Keratinocyte-derived defensins activate neutrophil-specific receptors Mrgpra2a/b to prevent skin dysbiosis and bacterial infection.

Healthy skin maintains a diverse microbiome and a potent immune system to fight off infections. Here, we discovered that the epithelial-cell-derived antimicrobial peptides defensins activated orphan G-protein-coupled receptors (GPCRs) Mrgpra2a/b on neutrophils. This signaling axis was required for effective neutrophil-mediated skin immunity and microbiome homeostasis. We generated mutant mouse lines lacking the entire Defensin (Def) gene cluster in keratinocytes or Mrgpra2a/b. Def and Mrgpra2 mutant animals both exhibited skin dysbiosis, with reduced microbial diversity and expansion of Staphylococcus species. Defensins and Mrgpra2 were critical for combating S. aureus infections and the formation of neutrophil abscesses, a hallmark of antibacterial immunity. Activation of Mrgpra2 by defensin triggered neutrophil release of IL-1ß and CXCL2 which are vital for proper amplification and propagation of the antibacterial immune response. This study demonstrated the importance of epithelial-neutrophil signaling via the defensin-Mrgpra2 axis in maintaining healthy skin ecology and promoting antibacterial host defense.

Capsular polysaccharide inhibits vaccine-induced O-antigen antibody binding and function across both classical and hypervirulent K2:O1 strains of Klebsiella pneumoniae.

Klebsiella pneumoniae presents as two circulating pathotypes: classical K. pneumoniae (cKp) and hypervirulent K. pneumoniae (hvKp). Classical isolates are considered urgent threats due to their antibiotic resistance profiles, while hvKp isolates have historically been antibiotic susceptible. Recently, however, increased rates of antibiotic resistance have been observed in both hvKp and cKp, further underscoring the need for preventive and effective immunotherapies. Two distinct surface polysaccharides have gained traction as vaccine candidates against K. pneumoniae: capsular polysaccharide and the O-antigen of lipopolysaccharide. While both targets have practical advantages and disadvantages, it remains unclear which of these antigens included in a vaccine would provide superior protection against matched K. pneumoniae strains. Here, we report the production of two bioconjugate vaccines, one targeting the K2 capsular serotype and the other targeting the O1 O-antigen. Using murine models, we investigated whether these vaccines induced specific antibody responses that recognize K2:O1 K. pneumoniae strains. While each vaccine was immunogenic in mice, both cKp and hvKp strains exhibited decreased O-antibody binding in the presence of capsule. Further, O1 antibodies demonstrated decreased killing in serum bactericidal assays with encapsulated strains, suggesting that the presence of K. pneumoniae capsule blocks O1-antibody binding and function. Finally, the K2 vaccine outperformed the O1 vaccine against both cKp and hvKp in two different murine infection models. These data suggest that capsule-based vaccines may be superior to O-antigen vaccines for targeting hvKp and some cKp strains, due to capsule blocking the O-antigen.

Ship traffic connects Antarctica's fragile coasts to worldwide ecosystems.

Antarctica, an isolated and long considered pristine wilderness, is becoming increasingly exposed to the negative effects of ship-borne human activity, and especially the introduction of invasive species. Here, we provide a comprehensive quantitative analysis of ship movements into Antarctic waters and a spatially explicit assessment of introduction risk for nonnative marine species in all Antarctic waters. We show that vessels traverse Antarctica's isolating natural barriers, connecting it directly via an extensive network of ship activity to all global regions, especially South Atlantic and European ports. Ship visits are more than seven times higher to the Antarctic Peninsula (especially east of Anvers Island) and the South Shetland Islands than elsewhere around Antarctica, together accounting for 88% of visits to Southern Ocean ecoregions. Contrary to expectations, we show that while the five recognized "Antarctic Gateway cities" are important last ports of call, especially for research and tourism vessels, an additional 53 ports had vessels directly departing to Antarctica from 2014 to 2018. We identify ports outside Antarctica where biosecurity interventions could be most effectively implemented and the most vulnerable Antarctic locations where monitoring programs for high-risk invaders should be established.

Heptavalent O-Antigen Bioconjugate Vaccine Exhibiting Differential Functional Antibody Responses Against Diverse Klebsiella pneumoniae Isolates.

Klebsiella pneumoniae is the leading cause of neonatal sepsis and is increasingly difficult to treat owing to antibiotic resistance. Vaccination represents a tractable approach to combat this resistant bacterium; however, there is currently not a licensed vaccine. Surface polysaccharides, including O-antigens of lipopolysaccharide, have long been attractive candidates for vaccine inclusion. Herein we describe the generation of a bioconjugate vaccine targeting 7 predominant O-antigen subtypes in K. pneumoniae. Each bioconjugate was immunogenic in isolation, with limited cross-reactivity among subtypes. Vaccine-induced antibodies demonstrated varying degrees of binding to a wide variety of K. pneumoniae strains. Furthermore, serum from vaccinated mice induced complement-mediated killing of many of these strains. Finally, increased capsule interfered with the ability of O-antigen antibodies to bind and mediate killing of some K. pneumoniae strains. Taken together, these data indicate that this novel heptavalent O-antigen bioconjugate vaccine formulation exhibits limited efficacy against some, but not all, K. pneumoniae isolates.

The impact of acute low salinity stress on Antarctic echinoderms.

Climate change is causing increased coastal freshening in Antarctica, leading to reduced salinity. For Antarctica's endemic echinoderms, adapted to the stable polar environment, the impact of rapid reductions in coastal salinity on physiology and behaviour is currently unknown. Six common Antarctic echinoderms (the sea urchin Sterechinus neumayeri; the sea star Odontaster validus; the brittle star Ophionotus victoriae; and three sea cucumbers Cucumaria georgiana, Echinopsolus charcoti and Heterocucumis steineni), were directly transferred from ambient salinity (34.5‰) to a range of salinity dilutions (29-9‰) for 24 h. All species showed reduced activity and the establishment of a temporary osmotic gradient between coelomic fluid and external seawater. Most species exhibited a depression in oxygen consumption across tolerated salinities; however, at very low salinities that later resulted in mortality, oxygen consumption increased to levels comparable to those at ambient. Low salinity tolerance varied substantially between species, with O. victoriae being the least tolerant (24 h LC50 (lethal for 50% of animals) = 19.9‰) while E. charcoti and C. georgiana demonstrated the greatest tolerance (24 h LC50 = 11.5‰). These findings demonstrate the species-specific response of Antarctica's endemic echinoderms to short-term hypoosmotic salinity events, providing valuable insight into this phylum's ability to respond to an underreported impact of climate change.

Climate and human stressors on global penguin hotspots: Current assessments for future conservation.

As charismatic and iconic species, penguins can act as "ambassadors" or flagship species to promote the conservation of marine habitats in the Southern Hemisphere. Unfortunately, there is a lack of reliable, comprehensive, and systematic analysis aimed at compiling spatially explicit assessments of the multiple impacts that the world's 18 species of penguin are facing. We provide such an assessment by combining the available penguin occurrence information from Global Biodiversity Information Facility (>800,000 occurrences) with three main stressors: climate-driven environmental changes at sea, industrial fisheries, and human disturbances on land. Our analyses provide a quantitative assessment of how these impacts are unevenly distributed spatially within species' distribution ranges. Consequently, contrasting pressures are expected among species, and populations within species. The areas coinciding with the greatest impacts for penguins are the coast of Perú, the Patagonian Shelf, the Benguela upwelling region, and the Australian and New Zealand coasts. When weighting these potential stressors with species-specific vulnerabilities, Humboldt (Spheniscus humboldti), African (Spheniscus demersus), and Chinstrap penguin (Pygoscelis antarcticus) emerge as the species under the most pressure. Our approach explicitly differentiates between climate and human stressors, since the more achievable management of local anthropogenic stressors (e.g., fisheries and land-based threats) may provide a suitable means for facilitating cumulative impacts on penguins, especially where they may remain resilient to global processes such as climate change. Moreover, our study highlights some poorly represented species such as the Northern Rockhopper (Eudyptes moseleyi), Snares (Eudyptes robustus), and Erect-crested penguin (Eudyptes sclateri) that need internationally coordinated efforts for data acquisition and data sharing to understand their spatial distribution properly.

Behavioural and physiological impacts of low salinity on the sea urchin Echinus esculentus.

Reduced seawater salinity as a result of freshwater input can exert a major influence on the ecophysiology of benthic marine invertebrates, such as echinoderms. While numerous experimental studies have explored the physiological and behavioural effects of short-term, acute exposure to low salinity in echinoids, surprisingly few have investigated the consequences of chronic exposure, or compared the two. In this study, the European sea urchin, Echinus esculentus, was exposed to low salinity over the short term (11‰, 16‰, 21‰, 26‰ and 31‰ for 24 h) and longer term (21, 26 and 31‰ for 25 days). Over the short term, oxygen consumption, activity coefficient and coelomic fluid osmolality were directly correlated with reduced salinity, with 100% survival at ≥21‰ and 0% at ≤16‰. Over the longer term at 21‰ (25 days), oxygen consumption was significantly higher, feeding was significantly reduced and activity coefficient values were significantly lower than at control salinity (31‰). At 26‰, all metrics were comparable to the control by the end of the experiment, suggesting acclimation. Furthermore, beneficial functional resistance (righting ability and metabolic capacity) to acute low salinity was observed at 26‰. Osmolality values were slightly hyperosmotic to the external seawater at all acclimation salinities, while coelomocyte composition and concentration were unaffected by chronic low salinity. Overall, E. esculentus demonstrate phenotypic plasticity that enables acclimation to reduced salinity around 26‰; however, 21‰ represents a lower acclimation threshold, potentially limiting its distribution in coastal areas prone to high freshwater input.

The SAGES masters program presents the 10 seminal articles for laparoscopic sleeve gastrectomy.

BACKGROUND: The Society of American Gastrointestinal and Endoscopic Surgeons (SAGES) Masters Program includes eight distinct clinical pathways. The Bariatric Surgery Pathway focuses on three anchoring procedures, including the laparoscopic sleeve gastrectomy (LSG) which is the most commonly performed bariatric procedure in the United States. In this article, we present and discuss the top 10 seminal articles regarding the LSG. METHODS: The literature was systematically searched to identify the most cited papers on LSG. The SAGES Metabolic and Bariatric Surgery committee reviewed the most cited article list, and using expert consensus elected the seminal articles deemed most pertinent to LSG. These articles were reviewed in detail by committee members and are presented here. RESULTS: The top 10 most cited sentinel papers on LSG focus on operative safety, outcomes, surgical technique, and physiologic changes after the procedure. A summary of each paper is presented, including expert appraisal and commentary. CONCLUSIONS: The seminal articles presented support the widespread acceptance and use of the LSG by bolstering the understanding of its mechanism of action and by demonstrating its safety and excellent patient outcomes. All bariatric surgeons should be familiar with these 10 landmark articles.

A comparative study of tissue protein synthesis rates in an Antarctic, Harpagifer antarcticus and a temperate, Lipophrys pholis teleost.

The affect of temperature on tissue protein synthesis rates has been reported in temperate and tropical, but not Antarctic fishes. Previous studies have generally demonstrated low growth rates in Antarctic fish species in comparison to temperate relatives and elevated levels of protein turnover. This study investigates how low temperatures effect tissue protein synthesis and hence tissue growth in a polar fish species. Groups of Antarctic, Harpagifer antarcticus and temperate, Lipophrys pholis, were acclimated to a range of overlapping water temperatures and protein synthesis was measure in white muscle (WM), liver and gastrointestinal tract (GIT). WM protein synthesis rates increased linearly with temperature in both species (H. antarcticus 0.16-0.23%.d-1, L. pholis, 0.31-0.76%.d-1), while liver (H. antarcticus 0.24-0.27%.d-1, L. pholis, 0.44-1.03%.d-1) and GIT were unaffected by temperature in H. antarcticus but increased non-linearly in L.pholis (H. antarcticus 0.22-0.26%.d-1, L. pholis, 0.40-0.86%.d-1). RNA to protein ratios were unaffected by temperature in H. antarcticus but increased weakly, in L.pholis WM and liver. In L.pholis, RNA translational efficiency increased significantly with temperature in all tissues, but only in liver in H. antarcticus. At the overlapping temperature of 3 °C, protein synthesis (WM 26%, Liver, 39%, GIT, 35%) and RNA translational efficiency (WM 273%, Liver, 271%, GIT, 300%) were significantly lower in H. antarcticus than L.pholis, while RNA to protein ratios were significantly higher (WM 270%, Liver 170%, GIT 186%). Tissue specific effects of temperature are detectable in both species. This study provides the first evidence, that tissue protein synthesis rates are constrained in Antarctic fishes.

Why do students choose to do an extended GP placement?

The drivers for medical students' decision making when considering which Student Selected Component (SSC) to undertake is poorly understood. Furthermore, it is unclear why students undertake a specific SSC allowing them to have an extended placement in GP in their final year. It is known that high quality GP placements encourage students to subsequently choose GP as their career, therefore if the decision-making process of students in this area can be better understood, then this may help inform medical school actions to encourage a greater uptake of these extended placements.Semi-structured interviews were conducted with final year medical students at a Scottish University. Students were selected to provide a mixture of those who had and had not chosen to undertake the extended placement. The data was transcribed and analysed using thematic analysis to generate themes which represented the data.This showed that career intention was a major factor driving SSC choice. Additionally, students sought peer feedback and tended to avoid specific SSCs if they felt a lack of internal motivation. Considering the choice for the extended placement, students tended, again, to choose based on career intentions, and they also based their decision on previous experiences of GP.Career intention and prior experience are key factors in student choice of SSC and whether to undertake an extended GP. In order to address the national shortage of GPs medical schools need to consider how they might influence these drivers.

Discovery and characterization of a new class of O-linking oligosaccharyltransferases from the Moraxellaceae family.

Bacterial protein glycosylation is commonly mediated by oligosaccharyltransferases (OTases) that transfer oligosaccharides en bloc from preassembled lipid-linked precursors to acceptor proteins. Natively, O-linking OTases usually transfer a single repeat unit of the O-antigen or capsular polysaccharide to the side chains of serine or threonine on acceptor proteins. Three major families of bacterial O-linking OTases have been described: PglL, PglS, and TfpO. TfpO is limited to transferring short oligosaccharides both in its native context and when heterologously expressed in glycoengineered Escherichia coli. On the other hand, PglL and PglS can transfer long-chain polysaccharides when expressed in glycoengineered E. coli. Herein, we describe the discovery and functional characterization of a novel family of bacterial O-linking OTases termed TfpM from Moraxellaceae bacteria. TfpM proteins are similar in size and sequence to TfpO enzymes but can transfer long-chain polysaccharides to acceptor proteins. Phylogenetic analyses demonstrate that TfpM proteins cluster in distinct clades from known bacterial OTases. Using a representative TfpM enzyme from Moraxella osloensis, we determined that TfpM glycosylates a C-terminal threonine of its cognate pilin-like protein and identified the minimal sequon required for glycosylation. We further demonstrated that TfpM has broad substrate tolerance and can transfer diverse glycans including those with glucose, galactose, or 2-N-acetyl sugars at the reducing end. Last, we find that a TfpM-derived bioconjugate is immunogenic and elicits serotype-specific polysaccharide IgG responses in mice. The glycan substrate promiscuity of TfpM and identification of the minimal TfpM sequon renders this enzyme a valuable additional tool for expanding the glycoengineering toolbox.

Crisaborole efficacy in murine models of skin inflammation and Staphylococcus aureus infection.

Phosphodiesterase 4 (PDE4) is highly expressed in keratinocytes and immune cells and promotes pro-inflammatory responses upon activation. The activity of PDE4 has been attributed to various inflammatory conditions, leading to the development and approval of PDE4 inhibitors as host-directed therapeutics in humans. For example, the topical PDE4 inhibitor, crisaborole, is approved for the treatment of mild-to-moderate atopic dermatitis and has shown efficacy in patients with psoriasis. However, the role of crisaborole in regulating the immunopathogenesis of inflammatory skin diseases and infection is not entirely known. Therefore, we evaluated the effects of crisaborole in multiple mouse models, including psoriasis-like dermatitis, AD-like skin inflammation with and without filaggrin mutations, and Staphylococcus aureus skin infection. We discovered that crisaborole dampens myeloid cells and itch in the skin during psoriasis-like dermatitis. Furthermore, crisaborole was effective in reducing skin inflammation in the context of filaggrin deficiency. Importantly, crisaborole reduced S. aureus skin colonization during AD-like skin inflammation. However, crisaborole was not efficacious in treating S. aureus skin infections, even as adjunctive therapy to antibiotics. Taken together, we found that crisaborole reduced itch during psoriasis-like dermatitis and decreased S. aureus skin colonization upon AD-like skin inflammation, which act as additional mechanisms by which crisaborole dampens the immunopathogenesis in mouse models of inflammatory skin diseases. Further examination is warranted to translate these preclinical findings to human disease.

Soluble adenylyl cyclase contributes to imiquimod-mediated inflammation and is a potential therapeutic target in psoriasis.

Cyclic AMP (cAMP) has a key role in psoriasis pathogenesis, as indicated by the therapeutic efficacy of phosphodiesterase inhibitors that prevent the degradation of cAMP. However, whether soluble adenylate cyclase (sAC) (encoded by the ADCY10 gene), which is an important source for cAMP, is involved in Th17 cell-mediated inflammation or could be an alternative therapeutic target in psoriasis is unknown. We have utilized the imiquimod model of murine psoriasiform dermatitis to address this question. Adcy10-/- mice had reduced erythema, scaling and swelling in the skin and reduced CD4+ IL17+ cell numbers in the draining lymph nodes, compared with wild-type mice after induction of psoriasiform dermatitis with imiquimod. Keratinocyte-specific knock out of Adcy10 had no effect on imiquimod-induced ear swelling suggesting keratinocyte sAC has no role in imiquimod-induced inflammation. During Th17 polarization in vitro, naive T cells from Adcy10-/- mice exhibited reduced IL17 secretion and IL-17+ T-cell proliferation suggesting that differentiation into Th17 cells is suppressed without sAC activity. Interestingly, loss of sAC did not impact the expression of Th17 lineage-defining transcription factors (such as Rorc and cMaf) but rather was required for CREB-dependent gene expression, which is known to support Th17 cell gene expression. Finally, topical application of small molecule sAC inhibitors (sACi) reduced imiquimod-induced psoriasiform dermatitis and Il17 gene expression in the skin. Collectively, these findings demonstrate that sAC is important for psoriasiform dermatitis in mouse skin. sACi may provide an alternative class of topical therapeutics for Th17-mediated skin diseases.

Glycan cross-feeding supports mutualism between Fusobacterium and the vaginal microbiota.

Women with bacterial vaginosis (BV), an imbalance of the vaginal microbiome, are more likely to be colonized by potential pathogens such as Fusobacterium nucleatum, a bacterium linked with intrauterine infection and preterm birth. However, the conditions and mechanisms supporting pathogen colonization during vaginal dysbiosis remain obscure. We demonstrate that sialidase activity, a diagnostic feature of BV, promoted F. nucleatum foraging and growth on mammalian sialoglycans, a nutrient resource that was otherwise inaccessible because of the lack of endogenous F. nucleatum sialidase. In mice with sialidase-producing vaginal microbiotas, mutant F. nucleatum unable to consume sialic acids was impaired in vaginal colonization. These experiments in mice also led to the discovery that F. nucleatum may also "give back" to the community by reinforcing sialidase activity, a biochemical feature of human dysbiosis. Using human vaginal bacterial communities, we show that F. nucleatum supported robust outgrowth of Gardnerella vaginalis, a major sialidase producer and one of the most abundant organisms in BV. These results illustrate that mutually beneficial relationships between vaginal bacteria support pathogen colonization and may help maintain features of dysbiosis. These findings challenge the simplistic dogma that the mere absence of "healthy" lactobacilli is the sole mechanism that creates a permissive environment for pathogens during vaginal dysbiosis. Given the ubiquity of F. nucleatum in the human mouth, these studies also suggest a possible mechanism underlying links between vaginal dysbiosis and oral sex.

Management of abdominal wall hernias in patients with severe obesity.

Obesity is a risk factor for abdominal wall hernia development and hernia recurrence. The management of these two pathologies is complex and often entwined. Bariatric and ventral hernia surgery require careful consideration of physiologic and technical components for optimal outcomes. In this review, a multidisciplinary group of Society of American Gastrointestinal and Endoscopic Surgeons' bariatric and hernia surgeons present the various weight loss modalities available for the pre-operative optimization of patients with severe obesity and concurrent hernias. The group also details the technical aspects of managing abdominal wall defects during weight loss procedures and suggests the optimal timing of definitive hernia repair after bariatric surgery. Since level one evidence is not available on some of the topics covered by this review, expert opinion was implemented in some instances. Additional high-quality research in this area will allow for better recommendations and therefore treatment strategies for these complex patients.

An oral caspase inhibitor as monotherapy or with antibiotics eradicates MRSA skin infections in mice.

Staphylococcus aureus is the leading cause of skin and soft tissue infections. With the emergence of antibiotic-resistant bacteria, there is an unmet clinical need to develop immune-based therapies to treat skin infections. Previously, we have shown pan-caspase inhibition as a potential host-directed immunotherapy against community-acquired methicillin-resistant S aureus (CA-MRSA) and other bacterial skin infections. Here, we evaluated the role of irreversible pan-caspase inhibitor emricasan as a monotherapy and an adjunctive with a standard-of-care antibiotic, doxycycline, as potential host-directed immunotherapies against S. aureus skin infections in vivo. We used the established CA-MRSA strain USA300 on the dorsum of WT C57BL/6J mice and monitored lesion size and bacterial burden noninvasively, and longitudinally over 14 days with in vivo bioluminescence imaging (BLI). Mice in four groups placebo (0.5% carboxymethyl cellulose [CMC] solution), placebo plus doxycycline (100 mg/kg), emricasan (40 mg/kg) plus doxycycline, and emricasan only were treated orally twice daily by oral gavage for 7 days, starting at 4 h after injection of S aureus. When compared with placebo, all three groups, placebo plus doxycycline, emricasan plus doxycycline, and emricasan treated group, exhibited biological effect, with reduction of both the lesion size (*p = .0277, ****p < .0001, ****p < .0001, respectively) and bacterial burden (***p = .003, ****p < .0001, ****p < .0001, respectively). Importantly, the efficacy of emricasan against S. aureus was not due to direct antibacterial activity. Collectively, pan-caspase inhibitor emricasan and emricasan plus doxycycline reduced both the lesion size and bacterial burden in vivo, and emricasan is a potential host-directed immunotherapy against MRSA skin infections in a preclinical mouse model.

BSHI/BTS guidance on crossmatching before deceased donor kidney transplantation.

All UK H&I laboratories and transplant units operate under a single national kidney offering policy, but there have been variations in approach regarding when to undertake the pre-transplant crossmatch test. In order to minimize cold ischaemia times for deceased donor kidney transplantation we sought to find ways to be able to report a crossmatch result as early as possible in the donation process. A panel of experts in transplant surgery, nephrology, specialist nursing in organ donation and H&I (all relevant UK laboratories represented) assessed evidence and opinion concerning five factors that relate to the effectiveness of the crossmatch process, as follows: when the result should be ready for reporting; what level of donor HLA typing is needed; crossmatch sample type and availability; fairness and equity; risks and patient safety. Guidelines aimed at improving practice based on these issues are presented, and we expect that following these will allow H&I laboratories to contribute to reducing CIT in deceased donor kidney transplantation.

Clonal Vγ6+Vδ4+ T cells promote IL-17-mediated immunity against Staphylococcus aureus skin infection.

T cell cytokines contribute to immunity against Staphylococcus aureus, but the predominant T cell subsets involved are unclear. In an S. aureus skin infection mouse model, we found that the IL-17 response was mediated by γδ T cells, which trafficked from lymph nodes to the infected skin to induce neutrophil recruitment, proinflammatory cytokines IL-1α, IL-1ß, and TNF, and host defense peptides. RNA-seq for TRG and TRD sequences in lymph nodes and skin revealed a single clonotypic expansion of the encoded complementarity-determining region 3 amino acid sequence, which could be generated by canonical nucleotide sequences of TRGV5 or TRGV6 and TRDV4 However, only TRGV6 and TRDV4 but not TRGV5 sequences expanded. Finally, Vγ6+ T cells were a predominant γδ T cell subset that produced IL-17A as well as IL-22, TNF, and IFNγ, indicating a broad and substantial role for clonal Vγ6+Vδ4+ T cells in immunity against S. aureus skin infections.

Neutrophil extracellular traps impair regeneration.

Fibrosis is a major health burden across diseases and organs. To remedy this, we study wound-induced hair follicle neogenesis (WIHN) as a model of non-fibrotic healing that recapitulates embryogenesis for de novo hair follicle morphogenesis after wounding. We previously demonstrated that TLR3 promotes WIHN through binding wound-associated dsRNA, the source of which is still unclear. Here, we find that multiple distinct contexts of high WIHN all show a strong neutrophil signature. Given the correlation between neutrophil infiltration and endogenous dsRNA release, we hypothesized that neutrophil extracellular traps (NETs) likely release nuclear spliceosomal U1 dsRNA and modulate WIHN. However, rather than enhance regeneration, we find mature neutrophils inhibit WIHN such that mice with mature neutrophil depletion exhibit higher WIHN. Similarly, Pad4 null mice, which are defective in NET production, show augmented WIHN. Finally, using single-cell RNA sequencing, we identify a dramatic increase in mature and activated neutrophils in the wound beds of low regenerating Tlr3-/- mice. Taken together, these results demonstrate that although mature neutrophils are stimulated by a common pro-regenerative cue, their presence and NETs hinder regeneration.

A minimal sequon sufficient for O-linked glycosylation by the versatile oligosaccharyltransferase PglS.

Bioconjugate vaccines, consisting of polysaccharides attached to carrier proteins, are enzymatically generated using prokaryotic glycosylation systems in a process termed bioconjugation. Key to bioconjugation are a group of enzymes known as oligosaccharyltransferases (OTases) that transfer polysaccharides to engineered carrier proteins containing conserved amino acid sequences known as sequons. The most recently discovered OTase, PglS, has been shown to have the broadest substrate scope, transferring many different types of bacterial glycans including those with glucose at the reducing end. However, PglS is currently the least understood in terms of the sequon it recognizes. PglS is a pilin-specific O-linking OTase that naturally glycosylates a single protein, ComP. In addition to ComP, we previously demonstrated that an engineered carrier protein containing a large fragment of ComP is also glycosylated by PglS. Here we sought to identify the minimal ComP sequon sufficient for PglS glycosylation. We tested >100 different ComP fragments individually fused to Pseudomonas aeruginosa exotoxin A (EPA), leading to the identification of an 11-amino acid sequence sufficient for robust glycosylation by PglS. We also demonstrate that the placement of the ComP sequon on the carrier protein is critical for stability and subsequent glycosylation. Moreover, we identify novel sites on the surface of EPA that are amenable to ComP sequon insertion and find that Cross-Reactive Material 197 fused to a ComP fragment is also glycosylated. These results represent a significant expansion of the glycoengineering toolbox as well as our understanding of bacterial O-linking sequons.