Engineered viral DNA polymerase with enhanced DNA amplification capacity: a proof-of-concept of isothermal amplification of damaged DNA

© The Author(s) 2020.The development of whole genome amplification (WGA) and related methods, coupled with the dramatic growth of sequencing capacities, has changed the paradigm of genomic and genetic analyses. This has led to a continual requirement of improved DNA amplification protocols and the elaboration of new tailored methods. As key elements in WGA, identification and engineering of novel, faithful and processive DNA polymerases is a driving force in the field. We have engineered the B-family DNA polymerase of virus Bam35 with a C-terminal fusion of DNA-binding motifs. The new protein, named B35-HhH, shows faithful DNA replication in the presence of magnesium or an optimised combination of magnesium and manganese divalent cofactors, which enhances the replication of damaged DNA substrates. Overall, the newly generated variant displays improved amplification performance, sensitivity, translesion synthesis and resistance to salt, which are of great interest for several applications of isothermal DNA amplification. Further, rolling-circle amplification of abasic site-containing minicircles provides a proof-of-concept for using B35-HhH for processive amplification of damaged DNA samples.This work was funded by grants from Spanish Ministry of Science, Innovation and Universities [PGC2018-093726-B-100 AEI/FEDER UE to M.S. and PGC2018-093723-A-100 to M.R.R.] and a ComFuturo Grant from Fundación General CSIC [NewPols4Biotech to M.R-R.]. C.D. O. and A. L. were holders of a PhD fellowship [FPU16/02665 and FPU15/05797, respectively] from the Spanish Ministry of Science, Innovation and Universities. An institutional grant from Fundación Ramón Areces and Banco Santander to the Centro de Biología Molecular Severo Ochoa is also acknowledged. Tis paper is dedicated to the memory of Prof. Margarita Salas, an irreplaceable researcher and mentor

Surface plasmon resonance biosensors for highly sensitive detection in real samples

In this work we summarize the main results obtained with the portable surface plasmon resonance (SPR) device developed in our group (commercialised by SENSIA, SL, Spain), highlighting its applicability for the real-time detection of extremely low concentrations of toxic pesticides in environmental water samples. In addition, we show applications in clinical diagnosis as, on the one hand, the real-time and label-free detection of DNA hybridization and single point mutations at the gene BRCA-1, related to the predisposition in women to develop an inherited breast cancer and, on the other hand, the analysis of protein biomarkers in biological samples (urine, serum) for early detection of diseases. Despite the large number of applications already proven, the SPR technology has two main drawbacks: (i) not enough sensitivity for some specific applications (where pM-fM or single-molecule detection are needed) (ii) low multiplexing capabilities. In order solve such drawbacks, we work in several alternative configurations as the Magneto-optical Surface Plasmon Resonance sensor (MOSPR) based on a combination of magnetooptical and ferromagnetic materials, to improve the SPR sensitivity, or the Localized Surface Plasmon Resonance (LSPR) based on nanostructures (nanoparticles, nanoholes,...), for higher multiplexing capabilities

Chemical sensors and biosensors in liquid environment based on microcantilevers with amplified quality factors

Póster presentado al 1st Senspol Workshop: SENSPOL European Thematic Network (EC Environmental and Climate Programma) Sensing Technologies for Contaminated Sites and Groundwater celebrado en Alcala de Henares (Madrid-España) en 2001.Peer reviewe

Femtosecond laser-controlled self-assembly of amorphous-crystalline nanogratings in silicon

8 págs.; 5 figs.; 1 tab.Self-assembly (SA) of molecular units to form regular, periodic extended structures is a powerful bottom-up technique for nanopatterning, inspired by nature. SA can be triggered in all classes of solid materials, for instance, by femtosecond laser pulses leading to the formation of laser-induced periodic surface structures (LIPSS) with a period slightly shorter than the laser wavelength. This approach, though, typically involves considerable material ablation, which leads to an unwanted increase of the surface roughness. We present a new strategy to fabricate high-precision nanograting structures in silicon, consisting of alternating amorphous and crystalline lines, with almost no material removal. The strategy can be applied to static irradiation experiments and can be extended into one and two dimensions by scanning the laser beam over the sample surface. We demonstrate that lines and areas with parallel nanofringe patterns can be written by an adequate choice of spot size, repetition rate and scan velocity, keeping a constant effective pulse number (N ) per area for a given laser wavelength. A deviation from this pulse number leads either to inhomogeneous or ablative structures. Furthermore, we demonstrate that this approach can be used with different laser systems having widely different wavelengths (1030 nm, 800 nm, 400 nm), pulse durations (370 fs, 100 fs) and repetition rates (500 kHz, 100 Hz, single pulse) and that the grating period can also be tuned by changing the angle of laser beam incidence. The grating structures can be erased by irradiation with a single nanosecond laser pulse, triggering recrystallization of the amorphous stripes. Given the large differences in electrical conductivity between the two phases, our structures could find new applications in nanoelectronics.This work has been supported by the LiNaBioFluid project of the H2020 program of the European Commission (FETOPEN- 665337) as well as by the Spanish TEC2014-52642-C2- 1-R. MG-L and JH-R acknowledge the grants respectively awarded by the Spanish Ministry of Education and the Spanish Ministry of Economy and Competiveness.Peer Reviewe

A systematic review on evidences supporting quality indicators of bilingual, plurilingual and multilingual programs in higher education

This systematic review intends to report on the strength of evidences supporting the quality indicators (predictors) attributed to higher education bilingual, plurilingual or multilingual practices and programs across four key dependent variables (outcomes) analyzed (i.e., student performance, second language proficiency, employment, and motivation and attitudes). The rapid growth of both offer and demand of this type of higher education and learning worldwide requires the implementation of high-quality evaluation strategies and techniques to measure potential causal links between interventions and results. To do so, a pre-specified systematic review protocol following the Campbell Collaboration (2015) recommendations is designed and implemented. The results suggest the urgent need to increase the primary research quality standards in this sub-discipline by reducing bias in the processes of designing, implementing and reporting research. Despite the scarcity of results sustained on statistical conclusions with the higher statistical power found in this review, specific results of the dependent variables indicate that this type of education benefits students’ performance and second language proficiency, with a higher impact on receptive skills. Although no results were obtained concerning student employment, other results point out that there is general satisfaction of participation with the programs. Finally, several recommendations on how to scale up those quality research standards in this sub-discipline are provided

Macroscopic and microscopic maturity stages. living working document for small tuna species.

Maturity ogives are usually estimated using different methods, including macroscopical and microscopical maturity data. Differences in maturity ogives estimations are found for species and by area/stock. So those differences may be a consequence of the use of different methodological techniques (criteria) or due to different spawning tactics. Taking this into account is essential to guarantee that the maturity criteria for each species are consistent across the laboratories and countries involved in stock assessment. The objective of this document is to show a large amount of detailed photos (macro and microphotographs) of the different gonad stages of Auxis rochei, Sarda sarda and Euthynnus alletteratus, for females and males, which will be an enhancement to the descriptions of maturity stages given in the maturity tables