Using an optimized transformation protocol we have studied the possible interactions between transforming plasmid DNA and the Hansenula polymorpha genome. Plasmids consisting only of a pBR322 replicon, an antibiotic resistance marker for Escherichia coli and the Saccharomyces cerevisiae LEU2 gene were shown to replicate autonomously in the yeast at an approximate copy number of 6 (copies per genome equivalent). This autonomous behaviour is probably due to an H. polymorpha replicon-like sequence present on the S. cerevisiae LEU2 gene fragment. Plasmids replicated as multimers consisting of monomers connected in a head-to-tail configuration. Two out of nine transformants analysed appeared to contain plasmid multimers in which one of the monomers contained a deletion. Plasmids containing internal or flanking regions of the genomic alcohol oxidase gene were shown to integrate by homologous single or double cross-over recombination. Both single- and multi-copy (two or three) tandem integrations were observed. Targeted integration occurred in 1-22% of the cases and was only observed with plasmids linearized within the genomic sequences, indicating that homologous linear ends are recombinogenic in H. polymorpha. In the cases in which no targeted integration occurred, double-strand breaks were efficiently repaired in a homology-independent way. Repair of double-strand breaks was precise in 50-68% of the cases. Linearization within homologous as well as nonhomologous plasmid regions stimulated transformation frequencies up to 15-fold.
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Carnitine/choline acyltransferases play diverse roles in energy metabolism and neuronal signalling. Our knowledge of their evolutionary relationships, important for functional understanding, is incomplete. Therefore, we aimed to determine the evolutionary relationships of these eukaryotic transferases. We performed extensivephylogenetic and intron position analyses. We found that mammalian intramitochondrial CPT2 is most closely related to cytosolic yeast carnitine transferases (Sc-YAT1 and 2), whereas the other members of the family are related to intraorganellar yeast Sc-CAT2. Therefore, the cytosolically active CPT1 more closely resembles intramitochondrial ancestors than CPT2. The choline acetyltransferase is closely related to carnitine acetyltransferase and shows lower evolutionary rates than long chain acyltransferases. In the CPT1 family several duplications occurred during animal radiation, leading to the isoforms CPT1A, CPT1B and CPT1C. In addition, we found five CPT1-like genes in Caenorhabditis elegans that strongly group to the CPT1 family. The long branch leading to mammalian brain isoform CPT1C suggests that either strong positive or relaxed evolution has taken place on this node. The presented evolutionary delineation of carnitine/choline acyltransferases adds to current knowledge on their functions and provides tangible leads for further experimental research.
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This article describes a relatively straightforward procedure to knock out the gene that encodes the invertase enzyme in baker’s yeast. The SUC2 gene, which encodes for the invertase enzyme, is knocked out by a single-step PCR knock out method. The knockout is subsequently confirmed at the genetic level by PCR and agarose gel electrophoresis. The knockout is confirmed at the biochemical level by measuring the activity of the invertase enzyme using a colorimetric assay. This tips and tools article describes an easily scalable, inexpensive, yet challenging research project helping undergraduate students at the Bachelor level to conceptualize the effect of the deletion of a gene encoding an enzyme.
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Op de HAS Hogeschool wordt al een aantal jaren voer voor vis en schaaldieren op basis van insecten vergeleken met regulier voer op basis van vismeel en/of visolie. Dit onderzoek wordt uitgevoerd in samenwerking met New Generation Nutrition. Resultaten laten zien dat garnalen gevoerd met voer op basis van insecten even goed groeien als bij regulier voer. Tot op heden is onbekend of het voer op basis van insecten gezondheidsrisico’s met zich meebrengt en dergelijk onderzoek komt na het aanstellen van Olga Haenen als lector Gezonde en Duurzame eiwitten in een stroomversnelling.
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Dairy products are known for their relatively low impact on the environment per unit of nutritional value. The carbon footprint of cheese from the Netherlands has been reduced in recent years by minimizing energy and water consumption. However,there are other options for further improving the sustainability of cheese production. The dairy research team at Van Hall Larenstein University of Applied Sciences is revealing new possibilities.
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There is a strong relation between food and identity. Especially when people move to another country, traditional food (or simply food from their country of origin), symbolizes a link with culture, communityand ethnic identity. As people move around the globe they introduce new foods in the places they land. Almere is becoming one of the largest majority minority cities of the Netherlands. Walking around thecity, the diversity of food ingredients and eating cultures as shown in shops and restaurants is immediately clear. The aim of this project was to get an insight into the diets of the residents of Almere so as to learn about eating patterns in a multicultural city and how multiculturality affects the diets of both newcomers and people who have been living here for generations.
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Five methods were compared to determine the best technique for accurate identification of coagulase-negative staphylococci (CoNS) (n=142 strains). MALDI-TOF MS showed the best results for rapid and accurate CoNS differentiation (correct identity in 99.3%). An alternative to this approach could be Vitek2 combined with partial tuf gene sequencing.
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Obesity and other lifestyle-related diseases are, amongst others, the result of an unbalanced diet and lifestyle. Excessive intake of energy, salt, saturated fat and sugar are leading to increased risk of chronic diseases, such as cardiovascular diseases, cancer and diabetes (WHO/FAO). Therefore, a healthier food intake (diet) is needed. But when is a food product healthier? From a nutritional perspective it is clear: the lower the levels of nutrients with a negative public health impact, the better the product fits in a healthy diet. However, when it comes to improving the health impact of the food supply through reformulation, other aspects are important as well. This article describes the ‘framework for product reformulation’, which integrates four essential disciplines: Nutrition & health, Foodtechnology, Legislation and Consumer perspective.
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This magazine presents the highlights of the applied research project “Inclusive and climate-smart business models in Ethiopian and Kenyan dairy valuechains (CSDEK)”. The CSDEK applied research project was conducted in six case study areas, three in Ethiopia and three in Kenya. At the time of publishing this magazine, research was still ongoing in some of the study areas. The projectteam and researchers hope to contribute to creating awareness of climatesmartdairy practices and development of the dairy sector in Ethiopia and Kenya. In two of the study areas, collaboration between VHL and dairy stakeholders will continue, preferably through local networks in a Living Lab approach.
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To accelerate differentiation between Staphylococcus aureus and Coagulase Negative Staphylococci (CNS), this study aimed to compare six different DNA extraction methods from 2 commonly used blood culture materials, i.e. BACTEC and Bact/ALERT. Furthermore, we analyzed the effect of reduced blood culture times for detection of Staphylococci directly from blood culture material. A real-time PCR duplex assay was used to compare 6 different DNA isolation protocols on two different blood culture systems. Negative blood culture material was spiked with MRSA. Bacterial DNA was isolated with: automated extractor EasyMAG (3 protocols), automated extractor MagNA Pure LC (LC Microbiology Kit MGrade), a manual kit MolYsis Plus, and a combination between MolYsis Plus and the EasyMAG. The most optimal isolation method was used to evaluate reduced bacterial culture times. Bacterial DNA isolation with the MolYsis Plus kit in combination with the specific B protocol on the EasyMAG resulted in the most sensitive detection of S.aureus, with a detection limit of 10 CFU/ml, in Bact/ALERT material, whereas using BACTEC resulted in a detection limit of 100 CFU/ml. An initial S.aureus load of 1 CFU/ml blood can be detected after 5 hours of culture in Bact/ALERT3D by combining the sensitive isolation method and the tuf LightCycler assay.
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