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SARS-CoV-2 remains a global threat to human health particularly as escape mutants emerge. There is an unmet need for effective treatments against COVID-19 for which neutralizing single domain antibodies (nanobodies) have significant potential. Their small size and stability mean that nanobodies are compatible with respiratory administration. We report four nanobodies (C5, H3, C1, F2) engineered as homotrimers with pmolar affinity for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Crystal structures show C5 and H3 overlap the ACE2 epitope, whilst C1 and F2 bind to a different epitope. Cryo Electron Microscopy shows C5 binding results in an all down arrangement of the Spike protein. C1, H3 and C5 all neutralize the Victoria strain, and the highly transmissible Alpha (B.1.1.7 first identified in Kent, UK) strain and C1 also neutralizes the Beta (B.1.35, first identified in South Africa). Administration of C5-trimer via the respiratory route showed potent therapeutic efficacy in the Syrian hamster model of COVID-19 and separately, effective prophylaxis. The molecule was similarly potent by intraperitoneal injection., Neutralizing nanobodies (Nb) are of considerable interest as therapeutic agents for COVID-19 treatment. Here, the authors functionally and structurally characterize Nbs that bind with high affinity to the receptor binding domain of the SARS-CoV-2 spike protein and show that an engineered homotrimeric Nb prevents disease progression in a Syrian hamster model of COVID-19 when administered intranasally.

Development of anti-drug antibodies (ADAs) can interfere with therapeutic monoclonal antibodies and may lead to drug neutralisation and clinical disease progression. Measurement of circulating drug levels and development of ADAs in the setting of anti-programmed cell death-1 agent pembrolizumab has not been well-studied. Enzyme-linked immunosorbent assays were used to measure pembrolizumab drug level and ADAs in 41 patients with melanoma at baseline, Time-point 1 (3 weeks) and Time-point 2 (21 weeks). Assay results were related to patient demographics and clinical outcome data at 6 months. The median pembrolizumab drug level at 3 weeks was 237 ng/μL and did not correlate with age, sex or body surface area.17/41 patients had an ADA detected at any timepoint, with the highest prevalence at Timepoint 1 (median concentration = 17 ng/μL). The presence of an ADA did not correlate with clinical progression at 6 months. 3/41 (7%) of patients displayed a falling pembrolizumab drug level and rising ADA titre between Timepoint 1 and 2 suggestive of a neutralising ADA. Pembrolizumab drug levels and ADAs can be readily measured. The rates of total and treatment-emergent ADAs may be higher in “real-word” settings than those previously reported. Larger studies are needed to determine effect of neutralising ADAs on long-term clinical outcome.

A novel coronavirus, SARS-CoV-2, has been identified as the causative agent of the current COVID-19 pandemic. Animal models, and in particular non-human primates, are essential to understand the pathogenesis of emerging diseases and to assess the safety and efficacy of novel vaccines and therapeutics. Here, we show that SARS-CoV-2 replicates in the upper and lower respiratory tract and causes pulmonary lesions in both rhesus and cynomolgus macaques. Immune responses against SARS-CoV-2 are also similar in both species and equivalent to those reported in milder infections and convalescent human patients. This finding is reiterated by our transcriptional analysis of respiratory samples revealing the global response to infection. We describe a new method for lung histopathology scoring that will provide a metric to enable clearer decision making for this key endpoint. In contrast to prior publications, in which rhesus are accepted to be the preferred study species, we provide convincing evidence that both macaque species authentically represent mild to moderate forms of COVID-19 observed in the majority of the human population and both species should be used to evaluate the safety and efficacy of interventions against SARS-CoV-2. Importantly, accessing cynomolgus macaques will greatly alleviate the pressures on current rhesus stocks., Non-human primates are important animal models for studying SARS-CoV-2 infection. Here, Salguero et al. directly compare rhesus and cynomolgus macaques and show that both species represent COVID-19 disease of mild clinical cases, and provide a lung histopathology scoring system.

Tripartite motif-containing proteins (TRIMs) play a variety of recently described roles in innate immunity. Although many TRIMs regulate type I IFN expression following cytosolic nucleic acid sensing of viruses, their contribution to innate immune signaling and gene expression during bacterial infection remains largely unknown. Because Mycobacterium tuberculosis is an activator of cGAS-dependent cytosolic DNA sensing, we set out to investigate a role for TRIM proteins in regulating macrophage responses to M. tuberculosis. In this study, we demonstrate that TRIM14, a noncanonical TRIM that lacks an E3 ubiquitin ligase RING domain, is a critical negative regulator of the type I IFN response in Mus musculus macrophages. We show that TRIM14 interacts with both cGAS and TBK1 and that macrophages lacking TRIM14 dramatically hyperinduce IFN stimulated gene (ISG) expression following M. tuberculosis infection, cytosolic nucleic acid transfection, and IFN-β treatment. Consistent with a defect in resolution of the type I IFN response, Trim14 knockout macrophages have more phospho-Ser754 STAT3 relative to phospho-Ser727 and fail to upregulate the STAT3 target Socs3, which is required to turn off IFNAR signaling. These data support a model whereby TRIM14 acts as a scaffold between TBK1 and STAT3 to promote phosphorylation of STAT3 at Ser727 and resolve ISG expression. Remarkably, Trim14 knockout macrophages hyperinduce expression of antimicrobial genes like Nos2 and are significantly better than control cells at limiting M. tuberculosis replication. Collectively, these data reveal an unappreciated role for TRIM14 in resolving type I IFN responses and controlling M. tuberculosis infection.

Immune checkpoint blockade (ICB) of PD-1 and CTLA-4 to treat metastatic melanoma (MM) has variable therapeutic benefit. To explore this in peripheral samples, we characterized CD8+ T cell gene expression across a cohort of patients with MM receiving anti-PD-1 alone (sICB) or in combination with anti-CTLA-4 (cICB). Whereas CD8+ transcriptional responses to sICB and cICB involve a shared gene set, the magnitude of cICB response is over fourfold greater, with preferential induction of mitosis- and interferon-related genes. Early samples from patients with durable clinical benefit demonstrated overexpression of T cell receptor–encoding genes. By mapping T cell receptor clonality, we find that responding patients have more large clones (those occupying >0.5% of repertoire) post-treatment than non-responding patients or controls, and this correlates with effector memory T cell percentage. Single-cell RNA-sequencing of eight post-treatment samples demonstrates that large clones overexpress genes implicated in cytotoxicity and characteristic of effector memory T cells, including CCL4, GNLY and NKG7. The 6-month clinical response to ICB in patients with MM is associated with the large CD8+ T cell clone count 21 d after treatment and agnostic to clonal specificity, suggesting that post-ICB peripheral CD8+ clonality can provide information regarding long-term treatment response and, potentially, facilitate treatment stratification. Transcriptomic analysis of peripheral CD8+ T cells in a cohort of patients with metastatic melanoma receiving checkpoint inhibitors shows that the number of large clones early post-treatment is strongly associated with six-month clinical outcome.

SUMMARYHuman mutations in mitochondrial-associated genes are associated with inflammatory diseases and susceptibility to infection. However, their mechanistic contributions to immune outcomes remain ill-defined. We discovered that the disease-associated gain-of-function allele Lrrk2G2019S (leucine-rich repeat kinase 2) promotes mitochondrial hyper-fission, depolarization, and oxidative stress in macrophages. In the presence of Lrrk2G2019S-dependent mitochondrial perturbations, AIM2 inflammasome activation promotes more cell death but not more pyroptotic IL-1b release. Instead, inflammasome activation in Lrrk2G2019S macrophages triggers gasdermin D (GSDMD)-mediated mitochondrial pores, driving up ROS-mediated RIPK1/RIPK3/MLKL dependent necroptosis. Consequently, infection of Lrrk2G2019S mice with Mycobacterium tuberculosis elicits hyperinflammation and immunopathology via enhanced neutrophil infiltration. By uncovering that GSDMD promotes non-pyroptotic cell death in Lrrk2G2019S macrophages, our findings demonstrate that altered mitochondrial function can reprogram cell death modalities to elicit distinct immune outcomes. This provides mechanistic insights into why mutations in LRRK2 are associated with susceptibility to chronic inflammatory and infectious diseases.HIGHLIGHTSAltered mitochondrial homeostasis reprograms cell death modalitiesGSDMD associates with and depolarizes mitochondrial membranes following AIM2 activationGSDMD initiates a shift from pyroptotic to necroptotic cell death in Lrrk2G2019S macrophagesLrrk2G2019S elicits hyperinflammation and susceptibility to infection in flies and mice

The global pandemic of coronavirus disease (COVID-19) caused by infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led to an international thrust to study pathogenesis and evaluate interventions. Experimental infection of hamsters and the resulting respiratory disease is one of the preferred animal models since clinical signs of disease and virus shedding are similar to more severe cases of human COVID-19. The main route of challenge has been direct inoculation of the virus via the intranasal route. To resemble the natural infection, we designed a bespoke natural transmission cage system to assess whether recipient animals housed in physically separate adjacent cages could become infected from a challenged donor animal in a central cage, with equal airflow across the two side cages. To optimise viral shedding in the donor animals, a low and moderate challenge dose were compared after direct intranasal challenge, but similar viral shedding responses were observed and no discernible difference in kinetics. The results from our natural transmission set-up demonstrate that most recipient hamsters are infected within the system developed, with variation in the kinetics and levels of disease between individual animals. Common clinical outputs used for the assessment in directly-challenged hamsters, such as weight loss, are less obvious in hamsters who become infected from naturally acquiring the infection. The results demonstrate the utility of a natural transmission model for further work on assessing the differences between virus strains and evaluating interventions using a challenge system which more closely resembles human infection.

Filoviruses cause high-consequence infections with limited approved medical countermeasures (MCMs). MCM development is dependent upon well-characterized animal models for the assessment of antiviral agents and vaccines. Following large-scale Ebola virus (EBOV) disease outbreaks in Africa, some survivors are left with long-term sequelae and persistent virus in immune-privileged sites for many years. We report the characterization of the ferret as a model for Ebola virus infection, reproducing disease and lethality observed in humans. The onset of clinical signs is rapid, and EBOV is detected in the blood, oral, and rectal swabs and all tissues studied. We identify viral RNA in the eye (a site of immune privilege) and report on specific genomic changes in EBOV present in this structure. Thus, the ferret model has utility in testing MCMs that prevent or treat long-term EBOV persistence in immune-privileged sites. IMPORTANCE Recent reemergence of Ebola in Guinea that caused over 28,000 cases between 2013 and 2016 has been linked to the original virus from that region. It appears the virus has remained in the region for at least 5 years and is likely to have been maintained in humans. Persistence of Ebola in areas of the body for extended periods of time has been observed, such as in the eye and semen. Despite the importance of reintroduction of Ebola from this route, such events are rare in the population, which makes studying medical interventions to clear persistent virus difficult. We studied various doses of Ebola in ferrets and detected virus in the eyes of most ferrets. We believe this model will enable the study of medical interventions that promote clearance of Ebola virus from sites that promote persistence.

Mycobacterium tuberculosis (Mtb) causes one of the deadliest infectious diseases worldwide. Upon infection, Mtb is phagocytosed by macrophages and uses its virulence-associated ESX-1 secretion system to modulate the host cell. We showed previously that the ESX-1 secretion system perturbs the Mtb-containing phagosome, and a population (∼30%) of intracellular Mtb is tagged with ubiquitin and targeted to selective autophagy. However, our understanding of how macrophages sense and respond to damaged Mtb-containing phagosomes remains incomplete. Here, we demonstrate that several cytosolic glycan-binding proteins called galectins recognize Mtb-containing phagosomes; in macrophage cell lines and in primary macrophages, galectin-3, -8, and -9 are all recruited to the same Mtb population that colocalizes with selective autophagy markers (ubiquitin, p62, and LC3). To test whether galectins are required for controlling Mtb replication in macrophages, we generated CRISPR/Cas9 knockouts and found that galectin-8−/− and galectin-3/8/9−/− macrophages were similarly defective in targeting Mtb to selective autophagy and controlling replication. This suggests galectin-8 plays a unique role in anti-Mtb autophagy. In investigating galectin-8's role, we identified a novel and specific interaction between galectin-8 and the selective autophagy adapter TAX1BP1 and found that this galectin-8/TAX1BP1 interaction was necessary for macrophages to efficiently target Mtb to selective autophagy. Remarkably, overexpressing galectin-8 increased targeting of Mtb to autophagy and limited Mtb replication. Taken together, these data demonstrate that while several galectins are capable of recognizing damaged Mtb-containing phagosomes, galectin-8 plays a privileged role in recruiting downstream autophagy machinery and may represent a promising target for host-directed tuberculosis therapies. IMPORTANCE Mycobacterium tuberculosis (Mtb) infects one-quarter of the global population and causes one of the deadliest infectious diseases worldwide. Macrophages are the first line of defense against Mtb infection and are typically incredibly efficient at destroying intracellular pathogens, but Mtb has evolved to survive and replicate in this harsh environment. Previous work has found that a portion of intracellular Mtb bacilli damage their phagosomes, leaving them vulnerable to detection by the host and delivery to an antibacterial pathway called selective autophagy. Here, we show that in macrophages, galectin-8 recognizes damaged Mtb-containing phagosomes and targets Mtb to selective autophagy; we found that galectin-8, unlike other highly similar and closely related galectins, is required for targeting and controlling Mtb in macrophages. The specific role for galectin-8 appears to stem from its interaction with TAX1BP1, a selective autophagy adapter protein. Interestingly, overexpressing galectin-8 helps macrophages target and control Mtb, highlighting the importance of galectin-8 in the innate immune response to Mtb.

Vaccines against SARS-CoV-2 are urgently required, but early development of vaccines against SARS-CoV-1 resulted in enhanced disease after vaccination. Careful assessment of this phenomena is warranted for vaccine development against SARS CoV-2. Here we report detailed immune profiling after ChAdOx1 nCoV-19 (AZD1222) and subsequent high dose challenge in two animal models of SARS-CoV-2 mediated disease. We demonstrate in rhesus macaques the lung pathology caused by SARS-CoV-2 mediated pneumonia is reduced by prior vaccination with ChAdOx1 nCoV-19 which induced neutralising antibody responses after a single intramuscular administration. In a second animal model, ferrets, ChAdOx1 nCoV-19 reduced both virus shedding and lung pathology. Antibody titre were boosted by a second dose. Data from these challenge models on the absence of enhanced disease and the detailed immune profiling, support the continued clinical evaluation of ChAdOx1 nCoV-19., Lambe, Spencer, Thomas, Gilbert and colleagues report on the detailed immune profile of rhesus macaques and ferrets vaccinated against SARS-CoV-2 under high dose challenge. Their findings indicate that the ChAdOx1 nCoV-19 (AZD1222) the vaccine induces immune responses and reduces disease symptoms in both models, including SARS-CoV-2 mediated pneumonia and virus shedding.

Background During the five decades since their discovery, filoviruses of four species have caused human hemorrhagic fever outbreaks: Marburg (MARV) marburgvirus, and Zaire (EBOV), Sudan (SUDV) and Bundybugyo (BDBV) ebolaviruses. The largest, devastating EBOV epidemic in West Africa in 2014-16, has been followed by outbreaks of MARV in Uganda, 2017, and EBOV in Democratic Republic of Congo, 2018, emphasizing the need to develop preparedness to diagnose all filoviruses. Objectives The aim of this study was to optimize a new filovirus RT-qPCR to detect all filoviruses, define its limits of detection (LOD) and perform a field evaluation with outbreak samples. Study design A pan-filovirus RT-qPCR targeting the L gene was developed and evaluated within the EbolaMoDRAD (Ebola virus: modern approaches for developing bedside rapid diagnostics) project. Specificity and sensitivity were determined and the effect of inactivation and PCR reagents (liquid and lyophilized format) were tested. Results The LODs for the lyophilized pan-filovirus L-RT-qPCR assay were 9.4 copies per PCR reaction for EBOV, 9.9 for MARV, 1151 for SUDV, 65 for BDBV and 289 for Tai Forest virus. The test was set at the Pasteur Institute, Dakar, Senegal, and 83 Ebola patient samples, with viral load ranging from 5 to 5 million copies of EBOV per reaction, were screened. The results for the patient samples were in 100% concordance with the reference EBOV-specific assay. Discussion Overall, the assay showed good sensitivity and specificity, covered all filoviruses known to be human pathogens, performed well both in lyophilized and liquid-phase formats and with EBOV outbreak clinical samples.

Safe and effective vaccines will provide essential medical countermeasures to tackle the COVID-19 pandemic. Here, we evaluate the safety, immunogenicity and efficacy of the intradermal delivery of INO-4800, a synthetic DNA vaccine candidate encoding the SARS-CoV-2 spike protein in the rhesus macaque model. Single and 2 dose vaccination regimens were evaluated. Vaccination induces both binding and neutralizing antibodies, along with IFN-γ-producing T cells against SARS-CoV-2. Upon administration of a high viral dose (5 x 106 pfu) via the intranasal and intratracheal routes we observe significantly reduced virus load in the lung and throat, in the vaccinated animals compared to controls. 2 doses of INO-4800 is associated with more robust vaccine-induced immune responses and improved viral protection. Importantly, histopathological examination of lung tissue provides no indication of vaccine-enhanced disease following SARS-CoV-2 challenge in INO-4800 immunized animals. This vaccine candidate is currently under clinical evaluation as a 2 dose regimen.

Within the last decade, we have learned that damaged mitochondria activate many of the same innate immune pathways that evolved to sense and respond to intracellular pathogens. These shared responses include cytosolic nucleic acid sensing and type I interferon (IFN) expression, inflammasome activation that leads to pyroptosis, and selective autophagy (called mitophagy when mitochondria are the cargo). Because mitochondria were once bacteria, parallels between how cells respond to mitochondrial and bacterial ligands are not altogether surprising. However, the potential for cross talk or synergy between bacterium- and mitochondrion-driven innate immune responses during infection remains poorly understood. This interplay is particularly striking, and intriguing, in the context of infection with the intracellular bacterial pathogen Mycobacterium tuberculosis (Mtb). Multiple studies point to a role for Mtb infection and/or specific Mtb virulence factors in disrupting the mitochondrial network in macrophages, leading to metabolic changes and triggering potent innate immune responses. Research from our laboratories and others argues that mutations in mitochondrial genes can exacerbate mycobacterial disease severity by hyperactivating innate responses or activating them at the wrong time. Indeed, growing evidence supports a model whereby different mitochondrial defects or mutations alter Mtb infection outcomes in distinct ways. By synthesizing the current literature in this minireview, we hope to gain insight into the molecular mechanisms driving, and consequences of, mitochondrion-dependent immune polarization so that we might better predict tuberculosis patient outcomes and develop host-directed therapeutics designed to correct these imbalances.

Summary Phagocytosis and autophagy play critical roles in immune defense. The human fungal pathogen Cryptococcus neoformans (Cn) subverts host autophagy-initiation complex (AIC)-related proteins, to promote its phagocytosis and intracellular parasitism of host cells. The mechanisms by which the pathogen engages host AIC-related proteins remain obscure. Here, we show that the recruitment of host AIC proteins to forming phagosomes is dependent upon the activity of CD44, a host cell surface receptor that engages fungal hyaluronic acid (HA). This interaction elevates intracellular Ca2+ concentrations and activates CaMKKβ and its downstream target AMPKα, which results in activation of ULK1 and the recruitment of AIC components. Moreover, we demonstrate that HA-coated beads efficiently recruit AIC components to phagosomes and CD44 interacts with AIC components. Taken together, these findings show that fungal HA plays a critical role in directing the internalization and productive intracellular membrane trafficking of a fungal pathogen of global importance., Graphical abstract, Highlights • Fungal HA drives non-canonical and ligand-induced autophagy in phagocytic cells • Cn recruits host CD44 to forming phagocytic cups to initiate fungal internalization • Fungal HA-CD44 interactions elevate intracellular Ca2+ levels and activate CaMKKβ • A Ca2+-CaMKKβ-AMPK-ULK1 signaling axis is involved in HA-CD44 induced autophagy, Immunology; Mycology

NP105–113-B*07:02-specific CD8+ T cell responses are considered among the most dominant in SARS-CoV-2-infected individuals. We found strong association of this response with mild disease. Analysis of NP105–113-B*07:02-specific T cell clones and single-cell sequencing were performed concurrently, with functional avidity and antiviral efficacy assessed using an in vitro SARS-CoV-2 infection system, and were correlated with T cell receptor usage, transcriptome signature and disease severity (acute n = 77, convalescent n = 52). We demonstrated a beneficial association of NP105–113-B*07:02-specific T cells in COVID-19 disease progression, linked with expansion of T cell precursors, high functional avidity and antiviral effector function. Broad immune memory pools were narrowed postinfection but NP105–113-B*07:02-specific T cells were maintained 6 months after infection with preserved antiviral efficacy to the SARS-CoV-2 Victoria strain, as well as Alpha, Beta, Gamma and Delta variants. Our data show that NP105–113-B*07:02-specific T cell responses associate with mild disease and high antiviral efficacy, pointing to inclusion for future vaccine design. Peng et al. find that immunodominant cytotoxic T lymphocytes (CTLs) specific for NP105–113-B*07:02 are associated with reduced COVID-19 severity. Mechanistically, NP105–113-B*07:02-specific CTLs show potent antiviral functionality and may represent rational T cell vaccine targets.

There is a vital need for authentic COVID-19 animal models to enable the pre-clinical evaluation of candidate vaccines and therapeutics. Here we report a dose titration study of SARS-CoV-2 in the ferret model. After a high (5 × 106 pfu) and medium (5 × 104 pfu) dose of virus is delivered, intranasally, viral RNA shedding in the upper respiratory tract (URT) is observed in 6/6 animals, however, only 1/6 ferrets show similar signs after low dose (5 × 102 pfu) challenge. Following sequential culls pathological signs of mild multifocal bronchopneumonia in approximately 5–15% of the lung is seen on day 3, in high and medium dosed groups. Ferrets re-challenged, after virus shedding ceased, are fully protected from acute lung pathology. The endpoints of URT viral RNA replication & distinct lung pathology are observed most consistently in the high dose group. This ferret model of SARS-CoV-2 infection presents a mild clinical disease., SARS-CoV-2 induces mild infection in ferret model. Here, Ryan et al. characterise optimal infection dosage inducing upper respiratory tract (UTR) viral shedding, progression time of viral shedding, and pathology in ferrets and finally provide evidence for protection after re-challenge.

Pathogen sensing via pattern recognition receptors triggers massive reprogramming of macro-phage gene expression. While the signaling cascades and transcription factors that activate these responses are well-known, the role of post-transcriptional RNA processing in modulating innate immune gene expression remains understudied. Recent phosphoproteomics analyses revealed that members of the SR and hnRNP families of splicing regulatory proteins are dynamically post-translationally modified in infected macrophages. To begin to test if these splicing factors play a privileged role in controlling the innate immune transcriptome, we analyzed steady state gene expression and alternatively spliced isoform production in ten SR/hnRNP knockdown RAW 264.7 macrophage cell lines following infection with the bacterial pathogen Salmonella enterica serovar Typhimurium (Salmonella). We identified thousands of transcripts whose abundance was increased or decreased by SR/hnRNP knockdown in macrophages. We observed that different SR/hnRNPs control the expression of distinct gene regulons in uninfected and Salmonella-infected macrophages, with several key innate immune genes (Nos2, Mx1, Il1a) relying on multiple SR/hnRNPs to maintain proper induction and/or repression. Knockdown of SR/hnRNPs promoted differential isoform usage (DIU) for a number of key immune sensors and signaling molecules and many of these splicing changes were again, distinct in uninfected and Salmonella-infected macrophages. Finally, after observing a surprising degree of similarity between the DEGs and DIUs in hnRNP K and U knockdown macrophages, we found that these cells are better able to restrict vesicular stomatitis virus replication than control cells, supporting a role for these hnRNPs in controlling infection outcomes in macrophages ex vivo. Based on these findings, we conclude that many innate immune genes have evolved to rely on one or more splicing regulatory factors to ensure the proper timing and magnitude of their induction, bolstering a model wherein pre-mRNA splicing is a critical regulatory node in the innate immune response.

Most bromodomain inhibitors mimic the interactions of the natural acetylated lysine (KAc) histone substrate through key interactions with conserved asparagine and tyrosine residues within the binding pocket. Herein we report the optimization of a series of phenyl sulfonamides that exhibit a novel mode of binding to non-bromodomain and extra terminal domain (non-BET) bromodomains through displacement of a normally conserved network of four water molecules. Starting from an initial hit molecule, we report its divergent optimization toward the ATPase family AAA domain containing 2 (ATAD2) and cat eye syndrome chromosome region, candidate 2 (CECR2) domains. This work concludes with the identification of (R)-55 (GSK232), a highly selective, cellularly penetrant CECR2 inhibitor with excellent physicochemical properties.

SummaryPhagocytosis and autophagy play critical roles in immune defense. Cryptococcus neoformans (Cn), a fungal pathogen that causes fatal infection, subverts the host autophagy initiation complex (AIC) and its upstream regulatory proteins, to promote its phagocytosis and intracellular parasitism of host cells. The mechanisms by which the pathogen engages host AIC proteins remain obscure. Here, we show that the recruitment of host AIC proteins to forming phagosomes is dependent upon the activity of CD44, a host cell surface receptor that engages fungal hyaluronic acid (HA). This interaction elevates intracellular Ca2+ concentrations and activates CaMKKβ and its downstream target AMPKα, which results in activation of ULK1 and the recruitment of AIC components. Moreover, we demonstrate that HA-coated beads efficiently recruit AIC components to phagosomes. Taken together, these findings show that fungal HA plays a critical role in directing the internalization and productive intracellular membrane trafficking of a fungal pathogen of global importance.Graphical AbstractIn BriefDing et al. reveal that interactions between fungal hyaluronic acid (HA) and host CD44 activate a Ca2+ - CaMKKβ-AMPK-ULK1 signaling pathway that recruits autophagy initiation complex components to forming phagosomes to drive fungal internalization.HighlightsFungal HA interactions with host cells drive a novel non-canonical, ligand-induced, autophagy pathway in phagocytic cellsCryptococcus neoformans recruits host CD44, together with AIC components and regulatory proteins, to forming phagocytic cups to initiate host cell internalizationFungal HA interactions with CD44 on host cell surfaces elevate intracellular Ca2+ concentrations, leading to activation of CaMKKβA Ca2+-CaMKKβ-AMPK-ULK1 signaling axis is involved in HA and CD44 induced autophagy protein recruitment during Cn internalization

The Parkinson’s disease (PD)-associated gene leucine-rich repeat kinase 2 (LRRK2) has been studied extensively in the brain. However, several studies have established that mutations in LRRK2 confer susceptibility to mycobacterial infection, suggesting LRRK2 also controls immunity. We demonstrate that loss of LRRK2 in macrophages induces elevated basal levels of type I interferon (IFN) and interferon stimulated genes (ISGs) and causes blunted interferon responses to mycobacterial pathogens and cytosolic nucleic acid agonists. Altered innate immune gene expression in Lrrk2 knockout (KO) macrophages is driven by a combination of mitochondrial stresses, including oxidative stress from low levels of purine metabolites and DRP1-dependent mitochondrial fragmentation. Together, these defects promote mtDNA leakage into the cytosol and chronic cGAS engagement. While Lrrk2 KO mice can control Mycobacterium tuberculosis (Mtb) replication, they have exacerbated inflammation and lower ISG expression in the lungs. These results demonstrate previously unappreciated consequences of LRRK2-dependent mitochondrial defects in controlling innate immune outcomes., eLife digest Parkinson’s disease is a progressive nervous system disorder that causes tremors, slow movements, and stiff and inflexible muscles. The symptoms are caused by the loss of cells known as neurons in a specific part of the brain that helps to regulate how the body moves. Researchers have identified mutations in several genes that are associated with an increased risk of developing Parkinson’s. The most common of these mutations occur in a gene called LRRK2. This gene produces a protein that has been shown to be important for maintaining cellular compartments known as mitochondria, which play a crucial role in generating energy. It remains unclear how these mutations lead to the death of neurons. Mutations in LRRK2 have also been shown to make individuals more susceptible to bacterial infections, suggesting that the protein that LRRK2 codes for may help our immune system. Weindel, Bell et al. set out to understand how this protein works in immune cells called macrophages, which ‘eat’ invading bacteria and produce type I interferons, molecules that promote immune responses. Mouse cells were used to measure the ability of normal macrophages and macrophages that lack the mouse equivalent to LRRK2 (referred to as Lrrk2 knockout macrophages) to make type I interferons. The experiments showed that the Lrrk2 knockout macrophages made type I interferons even when they were not infected with bacteria, suggesting they are subject to stress that triggers immune responses. It was possible to correct the behavior of the Lrrk2 knockout macrophages by repairing their mitochondria. When mice missing the gene equivalent to LRRK2 were infected with the bacterium that causes tuberculosis, they experienced more severe disease. The protein encoded by the LRRK2 gene is considered a potential target for therapies to treat Parkinson’s disease, and several drugs that inhibit this protein are being tested in clinical trials. The findings of Weindel, Bell et al. suggest that these drugs may have unintended negative effects on a patient’s ability to fight infection. This work also indicates that LRRK2 mutations may disrupt immune responses in the brain, where macrophage-like cells called microglia play a crucial role in maintaining healthy neurons. Future studies that examine how mutations in LRRK2 affect microglia may help us understand how Parkinson’s disease develops.

In last five years, the Africa has faced two outbreaks of Zaire ebolavirus. These outbreaks have been the largest so far, and latest outbreak is still ongoing and affecting the Democratic Republic of the Congo. We tested in parallel three different Zaire ebolavirus (EBOV) realtime RT-PCRs targeting the nucleoprotein gene (EBOV NP-RT-qPCRs) described by Trombley et al. (2010) ; Huang et al. (2012) and Weidmann et al. (2004) . These assays are used regularly in diagnostic laboratories. The limit of detection (LOD), intra-assay repeatability using different matrixes, sensitivity and specificity were determined. In addition, the primers and probes were aligned with the sequences available in ongoing and past outbreaks in order to check the mismatches. The specificity of all three EBOV NP-RT-qPCRs were excellent (100 %), and LODs were under or 10 copies per PCR reaction. Intra-assay repeatability was good in all assays, however the Ct-values were bit higher using the EDTA-blood based matrix. All of the primers and probes in EBOV NP-RT-qPCR assays have one or more mismatches in the probes and primers when the 2267 Zaire EBOV NP sequences, including strains Ituri from DRC outbreak (year 2018), was aligned. The EBOV strain of Bikoro (year 2018) circulating in DRC was 100 % match in Trombley and Weidmann assay, but had one mismatch in Huang assay.

The sudden and explosive expansion of Zika virus (ZIKV) from the African continent through Oceania and culminating in the outbreak in South America has highlighted the importance of new rapid point-of-care diagnostic tools for the control and prevention of transmission. ZIKV infection has devastating consequences, such as neurological congenital malformations in infants born to infected mothers and Guillain-Barré syndrome in adults. Additionally, its potential for transmission through vector bites, as well as from person to person through blood transfusions and sexual contact, are important considerations for prompt diagnosis. Recombinase polymerase amplification (RPA), an isothermal method, was developed as an alternative field-applicable assay to PCR. Here we report the development of a novel ZIKV real-time reverse transcriptase RPA (RT-RPA) assay capable of detecting a range of different ZIKV strains from a variety of geographical locations. The ZIKV RT-RPA was shown to be highly sensitive, being capable of detecting as few as five copies of target nucleic acid per reaction, and suitable for use with a battery-operated portable device. The ZIKV RT-RPA demonstrated 100 % specificity and 83 % sensitivity in clinical samples. Furthermore, we determined that the ZIKV RT-RPA is a versatile assay that can be applied to crude samples, such as saliva and serum, and can be used as a vector surveillance tool on crude mosquito homogenates. Therefore, the developed ZIKV RT-RPA is a useful diagnostic tool that can be transferred to a resource-limited location, eliminating the need for a specialized and sophisticated laboratory environment and highly trained staff.

Rhodococcus equi is a major cause of foal pneumonia and an opportunistic pathogen in immunocompromised humans. While alveolar macrophages constitute the primary replicative niche for R. equi, little is known about how intracellular R. equi is sensed by macrophages. Here, we discovered that in addition to previously characterized pro-inflammatory cytokines (e.g., Tnfa, Il6, Il1b), macrophages infected with R. equi induce a robust type I IFN response, including Ifnb and interferon-stimulated genes (ISGs), similar to the evolutionarily related pathogen, Mycobacterium tuberculosis. Follow up studies using a combination of mammalian and bacterial genetics demonstrated that induction of this type I IFN expression program is largely dependent on the cGAS/STING/TBK1 axis of the cytosolic DNA sensing pathway, suggesting that R. equi perturbs the phagosomal membrane and causes DNA release into the cytosol following phagocytosis. Consistent with this, we found that a population of ~12% of R. equi phagosomes recruits the galectin-3,-8 and -9 danger receptors. Interestingly, neither phagosomal damage nor induction of type I IFN require the R. equi’s virulence-associated plasmid. Importantly, R. equi infection of both mice and foals stimulates ISG expression, in organs (mice) and circulating monocytes (foals). By demonstrating that R. equi activates cytosolic DNA sensing in macrophages and elicits type I IFN responses in animal models, our work provides novel insights into how R. equi engages the innate immune system and furthers our understanding how this zoonotic pathogen causes inflammation and disease., Author summary Rhodococcus equi is a facultative intracellular bacterial pathogen of horses and other domestic animals, as well as an opportunistic pathogen of humans. In human patients, Rhodococcus pneumonia bears some pathological similarities to pulmonary tuberculosis, and poses a risk for misdiagnosis. In horses, R. equi infection has a major detrimental impact on the equine breeding industry due to a lack of an efficacious vaccine and its ubiquitous distribution in soil. Given the prevalence of subclinical infection and high false positive rate in current screening methods, there exists a critical need to identify factors contributing to host susceptibility. Here, we use a combination of bacterial genetics and animal models to investigate innate immune responses during R. equi infection. We found that R. equi modulates host immune sensing to elicit a type I interferon response in a manner resembling that of M. tuberculosis. We also found that the danger sensors galectin-3, -8, and -9 are recruited to a population of R. equi-containing vacuoles, independent of expression of VapA. Our research identifies innate immune sensing events and immune transcriptional signatures that may lead to biomarkers for clinical disease, more accurate screening methods, and insight into susceptibility to infection.

Tripartite motif-containing proteins (TRIMs) play a variety of recently described roles in innate immunity. While many TRIMs regulate type I interferon (IFN) expression following cytosolic nucleic acid sensing of viruses, their contribution to innate immune signaling and gene expression during bacterial infection remains largely unknown. Because Mycobacterium tuberculosis is a potent activator of cGAS-dependent cytosolic DNA sensing, we set out to investigate a role for TRIM proteins in regulating macrophage responses to M. tuberculosis. Here we demonstrate that TRIM14, a non-canonical TRIM that lacks an E3 ligase RING domain, is a critical negative regulator of the type I IFN response in macrophages. We show that TRIM14 physically interacts with both cGAS and TBK1 and that macrophages lacking TRIM14 dramatically hyperinduce interferon stimulated gene (ISG) expression following cytosolic nucleic acid transfection, IFN-β treatment, and M. tuberculosis infection. Consistent with a defect in resolution of the type I IFN response, Trim14 knockout (KO) macrophages have more phospho-Ser754 STAT3 relative to phospho-727 and fail to upregulate the STAT3 target Socs3 (Suppressor of Cytokine Signaling 3), which is required to turn off IFNAR signaling. These data support a model whereby TRIM14 acts as a scaffold between TBK1 and STAT3 to promote phosphorylation of STAT3 at Ser727 and enhance negative regulation of ISG expression. Remarkably, Trim14 KO macrophages hyperinduce antimicrobials like Inos2 and are significantly better than control cells at limiting M. tuberculosis replication. Collectively, these data reveal a previously unappreciated role for TRIM14 in resolving type I IFN responses and controlling M. tuberculosis infection.

Although 7-T MRI has recently received approval for use in clinical patient care, there are distinct safety issues associated with this relatively high magnetic field. Forces on metallic implants and radiofrequency power deposition and heating are safety considerations at 7 T. Patient bioeffects such as vertigo, dizziness, false feelings of motion, nausea, nystagmus, magnetophosphenes, and electrogustatory effects are more common and potentially more pronounced at 7 T than at lower field strengths. Herein the authors review safety issues associated with 7-T MRI. The rationale for safety concerns at this field strength are discussed as well as potential approaches to mitigate risk to patients and health care professionals.

SUMMARYDespite many connections between mutations in leucine-rich repeat kinase 2 (LRRK2) and susceptibility to mycobacterial infection, we know little about its function outside of the brain, where it is studied in the context of Parkinson’s Disease (PD). Here, we report that LRRK2 controls peripheral macrophages and brain-resident glial cells’ ability to respond to and express inflammatory molecules. LRRK2 KO macrophages express elevated basal levels of type I interferons, resulting from defective purine metabolism, mitochondrial damage, and engagement of mitochondrial DNA with the cGAS DNA sensing pathway. While LRRK2 KO mice can control Mycobacterium tuberculosis (Mtb) infection, they exhibit exacerbated lung inflammation and altered activation of glial cells in PD-relevant regions of the brain. These results directly implicate LRRK2 in peripheral immunity and support the “multiple-hit hypothesis” of neurodegenerative disease, whereby infection coupled with genetic defects in LRRK2 create an immune milieu that alters activation of glial cells and may trigger PD.

The optimisation of the azanaphthyridine series of Spleen Tyrosine Kinase inhibitors is described. The medicinal chemistry strategy was focused on optimising the human whole blood activity whilst achieving a sufficient margin over hERG activity. A good pharmacokinetic profile was achieved by modification of the pKa. Morpholine compound 32 is a potent SYK inhibitor showing moderate selectivity, good oral bioavailability and good efficacy in the rat Arthus model but demonstrated a genotoxic potential in the Ames assay.

Current methods for assessing the drug susceptibility of Mycobacterium tuberculosis are lengthy and do not capture information about viable organisms that are not immediately culturable under standard laboratory conditions as a result of antibiotic exposure. We have developed a rapid dual-fluorescence flow cytometry method using markers for cell viability and death. We show that the fluorescent marker calcein violet with an acetoxy-methyl ester group (CV-AM) can differentiate between populations of M. tuberculosis growing at different rates, while Sytox green (SG) can differentiate between live and dead mycobacteria. M. tuberculosis was exposed to isoniazid or rifampin at different concentrations over time and either dual stained with CV-AM and SG and analyzed by flow cytometry or plated to determine the viability of the cells. Although similar trends in the loss of viability were observed when the results of flow cytometry and the plate counting methods were compared, there was a lack of correlation between these two approaches, as the flow cytometry analysis potentially captured information about cell populations that were unable to grow under standard conditions. The flow cytometry approach had an additional advantage in that it could provide insights into the mode of action of the drug: antibiotics targeting the cell wall gave a flow cytometry profile distinct from those inhibiting intracellular processes. This rapid drug susceptibility testing method could identify more effective antimycobacterials, provide information about their potential mode of action, and accelerate their progress to the clinic.

The BRPF (Bromodomain and PHD Finger-containing) protein family are important scaffolding proteins for assembly of MYST histone acetyltransferase complexes. A selective benzimidazolone BRPF1 inhibitor showing micromolar activity in a cellular target engagement assay was recently described. Herein, we report the optimization of this series leading to the identification of a superior BRPF1 inhibitor suitable for in vivo studies.

Ebola virus (EBOV) is highly pathogenic, with a predisposition to cause outbreaks in human populations accompanied by significant mortality. Owing to the lack of approved therapies, screening programmes of potentially efficacious drugs have been undertaken. One of these studies has demonstrated the possible utility of chloroquine against EBOV using pseudotyped assays. In mouse models of EBOV disease there are conflicting reports of the therapeutic effects of chloroquine. There are currently no reports of its efficacy using the larger and more stringent guinea pig model of infection. In this study we have shown that replication of live EBOV is impaired by chloroquine in vitro. However, no protective effects were observed in vivo when EBOV-infected guinea pigs were treated with chloroquine. These results advocate that chloroquine should not be considered as a treatment strategy for EBOV.

This article describes the finding of substantial upregulation of mRNA and enzymes of the cytochrome P450 1A family during a lead optimization campaign for small molecule S1P1 agonists. Fold changes in mRNA up to 10,000-fold for CYP1A1 in vivo in rat and cynomolgus monkey and up to 45-fold for CYP1A1 and CYP1A2 in vitro in rat and human hepatocytes were observed. Challenges observed with correlating induction in vitro and induction in vivo resulted in the implementation of a short, 4 day in vivo screening study in the rat which successfully identified noninducers. Subtle structure-activity relationships in this series of S1P1 agonists are described extending beyond planarity and lipophilicity, and the impact and considerations of AhR and CYP1A induction in the context of drug development are discussed.

The widespread dissemination of CTX-M extended spectrum β-lactamases among Escherichia coli bacteria, both in nosocomial and community environments, is a challenge for diagnostic bacteriology laboratories. We describe a rapid and sensitive detection system for analysis of DNA containing the blaCTX-M-15 gene using isothermal DNA amplification by recombinase polymerase amplification (RPA) on a digital microfluidic platform; active matrix electrowetting-on-dielectric (AM-EWOD). The devices have 16,800 electrodes that can be independently controlled to perform multiple and simultaneous droplet operations. The device includes an in-built impedance sensor for real time droplet position and size detection, an on-chip thermistor for temperature sensing and an integrated heater for regulating the droplet temperature. Automatic dispensing of droplets (45 nL) from reservoir electrodes is demonstrated with a coefficient of variation (CV) in volume of approximately 2%. The RPA reaction is monitored in real-time using exonuclease fluorescent probes. Continuous mixing of droplets during DNA amplification significantly improves target DNA detection by at least 100 times compared to a benchtop assay, enabling the detection of target DNA over four-order-of-magnitude with a limit of detection of a single copy within ~15 minutes.

Nature is perceived and valued in starkly different and oftenconflicting ways. This paper presents the rationale for theinclusive valuation of nature's contributions to people (NCP) indecision making, as well as broad methodological steps fordoing so. While developed within the context of theIntergovernmental Platform on Biodiversity and Ecosystem Services (IPBES), this approach is more widely applicable toinitiatives at the knowledge?policy interface, which require apluralistic approach to recognizing the diversity of values. Weargue that transformative practices aiming at sustainablefutures would benefit from embracing such diversity, which require recognizing and addressing power relationships across stake holder groups that hold different values on human nature relations and NCP Fil: Pascual, Unai. University Of Cambridge; Reino Unido Fil: Balvanera, Patricia. Universidad Nacional Autónoma de México; México Fil: Díaz, Sandra Myrna. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; Argentina Fil: Pataki, György. Corvinus University of Budapest; Hungría Fil: Roth, Eva. University of Southern Denmark; Dinamarca Fil: Stenseke, Marie. University Goteborg; Suecia Fil: Watson, Robert T.. University of East Anglia; Reino Unido Fil: Basak Dessane, Esra. Project House; Turquía Fil: Islar, Mine. Lund University; Suecia Fil: Kelemen, Eszter. Environmental Social Science Research Group; Hungría Fil: Maris, Virginie. Centre d’Ecologie Fonctionnelle et Evolutive; Francia Fil: Quaas, Martin. Kiel University; Alemania Fil: Subramanian, Suneetha M. United Nations University; Japón Fil: Wittmer, Heidi. Helmholtz-Centre for Environmental Research; Alemania Fil: Adlan, Asia. University of Khartoum; Sudán Fil: Ahn, SoEun. Korea Environment Institute; Corea del Sur Fil: Al-Hafedh, Yousef S. King Abdulaziz City for Science & Technology; Arabia Saudita Fil: Amankwah, Edward. Center for Environmental Governance; Ghana Fil: Berry, Pam. University of Washington; Estados Unidos Fil: Bilgin, Adem. University of Oxford; Reino Unido Fil: Breslow, Sara J. Ministry of Forestry and Water Affairs of Turkey; Turquía Fil: Bullock, Craig. University College Dublin; Reino Unido Fil: Caceres, Daniel Mario. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Agropecuarias. Departamento de Desarrollo Rural; Argentina Fil: Daly-Hassen, Hamed. University of Carthage; Túnez Fil: Figueroa, Eugenio. Universidad de Chile; Chile Fil: van den Belt, Marjan. Victoria University of Wellington; Nueva Zelanda Fil: Verma, Madhu. Indian Institute of Forest Management; India Fil: Wickson, Fern. GenØk Centre for Biosafety, Siva innovasjonssenter; Noruega Fil: Yagi, Noboyuki. The University of Tokyo; Japón

The BRPF (bromodomain and PHD finger-containing) protein family are important scaffolding proteins for assembly of MYST histone acetyltransferase complexes. Here, we report the discovery, binding mode, and structure-activity relationship (SAR) of the first potent, selective series of inhibitors of the BRPF1 bromodomain.

SUMMARY While transcriptional control of innate immune gene expression is well characterized, almost nothing is known about how pre-mRNA splicing decisions influence, or are influenced by, macrophage activation. Here, we demonstrate that the splicing factor hnRNP M is a critical repressor of innate immune gene expression and that its function is regulated by pathogen sensing cascades. Loss of hnRNP M led to hyperinduction of a unique regulon of inflammatory and antimicrobial genes following diverse innate immune stimuli. While mutating specific serines on hnRNP M had little effect on its ability to control pre-mRNA splicing or transcript levels of housekeeping genes in resting macrophages, it greatly impacted the protein’s ability to dampen induction of specific innate immune transcripts following pathogen sensing. These data reveal a previously unappreciated role for pattern recognition receptor signaling in controlling splicing factor phosphorylation and establish pre-mRNA splicing as a critical regulatory node in defining innate immune outcomes., In Brief West et al. report that hnRNP M represses expression of a cohort of innate immune transcripts in infected macrophages. IL6 splicing repression is relieved when hnRNP M is phosphorylated at specific residues, demonstrating that post-translational modification of splicing factors downstream of pathogen sensing can control maturation of innate immune mRNAs., Graphical Abstract

Ubiquitin-mediated targeting of intracellular bacteria to the autophagy pathway is a key innate defence mechanism against invading microbes, including the important human pathogen Mycobacterium tuberculosis. However, the ubiquitin ligases responsible for catalysing ubiquitin chains that surround intracellular bacteria are poorly understood. The parkin protein is a ubiquitin ligase with a well-established role in mitophagy, and mutations in the parkin gene (PARK2) lead to increased susceptibility to Parkinson's disease. Surprisingly, genetic polymorphisms in the PARK2 regulatory region are also associated with increased susceptibility to intracellular bacterial pathogens in humans, including Mycobacterium leprae and Salmonella enterica serovar Typhi, but the function of parkin in immunity has remained unexplored. Here we show that parkin has a role in ubiquitin-mediated autophagy of M. tuberculosis. Both parkin-deficient mice and flies are sensitive to various intracellular bacterial infections, indicating parkin has a conserved role in metazoan innate defence. Moreover, our work reveals an unexpected functional link between mitophagy and infectious disease.

Answering large-scale questions in ecology can involve time-consuming data compilation. We show how networks of undergraduate classes can make these projects more manageable and provide an authentic research experience for students. With this approach, we examined the factors associated with plant species richness in US national wildlife refuges. We found that the richness of harmful invasive plants and the richness of native plants were positively correlated in mainland refuges but negatively correlated in island refuges. Nonnative richness and invasive richness were also positively correlated with colonization pressure as indicated by nonnative richness around each refuge. Associations between refuge characteristics and invasive plants varied substantially among regions, with refuge area and habitat diversity important predictors of invasion in some regions but not in others. Our results serve to identify the refuges that are most susceptible to plant invasion and demonstrate the potential value of a new model for education and research integration.

Datura arenicola, a new species originally collected and named by H. S. Gentry, is documented from Baja California Sur, Mexico. Following a study of Datura (Solanaceae) in western Mexico, the new species is placed in the new section Discola, with section Dutra emended and the revision of section Ceratocaulis proposed. Datura arenicola is found at elevations of 75–100 m on the eastern Vizcaino Desert, and is characterized by a short basal stem, decumbent habit, pubescent lobulate leaf, prismatic calyx, violet flower, and pendant globose spiny fruit, dehiscent in four parts. The new species is known from a very limited geographical area, 12 km across the range. A description of the Datura on the southern Baja California peninsula and a key to the comparable species are provided.

Background Despite several studies on the seroprevalence of antibodies against Crimean-Congo Haemorrhagic Fever virus (CCHFV) from humans and cattle in Nigeria, detailed investigation looking at IgG and IgM have not been reported. Additionally, there have been no confirmed cases of human CCHFV infection reported from Nigeria. Principal Findings Samples from sera (n = 1189) collected from four Local Government Areas in Borno State (Askira/Uba, Damboa, Jere and Maiduguri) were assessed for the presence of IgG and IgM antibodies. The positivity rates for IgG and IgM were 10.6% and 3.5%, respectively. Additionally, sera from undiagnosed febrile patients (n = 380) were assessed by RT-PCR assay for the presence of CCHFV RNA. One positive sample was characterised by further by next generation sequencing (NGS) resulting in complete S, M and L segment sequences. Conclusions This article provides evidence for the continued exposure of the human population of Nigeria to CCHFV. The genomic analysis provides the first published evidence of a human case of CCHFV in Nigeria and its phylogenetic context., Author Summary Crimean-Congo haemorrhagic fever (CCHF) is an acute tick-borne zoonotic disease. The causative agent, CCHF virus (CCHFV), has the most extensive geographical distribution of the medically important tick-borne viral diseases with a distribution over much of Asia, the Middle East, Africa and expanding areas of south-eastern Europe. Whilst the main route of human infection with CCHFV is predominantly through tick bite, it can also be spread via bodily fluids and it has a reputation for causing nosocomial outbreaks in healthcare settings. Whilst CCHFV has been identified in ticks from Nigeria since 1970, there is scarce information on human infection. Within this report, the prevalence of CCHFV-reactive antibodies has been assessed in human sera providing evidence of continued circulation of the virus in the human population of Borno state, Nigeria. Additionally, in one sample the presence of viral RNA was detected which allowed a full sequence of the CCHFV to be obtained. This is the first report of CCHFV being associated in a human case from Nigeria and the full genetic characterisation of the virus being completed. The evidence within supports the hypothesis that CCHFV is endemic in Nigeria and should be considered as an aetiological agent in febrile patients.

Accurate calibration of data is essential for the current generation of cosmic microwave background (CMB) experiments. Using data from the Very Small Array (VSA), we describe procedures which will lead to an accuracy of 1 per cent or better for experiments such as the VSA and CBI. Particular attention is paid to the stability of the receiver systems, the quality of the site and frequent observations of reference sources. At 30 GHz the careful correction for atmospheric emission and absorption is shown to be essential for achieving 1 per cent precision. The sources for which a 1 per cent relative flux density calibration was achieved included Cas A, Cyg A, Tau A and NGC 7027 and the planets Venus, Jupiter and Saturn. A flux density, or brightness temperature in the case of the planets, was derived at 33 GHz relative to Jupiter which was adopted as the fundamental calibrator. A spectral index at similar to 30 GHz is given for each. Cas A, Tau A, NGC 7027 and Venus were examined for variability. Cas A was found to be decreasing at 0.394 ± 0.019 per cent yr^(-1) over the period 2001 March to 2004 August. In the same period Tau A was decreasing at 0.22 ± 0.07 per cent yr^(-1). A survey of the published data showed that the planetary nebula NGC 7027 decreased at 0.16 ± 0.04 per cent yr^(-1) over the period 1967-2003. Venus showed an insignificant (1.5 ±1.3 per cent) variation with Venusian illumination. The integrated polarization of Tau A at 33 GHz was found to be 7.8 ± 0.6 per cent at position angle =148° ± 3°.

Zika virus (ZIKV) is a mosquito-borne pathogen which has recently spread beyond Africa and into Pacific and South American regions. Despite first being detected in 1947, very little information is known about the virus and its spread has been associated with increases in Guillain-Barre syndrome and microcephaly. There are currently no known vaccines or antivirals against ZIKV infection. Progress in assessing interventions will require the development of animal models to test efficacies; however, there are only limited reports on in vivo studies. The only susceptible murine models have involved intracerebral inoculations or juvenile animals, which do not replicate natural infection. Our report has studied the effect of ZIKV infection in type-I interferon receptor deficient (A129) mice and the parent strain (129Sv/Ev) after subcutaneous challenge in the lower leg to mimic a mosquito bite. A129 mice developed severe symptoms with viral RNA being detected widespread in the blood, brain, spleen, liver and ovaries. Histological changes were also striking in these animals. 129Sv/Ev mice developed no clinical symptoms or histological changes, despite viral RNA being detectable in the blood, spleen and ovaries, albeit at lower levels to those seen in A129 mice. Our results identify A129 mice as being highly susceptible to ZIKV and thus a suitable small animal model for the testing of vaccines and antivirals which are urgently required.

Adenosine possesses potent anti-inflammatory properties which are partly mediated by G(i) -coupled adenosine A3 receptors (A3Rs). A3R agonists have shown clinical benefit in a number of inflammatory conditions although some studies in A3R-deficient mice suggest a pro-inflammatory role. We hypothesised that, in addition to cell signalling effects, A3R compounds might inhibit neutrophil chemotaxis by disrupting the purinergic feedback loop controlling leukocyte migration. Human neutrophil activation triggered rapid upregulation of surface A3R expression which was disrupted by pre-treatment with either agonist (Cl-IB-MECA) or antagonist (MRS1220). Both compounds reduced migration velocity and neutrophil transmigration capacity without impacting the response to chemokines per se. Similar effects were observed in murine neutrophils, while cells from A3R-deficient mice displayed a constitutively impaired migratory phenotype indicating compound-induced desensitisation and genetic ablation had the same functional outcome. In a dextran sodium sulphate-induced colitis model, A3R-deficient mice exhibited reduced colon pathology and decreased tissue myeloperoxidase levels at day 8 - consistent with reduced neutrophil recruitment. However, A3R-deficient mice were unable to resolve the dextran sodium sulphate-induced inflammation and had elevated numbers of tissue-associated bacteria by day 21. Our data indicate that A3Rs play a role in neutrophil migration and disrupting this function has the potential to adversely affect innate immune responses.

SummaryEukaryotic cells sterilize the cytosol by using autophagy to route invading bacterial pathogens to the lysosome. During macrophage infection with Mycobacterium tuberculosis, a vacuolar pathogen, exogenous induction of autophagy can limit replication, but the mechanism of autophagy targeting and its role in natural infection remain unclear. Here we show that phagosomal permeabilization mediated by the bacterial ESX-1 secretion system allows cytosolic components of the ubiquitin-mediated autophagy pathway access to phagosomal M. tuberculosis. Recognition of extracelluar bacterial DNA by the STING-dependent cytosolic pathway is required for marking bacteria with ubiquitin, and delivery of bacilli to autophagosomes requires the ubiquitin-autophagy receptors p62 and NDP52 and the DNA-responsive kinase TBK1. Remarkably, mice with monocytes incapable of delivering bacilli to the autophagy pathway are extremely susceptible to infection. Our results reveal an unexpected link between DNA sensing, innate immunity, and autophagy and indicate a major role for this autophagy pathway in resistance to M. tuberculosis infection.

Datura gigantea, based on material grown from seed of decumbent plants originating in western Mexico, is shown to be a synonym of D. quercifolia, of which D. villosa is also a synonym.

The lead optimisation of the diaminopyrimidine carboxamide series of spleen tyrosine kinase inhibitors is described. The medicinal chemistry strategy was focused on optimising the human whole blood activity whilst achieving a sufficient margin over liability kinases and hERG activity. GSK143 is a potent and highly selective SYK inhibitor showing good efficacy in the rat Arthus model.

Intracellular bacterial pathogens secrete a repertoire of effector proteins into host cells that are required to hijack cellular pathways and cause disease. Despite decades of research, however, the molecular functions of most bacterial effectors remain unclear. To address this gap, we generated quantitative genetic interaction profiles between 36 validated and putative effectors from three evolutionarily divergent human bacterial pathogens and 4,190 yeast deletion strains. Correlating effector-generated profiles with those of yeast mutants, we recapitulated known biology for several effectors with remarkable specificity and predicted previously unknown functions for others. Biochemical and functional validation in human cells revealed a role for an uncharacterized component of the Salmonella SPI-2 translocon, SseC, in regulating maintenance of the Salmonella vacuole through interactions with components of the host retromer complex. These results exhibit the power of genetic interaction profiling to discover and dissect complex biology at the host-pathogen interface.

It is well known that insulin receptor substrates (IRS) act as a mediator for signal transduction of insulin, insulin-like growth factors, and several cytokines. To identify proteins that interact with IRS and modulate IRS-mediated signals, we performed yeast two-hybrid screening with IRS-1 as bait. Out of 109 cDNA-positive clones identified from a human placental cDNA library, two clones encoded 53BP2, p53-binding protein 2 (53BP2S), a short form splicing variant of the apoptosis-stimulating protein of p53 that possesses Src homology region 3 domain, and ankyrin repeats domain, and had been reported to interact with p53, Bcl-2, and NF-kappaB. Interaction of 53BP2S with IRS-1 was confirmed by glutathione S-transferase pull-down and co-immunoprecipitation assays in COS-7 cells and 3T3-L1 adipocytes. The Src homology region 3 domain and ankyrin repeats domain of 53BP2S were responsible for its interaction with IRS-1, whereas the phosphotyrosine binding domain and a central domain (amino acid residues 750-861) of IRS-1 were required for its interaction with 53BP2S. In CHO-C400 cells, expression of 53BP2S reduced insulin-stimulated IRS-1 tyrosine phosphorylation with a concomitant enhancement of IRS-2 tyrosine phosphorylation. In addition, the amount of the phosphatidylinositol 3-kinase regulatory p85 subunit associated with tyrosine-phosphorylated proteins, and activation of Akt was enhanced by 53BP2S expression. Although 53BP2S also enhanced Akt activation in 3T3-L1 adipocytes, insulin-induced glucose transporter 4 translocation was markedly inhibited in accordance with reduction of insulin-induced AS160 phosphorylation. Together these data demonstrate that 53BP2S interacts and modulates the insulin signals mediated by IRSs.

The insulin granule integral membrane protein marker phogrin-green fluorescent protein was co-localized with insulin in Min6B1 beta-cell secretory granules but did not undergo plasma membrane translocation following glucose stimulation. Surprisingly, although expression of a dominant-interfering dynamin mutant (Dyn/K44A) inhibited transferrin receptor endocytosis, it had no effect on phogringreen fluorescent protein localization in the basal or secretagogue-stimulated state. By contrast, co-expression of Dyn/K44A with human growth hormone as an insulin secretory marker resulted in a marked inhibition of human growth hormone release by glucose, KCl, and a combination of multiple secretagogues. Moreover, serial pulse depolarization stimulated an increase in cell surface capacitance that was also blocked in cells expressing Dyn/K44A. Similarly, small interference RNA-mediated knockdown of dynamin resulted in marked inhibition of glucose-stimulated insulin secretion. Together, these data suggest the presence of a selective kiss and run mechanism of insulin release. Moreover, these data indicate a coupling between endocytosis and exocytosis in the regulation of beta-cell insulin secretion.