Gem-induced cytoskeleton remodeling increases cellular migration of HTLV-1-infected cells, formation of infected-to-target T-cell conjugates and viral transmission

Efficient HTLV-1 viral transmission occurs through cell-to-cell contacts. The Tax viral transcriptional activator protein facilitates this process. Using a comparative transcriptomic analysis, we recently identified a series of genes up-regulated in HTLV-1 Tax expressing T-lymphocytes. We focused our attention towards genes that are important for cytoskeleton dynamic and thus may possibly modulate cell-to-cell contacts. We first demonstrate that Gem, a member of the small GTP-binding proteins within the Ras superfamily, is expressed both at the RNA and protein levels in Tax-expressing cells and in HTLV-1-infected cell lines. Using a series of ChIP assays, we show that Tax recruits CREB and CREB Binding Protein (CBP) onto a c-AMP Responsive Element (CRE) present in the gem promoter. This CRE sequence is required to drive Tax-activated gem transcription. Since Gem is involved in cytoskeleton remodeling, we investigated its role in infected cells motility. We show that Gem co-localizes with F-actin and is involved both in T-cell spontaneous cell migration as well as chemotaxis in the presence of SDF-1/CXCL12. Importantly, gem knock-down in HTLV-1-infected cells decreases cell migration and conjugate formation. Finally, we demonstrate that Gem plays an important role in cell-to-cell viral transmission

Plasmodium berghei Kinesin-5 Associates With the Spindle Apparatus During Cell Division and Is Important for Efficient Production of Infectious Sporozoites

Kinesin-5 motors play essential roles in spindle apparatus assembly during cell division, by generating forces to establish and maintain the spindle bipolarity essential for proper chromosome segregation. Kinesin-5 is largely conserved structurally and functionally in model eukaryotes, but its role is unknown in the Plasmodium parasite, an evolutionarily divergent organism with several atypical features of both mitotic and meiotic cell division. We have investigated the function and subcellular location of kinesin-5 during cell division throughout the Plasmodium berghei life cycle. Deletion of kinesin-5 had little visible effect at any proliferative stage except sporozoite production in oocysts, resulting in a significant decrease in the number of motile sporozoites in mosquito salivary glands, which were able to infect a new vertebrate host. Live-cell imaging showed kinesin-5-GFP located on the spindle and at spindle poles during both atypical mitosis and meiosis. Fixed-cell immunofluorescence assays revealed kinesin-5 co-localized with α-tubulin and centrin-2 and a partial overlap with kinetochore marker NDC80 during early blood stage schizogony. Dual-color live-cell imaging showed that kinesin-5 is closely associated with NDC80 during male gametogony, but not with kinesin-8B, a marker of the basal body and axonemes of the forming flagella. Treatment of gametocytes with microtubule-specific inhibitors confirmed kinesin-5 association with nuclear spindles and not cytoplasmic axonemal microtubules. Altogether, our results demonstrate that kinesin-5 is associated with the spindle apparatus, expressed in proliferating parasite stages, and important for efficient production of infectious sporozoites

Identifying Improved Sites for Heterologous Gene Integration Using ATAC-seq

Constructing efficient cellular factories often requires integration of heterologous pathways for synthesis of novel compounds and improved cellular productivity. Few genomic sites are routinely used, however, for efficient integration and expression of heterologous genes, especially in nonmodel hosts. Here, a data-guided framework for informing suitable integration sites for heterologous genes based on ATAC-seq was developed in the nonmodel yeast Komagataella phaffii. Single-copy GFP constructs were integrated using CRISPR/Cas9 into 38 intergenic regions (IGRs) to evaluate the effects of IGR size, intensity of ATAC-seq peaks, and orientation and expression of adjacent genes. Only the intensity of accessibility peaks was observed to have a significant effect, with higher expression observed from IGRs with low- to moderate-intensity peaks than from high-intensity peaks. This effect diminished for tandem, multicopy integrations, suggesting that the additional copies of exogenous sequence buffered the transcriptional unit of the transgene against effects from endogenous sequence context. The approach developed from these results should provide a basis for nominating suitable IGRs in other eukaryotic hosts from an annotated genome and ATAC-seq data

Mental health support through primary care during and after covid-19.

Editorial Materia

Network model of immune responses reveals key effectors to single and co-infection dynamics by a respiratory bacterium and a gastrointestinal helminth

Co-infections alter the host immune response but how the systemic and local processes at the site of infection interact is still unclear. The majority of studies on co-infections concentrate on one of the infecting species, an immune function or group of cells and often focus on the initial phase of the infection. Here, we used a combination of experiments and mathematical modelling to investigate the network of immune responses against single and co-infections with the respiratory bacterium Bordetella bronchiseptica and the gastrointestinal helminth Trichostrongylus retortaeformis. Our goal was to identify representative mediators and functions that could capture the essence of the host immune response as a whole, and to assess how their relative contribution dynamically changed over time and between single and co-infected individuals. Network-based discrete dynamic models of single infections were built using current knowledge of bacterial and helminth immunology; the two single infection models were combined into a co-infection model that was then verified by our empirical findings. Simulations showed that a T helper cell mediated antibody and neutrophil response led to phagocytosis and clearance of B. bronchiseptica from the lungs. This was consistent in single and co-infection with no significant delay induced by the helminth. In contrast, T. retortaeformis intensity decreased faster when co-infected with the bacterium. Simulations suggested that the robust recruitment of neutrophils in the co-infection, added to the activation of IgG and eosinophil driven reduction of larvae, which also played an important role in single infection, contributed to this fast clearance. Perturbation analysis of the models, through the knockout of individual nodes (immune cells), identified the cells critical to parasite persistence and clearance both in single and co-infections. Our integrated approach captured the within-host immuno-dynamics of bacteria-helminth infection and identified key components that can be crucial for explaining individual variability between single and co-infections in natural populations

A pragmatic cluster randomised trial evaluating three implementation interventions

Background Implementation research is concerned with bridging the gap between evidence and practice through the study of methods to promote the uptake of research into routine practice. Good quality evidence has been summarised into guideline recommendations to show that peri-operative fasting times could be considerably shorter than patients currently experience. The objective of this trial was to evaluate the effectiveness of three strategies for the implementation of recommendations about peri-operative fasting. Methods A pragmatic cluster randomised trial underpinned by the PARIHS framework was conducted during 2006 to 2009 with a national sample of UK hospitals using time series with mixed methods process evaluation and cost analysis. Hospitals were randomised to one of three interventions: standard dissemination (SD) of a guideline package, SD plus a web-based resource championed by an opinion leader, and SD plus plan-do-study-act (PDSA). The primary outcome was duration of fluid fast prior to induction of anaesthesia. Secondary outcomes included duration of food fast, patients' experiences, and stakeholders' experiences of implementation, including influences. ANOVA was used to test differences over time and interventions. Results Nineteen acute NHS hospitals participated. Across timepoints, 3,505 duration of fasting observations were recorded. No significant effect of the interventions was observed for either fluid or food fasting times. The effect size was 0.33 for the web-based intervention compared to SD alone for the change in fluid fasting and was 0.12 for PDSA compared to SD alone. The process evaluation showed different types of impact, including changes to practices, policies, and attitudes. A rich picture of the implementation challenges emerged, including inter-professional tensions and a lack of clarity for decision-making authority and responsibility. Conclusions This was a large, complex study and one of the first national randomised controlled trials conducted within acute care in implementation research. The evidence base for fasting practice was accepted by those participating in this study and the messages from it simple; however, implementation and practical challenges influenced the interventions' impact. A set of conditions for implementation emerges from the findings of this study, which are presented as theoretically transferable propositions that have international relevance. Trial registration ISRCTN18046709 - Peri-operative Implementation Study Evaluation (POISE

Developing and evaluating the implementation of a complex intervention: using mixed methods to inform the design of a randomised controlled trial of an oral healthcare intervention after stroke

<p>Abstract</p> <p>Background</p> <p>Many interventions delivered within the stroke rehabilitation setting could be considered complex, though some are more complex than others. The degree of complexity might be based on the number of and interactions between levels, components and actions targeted within the intervention. The number of (and variation within) participant groups and the contexts in which it is delivered might also reflect the extent of complexity. Similarly, designing the evaluation of a complex intervention can be challenging. Considerations include the necessity for intervention standardisation, the multiplicity of outcome measures employed to capture the impact of a multifaceted intervention and the delivery of the intervention across different clinical settings operating within varying healthcare contexts. Our aim was to develop and evaluate the implementation of a complex, multidimensional oral health care (OHC) intervention for people in stroke rehabilitation settings which would inform the development of a randomised controlled trial.</p> <p>Methods</p> <p>After reviewing the evidence for the provision of OHC following stroke, multi-disciplinary experts informed the development of our intervention. Using both quantitative and qualitative methods we evaluated the implementation of the complex OHC intervention across patients, staff and service levels of care. We also adopted a pragmatic approach to patient recruitment, the completion of assessment tools and delivery of OHC, alongside an attention to the context in which it was delivered.</p> <p>Results</p> <p>We demonstrated the feasibility of implementing a complex OHC intervention across three levels of care. The complementary nature of the mixed methods approach to data gathering provided a complete picture of the implementation of the intervention and a detailed understanding of the variations within and interactions between the components of the intervention. Information on the feasibility of the outcome measures used to capture impact across a range of components was also collected, though some process orientated uncertainties including eligibility and recruitment rates remain to be further explored within a Phase II exploratory trial.</p> <p>Conclusions</p> <p>Complex interventions can be captured and described in a manner which facilitates evaluation in the form of exploratory and subsequently definitive clinical trials. If effective, the evidence captured relating to the intervention context will facilitate translation into clinical practice.</p