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Fourteen proteins produced by three pathogenic Escherichia coli strains were identified using antibiotic induction, MALDI-TOF-TOF tandem mass spectrometry (MS/MS) and top-down proteomic analysis using software developed in-house. Host proteins as well as plasmid proteins were identified. Mature, intact protein ions were fragmented by post-source decay (PSD), and prominent fragment ions resulted from the aspartic acid effect fragmentation mechanism wherein polypeptide backbone cleavage (PBC) occurs on the C-terminal side of aspartic acid (D), glutamic acid (E) and asparagine (N) residues. These highly specific MS/MS-PSD fragment ions were compared to b- and y-type fragment ions on the C-terminal side of D-, E- and N-residues of in silico protein sequences derived from whole genome sequencing. Nine proteins were found to be post-translationally modified with either removal of an N-terminal methionine or a signal peptide. The protein sequence truncation algorithm of our software correctly identified all full and truncated protein sequences. Truncated sequences were compared to those predicted by SignalP. Nearly complete concurrence was obtained except for one protein where SignalP mis-identified the cleavage site by one residue. Two proteins had intramolecular disulfide bonds that were inferred by the absence of PBC on the C-terminal side of a D-residue located within the disulfide loop. These results demonstrate the utility of MALDI-TOF-TOF for identification of full and truncated bacterial proteins.

Two vital functions of the innate immune system are to initiate inflammation and redistribute micronutrients in favor of the host. Zinc is an essential micronutrient used in host defense. The zinc importer ZIP8 is uniquely induced through stimulation of the NF-κB pathway by LPS in monocytes and functions to regulate inflammation in a zinc-dependent manner. Herein we determined the impact of zinc metabolism following LPS-induced inflammation in human macrophages. We observed that ZIP8 is constitutively expressed in resting macrophages and strikingly elevated following LPS exposure, a response that is unique compared to the 13 other known zinc import proteins. During LPS exposure, extracellular zinc concentrations within the physiological range markedly reduced IL-10 mRNA expression and protein release but increased mRNA expression of TNFα, IL-8, and IL-6. ZIP8 knockdown inhibited LPS-driven cellular accumulation of zinc and prevented zinc-dependent reduction of IL-10 release. Further, zinc supplementation reduced nuclear localization and activity of C/EBPβ, a transcription factor known to drive IL-10 expression. These studies demonstrate for the first time that zinc regulates LPS-mediated immune activation of human macrophages in a ZIP8-dependent manner, reducing IL-10. Based on these findings we predict that macrophage zinc metabolism is important in host defense against pathogens.

Macrophages employ an array of pattern recognition receptors to detect and eliminate intracellular pathogens that access the cytosol. The cytosolic carbohydrate sensors Galectin-3, -8, and -9 (Gal-3, Gal-8, and Gal-9) recognize damaged pathogen-containing phagosomes, and Gal-3 and Gal-8 are reported to restrict bacterial growth via autophagy in cultured cells. However, the contribution of these galectins to host resistance during bacterial infection in vivo remains unclear. We found that Gal-9 binds directly to Mycobacterium tuberculosis ( Mtb ) and Salmonella enterica serovar Typhimurium ( Stm ) and localizes to Mtb in macrophages. To determine the combined contribution of membrane damage-sensing galectins to immunity, we generated Gal-3, -8, and -9 triple knockout (TKO) mice. Mtb infection of primary macrophages from TKO mice resulted in defective autophagic flux but normal bacterial replication. Surprisingly, these mice had no discernable defect in resistance to acute infection with Mtb , Stm or Listeria monocytogenes , and had only modest impairments in bacterial growth restriction and CD4 T cell activation during chronic Mtb infection. Collectively, these findings indicate that while Gal-3, -8, and -9 respond to an array of intracellular pathogens, together these membrane damage-sensing galectins play a limited role in host resistance to bacterial infection.

The development of novel antibiotics is essential because the current arsenal of antimicrobials will soon be ineffective due to the widespread occurrence of antibiotic resistance. The development of naturally occurring cationic antimicrobial peptides (CAMPs) for therapeutics to combat antibiotic resistance has been hampered by high production costs and protease sensitivity, among other factors. Ceragenins are a family of synthetic amphipathic molecules designed to mimic the properties of naturally occurring cationic antimicrobial peptides (CAMPs). Although ceragenins have potent antimicrobial activity, whether their mode of action is similar to that of CAMPs has remained elusive. Here, we reported the results of a comparative study of the bacterial responses to two well-studied CAMPs, LL37 and colistin, and two ceragenins with related structures, CSA13 and CSA131. Using transcriptomic and proteomic analyses, we found that Escherichia coli responded similarly to both CAMPs and ceragenins by inducing a Cpx envelope stress response. However, whereas E. coli exposed to CAMPs increased expression of genes involved in colanic acid biosynthesis, bacteria exposed to ceragenins specifically modulated functions related to phosphate transport, indicating distinct mechanisms of action between these two classes of molecules. Although traditional genetic approaches failed to identify genes that confer high-level resistance to ceragenins, using a Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) approach we identified E. coli essential genes that when knocked down modify sensitivity to these molecules. Comparison of the essential gene-antibiotic interactions for each of the CAMPs and ceragenins identified both overlapping and distinct dependencies for their antimicrobial activities. Overall, this study indicated that, while some bacterial responses to ceragenins overlap those induced by naturally occurring CAMPs, these synthetic molecules target the bacterial envelope using a distinctive mode of action. IMPORTANCE The development of novel antibiotics is essential because the current arsenal of antimicrobials will soon be ineffective due to the widespread occurrence of antibiotic resistance. The development of naturally occurring cationic antimicrobial peptides (CAMPs) for therapeutics to combat antibiotic resistance has been hampered by high production costs and protease sensitivity, among other factors. The ceragenins are a family of synthetic CAMP mimics that kill a broad spectrum of bacterial species but are less expensive to produce, resistant to proteolytic degradation, and seemingly resistant to the development of high-level resistance. Determining how ceragenins function may identify new essential biological pathways of bacteria that are less prone to the development of resistance and will further our understanding of the design principles for maximizing the effects of synthetic CAMPs.

Two vital functions of the innate immune system are to initiate inflammation and redistribute micronutrients in favor of the host. Zinc is an essential micronutrient used in host defense. The zinc importer ZIP8 is uniquely induced through stimulation of the NF-[kappa]B pathway by LPS in monocytes and functions to regulate inflammation in a zinc-dependent manner. Herein we determined the impact of zinc metabolism following LPS-induced inflammation in human macrophages. We observed that ZIP8 is constitutively expressed in resting macrophages and strikingly elevated following LPS exposure, a response that is unique compared to the 13 other known zinc import proteins. During LPS exposure, extracellular zinc concentrations within the physiological range markedly reduced IL-10 mRNA expression and protein release but increased mRNA expression of TNF[alpha], IL-8, and IL-6. ZIP8 knockdown inhibited LPS-driven cellular accumulation of zinc and prevented zinc-dependent reduction of IL-10 release. Further, zinc supplementation reduced nuclear localization and activity of C/EBP[beta], a transcription factor known to drive IL-10 expression. These studies demonstrate for the first time that zinc regulates LPS-mediated immune activation of human macrophages in a ZIP8-dependent manner, reducing IL-10. Based on these findings we predict that macrophage zinc metabolism is important in host defense against pathogens.

ABSTRACT Ceragenins are a family of synthetic amphipathic molecules designed to mimic the properties of naturally-occurring cationic antimicrobial peptides (CAMPs). Although ceragenins have potent antimicrobial activity, whether their mode of action is similar to that of CAMPs has remained elusive. Here we report the results of a comparative study of the bacterial responses to two well-studied CAMPs, LL37 and colistin, and two ceragenins with related structures, CSA13 and CSA131. Using transcriptomic and proteomic analyses, we found that Escherichia coli responds similarly to both CAMPs and ceragenins by inducing a Cpx envelope stress response. However, whereas E. coli exposed to CAMPs increased expression of genes involved in colanic acid biosynthesis, bacteria exposed to ceragenins specifically modulated functions related to phosphate transport, indicating distinct mechanisms of action between these two classes of molecules. Although traditional genetic approaches failed to identify genes that confer high-level resistance to ceragenins, using a Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) approach we identified E. coli essential genes that when knocked down modify sensitivity to these molecules. Comparison of the essential gene-antibiotic interactions for each of the CAMPs and ceragenins identified both overlapping and distinct dependencies for their antimicrobial activities. Overall, this study indicates that while some bacterial responses to ceragenins overlap with those induced by naturally-occurring CAMPs, these synthetic molecules target the bacterial envelope using a distinctive mode of action. IMPORTANCE The development of novel antibiotics is essential since the current arsenal of antimicrobials will soon be ineffective due to the widespread occurrence of antibiotic resistance. Development of naturally-occurring cationic antimicrobial peptides (CAMPs) for therapeutics to combat antibiotic resistance has been hampered by high production costs and protease sensitivity, among other factors. The ceragenins are a family of synthetic CAMP mimics that kill a broad spectrum of bacterial species but are less expensive to produce, resistant to proteolytic degradation and have been associated with low levels of resistance. Determining how ceragenins function may identify new essential biological pathways of bacteria that are less prone to development of resistance and will further our understanding of the design principles for maximizing the effects of synthetic CAMPs. Competing Interest Statement The authors have declared no competing interest.

Macrophages employ an array of pattern recognition receptors to detect and eliminate intracellular pathogens that access the cytosol. The cytosolic carbohydrate sensors Galectin-3, -8, and -9 (Gal-3, Gal-8, and Gal-9) recognize damaged pathogen-containing phagosomes, and Gal-3 and Gal-8 are reported to restrict bacterial growth via autophagy in cultured cells. However, the contribution of these galectins to host resistance during bacterial infection remains unclear. We found that Gal-9 binds directly to Mycobacterium tuberculosis (Mtb) and Salmonella enterica serovar Typhimurium (Stm) and localizes to Mtb in macrophages. To determine the combined contribution of membrane damage-sensing galectins to immunity in vivo, we generated Gal-3, -8, and -9 triple knockout (TKO) mice. Mtb infection of primary macrophages from TKO mice resulted in defective lysosomal trafficking but normal bacterial replication. Surprisingly, these mice had no discernable defect in resistance to acute infection with Mtb, Stm or Listeria monocytogenes, and had only modest impairments in bacterial growth restriction and CD4 T cell activation during chronic Mtb infection. Collectively, these findings indicate that while Gal-3, -8, and -9 respond to an array of intracellular pathogens, together these membrane damage-sensing galectins play a limited role in host resistance to bacterial infection.Competing Interest StatementREV is on the Scientific Advisory Board of Tempest Therapeutics. NJK has consulting agreements with the Icahn School of Medicine at Mount Sinai, New York, Maze Therapeutics, and Interline Therapeutics. He is a shareholder in Tenaya Therapeutics, Maze Therapeutics and Interline Therapeutics, and is financially compensated by GEn1E Lifesciences, Inc. and Twist Bioscience Corp. The Krogan Laboratory has received research support from Vir Biotechnology and F. Hoffmann-La Roche. DAP has a financial interest in Laguna Biotherapeutics and both he and the company stand to benefit from commercialization of the results of this research.

North Atlantic Deep Water (NADW), the return flow component of the Atlantic Meridional Overturning Circulation (AMOC), is a major inter-hemispheric ocean water mass with strong climate effects but the evolution of its source components on million-year timescales is poorly known. Today, two major NADW components that flow southward over volcanic ridges to the east and west of Iceland are associated with distinct contourite drift systems that are forming off the coast of Greenland and on the eastern flank of the Reykjanes (mid-Atlantic) Ridge. Here we provide direct records of the early history of this drift sedimentation based on cores collected during International Ocean Discovery Programme (IODP) Expeditions 395C and 395. We find rapid acceleration of drift deposition linked to the eastern component of NADW, known as Iceland–Scotland Overflow Water at 3.6 million years ago (Ma). In contrast, the Denmark Strait Overflow Water feeding the western Eirik Drift has been persistent since the Late Miocene. These observations constrain the long-term evolution of the two NADW components, revealing their contrasting independent histories and allowing their links with climatic events such as Northern Hemisphere cooling at 3.6 Ma, to be assessed. This study examines the history of North Atlantic deep-water masses, as recorded in marine sediments. Major lithological changes and increased rate of deposition reveal that stronger deep-ocean circulation initiated 3.6 million years ago.

Background Covid-19 healthcare worker testing, isolation and quarantine policies had to balance risks to patients from the virus and from staff absence. The emergence of the Omicron variant led to dangerous levels of key-worker absence globally. We evaluated whether using two manufacturers’ lateral flow tests (LFTs) concurrently improved SARS-CoV-2 Omicron detection significantly and was acceptable to hospital staff. In a nested study, to understand risks of return to work after a 5-day isolation/quarantine period, we examined virus culture 5–7 days after positive test or significant exposure. Methods Fully-vaccinated Liverpool (UK) University Hospitals staff participated (February-May 2022) in a random-order, open-label trial testing whether dual LFTs improved SARS-CoV-2 detection, and whether dual swabbing was acceptable to users. Participants used nose-throat swab Innova and nose-only swab Orient Gene LFTs in daily randomised order for 10 days. A user-experience questionnaire was administered on exit. Selected participants gave swabs for viral culture on days 5–7 after symptom onset or first positive test. Cultures were considered positive if cytopathic effect was apparent or SARS-CoV-2 N gene sub-genomic RNA was detected. Results Two hundred and twenty-six individuals reported 1466 pairs of LFT results. Tests disagreed in 127 cases (8.7%). Orient Gene was more likely (78 cf. 49; OR: 2.1, 1.1–4.1; P  = 0.03) to be positive. If Innova was swabbed second, it was less likely to agree with a positive Orient Gene result (OR: 2.7, 1.3–5.2; P  = 0.005); swabbing first with Innova made no significant difference (OR: 1.1, 0.5–2.3; P  = 0.85). Orient Gene positive Innova negative result-pairs became more frequent over time (OR: 1.2, 1.1–1.3; P  < 0.001). Of individuals completing the exit questionnaire, 90.7% reported dual swabbing was easy, 57.1% said it was no barrier to their daily routine and 65.6% preferred dual testing. Respondents had more confidence in dual versus single test results. Viral cultures from days 5–7 were positive for 6/31 (19.4%, 7.5%-37.5%) and indeterminate for 11/31 (35.5%, 19.2%-54.6%) LFT-positive participants, indicating they were likely still infectious. Conclusions Dual brand testing increased LFT detection of SARS-CoV-2 antigen by a small but meaningful margin and was acceptable to hospital workers. Viral cultures demonstrated that policies recommending safe return to work ~ 5 days after Omicron infection/exposure were flawed. Key-workers should be prepared for dynamic self-testing protocols in future pandemics. Trial registration https://www.isrctn.com/ISRCTN47058442 (26 January 2022).

Background Identifying and supporting young children who are at risk of developing anxiety disorders would benefit children, families, and wider society. Elevated anxiety symptoms, inhibited temperament, and high parental anxiety are established risk factors for later anxiety disorders, but it remains unclear who is most likely to benefit from prevention and early intervention programmes. Delivering an online intervention through schools to parents of young children who have one or more of these risks could maximise reach. The primary aim of this trial is to evaluate the effectiveness and cost-effectiveness of delivering an online parent-led intervention, compared with usual school provision only, for children (aged 4–7) identified as at risk for anxiety disorders on the basis of at least one risk factor. We also aim to identify the characteristics of children who do and do not benefit from intervention and mechanisms of change from the intervention. Methods The design will be a parallel group, superiority cluster randomised controlled trial, with schools (clusters) randomised to intervention or usual school practice arms in a 1:1 ratio stratified according to level of deprivation within the school. The study will recruit and randomise at least 60 primary/infant schools in England, and on the basis of recruiting 60 schools, we will recruit 1080 trial participants (540 per arm). Parents of all children (aged 4–7) in sampled Reception, Year 1, and Year 2 classes will be invited to complete screening questionnaires. Children who screen positive on the basis of anxiety symptoms, and/or behavioural inhibition, and/or parent anxiety symptoms will be eligible for the trial. Parents/carers of children in schools allocated to the intervention arm will be offered a brief online intervention; schools in both arms will continue to provide any usual support for children and parents throughout the trial. Assessments will be completed at screening, baseline (before randomisation), 6 weeks, 12 weeks, and 12 months post-randomisation. The primary outcome will be the absence/presence of an anxiety disorder diagnosis at 12 months. Discussion The trial will determine if delivering an online intervention for parents of young children at risk of anxiety disorders identified through screening in schools is effective and cost-effective. Trial registration ISRCTN 82398107 . Prospectively registered on Jan. 14, 2021.