Otoferlin gene editing in sheep via CRISPR-assisted ssODN-mediated Homology Directed Repair.

Different mutations of the OTOF gene, encoding for otoferlin protein expressed in the cochlear inner hair cells, induces a form of deafness that is the major cause of nonsyndromic recessive auditory neuropathy spectrum disorder in humans. We report the generation of the first large animal model of OTOF mutations using the CRISPR system associated with different Cas9 components (mRNA or protein) assisted by single strand oligodeoxynucleotides (ssODN) to induce homology-directed repair (HDR). Zygote microinjection was performed with two sgRNA targeting exon 5 and 6 associated to Cas9 mRNA or protein (RNP) at different concentrations in a mix with an ssODN template targeting HDR in exon 5 containing two STOP sequences. A total of 73 lambs were born, 13 showing indel mutations (17.8%), 8 of which (61.5%) had knock-in mutations by HDR. Higher concentrations of Cas9-RNP induced targeted mutations more effectively, but negatively affected embryo survival and pregnancy rate. This study reports by the first time the generation of OTOF disrupted sheep, which may allow better understanding and development of new therapies for human deafness related to genetic disorders. These results support the use of CRISPR/Cas system assisted by ssODN as an effective tool for gene editing in livestock.

CRISPR in livestock: From editing to printing.

Precise genome editing of large animals applied to livestock and biomedicine is nowadays possible since the CRISPR revolution. This review summarizes the latest advances and the main technical issues that determine the success of this technology. The pathway from editing to printing, from engineering the genome to achieving the desired animals, does not always imply an easy, fast and safe journey. When applied in large animals, CRISPR involves time- and cost-consuming projects, and it is mandatory not only to choose the best approach for genome editing, but also for embryo production, zygote microinjection or electroporation, cryopreservation and embryo transfer. The main technical refinements and most frequent questions to improve this disruptive biotechnology in large animals are presented. In addition, we discuss some CRISPR applications to enhance livestock production in the context of a growing global demand of food, in terms of increasing efficiency, reducing the impact of farming on the environment, enhancing pest control, animal welfare and health. The challenge is no longer technical. Controversies and consensus, opportunities and threats, benefits and risks, ethics and science should be reconsidered to enter into the CRISPR era.

Los Comités de Ética Asistencial y los Comités de Ética de la Investigación en España: organización, regulación y funciones / Healthcare Ethics Committees and Institutional Review Boards in Spain: organization, regulation and functions

En las últimas décadas hemos asistido a una evolución en la medicina que obliga a una reflexión ética más cuidadosa y a una organización e institucionalización de los procesos de toma de decisiones que puedan resultar controvertidos. Los Comités de Ética Asistencial se constituyen para analizar y asesorar en la resolución de los conflictos éticos que se producen durante la práctica clínica asistencial y garantizar que los pacientes estén informados y puedan tomar las decisiones referentes a su salud, de acuerdo con su libre y propia voluntad. El objetivo de los Comités de Ética de la Investigación es la protección y el respeto de las personas participantes en estudios de investigación. En ambos casos, juegan un papel importante en la protección de los derechos, la equidad y la dignidad de la personas. Un marco normativo adecuado permite dar respuesta a los nuevos retos científicos y garantiza la protección de los ciudadanos en el ámbito de la salud. Sin embargo, la extensa red de comités locales, autonómicos y estatales en España, puede comprometer la eficiencia en la gestión administrativa de los procedimientos de evaluación y en la resolución de conflictos y dar lugar a duplicidades o incoherencias en los mismos. En este artículo se realiza una revisión y descripción de los diferentes comités de ética asistencial y de la investigación en España y se analizan aspectos relevantes en relación a su organización, regulación y ámbito de actuación

Recent decades have witnessed major advances in medical science that require a careful ethical reflection, organization and institutionalization of controversial decision-making processes. The Healthcare Ethics Committees (HECs) are independent interdisciplinary bodies to provide support in ethical dilemmas in healthcare settings, ensuring that patients are informed about their treatments and procedures and make their own decisions. The Institutional Review Boards (IRBs) are focus on the rights and welfare of the potential participants in the research. Nevertheless both HECs and IRBs play an important role in promoting respect and protection of dignity and human right in an attempt to achieve health equity and social justice. The regulation of HECs and IRBs provides an appropriate framework to address new scientific challenges in the field of health. Nevertheless the large number of local, regional and national HECs and IRBs in Spain might compromise the efficiency in the assessment procedures leading to duplication of reports and inconsistent opinions. In this article a review and description of the different HECs and IRBs in Spain is performed and relevant issues related to their organization, regulation and functions are also analyzed

Inactivation of the microbiota and effect on the quality attributes of pineapple juice using a continuous flow ultrasound-assisted supercritical carbon dioxide system.

Supercritical carbon dioxide inactivation technology represents a promising nonthermal processing method, as it causes minimum impact on the nutritional food properties. The aim of this study was to analyze the combined effect of supercritical carbon dioxide and high-power ultrasound on the inactivation of natural microbiota and the quality attributes of pineapple juice treated in a continuous flow system. Different juice residence times (3.06-4.6 min), at 100 bar and 31.5 ℃, were used. The results indicated that the microbiota inactivation was complete and the differences obtained in the quality attributes (2.2% for pH, 4.8% for °Brix, 2% for vitamin C) were minimal. During storage, microorganisms were not able to recover and the vitamin C decrease could be limited to 8.2% after four weeks. The results demonstrated that the supercritical carbon dioxide-high-power ultrasound technique could be an excellent alternative for the cold pasteurization of pineapple juice.

Inhibition of serotonin receptor type 1 in acute myeloid leukemia impairs leukemia stem cell functionality: a promising novel therapeutic target.

Acute myeloid leukemia (AML) is a clinically and molecularly heterogeneous neoplasia with poor outcome, organized as a hierarchy initiated and maintained by a sub-population with differentiation and self-renewal capacities called leukemia stem cells (LSCs). Although currently used chemotherapy is capable of initially reducing the tumor burden producing a complete remission, most patients will ultimately relapse and will succumb to their disease. As such, new therapeutic strategies are needed. AML cells differentially expressed serotonin receptor type 1 (HTR1) compared with healthy blood cells and the most primitive hematopoietic fraction; in fact, HTR1B expression on AML patient samples correlated with clinical outcome. Inhibition of HTR1s activated the apoptosis program, induced differentiation and reduced the clonogenic capacity, while minimal effect was observed on healthy blood cells. In vivo regeneration capacity of primary AML samples was disrupted upon inhibition of HTR1. The self-renewal capacity remaining in AML cells upon in vivo treatment was severely reduced as demonstrated by serial transplantation. Thus, treatment with HTR1 antagonists showed antileukemia effect, especially anti-LSC activity while sparing healthy blood cells. Our results highlight the importance of HTR1 in leukemogenesis and LSC survival and identify this receptor family as a new target for therapy in AML with prognostic value.

RAPID COMMUNICATION: Nerve growth factor influences cleavage rate and embryo development in sheep.

Recent information about Nerve growth factor (NGF), a protein traditionally associated to the nervous system that regulates survival and maturation of developing neurons, suggests that it may exert action also on different levels in the reproductive system. The aim of this study was to evaluate the effect of NGF added during in vitro oocyte maturation, fertilization or in vitro embryo development in sheep. Nerve growth factor was supplemented to the culture medium at 0, 100, or 1,000 ng/mL, during either in vitro maturation (Exp. 1), in vitro fertilization (Exp. 2), or in vitro culture (Exp. 3). In addition, NGF mRNA expression was determined in cumulus cells and oocytes. Nerve growth factor induced early cleavage when added during oocyte maturation or fertilization, improved embryo development when added during fertilization, and had no significant effect when added during embryo culture. In general, the effect was more evident with 100 rather than 1,000 ng/mL (P < 0.05). Expression of endogenous NGF was not detected in oocytes, and increased in cumulus cells when 1,000 ng/mL of NGF was added during fertilization, but not during maturation and embryo culture. In conclusion, the addition of NGF during oocyte maturation and fertilization affects in vitro cleavage and embryo development in sheep. We suggest a possible effect of this growth factor on oocyte maturation and mainly on the fertilization process.

Efficient Generation of Myostatin Knock-Out Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes.

While CRISPR/Cas9 technology has proven to be a valuable system to generate gene-targeted modified animals in several species, this tool has been scarcely reported in farm animals. Myostatin is encoded by MSTN gene involved in the inhibition of muscle differentiation and growth. We determined the efficiency of the CRISPR/Cas9 system to edit MSTN in sheep and generate knock-out (KO) animals with the aim to promote muscle development and body growth. We generated CRISPR/Cas9 mRNAs specific for ovine MSTN and microinjected them into the cytoplasm of ovine zygotes. When embryo development of CRISPR/Cas9 microinjected zygotes (n = 216) was compared with buffer injected embryos (n = 183) and non microinjected embryos (n = 173), cleavage rate was lower for both microinjected groups (P<0.05) and neither was affected by CRISPR/Cas9 content in the injected medium. Embryo development to blastocyst was not affected by microinjection and was similar among the experimental groups. From 20 embryos analyzed by Sanger sequencing, ten were mutant (heterozygous or mosaic; 50% efficiency). To obtain live MSTN KO lambs, 53 blastocysts produced after zygote CRISPR/Cas9 microinjection were transferred to 29 recipient females resulting in 65.5% (19/29) of pregnant ewes and 41.5% (22/53) of newborns. From 22 born lambs analyzed by T7EI and Sanger sequencing, ten showed indel mutations at MSTN gene. Eight showed mutations in both alleles and five of them were homozygous for indels generating out-of frame mutations that resulted in premature stop codons. Western blot analysis of homozygous KO founders confirmed the absence of myostatin, showing heavier body weight than wild type counterparts. In conclusion, our results demonstrate that CRISPR/Cas9 system was a very efficient tool to generate gene KO sheep. This technology is quick and easy to perform and less expensive than previous techniques, and can be applied to obtain genetically modified animal models of interest for biomedicine and livestock.

Embryo development, fetal growth and postnatal phenotype of eGFP lambs generated by lentiviral transgenesis.

Lentiviral technology has been recently proposed to generate transgenic farm animals more efficiently and easier than traditional techniques. The objective was to evaluate several parameters of lambs obtained by lentiviral transgenesis in comparison with non-transgenic counterparts. In vitro produced embryos were microinjected (TG group) at two-cell stage with a lentiviral construct containing enhanced green fluorescent protein (eGFP) gene, while embryos produced by in vitro fertilization (IVF group) or intrauterine insemination (IUI group) were not microinjected. Microinjection technique efficiently generated eight-cell transgenic embryos (97.4%; 114/117). Development rate on day 5 after fertilization was similar for TG (39.3%, 46/117) and IVF embryos (39.6%, 44/111). Pregnancy rate was detected in 50.0% (6/12) of recipient ewes with TG embryos, in 46.7% (7/15) with IVF embryos, and in 65.0% (13/20) of IUI ewes (P = NS). Nine lambs were born in TG group, six lambs in IVF group, and 16 lambs in IUI group. All TG lambs (9/9) were GFP positive to real-time PCR and eight (88.9%) showed a strong and evident GFP expression in mucosae, eyes and keratin tissues. Fetal growth monitored every 15 day by ultrasonography did not show significant differences. Transgenic lambs neither differ in morphometric variables in comparison with non transgenic IVF lambs within 3 months after birth. Transmission of the transgene to the progeny was observed in green fluorescent embryos produced by IVF using semen from the TG founder lambs. In conclusion, this study demonstrates the high efficiency of lentiviral technology to produce transgenic sheep, with no clinic differences in comparison with non transgenic lambs.

Ultrasonic assessment of textural changes in vacuum packaged sliced Iberian ham induced by high pressure treatment or cold storage.

Ultrasonic measurements were used to characterize the effect of high pressure treatment (HPT) (600 MPa/6 min) or cold storage (6°C/120 days) on the textural properties of vacuum packaged dry-cured ham. The ultrasonic velocity, textural properties and fat content were determined in the ham packages. The ultrasonic velocity was related to the ham hardness, which depends on the sample composition. HPT induced molecular alterations which resulted in an average increase in the hardness of lean tissue of 0.2N and one of 0.3N in that of fatty tissue. These textural changes give rise to a velocity increase (8m/s for lean and 17 m/s for fatty tissue). The cold storage of the Iberian ham also led to an increase in hardness (average 1.10 N) and ultrasonic velocity (average 70 m/s). Therefore, the non-destructive ultrasonic technique could be a reliable method with which to assess the textural changes induced by HPT or cold storage on packaged dry-cured ham.

Modeling of the process of moisture loss during the storage of dried apricots.

Moisture content is a reference parameter for dried food because the growth of most microorganisms is inhibited below certain water activity levels. In addition, it has a determining influence on the evolution of important parameters, such as color and flavor, and on other properties and deterioration reactions, such as texture, oxidation processes and nutritional value. During the storage of some dried fruits, moisture is produced due to Maillard reactions and exchanged with the surrounding environment through the packaging. The evolution of dried foods during their shelf life depends on the storage conditions. The aim of this study is to analyze the evolution of the moisture content in dried apricots packaged in different types of containers, namely glass and thermosealed polypropylene trays. The samples were stored at constant temperatures: 5, 15, 25 and 35 °C and were analyzed periodically over a period of 12 months. The sorption isotherms of apricots used in this study were also determined. In order to model how the moisture evolved, an empirical kinetic model was tested. This model considers both water transfer from the fruit and also water production as a result of the Maillard processes. The explained variance was higher than 95% in the samples stored in trays, which were thermosealed with film.

Ultrasonic characterisation of B. femoris from Iberian pigs of different genetics and feeding systems.

Ultrasonic velocity was used to characterise the differences in composition and texture of Biceps Femoris muscles from four batches of pigs of different genetics (Iberian and Iberian × Duroc) and feeding systems ("montanera" and concentrate). Significant differences (p<0.05) were found for the ultrasonic velocity in samples with different genetics and feeding systems. These differences were dependent on the temperature of the measurements and were related to the intramuscular fat content (IMF) of the samples and, therefore, to the meat quality. The ultrasonic velocities at 0 and 20 °C were related to the IMF (R=0.77 and 0.65, respectively). A discriminant analysis, including ultrasonic velocity at temperatures from 0 to 20 °C, allowed 87.0% of the samples to be correctly classified in the batches. Therefore, ultrasonics could be useful in the characterisation and differentiation of B. femoris muscles of Iberian pigs with different genetics and from different feeding systems.

Drying modelling of defrosted pork meat under forced convection conditions.

Drying is the lengthiest and the most energy consuming step during the production of dry-cured ham, affecting also the curing process and consequently product quality. In order to manage the drying process, it is quite interesting to establish the complexity of model needed. For that purpose, pork meat cylinders (Biceps femoris and Semimembranosus muscles) were dehydrated under forced convection conditions (25°C and air velocity 0.6±0.1, 2.0±0.1 and 2.8±0.1 m/s). Experimental drying kinetics were modelled by means of 4 diffusion models: model 1 (not considering shrinkage and no external resistance), model 2 (considering shrinkage and no external resistance), model 3 (not considering shrinkage and considering external resistance) and model 4 (considering both shrinkage and external resistance). From the effective diffusivity values identified, it was concluded that when external resistance was negligible (air velocity 2.0±0.1 and 2.8±0.1 m/s), the results obtained for D(e) with the four models were the same. Nevertheless, when external resistance was not negligible (0.6±0.1 m/s) the D(e) identified was influenced by the model due to the fact that models 1 and 2 neglect that resistance and for that reason they do not describe experimental conditions properly. The effect of shrinkage did not influence the identified D(e) values for the drying conditions considered. In order to model water losses in meat curing chambers, external resistance must be considered.

Ultrasonic assessment of the melting behaviour in fat from Iberian dry-cured hams.

The feasibility of using ultrasounds to characterize the melting properties of fat from Iberian dry-cured hams was evaluated. Differential Scanning Calorimetry (DSC) and ultrasonic measurements were used to characterize the fat melting. The ultrasonic velocity in fat decreased with the increase in temperature, showing four different sections (0-4 degrees C, 4-10 degrees C, 10-20 degrees C and 20-24 degrees C). Ultrasonic velocity was related (R(2)=0.99) to the percentage of melted fat (%MEF) showing an increase of 5.4 ms(-1) for 1% increase of melted fat (%MEF above 60%). The thermal history did not affect the ultrasonic measurements from 10 to 25 degrees C and, consequently, this range was the most suitable for classifying Iberian dry-cured products with different genetics and feeding backgrounds. Ultrasonic measurements could be a reliable technique to estimate the %MEF and subsequently the related sensory attributes in Iberian dry-cured ham at 10-25 degrees C, which is the common temperature range for the consumption of Iberian dry-cured products.

Potato shrinkage during hot air drying.

Shrinkage is one of the most important physical changes that occur during the dehydration of foods. In this work, the effect of the temperature (35, 40, 50, 60 and 70 °C) and air velocity (7, 8, 9 and 10 m/s) on bulk volumetric shrinkage was investigated. Volume changes were evaluated by image analysis. It was found that neither temperature nor air velocity had any significant effects on bulk shrinkage in this system. The bulk shrinkage of the potato cubes was well correlated with the moisture content of the sample during drying (R(2) = 97.28). Volume varied linearly with the moisture content changes under the studied conditions. The volume of lost water and the decrease in volume of the samples during dehydration were similar.

Treatment with cinacalcet of secondary hyperparathyroidism after renal transplantation.

BACKGROUND: The treatment of posttransplant secondary hyperparathyroidism (SHP) with vitamin D analogues is determined by their effectiveness to reverse hypercalcemia. Calcimimetics inhibit parathyroid hormone (PTH) secretion by modulating the calcium-sensing receptor in the parathyroid gland. Cinacalcet, a calcimimetic drug, has proven its effectiveness for the treatment of SHP among patients in phase V of chronic renal disease. PATIENTS AND METHODS: This retrospective analysis included 48 patients with SHP who were treated with cinacalcet. The initial dose of 30 mg/d could be increased to 180 mg, administering calcitriol also, depending on the serum calcium and PTH levels. The objectives were a PTH level between 75 and 125 pg/mL or a decrease >40%, and a serum calcium level below 10.5 mg/dL. RESULTS: The average PTH at baseline was 244 pg/mL, decreasing to 131 pg/mL at 1 year (P < .01). The average calcium at baseline was 10.1 mg/dL descending to 9.2 mg/dL at 1 year (P < .01). Among patients with hypercalcemia, the calcium decreased from 11 to 9.6 mg/dL at 1 year (P < .01). Seventy percent of patients without hypercalcemia reached the desired value of PTH, and 100% of those with hypercalcemia. Among patients with hypercalcemia, the desired calcium level was reached in 91% of cases. Ten patients developed hypocalcemia. In 3 cases we stopped the treatment with cinacalcet due to digestive intolerance. CONCLUSIONS: Treatment with cinacalcet controlled hyperparathyroidism and hypercalcemia among patients with posttransplant SHP. It was a safe drug, with a low incidence of side effects.

Desorption isotherms and isosteric heat of desorption of previously frozen raw pork meat.

Some meat products involve drying previously frozen pork meat, which makes the knowledge of sorption characteristics very important for the design and management of meat dehydration processes. The sorption isotherms of raw pork meat from the Biceps femoris and Semimembranosus muscles were determined at four temperatures: 25, 30, 35 and 40°C. The experimental results were modelled using the GAB (Guggenheim, Anderson and De Boer) model. The effect of temperature was also taken into account to model the experimental sorption isotherms using four models (GAB, Oswin, Halsey and Henderson). The best results were provided by the GAB model. From the experimental sorption isotherms the isosteric heats of sorption were determined. For a moisture content higher than 0.15kgwater/kgdm, the isosteric heat of meat was similar to the latent heat of vaporization for pure water. For a lower moisture content, an increase in the isosteric heat was observed when the moisture content decreased.

Rehydration of freeze-dried and convective dried boletus edulis mushrooms: effect on some quality parameters.

Quality of rehydrated products is a key aspect linked to rehydration conditions. To assess the effect of rehydration temperature on some quality parameters, experiments at 20 and 70 degrees C were performed with convective dried and freeze-dried Boletus edulis mushrooms. Rehydration characteristics (through Peleg's parameter, k(1), and equilibrium moisture, W(e)), texture (Kramer), and microstructure (Cryo-Scanning Electron Microscopy) were evaluated. Freeze-dried samples absorbed water more quickly and attained higher W(e) values than convective dried ones. Convective dehydrated samples rehydrated at 20 degrees C showed significantly lower textural values (11.9 +/- 3.3 N/g) than those rehydrated at 70 degrees C (15.7 +/- 1.2 N/g). For the freeze-dried Boletus edulis, the textural values also exhibited significant differences, being 8.2 +/- 1.3 and 10.5 +/- 2.3 N/g for 20 and 70 degrees C, respectively. Freeze-dried samples showed a porous structure that allows rehydration to take place mainly at the extracellular level. This explains the fact that, regardless of temperature, freeze-dried mushrooms absorbed water more quickly and reached higher W(e) values than convective dried ones. Whatever the dehydration technique used, rehydration at 70 degrees C produced a structural damage that hindered water absorption; consequently lower W(e) values and higher textural values were attained than when rehydrating at 20 degrees C.

Detection of internal cracks in Manchego cheese using the acoustic impulse-response technique and ultrasounds.

Nowadays, due to the more global nature of markets, the commercialization of cheese relies on the high quality of the product. Internal defects such as cracks or flaws may affect quality. Two different nondestructive inspection techniques (ultrasonic and acoustic experiments) were used to detect cracks in Manchego cheese. The existence of small eyes in this type of cheese limited the use of ultrasonic pulse-echo experiments due to high scattering, and only cracks close to the surface of the cheese could be detected. The acoustic impulse-response technique, however, allowed us to study wheel pieces with cracks located elsewhere in the cheese. Two different impact probes (A and B) were assayed. The energy content of the acoustic spectrum was higher for cracked wheel pieces (7,116 and 17,520 V Hz(1/2) for probes A and B, respectively) than for normal ones (6,841 and 16,821 V Hz(1/2)). The differences were mainly found for frequencies higher than 150 Hz, which made the centroid for cracked pieces higher (162 and 170 Hz for probes A and B, respectively) than that for normal cheeses (132 and 148 Hz for probes A and B, respectively). Discriminant functions were developed to classify wheel pieces, and the input variables used were the acoustic parameters from the spectrum and the principal components extracted from the whole spectrum. The best classification procedure used the principal components from the principal components analysis of the spectrum for probe B. In this case, the 50 wheel pieces used in this study were correctly classified. These results showed that a simple and low-cost acoustic impulse-response technique could be used to detect cheese cracks, formed at different moments of Manchego cheese maturation.