We have sequenced and annotated the genome of ®ssion yeast (Schizosaccharomyces pombe), which contains the smallest number of protein-coding genes yet recorded for a eukaryote: 4,824. The centromeres are between 35 and 110 kilobases (kb) and contain related repeats including a highly conserved 1.8-kb element. Regions upstream of genes are longer than in budding yeast (Saccharomyces cerevisiae), possibly re¯ecting more-extended control regions. Some 43% of the genes contain introns, of which there are 4,730. Fifty genes have signi®cant similarity with human disease genes; half of these are cancer related. We identify highly conserved genes important for eukaryotic cell organization including those required for the cytoskeleton, compartmentation, cell-cycle control, proteolysis, protein phosphorylation and RNA splicing. These genes may have originated with the appearance of eukaryotic life. Few similarly conserved genes that are important for multicellular organization were identi®ed, suggesting that the transition from prokaryotes to eukaryotes required more new genes than did the transition from unicellular to multicellular organization.We report here the completion of the fully annotated genome sequence of the simple eukaryote Schizosaccharomyces pombe, a ®ssion yeast. It becomes the sixth eukaryotic genome to be sequenced, following Saccharomyces cerevisiae 1 , Caenorhabditis elegans 2 , Drosophila melanogaster 3 , Arabidopsis thaliana 4 and Homo sapiens 5,6 . The entire sequence of the unique regions of the three chromosomes is complete, with gaps in the centromeric regions of about 40 kb, and about 260 kb in the telomeric regions. The completion of this sequence, the availability of sophisticated research methodologies, and the expanding community working on S. pombe, will accelerate the use of S. pombe for functional and comparative studies of eukaryotic cell processes.

he COVID-19 pandemic, caused by SARS-CoV-2, has resulted in a worldwide health crisis 1 and few effective drugs are available to treat patients with COVID-19. Although remdesivir initially seemed promising for severe cases 2 , the World Health Organization's Solidarity trial showed that it has no definite impact on mortality 3. Dexamethasone can reduce mortality by a third among critically ill patients with COVID-19, by suppressing the hyperactive immune response 4. However, as treatment benefits severe cases only to a limited extent, efficient and safe therapeutics are urgently required while awaiting the worldwide implementation of vaccines. Coronaviruses cause respiratory and intestinal infections in a broad range of mammals and birds. Seven human coronaviruses (HCoVs) are known, which probably all emerged as zoonoses from bats, mice or domestic animals 5. The four so-called 'common cold HCoVs'-229E, NL63, OC43 and HKU1-cause mild upper respiratory tract illnesses 6. In contrast, SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV) and the recently emerged SARS-CoV-2 are highly pathogenic and cause severe, potentially lethal respiratory infections. As numerous coronaviruses reside in animal reservoirs and interspecies transmission frequently occurs 5,7,8 , there is a constant risk of new pathogenic coronaviruses spreading into the human population, as exemplified by the recent SARS-CoV-2 pandemic. Nevertheless, our options to prevent or treat coronavirus infections remain limited. Hence, the development of broad-spectrum anti-coronavirus drugs could help not only to address the current high medical need, but also to quickly contain zoonotic events in the future. Common host factors essential for replication of multiple coronaviruses represent attractive targets for broad-spectrum antiviral drugs. To develop such drugs, it is crucial to understand which host factors coronaviruses require to infect a cell, because each step of the coronavirus replication cycle (receptor binding, endocytosis, fusion, viral protein translation, genome replication, virion assembly and release) may serve as a target for intervention. Although the entry step of coronaviruses has been relatively well characterized, the host-virus interplay in later steps of the viral life cycle remains largely elusive. For SARS-CoV-2, previous studies have shown that the protein angiotensin-converting enzyme 2 (ACE2) can serve as a receptor in Vero E6 cells 9 or in human cells overexpressing ACE2 (refs. 10-12). In addition, it was shown that the SARS-CoV-2 spike (S) can be primed for fusion by cellular proteases such as furin, transmembrane serine protease 2 (TMPRSS2) or cathepsin B or L, depending on the target cell type 10,13. In the present study, we performed a series of genome-wide CRISPR (clustered regularly interspaced short palindromic repeats)-based genetic screens to identify host factors required for SARS-CoV-2 and HCoV-229E infection. We identified phosphoinositide 3-kinase (PI3K) type 3 as a common host factor for SARS-CoV-2,...

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Malaria-associated acute respiratory distress syndrome (MA-ARDS) is a deadly complication of malaria, and its pathophysiology is insufficiently understood. Both in humans and in murine models, MA-ARDS is characterized by marked pulmonary inflammation. We investigated the role of hemozoin in MA-ARDS in C57Bl/6 mice infected with Plasmodium berghei NK65, P. berghei ANKA, and P. chabaudi AS. By quantifying hemozoin in the lungs and measuring the disease parameters of MA-ARDS, we demonstrated a highly significant correlation between pulmonary hemozoin concentrations, lung weights, and alveolar edema. Histological analysis of the lungs demonstrated that hemozoin is localized in phagocytes and infected erythrocytes, and only occasionally in granulocytes. Species-specific differences in hemozoin production, as measured among individual schizonts, were associated with variations in pulmonary pathogenicity. Furthermore, both pulmonary hemozoin and lung pathology were correlated with the number of infiltrating inflammatory cells, an increased pulmonary expression of cytokines, chemokines, and enzymes, and concentrations of alveolar vascular endothelial growth factor. The causal relationship between hemozoin and inflammation was investigated by injecting P. falciparum-derived hemozoin intravenously into malaria-free mice. Hemozoin potently induced the pulmonary expression of proinflammatory chemokines (interferon-γ inducible protein-10/CXC-chemokine ligand (CXCL)10, monocyte chemotactic protein-1/CC-chemokine ligand 2, and keratinocyte-derived chemokine/CXCL1), cytokines (IL-1β, IL-6, IL-10, TNF, and transforming growth factor-β), and other inflammatory mediators (inducible nitric oxide synthase, heme oxygenase-1, nicotinamide adenine dinucleotide phosphate- oxidase-2, and intercellular adhesion molecule-1). Thus, hemozoin correlates with MA-ARDS and induces pulmonary inflammation.

Validation of drug-target interaction is essential in drug discovery and development. The ultimate proof for drug-target validation requires the introduction of mutations that confer resistance in cells, an approach that is not straightforward in mammalian cells. Using CRISPR/Cas9 genome editing, we show that a homozygous genomic C528S mutation in the XPO1 gene confers cells with resistance to selinexor (KPT-330). Selinexor is an orally bioavailable inhibitor of exportin-1 (CRM1/XPO1) with potent anticancer activity and is currently under evaluation in human clinical trials. Mutant cells were resistant to the induction of cytotoxicity, apoptosis, cell cycle arrest, and inhibition of XPO1 function, including direct binding of the drug to XPO1. These results validate XPO1 as the prime target of selinexor in cells and identify the selectivity of this drug toward the cysteine 528 residue of XPO1. Our findings demonstrate that CRISPR/Cas9 genome editing enables drug-target validation and drug-target selectivity studies in cancer cells.

The human immunodeficiency virus, type 1 (HIV-1)-encoded Rev protein is essential for the expression of late viral mRNAs. Rev forms a large organized multimeric protein-protein complex on the Rev response element of these viral mRNA species and transports them from the nucleus to the cytoplasm, exploiting the CRM1-mediated cellular machinery. Here we report the selection of a nanobody, derived from a llama heavy-chain only antibody, that efficiently blocks the assembly of Rev multimers. The nanobody inhibits HIV-1 replication in cells and specifically suppresses the Rev-dependent expression of partially spliced and unspliced HIV-1 RNA. In HIV-susceptible cells, this nanobody thus has potential as an effective anti-HIV agent using genetic immunization strategies. Its binding site was mapped to Rev residues Lys-20 and Tyr-23 located in the N-terminal ␣-helical multimerization domain. In the presence of this nanobody, we observed an accumulation of dimeric Rev species, supporting a head-to-head/tail-to-tail molecular model for Rev assembly. The results indicate that the oligomeric assembly of Rev follows an ordered stepwise process and identify a new epitope within Rev that could guide strategies for the development of novel HIV inhibitors.Nuclear export of viral mRNA is critical for the life cycle of the human immunodeficiency virus (HIV).2 Fully spliced viral mRNA is exported to the cytoplasm of the infected cell through cellular mechanisms. However, in contrast to cellular systems, HIV uses a sophisticated molecular machinery to transport unspliced and partially spliced forms of its viral mRNA. The nuclear export of these late viral messengers is required for both the expression of late viral genes and packaging of genomic RNA and is mediated by the HIV-encoded regulatory protein Rev (1). The viral Rev protein forms a multimeric complex on a secondary structured RNA element (the Rev response element (RRE)) present in all unspliced and partially spliced mRNAs (2-4) and exploits the CRM1-mediated cellular machinery to transport these RNAs from the nucleus to the cytoplasm (5-7).Rev is a small protein of 116 amino acid residues. Under steady state conditions, Rev localizes mainly to the nucleoli of cells (8). However, different functional elements cause Rev to continuously shuttle between the nucleus and the cytoplasm (9 -11). A short stretch of basic amino acids characterized by 10 arginine residues serves both as a nuclear localization signal for the nuclear import of Rev and as an RNA-binding domain during the export of RNA-Rev complexes (4). This arginine-rich region is flanked on both sides by sequences that contribute to the oligomerization of Rev on the RRE. Its strong tendency to oligomerize has hampered the structure determination of Rev by x-ray crystallography or NMR spectroscopy. However, circular dichroism, nuclear magnetic resonance, and Raman spectroscopy studies strongly suggest that the oligomerization domains and the nuclear localization signal are components of a helix-turn-helix motif (12-14). A...

2-DE was applied to study core breakdown disorder in controlled atmosphere stored 'Conference' pears. This physiological disorder is characterized by internal browning of the fruit tissue and the development of cavities. Suitable protein phenol extraction/ammonium acetate-methanol precipitation and 2-DE protocols for a wide pH range were established for pear tissue. The protein expression profiles of healthy, sound (intact tissue of pears with core breakdown) and brown tissue were analyzed with the univariate non-parametric Kolmogorov-Smirnov test and multivariate statistical techniques such as principal component analysis and partial least square discriminant analysis. Both statistical approaches revealed interesting differentially expressed proteins between healthy and disordered pears. LC-ESI-MS/MS identification of differentially expressed proteins between healthy and sound tissue revealed their participation in the energy metabolism, the antioxidant system and ethylene biosynthesis. Up-regulated characteristic proteins in brown tissue were mainly involved in energy metabolism and defense mechanisms. Proteomics coupled to univariate and multivariate statistical techniques seems to be an efficient approach to get a better insight into the different mechanisms and pathways leading to the core breakdown disorder.

To our knowledge this is the first study of 2 main interstitial cell populations in the upper and deeper lamina propria of the human bladder with distinct ultrastructural and immunohistochemical phenotypes. Future research is needed to elucidate whether these morphological findings reflect different roles for upper and deeper lamina propria interstitial cells in bladder physiology.