Usp18 Driven Enforced Viral Replication in Dendritic Cells Contributes to Break of Immunological Tolerance in Autoimmune Diabetes

Infection with viruses carrying cross-reactive antigens is associated with break of immunological tolerance and induction of autoimmune disease. Dendritic cells play an important role in this process. However, it remains unclear why autoimmune-tolerance is broken during virus infection, but usually not during exposure to non-replicating cross-reactive antigens. Here we show that antigen derived from replicating virus but not from non-replicating sources undergoes a multiplication process in dendritic cells in spleen and lymph nodes. This enforced viral replication was dependent on Usp18 and was essential for expansion of autoreactive CD8⁺ T cells. Preventing enforced virus replication by depletion of CD11c⁺ cells, genetically deleting Usp18, or pharmacologically inhibiting of viral replication blunted the expansion of autoreactive CD8⁺ T cells and prevented autoimmune diabetes. In conclusion, Usp18-driven enforced viral replication in dendritic cells can break immunological tolerance and critically influences induction of autoimmunity.     

Structural Control of Metabolic Flux

Organisms have to continuously adapt to changing environmental conditions or undergo developmental transitions. To meet the accompanying change in metabolic demands, the molecular mechanisms of adaptation involve concerted interactions which ultimately induce a modification of the metabolic state, which is characterized by reaction fluxes and metabolite concentrations. These state transitions are the effect of simultaneously manipulating fluxes through several reactions. While metabolic control analysis has provided a powerful framework for elucidating the principles governing this orchestrated action to understand metabolic control, its applications are restricted by the limited availability of kinetic information. Here, we introduce structural metabolic control as a framework to examine individual reactions'' potential to control metabolic functions, such as biomass production, based on structural modeling. The capability to carry out a metabolic function is determined using flux balance analysis (FBA). We examine structural metabolic control on the example of the central carbon metabolism of Escherichia coli by the recently introduced framework of functional centrality (FC). This framework is based on the Shapley value from cooperative game theory and FBA, and we demonstrate its superior ability to assign “share of control” to individual reactions with respect to metabolic functions and environmental conditions. A comparative analysis of various scenarios illustrates the usefulness of FC and its relations to other structural approaches pertaining to metabolic control. We propose a Monte Carlo algorithm to estimate FCs for large networks, based on the enumeration of elementary flux modes. We further give detailed biological interpretation of FCs for production of lactate and ATP under various respiratory conditions.     

A Foraminiferal Parasite on the Sea Urchin Echinocorys: Ichnological Evidence from the Late Cretaceous (Lower Maastrichtian,Northern Germany)

Archaeonassa is a poorly known ichnogenus, originally described from the Cambrian of North America, which is a member of the Scolicia ’group’, but can be regarded as distinct from Scolicia. Archaeonassa was originally based on modern material, and therefore falls into a taxonomic grey zone concerning its availability, but as A. fossulata was erected on fossil material the ichnogenus is here retained. Although exhibiting a degree of morphological plasticity in its style of ornament, Archaeonassa is considered to be monospecific, with only the ichnospecies A. fossulata recognised. The ichnogenus appears to be restricted to the Paleozoic, although similar material is well documented from modern environments. Thus the ichnogenus is expected to be geographically and stratigraphically more widespread than it otherwise appears. Archaeonassa is a valuable tool for environmental reconstruction within the Paleozoic as it occurs within intertidal deposits. The ichnogenus is interpreted chiefly as the work of gastropods, although in many cases may have been produced by trilobites or even echinoids, and represents the exogenic expression of either surface or shallow subsurface locomotion.     

Focus: Preventive Medicine: Who is Biking for? Urban Bikeshare Networks’ Responses to the COVID-19 Pandemic,Disparities in Bikeshare Access,and a Way Forward

Black, Latinx, and Indigenous people have contracted the SARS-CoV-2 virus and died of COVID-19 at higher rates than White people. Individuals rated public transit, taxis, and ride-hailing as the modes of transportation putting them at greatest risk of COVID-19 infection. Cycling may thus be an attractive alternative for commuting. Amid the increase in bikeshare usage during the early months of the pandemic, bikeshare companies made changes to membership requirements to increase accessibility, targeting especially essential workers. Essential workers in the United States are disproportionately Black and Latinx, underpaid, and reliant on public transit to commute to work. We document changes made by bikeshare companies, including benefits to various groups of essential workers, and we discuss such changes in the context of longstanding racial disparities in bikeshare access. While well intended, the arbitrary delineation in eligibility for such benefits by class of essential workers unwittingly curtailed access for many who may have benefited most. Given that equity in bikeshare is an important tool to improve access to safe transportation, critical changes in the distribution, accessibility, and usability of bikeshare networks is essential. Bikeshare companies, city planners, and policy makers should collaborate with community-based bike advocates to implement changes, as vocalized by those most in need of alternative forms of transportation.     

An expanded auxin-inducible degron toolkit for Caenorhabditis elegans

The auxin-inducible degron (AID) system has emerged as a powerful tool to conditionally deplete proteins in a range of organisms and cell types. Here, we describe a toolkit to augment the use of the AID system in Caenorhabditis elegans. We have generated a set of single-copy, tissue-specific (germline, intestine, neuron, muscle, pharynx, hypodermis, seam cell, anchor cell) and pan-somatic TIR1-expressing strains carrying a co-expressed blue fluorescent reporter to enable use of both red and green channels in experiments. These transgenes are inserted into commonly used, well-characterized genetic loci. We confirmed that our TIR1-expressing strains produce the expected depletion phenotype for several nuclear and cytoplasmic AID-tagged endogenous substrates. We have also constructed a set of plasmids for constructing repair templates to generate fluorescent protein::AID fusions through CRISPR/Cas9-mediated genome editing. These plasmids are compatible with commonly used genome editing approaches in the C. elegans community (Gibson or SapTrap assembly of plasmid repair templates or PCR-derived linear repair templates). Together these reagents will complement existing TIR1 strains and facilitate rapid and high-throughput fluorescent protein::AID tagging of genes. This battery of new TIR1-expressing strains and modular, efficient cloning vectors serves as a platform for straightforward assembly of CRISPR/Cas9 repair templates for conditional protein depletion.     

High-content imaging-based pooled CRISPR screens in mammalian cells

CRISPR (clustered regularly interspaced short palindromic repeats)-based gene inactivation provides a powerful means for linking genes to particular cellular phenotypes. CRISPR-based screening typically uses large genomic pools of single guide RNAs (sgRNAs). However, this approach is limited to phenotypes that can be enriched by chemical selection or FACS sorting. Here, we developed a microscopy-based approach, which we name optical enrichment, to select cells displaying a particular CRISPR-induced phenotype by automated imaging-based computation, mark them by photoactivation of an expressed photoactivatable fluorescent protein, and then isolate the fluorescent cells using fluorescence-activated cell sorting (FACS). A plugin was developed for the open source software μManager to automate the phenotypic identification and photoactivation of cells, allowing ∼1.5 million individual cells to be screened in 8 h. We used this approach to screen 6,092 sgRNAs targeting 544 genes for their effects on nuclear size regulation and identified 14 bona fide hits. These results present a scalable approach to facilitate imaging-based pooled CRISPR screens.