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Summary: Here, we exploit a deep serological profiling strategy coupled with an integrated, computational framework for the analysis of SARS-CoV-2 humoral immune responses. Applying a high-density peptide array (HDPA) spanning the entire proteomes of SARS-CoV-2 and endemic human coronaviruses allowed identification of B cell epitopes and relate them to their evolutionary and structural properties. We identify hotspots of pre-existing immunity and identify cross-reactive epitopes that contribute to increasing the overall humoral immune response to SARS-CoV-2. Using a public dataset of over 38,000 viral genomes from the early phase of the pandemic, capturing both inter- and within-host genetic viral diversity, we determined the evolutionary profile of epitopes and the differences across proteins, waves, and SARS-CoV-2 variants. Lastly, we show that mutations in spike and nucleocapsid epitopes are under stronger selection between than within patients, suggesting that most of the selective pressure for immune evasion occurs upon transmission between hosts.

Papillary thyroid carcinoma (PTC) is the most common malignancy of the thyroid gland and early stages are curable. However, a subset of PTCs shows an unusually aggressive phenotype with extensive lymph node metastasis and higher incidence of locoregional recurrence. In this study, we investigated a large cohort of PTC cases with an unusual aggressive phenotype using a high-throughput RNA sequencing (RNA-Seq) to identify differentially regulated genes associated with metastatic PTC. All metastatic PTC with mutated BRAF (V600E) but not BRAF wild-type expressed an up-regulation of R-Spondin Protein 4 (RSPO4) concomitant with an upregulation of genes involved in focal adhesion and cell-extracellular matrix signaling. Further immunohistochemistry validation confirmed the upregulation of these target genes in metastatic PTC cases. Preclinical studies using established PTC cell lines support that RSPO4 overexpression is associated with BRAF V600E mutation and is a critical upstream event that promote activation of kinases of focal adhesion signaling known to drive cancer cell locomotion and invasion. This finding opens up the potential of co-targeting B-Raf, RSPO and focal adhesion proteins as a pharmacological approach for aggressive BRAF V600E PTC.

Abstract Fluorescent proteins (FPs) are commonly used in pairs to monitor dynamic biomolecular events through changes in proximity via distance dependent processes such as Förster resonance energy transfer (FRET). The impact of FP association is assessed by predicting dimerization sites in silico and stabilizing the dimers by bio‐orthogonal covalent linkages. In each tested case dimerization changes inherent fluorescence, including FRET. GFP homodimers demonstrate synergistic behavior with the dimer being brighter than the sum of the monomers. The homodimer structure reveals the chromophores are close with favorable transition dipole alignments and a highly solvated interface. Heterodimerization (GFP with Venus) results in a complex with ≈87% FRET efficiency, significantly below the 99.7% efficiency predicted. A similar efficiency is observed when the wild‐type FPs are fused to a naturally occurring protein–protein interface system. GFP complexation with mCherry results in loss of mCherry fluorescence. Thus, simple assumptions used when monitoring interactions between proteins via FP FRET may not always hold true, especially under conditions whereby the protein–protein interactions promote FP interaction.

Pathogen monitoring, detection and removal are essential to public health and outbreak management. Systems are in place for monitoring the microbial load of hospitals and public health facilities with strategies to mitigate pathogen spread. However, no such strategies are in place for ambulances, which are tasked with transporting at-risk individuals in immunocompromised states. As standard culturing techniques require a laboratory setting, and are time consuming and labour intensive, our approach was designed to be portable, inexpensive and easy to use based on the MinION third-generation sequencing platform from Oxford Nanopore Technologies. We developed a transferable sampling-to-analysis pipeline to characterize the microbial community in emergency medical service vehicles. Our approach identified over sixty-eight organisms in ambulances to the genera level, with a proportion of these being connected with health-care associated infections, such as Clostridium spp. and Staphylococcus spp. We also monitored the microbiome of different locations across three ambulances over time, and examined the dynamic community of microorganisms found in emergency medical service vehicles. Observed differences identified hot spots, which may require heightened monitoring and extensive cleaning. Through metagenomics analysis it is also possible to identify how microorganisms spread between patients and colonize an ambulance over time. The sequencing results aid in the development of practices to mitigate disease spread, while also providing a useful tool for outbreak prediction through ongoing analysis of the ambulance microbiome to identify new and emerging pathogens. Overall, this pipeline allows for the tracking and monitoring of pathogenic microorganisms of epidemiological interest, including those related to health-care associated infections.