The host machinery in the secretory organelles, the endoplasmic reticulum and the Golgi network, is pivotal in directing the biogenesis of enveloped virus proteins during natural infection and vaccination. Secretory trafficking of the viral proteins in these organelles is accompanied by post-translational modifications (PTMs), which modify interactions with host receptors and antibodies. Hence, elucidating the fundamental basis of PTM modulation by secretory trafficking is essential for designing genetic vaccines that encode stable and immunogenic viral proteins. For instance, the spike protein in COVID-19 mRNA vaccines undergoes bidirectional secretory trafficking after export from the endoplasmic reticulum to the cis-Golgi. This supplies the spike from the cis-Golgi to the coronavirus assembly site in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) by retrograde trafficking and to the plasma membrane by anterograde trafficking for coronavirus transmission and for immune display. However, surprisingly, little is known about the modulation of spike PTMs by this secretory recycling and retention in the endoplasmic reticulum-ERGIC-Golgi pathway before export to the plasma membrane. Since PTMs such as N-glycans modulate the conformations of immunogenic epitopes in the spike protein, addressing this knowledge gap in secretory routing is critical for understanding spike-immune system interactions. Furthermore, the bidirectional trafficking of the spike protein engages and diverts the secretory machinery, which can cause secretory stress, inflammation, and immune dysfunction. Hence, elucidating the fundamental interactions that govern spike biogenesis and trafficking will expedite the design of next- generation genetic vaccines that interfere minimally with the secretory trafficking machinery while undergoing proper PTMs for stability and efficient delivery to the plasma membrane. Here, we report atomic-level insights into the PTM and structural biology of novel spike protein constructs arrested in various stages of secretory trafficking. Using a combination of mass spectrometry, single particle cryoEM, and MD simulations on these differently arrested spike constructs, we identified clusters of N-glycans whose remodeling is intimately linked to spike recycling and secretory routing. These spike constructs demonstrate robust binding to a panel of neutralizing antibodies despite differences in their N-glycans and secretory routing to the plasma membrane. Finally, building on these structure-function insights, we engineered a novel spike vaccine candidate with enhanced secretion to the cell surface. We determined its structures in various conformational states by single particle cryoEM, demonstrating the presentation of immunogenic epitopes. Thus, our investigation provides novel strategies for designing a new generation of genetic vaccines with enhanced secretion and immune display at the host cell plasma membrane. Bottom of Form
Reintroduction of grazing to counteract insect decline will lead to an increase of semi-open habitats. Semi-open habitats are highly heterogeneous, consisting of a mosaic of patches of trees or shrubs embedded in a matrix dominated by dwarf shrubs or grasses. Despite a lack of evidence, structural heterogeneity is expected to allow many species to co-occur, making semi-open habitats appear highly valuable for invertebrate conservation. We studied ground beetles in eight sites in two landscapes of Central Europe each encompassing semi-open, open, and forest habitats. Rapid response to environmental change and limited dispersal abilities make ground beetles an excellent model taxon to evaluate the effectiveness of such conservations measures. In both studied landscapes, ground beetle assemblages in semi-open habitats tended to be distinct and intermediate between those from the forest and open habitats. Species richness and functional diversity in semi-open habitats were similar to open habitats at site level. The majority of species entered the semi-open habitats, except for most threatened species, yet, few species were exclusively associated with semi-open habitats. We conclude that the continuous presence of many species in semi-open habitats likely results from mass effects rather than habitat heterogeneity per se . Our findings underline the conservation value of the existing forest, heathland, and grassland habitats over semi-open habitats which can, however, function as dispersal habitats and increase landscape connectivity. Strategies aiming at promoting semi-open habitats to counteract insect decline should target enhancing connectivity rather than the creation of habitats only.
RESUMO – Estudo sobre questões epistemológicas referentes à história do direito, em especial o problema da historicidade. O trabalho chama a atenção para dificuldades na abordagem do tema, como o risco do anacronismo e a dupla compreensão do direito como construção intelectual e prática social. Procura-se definir o território da historicidade com foco nas relações jurídicas e na desconstrução de abordagens ahistóricas com efeitos como a naturalização e a reificação dos conceitos. A dinâmica da historicidade é considerada nas perspectivas do conhecimento histórico (reflexão historiográfica, Historizität) e do processo histórico (Geschichtlichkeit, exemplificada com a questão da ruptura e continuidade).
BACKGROUND:The emergence of Mycobacterium tuberculosis with complex drug resistance profiles necessitates a rapid and comprehensive drug susceptibility test for guidance of patient treatment. We developed two targeted-sequencing workflows based on Illumina MiSeq and Nanopore MinION for the prediction of drug resistance in M. tuberculosis toward 12 antibiotics.METHODS:A total of 163 M. tuberculosis isolates collected from Hong Kong and Ethiopia were subjected to a multiplex PCR for simultaneous amplification of 19 drug resistance-associated genetic regions. The amplicons were then barcoded and sequenced in parallel on MiSeq and MinION in respective batch sizes of 24 and 12 samples. A web-based bioinformatics pipeline, BacterioChek-TB, was developed to translate the raw datasets into clinician-friendly reports.RESULTS:Both platforms successfully sequenced all samples with mean read depths of 1,127× and 1,649×, respectively. The variant calling by MiSeq and MinION could achieve 100% agreement if variants with an allele frequency of <40% reported by MinION were excluded. Both workflows achieved a mean clinical sensitivity of 94.8% and clinical specificity of 98.0% when compared with phenotypic drug susceptibility test (pDST). Turnaround times for the MiSeq and MinION workflows were 38 and 15 h, facilitating the delivery of treatment guidance at least 17-18 days earlier than pDST, respectively. The higher cost per sample on the MinION platform ($71.56) versus the MiSeq platform ($67.83) was attributed to differences in batching capabilities.CONCLUSION:Our study demonstrates the interchangeability of MiSeq and MinION platforms for generation of accurate and actionable results for the treatment of tuberculosis.
