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Explanation and example analysis pipeline used to quantitate changes in cell size and other parameters. Supplemental Figure 1: (A) Sample images of immunofluorescence of F-actin (red) and DAPI (blue) before and after IC50treatment with CX-5461. Images were taken at random at 200x magnification. Thresholding was performed to identify DAPI stained nuclei using the Mixture of Gaussian (MoG) technique to bin pixels into foreground and background classes. Cytoplasm, marked by F-actin, was segmented using the watershed technique after Sobel transformation followed by Otsu thresholding. All image segmentation and quantitation was performed by CellProfiler. (B) Summary quantitation in representative images of changes to segmented cells' parameters before and after treatment.

Purpose: A hallmark of neoplasia is increased ribosome biogenesis, and targeting this process with RNA polymerase I (Pol I) inhibitors has shown some efficacy. We examined the contribution and potential targeting of ribosomal machinery in chemotherapy-resistant and -sensitive models of ovarian cancer.Experimental Design: Pol I machinery expression was examined, and subsequently targeted with the Pol I inhibitor CX-5461, in ovarian cancer cell lines, an immortalized surface epithelial line, and patient-derived xenograft (PDX) models with and without chemotherapy. Effects on viability, Pol I occupancy of rDNA, ribosomal content, and chemosensitivity were examined.Results: In PDX models, ribosomal machinery components were increased in chemotherapy-treated tumors compared with controls. Thirteen cell lines were sensitive to CX-5461, with IC50s 25 nmol/L–2 μmol/L. Interestingly, two chemoresistant lines were 10.5- and 5.5-fold more sensitive than parental lines. CX-5461 induced DNA damage checkpoint activation and G2–M arrest with increased γH2AX staining. Chemoresistant cells had 2- to 4-fold increased rDNA Pol I occupancy and increased rRNA synthesis, despite having slower proliferation rates, whereas ribosome abundance and translational efficiency were not impaired. In five PDX models treated with CX-5461, one showed a complete response, one a 55% reduction in tumor volume, and one maintained stable disease for 45 days.Conclusions: Pol I inhibition with CX-5461 shows high activity in ovarian cancer cell lines and PDX models, with an enhanced effect on chemoresistant cells. Effects occur independent of proliferation rates or dormancy. This represents a novel therapeutic approach that may have preferential activity in chemoresistant populations. Clin Cancer Res; 23(21); 6529–40. ©2017 AACR.

Since the outbreak of the COVID-19 pandemic, widespread infections have allowed SARS-CoV-2 to evolve in human, leading to the emergence of multiple circulating variants. Some of these variants show increased resistance to vaccines, convalescent plasma, or monoclonal antibodies. In particular, mutations in the SARS-CoV-2 spike have drawn attention. To facilitate the isolation of neutralizing antibodies and the monitoring the vaccine effectiveness against these variants, we designed and produced biotin-labeled molecular probes of variant SARS-CoV-2 spikes and their subdomains, using a structure-based construct design that incorporated an N-terminal purification tag, a specific amino acid sequence for protease cleavage, the variant spike-based region of interest, and a C-terminal sequence targeted by biotin ligase. These probes could be produced by a single step using in-process biotinylation and purification. We characterized the physical properties and antigenicity of these probes, comprising the N-terminal domain (NTD), the receptor-binding domain (RBD), the RBD and subdomain 1 (RBD-SD1), and the prefusion-stabilized spike ectodomain (S2P) with sequences from SARS-CoV-2 variants of concern or of interest, including variants Alpha, Beta, Gamma, Epsilon, Iota, Kappa, Delta, Lambda, Mu, and Omicron. We functionally validated probes by using yeast expressing a panel of nine SARS-CoV-2 spike-binding antibodies and confirmed sorting capabilities of variant probes using yeast displaying libraries of plasma antibodies from COVID-19 convalescent donors. We deposited these constructs to Addgene to enable their dissemination. Overall, this study describes a matrix of SARS-CoV-2 variant molecular probes that allow for assessment of immune responses, identification of serum antibody specificity, and isolation and characterization of neutralizing antibodies.

The study of organic matter content and composition in source rocks using the methods of organic geochemistry is an important part of unconventional reservoir characterization. The aim of this work was the structural group analysis of organic matter directly in the source rock in combination with a quantitative assessment and surface distribution analysis of the rock sample by FTIR spectroscopy and FTIR microscopy. We have developed new experimental procedures for semi-quantitative assessment of the organic matter content, composition and distribution in the source rocks and applied these procedures for the study of the samples from the Bazhenov shale formation (West Siberia, Russia). The results have been verified using the data from the study of organic matter obtained by Rock-Eval pyrolysis and differential thermal analysis. The obtained results demonstrate the prospects of FTIR spectroscopy and FTIR microscopy application for non-destructive and express analysis of the chemical structure and distribution of organic matter in rocks.