All-in-one construct for genome engineering using Cre-lox technology

Mycoplasma genitalium is an appealing model of a minimal cell and synthetic biology study, and it was one of the first organisms whose genome was fully sequenced and chemically synthesized. Despite its usefulness as a model organism, many genetic tools well established for other microorganisms are not currently available in mycoplasmas. We have developed several vectors to adapt the Cre-lox technology for genome engineering in M. genitalium, providing an all-in-one construct that could be also useful to obtain unmarked genetic modifications in many other slow growing microorganisms. This construct contains a modified promoter sequence based in TetR system that exhibits an enhanced control on Cre recombinase expression, virtually abolishing the presence of this recombinase in the absence of inducer. This allows to introduce the Cre recombinase gene and the desired genetic modification in a single transformation step. In addition, this inducible promoter may be a very promising tool for a wide range of molecular applications

Maternal behaviour and welfare of the domestic and wild rabbit doe and its litter

The European rabbit (Oryctolagus cuniculus), in addition to its faunal interest in the western Mediterranean, is a relevant species which in several European countries is the basis of a meat-oriented, industrial livestock subsector, while in many developing countries rabbits are raised under alternative systems aimed at income integration and food security. In addition to meat production, other productive orientations exist that generate a variety of rabbit production systems. This paper reviews the ethology of maternal behaviour of the breeding doe and her litter, including its endocrine regulation, both in wild animal and in industrial and alternative farming systems, and its relation to management factors, productivity and performance as well as the welfare of the species. It also discusses the implications of the regulations concerning animal welfare on housing, management and satisfaction of behavioural needs of breeding does and their litters, which in some countries tend to provide more space and environmental enrichment in cages

Development of in vitro systems to study IFN signalling in gilthead seabream (Sparus aurata)

Type I interferon (IFN I) triggers specific signalling pathways leading to the activation of the innate immune defence of vertebrates against viral infections. In contrats, type II IFN (IFN II) is generally accepted to be part of the adaptive response. Among IFN I-stimulated genes, those coding the Mx proteins play a main role due to the direct antiviral activity of these proteins. The study of Mx genes in gilthead seabream, one of the most important species in the Mediterranean aquaculture, is especially interesting, as this species displays a high natural resistance to viral diseases, and behaves as asymptomatic carrier and/or reservoir of several viruses, such as viral nervous necrosis virus (VNNV), infectious pancreatic necrosis virus (IPNV), and viral haemorrhagic septicaemia virus (VHSV), which are pathogenic to other fish species. Three Mx genes (Mx1, Mx2, and Mx3) have been identified in S. aurata, showing the three proteins a wide spectrum of antiviral activity. The structure of the three promoters (pMx1, pMx2 and pMx3) has been disclosed, and their response to IFN I, IPNV and VHSV indicated a clear induction of the three promoters, with some differences in the kinetics and magnitude of the response. Several studies evidenced the important role of Mx transcription regulation on virus-host interaction: i) Mx promoters can respond to both IFN I and IFN II, thus Mx might be the link between innate and adaptive immunity; ii) Mx activation is blocked by several viruses, thus Mx transcription is the target of their IFN I antagonistic activity; and iii) A fish cell line modified with the promoter of a fish Mx gene was used to measure viraemia in serum with high sensitivity. Therefore, assessing the regulatory mechanisms controlling the transcription of fish Mx genes could significantly contribute to both, understanding virus-host interactions, and designing strategies to control viral infections. In our case, this approach can also give light to understand the successful antiviral strategies developed by gilthead seabream in nature. Thus, the purpose of the present work was to develop three stable transgenic cell lines expressing the firefly luciferase gene under the control of the gilthead seabream Mx promoters. These in vitro systems were established and their response to poly I:C, and to two viral infections was characterized. In the case of IPNV, a clear antagonistic activity was observed for pMx2, as the activity of the promoter was 78.53% lower, however, this effect was not observed for pMx1 and pMx3. When cells were infected with VHSV, no changes in the promoters’ activity were detected, thus indicating that seabream Mx promoters are not targeted by VHSV antagonistic activity. These results confirm the specificity of the interactions between each virus/promoter combination, and support the use of the three cell lines developed as useful tools to characterize virus-host interactions in this species. Further studies aimed at the identification of the molecular mechanisms behind our observations will allow us to get more insight into this complex system.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Beta cell functionality and hepatic insulin resistance are major contributors to type 2 diabetes remission and starting pharmacological therapy: from CORDIOPREV randomized controlled trial

In order to assess whether previous hepatic IR (Hepatic-IR fasting) and beta-cell functionality could modulate type 2 diabetes remission and the need for starting glucose- lowering treatment, newly-diagnosed type 2 diabetes participants who had never received glucose-lowering treatment (190 out of 1002) from the CORonary Diet Intervention with Olive oil and cardiovascular PREVention study (a prospective, randomized and controlled clinical trial), were randomized to consume a Mediterranean or a low-fat diet. Type 2 diabetes remission was defined according to the American Diabetes Association recommendation for levels of HbA1c, fasting plasma glucose and 2h plasma glucose after oral glucose tolerance test, and having maintained them for at least 2 consecutive years. Patients were classified according to the median of Hepatic-IR fasting and beta-cell functionality, measured as the disposition index (DI) at baseline. Cox proportional hazards regression determined the potential for Hepatic-IR fasting and DI indexes as predictors of diabetes remission and the probability of starting pharmacological treatment after a 5-year follow-up. Low-Hepatic-IR fasting or high-DI patients had a higher probability of diabetes remission than high-Hepatic-IR fasting or low-DI subjects (HR:1.79; 95% CI 1.06_3.05; and HR:2.66; 95% CI 1.60_4.43, respectively) after a dietary intervention with no pharmacological treatment and no weight loss. The combination of low- Hepatic-IR fasting and high-DI presented the highest probability of remission (HR:4.63; 95% CI 2.00_10.70). Among patients maintaining diabetes, those with high- Hepatic-IR fasting and low-DI showed the highest risk of starting glucose-lowerin

Differential induction of the gilthead seabream Mx1, Mx2 and Mx3 promoters by IPNV and VHSV

Type I interferon (IFN I) system triggers specific signalling pathways leading to the activation of the innate immune defence of vertebrates against viral infections. The complex expression regulation of Interferon Stimulated Genes (ISGs) is responsible for the control of the IFN I response. Hence, one of the key issues in understanding virus-host interactions relies on the knowledge of the regulatory mechanisms governing ISGs expression. Among ISGs, the Mx proteins play a main role due to their direct antiviral activity. The study of Mx genes in the farmed fish gilthead seabream is especially interesting, since this species displays an unusually high natural resistance to viral diseases, and behaves as an asymptomatic carrier and/or reservoir of several viruses, such as infectious pancreatic necrosis virus (IPNV) and viral haemorrhagic septicaemia virus (VHSV), pathogenic to other fish species. Three independent Mx genes (Mx1, Mx2, and Mx3) have been identified in this species, showing the three proteins a wide spectrum of antiviral activity. The structure of the three promoters (pMx1, pMx2 and pMx3) has been disclosed, and their response to poly I:C characterized in RTG-2 cells, where a clear induction of the three promoters, although with some differences in the kinetics and magnitude of the response, was observed. To further analyse these promoters, the response of pMx1, pMx2 and pMx3 to two viral infections has been evaluated in the present study. For that purpose, RTG-2 cells were transiently transfected with plasmids containing each promoter driving the luciferase gene, and subsequently inoculated with either IPNV or VHSV. Although the three promoters were induced by IPNV and VHSV, several differences were recorded. In general, the response was stronger in cells inoculated with VHSV compared to IPNV-inoculated cells, and the fold induction was higher for pMx2. These results highlight the specific regulation that controls the activity of each promoter, and support the idea that a complex interaction between host cells, specific Mx promoters, and viruses, is the responsible of the final outcome of a viral infection, in terms of Mx induction. The authors want to thank Dr. C. P. Dopazo (University of Santiago de Compostela, Spain) for supplying the VHSV isolate used in this work.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Gentamicin sulphate permeation through porcine intestinal epithelial cell monolayer

Gentamicin is an aminoglycoside antibiotic widely used in combination with dimethyl sulphoxide (DMSO) in topical drug formulations. It is not known, however, whether DMSO can enhance the permeation of gentamicin through biological membranes, leading to oto- and nephrotoxic side effects. A simple and reliable high-performance liquid chromatographic (HPLC) method was applied for the quantitative determination of gentamicin collected from the apical and basolateral compartments of the porcine intestinal epithelial cell line IPEC-J2 cell monolayer using fluorometric derivatisation of the analyte with fluorenylmethyloxycarbonyl chloride (FMOC) prior to chromatographic run in the presence and absence of 1% DMSO. The lack of change in transepithelial electrical resistance (TER) demonstrated that gentamicin and 1% DMSO did not affect IPEC-J2 cell monolayer integrity via the disruption of cell membranes. Chromatographic data also ascertained that gentamicin penetration across the cell monolayer even in the presence of 1% DMSO was negligible at 6 h after the beginning of apical gentamicin administration. This study further indicates that the addition of this organic solvent does not increase the incidence of toxic effects related to gentamicin permeation

Consens interdisciplinari sobre l’abordatge de la persona amb malaltia renal crònica avançada: pla operatiu de la malaltia renal crònica

Malalts crònics; Malaltia renal crònica; AbordatgeEnfermos crónicos; Enfermedad renal crónica; AbordajeChronically ill; Chronic kidney disease; ApproachEl present consens té per voluntat millorar l’atenció en aquesta fase de l’MRC, donar eines als professionals de cara a la valoració preventiva prèvia a la decisió del tractament que cal seguir en la fase d’MRCA i l’homogeneïtzació de l’atenció específica a partir de la decisió d’instaurar un tractament conservador aprofitant les eines establertes al Departament de Salut per a l’atenció a les persones amb malalties cròniques avançades (MACA)

A Novel Circulating MicroRNA for the Detection of Acute Myocarditis.

The diagnosis of acute myocarditis typically requires either endomyocardial biopsy (which is invasive) or cardiovascular magnetic resonance imaging (which is not universally available). Additional approaches to diagnosis are desirable. We sought to identify a novel microRNA for the diagnosis of acute myocarditis. To identify a microRNA specific for myocarditis, we performed microRNA microarray analyses and quantitative polymerase-chain-reaction (qPCR) assays in sorted CD4+ T cells and type 17 helper T (Th17) cells after inducing experimental autoimmune myocarditis or myocardial infarction in mice. We also performed qPCR in samples from coxsackievirus-induced myocarditis in mice. We then identified the human homologue for this microRNA and compared its expression in plasma obtained from patients with acute myocarditis with the expression in various controls. We confirmed that Th17 cells, which are characterized by the production of interleukin-17, are a characteristic feature of myocardial injury in the acute phase of myocarditis. The microRNA mmu-miR-721 was synthesized by Th17 cells and was present in the plasma of mice with acute autoimmune or viral myocarditis but not in those with acute myocardial infarction. The human homologue, designated hsa-miR-Chr8:96, was identified in four independent cohorts of patients with myocarditis. The area under the receiver-operating-characteristic curve for this novel microRNA for distinguishing patients with acute myocarditis from those with myocardial infarction was 0.927 (95% confidence interval, 0.879 to 0.975). The microRNA retained its diagnostic value in models after adjustment for age, sex, ejection fraction, and serum troponin level. After identifying a novel microRNA in mice and humans with myocarditis, we found that the human homologue (hsa-miR-Chr8:96) could be used to distinguish patients with myocarditis from those with myocardial infarction. (Funded by the Spanish Ministry of Science and Innovation and others.).Supported by a grant (PI19/00545, to Dr. Martín) from the Ministry of Science and Innovation through the Carlos III Institute of Health–Fondo de Investigación Sanitaria; by a grant from the Biomedical Research Networking Center on Cardiovascular Diseases (to Drs. Martín, Sánchez-Madrid, and Ibáñez); by grants (S2017/BMD-3671-INFLAMUNE-CM, to Drs. Martín and Sánchez-Madrid; and S2017/BMD-3867-RENIM-CM, to Dr. Ibáñez) from Comunidad de Madrid; by a grant (20152330 31, to Drs. Martín, Sánchez-Madrid, and Alfonso) from Fundació La Marató de TV3; by grants (ERC-2011-AdG 294340-GENTRIS, to Dr. Sánchez-Madrid; and ERC-2018-CoG 819775-MATRIX, to Dr. Ibáñez) from the European Research Council; by grants (SAF2017-82886R, to Dr. Sánchez-Madrid; RETOS2019-107332RB-I00, to Dr. Ibáñez; and SAF2017-90604-REDT-NurCaMeIn and RTI2018-095928-BI00, to Dr. Ricote) from the Ministry of Science and Innovation; by Fondo Europeo de Desarrollo Regional (FEDER); and by a 2016 Leonardo Grant for Researchers and Cultural Creators from the BBVA Foundation to Dr. Martín. The National Center for Cardiovascular Research (CNIC) is supported by the Carlos III Institute of Health, the Ministry of Science and Innovation, the Pro CNIC Foundation, and by a Severo Ochoa Center of Excellence grant (SEV-2015-0505). Mr. Blanco-Domínguez is supported by a grant (FPU16/02780) from the Formación de Profesorado Universitario program of the Spanish Ministry of Education, Culture, and Sports. Ms. Linillos-Pradillo is supported by a fellowship (PEJD-2016/BMD-2789) from Fondo de Garantía de Empleo Juvenil de Comunidad de Madrid. Dr. Relaño is supported by a grant (BES-2015-072625) from Contratos Predoctorales Severo Ochoa para la Formación de Doctores of the Ministry of Economy and Competitiveness. Dr. Alonso-Herranz is supported by a fellowship from La Caixa–CNIC. Dr. Caforio is supported by Budget Integrato per la Ricerca dei Dipartimenti BIRD-2019 from Università di Padova. Dr. Das is supported by grants (UG3 TR002878 and R35 HL150807) from the National Institutes of Health and the American Heart Association through its Strategically Focused Research Networks.S

Toxicogenomic and Phenotypic Analyses of Bisphenol-A Early-Life Exposure Toxicity in Zebrafish

Bisphenol-A is an important environmental contaminant due to the increased early-life exposure that may pose significant health-risks to various organisms including humans. This study aimed to use zebrafish as a toxicogenomic model to capture transcriptomic and phenotypic changes for inference of signaling pathways, biological processes, physiological systems and identify potential biomarker genes that are affected by early-life exposure to bisphenol-A. Phenotypic analysis using wild-type zebrafish larvae revealed BPA early-life exposure toxicity caused cardiac edema, cranio-facial abnormality, failure of swimbladder inflation and poor tactile response. Fluorescent imaging analysis using three transgenic lines revealed suppressed neuron branching from the spinal cord, abnormal development of neuromast cells, and suppressed vascularization in the abdominal region. Using knowledge-based data mining algorithms, transcriptome analysis suggests that several signaling pathways involving ephrin receptor, clathrin-mediated endocytosis, synaptic long-term potentiation, axonal guidance, vascular endothelial growth factor, integrin and tight junction were deregulated. Physiological systems with related disorders associated with the nervous, cardiovascular, skeletal-muscular, blood and reproductive systems were implicated, hence corroborated with the phenotypic analysis. Further analysis identified a common set of BPA-targeted genes and revealed a plausible mechanism involving disruption of endocrine-regulated genes and processes in known susceptible tissue-organs. The expression of 28 genes were validated in a separate experiment using quantitative real-time PCR and 6 genes, ncl1, apoeb, mdm1, mycl1b, sp4, U1SNRNPBP homolog, were found to be sensitive and robust biomarkers for BPA early-life exposure toxicity. The susceptibility of sp4 to BPA perturbation suggests its role in altering brain development, function and subsequently behavior observed in laboratory animals exposed to BPA during early life, which is a health-risk concern of early life exposure in humans. The present study further established zebrafish as a model for toxicogenomic inference of early-life chemical exposure toxicity

The SuperCam Instrument Suite on the Mars 2020 Rover: Science Objectives and Mast-Unit Description

On the NASA 2020 rover mission to Jezero crater, the remote determination of the texture, mineralogy and chemistry of rocks is essential to quickly and thoroughly characterize an area and to optimize the selection of samples for return to Earth. As part of the Perseverance payload, SuperCam is a suite of five techniques that provide critical and complementary observations via Laser-Induced Breakdown Spectroscopy (LIBS), Time-Resolved Raman and Luminescence (TRR/L), visible and near-infrared spectroscopy (VISIR), high-resolution color imaging (RMI), and acoustic recording (MIC). SuperCam operates at remote distances, primarily 2-7 m, while providing data at sub-mm to mm scales. We report on SuperCam's science objectives in the context of the Mars 2020 mission goals and ways the different techniques can address these questions. The instrument is made up of three separate subsystems: the Mast Unit is designed and built in France; the Body Unit is provided by the United States; the calibration target holder is contributed by Spain, and the targets themselves by the entire science team. This publication focuses on the design, development, and tests of the Mast Unit; companion papers describe the other units. The goal of this work is to provide an understanding of the technical choices made, the constraints that were imposed, and ultimately the validated performance of the flight model as it leaves Earth, and it will serve as the foundation for Mars operations and future processing of the data.In France was provided by the Centre National d'Etudes Spatiales (CNES). Human resources were provided in part by the Centre National de la Recherche Scientifique (CNRS) and universities. Funding was provided in the US by NASA's Mars Exploration Program. Some funding of data analyses at Los Alamos National Laboratory (LANL) was provided by laboratory-directed research and development funds