A live RSV vaccine with engineered thermostability is immunogenic in cotton rats despite high attenuation

Respiratory syncytial virus (RSV) is a leading cause of infant hospitalization and there remains no pediatric vaccine. RSV live-attenuated vaccines (LAVs) have a history of safe testing in infants; however, achieving an effective balance of attenuation and immunogenicity has proven challenging. Here we seek to engineer an RSV LAV with enhanced immunogenicity. Genetic mapping identifies strain line 19 fusion (F) protein residues that correlate with pre-fusion antigen maintenance by ELISA and thermal stability of infectivity in live RSV. We generate a LAV candidate named OE4 which expresses line 19F and is attenuated by codon-deoptimization of non-structural (NS1 and NS2) genes, deletion of the small hydrophobic (SH) gene, codon-deoptimization of the attachment (G) gene and ablation of the secreted form of G. OE4 (RSV-A2-dNS1-dNS2-ΔSH-dGm-Gsnull-line19F) exhibits elevated pre-fusion antigen levels, thermal stability, immunogenicity, and efficacy despite heavy attenuation in the upper and lower airways of cotton rats

Non-affirmative Theory of Education as a Foundation for Curriculum Studies, Didaktik and Educational Leadership

This chapter presents non-affirmative theory of education as the foundation for a new research program in education, allowing us to bridge educational leadership, curriculum studies and Didaktik. We demonstrate the strengths of this framework by analyzing literature from educational leadership and curriculum theory/didaktik. In contrast to both socialization-oriented explanations locating curriculum and leadership within existing society, and transformation-oriented models viewing education as revolutionary or super-ordinate to society, non-affirmative theory explains the relation between education and politics, economy and culture, respectively, as non-hierarchical. Here critical deliberation and discursive practices mediate between politics, culture, economy and education, driven by individual agency in historically developed cultural and societal institutions. While transformative and socialization models typically result in instrumental notions of leadership and teaching, non-affirmative education theory, previously developed within German and Nordic education, instead views leadership and teaching as relational and hermeneutic, drawing on ontological core concepts of modern education: recognition; summoning to self-activity and Bildsamkeit. Understanding educational leadership, school development and teaching then requires a comparative multi-level approach informed by discursive institutionalism and organization theory, in addition to theorizing leadership and teaching as cultural-historical and critical-hermeneutic activity. Globalisation and contemporary challenges to deliberative democracy also call for rethinking modern nation-state based theorizing of education in a cosmopolitan light. Non-affirmative education theory allows us to understand and promote recognition based democratic citizenship (political, economical and cultural) that respects cultural, ethical and epistemological variations in a globopolitan era. We hope an American-European-Asian comparative dialogue is enhanced by theorizing education with a non-affirmative approach

Pleomorphic Structures in Human Blood Are Red Blood Cell-Derived Microparticles, Not Bacteria

<div><p>Background</p><p>Red blood cell (RBC) transfusions are a common, life-saving therapy for many patients, but they have also been associated with poor clinical outcomes. We identified unusual, pleomorphic structures in human RBC transfusion units by negative-stain electron microscopy that appeared identical to those previously reported to be bacteria in healthy human blood samples. The presence of viable, replicating bacteria in stored blood could explain poor outcomes in transfusion recipients and have major implications for transfusion medicine. Here, we investigated the possibility that these structures were bacteria.</p><p>Results</p><p>Flow cytometry, miRNA analysis, protein analysis, and additional electron microscopy studies strongly indicated that the pleomorphic structures in the supernatant of stored RBCs were RBC-derived microparticles (RMPs). Bacterial 16S rDNA PCR amplified from these samples were sequenced and was found to be highly similar to species that are known to commonly contaminate laboratory reagents.</p><p>Conclusions</p><p>These studies suggest that pleomorphic structures identified in human blood are RMPs and not bacteria, and they provide an example in which laboratory contaminants may can mislead investigators.</p></div

Vesicles isolated from supernatant of RBC storage units are membrane-bound, intact, and contain RBC surface antigen and RBC-specific miRNA.

<p><b>A</b>, Unstained vesicles (red, bottom left) or vesicles co-stained with calcein-AM and fluorescent anti-GPA (blue, top right) were analyzed by flow cytometry. >99% of the vesicles were positive for calcein-AM and anti-GPA. <b>B</b>, RNA from vesicles was analyzed by Agilent Bioanalyzer. Peak on electropherogram at 25 nt is internal standard and small peak to the right reflects small RNA. This electropherogram is representative of Bioanlyzer data from six different RBC storage units. <b>C</b>, Levels (Ct values) of miR-451 were assessed by qRT-PCR in stored RBCs, the RMP pellet, and in human aortic endothelial cells (HAECs, negative control). <b>D</b>, Hemoglobin-alpha content of RMP pellet and stored RBCs, as assessed by Western blot. Blot is representative of six different RBC storage units.</p

Representative 3D cryo-electron tomograpahy data and visual characterization of RBC-derived microparticles (RMPs).

<p><b>A,</b> Slices through 3D volumes of RMPs. <b>B,</b> RMPs were measured and were characterized as either round or pleiomorphic, and as either full or empty. The number of each particle type, percent of the total, mean diameter, and standard deviation are presented in the table.</p