SARS-CoV-2 infection of primary human lung epithelium for COVID-19 modeling and drug discovery.

Coronavirus disease 2019 (COVID-19) is the latest respiratory pandemic resulting from zoonotic transmission of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). Severe symptoms include viral pneumonia secondary to infection and inflammation of the lower respiratory tract, in some cases causing death. We developed primary human lung epithelial infection models to understand responses of proximal and distal lung epithelium to SARS-CoV-2 infection. Differentiated air-liquid interface cultures of proximal airway epithelium and 3D organoid cultures of alveolar epithelium were readily infected by SARS-CoV-2 leading to an epithelial cell-autonomous proinflammatory response. We validated the efficacy of selected candidate COVID-19 drugs confirming that Remdesivir strongly suppressed viral infection/replication. We provide a relevant platform for studying COVID-19 pathobiology and for rapid drug screening against SARS-CoV-2 and future emergent respiratory pathogens. ONE SENTENCE SUMMARY: A novel infection model of the adult human lung epithelium serves as a platform for COVID-19 studies and drug discovery.

High-coercivity FePt nanoparticle assemblies embedded in silica thin films.

The ability to process assemblies using thin film techniques in a scalable fashion would be a key to transmuting the assemblies into manufacturable devices. Here, we embed FePt nanoparticle assemblies into a silica thin film by sol-gel processing. Annealing the thin film composite at 650 degrees C transforms the chemically disordered fcc FePt phase into the fct phase, yielding magnetic coercivity values H(c)>630 mT. The positional order of the particles is retained due to the protection offered by the silica host. Such films with assemblies of high-coercivity magnetic particles are attractive for realizing new types of ultra-high-density data storage devices and magneto-composites.

A versatile computational pipeline for bacterial genome annotation improvement and comparative analysis, with Brucella as a use case.

We present a bacterial genome computational analysis pipeline, called GenVar. The pipeline, based on the program GeneWise, is designed to analyze an annotated genome and automatically identify missed gene calls and sequence variants such as genes with disrupted reading frames (split genes) and those with insertions and deletions (indels). For a given genome to be analyzed, GenVar relies on a database containing closely related genomes (such as other species or strains) as well as a few additional reference genomes. GenVar also helps identify gene disruptions probably caused by sequencing errors. We exemplify GenVar's capabilities by presenting results from the analysis of four Brucella genomes. Brucella is an important human pathogen and zoonotic agent. The analysis revealed hundreds of missed gene calls, new split genes and indels, several of which are species specific and hence provide valuable clues to the understanding of the genome basis of Brucella pathogenicity and host specificity.

PATRIC: the VBI PathoSystems Resource Integration Center.

The PathoSystems Resource Integration Center (PATRIC) is one of eight Bioinformatics Resource Centers (BRCs) funded by the National Institute of Allergy and Infection Diseases (NIAID) to create a data and analysis resource for selected NIAID priority pathogens, specifically proteobacteria of the genera Brucella, Rickettsia and Coxiella, and corona-, calici- and lyssaviruses and viruses associated with hepatitis A and E. The goal of the project is to provide a comprehensive bioinformatics resource for these pathogens, including consistently annotated genome, proteome and metabolic pathway data to facilitate research into counter-measures, including drugs, vaccines and diagnostics. The project's curation strategy has three prongs: 'breadth first' beginning with whole-genome and proteome curation using standardized protocols, a 'targeted' approach addressing the specific needs of researchers and an integrative strategy to leverage high-throughput experimental data (e.g. microarrays, proteomics) and literature. The PATRIC infrastructure consists of a relational database, analytical pipelines and a website which supports browsing, querying, data visualization and the ability to download raw and curated data in standard formats. At present, the site warehouses complete sequences for 17 bacterial and 332 viral genomes. The PATRIC website (https://patric.vbi.vt.edu) will continually grow with the addition of data, analysis and functionality over the course of the project.

Effect of nanoparticles on sessile droplet contact angle.

This paper investigates the change in contact angle of droplets of fluid containing dispersed nanoparticles (nanofluid) functionalized with thioglycolic acid molecules as a function of the concentration and size of nanoparticles, and the quality and composition of the substrate material. Bismuth telluride nanoparticles with an average size ranging from 2.5 to 10.4 nm and functionalized with thioglycolic acid groups were grown by a microemulsion method and dispersed in water. Experimental measurements of the contact angle of nanofluid droplets cast on smooth glass and silicon substrates show that the contact angle depends strongly on nanoparticle concentration. Moreover, smaller size nanoparticles lead to larger changes in contact angle at the same mass concentration. These findings contribute to understanding the role of functionalized nanoparticles in surface wettability.

Spontaneous thrombosis in mice carrying the factor V Leiden mutation.

A polymorphism in coagulation factor V, factor V Leiden (FVL), is the major known genetic risk factor for thrombosis in humans. Approximately 10% of mutation carriers experience clinically significant thrombosis in their lifetime. In a small subset of patients, thrombosis is associated with coinheritance of other prothrombotic gene mutations. However, the potential contribution of additional genetic risk factors in the majority of patients remains unknown. To gain insight into the molecular basis for the variable expressivity of FVL, mice were generated carrying the homologous mutation (R504Q [single-letter amino acid codes]) inserted into the endogenous murine Fv gene. Adult heterozygous (FvQ/+) and homozygous (FvQ/Q) mice are viable and fertile and exhibit normal survival. Compared with wild-type mice, adult FvQ/Q mice demonstrate a marked increase in spontaneous tissue fibrin deposition. No differences in fetal development or survival are observed among FvQ/Q, FvQ/+ or control littermates on the C57BL/6J genetic background. In contrast, on a mixed 129Sv-C57BL/6J genetic background, FvQ/Q mice develop disseminated intravascular thrombosis in the perinatal period, resulting in significant mortality shortly after birth. These results may explain the high degree of conservation of the R504/R506 activated protein C cleavage site within FV among mammalian species and suggest an important contribution of other genetic factors to the thrombosis associated with FVL in humans. (Blood. 2000;96:4222-4226)

Fluorescence-based detection of lacZ reporter gene expression in intact and viable bacteria including Mycobacterium species.

A variety of fluorescein di-beta-D-galactopyranoside (FDG)-based substrates were evaluated for measuring beta-galactosidase expression in bacteria. One substrate, 5-acetylamino-FDG (C2FDG), performed well in all bacteria tested, including the slow growing mycobacterium, Mycobacterium bovis BCG. The sensitivity of C2FDG in intact, viable BCG was similar to that of o-nitrophenyl-beta-D-galactopyranoside in cell lysates when used to measure lacZ reporter gene activity. C2FDG was approximately 70-fold more sensitive than green fluorescent protein (GFP) in BCG when assayed in a fluorescence plate reader, and comparable to GFP when measured by flow cytometry. These assays provide an important new alternative for the rapid measurement of reporter gene expression in viable bacteria.

Mvwf, a dominant modifier of murine von Willebrand factor, results from altered lineage-specific expression of a glycosyltransferase.

We have identified altered lineage-specific expression of an N-acetylgalactosaminyltransferase gene, Galgt2, as the gain-of-function mechanism responsible for the action of the Mvwf locus, a major modifier of plasma von Willebrand factor (VWF) level in RIIIS/J mice. A switch of Galgt2 gene expression from intestinal epithelial cell-specific to a pattern restricted to the vascular endothelial cell bed leads to aberrant posttranslational modification and rapid clearance of VWF from plasma. Transgenic expression of Galgt2 directed to vascular endothelial cells reproduces the low VWF phenotype, confirming this switch in lineage-specific gene expression as the likely molecular mechanism for Mvwf. These findings identify alterations in glycosyltransferase function as a potential general mechanism for the genetic modification of plasma protein levels.

Comparative mapping of distal murine chromosome 11 and human 17q21.3 in a region containing a modifying locus for murine plasma von Willebrand factor level.

Type 1 von Willebrand disease (VWD) is a common inherited disorder characterized by mild to moderate bleeding and reduced levels of von Willebrand factor (VWF). An animal model for human type 1 VWD, the RIIIS/J mouse strain, exhibits a prolonged bleeding time and reduced plasma VWF levels. We have previously mapped the defect in RIIIS/J to distal mouse Chr 11, distinct from the Vwf locus on Chr 6. This locus, Mvwf, was localized to an approximately 0.5-cM interval, tightly linked to Gip, distal to Ngfr, and proximal to Hoxb. We have now used these genetic markers to construct a contig of yeast and bacterial artificial chromosomes and bacteriophage P1 clones spanning the approximately 300-kb Mvwf nonrecombinant interval. In a comparative mapping approach, mouse homologues of mapped human expressed sequence tags (ESTs) were localized relative to the candidate interval. Twenty-one sequence-tagged sites and ESTs from the corresponding human syntenic region 17q21.3 were ordered using the high-resolution Stanford TNG3 radiation hybrid panel. Based on the resulting radiation hybrid map and our mouse genetic and physical maps, the order of human and mouse genes in a >0.7-cM region appears to be conserved. Six genes localized to the Mvwf nonrecombinant interval by comparative mapping included orthologs of GNGT2, ATP6N1, and a nuclear domain protein. Seven other genes or ESTs were excluded from the candidate interval, including orthologs of PHB, PDK2, a speckle-type protein, and a UDP-galactose transporter. Using exon trapping, 10 additional putative expressed sequences were identified within the Mvwf nonrecombinant interval, including a previously cloned murine glycosyltransferase as well as exons showing sequence similarity to genes for Caenorhabditis elegans and Saccharomyces cerevisiae predicted proteins, an Arabidopsis thaliana ubiquitin-conjugating enzyme, and a Gallus gallus mRNA zipcode-binding protein. Further characterization of these putative genes could identify the dominant mutation responsible for low plasma VWF levels in RIIIS/J mice. These data may also aid in the localization of other disease loci mapped to this region, including the gene for tricho-dento-osseous syndrome and a murine locus for susceptibility to ozone-induced acute lung injury.

Fatal haemorrhage and incomplete block to embryogenesis in mice lacking coagulation factor V.

Coagulation factor V is a critical cofactor for the activation of prothrombin to thrombin, the penultimate step in the generation of a fibrin blood clot. Genetic deficiency of factor V results in a congenital bleeding disorder (parahaemophilia), whereas inheritance of a mutation rendering factor V resistant to inactivation is an important risk factor for thrombosis. We report here that approximately half of homozygous embryos deficient in factor V (Fv-/-), which have been generated by gene targeting, die at embryonic day (E) 9-10, possibly as a result of an abnormality in the yolk-sac vasculature. The remaining Fv-/- mice progress normally to term, but die from massive haemorrhage within 2 hours of birth. Considered together with the milder phenotypes generally associated with deficiencies of other clotting factors, our findings demonstrate the primary role of the common coagulation pathway and the absolute requirement for functional factor V for prothrombinase activity. They also provide direct evidence for the existence of other critical haemostatic functions for thrombin in addition to fibrin clot formation, and identify a previously unrecognized role for the coagulation system in early mammalian development.