DDX3 directly regulates TRAF3 ubiquitination and acts as a scaffold to coordinate assembly of signalling complexes downstream of MAVS

The human DEAD-box helicase 3 (DDX3) has been shown to contribute to type I interferon induction downstream of anti-viral pattern recognition receptors (PRRs). It binds to TANK-binding kinase 1 (TBK1) and IB-kinase- (IKK, the two key kinases mediating activation of Interferon regulatory factor (IRF) 3 and IRF7. We previously demonstrated that DDX3 facilitates IKK activation downstream of RIG-I and then links the activated kinase to IRF3. In this study, we probed the interactions between DDX3 and other key signalling molecules in the RIG-I pathway and identified a novel direct interaction between DDX3 and TRAF3 mediated by a TRAF-interaction motif in the N-terminus of DDX3, which was required for TRAF3 ubiquitination. Interestingly, we observed two waves of K63-linked TRAF3 ubiquitination following RIG-I activation by Sendai Virus infection (SeV), both of which were suppressed by DDX3 knockdown. We also investigated the spatiotemporal formation of endogenous downstream signalling complexes containing the MAVS adaptor, DDX3, IKK, TRAF3 and IRF3. DDX3 was recruited to MAVS early after SeV infection, suggesting it might mediate subsequent recruitment of other molecules. Indeed, knockdown of DDX3 prevented formation of TRAF3-MAVS and TRAF3-IKKcomplexes. Based on our data, we propose that early TRAF3 ubiquitination is required for formation of a stable MAVS-TRAF3 complex, while the second wave of TRAF3 ubiquitination mediates IRF3 recruitment and activation. Our study characterises DDX3 as a multifunctional adaptor molecule that coordinates assembly of different TRAF3, IKK and IRF3-containing signalling complexes downstream of MAVS. Additionally, it provides novel insights into the role of TRAF3 in RIG-I signalling

DDX3 directly regulates TRAF3 ubiquitination and acts as a scaffold to coordinate assembly of signalling complexes downstream of MAVS

The human DEAD-box helicase 3 (DDX3) has been shown to contribute to type I interferon induction downstream of anti-viral pattern recognition receptors (PRRs). It binds to TANK-binding kinase 1 (TBK1) and IB-kinase- (IKK, the two key kinases mediating activation of Interferon regulatory factor (IRF) 3 and IRF7. We previously demonstrated that DDX3 facilitates IKK activation downstream of RIG-I and then links the activated kinase to IRF3. In this study, we probed the interactions between DDX3 and other key signalling molecules in the RIG-I pathway and identified a novel direct interaction between DDX3 and TRAF3 mediated by a TRAF-interaction motif in the N-terminus of DDX3, which was required for TRAF3 ubiquitination. Interestingly, we observed two waves of K63-linked TRAF3 ubiquitination following RIG-I activation by Sendai Virus infection (SeV), both of which were suppressed by DDX3 knockdown. We also investigated the spatiotemporal formation of endogenous downstream signalling complexes containing the MAVS adaptor, DDX3, IKK, TRAF3 and IRF3. DDX3 was recruited to MAVS early after SeV infection, suggesting it might mediate subsequent recruitment of other molecules. Indeed, knockdown of DDX3 prevented formation of TRAF3-MAVS and TRAF3-IKKcomplexes. Based on our data, we propose that early TRAF3 ubiquitination is required for formation of a stable MAVS-TRAF3 complex, while the second wave of TRAF3 ubiquitination mediates IRF3 recruitment and activation. Our study characterises DDX3 as a multifunctional adaptor molecule that coordinates assembly of different TRAF3, IKK and IRF3-containing signalling complexes downstream of MAVS. Additionally, it provides novel insights into the role of TRAF3 in RIG-I signalling

Fibronectin-binding protein B variation in Staphylococcus aureus

BACKGROUND: Fibronectin binding proteins A and B (FnBPA and FnBPB) mediate adhesion of S. aureus to fibrinogen, elastin and fibronectin. We previously identified seven different isotypes of FnBPA based on divergence in the fibrinogen- and elastin-binding A domains. The variation created differences in antigenicity while ligand binding functions were retained. Here, FnBPB variation was examined in both human and bovine isolates and compared to that of FnBPA. RESULTS: Seven different fnbB allelic variants were identified. Some strains that cluster by phylogenetic analysis contain different fnbB variants, whereas more divergent strains contain the same fnbB variant. The phylogeny of fnbB alleles does not match the phylogeny of fnbA alleles. Some FnBPA and FnBPB isotypes that are specified by human S. aureus strains are also found in bovine strains. The seven fnbB allelic variants encode seven distinct isotypes of the FnBPB A domain that are 61 to 85% identical in amino acid sequence. Variant amino acid residues were mapped on a three-dimensional model of the FnBPB A domain and were predicted to be surface-exposed. They are responsible for the antigenic diversity detected with polyclonal antibody and a monoclonal antibody raised against isotype I. Ligand binding by recombinant FnBPB N23 isotypes was compared by ELISA-based solid phase assays and surface plasmon resonance. Each bound to immobilized fibrinogen, elastin and fibronectin dose-dependently and saturably with similar affinities. Binding to fibronectin was surprising because the A domains do not contain any known motifs that mediate binding to fibronectin. This raises the possibility that the A domain of FnBPB contains a novel fibronectin binding motif that binds fibronectin by a novel mechanism. CONCLUSIONS: Seven different isoforms of FnBPB A domain retain ligand-binding functions but are antigenically distinct. The variation in FnBPA and FnBPB occurs in human and bovine S. aureus strains and may act as an immune evasion mechanism. All seven isotypes of FnBPB are capable of binding fibronectin though none contain any known fibronectin-binding motifs. These results have implications for the development of vaccines or immunotherapeutics that target FnBPB

Protein with negative surface charge distribution, Bnr1, shows characteristics of aDNA‐mimic protein andmay be involved in the adaptation of Burkholderia cenocepacia

Adaptation of opportunistic pathogens to their host environment requires reprogramming of a vast array of genes to facilitate survival in the host. Burkholderia cenocepacia, a Gram-negative bacterium with a large genome of ∼8 Mb that colonizes environmental niches, is exquisitely adaptable to the hypoxic environment of the cystic fibrosis lung and survives in macrophages. We previously identified an immunoreactive acidic protein encoded on replicon 3, BCAS0292. Deletion of the BCAS0292 gene significantly altered the abundance of 979 proteins by 1.5-fold or more; 19 proteins became undetectable while 545 proteins showed ≥1.5-fold reduced abundance, suggesting the BCAS0292 protein is a global regulator. Moreover, the ∆BCAS0292 mutant showed a range of pleiotropic effects: virulence and host-cell attachment were reduced, antibiotic susceptibility was altered, and biofilm formation enhanced. Its growth and survival were impaired in 6% oxygen. In silico prediction of its three-dimensional structure revealed BCAS0292 presents a dimeric β-structure with a negative surface charge. The ΔBCAS0292 mutant displayed altered DNA supercoiling, implicated in global regulation of gene expression. Three proteins were identified in pull-downs with FLAG-tagged BCAS0292, including the Histone H1-like protein, HctB, which is recognized as a global transcriptional regulator. We propose that BCAS0292 protein, which we have named Burkholderia negatively surface-charged regulatory protein 1 (Bnr1), acts as a DNA-mimic and binds to DNA-binding proteins, altering DNA topology and regulating the expression of multiple genes, thereby enabling the adaptation of B. cenocepacia to highly diverse environments

Point of care detection of SARS-CoV-2 antibodies and neutralisation capacity—lateral flow immunoassay evaluation compared to commercial assay to inform potential role in therapeutic and surveillance practices

IntroductionAs the COVID-19 pandemic moves towards endemic status, testing strategies are being de-escalated. A rapid and effective point of care test (POCT) assessment of SARS-CoV-2 immune responses can inform clinical decision-making and epidemiological monitoring of the disease. This cross-sectional seroprevalence study of anti-SARS-CoV-2 antibodies in Irish healthcare workers assessed how rapid anti-SARS-CoV-2 antibody testing can be compared to a standard laboratory assay, discusses its effectiveness in neutralisation assessment and its uses into the future of the pandemic.MethodsA point of care lateral flow immunoassay (LFA) detecting anti-SARS-CoV-2 spike (S)-receptor binding domain (RBD) neutralising antibodies (Healgen SARS-CoV-2 neutralising Antibody Rapid Test Cassette) was compared to the Roche Elecsys/-S anti-SARS-CoV-2 antibody assays and an in vitro surrogate neutralisation assay. A correlation between anti-spike (S), anti-nucleocapsid (N) titres, and in vitro neutralisation was also assessed.Results1,777 serology samples were tested using Roche Elecsys/-S anti-SARS-CoV-2 assays to detect total anti-N/S antibodies. 1,562 samples were tested using the POC LFA (including 50 negative controls), and 90 samples were tested using an in vitro ACE2-RBD binding inhibition surrogate neutralisation assay. The POCT demonstrated 97.7% sensitivity, 100% specificity, a positive predictive value (PPV) of 100%, and a negative predictive value (NPV) of 61% in comparison to the commercial assay. Anti-S antibody titres determined by the Roche assay stratified by the POC LFA result groups demonstrated statistically significant differences between the “Positive” and “Negative” LFA groups (p < 0.0001) and the “Weak Positive” and “Positive” LFA groups (p < 0.0001). No statistically significant difference in ACE2-RBD binding inhibition was demonstrated when stratified by the LFA POC results. A positive, statistically significant correlation was demonstrated between the in vitro pseudo-neutralisation assay results and anti-S antibody titres (rho 0.423, p < 0.001) and anti-N antibody titres (rho = 0.55, p < 0.0001).ConclusionHigh sensitivity, specificity, and PPV were demonstrated for the POC LFA for the detection of anti-S-RBD antibodies in comparison to the commercial assay. The LFA was not a reliable determinant of the neutralisation capacity of identified antibodies. POC LFA are useful tools in sero-epidemiology settings, pandemic preparedness and may act as supportive tools in treatment decisions through the rapid identification of anti-Spike antibodies

Phylogenetic Relationships of the Marine Haplosclerida (Phylum Porifera) Employing Ribosomal (28S rRNA) and Mitochondrial (cox1, nad1) Gene Sequence Data

The systematics of the poriferan Order Haplosclerida (Class Demospongiae) has been under scrutiny for a number of years without resolution. Molecular data suggests that the order needs revision at all taxonomic levels. Here, we provide a comprehensive view of the phylogenetic relationships of the marine Haplosclerida using many species from across the order, and three gene regions. Gene trees generated using 28S rRNA, nad1 and cox1 gene data, under maximum likelihood and Bayesian approaches, are highly congruent and suggest the presence of four clades. Clade A is comprised primarily of species of Haliclona and Callyspongia, and clade B is comprised of H. simulans and H. vansoesti (Family Chalinidae), Amphimedon queenslandica (Family Niphatidae) and Tabulocalyx (Family Phloeodictyidae), Clade C is comprised primarily of members of the Families Petrosiidae and Niphatidae, while Clade D is comprised of Aka species. The polyphletic nature of the suborders, families and genera described in other studies is also found here

Analysis of the function of the N1 subdomain of MSCRAMMs of Staphylococcus aureus

THESIS 10364The Microbial Surface Component Recognising Adhesive Matrix Molecules (MSCRAMMs) family of cell wall-associated proteins have recently been reclassified based on structure-function analysis. Under this new regime the MSCRAMM family is defined by the presence of at least two tandemly-linked IgG-like folded domains. Clumping factor A (ClfA) is the archetypal fibrinogen-binding MSCRAMM of Staphylococcus aureus and an important virulence factor. An N-terminal signal sequence directs export by the Sec pathway and the C-terminal cell wall-anchoring domain allows covalent attachment of ClfA to peptidoglycan by sortase. Region A of ClfA comprises three independently folded subdomains N1, N2 and N3. Subdomains N2N3 comprise IgG-like folds and promote fibrinogen binding. However the function of subdomain N1 has, until now, remained elusive

Fibronectin-binding protein B variation in Staphylococcus aureus

Background: Fibronectin binding proteins A and B (FnBPA and FnBPB) mediate adhesion of S.aureus to fibrinogen, elastin and fibronectin. We previously identified seven different isotopes of FnBPA based on divergence in the fibrinogen- and elastin-binding A domains. The variation created differences in antigenicity while ligand binding functions were retained. Here, FnBPB variation was examined in both human and bovine isolates and compared to that of FnBPA. Results: Seven different fnbB allelic variants were identified. Some strains that cluster by phylogenetic analysis contain different fnbB variants, whereas more divergent strains contain the same fnbB variant. The phylogeny of fnbB alleles does not match the phylogeny of fnbA alleles. Some FnBPA and FnBPB isotypes that are specified by human S. aureus strains are also found in bovine strains. The seven fnbB allelic variants encode seven distinct isotopes of the FnBPB A domain that are 61 to 85 % identical in amino acid sequence. Variant amino acid residues were mapped on a three-dimensional model of the FnBPB A domain and were predicted to be surface-exposed. They are responsible for the antigenic diversity detected with polyclonal antibody and a monoclonal antibody raised against isotype I. Ligand binding by recombinant FnBPB N23 isotypes was compared by ELISA-based solid phase assays and surface plasmon resonance. Each bound to immobilized fibrinogen, elastin and fibronectin dose-dependently and saturably with similar affinities. Binding to fibronectin was surprising because the A domains do not contain any known motifs that mediate binding to fibronectin. This raises the possibility that the A domain of FnBPB contains a novel fibronectin binding motif that binds fibronectin by a novel mechanism. Conclusions: Seven different isoforms of FnBPB A domain retain ligand-binding functions but are antigenically distinct. The variation in FnBPA and FnBPB occurs in human and bovine S. aureus strains and may act as an immune evasion mechanism

How context influences person-centred practice: a critical-creative case study examining the use of research evidence in occupational therapy with people living with dementia.

From PubMed via Jisc Publications RouterPublication status: aheadofprintOccupational therapists are encouraged to use research evidence to guide therapeutic interventions that holistically address the consequences of dementia. Recent efforts to use research evidence in practice have emphasized the challenges of doing so in ways aligned to person-centred and professional principles. Using research evidence is a complex process influenced by multiple contextual factors and layers. The influence of context in occupational therapy for dementia is currently unclear. To explore the contextual complexities of using research evidence in practice with people with dementia, and to develop knowledge to improve the approach to using evidence in person-centred, occupation-focused practice. A case study methodology was used, in which the contextual conditions of practice were clarified through the facilitation of critical and creative reflection using the following methods - Think Aloud, practice observation, creative expression and reflective dialogue. Cultural beliefs that affected evidence use included technically-orientated understandings of evidence-based practice. These were underpinned by apprehensions about losing professional identity and taking risks when processes derived from research evidence were adjusted to incorporate a persons' occupations. These cultural factors were perpetuated at the organizational layers of context, where systemic priorities and other team members' needs disproportionately influenced occupational therapists' decisions. Occupational therapists' potential to make reflexive and responsive decisions by adjusting evidence-based processes can be affected by their perceived freedom to address organizational tensions. Raising consciousness of the influence of the organizational context on decision-making about evidence use could adjust occupational therapists' perceptions of their freedom and ability to be person-centred. Intentionality in reflective processes in practice are required to foster reflexivity

The effects of prolyl hydroxylase inhibition during and post, hypoxia, oxygen glucose deprivation and oxidative stress, in isolated rat hippocampal slices

The contributions of hypoxia and oxidative stress to the pathophysiology of acute ischemic stroke are well established and can lead to disruptions in synaptic signaling. Hypoxia and oxidative stress lead to the neurotoxic overproduction of reactive oxygen species (ROS) and the stabilization of hypoxia inducible factors (HIF). Compounds such as prolyl-4-hydroxylase domain enzyme inhibitors (PHDIs) have been shown to have a preconditioning and neuroprotective effect against ischemic insults such as hypoxia, anoxia, oxygen glucose deprivation (OGD) or H2O2. Therefore, this study explored the effects of two PHDIs, JNJ-42041935 (10 µM) and roxadustat (100 µM) on cell viability using organotypic hippocampal slice cultures. We also assessed the effects of these compounds on synaptic transmission during and post hypoxia, OGD and H2O2 application in isolated rat hippocampal slices using field recording electrophysiological techniques and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit trafficking using immunohistochemistry. Our organotypic data demonstrated a protective role for both inhibitors, where slices had significantly less cell death post anoxia and OGD compared to controls. We also report a distinct modulatory role for both JNJ-42041935 and roxadustat on fEPSP slope post hypoxia and OGD but not H2O2. In addition, we report that application of roxadustat impaired long-term potentiation, but only when applied post-hypoxia. This inhibitory effect was not reversed with co-application of the cyclin-dependent kinase 5 (CDK-5) inhibitor, roscovitine (10 µM), suggesting a CDK-5 independent synaptic AMPAR trafficking mechanism. Both hypoxia and OGD induced a reduction in synaptic AMPA GluA2 subunits, the OGD effect being reversed by prior treatment with both JNJ-42041935 and roxadustat. These results suggest an important role for PHDs in synaptic signaling and plasticity during episodes of ischemic stress