Toxicologic Pathology Forum: A Roadmap for Building State-of-the-Art Digital Image Data Resources for Toxicologic Pathology in the Pharmaceutical Industry.

Digitization of histologic slides brings with it the promise of enhanced toxicologic pathology practice through the increased application of computational methods. However, the development of these advanced methods requires access to substrate image data, that is, whole slide images (WSIs). Deep learning methods, in particular, rely on extensive training data to develop robust algorithms. As a result, pharmaceutical companies interested in leveraging computational methods in their digital pathology workflows must first invest in data infrastructure to enable data access for both data scientists and pathologists. The process of building robust image data resources is challenging and includes considerations of generation, curation, and storage of WSI files, and WSI access including via linked metadata. This opinion piece describes the collective experience of building resources for WSI data in the Roche group. We elaborate on the challenges encountered and solutions developed with the goal of providing examples of how to build a data resource for digital pathology analytics in the pharmaceutical industry.

Host-derived fatty acids activate type VII secretion in Staphylococcus aureus.

The type VII secretion system (T7SS) of Staphylococcus aureus is a multiprotein complex dedicated to the export of several virulence factors during host infection. This virulence pathway plays a key role in promoting bacterial survival and the long-term persistence of staphylococcal abscess communities. The expression of the T7SS is activated by bacterial interaction with host tissues including blood serum, nasal secretions, and pulmonary surfactant. In this work we identify the major stimulatory factors as host-specific cis-unsaturated fatty acids. Increased T7SS expression requires host fatty acid incorporation into bacterial biosynthetic pathways by the Saureus fatty acid kinase (FAK) complex, and FakA is required for virulence. The incorporated cis-unsaturated fatty acids decrease Saureus membrane fluidity, and these altered membrane dynamics are partially responsible for T7SS activation. These data define a molecular mechanism by which Saureus cells sense the host environment and implement appropriate virulence pathways.

Reversible antibiotic tolerance induced in Staphylococcus aureus by concurrent drug exposure.

UNLABELLED: Resistance of Staphylococcus aureus to beta-lactam antibiotics has led to increasing use of the glycopeptide antibiotic vancomycin as a life-saving treatment for major S. aureus infections. Coinfection by an unrelated bacterial species may necessitate concurrent treatment with a second antibiotic that targets the coinfecting pathogen. While investigating factors that affect bacterial antibiotic sensitivity, we discovered that susceptibility of S. aureus to vancomycin is reduced by concurrent exposure to colistin, a cationic peptide antimicrobial employed to treat infections by Gram-negative pathogens. We show that colistin-induced vancomycin tolerance persists only as long as the inducer is present and is accompanied by gene expression changes similar to those resulting from mutations that produce stably inherited reduction of vancomycin sensitivity (vancomycin-intermediate S. aureus [VISA] strains). As colistin-induced vancomycin tolerance is reversible, it may not be detected by routine sensitivity testing and may be responsible for treatment failure at vancomycin doses expected to be clinically effective based on such routine testing. IMPORTANCE: Commonly, antibiotic resistance is associated with permanent genetic changes, such as point mutations or acquisition of resistance genes. We show that phenotypic resistance can arise where changes in gene expression result in tolerance to an antibiotic without any accompanying genetic changes. Specifically, methicillin-resistant Staphylococcus aureus (MRSA) behaves like vancomycin-intermediate S. aureus (VISA) upon exposure to colistin, which is currently used against infections by Gram-negative bacteria. Vancomycin is a last-resort drug for treatment of serious S. aureus infections, and VISA is associated with poor clinical prognosis. Phenotypic and reversible resistance will not be revealed by standard susceptibility testing and may underlie treatment failure.

Vaccine candidate P6 of nontypable Haemophilus influenzae is not a transmembrane protein based on protein structural analysis.

P6 has been a vaccine candidate for nontypable Haemophilus influenzae (NTHi) based on its location on the outer membrane and immunogenicity. Because P6 is attached to the inner peptidoglycan layer of NTHi, and is putatively surface exposed, it must be a transmembrane protein. We examined the P6 structure using computational modeling, site-directed mutagenesis, and nuclear magnetic resonance spectroscopy. We found that P6 cannot be a transmembrane protein, and therefore may not be surface exposed. We conclude that there may be another protein on the surface of NTHi that has epitopes similar if not identical to P6.

The peculiarities of large intron splicing in animals.

In mammals a considerable 92% of genes contain introns, with hundreds and hundreds of these introns reaching the incredible size of over 50,000 nucleotides. These "large introns" must be spliced out of the pre-mRNA in a timely fashion, which involves bringing together distant 5' and 3' acceptor and donor splice sites. In invertebrates, especially Drosophila, it has been shown that larger introns can be spliced efficiently through a process known as recursive splicing-a consecutive splicing from the 5'-end at a series of combined donor-acceptor splice sites called RP-sites. Using a computational analysis of the genomic sequences, we show that vertebrates lack the proper enrichment of RP-sites in their large introns, and, therefore, require some other method to aid splicing. We analyzed over 15,000 non-redundant, large introns from six mammals, 1,600 from chicken and zebrafish, and 560 non-redundant large introns from five invertebrates. Our bioinformatic investigation demonstrates that, unlike the studied invertebrates, the studied vertebrate genomes contain consistently abundant amounts of direct and complementary strand interspersed repetitive elements (mainly SINEs and LINEs) that may form stems with each other in large introns. This examination showed that predicted stems are indeed abundant and stable in the large introns of mammals. We hypothesize that such stems with long loops within large introns allow intron splice sites to find each other more quickly by folding the intronic RNA upon itself at smaller intervals and, thus, reducing the distance between donor and acceptor sites.