Speed matters: How subtle changes in DNA end resection rate affect repair

The contribution of BRCA1 (breast cancer 1) to the repair of broken DNA is well established, but its real role at the molecular level is less well understood. By developing a new high-resolution, single-molecule technique, we have now shown that BRCA1 accelerates the processing of DNA breaks that subsequently engage in homologous recombination.España, Ministerio de Economía y Competitividad, SAF2010-1487

Studying DNA Double-Strand Break Repair: An Ever-Growing Toolbox

To ward off against the catastrophic consequences of persistent DNA double-strand breaks (DSBs), eukaryotic cells have developed a set of complex signaling networks that detect these DNA lesions, orchestrate cell cycle checkpoints and ultimately lead to their repair. Collectively, these signaling networks comprise the DNA damage response (DDR). The current knowledge of the molecular determinants and mechanistic details of the DDR owes greatly to the continuous development of ground-breaking experimental tools that couple the controlled induction of DSBs at distinct genomic positions with assays and reporters to investigate DNA repair pathways, their impact on other DNA-templated processes and the specific contribution of the chromatin environment. In this review, we present these tools, discuss their pros and cons and illustrate their contribution to our current understanding of the DDR.European Research Council (ERC-2014-CoG 647344

The ubiquitin E3/E4 ligase, UBE4A, fine-tunes protein ubiquitylation and accumulation at sites of DNA damage facilitating double-strand break repair

Double-strand breaks (DSBs) are critical DNA lesions that robustly activate the elaborate DNA damage response (DDR) network. We identified a critical player in DDR fine-tuning - the E3/E4 ubiquitin ligase, UBE4A. UBE4A’s recruitment to sites of DNA damage is dependent on primary E3 ligases in the DDR and promotes enhancement and sustainment of K48- and K63-linked ubiquitin chains at these sites. This step is required for timely recruitment of the RAP80 and BRCA1 proteins and proper organization of RAP80- and BRCA1-associated protein complexes at DSB sites. This pathway is essential for optimal end-resection at DSBs, and its abrogation leads to up-regulation of the highly mutagenic alternative end-joining repair at the expense of error-free homologous recombination repair. Our data uncover a critical regulatory level in the DSB response and underscore the importance of fine-tuning of the complex DDR network for accurate and balanced execution of DSB repai

Data-Analytics Modeling of Electrical Impedance Measurements for Cell Culture Monitoring

High-throughput data analysis challenges in laboratory automation and lab-on-a-chip devices’ applications are continuously increasing. In cell culture monitoring, specifically, the electrical cell-substrate impedance sensing technique (ECIS), has been extensively used for a wide variety of applications. One of the main drawbacks of ECIS is the need for implementing complex electrical models to decode the electrical performance of the full system composed by the electrodes, medium, and cells. In this work we present a new approach for the analysis of data and the prediction of a specific biological parameter, the fill-factor of a cell culture, based on a polynomial regression, data-analytic model. The method was successfully applied to a specific ECIS circuit and two different cell cultures, N2A (a mouse neuroblastoma cell line) and myoblasts. The data-analytic modeling approach can be used in the decoding of electrical impedance measurements of different cell lines, provided a representative volume of data from the cell culture growth is available, sorting out the difficulties traditionally found in the implementation of electrical models. This can be of particular importance for the design of control algorithms for cell cultures in tissue engineering protocols, and labs-on-a-chip and wearable devices applicationsEspaña, Ministerio de Ciencia e Innovación y Universidades project RTI2018-093512-B-C2

MRGBP, a member of the NuA4 complex, inhibits DNA double-strand break repair

The repair of DNA breaks takes place in the context of chromatin and thus involves the activity of chromatin remodelers. The nucleosome acetyltransferase of H4 (NuA4) remodeler complex enables DNA break repair by relaxing flanking chromatin. Here, we show that MRG domain binding protein (MRGBP), a member of this complex, acts as a general inhibitor of DNA double-strand break repair. Upon its downregulation, repair is generally increased. This is particularly evident for the stimulation of early events of homologous recombination. Thus, MRGBP has an opposing role to the main catalytic subunits of the NuA4 complex. Our data suggest that MRGBP acts by limiting the activity of this complex in DNA repair, specifically by narrowing the extent of DNA-end resection.Ministerio de Economía y Competitividad SAF2016-74855-

A plethysmographic sensor for monitoring volume changes in cardiovascular pathologies

This paper presents a capacitive system capable of performing leg volume change monitorization in patients suffering from Heart Failure (HF) conditions. The body volume evolution serves as a prognosis marker for this kind of patients, such patients can benefit from a wearable monitorization system. The proposal is based on a contactless capacitive wearable structure implemented by a two-plate plane-parallel capacitor geometry. The system exhibits sensitivity to leg volume change and the sensor curves are provided. A 2.5x2.5cm capacitive electrode design will generate capacitive values within the range [1-2] pF. Acquisition of the capacitance value is performed via an electronic differentiator implemented using op-amps, illustrating good results in simulated volume analysis implemented using pspice.Ministerio de Ciencia, Innovación y Universidades RTI2018-093512-B-C2

Cell-Culture Measurements Using Voltage Oscillations

A comprehensive system for real-time monitoring of a set of cell-cultures using a Voltage Oscillation (VO) methodology is proposed. The main idea is to connect the bio-electrical elements (electrodes & cell-culture) in such a way that sequentially a suitable electrical oscillator, which only uses a DC power source, is built. Using the employed electrical models given in [1, 2], the attained oscillation parameters (frequency and amplitude) can be directly related to the biological test. A digital circuitry is developed to pick-up the experimental measurements, which are gathered and shown in real-time in a web application.Ministerio de Economía y Competitividad TEC2013-46242-C3-1-

Towards Bio-impedance Based Labs: A Review

In this article, some of the main contributions to BI (Bio-Impedance) parameter-based systems for medical, biological and industrial fields, oriented to develop micro laboratory systems are summarized. These small systems are enabled by the development of new measurement techniques and systems (labs), based on the impedance as biomarker. The electrical properties of the life mater allow the straightforward, low cost and usually non-invasive measurement methods to define its status or value, with the possibility to know its time evolution. This work proposes a review of bio-impedance based methods being employed to develop new LoC (Lab-on-a-Chips) systems, and some open problems identified as main research challenges, such as, the accuracy limits of measurements techniques, the role of the microelectrode-biological impedance modeling in measurements and system portability specifications demanded for many applications.Spanish founded Project: TEC 2013-46242-C3-1-P: Integrated Microsystem for Cell Culture AssaysFEDE

New Tools to Study DNA Double-Strand Break Repair Pathway Choice

A broken DNA molecule is difficult to repair, highly mutagenic, and extremely cytotoxic. Such breaks can be repaired by homology-independent or homology-directed mechanisms. Little is known about the network that controls the repair pathway choice except that a licensing step for homology-mediated repair exists, called DNA-end resection. The choice between these two repair pathways is a key event for genomic stability maintenance, and an imbalance of the ratio is directly linked with human diseases, including cancer. Here we present novel reporters to study the balance between both repair options in human cells. In these systems, a double-strand break can be alternatively repaired by homology-independent or -dependent mechanisms, leading to the accumulation of distinct fluorescent proteins. These reporters thus allow the balance between both repair pathways to be analyzed in different experimental setups. We validated the reporters by analyzing the effect of protein downregulation of the DNA end resection and non-homologous end-joining pathways. Finally, we analyzed the role of the DNA damage response on double-strand break (DSB) repair mechanism selection. Our reporters could be used in the future to understand the roles of specific factors, whole pathways, or drugs in DSB repair pathway choice, or for genome-wide screening. Moreover, our findings can be applied to increase gene-targeting efficiency, making it a beneficial tool for a broad audience in the biological sciences. © 2013 Gomez-Cabello et al.This work has been funded by a R+D+I grant from the Spanish Ministry of Economy and Competitivity (SAF2010-14877) and an European Research Council (ERC) Starting Grant (DSBRECA).Peer Reviewe

New tools to study DNA double-strand break repair pathway choice

A broken DNA molecule is difficult to repair, highly mutagenic, and extremely cytotoxic. Such breaks can be repaired by homology-independent or homology-directed mechanisms. Little is known about the network that controls the repair pathway choice except that a licensing step for homology-mediated repair exists, called DNA-end resection. The choice between these two repair pathways is a key event for genomic stability maintenance, and an imbalance of the ratio is directly linked with human diseases, including cancer. Here we present novel reporters to study the balance between both repair options in human cells. In these systems, a double-strand break can be alternatively repaired by homology-independent or -dependent mechanisms, leading to the accumulation of distinct fluorescent proteins. These reporters thus allow the balance between both repair pathways to be analyzed in different experimental setups. We validated the reporters by analyzing the effect of protein downregulation of the DNA end resection and non-homologous end-joining pathways. Finally, we analyzed the role of the DNA damage response on double-strand break (DSB) repair mechanism selection. Our reporters could be used in the future to understand the roles of specific factors, whole pathways, or drugs in DSB repair pathway choice, or for genome-wide screening. Moreover, our findings can be applied to increase gene-targeting efficiency, making it a beneficial tool for a broad audience in the biological sciences