A rapid multiplex cell-free assay on biochip to evaluate functional aspects of double-strand break repair.

The repair of DNA double-strand breaks (DSBs) involves interdependent molecular pathways, of which the choice is crucial for a cell's fate when facing a damage. Growing evidence points toward the fact that DSB repair capacities correlate with disease aggressiveness, treatment response and treatment-related toxicities in cancer. Scientific and medical communities need more easy-to-use and efficient tools to rapidly estimate DSB repair capacities from a tissue, enable routine-accessible treatment personalization, and hopefully, improve survival. Here, we propose a new functional biochip assay (NEXT-SPOT) that characterizes DSB repair-engaged cellular pathways and provides qualitative and quantitative information on the contribution of several pathways in less than 2 h, from 10 mg of cell lysates. We introduce the NEXT-SPOT technology, detail the molecular characterizations of different repair steps occurring on the biochip, and show examples of DSB repair profiling using three cancer cell lines treated or not with a DSB-inducer (doxorubicin) and/or a DNA repair inhibitor (RAD51 inhibitor; DNA-PK inhibitor; PARP inhibitor). Among others, we demonstrate that NEXT-SPOT can accurately detect decreased activities in strand invasion and end-joining mechanisms following DNA-PK or RAD51 inhibition in DNA-PK-proficient cell lines. This approach offers an all-in-one reliable strategy to consider DSB repair capacities as predictive biomarkers easily translatable to the clinic.

Evaluation of DNA double-strand break repair capacity in human cells: Critical overview of current functional methods.

DNA double-strand breaks (DSBs) are highly deleterious lesions, responsible for mutagenesis, chromosomal translocation or cell death. DSB repair (DSBR) is therefore a critical part of the DNA damage response (DDR) to restore molecular and genomic integrity. In humans, this process is achieved through different pathways with various outcomes. The balance between DSB repair activities varies depending on cell types, tissues or individuals. Over the years, several methods have been developed to study variations in DSBR capacity. Here, we mainly focus on functional techniques, which provide dynamic information regarding global DSB repair proficiency or the activity of specific pathways. These methods rely on two kinds of approaches. Indirect techniques, such as pulse field gel electrophoresis (PFGE), the comet assay and immunofluorescence (IF), measure DSB repair capacity by quantifying the time-dependent decrease in DSB levels after exposure to a DNA-damaging agent. On the other hand, cell-free assays and reporter-based methods directly track the repair of an artificial DNA substrate. Each approach has intrinsic advantages and limitations and despite considerable efforts, there is currently no ideal method to quantify DSBR capacity. All techniques provide different information and can be regarded as complementary, but some studies report conflicting results. Parameters such as the type of biological material, the required equipment or the cost of analysis may also limit available options. Improving currently available methods measuring DSBR capacity would be a major step forward and we present direct applications in mechanistic studies, drug development, human biomonitoring and personalized medicine, where DSBR analysis may improve the identification of patients eligible for chemo- and radiotherapy.

Associations Between Sugars Intakes and Urinary Sugars Excretion and Carbon Stable Isotope Ratios in Red Blood Cells as Biomarkers of Sugars Intake in a Predominantly Maori Population.

Determining the extent to which added sugars intake contribute to non-communicable disease in various populations is challenging because it is difficult to accurately measure intakes. Biomarkers may provide a reliable and easily measured method of assessing intakes. In a predominantly Maori population we compared various sugars intake estimates derived from a 36 item sugar-specific food frequency questionnaire (FFQ) with biomarkers of sugars intake; urinary sugars excretion in random spot collections (n = 153) and carbon stable isotope ratios (n = 36) in red blood cells (RBCs, δ13CRBC) and in the alanine fraction of the RBCs (δ13Calanine). Estimated 24 h urinary sucrose+fructose excretion was statistically significantly correlated with intakes of total sugars (r = 0.23), sucrose (r = 0.26) and added sugars from sugar-sweetened beverages (SSBs; r = 0.26). δ13Calanine was correlated with added sugars (r = 0.40). In log linear multiple regression models adjusted with HbA1C and eGFR δ13Calanine predicted added sugars intakes (r 2 = 0.29) and estimated 24 h urinary sucrose+fructose excretion predicted intakes of total sugars (r 2 = 0.14), sucrose (r 2 = 0.17), added sugars (r 2 = 0.17) and sugars from SSBs (r 2 = 0.14). These biomarkers have potential for improving assessment of sugars intake in New Zealand populations enabling monitoring of the effectiveness of sugar reduction strategies designed to reduce risk of NCDs. However, further validation is required to confirm these preliminary findings.