Evaluation of antibody-based single cell type imaging techniques coupled to multiplexed imaging of N-glycans and collagen peptides by matrix-assisted laser desorption/ionization mass spectrometry imaging.

The integration of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) with single cell spatial omics methods allows for a comprehensive investigation of single cell spatial information and matrisomal N-glycan and extracellular matrix protein imaging. Here, the performance of the antibody-directed single cell workflows coupled with MALDI-MSI are evaluated. Miralys™ photocleavable mass-tagged antibody probes (MALDI-IHC, AmberGen, Inc.), GeoMx DSP® (NanoString, Inc.), and Imaging Mass Cytometry (IMC, Standard BioTools Inc.) were used in series with MALDI-MSI of N-glycans and extracellular matrix peptides on formalin-fixed paraffin-embedded tissues. Single cell omics protocols were performed before and after MALDI-MSI. The data suggests that for each modality combination, there is an optimal order for performing both techniques on the same tissue section. An overall conclusion is that MALDI-MSI studies may be completed on the same tissue section as used for antibody-directed single cell modalities. This work increases access to combined cellular and extracellular information within the tissue microenvironment to enhance research on the pathological origins of disease.

The consequences of tetraploidy on Caenorhabditis elegans physiology and sensitivity to chemotherapeutics.

Polyploid cells contain more than two copies of each chromosome. Polyploidy has important roles in development, evolution, and tissue regeneration/repair, and can arise as a programmed polyploidization event or be triggered by stress. Cancer cells are often polyploid. C. elegans nematodes are typically diploid, but stressors such as heat shock and starvation can trigger the production of tetraploid offspring. In this study, we utilized a recently published protocol to generate stable tetraploid strains of C. elegans and compared their physiological traits and sensitivity to two DNA-damaging chemotherapeutic drugs, cisplatin and doxorubicin. As prior studies have shown, tetraploid worms are approximately 30% longer, shorter-lived, and have a smaller brood size than diploids. We investigated the reproductive defect further, determining that tetraploid worms have a shorter overall germline length, a higher rate of germ cell apoptosis, more aneuploidy in oocytes and offspring, and larger oocytes and embryos. We also found that tetraploid worms are modestly protected from growth delay from the chemotherapeutics but are similarly or more sensitive to reproductive toxicity. Transcriptomic analysis revealed differentially expressed pathways that may contribute to sensitivity to stress. This study reveals phenotypic consequences of whole-animal tetraploidy that make C. elegans an excellent model for ploidy differences.

The consequences of tetraploidy on Caenorhabditis elegans physiology and sensitivity to chemotherapeutics.

Polyploid cells contain more than two copies of each chromosome. Polyploidy has important roles in development, evolution, and tissue regeneration/repair, and can arise as a programmed polyploidization event or be triggered by stress. Cancer cells are often polyploid. C. elegans nematodes are typically diploid, but stressors such as heat shock and starvation can trigger the production of tetraploid offspring. In this study, we utilized a recently published protocol to generate stable tetraploid strains of C. elegans and compared their physiological traits and sensitivity to two DNA-damaging chemotherapeutic drugs, cisplatin and doxorubicin. As prior studies have shown, tetraploid worms are approximately 30% longer, shorter-lived, and have a smaller brood size than diploids. We investigated the reproductive defect further, determining that tetraploid worms have a shorter overall germline length, a higher rate of germ cell apoptosis, more aneuploidy in oocytes and offspring, and larger oocytes and embryos. We also found that tetraploid worms are modestly protected from growth delay from the chemotherapeutics but are similarly or more sensitive to reproductive toxicity. Transcriptomic analysis revealed differentially expressed pathways that may contribute to sensitivity to stress. Overall, this study reveals the phenotypic consequences of whole-animal tetraploidy in C. elegans.

Spatial metabolomics reveals glycogen as an actionable target for pulmonary fibrosis.

Matrix assisted laser desorption/ionization imaging has greatly improved our understanding of spatial biology, however a robust bioinformatic pipeline for data analysis is lacking. Here, we demonstrate the application of high-dimensionality reduction/spatial clustering and histopathological annotation of matrix assisted laser desorption/ionization imaging datasets to assess tissue metabolic heterogeneity in human lung diseases. Using metabolic features identified from this pipeline, we hypothesize that metabolic channeling between glycogen and N-linked glycans is a critical metabolic process favoring pulmonary fibrosis progression. To test our hypothesis, we induced pulmonary fibrosis in two different mouse models with lysosomal glycogen utilization deficiency. Both mouse models displayed blunted N-linked glycan levels and nearly 90% reduction in endpoint fibrosis when compared to WT animals. Collectively, we provide conclusive evidence that lysosomal utilization of glycogen is required for pulmonary fibrosis progression. In summary, our study provides a roadmap to leverage spatial metabolomics to understand foundational biology in pulmonary diseases.

SARS-CoV-2 Sequencing for Variant Surveillance.

In this chapter, next-generation sequencing of the entire viral genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is described. Successful sequencing of the SARS-CoV-2 virus is dependent upon quality of the specimen, adequate coverage of the entire genome, and up-to-date annotation. Some of the advantages of performing SARS-CoV-2 surveillance using next-generation sequencing are scalability, high-throughput, cost, and full genome analysis. Some of the disadvantages can be expensive instrumentation, large upfront reagent and supply costs, increased time-to-result, computational needs, and complicated bioinformatics. This chapter will provide an overview of a modified FDA Emergency Use Authorization procedure for the genomic sequencing of SARS-CoV-2. The procedure is also referred to as the research use only (RUO) version.

Cellular and Molecular Mechanisms of MEK1 Inhibitor-Induced Cardiotoxicity.

Background: Trametinib is a MEK1 (mitogen-activated extracellular signal-related kinase kinase 1) inhibitor used in the treatment of BRAF (rapid accelerated fibrosarcoma B-type)-mutated metastatic melanoma. Roughly 11% of patients develop cardiomyopathy following long-term trametinib exposure. Although described clinically, the molecular landscape of trametinib cardiotoxicity has not been characterized. Objectives: The aim of this study was to test the hypothesis that trametinib promotes widespread transcriptomic and cellular changes consistent with oxidative stress and impairs cardiac function. Methods: Mice were treated with trametinib (1 mg/kg/d). Echocardiography was performed pre- and post-treatment. Gross, histopathologic, and biochemical assessments were performed to probe for molecular and cellular changes. Human cardiac organoids were used as an in vitro measurement of cardiotoxicity and recovery. Results: Long-term administration of trametinib was associated with significant reductions in survival and left ventricular ejection fraction. Histologic analyses of the heart revealed myocardial vacuolization and calcification in 28% of animals. Bulk RNA sequencing identified 435 differentially expressed genes and 116 differential signaling pathways following trametinib treatment. Upstream gene analysis predicted interleukin-6 as a regulator of 17 relevant differentially expressed genes, suggestive of PI3K/AKT and JAK/STAT activation, which was subsequently validated. Trametinib hearts displayed elevated markers of oxidative stress, myofibrillar degeneration, an 11-fold down-regulation of the apelin receptor, and connexin-43 mislocalization. To confirm the direct cardiotoxic effects of trametinib, human cardiac organoids were treated for 6 days, followed by a 6-day media-only recovery. Trametinib-treated organoids exhibited reductions in diameter and contractility, followed by partial recovery with removal of treatment. Conclusions: These data describe pathologic changes observed in trametinib cardiotoxicity, supporting the exploration of drug holidays and alternative pharmacologic strategies for disease prevention.

Application of Pharmacokinetic Prediction Platforms in the Design of Optimized Anti-Cancer Drugs.

Cancer is the second most common cause of death in the United States, accounting for 602,350 deaths in 2020. Cancer-related death rates have declined by 27% over the past two decades, partially due to the identification of novel anti-cancer drugs. Despite improvements in cancer treatment, newly approved oncology drugs are associated with increased toxicity risk. These toxicities may be mitigated by pharmacokinetic optimization and reductions in off-target interactions. As such, there is a need for early-stage implementation of pharmacokinetic (PK) prediction tools. Several PK prediction platforms exist, including pkCSM, SuperCypsPred, Pred-hERG, Similarity Ensemble Approach (SEA), and SwissADME. These tools can be used in screening hits, allowing for the selection of compounds were reduced toxicity and/or risk of attrition. In this short commentary, we used PK prediction tools in the optimization of mitogen activated extracellular signal-related kinase kinase 1 (MEK1) inhibitors. In doing so, we identified MEK1 inhibitors with retained activity and optimized predictive PK properties, devoid of hERG inhibition. These data support the use of publicly available PK prediction platforms in early-stage drug discovery to design safer drugs.

Comparative analysis of antibodies to SARS-CoV-2 between asymptomatic and convalescent patients.

The SARS-CoV-2 viral pandemic has induced a global health crisis, which requires more in-depth investigation into immunological responses to develop effective treatments and vaccines. To understand protective immunity against COVID-19, we screened over 60,000 asymptomatic individuals in the Southeastern United States for IgG antibody positivity against the viral Spike protein, and approximately 3% were positive. Of these 3%, individuals with the highest anti-S or anti-RBD IgG level showed a strong correlation with inhibition of ACE2 binding and cross-reactivity against non-SARS-CoV-2 coronavirus S-proteins. We also analyzed samples from 94 SARS-CoV-2 patients and compared them with those of asymptomatic individuals. SARS-CoV-2 symptomatic patients had decreased antibody responses, ACE2 binding inhibition, and antibody cross-reactivity. Our study shows that healthy individuals can mount robust immune responses against SARS-CoV-2 without symptoms. Furthermore, IgG antibody responses against S and RBD may correlate with high inhibition of ACE2 binding in individuals tested for SARS-CoV-2 infection or post vaccination.