystine Lactose Electrolyte Deficient (CLED) to isolate UTI bacterial etiologies, while antibiotic sensitivity testing was done using the Kirby-Bauer disk diffusion using CLSI guidelines and interpretive criteria. Results A total of 1,027(54%) uropathogens were isolated from the urine samples of 1898 participants. Staphylococcus spp. and Escherichia coli were the main uropathogens at 37.6% and 30.9%, respectively. The percentage resistance to commonly used drugs for the treatment of UTI were as follows: trimethoprim (64%), sulfamethoxazole (57%), nalidixic acid(57%), ciprofloxacin (27%), amoxicillin-clavulanic acid (5%), and nitrofurantoin (9%) and cefixime (9%). Resistance rates to broad-spectrum antimicrobials, such as ceftazidime, gentamicin, and ceftriaxone, were 15%, 14%, and 11%, respectively. Additionally, the proportion of Multidrug-resistant (MDR) bacteria was 66%. Conclusion High resistance rates toward fluoroquinolones, sulfamethoxazole, and trimethoprim were reported. These antibiotics are commonly used drugs as they are inexpensive and readily available. Based on these findings, more robust standardised surveillance is needed to confirm the patterns observed while recognising the potential impact of sampling biases on observed resistance rates. Publisher PDF

Funding: The authors gratefully acknowledge the China Scholarship Council for funding a PhD scholarship for D. W. This work was also funded by the Leverhulme Trust (grant no. RPG-2017-214). I. G. S., W. M. and M. G. were funded by NIH grant R01-GM132595 and Harrison Family Funds. M. P. C. acknowledges the support of a Robert R. Wagner Fellowship at the University of Virginia. R. F. was supported by the Biological and Biotechnological Sciences Research Council (grant no. BB/V014684/1). Serum albumin–Co2+ interactions are of clinical importance. They play a role in mediating the physiological effects associated with cobalt toxicity and are central to the albumin cobalt binding (ACB) assay for diagnosis of myocardial ischemia. To further understand these processes, a deeper understanding of albumin–Co2+ interactions is required. Here, we present the first crystallographic structures of human serum albumin (HSA; three structures) and equine serum albumin (ESA; one structure) in complex with Co2+. Amongst a total of sixteen sites bearing a cobalt ion across the structures, two locations were prominent, and they relate to metal-binding sites A and B. Site-directed mutagenesis and isothermal titration calorimetry (ITC) were employed to characterise sites on HSA. The results indicate that His9 and His67 contribute to the primary (putatively corresponding to site B) and secondary Co2+-binding sites (site A), respectively. The presence of additional multiple weak-affinity Co2+ binding sites on HSA was also supported by ITC studies. Furthermore, addition of 5 molar equivalents of the non-esterified fatty acid palmitate (C16:0) reduced the Co2+-binding affinity at both sites A and B. The presence of bound myristate (C14:0) in the HSA crystal structures provided insight into the fatty acid-mediated structural changes that diminish the affinity of the protein toward Co2+. Together, these data provide further support for the idea that ischemia-modified albumin corresponds to albumin with excessive fatty-acid loading. Collectively, our findings provide a comprehensive understanding of the molecular underpinnings governing Co2+ binding to serum albumin. Publisher PDF

This study was funded by the Holistic Approach to Unravel Antibacterial Resistance in East Africa (HATUA) project funded by the National Institute for Health Research, Medical Research Council and the Department of Health and Social Care, Award (MR/S004785/1). Background: The dissemination of the extended spectrum β-lactamases (ESBL) producing E. coli poses a significant public health problem. Understanding the efficiency and frequency of horizontal gene transfer via conjugation of ESBL producing E. coli is imperative towards devising prevention and control measures. This study compared the frequencies and efficiencies of horizontal blaCTX-M gene transfer via conjugation among Escherichia coli isolates from urine and gastrointestinal tract (GIT) of patients with urinary tract infection (UTI), their animals and environment. Methods: Horizontal blaCTX-M gene transfer via conjugation by a broth mating experiment was performed using 50 confirmed ESBL producing E. coli isolates as donors and Escherichia coli J53 (F−, met, pro, Azr), as the recipient. The transconjugants were detected and their frequencies and efficiencies of conjugation were measured and compared between ESBL producing E. coli isolates multi-sourced from urine, GIT, animals and environment. Antimicrobial susceptibility testing of all resulting transconjugants was performed. DNA was extracted from all transconjugants to confirm the presence and the acquisition of blaCTX-M gene. Results: Out of 50 ESBL producing E. coli isolates harboring blaCTX-M gene, 37 (74.0%) successfully exercised horizontal gene transfer through conjugation. All transconjugants were confirmed phenotypically and genotypically by PCR. Of note, all of the isolates from environment 100.0% (7/7) performed conjugation, exhibiting the highest transfer efficiency, followed by isolates from urine and animals, with the conjugation transfer efficiency of 77.8% (14/18) and 76.1% (10/13), respectively. The isolates from the environment conjugated with a significant more efficiency than those from the GIT [Two-sample test of proportions; p-value = 0.0119]. The overall conjugation transfer frequencies ranged from 0.4 × 10-14 – 5.5 × 10-11 per donor cells with the highest median conjugation transfer frequency observed among isolates from animal (3.23 × 10-12 [IQR: 0.70 × 10-12 – 7.22 × 10-12]) followed by that of isolates from the environment (1.60 × 10-12 [IQR: 0.30 × 10-12 – 5.0 × 10-12]). Conclusion: ESBL producing E. coli from human, animals and environment exercises horizontal blaCTX-M gene transfer efficiently with the highest occurrence among isolates from the environment and animals. The antimicrobial resistance control and prevention strategies should be widened up to explore strategies to prevent horizontal AMR gene transfer. Publisher PDF

Funding: R.F. is supported by the Biological and Biotechnological Sciences Research Council. Magnesium (Mg2+) has many physiological functions within the body. These include important roles in maintaining cardiovascular functioning, where it contributes to the regulation of cardiac excitation–contraction coupling, endothelial functioning and haemostasis. The haemostatic roles of Mg2+ impact upon both the protein and cellular arms of coagulation. In this review, we examine how Mg2+ homeostasis is maintained within the body and highlight the various molecular roles attributed to Mg2+ in the cardiovascular system. In addition, we describe how nutritional and/or disease-associated magnesium deficiency, seen in some metabolic conditions, has the potential to influence cardiac and vascular outcomes. Finally, we also examine the potential for magnesium supplements to be employed in the prevention and treatment of cardiovascular disorders and in the management of cardiometabolic health. Publisher PDF

Funding: UKHSA funded the metagenomics and HAdV sequencing. The work was part funded by the NIHR Blood and Transplant Research Unit in Genomics to Enhance Microbiology Screening (GEMS), the National Institute for Health and Care Research (CO-CIN-01) or jointly by NIHR and UK Research and Innovation (CV220-169, MC_PC_19059). S. Morfopoulou is funded by a W.T. Henry Wellcome fellowship (206478/Z/17/Z). S.B. and O.E.T.M. are funded by the NIHR Blood and Transplant Research Unit (GEMS). M.M.M. and M.L. are supported in part by the NIHR Biomedical Research Centre of Imperial College NHS Trust. J.B. receives NIHR Senior Investigator Funding. M.N. and J.B. are supported by the Wellcome Trust (207511/Z/17/Z and 203268/Z/16/Z). M.N., J.B. and G.P. are supported by the NIHR University College London Hospitals Biomedical Research Centre. P. Simmonds is supported by the NIHR (NIHR203338). T.S.J. is grateful for funding from the Brain Tumour Charity, Children with Cancer UK, GOSH Children’s Charity, Olivia Hodson Cancer Fund, Cancer Research UK and the NIHR. DIAMONDS is funded by the European Union (Horizon 2020; grant 848196). PERFORM was funded by the European Union (Horizon 2020; grant 668303). Since its first identification in Scotland, over 1000 cases of unexplained pediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator subjects, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in liver, blood, plasma or stool from 27/28 cases. We found low levels of Adenovirus (HAdV) and Human Herpesvirus 6B (HHV-6B), in 23/31 and 16/23 respectively of the cases tested. In contrast, AAV2 was infrequently detected at low titre in blood or liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T-cells and B-lineage cells. Proteomic comparison of liver tissue from cases and healthy controls, identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and in severe cases HHV-6B, may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children. Publisher PDF

Funding: For the purpose of open access, the authors have applied a Creative Commons Attribution (CC BY) licence to any Accepted Author Manuscript version arising. The authors thank the StAnD (St Andrews and Dundee) EPR group for long-standing support and in particular Dr El Mkami for assistance with PDS experiments. J. L.W. acknowledges support by the BBSRC DTP Eastbio (BB/M010996/1). A. G. acknowledges the EPSRC-funded Centre for Doctoral Training in ‘integrated magnetic resonance’, iMR-CDT (EP/J500045/1) B. E. B. and K. A. acknowledge support by the Leverhulme Trust (RPG-2018-397). B. E. B. acknowledges equipment funding by BBSRC (BB/R013780/1 and BB/T017740/1). Pulse dipolar spectroscopy (PDS) measurements are an important complementary tool in structural biology and are increasingly applied to macromolecular assemblies implicated in human health and disease at physiological concentrations. This requires ever higher sensitivity, and recent advances have driven PDS measurements into the mid-nanomolar concentration regime, though optimization and acquisition of such measurements remains experimentally demanding and time expensive. One important consideration is that constant-time acquisition represents a hard limit for measurement sensitivity, depending on the maximum measured distance. Determining this distance a priori has been facilitated by machine-learning structure prediction (AlphaFold2 and RoseTTAFold) but is often confounded by non-representative behaviour in frozen solution that may mandate multiple rounds of optimization and acquisition. Herein, we endeavour to simultaneously enhance sensitivity and streamline PDS measurement optimization to one-step by benchmarking a variable-time acquisition RIDME experiment applied to CuII-nitroxide and CuII-CuII model systems. Results demonstrate marked sensitivity improvements of both 5- and 6-pulse variable-time RIDME of between 2- and 5-fold over the constant-time analogues. Publisher PDF

Funding: This work was supported by the National Institutes of Health (AI139201 and AI137767 to GEW, each including salary support; GM141743 to DMW, including salary support; F31AI145214 to RVS, including predoctoral fellowship stipend support; and T32AI055402 to GEW, including predoctoral fellowship stipend support for AKS). The work was also supported by the Canadian Institutes of Health Research (148596 to MJB), the Canada Research Chair program (to MJB, salary support) and the American Heart Association (20POST35220017 to RSK, including postdoctoral fellowship stipend support). Toxoplasma gondii is a widespread apicomplexan parasite that can cause severe disease in its human hosts. The ability of T. gondii and other apicomplexan parasites to invade into, egress from, and move between cells of the hosts they infect is critical to parasite virulence and disease progression. An unusual and highly conserved parasite myosin motor (TgMyoA) plays a central role in T. gondii motility. The goal of this work was to determine whether the parasite's motility and lytic cycle can be disrupted through pharmacological inhibition of TgMyoA, as an approach to altering disease progression in vivo. To this end, we first sought to identify inhibitors of TgMyoA by screening a collection of 50,000 structurally diverse small molecules for inhibitors of the recombinant motor's actin-activated ATPase activity. The top hit to emerge from the screen, KNX-002, inhibited TgMyoA with little to no effect on any of the vertebrate myosins tested. KNX-002 was also active against parasites, inhibiting parasite motility and growth in culture in a dose-dependent manner. We used chemical mutagenesis, selection in KNX-002, and targeted sequencing to identify a mutation in TgMyoA (T130A) that renders the recombinant motor less sensitive to compound. Compared to wild-type parasites, parasites expressing the T130A mutation showed reduced sensitivity to KNX-002 in motility and growth assays, confirming TgMyoA as a biologically relevant target of KNX-002. Finally, we present evidence that KNX-002 can slow disease progression in mice infected with wild-type parasites, but not parasites expressing the resistance-conferring TgMyoA T130A mutation. Taken together, these data demonstrate the specificity of KNX-002 for TgMyoA, both in vitro and in vivo, and validate TgMyoA as a druggable target in infections with T. gondii. Since TgMyoA is essential for virulence, conserved in apicomplexan parasites, and distinctly different from the myosins found in humans, pharmacological inhibition of MyoA offers a promising new approach to treating the devastating diseases caused by T. gondii and other apicomplexan parasites. Publisher PDF

Funding: this work was supported by several funding agencies. The PanACEA MAMS study was funded by the European and Developing Countries Clinical Trials Partnership (EDCTP1); Grant No. IP.2007.32011.013, IP.2007.32011.012, and IP.2007.32011.011 as well as by the German Ministry of Education and Research (01KA0901). This analysis was funded by the German Center for Infection Research (DZIF); grant no. 02.702. Bacterial killing in patients with tuberculosis (TB) relapse was compared to that in patients achieving cure, measured by TB molecular bacterial load assay (TB-MBLA) or mycobacteria growth indicator tube (MGIT) time to positivity (TTP). TB-MBLA in 4 relapsed patients was significantly different compared to 132 cured patients after 2 weeks of treatment; MGIT TTP showed a significant difference from week 8. Publisher PDF

Authors thank the Chinese Scholarship Council for funding a Studentship (No. 202008060063) at the University of St. Andrews, U.K. C-2 fluorinated and methylated stereoisomers of the fragrance citronellol 1 and its oxalate esters were prepared from (R)-pulegone 11 and explored as agonists of the human olfactory receptor OR1A1 and assayed also against site-specific mutants. There were clear isomer preferences and C-2 difluorination as in 18 led to the most active compound suggesting an important hydrogen bond donor role for citronellol 1. C-2 methylation and the corresponding oxalate ester analogues were less active. Publisher PDF

Funding: This work was supported by the British Heart Foundation (grant code: FS/20/3/34956). Obesity enhances the risk of type-2 diabetes, cardiovascular disease and inflammatory conditions and often leads to metal dyshomeostasis, which contributes to the negative health aspects associated with the disease. In severe cases, bariatric surgery can be recommended to achieve sustained weight loss and improvement in health. Here, magnesium, zinc, copper and selenium concentrations were examined in 24 obese patients (7 males; 17 females) before and 9 months after undergoing Roux-en-Y gastric bypass surgery. All patients lost weight over this period, with the mean BMI reducing from 51.2±7.1 kg/m2 to 37.2±5.5 kg/m2. Moreover, whole-blood glycated haemoglobin (HbA1c), as a marker of average glycaemia, was also measured and a correlative analysis of this parameter with metal concentrations performed. Significant alterations in the plasma concentrations of magnesium, zinc (both increased by 13.2% and 25.2% respectively) and copper (decreased by 7.9%) were observed over this period (plasma selenium concentration was unchanged), with BMI values correlating with plasma magnesium (p=0.004) and zinc (p=0.022) concentrations. At 9 months post-surgery, an increase in mean zinc/copper ratio was observed (0.86±0.29 compared to 0.63±0.14 pre-surgery). Comparison of whole-blood HbA1c concentrations pre- and post-surgery revealed a reduction from 6.50±1.28% pre-surgery to 5.51±0.49% post-surgery. Differences in plasma HbA1c and magnesium at either pre- and post-surgery correlated significantly, as did HbA1c and magnesium levels when pre- and post-surgery values were analysed together. Collectively, this work reveals that bariatric surgery, in conjunction with lifestyle/dietary changes, lead to improvements in the nutritional status of magnesium, zinc and copper. Furthermore, the observed improvements in magnesium and zinc were associated with weight loss and in the case of magnesium, to better glycaemic control. Publisher PDF

Funding: The authors thank the EPSRC Centre for Doctoral Training in Critical Resource Catalysis (CRITICAT) for financial support [Ph.D. studentship to S.M.; Grant code: EP/L016419/1]. Aqueous transformations confer many advantages, including decreased environmental impact and increased opportunity for biomolecule modulation. Although several studies have been conducted to enable the cross-coupling of aryl halides in aqueous conditions, until now a process for the cross-coupling of primary alkyl halides in aqueous conditions was missing from the catalytic toolbox and considered impossible. Alkyl halide coupling in water suffers from severe problems. The reasons for this include the strong propensity for β-hydride elimination, the need for highly air- and water-sensitive catalysts and reagents, and the intolerance of many hydrophilic groups to cross-coupling conditions. Here, we report a broadly applicable and readily accessible process for the cross-coupling of water-soluble alkyl halides in water and air by using simple and commercially available bench-stable reagents. The trisulfonated aryl phosphine TXPTS in combination with a water-soluble palladium salt Na2PdCl4 allowed for the Suzuki−Miyaura coupling of water-soluble alkyl halides with aryl boronic acids, boronic esters, and borofluorate salts in mild, fully aqueous conditions. Multiple challenging functionalities, including unprotected amino acids, an unnatural halogenated amino acid within a peptide, and herbicides can be diversified in water. Structurally complex natural products were used as testbeds to showcase the late-stage tagging methodology of marine natural products to enable liquid chromatography−mass spectrometry (LC−MS) detection. This enabling methodology therefore provides a general method for the environmentally friendly and biocompatible derivatization of sp3 alkyl halide bonds. Publisher PDF

Funding: This study was funded by an Investigator Award from the Wellcome Trust (206369/Z/17/Z). Additional funding was provided by the MRC (MC_UU_12014/10; MC_UU_12014/12). Infected hosts possess two alternative strategies to protect themselves against the negative impact of virus infections: resistance, used to abrogate virus replication, and disease tolerance, used to avoid tissue damage without controlling viral burden. The principles governing pathogen resistance are well understood, while less is known about those involved in disease tolerance. Here, we studied bluetongue virus (BTV), the cause of bluetongue disease of ruminants, as a model system to investigate the mechanisms of virus-host interactions correlating with disease tolerance. BTV induces clinical disease mainly in sheep, while cattle are considered reservoirs of infection, rarely exhibiting clinical symptoms despite sustained viremia. Using primary cells from multiple donors, we show that BTV consistently reaches higher titers in ovine cells than cells from cattle. The variable replication kinetics of BTV in sheep and cow cells were mostly abolished by abrogating the cell type I interferon (IFN) response. We identified restriction factors blocking BTV replication, but both the sheep and cow orthologues of these antiviral genes possess anti-BTV properties. Importantly, we demonstrate that BTV induces a faster host cell protein synthesis shutoff in primary sheep cells than cow cells, which results in an earlier downregulation of antiviral proteins. Moreover, by using RNA sequencing (RNA-seq), we also show a more pronounced expression of interferon-stimulated genes (ISGs) in BTV-infected cow cells than sheep cells. Our data provide a new perspective on how the type I IFN response in reservoir species can have overall positive effects on both virus and host evolution. Publisher PDF

Funding: Financial support for this work was provided by the University of St Andrews and EaStCHEM, and the Leverhulme Trust [Grant RPG-2019-155]. Transient states maintained by energy dissipation are an essential feature of dynamic systems where structures and functions are regulated by fluxes of energy and matter through chemical reaction networks. Perfected in biology, chemically fueled dissipative networks incorporating nanoscale components allow the unique properties of nanomaterials to be bestowed with spatiotemporal adaptability and chemical responsiveness. We report the transient dispersion of gold nanoparticles in water, powered by dissipation of a chemical fuel. A dispersed state that is generated under nonequilibrium conditions permits fully reversible solid–liquid or liquid–liquid phase transfer. The molecular basis of the out-of-equilibrium process is reversible covalent modification of nanoparticle-bound ligands by a simple inorganic activator. Activator consumption by a coupled dissipative reaction network leads to autonomous cycling between phases. The out-of-equilibrium lifetime is tunable by adjusting pH, and reversible phase cycling is reproducible over several cycles. Publisher PDF

Funder: COG-UK, Objectives: The SARS-CoV-2 Alpha variant was associated with increased transmission relative to other variants present at the time of its emergence and several studies have shown an association between Alpha variant infection and increased hospitalisation and 28-day mortality. However, none have addressed the impact on maximum severity of illness in the general population classified by the level of respiratory support required, or death. We aimed to do this. Methods: In this retrospective multi-centre clinical cohort sub-study of the COG-UK consortium, 1475 samples from Scottish hospitalised and community cases collected between 1st November 2020 and 30th January 2021 were sequenced. We matched sequence data to clinical outcomes as the Alpha variant became dominant in Scotland and modelled the association between Alpha variant infection and severe disease using a 4-point scale of maximum severity by 28 days: 1. no respiratory support, 2. supplemental oxygen, 3. ventilation and 4. death. Results: Our cumulative generalised linear mixed model analyses found evidence (cumulative odds ratio: 1.40, 95% CI: 1.02, 1.93) of a positive association between increased clinical severity and lineage (Alpha variant versus pre-Alpha variants). Conclusions: The Alpha variant was associated with more severe clinical disease in the Scottish population than co-circulating lineages.

Multiplexed, specific and sensitive detection of antigens is critical for the rapid and accurate diagnosis of disease and the informed development of personalized treatment plans. Here, we show that polymer microsphere lasers can be used as photonic sensors to monitor and quantify direct surface binding of biomolecules via changes in the refractive index. The unique spectral signature of each individual laser can be used to find their size and effective refractive index which adds a new encoding dimension when compared to conventional fluorescent beads. We utilize antibody-functionalized microlasers to selectively detect protein binding. Different stages of the multilayer surface modification can be resolved, and protein binding is demonstrated for two different proteins, IgG and CRP. Moreover, by continuously monitoring single lasers, we demonstrate the possibility of real-time monitoring of binding dynamics between antigens in solution phase and the immobilized antibodies. For multiplexed detection, the microlasers are employed in a flow cytometer configuration, with fast spectral detection and identification of microlasers with and without antigen binding. We envision that by combining microlasers with well-established surface modification chemistries and flow geometries, the multiplexing ability of microbead immunoassays can be strongly increased while also opening avenues for single cell profiling within heterogenous cell populations., Comment: 18 pages 5 figures and SI

Funding: This study is part of the Holistic Approach to Unravel Antibacterial Resistance in East Africa (HATUA) project funded by the National Institute for Health Research, Medical Research Council and the Department of Health and Social Care, Award (MR/S004785/1). Background Urogenital pathogens such as Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium and Trichomonas vaginalis have been reported to cause pyuria, however they are not routinely cultured from urine samples of patients clinically diagnosed to have urinary tract infections (UTI). In this study, pathogen specific PCR was done to identify the urogenital pathogens in the urine samples among clinically diagnosed UTI patients with negative routine urine culture. Methods A cross-sectional study was conducted involving 227 archived urine samples from clinically diagnosed UTI patients with positive leucocyte esterase but negative urine culture results. The urogenital pathogens were detected using pathogen specific singleplex PCR. Data were cleaned and analyzed using STATA version 15. Results The median age of patients was 31[IQR 23 – 51] years and the majority (174, 76.7%) were females. Two thirds of patients had history of antibiotic use two weeks prior to recruitment (154, 67.8%). A total of 62(27.3%) urine samples were positive for at least one urogenital pathogen. Of 62 positive samples, 9 had two urogenital pathogens and 1 had three urogenital pathogens. The most predominant urogenital pathogen detected was Neisseria gonorrhoeae 25(34.2%) and Trichomonas vaginalis 24(32.9%). Being female (aOR 2.4; 95% CI: 1.04 – 5.49; p-value 0.039) and having history of using antibiotics in the past two weeks (aOR 1.9; 95%CI: 1.04 – 3.60; p-value 0.036) was independently associated with the presence of urogenital pathogens. Conclusion More than a quarter of female patients with clinical symptoms of UTI and routine urine culture negative results were infected with urogenital pathogens mainly Neisseria gonorrhoeae and Trichomonas vaginalis. Further research with a larger sample set in a range of settings is required to understand the implications of these finding generally. Publisher PDF

Funding: The authors acknowledge the Universiti Teknologi MARA for funding under the UMP-IIUM-UiTM Sustainable Research Collaboration Grant [600–RMC/SRC/5/3(043/2020)]. A new isoxazole-based iodo-noium salt, C13 H13 INO5+ ·C2 F3 O2- , has been synthesized and structurally characterized. In the crystal, ions are linked by short I⋯O contacts to form a neutral tetra-ion aggregate. These combine with C-H⋯F and C-H⋯O inter-actions to form double-layered two-dimensional sheets in the (001) plane. Publisher PDF

Funding: For the purpose of open access, the authors have applied a Creative Commons Attribution (CC BY) license to any Accepted Author Manuscript version arising. They acknowledge support by the Wellcome Trust (204821/Z/16/Z), the British Heart Foundation (PG/15/9/31270 and FS/15/42/31556), and the Leverhulme Trust (RPG-2018–397). J.L.W. acknowledges support by the BBSRC DTP Eastbio. B.E.B. acknowledges equipment funding by BBSRC (BB/R013780/1 and BB/T017740/1). Mammalian histidine-rich glycoprotein (HRG) is a highly versatile and abundant blood plasma glycoprotein with a diverse range of ligands that is involved in regulating many essential biological processes, including coagulation, cell adhesion, and angiogenesis. Despite its biomedical importance, structural information on the multi-domain protein is sparse, not least due to intrinsically disordered regions that elude high-resolution structural characterization. Binding of divalent metal ions, particularly ZnII, to multiple sites within the HRG protein is of critical functional importance and exerts a regulatory role. However, characterization of the ZnII binding sites of HRG is a challenge; their number and composition as well as their affinities and stoichiometries of binding are currently not fully understood. In this study, we explored modern electron paramagnetic resonance (EPR) spectroscopy methods supported by protein secondary and tertiary structure prediction to assemble a holistic picture of native HRG and its interaction with metal ions. To the best of our knowledge, this is the first time that this suite of EPR techniques has been applied to count and characterize endogenous metal ion binding sites in a native mammalian protein of unknown structure. Publisher PDF

Funding: This work was supported by the European Research Council (Consolidator Grant 101002326 to J.K.). Ribosomally synthesized and post-translationally modified peptide natural products have provided many highly unusual scaffolds. This includes the intriguing alkaloids crocagins, which possess a tetracyclic core structure and whose biosynthesis has remained enigmatic. Here we use in vitro experiments to demonstrate that three proteins, CgnB, CgnC and CgnE, are sufficient for the production of the hallmark tetracyclic crocagin core from the precursor peptide CgnA. The crystal structures of the homologues CgnB and CgnE reveal them to be the founding members of a peptide-binding protein family and allow us to rationalize their distinct functions. We further show that the hydrolase CgnD liberates the crocagin core scaffold, which is subsequently N-methylated by CgnL. These insights allow us to propose a biosynthetic scheme for crocagins. Bioinformatic analyses based on these data led to the discovery of related biosynthetic pathways that may provide access to a structurally diverse family of peptide-derived pyrroloindoline alkaloids. Publisher PDF

Funding: The work was funded by Public Health Scotland, the National Institute for Health Research (NIHR; award CO-CIN-01) and the Medical Research Council (MRC; grants MR/X010252/1, MC_UU_1201412, MC_UU_12018/12, MC_PC_19059, MC_PC_19025 & MC_PC_22004). DIAMONDS is funded by the European Union Horizon 2020 programme; grant 848196). MP acknowledges funding support from the Wellcome Trust (206369/Z/17/Z). MGS gratefully acknowledges funding support from The Pandemic Institute, Liverpool and the NIHR Health Protection Research Unit (HPRU) in Emerging and Zoonotic Infections at University of Liverpool, and United Kingdom Health Security Agency (United KingdomHSA). JKB gratefully acknowledges funding support from a Wellcome Trust Senior Research Fellowship (223164/Z/21/Z), and MC_PC_20029, Sepsis Research (Fiona Elizabeth Agnew Trust), a BBSRC Institute Strategic Programme Grant to the Roslin Institute (BB/P013732/1, BB/P013759/1), and the United Kingdom Intensive Care Society. An outbreak of acute hepatitis of unknown aetiology in children was reported in Scotland in April 20221 and has now been identified in 35 countries2. Several recent studies have suggested an association with human adenovirus (HAdV), a virus not commonly associated with hepatitis. Here we report a detailed case-control investigation and find an association between adeno-associated virus (AAV2) infection and host genetics in disease susceptibility. Using next-generation sequencing (NGS), reverse transcription-polymerase chain reaction (RT-PCR), serology and in situ hybridisation (ISH), we detected recent infection with AAV2 in the plasma and liver samples of 26/32 (81%) hepatitis cases versus 5/74 (7%) of controls. Further, AAV2 was detected within ballooned hepatocytes alongside a prominent T cell infiltrate in liver biopsies. In keeping with a CD4+ T-cell-mediated immune pathology, the Human Leucocyte Antigen (HLA) class II DRB1*04:01 allele was identified in 25/27 cases (93%), compared with a background frequency of 10/64 (16%; p=5.49 x 10-12). In summary, we report an outbreak of acute paediatric hepatitis associated with AAV2 infection (most likely acquired as a coinfection with HAdV which is required as a "helper virus" to support AAV2 replication) and HLA class II-related disease susceptibility. Postprint

Funding: This work was performed by funding from The University of Sydney (CIA Jenkins) and funds provided by the National Health and Medical Research Council (Australia) APP1147897. Purpose: Robust, affordable plasma proteomic biomarker workflows are needed for large-scale clinical studies. We evaluated aspects of sample preparation to allow LC-MS analysis of more than 1500 samples from the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) trial of adults with type 2 diabetes. Methods: Using LC-MS with data-independent acquisition we evaluated four variables: plasma protein depletion, EDTA or citrated anti-coagulant blood collection tubes, plasma lipid depletion strategies and plasma freeze-thaw cycles. Optimised methods were applied in a pilot study of FIELD participants. Results: LC-MS of undepleted plasma conducted over a 45 min gradient yielded 172 proteins after excluding immunoglobulin isoforms. Cibachrome-blue-based depletion yielded additional proteins but with cost and time expenses, while immunodepleting albumin and IgG provided few additional identifications. Only minor variations were associated with blood collection tube type, delipidation methods and freeze-thaw cycles. From 65 batches involving over 1500 injections, the median intra-batch quantitative differences in the top 100 proteins of the plasma external standard was less than 2%. Fenofibrate altered seven plasma proteins. Conclusions and Clinical Relevance: A robust plasma handling and LC-MS proteomics workflow for abundant plasma proteins has been developed for large-scale biomarker studies that balances proteomic depth with time and resource costs. Publisher PDF

Funding: Engineering and Physical Sciences Research Council (EP/P030017/1, EP/R004854/1); Medical Research Scotland (PhD-873-2015); Australian Research Council (FL210100099). T cells of the adaptive immune system provide effective protection to the human body against numerous pathogenic challenges. Current labelling methods of detecting these cells, such as flow cytometry or magnetic bead labelling, are time consuming and expensive. To overcome these limitations, the label-free method of digital holographic microscopy (DHM) combined with deep learning has recently been introduced which is both time and cost effective. In this study, we demonstrate the application of digital holographic microscopy with deep learning to classify the key CD4+ and CD8+ T cell subsets. We show that combining DHM of varying fields of view, with deep learning, can potentially achieve a classification throughput rate of 78,000 cells per second with an accuracy of 76.2% for these morphologically similar cells. This throughput rate is 100 times faster than the previous studies and proves to be an effective replacement for labelling methods. Publisher PDF

Lipids may serve as energy reserves to support coral calcification, allow acclimation to higher temperatures, and are implicated in the control of CaCO3 precipitation. Here, we report the lipid composition of the soft tissues (including host and symbionts) of 7 massive Porites spp. coral colonies (4 × P. lutea and 3 × P. murrayensis), which were cultured under different pCO2 concentrations (180, 260, 400 and 750 µatm) and at two temperatures (25 ℃ and 28 ℃), below the thermal stress threshold. We report the fatty acid methyl esters (FAME), free fatty acid (FFA) to total fatty acid content, sterol and wax ester profiles, and identify two ketones (n-alkanone) and three long chain aldehyde (n-alkanal) derivatives. Increasing seawater temperature significantly increases the contributions of FFAs to the total lipids, of C18:2 and C20:0 to the total FFA pool, of C14:0 to total FAME, and of campesterol to total sterol. The temperature increase also reduces the contributions of unusual fatty acid derivatives to total lipids, of C14:0, C15:0, C16:0 and C17:0 saturated free fatty acids to total FFAs, and of C16:0 FA to total FAME. Fatty acids are implicated in the control of membrane structure fluidity and the observed changes may promote acclimation and thermostability as temperature varies. Seawater pCO2 has no significant effect on the composition of tissue lipids with the exception that the contribution of C14:0 FA to total lipid content is significantly lower at 180 µatm compared to 260 and 750 µatm. Decreased contribution of total sterols and unusual fatty acid derivatives and increased contribution of total FFAs to total lipids are observed in the fastest calcifying coral (a P. lutea specimen) compared to the other corals, under all pCO2 and temperature conditions. Although a rapid calcifier this genotype has been shown previously to exhibit pronounced abnormal changes in skeletal morphology in response to decreased seawater pCO2. Variations in tissue lipid composition between coral genotypes may influence their resilience to future climate change.

This work was funded by the Scottish Government Rural and Environment Science and Analytical Services Strategic Research Programme 2022–2027 (Project Ref. JHI-B1-1). Effective screening of plant germplasm collections for resistance to plant viruses requires that there is a rapid and efficient system in place to challenge individual plants with the virus. Potato leafroll virus (PLRV), a commercially important pathogen of potato, is able naturally to infect only the phloem-associated tissue of plants and is delivered to this tissue by feeding aphids. Mechanical (non-vector-mediated) infection by PLRV does not occur thus screening for PLRV resistance is currently laborious and time consuming. We constructed an infectious cDNA clone of a new (Hutton) isolate of PLRV in the binary vector pDIVA and transformed it into Agrobacterium tumefaciens strain LBA4404. Infiltration of this culture into leaves of Nicotiana benthamiana, a highly susceptible model plant, produced a systemic infection with PLRV, although this approach was not successful for potato. However, a very efficient and reproducible systemic infection of potato was achieved when we submerged cut stems of the plant into the agrobacterium cell suspension and then transplanted the stems into compost to grow roots and new apical leaves. Using a standardised protocol developed for this new PLRV inoculation method we have confirmed the previously described resistance to the virus in the JHI breeding line G8107(1) and identified 62 plant accessions from the Commonwealth Potato Collection in which no PLRV infection was detected. Publisher PDF

Funding: This work was partly funded by the Perry foundation (JAP) (https://www.perryfoundation.co.uk/), The Rural and Environment Science and Analytical Services (RESAS) division of the Scottish Government (JAP & JTJ) (https://www.gov.scot/) and BBSRC award BB/V00249X/1 (JAP, TS & JTJ) (https://www.ukri.org/councils/bbsrc/). Host-specific plant pathogens must coordinate their life cycles with the availability of a host plant. Although this is frequently achieved through a response to specific chemical cues derived from the host plant, little is known about the molecular basis of the response to such cues and how these are used to trigger activation of the life cycle. In host-specific plant-parasitic cyst nematodes, unhatched juvenile nematodes lie dormant in the eggshell until chemical cues from a suitable host plant are detected and the hatching process is initiated. The molecular mechanisms by which hatch is linked to the presence of these chemical cues is unknown. We have identified a novel annexin-like protein that is localised to the eggshell of the potato cyst nematode Globodera rostochiensis. This annexin is unique in having a short peptide insertion that structural modelling predicts is present in one of the calcium-binding sites of this protein. Host-induced gene silencing of the annexin impacts the ability of the nematode to regulate and control permeability of the eggshell. We show that in the presence of the chemicals that induce hatching annexin lipid binding capabilities change, providing the first molecular link between a nematode eggshell protein and host-derived cues. This work demonstrates how a protein from a large family has been recruited to play a critical role in the perception of the presence of a host and provides a new potential route for control of cyst nematodes that impact global food production. Publisher PDF

Background Over-the-counter antibiotic access is common in low-and-middle-income countries and this may accelerate antimicrobial resistance. Our study explores critical aspects of the drug seller–client interaction and antibiotic dispensing patterns for simulated COVID-19 symptoms during the pandemic in two study sites in Tanzania and Uganda, countries with different government responses to the pandemic. Methods Research assistants posing as clients approached different types of drug sellers such as pharmacies (Pharms), drug shops (DSs), and accredited drug dispensing outlets (ADDOs) in Mwanza, Tanzania (nPharms = 415, nADDOs = 116) and Mbarara, Uganda (nPharms = 440, nDSs = 67), from June 10 to July 30, 2021. The mystery clients held no prescription and sought advice for simulated COVID-19 symptoms from the drug sellers. They documented the quality of their interaction with sellers and the type of drugs dispensed. Results Adherence to COVID-19 preventive measures and vigilance to COVID-19 symptoms was low in both sites but significantly higher in Uganda than in Tanzania. A higher percentage of drug sellers in Mbarara (Pharms = 36%, DSs = 35%, P-value = 0.947) compared to Mwanza (Pharms = 9%, ADDOs = 4%, P-value = 0.112) identified the client’s symptoms as possibly COVID-19. More than three-quarters of drug sellers that sold prescription-only medicines in both Mbarara (Pharms = 86%, DSs = 89%) and Mwanza (Pharms = 93%, ADDOs = 97%) did not ask the MCs for a prescription. A relatively high percentage of drug sellers that sold prescription-only medicines in Mwanza (Pharms = 51%, ADDOs = 67%) compared to Mbarara (Pharms = 31%, DSs = 42%) sold a partial course without any hesitation. Of those who sold antibiotics, a higher proportion of drug sellers in Mbarara (Pharms = 73%, DSs = 78%, P-value = 0.580) compared to Mwanza (Pharms = 40% ADDOs = 46%, P-value = 0.537) sold antibiotics relevant for treating secondary bacterial infections in COVID-19 patients. Conclusion Our study highlights low vigilance towards COVID-19 symptoms, widespread propensity to dispense prescription-only antibiotics without a prescription, and to dispense partial doses of antibiotics. This implies that drug dispensing related to COVID-19 may further drive AMR. Our study also highlights the need for more efforts to improve antibiotic stewardship among drug sellers in response to COVID-19 and to prepare them for future health emergencies.

The zoonotic Plasmodium knowlesi parasite is a growing public health concern in Southeast Asia, especially in Malaysia, where elimination of P. falciparum and P. vivax malaria has been the focus of control efforts. Understanding of the genetic diversity of P. knowlesi parasites can provide insights into its evolution, population structure, diagnostics, transmission dynamics, and the emergence of drug resistance. Previous work has revealed that P. knowlesi fall into three main sub-populations distinguished by a combination of geographical location and macaque host (Macaca fascicularis and M. nemestrina). It has been shown that Malaysian Borneo groups display profound heterogeneity with long regions of high or low divergence resulting in mosaic patterns between sub-populations, with some evidence of chromosomal-segment exchanges. However, the genetic structure of non-Borneo sub-populations is less clear. By gathering one of the largest collections of P. knowlesi whole-genome sequencing data, we studied structural genomic changes across sub-populations, with the analysis revealing differences in Borneo clusters linked to mosquito-related stages of the parasite cycle, in contrast to differences in host-related stages for the Peninsular group. Our work identifies new genetic exchange events, including introgressions between Malaysian Peninsular and M. nemestrina-associated clusters on various chromosomes, including in parasite invasion genes (DBP$$\beta$$ β , NBPX$$\alpha$$ α and NBPX$$\beta$$ β ), and important proteins expressed in the vertebrate parasite stages. Recombination events appear to have occurred between the Peninsular and M. fascicularis-associated groups, including in the DBP$$\beta$$ β and DBP$$\gamma$$ γ invasion associated genes. Overall, our work finds that genetic exchange events have occurred among the recognised contemporary groups of P. knowlesi parasites during their evolutionary history, leading to apparent mosaicism between these sub-populations. These findings generate new hypotheses relevant to parasite evolutionary biology and P. knowlesi epidemiology, which can inform malaria control approaches to containing the impact of zoonotic malaria on human communities.

Funding information: M.G. and J.L.S.B. are funded by the Deutsche Forschungsgemeinschaft under Germany’s Excellence Strategy–EXC2151–390873048. M.F.W. acknowledges a European Research Council Advanced Grant (grant number 101018608) and the China Scholarship Council (REF: 202008420207 to H.C.). G.H. is grateful for funding by the Deutsche Forschungsgemeinschaft (grant number HA6805/6-1). CRISPR defense systems such as the well-known DNA-targeting Cas9 and the RNA-targeting type III systems are widespread in prokaryotes1,2. The latter can orchestrate a complex antiviral response that is initiated by the synthesis of cyclic oligoadenylates (cOAs) upon foreign RNA recognition3-5. Among a large set of proteins that were linked to type III systems and predicted to bind cOAs6,7, a CRISPR associated Lon protease (CalpL) stood out to us. The protein contains a sensor domain of the SAVED (SMODS-associated and fused to various effector domains) family7, fused to a Lon protease effector domain. However, the mode of action of this effector was unknown. Here, we report the structure and function of CalpL and show that the soluble protein forms a stable tripartite complex with two further proteins, CalpT and CalpS, that are encoded in the same operon. Upon activation by cA4, CalpL oligomerizes and specifically cleaves the MazF-homolog CalpT, releasing the extracytoplasmic function (ECF) sigma factor CalpS from the complex. This provides a direct connection between CRISPR-based foreign nucleic acid detection and transcriptional regulation. Furthermore, the presence of a cA4-binding SAVED domain in a CRISPR effector reveals an unexpected link to the cyclic oligonucleotide-based antiphage signaling system (CBASS). Postprint

We thank the EPSRC for a studentship (TJP) through the CRITICAT Doctoral Training Centre. FAPESP is also gratefully acknowledged for a studentship (BAP, #2021/09716-5) and a Young Researcher Award (RAC, #2018/03910-1). Concise and general synthesis protocols are reported to generate all-syn mono-, di- and tri-alkylated cyclohexanes where a single fluorine is located on the remaining carbons of the ring. The alkyl groups are positioned to lie equatorially and to have triaxial C–F bonds imparting polarity to these ring systems. Intermolecular electrostatic interactions in the solid-state structure of the trialkylated systems are explored and the resultant supramolecular order opens up prospects for design in soft materials. Publisher PDF

This work was financially supported by the Leverhulme Trust (RPG-2017-214) and BBSRC (BB/J006467/1 and BB/V014684/1). We thank Prof. Andrew Riches (University of St. Andrews) for provision of materials, and Dr. Elizabeth Bolitho (University of Warwick) for assistance with cell culture experiments. The role of the extracellular medium in influencing metal uptake into cells has not been described quantitatively. In a chemically defined model system containing albumin, zinc influx into endothelial cells correlates with the extracellular free zinc concentration. Allosteric inhibition of zinc-binding to albumin by free fatty acids increased zinc flux. Publisher PDF