Tick-borne flavivirus NS5 antagonizes interferon signaling by inhibiting the catalytic activity of TYK2.

The mechanisms utilized by different flaviviruses to evade antiviral functions of interferons are varied and incompletely understood. Using virological approaches, biochemical assays, and mass spectrometry analyses, we report here that the NS5 protein of tick-borne encephalitis virus (TBEV) and Louping Ill virus (LIV), two related tick-borne flaviviruses, antagonize JAK-STAT signaling through interactions with the tyrosine kinase 2 (TYK2). Co-immunoprecipitation (co-IP) experiments, yeast gap-repair assays, computational protein-protein docking and functional studies identify a stretch of 10 residues of the RNA dependent RNA polymerase domain of tick-borne flavivirus NS5, but not mosquito-borne NS5, that is critical for interactions with the TYK2 kinase domain. Additional co-IP assays performed with several TYK2 orthologs reveal that the interaction is conserved across mammalian species. In vitro kinase assays show that TBEV and LIV NS5 reduce the catalytic activity of TYK2. Our results thus illustrate a novel mechanism by which viruses suppress the interferon response.

CD94+ Natural Killer cells potentiate pulmonary ischemia-reperfusion injury.

Pulmonary ischemia-reperfusion injury (IRI) is a major contributor to poor lung transplant outcomes. We recently demonstrated a central role of airway-centered NK cells in mediating IRI; however, there are no existing effective therapies for directly targeting NK cells in humans. We hypothesized that a depleting anti-CD94 monoclonal antibody (mAb) would provide therapeutic benefit in mouse and human models of IRI based on high levels of KLRD1 (CD94) transcripts in bronchoalveolar lavage samples from lung transplant patients. We found that CD94 is highly expressed on mouse and human NK cells, with increased expression during IRI. Anti-mouse and anti-human mAbs against CD94 showed effective NK cell depletion in mouse and human models and blunted lung damage and airway epithelial killing. In two different allogeneic orthotopic lung transplant mouse models, anti-CD94 treatment during induction reduced early lung injury and chronic inflammation relative to control therapies. Anti-CD94 did not increase donor antigen-presenting cells that could alter long-term graft acceptance. Lung transplant induction regimens incorporating anti-CD94 treatment may safely improve early clinical outcomes.

An equine iPSC-based phenotypic screening platform identifies pro- and anti-viral molecules against West Nile virus.

Outbreaks of West Nile virus (WNV) occur periodically, affecting both human and equine populations. There are no vaccines for humans, and those commercialised for horses do not have sufficient coverage. Specific antiviral treatments do not exist. Many drug discovery studies have been conducted, but since rodent or primate cell lines are normally used, results cannot always be transposed to horses. There is thus a need to develop relevant equine cellular models. Here, we used induced pluripotent stem cells to develop a new in vitro model of WNV-infected equine brain cells suitable for microplate assay, and assessed the cytotoxicity and antiviral activity of forty-one chemical compounds. We found that one nucleoside analog, 2'C-methylcytidine, blocked WNV infection in equine brain cells, whereas other compounds were either toxic or ineffective, despite some displaying anti-viral activity in human cell lines. We also revealed an unexpected proviral effect of statins in WNV-infected equine brain cells. Our results thus identify a potential lead for future drug development and underscore the importance of using a tissue- and species-relevant cellular model for assessing the activity of antiviral compounds.

Pathological modeling of TBEV infection reveals differential innate immune responses in human neurons and astrocytes that correlate with their susceptibility to infection.

BACKGROUND: Tick-borne encephalitis virus (TBEV) is a member of the Flaviviridae family, Flavivirus genus, which includes several important human pathogens. It is responsible for neurological symptoms that may cause permanent disability or death, and, from a medical point of view, is the major arbovirus in Central/Northern Europe and North-Eastern Asia. TBEV tropism is critical for neuropathogenesis, yet little is known about the molecular mechanisms that govern the susceptibility of human brain cells to the virus. In this study, we sought to establish and characterize a new in vitro model of TBEV infection in the human brain and to decipher cell type-specific innate immunity and its relation to TBEV tropism and neuropathogenesis. METHOD: Human neuronal/glial cells were differentiated from neural progenitor cells and infected with the TBEV-Hypr strain. Kinetics of infection, cellular tropism, and cellular responses, including innate immune responses, were characterized by measuring viral genome and viral titer, performing immunofluorescence, enumerating the different cellular types, and determining their rate of infection and by performing PCR array and qRT-PCR. The specific response of neurons and astrocytes was analyzed using the same approaches after enrichment of the neuronal/glial cultures for each cellular subtype. RESULTS: We showed that infection of human neuronal/glial cells mimicked three major hallmarks of TBEV infection in the human brain, namely, preferential neuronal tropism, neuronal death, and astrogliosis. We further showed that these cells conserved their capacity to mount an antiviral response against TBEV. TBEV-infected neuronal/glial cells, therefore, represented a highly relevant pathological model. By enriching the cultures for either neurons or astrocytes, we further demonstrated qualitative and quantitative differential innate immune responses in the two cell types that correlated with their particular susceptibility to TBEV. CONCLUSION: Our results thus reveal that cell type-specific innate immunity is likely to contribute to shaping TBEV tropism for human brain cells. They describe a new in vitro model for in-depth study of TBEV-induced neuropathogenesis and improve our understanding of the mechanisms by which neurotropic viruses target and damage human brain cells.

Molecular Determinants of West Nile Virus Virulence and Pathogenesis in Vertebrate and Invertebrate Hosts.

West Nile virus (WNV), like the dengue virus (DENV) and yellow fever virus (YFV), are major arboviruses belonging to the Flavivirus genus. WNV is emerging or endemic in many countries around the world, affecting humans and other vertebrates. Since 1999, it has been considered to be a major public and veterinary health problem, causing diverse pathologies, ranging from a mild febrile state to severe neurological damage and death. WNV is transmitted in a bird-mosquito-bird cycle, and can occasionally infect humans and horses, both highly susceptible to the virus but considered dead-end hosts. Many studies have investigated the molecular determinants of WNV virulence, mainly with the ultimate objective of guiding vaccine development. Several vaccines are used in horses in different parts of the world, but there are no licensed WNV vaccines for humans, suggesting the need for greater understanding of the molecular determinants of virulence and antigenicity in different hosts. Owing to technical and economic considerations, WNV virulence factors have essentially been studied in rodent models, and the results cannot always be transported to mosquito vectors or to avian hosts. In this review, the known molecular determinants of WNV virulence, according to invertebrate (mosquitoes) or vertebrate hosts (mammalian and avian), are presented and discussed. This overview will highlight the differences and similarities found between WNV hosts and models, to provide a foundation for the prediction and anticipation of WNV re-emergence and its risk of global spread.

PSM Peptides of Staphylococcus aureus Activate the p38-CREB Pathway in Dendritic Cells, Thereby Modulating Cytokine Production and T Cell Priming.

The challenging human pathogen Staphylococcus aureus has highly efficient immune evasion strategies for causing a wide range of diseases, from skin and soft tissue to life-threatening infections. Phenol-soluble modulin (PSM) peptides are major pathogenicity factors of community-associated methicillin-resistant S. aureus strains. In previous work, we demonstrated that PSMs in combination with TLR2 ligand from S. aureus induce tolerogenic dendritic cells (DCs) characterized by the production of high amounts of IL-10, but no proinflammatory cytokines. This in turn promotes the activation of regulatory T cells while impairing Th1 response; however, the signaling pathways modulated by PSMs remain elusive. In this study, we analyzed the effects of PSMs on signaling pathway modulation downstream of TLR2. TLR2 stimulation in combination with PSMα3 led to increased and prolonged phosphorylation of NF-κB, ERK, p38, and CREB in mouse bone marrow-derived DCs compared with single TLR2 activation. Furthermore, inhibition of p38 and downstream MSK1 prevented IL-10 production, which in turn reduced the capacity of DCs to activate regulatory T cells. Interestingly, the modulation of the signaling pathways by PSMs was independent of the known receptor for PSMs, as shown by experiments with DCs lacking the formyl peptide receptor 2. Instead, PSMs penetrate the cell membrane most likely by transient pore formation. Moreover, colocalization of PSMs and p38 was observed near the plasma membrane in the cytosol, indicating a direct interaction. Thus, PSMs from S. aureus directly modulate the signaling pathway p38-CREB in DCs, thereby impairing cytokine production and in consequence T cell priming to increase the tolerance toward the pathogen.

Borna disease virus phosphoprotein impairs the developmental program controlling neurogenesis and reduces human GABAergic neurogenesis.

It is well established that persistent viral infection may impair cellular function of specialized cells without overt damage. This concept, when applied to neurotropic viruses, may help to understand certain neurologic and neuropsychiatric diseases. Borna disease virus (BDV) is an excellent example of a persistent virus that targets the brain, impairs neural functions without cell lysis, and ultimately results in neurobehavioral disturbances. Recently, we have shown that BDV infects human neural progenitor cells (hNPCs) and impairs neurogenesis, revealing a new mechanism by which BDV may interfere with brain function. Here, we sought to identify the viral proteins and molecular pathways that are involved. Using lentiviral vectors for expression of the bdv-p and bdv-x viral genes, we demonstrate that the phosphoprotein P, but not the X protein, diminishes human neurogenesis and, more particularly, GABAergic neurogenesis. We further reveal a decrease in pro-neuronal factors known to be involved in neuronal differentiation (ApoE, Noggin, TH and Scg10/Stathmin2), demonstrating that cellular dysfunction is associated with impairment of specific components of the molecular program that controls neurogenesis. Our findings thus provide the first evidence that a viral protein impairs GABAergic human neurogenesis, a process that is dysregulated in several neuropsychiatric disorders. They improve our understanding of the mechanisms by which a persistent virus may interfere with brain development and function in the adult.

Staphylococcus aureus PSM peptides induce tolerogenic dendritic cells upon treatment with ligands of extracellular and intracellular TLRs.

Dendritic cells (DCs) are key players of the immune system and thus a target for immune evasion by pathogens. We recently showed that the virulence factor phenol-soluble modulin (PSM) produced by community-associated methicillin-resistant Staphylococcus aureus strains induces tolerogenic DCs upon Toll-like receptor (TLR) 2 activation via the p38-CREB-IL-10 pathway. Here, we addressed the question whether this tolerogenic phenotype of DCs induced by PSMs is specific for TLR2 activation. Therefore, bone marrow-derived DCs were treated with various ligands for extracellular and intracellular TLRs simultaneously with PSMα3. We show that PSMα3 modulates antigen uptake, maturation and cytokine production of DCs activated by TLR1/2, TLR2/6, TLR4, TLR7, and TLR9. Pre-incubation of DCs with a p38 MAP kinase inhibitor prevented the PSMα3-induced IL-10 secretion, as well as MHC class II up-regulation upon TLR activation. In consequence, the tolerogenic DCs induced by PSMα3 in response to several TLR ligands promoted priming of regulatory T cells. Thus, PSMs could be useful as inducers of tolerogenic DCs upon TLR ligand stimulation for therapeutic applications.

Parietal hyper-connectivity, aberrant brain organization, and circuit-based biomarkers in children with mathematical disabilities.

Mathematical disabilities (MD) have a negative life-long impact on professional success, employment, and health outcomes. Yet little is known about the intrinsic functional brain organization that contributes to poor math skills in affected children. It is now increasingly recognized that math cognition requires coordinated interaction within a large-scale fronto-parietal network anchored in the intraparietal sulcus (IPS). Here we characterize intrinsic functional connectivity within this IPS-network in children with MD, relative to a group of typically developing (TD) children who were matched on age, gender, IQ, working memory, and reading abilities. Compared to TD children, children with MD showed hyper-connectivity of the IPS with a bilateral fronto-parietal network. Importantly, aberrant IPS connectivity patterns accurately discriminated children with MD and TD children, highlighting the possibility for using IPS connectivity as a brain-based biomarker of MD. To further investigate regional abnormalities contributing to network-level deficits in children with MD, we performed whole-brain analyses of intrinsic low-frequency fluctuations. Notably, children with MD showed higher low-frequency fluctuations in multiple fronto-parietal areas that overlapped with brain regions that exhibited hyper-connectivity with the IPS. Taken together, our findings suggest that MD in children is characterized by robust network-level aberrations, and is not an isolated dysfunction of the IPS. We hypothesize that intrinsic hyper-connectivity and enhanced low-frequency fluctuations may limit flexible resource allocation, and contribute to aberrant recruitment of task-related brain regions during numerical problem solving in children with MD.

Stratification of Treatment in a Community-Based Musculoskeletal Service: A Mixed-Methods Study to Assess Predictors of Requiring Complex Care.

OBJECTIVE: To explore factors that might be relevant when designing a triage tool. DESIGN: A mixed-methods study using multivariable logistic regression analysis to identify significant factors associated with requiring different levels of care, and qualitative focus groups exploring views of patients and physiotherapy clinicians regarding case complexity. SETTING: A community-based adult musculoskeletal service delivering tier 1 (standard physiotherapy) and tier 2 care (complex care beyond the scope of standard physiotherapy) and providing onward referral to orthopedic clinics (tier 3). PARTICIPANTS: Quantitative data were extracted from a random sample of patients (N=484) who had received treatment for musculoskeletal conditions. Patients and physiotherapists who had received care or who worked in the service participated in focus groups. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Themes that emerged from focus groups were compared against predictors of requiring complex care found to be significant (P<.05) after quantitative data analysis. RESULTS: A total of 184 patients (38.0%; 95% confidence interval, 33.8-42.4) received complex care. Peripheral joint problems, unclear diagnosis, and symptoms affecting sleep were significant independent predictors of requiring complex care. These data supported some of the main themes raised at focus groups. CONCLUSIONS: A substantial proportion of patients receive tier 2 complex care. Further studies are needed to evaluate whether the predictive factors found to be significant in our study might be useful for developing a tool for more effective triage to the most appropriate tier of musculoskeletal care.

Glucocorticoid receptor is required for foetal heart maturation.

Glucocorticoids are vital for the structural and functional maturation of foetal organs, yet excessive foetal exposure is detrimental to adult cardiovascular health. To elucidate the role of glucocorticoid signalling in late-gestation cardiovascular maturation, we have generated mice with conditional disruption of glucocorticoid receptor (GR) in cardiomyocytes and vascular smooth muscle cells using smooth muscle protein 22-driven Cre recombinase (SMGRKO mice) and compared them with mice with global deficiency in GR (GR(-/-)). Echocardiography shows impaired heart function in both SMGRKO and GR(-/-) mice at embryonic day (E)17.5, associated with generalized oedema. Cardiac ultrastructure is markedly disrupted in both SMGRKO and GR(-/-) mice at E17.5, with short, disorganized myofibrils and cardiomyocytes that fail to align in the compact myocardium. Failure to induce critical genes involved in contractile function, calcium handling and energy metabolism underpins this common phenotype. However, although hearts of GR(-/-) mice are smaller, with 22% reduced ventricular volume at E17.5, SMGRKO hearts are normally sized. Moreover, while levels of mRNA encoding atrial natriuretic peptide are reduced in E17.5 GR(-/-) hearts, they are normal in foetal SMGRKO hearts. These data demonstrate that structural, functional and biochemical maturation of the foetal heart is dependent on glucocorticoid signalling within cardiomyocytes and vascular smooth muscle, though some aspects of heart maturation (size, ANP expression) are independent of GR at these key sites.

The Academy for Future Science Faculty: randomized controlled trial of theory-driven coaching to shape development and diversity of early-career scientists.

BACKGROUND: Approaches to training biomedical scientists have created a talented research community. However, they have failed to create a professional workforce that includes many racial and ethnic minorities and women in proportion to their representation in the population or in PhD training. This is particularly true at the faculty level. Explanations for the absence of diversity in faculty ranks can be found in social science theories that reveal processes by which individuals develop identities, experiences, and skills required to be seen as legitimate within the profession. METHODS/DESIGN: Using the social science theories of Communities of Practice, Social Cognitive Career Theory, identity formation, and cultural capital, we have developed and are testing a novel coaching-based model to address some of the limitations of previous diversity approaches. This coaching intervention (The Academy for Future Science Faculty) includes annual in-person meetings of students and trained faculty Career Coaches, along with ongoing virtual coaching, group meetings and communication. The model is being tested as a randomized controlled trial with two cohorts of biomedical PhD students from across the U.S., one recruited at the start of their PhDs and one nearing completion. Stratification into the experimental and control groups, and to coaching groups within the experimental arms, achieved equal numbers of students by race, ethnicity and gender to the extent possible. A fundamental design element of the Academy is to teach and make visible the social science principles which highly influence scientific advancement, as well as acknowledging the extra challenges faced by underrepresented groups working to be seen as legitimate within the scientific communities. DISCUSSION: The strategy being tested is based upon a novel application of the well-established principles of deploying highly skilled coaches, selected and trained for their ability to develop talents of others. This coaching model is intended to be a complement, rather than a substitute, for traditional mentoring in biomedical research training, and is being tested as such.

What is Effective in Massage Therapy? Well, "It Depends ": a Qualitative Study of Experienced Orthopaedic Massage Therapists.

Background: Massage has been used as a treatment for musculoskeletal pain throughout history and across cultures, and yet most meta-analyses have only shown weak support for the efficacy of massage. There is a recognised need for more research in foundational questions including: how massage treatments are constructed; what therapists actually do within a treatment, including their clinical reasoning; and what role therapists play in determining the effectiveness of a massage treatment. Purpose: The aim of this study was to explore what experienced orthopaedic massage therapists consider to be the aspects of their work that contribute to effectiveness. Setting and Participants: Semi-structured interviews were conducted via Zoom with six experienced orthopaedic massage therapists in Australia. Research Design: The interviews were analysed using inductive thematic analysis, seeking insights that might be practically applied, rather than theory-driven interpretations. Results: The participants focused on the underlying differences between clients, between therapists, and between treatments, and clearly indicated that this concept of "difference" was foundational to their view of their work and was the underlying context for the comments they made. Within that frame of "difference", three key themes were interpreted from the data: (1) "Everyone is different so every treatment is different": how they individualised treatment based on these differences; (2) "How therapists cope with difference": how they managed the challenges of working in this context; and (3) "What makes a difference": the problem-solving processes they used to target each treatment to meeting the client's needs. Conclusions: Participants did not identify specific techniques or modalities as "effective" or not. Rather, a therapist's ability to provide effective treatment was based on an iterative process of treatment and assessment that allowed them to focus on the individual needs of the client. In this case "effectiveness" could be considered a process rather than a specific massage technique.

Borna disease virus infects human neural progenitor cells and impairs neurogenesis.

Understanding the complex mechanisms by which infectious agents can disrupt behavior represents a major challenge. The Borna disease virus (BDV), a potential human pathogen, provides a unique model to study such mechanisms. Because BDV induces neurodegeneration in brain areas that are still undergoing maturation at the time of infection, we tested the hypothesis that BDV interferes with neurogenesis. We showed that human neural stem/progenitor cells are highly permissive to BDV, although infection does not alter their survival or undifferentiated phenotype. In contrast, upon the induction of differentiation, BDV is capable of severely impairing neurogenesis by interfering with the survival of newly generated neurons. Such impairment was specific to neurogenesis, since astrogliogenesis was unaltered. In conclusion, we demonstrate a new mechanism by which BDV might impair neural function and brain plasticity in infected individuals. These results may contribute to a better understanding of behavioral disorders associated with BDV infection.

Epidemiology, molecular virology and diagnostics of Schmallenberg virus, an emerging orthobunyavirus in Europe.

After the unexpected emergence of Bluetongue virus serotype 8 (BTV-8) in northern Europe in 2006, another arbovirus, Schmallenberg virus (SBV), emerged in Europe in 2011 causing a new economically important disease in ruminants. The virus, belonging to the Orthobunyavirus genus in the Bunyaviridae family, was first detected in Germany, in The Netherlands and in Belgium in 2011 and soon after in the United Kingdom, France, Italy, Luxembourg, Spain, Denmark and Switzerland. This review describes the current knowledge on the emergence, epidemiology, clinical signs, molecular virology and diagnosis of SBV infection.

Middle school special education teachers' perceptions and use of assistive technology in literacy instruction.

In this research the authors examined middle school special education teachers' perceptions of assistive technology during literacy instruction with students with high incidence disabilities. A survey explored the use, effectiveness, and factors impacting use or effectiveness of assistive technology for literacy teaching and learning. Results suggested teachers' perceived assistive technology to be an effective tool for literacy, but use it minimally. When assistive technology was used, teachers indicated it was an effective literacy support. Teachers also reported barriers to using assistive technology in literacy including cost, usability, and lack of training/experience. However, factors such as previous successful experiences with assistive technology and assistive technology supporting students' learning encouraged assistive technology use. The consistency of teachers' reports of needing more experience and knowledge in assistive technology to fully use it suggests implications for preservice preparation such as providing additional experiences and information on assistive technology.

The Process of Developing a Digital Repository for Online Teaching Using Design-Based Research.

The Purdue Repository for Online Teaching and Learning (PoRTAL) was developed as an Open Educational Resource (OER) for graduate students and faculty in higher education settings to enhance their online teaching skills and strategies. The PoRTAL team used a design-based research approach (DBR; Wang & Hannafin, Educational Technology Research and Development, 53(4), 5-23, 2005). In this study context, we used Van Tiem et al.'s (2012) model to identify problems faced by instructors who struggled with or were new to online teaching from a Human Performance Technology (HPT) standpoint. To address the identified needs, we created resources for online teaching and embedded our research within practical activities to further study our design process. Our efforts resulted in an HPT-OER Model for Designing Digital Repositories. The purpose of this paper is to share the DBR process that we used to develop an OER repository within an HPT model.

Erratum to "Impact of pharmacist intervention to deprescribe inappropriate aspirin therapy in an outpatient anticoagulation clinic at a community hospital" [Am. Heart J. Plus: Cardiol. Res. Pract. volume 17, May 2022, 100165].

[This corrects the article DOI: 10.1016/j.ahjo.2022.100165.].

Comparison of Radiography and Computed Tomography for Evaluation of Third Carpal Bone Fractures in Horses.

Radiographs underestimate the extent of bone injury in horses with third carpal bone (C3) fractures (Fx). We aimed to describe bone pathologies identified using computed tomography (CT) and compare the diagnostic value of digital radiography (DR) and CT in horses with C3 Fx. CT images of 15 racehorses with C3 Fx and 10 controls were reviewed (Part 1) then DR and CT images of 26 racehorses (24 Thoroughbred, 2 Standardbred) with C3 Fx (Part 2) were evaluated. Agreement on fracture geometry and concomitant bone lesions was tested between DR and CT using the kappa statistic (Part 2). For agreement analysis, 38 limbs were used (27 Fx carpi from 26 horses and 11 contralateral carpi). Intermodality agreement was good for recognition of displacement, fair for comminution, articular surface bone loss and osseous fragmentation, and poor-slight for recognition of whether the Fx was complete, additional fissures and lucencies. CT provides more detailed information than DR regarding bone pathology and fracture configuration in horses with C3 fracture. Correlation of CT findings with clinical information and outcome needs to be explored; however, the more accurate diagnosis possible with CT is likely valuable when deciding on the most appropriate management and for surgical planning.