Generation of Calpain-3 knock-out porcine embryos by CRISPR-Cas9 electroporation and intracytoplasmic microinjection of oocytes before insemination

Limb girdle muscular dystrophy type R1 (LGMDR1) is an autosomal recessive myopathy described in humans resulting from a deficiency of calpain-3 protein (CAPN3). This disease lacks effective treatment and an appropriate model, so the generation of KO pigs by CRISPR-Cas9 offers a way to better understand disease ethology and to develop novel therapies. Microinjection is the main method described for gene editing by CRISPR-Cas9 in porcine embryo, but electroporation, which allows handling more embryos faster and easier, has also recently been reported. The objective of the current study was to optimize porcine oocyte electroporation to maximize embryo quality and mutation rate in order to efficiently generate LGMDR1 porcine models. We found that the efficiency of generating CAPN3 KO embryos was highest with 4 electroporation pulses and double sgRNA concentration than microinjection. Direct comparison between microinjection and electroporation demonstrated similar rates of embryo development and mutation parameters. The results of our study demonstrate that oocyte electroporation, an easier and faster method than microinjection, is comparable to standard approaches, paving the way for democratization of transgenesis in pigs

ApiDB: integrated resources for the apicomplexan bioinformatics resource center

ApiDB () represents a unified entry point for the NIH-funded Apicomplexan Bioinformatics Resource Center (BRC) that integrates numerous database resources and multiple data types. The phylum Apicomplexa comprises numerous veterinary and medically important parasitic protozoa including human pathogenic species of the genera Cryptosporidium, Plasmodium and Toxoplasma. ApiDB serves not only as a database in its own right, but as a single web-based point of entry that unifies access to three major existing individual organism databases (, and CryptoDB.org), and integrates these databases with data available from additional sources. Through the ApiDB site, users may pose queries and search all available apicomplexan data and tools, or they may visit individual component organism databases

Comprehensive analysis of the major histocompatibility complex in systemic sclerosis identifies differential HLA associations by clinical and serological subtypes

Objective: The greatest genetic effect reported for systemic sclerosis (SSc) lies in the major histocompatibility complex (MHC) locus. Leveraging the largest SSc genome-wide association study, we aimed to fine-map this region to identify novel human leucocyte antigen (HLA) genetic variants associated with SSc susceptibility and its main clinical and serological subtypes. Methods: 9095 patients with SSc and 17 584 controls genome-wide genotyped were used to impute and test single-nucleotide polymorphisms (SNPs) across the MHC, classical HLA alleles and their composite amino acid residues. Additionally, patients were stratified according to their clinical and serological status, namely, limited cutaneous systemic sclerosis (lcSSc), diffuse cutaneous systemic sclerosis (dcSSc), anticentromere (ACA), antitopoisomerase (ATA) and anti-RNApolIII autoantibodies (ARA). Results: Sequential conditional analyses showed nine SNPs, nine classical alleles and seven amino acids that modelled the observed associations with SSc. This confirmed previously reported associations with HLA-DRB1∗11:04 and HLA-DPB1∗13:01, and revealed a novel association of HLA-B∗08:01. Stratified analyses showed specific associations of HLA-DQA1∗02:01 with lcSSc, and an exclusive association of HLA-DQA1∗05:01 with dcSSc. Similarly, private associations were detected in HLA-DRB1∗08:01 and confirmed the previously reported association of HLA-DRB1∗07:01 with ACA-positive patients, as opposed to the HLA-DPA1∗02:01 and HLA-DQB1∗03:01 alleles associated with ATA presentation. Conclusions: This study confirms the contribution of HLA class II and reveals a novel association of HLA class I with SSc, suggesting novel pathways of disease pathogenesis. Furthermore, we describe specific HLA associations with SSc clinical and serological subtypes that could serve as biomarkers of disease severity and progression

Utilising Drone Technology in Primatology for 3D Mapping

Emergent Unmanned Aerial System (or drone) technology allows the 3-dimensional mapping of forest landscapes, allowing a new perspective of arboreal primate habitat use. Utilising UASs in primatological studies enables the assessment of habitat quality for different arboreal primate species, the identification of discreet forms of anthropogenic disturbance (such as historical selective logging), and detailed investigation of canopy use by arboreal primate species. Combining 3D canopy structure with microclimate measurements, we can see how canopy structure buffers solar radiation and how arboreal species may be affected by future climate change. We present data on a study of the arboreal primate community in a lowland section of the Gunung Leuser Ecosystem in northern Sumatra, focusing on how 3D canopy structure effects ranging (siamang, Symphalangus syndactylus), different primate species’ population densities (lar gibbon, Hylobates lar, siamang and Thomas langur, Presbytis thomasi) and habitat selection (orang-utan, Pongo abelii and siamang) and how UAS technology can be utilised in other future studies; the potential opportunities, challenges and pitfalls

Unexplored olive cultivars from the Valencian Community (Spain): some chemical characteristics as a valorization strategy

[EN] The olive processing industry has till date been dominated by a small group of cultivars, leading to the possibility of some olive cultivars becoming extinct in the near future. In this study, we determined the composition of some chemical components in the olive oils from 31 minor olive cultivars of the Valencian Community. Our main aim was to identify suitable cultivars, which could produce differentiated olive oils, thus aiming towards their valorization. The average oil content of minor olive cultivars was found to be good, with some of them reporting approximately 60% (dry basis). On average, the total phenolic content was 229mg kg(-1), with cv. Mas Blanc reporting the highest content (570mg kg(-1)). Among the various tocopherols found in olives, -tocopherol was the main constituent, with a maximum concentration of 290.6mg kg(-1). Linoleic acid was the main polyunsaturated fatty acid and varied between 3.4% (cv. Del Pomet) and 16.9% (cv. Blanqueta Enguera). Special attention needs to be paid to the composition of sterols, since some olive oils exceeded the limits established for some sterols by the current European legislation. Some of the cultivars studied were highly productive, and originated differentiated olive oils with a rich composition of antioxidants and essential fatty acids. In some cases, these beneficial compounds were higher than those of commercial oils obtained from the most common cultivars worldwide. These results could contribute to the commercial exploitation of some of the studied cultivars.Salazar-García, DC.; Malheiro, R.; Pereira, JA.; López- Cortés, I. (2019). Unexplored olive cultivars from the Valencian Community (Spain): some chemical characteristics as a valorization strategy. European Food Research and Technology. 245(2):325-334. https://doi.org/10.1007/s00217-018-3164-7S3253342452Avidan B, Birger R, Abed-El-Hadi F, Salmon O, Hekster O, Friedman Y, Lavee S (2011) Adopting vigorous olive cultivars to high density hedgerow cultivation by soil applications of uniconazole, a gibberellin synthesis inhibitor. Span J Agric Res 9:821–830Barranco D, Rallo L (2000) Olive cultivars in Spain. HortTechnology 10:107–110Navero DB (2000) World catalogue of olive varieties. International Olive Oil Council, MadridBorges TH, Pereira JA, Cabrera-Vique C, Lara L, Oliveira AF, Seiquer I (2017) Characterization of Arbequina virgin olive oils produced in different regions of Brazil and Spain: physicochemical properties, oxidative stability and fatty acid profile. Food Chem 215:454–462Laroussi-Mezghani S, Le Dréau Y, Molinet J, Hammami M, Grati-Kamoun N, Artaud J (2016) Biodiversity of Tunisian virgin olive oils: varietal origin classification according to their minor compounds. Eur Food Res Technol 242:1087–1099Kosma I, Vavoura M, Kontakos S, Karabagias I, Kontominas M, Apostolos K, Badeka A (2016) Characterization and classification of extra virgin olive oil from five less well-known Greek olive cultivars. J Am Oil Chem Soc 93:837–848Reboredo-Rodríguez P, González-Barreiro C, Cancho-Grande B, Valli E, Bendini A, Toschi TG, Simal-Gandara J (2016) Characterization of virgin olive oils produced with autochthonous Galician varieties. Food Chem 212:162–171Kyçyk O, Aguillera MP, Gaforio JJ, Jiménez A, Beltrán G (2016) Sterol composition of virgin olive oil of forty-three olive cultivars from the World Collection Olive Germplasm Bank of Cordoba. J Sci Food Agric 96:4143–4150Ruiz-Domínguez ML, Raigón MD, Prohens J (2013) Diversity for olive oil composition in a collection of varieties from the region of Valencia (Spain). Food Res Int 54:1941–1949Mateos R, Dominguez MM, Espartero JL, Cert A (2003) Antioxidant effect of phenolic compounds, α-tocopherol, and other minor components in virgin olive oil. J Agric Food Chem 51:7170–7175Hermoso M, Uceda M, García A, Morales B, Frias ML, Fernández A (1991) Elaboración de Aceite de Calidad. Consejeria de Agricultura y Pesca, SevillaMalheiro R, Rodrigues N, Bissaro C, Leimann F, Casal S, Ramalhosa E, Pereira JA (2017) Improvement of sensorial and volatile profiles of olive oil by addition of olive leaves. Eur J Lipid Sci Technol 119:1700177Commission Delegated Regulation (EU) 2016/2095 amending Regulation (EEC) No 2568/91 on the characteristics of olive oil and olive-residue oil and on the relevant methods of analysis. Off J Eur Union L:326Slover HT, Thompson RH, Merola GV (1983) Tocopherol and sterol determination by capillary gas chromatography. J Am Oil Chem Soc 60:1524–1528Sousa A, Casal S, Malheiro R, Lamas H, Bento A, Pereira JA (2015) Aromatized olive oil: Influence of flavouring in quality, composition, stability, antioxidants, and antiradical potential. LWT Food Sci Technol 60:22–28Limón P, Malheiro R, Casal S, Acién-Fernández FG, Fernández-Sevilla JM, Rodrigues N, Cruz R, Bermejo R, Pereira JA (2015) Improvement of stability and carotenoids fraction of virgin olive oil by addition of microalgae Scenedesmus almeriensis extracts. Food Chem 175:203–211Motilva MJ, Tovar MJ, Romero MP, Alegre S, Girona J (2000) Influence of regulated deficit irrigation strategies applied to olive trees (Arbequina cultivar) on oil yield and oil composition during the fruit ripening period. J Sci Food Agric 80:2037–2043Palese AM, Nuzzo V, Favati F, Pietrafesa A, Celano G, Xiloyannis C (2010) Effects of water deficit on the vegetative response, yield and oil quality of olive trees (Olive europaea L., cv Coratina) grown under intensive cultivation. Sci Hortic 125:222–229Allalout A, Krichèn D, Methenni K, Taamalli A, Oueslati I, Daoud D, Zarrouk M (2009) Characterization of virgin olive oil from Super Intensive Spanish and Greek varieties grown in northern Tunisia. Sci Hortic 120:77–83Simopoulos AP, DiNicolantonio JJ (2016) The importance of a balanced ω-6 to ω-3 ratio in the prevention and management of obesity. Open Heart 3:e000385Marongui B, Özcan MM, Rosa A, Dessi MA, Piras A, AlJuhaimi F (2015) Monitoring of the fatty acid compositions of some olive oils. Riv Ital Sostanze Grasse 92:39–42Paiva-Martins F, Kiritsakis A (2017) Olive fruit and olive oil composition and their functionalcompounds. In: Kiritsakis A, Shahidi F (eds) Olives and olive oil as functional foods. Bioactivity, chemistry and processing. Wiley, Hoboken, pp 81–116Shahzad N, Khan W, Shadab MD, Ali A, Saluja SS, Sharma S, Al-Allaf FA, Abduljaleel Z, Ibrahim IAA (2017) Phytosterols as a natural anticancer agent: current status and future perspective. Biomed Pharmacol 88:786–794Covas MI, Ruiz-Gutiérrez V, de la Torre R, Kafatos A, Lamuela-Raventós RM, Osada J, Owen RW, Visioli F (2006) Minor components of olive oil: evidence to date of health benefits in humans. Nutr Rev 64:S20–S30Pirodi M, Albini A, Fabiani R, Giovannelli L, Luceri C, Natella F, Rosignoli P, Rossi T, Taticchi A, Servili M, Galli F (2017) Nutrigenomics of extra-virgin olive oil: a review. Biofactors 43:17–41Franco MN, Galeano-Díaz T, Sánchez J, De Miguel C, Martín-Vertedor D (2014) Total phenolic compounds and tocopherols profiles of seven olive oil varieties grown in the South-West of Spain. J Oleo Sci 63:115–125Aparicio R, Roda L, Albi MA, Gutiérrez F (1999) Effect of various compounds on virgin olive oil stability measured by Rancimat. J Agric Food Chem 47:4150–4155Bullota S, Celano M, Lepore SM, Montalcini T, Pujia A, Russo D (2014) Beneficial effects of the olive oil phenolic components oleuropein and hydroxytyrosol: focus on protection against cardiovascular and metabolic diseases. J Transl Med 12:1–9Krychene D, Salvador MD, Fregapane G (2015) Stability of virgin olive oil phenolic compounds during long-term storage (18 months) at temperatures of 5–50 °C. J Agric Food Chem 63:6779–6786Aparicio-Ruiz R, García-González DL, Oliver-Pozo C, Tena N, Morales MT, Aparicio A (2016) Phenolic profile of virgin olive oils with and without sensory defects: oils with non-oxidative defects exhibit a considerable concentration of phenols. Eur J Lipid Sci Technol 118:299–307Yorulmaz A, Poyrazoğlu ES, Özcan MM, Tekin A (2012) Phenolic profiles of Turkish olives and olive oils. Eur J Lipid Sci Technol 14:1083–1093Arslan A, Özcan MM (2011) Phenolic profile and antioxidant activity of olive fruits of the Turkish variety “Sarıulak” from different locations. Grasas Aceites 64:453–461Dağdelen A, Tümen G, Özcan MM, Dündar E (2013) Phenolics profiles of olive fruits (Olea europaea L.) and oils from Ayvalık, Domat and Gemlik varieties at different ripening stages. Food Chem 136:41–45Malheiro R, Rodrigues N, Pereira JA (2015). In: Boskou D (ed) Olive and olive oil bioactive constituents. AOCS Press, UrbanaCriado MN, Morelló JR, Motilva MJ, Romero MP (2004) Effect of growing area on pigment and phenolic fractions of virgin olive oils of the Arbequina variety in Spain. J Am Oil Chem Soc 81:633–640Gómez-Rico A, Fregapane G, Salvador MD (2008) Effect of cultivar and ripening on minor components in Spanish olive fruits and their corresponding virgin olive oils. Food Res Int 41:433–440Parkinson L, Cicerale S (2016) The health benefiting mechanisms of virgin olive oil phenolic compounds. Molecules 21:1734Lerma-García MJ, Herrero-Martínez JM, Ramis-Ramos G, Simó-Alfonso EF (2008) Prediction of the genetic variety of Spanish extra virgin olive oils using fatty acid and phenolic compound profiles established by direct infusion mass spectrometry. Food Chem 108:1142–1148Luna G, Morales MT, Aparicio R (2006) Characterisation of 39 varietal virgin olive oils by their volatile compositions. Food Chem 98:243–252Arslan A, Özcan MM (2011) Influence of growing area and harvest date on the organic acid composition of olive fruits from Gemlik variety. Sci Hortic 130:633–64

Amputation-free survival in 17,353 people at high risk for foot ulceration in diabetes:a national observational study

Acknowledgements Some of the data were presented as an abstract at the Diabetes UK Professional Conference in 2017. Diabetes data for Scotland are available for analysis by members of the Scottish Diabetes Research Network (SDRN) thanks to the hard work and dedication of NHS staff across Scotland who enter the data and people and organisations (the Scottish Care Information –Diabetes Collaboration (SCI-DC) Steering Group, the Scottish Diabetes Group, the Scottish Diabetes Survey Group, the managed clinical network managers and staff in each Health Board) involved in setting up, maintaining and overseeing SCI-DC. The SDRN receives core support from the Chief Scientist’s Office at the Scottish Government Health Department. Members of the Scottish Diabetes Research Network Epidemiology Group who do not qualify for authorship but who contributed to data collection include R. Lindsay (Institute of Cardiovascular and Medical Sciences, University of Glasgow, UK); J. McKnight (Western General Hospital, Edinburgh, UK); S. Philip (Institute of Applied Health Sciences, University of Aberdeen, UK); Members of the Scottish Diabetes Research Network Epidemiology Group who do not qualify for authorship but who contributed to data management include L. Blackbourn (Institute of Genetics and Molecular Medicine, University of Edinburgh, UK); B. Farran (Institute of Genetics and Molecular Medicine, University of Edinburgh, UK); D. McAllister (Institute of Health and Wellbeing, University of Glasgow, UK); P. McKeigue (Usher Institute of Population Health Sciences, University of Edinburgh, UK); S. Read (Usher Institute of Population Health Sciences, University of Edinburgh, UK).Peer reviewedPublisher PD

Potassium Starvation in Yeast: Mechanisms of Homeostasis Revealed by Mathematical Modeling

The intrinsic ability of cells to adapt to a wide range of environmental conditions is a fundamental process required for survival. Potassium is the most abundant cation in living cells and is required for essential cellular processes, including the regulation of cell volume, pH and protein synthesis. Yeast cells can grow from low micromolar to molar potassium concentrations and utilize sophisticated control mechanisms to keep the internal potassium concentration in a viable range. We developed a mathematical model for Saccharomyces cerevisiae to explore the complex interplay between biophysical forces and molecular regulation facilitating potassium homeostasis. By using a novel inference method (“the reverse tracking algorithm”) we predicted and then verified experimentally that the main regulators under conditions of potassium starvation are proton fluxes responding to changes of potassium concentrations. In contrast to the prevailing view, we show that regulation of the main potassium transport systems (Trk1,2 and Nha1) in the plasma membrane is not sufficient to achieve homeostasis

NQO1-Dependent Redox Cycling of Idebenone: Effects on Cellular Redox Potential and Energy Levels

Short-chain quinones are described as potent antioxidants and in the case of idebenone have already been under clinical investigation for the treatment of neuromuscular disorders. Due to their analogy to coenzyme Q10 (CoQ10), a long-chain quinone, they are widely regarded as a substitute for CoQ10. However, apart from their antioxidant function, this provides no clear rationale for their use in disorders with normal CoQ10 levels. Using recombinant NAD(P)H:quinone oxidoreductase (NQO) enzymes, we observed that contrary to CoQ10 short-chain quinones such as idebenone are good substrates for both NQO1 and NQO2. Furthermore, the reduction of short-chain quinones by NQOs enabled an antimycin A-sensitive transfer of electrons from cytosolic NAD(P)H to the mitochondrial respiratory chain in both human hepatoma cells (HepG2) and freshly isolated mouse hepatocytes. Consistent with the substrate selectivity of NQOs, both idebenone and CoQ1, but not CoQ10, partially restored cellular ATP levels under conditions of impaired complex I function. The observed cytosolic-mitochondrial shuttling of idebenone and CoQ1 was also associated with reduced lactate production by cybrid cells from mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) patients. Thus, the observed activities separate the effectiveness of short-chain quinones from the related long-chain CoQ10 and provide the rationale for the use of short-chain quinones such as idebenone for the treatment of mitochondrial disorders