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The objective of this study was to demonstrate that RNA interference (RNAi) and somatic cell nuclear transfer (SCNT) technologies can be used to attenuate the expression of specific genes in tissues of swine, a large animal species. Apolipoprotein E (apoE), a secreted glycoprotein known for its major role in lipid and lipoprotein metabolism and transport, was selected as the target gene for this study. Three synthetic small interfering RNAs (siRNA) targeting the porcine apoE mRNA were tested in porcine granulosa cells in primary culture and reduced apoE mRNA abundance ranging from 45-82% compared to control cells. The most effective sequence was selected for cloning into a short hairpin RNA (shRNA) expression vector under the control of RNA polymerase III (U6) promoter. Stably transfected fetal porcine fibroblast cells were generated and used to produce embryos with in vitro matured porcine oocytes, which were then transferred into the uterus of surrogate gilts. Seven live and one stillborn piglet were born from three gilts that became pregnant. Integration of the shRNA expression vector into the genome of clone piglets was confirmed by PCR and expression of the GFP transgene linked to the expression vector. Analysis showed that apoE protein levels in the liver and plasma of the clone pigs bearing the shRNA expression vector targeting the apoE mRNA was significantly reduced compared to control pigs cloned from non-transfected fibroblasts of the same cell line. These results demonstrate the feasibility of applying RNAi and SCNT technologies for introducing stable genetic modifications in somatic cells for eventual attenuation of gene expression in vivo in large animal species.

The mobility project concerns all the Spanish universities. Within the whole range of exchanges the broadest and best known is the Erasmus Programme. The new teaching system of Europe is rooted in it. Accordingly, this paper presents the experience at the Faculty of Economics and Business Studies of the Universidad de León (Spain) where an opinion poll has been addressed to students (both Spanish and Foreign) who have taken part in the programme during the academic years 2003-2004 to 2008-2009. The objective data show that the Faculty of Economics is the one which has the biggest exchange rate in the Universidad de León's total figures together with the largest list of signed agreements with other European universities. The received answers to the poll highlight a positive assessment of their stays both in academic and personal terms. On the other hand, some faults have been noted such as poor financial funding; an urgent need to adapt information technologies to teaching resources; bigger information about the contents of the subjects and a clearer explanation about the validating system. [PUBLICATION ABSTRACT]

Many factors can affect the prolificity of a pig population, such as ovulation rate, embryonic attachment, placental formation and embryonic survival. Here we have chosen to investigate whether the development of somatic cell nuclear transfer embryos would be different when using nuclei derived from sows at extremes of fertility. Mesenchymal cell cultures were established from hyper-prolific (those that had an average of >15 piglets/litter for at least 3 consecutive farrowings) and hypo-prolific (those that had an average of <10 piglets/litter for at least 3 consecutive farrowings) sows by aspirating the humerus bone marrow within 5 minutes after slaughter. Mesenchymal cells were cultured for several (3-5) passages and then frozen or used for embryo reconstruction. Host oocytes were collected from 3-6 mm follicles of pre-pubertal gilts ovaries recovered at slaughterhouse and matured in vitro for 44-46 h using standard procedures, and then selected for the presence of a polar body. After enucleation and fusion, reconstructed oocytes were activated using ionomycin and strontium chloride, and then cultured for 5-7 days in porcine zygote (PZM-3) medium (Che et al. 2007, Theriogenolgy 67:1297-304). Three different cell lines were used from hypo- and hyper-prolific sows and at least 4 nuclear transfer replications were performed for each cell line. The total cleavage (79.6% vs. 78.9%) and blastocyst (34.4% vs. 31.6%) rates, and the average number of nuclei per blastocyst (33.6±3.9 vs. 33.8±4.4) were similar when comparing embryos derived from hyper- vs. hypo-prolific cell lines. There was also no difference between cell lines within the groups on both cleavage (75.3, 84.0 and 83.1%) and (79.4, 83.2 and 75.1%), and blastocyst (40.5, 34.4 and 32.4%) and (29.8, 34.8 and 34.9%) rates in embryos reconstructed from hyper- and hypo-prolific cell lines, respectively. Developing blastocysts at day 5-6 of culture were selected and surgically transferred to 8 recipient gilts that were estrus synchronized with oral progesterone for 12 days and then 1000 IU eCG and 500 IU hCG injected 72 later. Four females were transferred with hypo- and 4 with hyper-prolific cell lines derived blastocysts. The total number of blastocysts transferred to each recipient ranged from 35 to 70. Each recipient female received either embryos derived from the 3 hyper- or hypo-prolific cell lines in the same proportion. Two females transferred with embryos derived from the hypo-prolific cell lines were detected pregnant by ultrassonography and are presently in the last third of gestation. These findings suggest that the prolificity of animals used as source of nuclear donor cells do not affect the development of embryos produced by somatic cell nuclear transfer. Supported by NSERC.

Treatment with trichostatin A (TSA), an inhibitor of histone deacethylases, was reported to increase pre- and post-implantation development in mice embryos produced by somatic cell nuclear transfer (SCNT) (Kishigami et al. 2006, Biochem Biophys Res Commun. 340:183-9), and also the in vitro development of porcine embryos (Zhang et al. 2007, Cloning and Stem Cells 9: 357-63). The present study assessed changes in epigenetic markers and pre- and post-implantation development in porcine SCNT embryos after TSA treatment. Host oocytes were produced in vitro using standard procedures. Embryos were generated either using a male fetal fibroblast (FF) cell line or bone marrow cell (BMC) lines derived from adult females. Reconstructed oocytes were either exposed or not to 10 ng/ml TSA for 10 h starting 1 h after cell fusion. Activation was performed using ionomycin and strontium chloride and embryos were cultured for 5-7 days in porcine zygote (PZM-3) medium (Che et al. 2007, Theriogenolgy 67:1297-304). Reconstructed oocytes from FF cells were fixed at 18-20 h and immunocytochemically processed to assess H3K14 acetylation, H3K9 di-methylation and 5-methylcytidine (DNA methylation) using specific primary antibodies and Alexa Fluor® 488 secondary antibodies. Immunofluorescent images were acquired using a Retiga 2000R monochromo digital camera (Qimaging, BC, Canada) and the grayscale signal on the chromatin was compared between TSA and control embryos using the SimplePCI Imaging Software (Complix Inc., Sewickley, PA). The chromatin signal for H3K14 acetylated and H3K9 di-methylated was higher in TSA treated samples but there was no effect on DNA methylation. There was no effect of TSA on the cleavage rate in embryos produced using either FF or BMC. TSA treatment resulted in higher development to blastocyst (45.2%) compared to control (23.9%) in FF-derived embryos, but there was no difference between treated (34.7%) and control (34.9%) groups derived from BMC. Day 5-6 blastocysts were transferred to 8 gilts that were estrus synchronized with oral progesterone for 12 days and then 1000 IU eCG and 500 IU hCG injected 72 later. Two females were transferred with FF-TSA treated (n=53 and 72) and two with FF-control (n=81 and 50) embryos. Both controls and one TSA treated females farrowed naturally after PGF2a injection given at D-115 after estrus. Eleven (5 and 6) and 6 piglets were born from control and TSA treated embryos, respectively. The average birth weight was higher but not significantly different between TSA (1020+173 gr) vs. control (874+102 gr) groups. One of the 2 females receiving BMC-TSA embryos (n=60 and 60) became pregnant but both females transferred with BMC-control (n=60 and 45) returned to estrus. These findings suggest that TSA treatment alters epigenetic reprogramming and in vitro development in SCNT swine embryos but it seems not to improve long term development after embryo transfer to recipient females.

The objective of this study was to demonstrate that RNA interference (RNAi) and somatic cell nuclear transfer (SCNT) technologies can be used to attenuate the expression of specific genes in tissues of swine, a large animal species. Apolipoprotein E (apoE), a secreted glycoprotein known for its major role in lipid and lipoprotein metabolism and transport, was selected as the target gene for this study. Three synthetic small interfering RNAs (siRNA) targeting the porcine apoE mRNA were tested in porcine granulosa cells in primary culture and reduced apoE mRNA abundance ranging from 45-82% compared to control cells. The most effective sequence was selected for cloning into a short hairpin RNA (shRNA) expression vector under the control of RNA polymerase III (U6) promoter. Stably transfected fetal porcine fibroblast cells were generated and used to produce embryos with in vitro matured porcine oocytes, which were then transferred into the uterus of surrogate gilts. Seven live and one stillborn piglet were born from three gilts that became pregnant. Integration of the shRNA expression vector into the genome of clone piglets was confirmed by PCR and expression of the GFP transgene linked to the expression vector. Analysis showed that apoE protein levels in the liver and plasma of the clone pigs bearing the shRNA expression vector targeting the apoE mRNA was significantly reduced compared to control pigs cloned from non-transfected fibroblasts of the same cell line. These results demonstrate the feasibility of applying RNAi and SCNT technologies for introducing stable genetic modifications in somatic cells for eventual attenuation of gene expression in vivo in large animal species.

Information theory allows us to investigate information processing in neural systems in terms of information transfer, storage and modification. Especially the measure of information transfer, transfer entropy, has seen a dramatic surge of interest in neuroscience. Estimating transfer entropy from two processes requires the observation of multiple realizations of these processes to estimate associated probability density functions. To obtain these necessary observations, available estimators typically assume stationarity of processes to allow pooling of observations over time. This assumption however, is a major obstacle to the application of these estimators in neuroscience as observed processes are often non-stationary. As a solution, Gomez-Herrero and colleagues theoretically showed that the stationarity assumption may be avoided by estimating transfer entropy from an ensemble of realizations. Such an ensemble of realizations is often readily available in neuroscience experiments in the form of experimental trials. Thus, in this work we combine the ensemble method with a recently proposed transfer entropy estimator to make transfer entropy estimation applicable to non-stationary time series. We present an efficient implementation of the approach that is suitable for the increased computational demand of the ensemble method's practical application. In particular, we use a massively parallel implementation for a graphics processing unit to handle the computationally most heavy aspects of the ensemble method for transfer entropy estimation. We test the performance and robustness of our implementation on data from numerical simulations of stochastic processes. We also demonstrate the applicability of the ensemble method to magnetoencephalographic data. While we mainly evaluate the proposed method for neuroscience data, we expect it to be applicable in a variety of fields that are concerned with the analysis of information transfer in complex biological, social, and artificial systems.

This work was supported by the Spanish Association Against Cancer (PROYE18061FERN to M.F.F.), the Asturias Government (PCTI) co-funding 2018-2023/FEDER (IDI/2018/146 and IDI/2021/000077 to M.F.F.), the Health Institute Carlos III (Plan Nacional de I+D+I) co-funding FEDER (PI18/01527 and PI21/01067 to M.F.F and A.F.F.), CIBERER Acciones Cooperativas y Complementarias Intramurales (ACCI20-35 to M.F.F.), the Fundación General CSIC (0348_CIE_6_E to M.F.F.), the ISCIII (COV00624 to J.R.T. and M.F.F.), ISPA and the Asociación Galbán (2021-052-INTRAMUR GALBAN-GOURR to R.G.U.), ISPA-Jannsen (2021-048-INTRAMURAL NOV-TEVAR to J.R.T.), CSIC (202020E092 to M.F.F), and the European Commission NextGenerationEU, through CSIC’s Global Health Platform (PTI Salud Global) and the Spanish Ministry of Science and Innovation through the Recovery, Transformation and Resilience Plan (SGL2021-03-39 and SGL2021-03-040).

Protein oxidation is a topic of indisputable scientific interest given the impact of oxidized proteins on food quality and safety. Carbonylation is regarded as one of the most notable post-translational modifications in proteins and yet, this reaction and its consequences are poorly understood. From a mechanistic perspective, primary protein carbonyls (i.e. α-aminoadipic and γ-glutamic semialdehydes) have been linked to radical-mediated oxidative stress, but recent studies emphasize the role alternative carbonylation pathways linked to the Maillard reaction. Secondary protein carbonyls are introduced in proteins via covalent linkage of lipid carbonyls (i.e. protein-bound malondialdehyde). The high reactivity of protein carbonyls in foods and other biological systems indicates the intricate chemistry of these species and urges further research to provide insight into these molecular mechanisms and pathways. In particular, protein carbonyls are involved in the formation of aberrant and dysfunctional protein aggregates, undergo further oxidation to yield carboxylic acids of biological relevance and establish interactions with other biomolecules such as oxidizing lipids and phytochemicals. From a methodological perspective, the routine dinitrophenylhydrazine (DNPH) method is criticized not only for the lack of accuracy and consistency but also authors typically perform a poor interpretation of DNPH results, which leads to misleading conclusions. From a practical perspective, the biological relevance of protein carbonyls in the field of food science and nutrition is still a topic of debate. Though the implication of carbonylation on impaired protein functionality and poor protein digestibility is generally recognized, the underlying mechanism of such connections requires further clarification. From a medical perspective, protein carbonyls are highlighted as markers of protein oxidation, oxidative stress and disease. Yet, the specific role of specific protein carbonyls in the onset of particular biological impairments needs further investigations. Recent studies indicates that regardless of the origin (in vivo or dietary) protein carbonyls may act as signalling molecules which activate not only the endogenous antioxidant defences but also implicate the immune system. The present paper concisely reviews the most recent advances in this topic to identify, when applicable, potential fields of interest for future studies.

Neural preconfigured activity patterns (nPAPs), conceptualized as organized activity parcellated into groups of neurons, have been proposed as building blocks for cognitive and sensory processing. However, their existence and function in motor networks have been scarcely studied. Here, we explore the possibility that nPAPs are present in the motor thalamus (VL/VM) and their potential contribution to motor-related activity. To this end, we developed a preparation where VL/VM multiunitary activity could be robustly recorded in mouse behavior evoked by primary motor cortex (M1) optogenetic stimulation and forelimb movements. VL/VM-evoked activity was organized as rigid stereotypical activity patterns at the single and population levels. These activity patterns were unable to dynamically adapt to different temporal architectures of M1 stimulation. Moreover, they were experience-independent, present in virtually all animals, and pairs of neurons with high correlations during M1-stimulation also presented higher correlations during spontaneous activity, confirming their preconfigured nature. Finally, subpopulations expressing specific M1-evoked patterns also displayed specific movement-related patterns. Our data demonstrate that the behaviorally related identity of specific neural subpopulations is tightly linked to nPAPs. Cortico-thalamic interactions have been implicated in a variety of motor functions, including movement planning, initiation, and moment-to-moment control. Here the authors present evidence suggesting that these interactions rest on the existence of preconfigured neural activity patterns that constrain movement representation and execution.