32 research outputs found
PRODUCTION OF GENETICALLY MODIFIED PORCINE EMBRYOS BY LIPOFECTION OF IN VITRO MATURED OOCYTES USING CRISPR/CAS9 SYSTEM
P-109 Nanoflakes of molybdenum disulfide functionalized with catechins modulate the sperm capacitation resulting in an improvement of the IVF outcomes in a swine in vitro model
Metabolic Networks of Sodalis glossinidius: A Systems Biology Approach to Reductive Evolution
Background: Genome reduction is a common evolutionary process affecting bacterial lineages that establish symbiotic or pathogenic associations with eukaryotic hosts. Such associations yield highly reduced genomes with greatly streamlined metabolic abilities shaped by the type of ecological association with the host. Sodalis glossinidius, the secondary endosymbiont of tsetse flies, represents one of the few complete genomes available of a bacterium at the initial stages of this process. In the present study, genome reduction is studied from a systems biology perspective through the reconstruction and functional analysis of genome-scale metabolic networks of S. glossinidius. Results: The functional profile of ancestral and extant metabolic networks sheds light on the evolutionary events underlying transition to a host-dependent lifestyle. Meanwhile, reductive evolution simulations on the extant metabolic network can predict possible future evolution of S. glossinidius in the context of genome reduction. Finally, knockout simulations in different metabolic systems reveal a gradual decrease in network robustness to different mutational events for bacterial endosymbionts at different stages of the symbiotic association. Conclusions: Stoichiometric analysis reveals few gene inactivation events whose effects on the functionality of S. glossinidius metabolic systems are drastic enough to account for the ecological transition from a free-living to hostdependent lifestyle. The decrease in network robustness across different metabolic systems may be associated with th
Roflumilast in moderate-to-severe chronic obstructive pulmonary disease treated with longacting bronchodilators: two randomised clinical trials
Background Patients with chronic obstructive pulmonary disease (COPD) have few options for treatment. The efficacy and safety of the phosphodiesterase-4 inhibitor roflumilast have been investigated in studies of patients with moderate-to-severe COPD, but not in those concomitantly treated with longacting inhaled bronchodilators. The effect of roflumilast on lung function in patients with COPD that is moderate to severe who are already being treated with salmeterol or tiotropium was investigated. Methods In two double-blind, multicentre studies done in an outpatient setting, after a 4-week run-in, patients older than 40 years with moderate-to-severe COPD were randomly assigned to oral roflumilast 500 mu g or placebo once a day for 24 weeks, in addition to salmeterol (M2-127 study) or tiotropium (M2-128 study). The primary endpoint was change in prebronchodilator forced expiratory volume in 1s (FEV(1)). Analysis was by intention to treat. The studies are registered with ClinicalTrials.gov, number NCT00313209 for M2-127, and NCT00424268 for M2-128. Findings In the salmeterol plus roflumilast trial, 466 patients were assigned to and treated with roflumilast and 467 with placebo; in the tiotropium plus roflumilast trial, 371 patients were assigned to and treated with roflumilast and 372 with placebo. Compared with placebo, roflumilast consistently improved mean prebronchodilator FEV(1) by 49 mL (p<0.0001) in patients treated with salmeterol, and 80 mL (p<0.0001) in those treated with tiotropium. Similar improvement in postbronchodilator FEV(1) was noted in both groups. Furthermore, roflumilast had beneficial effects on other lung function measurements and on selected patient-reported outcomes in both groups. Nausea, diarrhoea, weight loss, and, to a lesser extent, headache were more frequent in patients in the roflumilast groups. These adverse events were associated with increased patient withdrawal. Interpretation Roflumilast improves lung function in patients with COPD treated with salmeterol or tiotropium, and could become an important treatment for these patients
Global patient outcomes after elective surgery: prospective cohort study in 27 low-, middle- and high-income countries.
BACKGROUND: As global initiatives increase patient access to surgical treatments, there remains a need to understand the adverse effects of surgery and define appropriate levels of perioperative care. METHODS: We designed a prospective international 7-day cohort study of outcomes following elective adult inpatient surgery in 27 countries. The primary outcome was in-hospital complications. Secondary outcomes were death following a complication (failure to rescue) and death in hospital. Process measures were admission to critical care immediately after surgery or to treat a complication and duration of hospital stay. A single definition of critical care was used for all countries. RESULTS: A total of 474 hospitals in 19 high-, 7 middle- and 1 low-income country were included in the primary analysis. Data included 44 814 patients with a median hospital stay of 4 (range 2-7) days. A total of 7508 patients (16.8%) developed one or more postoperative complication and 207 died (0.5%). The overall mortality among patients who developed complications was 2.8%. Mortality following complications ranged from 2.4% for pulmonary embolism to 43.9% for cardiac arrest. A total of 4360 (9.7%) patients were admitted to a critical care unit as routine immediately after surgery, of whom 2198 (50.4%) developed a complication, with 105 (2.4%) deaths. A total of 1233 patients (16.4%) were admitted to a critical care unit to treat complications, with 119 (9.7%) deaths. Despite lower baseline risk, outcomes were similar in low- and middle-income compared with high-income countries. CONCLUSIONS: Poor patient outcomes are common after inpatient surgery. Global initiatives to increase access to surgical treatments should also address the need for safe perioperative care. STUDY REGISTRATION: ISRCTN5181700
Production of genetically modified porcine embryos by lipofection of zona pellucida-intact oocytes using the CRISPR/Cas9 system
The generation of genetically modified pigs has an important impact thanks its applications in basic research, biomedicine, and meat production. Cloning was the first technique used for this production, although easier and cheaper methods were developed, such as the microinjection, electroporation, or lipofection of oocytes and zygotes. In this study, we analyzed the production of genetically modified embryos via lipofection of zona-pellucida-intact oocytes using LipofectamineTM CRISPRMAXTM Cas9 in comparison with the electroporation method. Two factors were evaluated: (i) the increment in the concentration of the lipofectamine–ribonucleoprotein complexes (LRNPC) (5% vs. 10%) and (ii) the concentration of ribonucleoprotein within the complexes (1xRNP vs. 2xRNP).
We found that the increment in the concentration of the LRNPC had a detrimental effect on embryo development and a subsequent effect on the number of mutant embryos. The 5% group had a similar mutant blastocyst rate to the electroporation method (5.52% and 6.38%, respectively, p > 0.05). The increment in the concentration of the ribonucleoprotein inside the complexes had no effect on the blastocyst rate and mutation rate, with the mutant blastocyst rate being similar in both the 1xRNP and 2xRNP lipofection groups and the electroporation group (1.75%, 3.60%, and 3.57%, respectively, p > 0.05). Here, we showed that it is possible to produce knock-out embryos via lipofection of zona-pellucida-intact porcine oocytes with similar efficiencies as with electroporation, although more optimization is needed, mainly in terms of the use of more efficient vesicles for encapsulation with different compositions
Previous fire occurrence, but not fire recurrence, modulates the effect of charcoal and ash on soil C and N dynamics in Pinus pinaster Aiton forests
Understanding the effects of fire history on soil processes is key to characterise their resistance and resilience under future fire events. Wildfires produce pyrogenic carbonaceous material (PCM) that is incorporated into the soil, playing a critical role in the global carbon (C) cycle, but its interactions with soil processes are poorly understood. We evaluated if the previous occurrence of wildfires modulates the dynamic of soil C and nitrogen (N) and microbial community by soil ester linked fatty acids, after a new simulated low-medium intensity fire. Soils with a different fire history (none, one, two or three fires) were heat-shocked and amended with charcoal and/or ash derived from Pinus pinaster. Soil C and N mineralization rates were measured under controlled conditions, with burned soils showing lower values than unburned (without fire for more than sixty years). In general, no effects of fire recurrence were observed for any of the studied variables. Microbial biomass was lower in burned, with a clear dominance of Gram-positive bacteria in these soils. PCM amendments increased cumulative carbon dioxide (CO2) production only in previously burned soils, especially when ash was added. This contrasted response to PCM between burned and unburned soils in CO2 production could be related to the effect of the previous wildfire history on soil microorganisms. In burned soils some microorganisms might have been adapted to the resulting conditions after a new fire event. Burned soils showed a significant positive priming effect after PCM amendment, mainly ash, probably due to an increased pH and phosphorous availability. Our results reveal the role of different PCMs as drivers of C and N mineralization processes in burned soils when a new fire occurs. This is relevant for improving models that evaluate the net impact of fire in C cycling and to reduce uncertainties under future changing fire regimes scenarios
Catechin versus MoS2 Nanoflakes Functionalized with Catechin: Improving the Sperm Fertilizing Ability—An In Vitro Study in a Swine Model
Nowadays, the adoption of In Vitro Fertilization (IVF) techniques is undergoing an impressive increase. In light of this, one of the most promising strategies is the novel use of non-physiological materials and naturally derived compounds for advanced sperm preparation methods. Here, sperm cells were exposed during capacitation to MoS2/Catechin nanoflakes and catechin (CT), a flavonoid with antioxidant properties, at concentrations of 10, 1, 0.1 ppm. The results showed no significant differences in terms of sperm membrane modifications or biochemical pathways among the groups, allowing the hypothesis that MoS2/CT nanoflakes do not induce any negative effect on the parameters evaluated related to sperm capacitation. Moreover, the addition of CT alone at a specific concentration (0.1 ppm) increased the spermatozoa fertilizing ability in an IVF assay by increasing the number of fertilized oocytes with respect to the control group. Our findings open interesting new perspectives regarding the use of catechins and new materials obtained using natural or bio compounds, which could be used to implement the current strategies for sperm capacitation
Genomic Responses of Pseudomonas putida to Aromatic Hydrocarbons
When Pseudomonas putida mt-2 cells face aromatic hydrocarbons, they must make a number of decisions that exemplify virtually every challenge that environmental bacteria need to overcome for thriving in polluted sites. It is thus no surprise that the genome-wide responses of P. putida to a variety conditions have been studied in considerable detail. One long-standing issue is how P. putida handles nutrient choice when a mixture of substrates coexist in the same site. A complex mechanism reminiscent of catabolite repression is in place to ensure that hydrocarbons are tackled only after virtually all other easy nutrients are depleted. Once this occurs, typical components of the lighter aromatic fractions of petroleum (e.g., toluene and xylenes) are not only potential nutrients but also strong stressors for P. putida. For handling this scenario, simultaneous processing of their presence both as an edible carbon source to benefit from and as a detrimental hassle to keep at bay becomes necessary. A second issue is the metabolic jam caused by the coincident presence in the same cells of competing pathways for ortho-cleavage and meta-cleavage of the intermediate catechols that degradation of aromatics must necessarily go through. Availability of a suite of genomic, proteomic and reporter technologies has shed light on these questions and exposed a number of surprising solutions that would otherwise look like intractable biochemical and regulatory problems. These include not only specific biochemical and transcriptional devices that overcome intracellular conflicts but also diversification of the population for acquiring different phenotypic roles.The work in VdL’s Laboratory was funded by the HELIOS Project of the Spanish Ministry of Science BIO 2015-66960-C3-2-R (MINECO/FEDER), the ARISYS (ERC-2012-ADG-322797), EmPowerPutida (EU-H2020-BIOTEC-2014-2015-6335536) and MADONNA (H2020-FET-OPEN-RIA-2017-1-766975) Contracts of the European Union, and the InGEMICS-CM (B2017/BMD-3691) contract of the Comunidad de Madrid (FSE, FECER).Peer reviewe