Screening of Bacteriophage Encoded Toxic Proteins with a Next Generation Sequencing-Based Assay

Bacteriophage vB_EcoM_fHy-Eco03 (fHy-Eco03 for short) was isolated from a sewage sample based on its ability to infect an Escherichia coli clinical blood culture isolate. Altogether, 32 genes encoding hypothetical proteins of unknown function (HPUFs) were identified from the genomic sequence of fHy-Eco03. The HPUFs were screened for toxic properties (toxHPUFs) with a novel, Next Generation Sequencing (NGS)-based approach. This approach identifies toxHPUF-encoding genes through comparison of gene-specific read coverages in DNA from pooled ligation mixtures before electroporation and pooled transformants after electroporation. The performance and reliability of the NGS screening assay was compared with a plating efficiency-based method, and both methods identified the fHy-Eco03 gene g05 product as toxic. While the outcomes of the two screenings were highly similar, the NGS screening assay outperformed the plating efficiency assay in both reliability and efficiency. The NGS screening assay can be used as a high throughput method in the search for new phage-inspired antimicrobial molecules.

The clonogenic assay: robustness of plating efficiency-based analysis is strongly compromised by cellular cooperation

Background The clonogenic assay is a versatile and frequently used tool to quantify reproductive cell survival in vitro. Current state-of-the-art analysis relies on plating efficiency-based calculations which assume a linear correlation between the number of cells seeded and the number of colonies counted. The present study was designed to test the validity of this assumption and to evaluate the robustness of clonogenic survival results obtained. Methods A panel of 50 established cancer cell lines was used for comprehensive evaluation of the clonogenic assay procedure and data analysis. We assessed the performance of plating efficiency-based calculations and examined the influence of critical experimental parameters, such as cell density seeded, assay volume, incubation time, as well as the cell line-intrinsic factor of cellular cooperation by auto-/paracrine stimulation. Our findings were integrated into a novel mathematical approach for the analysis of clonogenic survival data. Results For various cell lines, clonogenic growth behavior failed to be adequately described by a constant plating efficiency, since the density of cells seeded severely influenced the extent and the dynamics of clonogenic growth. This strongly impaired the robustness of survival calculations obtained by the current state-of-the-art method using plating efficiency-based normalization. A novel mathematical approach utilizing power regression and interpolation of matched colony numbers at different irradiation doses applied to the same dataset substantially reduced the impact of cell density on survival results. Cellular cooperation was observed to be responsible for the non-linear clonogenic growth behavior of a relevant number of cell lines and the impairment of survival calculations. With 28/50 cell lines of different tumor entities showing moderate to high degrees of cellular cooperation, this phenomenon was found to be unexpectedly common. Conclusions Our study reveals that plating efficiency-based analysis of clonogenic survival data is profoundly compromised by cellular cooperation resulting in strongly underestimated assay-intrinsic errors in a relevant proportion of established cancer cell lines. This severely questions the use of plating efficiency-based calculations in studies aiming to achieve more than semiquantitative results. The novel approach presented here accounts for the phenomenon of cellular cooperation and allows the extraction of clonogenic survival results with clearly improved robustness.

The Clonogenic Assay: Robustness of Plating-Efficiency-Based Analysis is Strongly Compromised by Cellular Cooperation

Background:The clonogenic assay is a versatile and frequently used tool to quantify reproductive cell survival in vitro. Current state-of-the-art analysis relies on plating efficiency-based calculations which assume a linear correlation between the number of cells seeded and the number of colonies counted. The present study was designed to test the validity of this assumption and to evaluate the robustness of clonogenic survival results obtained.Methods:A panel of 50 established cancer cell lines was used for comprehensive evaluation of the clonogenic assay procedure and data analysis. We assessed the performance of plating efficiency-based calculations and examined the influence of critical experimental parameters, such as cell density seeded, assay volume, incubation time, as well as the cell line-intrinsic factor of cellular cooperation by auto‑/paracrine stimulation. Our findings were integrated into a novel mathematical approach for the analysis of clonogenic survival data.Results:For various cell lines, clonogenic growth behavior failed to be adequately described by a constant plating efficiency, since the density of cells seeded severely influenced the extent and the dynamics of clonogenic growth. This strongly impaired the robustness of survival calculations obtained by the current state-of-the-art method using plating efficiency-based normalization. A novel mathematical approach utilizing power regression and interpolation of matched colony numbers at different irradiation doses applied to the same dataset substantially reduced the impact of cell density on survival results. Cellular cooperation was observed to be responsible for the non-linear clonogenic growth behavior of a relevant number of cell lines and the impairment of survival calculations. With 28/50 cell lines of different tumor entities showing moderate to high degrees of cellular cooperation, this phenomenon was found to be unexpectedly common.Conclusions:Our study reveals that plating efficiency-based analysis of clonogenic survival data is profoundly compromised by cellular cooperation resulting in strongly underestimated assay-intrinsic errors in a relevant proportion of established cancer cell lines. This severely questions the use of plating efficiency-based calculations in studies aiming to achieve more than semiquantitative results. The novel approach presented here accounts for the phenomenon of cellular cooperation and allows the extraction of clonogenic survival results with clearly improved robustness.

Progress towards sugar beet improvement through somatic hybridization I. Inactivation of nuclei and cytoplasm in donor and recipient protoplasts

The isolation and culture of suspension-derived protoplasts from two sugar beet (<em>Beta vulgaris</em> L.) genotypes are described. Immobilization of protoplasts in agarose resulted in high frequency divisions and microcallus regeneration, with plating efficiency (PE) being clearly genotype-dependent. In further studies towards asymmetric fusion experiments, the effect of different doses of ultraviolet radiation (UV) and iodoacetic acid (IA) on protoplast physiology was assessed. Viability of both treated (UV, IA) and untreated protoplasts (control) was determined by FDA staining, and the biological effect was evaluated by testing the ability of protoplasts to divide and to form calli. The results are discussed in terms of the applicability of the methods for the production of asymmetric protoplasts suitable for somatic hybridization within the genus <em>Beta</em>.