Better understanding on interactions between SARS‐CoV‐2 and host cells should help to identify host factors that may be targetable to combat infection and COVID‐19 pathology. To this end, we have ...conducted a genome‐wide CRISPR/Cas9‐based loss‐of‐function screen in human lung cancer cells infected with SARS‐CoV‐2‐pseudotyped lentiviruses. Our results recapitulate many findings from previous screens that used full SARS‐CoV‐2 viruses, but also unveil two novel critical host factors: the lysosomal efflux transporter SPNS1 and the plasma and lysosomal membrane protein PLAC8. Functional experiments with full SARS‐CoV‐2 viruses confirm that loss‐of‐function of these genes impairs viral entry. We find that PLAC8 is a key limiting host factor, whose overexpression boosts viral infection in eight different human lung cancer cell lines. Using single‐cell RNA‐Seq data analyses, we demonstrate that PLAC8 is highly expressed in ciliated and secretory cells of the respiratory tract, as well as in gut enterocytes, cell types that are highly susceptible to SARS‐CoV‐2 infection. Proteomics and cell biology studies suggest that PLAC8 and SPNS1 regulate the autophagolysosomal compartment and affect the intracellular fate of endocytosed virions.
Synopsis
Identifying targetable host factors to fight COVID‐19 requires full understanding of virus‐host cell interactions. Here, a genome‐wide loss‐of‐function screen identifies new regulators of the intracellular fate of endocytosed SARS‐CoV‐2 virions.
A genome‐wide CRISPR/Cas9‐based screen in human lung cancer cells identifies lysosomal membrane proteins PLAC8 and SPNS1 as required for viral entry.
PLAC8 loss reduces SARS‐CoV‐2 infection efficiency, while overexpression boosts it in several lung cancer lines
Single‐cell RNA‐Seq analyses reveal a strong enrichment of PLAC8 expression in lung and colon SARS‐CoV‐2 target cells
PLAC8 colocalizes with ACE2, but its loss‐of‐function does not affect binding or endocytosis in experiments with recombinant SPIKE receptor binding domain
PLAC8 loss‐of‐function causes an expansion of the autophagolysosomal compartment
Genome‐wide CRISPR screens identify lysosomal efflux transporter SPNS1 and transmembrane protein PLAC8 as regulators of the intracellular fate of endocytosed SARS‐CoV‐2 virions.
Abstract
Motivation
Venn and Euler diagrams are extensively used for the visualization of relationships between experiments and datasets. However, representing more than three datasets while keeping ...the proportions of each region is still not feasible with existing tools.
Results
We present an algorithm to render all the regions of a generalized n-dimensional Venn diagram, while keeping the area of each region approximately proportional to the number of elements included. In addition, missing regions in Euler diagrams lead to simplified representations. The algorithm generates an n-dimensional Venn diagram and inserts circles of given areas in each region. Then, the diagram is rearranged with a dynamic, self-correcting simulation in which each set border is contracted until it contacts the circles inside. This algorithm is implemented in a C++ tool (nVenn) with or without a web interface. The web interface also provides the ability to analyze the regions of the diagram.
Availability and implementation
The source code and pre-compiled binaries of nVenn are available at https://github.com/vqf/nVenn. A web interface for up to six sets can be accessed at http://degradome.uniovi.es/cgi-bin/nVenn/nvenn.cgi.
Supplementary information
Supplementary data are available at Bioinformatics online.
Through its ability to open pores in cell membranes, perforin-1 plays a key role in the immune system. Consistent with this role, the gene encoding perforin shows hallmarks of complex evolutionary ...events, including amplification and pseudogenization, in multiple species. A large proportion of these events occurred in phyla for which scarce genomic data were available. However, recent large-scale genomics projects have added a wealth of information on those phyla. Using this input, we annotated perforin-1 homologs in more than eighty species including mammals, reptiles, birds, amphibians and fishes.
We have annotated more than 400 perforin genes in all groups studied. Most mammalian species only have one perforin locus, which may contain a related pseudogene. However, we found four independent small expansions in unrelated members of this class. We could reconstruct the full-length coding sequences of only a few avian perforin genes, although we found incomplete and truncated forms of these gene in other birds. In the rest of reptilia, perforin-like genes can be found in at least three different loci containing up to twelve copies. Notably, mammals, non-avian reptiles, amphibians, and possibly teleosts share at least one perforin-1 locus as assessed by flanking genes. Finally, fish genomes contain multiple perforin loci with varying copy numbers and diverse exon/intron patterns. We have also found evidence for shorter genes with high similarity to the C2 domain of perforin in several teleosts. A preliminary analysis suggests that these genes arose at least twice during evolution from perforin-1 homologs.
The assisted annotation of new genomic assemblies shows complex patterns of birth-and-death events in the evolution of perforin. These events include duplication/pseudogenization in mammals, multiple amplifications and losses in reptiles and fishes and at least one case of partial duplication with a novel start codon in fishes.
Abstract
Intravesical administration of Bacillus Calmette-Guérin (BCG) was one of the first FDA-approved immunotherapies and remains a standard treatment for bladder cancer. Previous studies have ...demonstrated that intravenous (IV) administration of BCG is well-tolerated and effective in preventing tuberculosis infection in animals. Here, we examine IV BCG in several preclinical lung tumor models. Our findings demonstrate that BCG inoculation reduced tumor growth and prolonged mouse survival in models of lung melanoma metastasis and orthotopic lung adenocarcinoma. Moreover, IV BCG treatment was well-tolerated with no apparent signs of acute toxicity. Mechanistically, IV BCG induced tumor-specific CD8
+
T cell responses, which were dependent on type 1 conventional dendritic cells, as well as NK cell-mediated immunity. Lastly, we also show that IV BCG has an additive effect on anti-PD-L1 checkpoint inhibitor treatment in mouse lung tumors that are otherwise resistant to anti-PD-L1 as monotherapy. Overall, our study demonstrates the potential of systemic IV BCG administration in the treatment of lung tumors, highlighting its ability to enhance immune responses and augment immune checkpoint blockade efficacy.
The genomic sequencing of chronic lymphocytic leukemia (CLL) samples has provided exciting new venues for the understanding and treatment of this prevalent disease. This feat is possible thanks to ...high-throughput sequencing methods, such as Illumina sequencing. The interpretation of these data sources requires not only appropriate software and hardware, but also understanding the biology and technology behind the sequencing process. Here, we provide a primer to understand each step in the analysis of point mutations from whole-genome or whole-exome sequencing experiments of tumor and normal samples.
Giant tortoises are among the longest-lived vertebrate animals and, as such, provide an excellent model to study traits like longevity and age-related diseases. However, genomic and molecular ...evolutionary information on giant tortoises is scarce. Here, we describe a global analysis of the genomes of Lonesome George-the iconic last member of Chelonoidis abingdonii-and the Aldabra giant tortoise (Aldabrachelys gigantea). Comparison of these genomes with those of related species, using both unsupervised and supervised analyses, led us to detect lineage-specific variants affecting DNA repair genes, inflammatory mediators and genes related to cancer development. Our study also hints at specific evolutionary strategies linked to increased lifespan, and expands our understanding of the genomic determinants of ageing. These new genome sequences also provide important resources to help the efforts for restoration of giant tortoise populations.
Exact tests greatly improve the analysis of contingency tables when marginals are low. For instance, researchers often use Fisher's exact test, which is conditional, or Barnard's test, which is ...unconditional but needs to deal with a nuisance parameter. Here, we describe the m-test, an exact, unconditional test for the study of d x m binomial contingency tables. When comparing binomial trials, the m-test is related to Barnard's exact test. However, the nuisance parameter is integrated over all its possible values, instead of maximized or otherwise estimated. According to Monte Carlo simulations, this strategy yields a higher statistical power than other exact tests. We also provide a package to perform the m-test in R.
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