Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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Interactions and functionalities of the gut revealed by computational approaches
Benis, Nirupama - \ 2017
Wageningen University. Promotor(en): M.A. Smits; V.A.P. Martins dos Santos, co-promotor(en): D. Schokker; M. Suarez-Diez. - Wageningen : Wageningen University - ISBN 9789463434546 - 247
pigs - mice - digestive tract - digestive system - intestinal microorganisms - intestinal mucosa - computational science - immune system - feeds - animal nutrition - nutrition physiology - animal health - varkens - muizen - spijsverteringskanaal - spijsverteringsstelsel - darmmicro-organismen - darmslijmvlies - computational science - immuunsysteem - voer - diervoeding - voedingsfysiologie - diergezondheid

The gastrointestinal tract is subject of much research for its role in an organism’s health owing to its role as gatekeeper. The tissue acts as a barrier to keep out harmful substances like pathogens and toxins while absorbing nutrients that arise from the digestion of dietary components in in the lumen. There is a large population of microbiota that plays an important role in the functioning of the gut. All these sub-systems of the gastrointestinal tract contribute to the normal functioning of the gut. Due to its various functionalities, the gut is able to respond to different types of stimuli and bring the system back to homeostasis after perturbations.

The work done in this thesis uses several bioinformatic tools to improve our understanding of the functioning of the gut. This was achieved with data from model animals, mice and pigs which were subjected to changing environments before their gastrointestinal response was measured. Different types of stimuli were studied (eg, antibiotic exposure, changing diets and infection with pathogens) in order to understand the response of the gut to varying environments. This data was analysed using different data integration techniques that provide a holistic view of the gut response.

Vertical data integration techniques look for associations between different types of ~omics data to highlight possible interactions between the measured variables. Lateral integration techniques allow the study of one type of ~omics data over several time points or several experimental conditions. Using these techniques, we show proof of interactions between different sub-systems of the gut and the functional plasticity of the gut. Of the several hypotheses generated in this thesis we have validated several using existing literature and one using an in-vitro system. Further validation of these hypotheses will increase understanding of the responses of the gut and the interactions involved.

Ecophysiology of novel intestinal butyrate-producing bacteria
Bui, Thi Phuong Nam - \ 2016
Wageningen University. Promotor(en): Willem de Vos, co-promotor(en): Caroline Plugge. - Wageningen : Wageningen University - ISBN 9789462577015 - 202
butyrates - butyric acid bacteria - intestines - microbial interactions - faecal examination - mice - man - infants - genomics - intestinal physiology - microbial physiology - biochemical pathways - lysine - sugar - butyraten - boterzuurbacteriën - darmen - microbiële interacties - fecesonderzoek - muizen - mens - zuigelingen - genomica - darmfysiologie - microbiële fysiologie - biochemische omzettingen - lysine - suiker

The human intestinal tract harbours a trillion on microbial cells, predominantly anaerobes. The activity and physiology of these anaerobes is strongly associated with health and disease. This association has been investigated for a long time.However, this has not been fully understood. One of the reasons is the limited availability of cultured representatives. It is estimated that there may be more than 3000 species colonised in the gut of healthy individuals, however, only a bit over 1000 species have been isolated and characterised. Among the intestinal microbes, butyrate-producing bacteria are of special interest as the butyrate produced, is crucial to maintain a healthy gut. In addition, butyrate-producing bacteria have shown a reverse correlation with several intestinal diseases. In Chapter 2 we described a novel species Anaerostipes rhamnosivorans 1y2T isolated from an infant stool. This strain belonged to genus Anaerostipes within Clostridium cluster XIVa. A. rhamnosivorans had a capability of converting rhamnose into butyrate that is unique within intestinal butyrate-producing bacteria. The genomic analysis also revealed the entire rhamnose fermentation pathway as well as the acetyl-CoA pathway for butyrate production. This bacterium is able to produce butyrate from a wide range of sugars as well as lactate plus acetate. In Chapter 3, we described the microbial interactions between A. rhamnosivorans and Bacteriodes thetaiotaomicron in dietary pectins; Blautia hydrogenotrophica in lactate and small amount of acetate; Methanobrevibacter smithii in glucose. We observed that A. rhamnosivorans was able to benefit from its partners in all cocultures for butyrate production. This is likely due to its high metabolic flexibility. While the interaction between A. rhamnosivorans and B. thetaiotaomicron appeared as syntrophy, the interaction between A. rhamnosivorans and hydrogenotrohic microbes were cross-feeding type where hydrogen was transferred between two species. The latter resulted in an increase in butyrate level. In Chapter 4 we described a novel species Intestinimonas butyriciproducens SRB521T representing a novel genus Intestinimonas from a mouse caecum within Clostridium cluster IV. This bacterium produced butyrate and acetate as end products from Wilkins-Chalgren-Anaerobe broth.

Butyrate production is assumed to derive from carbohydrate employing acetyl-CoA pathway. No gut bacterium is known to convert proteins or amino acids to butyrate although butyrogenic pathways from amino acid degradation have been detected in the human gut using metagenomic approach. In Chapter 5 we discovered a novel butyrate synthesis pathway from the amino acid lysine and the Amadori product fructoselysine in Intestinimonas butyriciproducens AF211 that was isolated from human stool. This strain appeared to grow much better in lysine as compared to sugars although lysine and acetyl-CoA pathways were both detected in its complete genome. Moreover, the strain AF211 was able to metabolise efficiently fructoselysine into butyrate, and acetate was found to affect the fructoselysine fermentation, representing the impact of the environmental conditions where acetate is abundant in the gut. While the lysine pathway was found in the gut of many individuals, the fructoselysine pathway was present in only half of those samples. The finding that strain I. butyriciproducens AF211 is capable of the butyrogenic conversion of amino acid lysine and fructoselysine, an Amadori product formed in heated foods via the Maillard reaction, indicated a missing link that coupling protein metabolism and butyrate formation. As this Amadori product has been implicated to play a role in aging process, the use of strain AF211 as fructoselysine clearance in the gut needs further investigation. In Chapter 6 we performed genomic and physiological comparison between the I. butyriciproducens strain AF211 (human isolate) and SRB521T (mouse isolate). I. butyriciproducens was the most abundant species within the Intestinimonas genus and highly prevalent in humans based on metadata analysis on 16S amplicons. We confirmed that the butyrogenesis from lysine was a shared characteristic between the two I. butyriciproducens strains. We also observed the host specific features including tolerance to bile, cellular fatty acid composition, more efficient capability of converting sugars into butyrate, especially galactose and arabinose, in the human strain AF211. In addition, genomic rearrangements as well as variations in bacteriophages differed among strains.

Terugkerende muizenplagen in Nederland : inventarisatie, sturende factoren en beheersing
Wymenga, E. ; Latour, J. ; Beemster, N. ; Bos, D. ; Bosma, N. ; Haverkamp, J. ; Hendriks, R.F.A. ; Roerink, G.J. ; Kasper, G.J. ; Roelsma, J. ; Scholten, S. ; Wiersma, P. ; Zee, E. van der - \ 2016
Altenburg & Wymenga (A&W-rapport 2123) - 137 p.
muizen - woelmuizen - plagen - schadelijke dieren - schade - graslanden - knaagdierenbestrijding - dijken - waterschappen - mice - voles - pests - noxious animals - damage - grasslands - rodent control - dykes - polder boards
2014 en 2015 zullen de geschiedenis in gaan als jaren met een uitzonderlijk grote veldmuizenplaag in Fryslân en op beperkte schaal elders in Nederland. In de loop van de zomer van 2014 meldden agrariërs schade aan graslanden. De werkelijke omvang bleek pas in het najaar en de winter van 2014-2015 en was zonder precedent. Ook uit andere provincies kwamen dergelijke meldingen. De economische schade werd door LTO Noord berekend op 73 miljoen euro (de Boer 2015). Uit een schouw van Wetterskip Fryslân bleek dat ook veel waterkeringen te maken hadden met muizenschade. Daarnaast was het waterschap beducht op een mogelijk verhoogde uitspoeling van meststoffen vanuit polders naar het oppervlaktewater. Bovengenoemde zaken waren aanleiding voor verschillende organisaties om in samenwerking met LTO Noord en het Actiecomité een onderzoek te starten naar de achtergronden van de terugkerende muizenplagen en de mogelijke maatregelen om die te beheersen. Ook de STOWA - Stichting Toegepast Onderzoek Waterbeheer – richtte haar vizier op de muizenplaag, om uit te zoeken in hoeverre de recente muizenplaag voor waterschappen relevant was. De overkoepelende doelstelling van beide onderzoeken was om kennis en bouwstenen te leveren voor een strategie om in de toekomst muizenplagen vroegtijdig te signaleren en de schade te beheersen.
Bosmuizen verlicht [door studenten / promovendi]
Sindram, Janneke - \ 2015
De Levende Natuur 116 (2015)2. - ISSN 0024-1520 - p. 71 - 72.
forest ecology - artificial light - fauna - mice - adverse effects - bosecologie - kunstlicht - muizen - nadelige gevolgen
Kunstlicht heeft op allerlei dieren invloed. Daar zijn veel voorbeelden van te verzinnen. Gecombineerd met het feit dat het in Europa 's nachts steeds lichter is geworden, mag de conclusie getrokken worden dat de natuur hier veel gevolgen van ondervindt. Nader onderzoek wordt verricht door Janneke Sandram, begeleid vanuit Wageningen Universiteit en NIOO.
Hypothalamic regulation of food intake during cancer
Dwarkasing, J.T. - \ 2015
Wageningen University. Promotor(en): Renger Witkamp, co-promotor(en): Klaske van Norren; Mark Boekschoten. - Wageningen : Wageningen University - ISBN 9789462575486 - 147
hypothalamische regulatie - anorexia - eetlustcontrole - voedselopname - cancer - chronische ziekten - diermodellen - muizen - serotonine - hypothalamic regulation - anorexia - appetite control - food intake - cancer - chronic diseases - animal models - mice - serotonin

Appetite is often reduced in patients with chronic illness, including cancer.

Cancer anorexia, loss of appetite, frequently co-exists with cachexia, and the combined clinical picture is known as anorexia-cachexia syndrome. In patients suffering from this syndrome, anorexia considerably contributes to the progression of cachexia, and strongly impinges on quality of life. Inflammatory processes in the hypothalamus are considered to play a crucial role in the development of disease-related anorexia.

The main aim of this thesis was to further elucidate crucial processes involved in the pathogenesis of anorexia in cancer. To investigate mechanisms specifically involved in cancer anorexia, we used two tumour mouse models with opposing food intake behaviours: a C26-colon adenocarcinoma model with increased food intake and a Lewis lung carcinoma model with decreased food intake. In both models, tumour-induced cachexia (body wasting) was strongly present. The contrast in food intake behaviour between tumour-bearing (TB) mice in response to growth of the two different tumours was used to distinguish processes involved in cachexia from those specifically involved in anorexia.

The hypothalamus was used for transcriptomic analysis (Affymetrix chips). We found expression of genes involved in serotonin signalling in the hypothalamus to be differentially regulated between the two tumour models. Furthermore, transcriptional activity of genes involved in serotonin signalling were inversely associated with food intake behaviour. Surprisingly, we also found a strong increase in gene expression of NPY and AgRP, potent orexigenic neuropeptides, in both models, meaning that their expression did not reflect food intake behaviour. However, NPY has also been described to regulate energy storage. Therefore, we hypothesized that this upregulation of NPY/AgRP corresponded to weight loss, which was severe in both tumour models.

Using hypothalamic cell lines we further explored how serotonin might act on food intake regulatory pathways. We showed that serotonin was able to inhibit neuronal NPY secretion, while not affecting gene expression. Inflammatory markers IL-6 and TNFα were also measured in plasma and it was found that C26 TB mice had a lower inflammatory response than LL TB mice. These differences in inflammatory response could be implicated in the differences in feeding behaviour and serotonin signalling between C26 and LL TB mice. We therefore investigated the direct influence of inflammation on hypothalamic serotonin turnover and its contribution to the development of anorexia. To this end, different doses of TNF and IL-6 were administered by injection to healthy mice, inducing an acute inflammatory response. The injected cytokine doses were estimated from their corresponding plasma levels measured in tumour bearing (TB) mice. Also in this cytokine induced-anorexia model, where anorexia was exclusively induced by an inflammatory response, serotonin metabolism in the hypothalamus was affected. Both TNF and IL-6 increased hypothalamic serotonin turnover while also inducing anorectic behaviour. Furthermore, the effect of cytokines on increasing serotonin turnover was supported by in vitro experiments with hypothalamic neuronal cell lines.

In conclusion, we identified hypothalamic serotonin signalling to play a major role in the decrease in food intake during cancer. Serotonin signalling itself is modulated by inflammatory mediators. Therefore, hypothalamic inflammation is an important trigger in the failure of hypothalamic food-intake regulation, probably by affecting serotonergic signalling, which acts as an upstream modulator of various orexigenic and anorexigenic systems.

Mucus and gut barrier in health and disease
Sovran, B. - \ 2015
Wageningen University. Promotor(en): Jerry Wells; P. de Vos, co-promotor(en): J. Dekker. - Wageningen : Wageningen University - ISBN 9789462574892 - 233
slijm - spijsverteringskanaal - darmen - muizen - probiotica - eilandjes van peyer - colitis - transcriptomen - immunohistologie - veroudering - geslacht (sex) - homeostase - gezondheid - ziekten - mucus - digestive tract - intestines - mice - probiotics - peyer patches - colitis - transcriptomes - immunohistology - senescence - sex - homeostasis - health - diseases

This publication describes his work as a PhD student in the Host-Microbe Interactomics Chair group at Wageningen University within the Gastrointestinal Health theme. It has been completed under the supervision of Prof. Dr Jerry M Wells, Dr Jan Dekker and the TIFN project leader, Prof. Dr Paul de Vos.

Mucus serves as a protective layer between the intestinal content and the intestinal wall. It facilitates the passage of the luminal content through the intestine, reducing the risk of mechanical damage to the intestinal epithelium. The overarching goal of this thesis was to investigate the role of mucus in the maintenance of the intestinal immune barrier and the effects of ageing and gender differences on mucus production and the gut barrier.

We found by using a mouse model that decreased mucus production leads to changes in microbiota and mucosal stress responses, without the appearance of pathology, demonstrating the importance of mucus in intestinal homeostasis. The mucus barrier was shown to deteriorate during aging but this could be prevented with specific probiotics. Furthermore gender-specific differences in the effects of ageing on the mucosal barrier were found. Increased knowledge on these mechanisms might contribute significantly to disease prevention and treatment, for instance by optimizing gender-specific dietary and pharmacological requirements.

The study presented in this thesis was performed within the framework of Top Institute Food and Nutrition, within the GH002 project.

Gut microbiota facilitates dietary heme-induced epithelial hyperproliferation by opening the mucus barrier in colon
IJssennagger, N. ; Belzer, C. ; Hooiveld, G.J.E.J. ; Dekker, J. ; Mil, S.W.C. ; Müller, M.R. ; Kleerebezem, M. ; Meer, R. van der - \ 2015
Proceedings of the National Academy of Sciences of the United States of America 112 (2015)32. - ISSN 0027-8424 - p. 10038 - 10043.
colorectal-cancer - red meat - mice - mucin - fat - susceptibility - cytotoxicity - expression - inhibitor - bacterial
Colorectal cancer risk is associated with diets high in red meat. Heme, the pigment of red meat, induces cytotoxicity of colonic contents and elicits epithelial damage and compensatory hyperproliferation, leading to hyperplasia. Here we explore the possible causal role of the gut microbiota in heme-induced hyperproliferation. To this end, mice were fed a purified control or heme diet (0.5 µmol/g heme) with or without broad-spectrum antibiotics for 14 d. Heme-induced hyperproliferation was shown to depend on the presence of the gut microbiota, because hyperproliferation was completely eliminated by antibiotics, although heme-induced luminal cytotoxicity was sustained in these mice. Colon mucosa transcriptomics revealed that antibiotics block heme-induced differential expression of oncogenes, tumor suppressors, and cell turnover genes, implying that antibiotic treatment prevented the heme-dependent cytotoxic micelles to reach the epithelium. Our results indicate that this occurs because antibiotics reinforce the mucus barrier by eliminating sulfide-producing bacteria and mucin-degrading bacteria (e.g., Akkermansia). Sulfide potently reduces disulfide bonds and can drive mucin denaturation and microbial access to the mucus layer. This reduction results in formation of trisulfides that can be detected in vitro and in vivo. Therefore, trisulfides can serve as a novel marker of colonic mucolysis and thus as a proxy for mucus barrier reduction. In feces, antibiotics drastically decreased trisulfides but increased mucin polymers that can be lysed by sulfide. We conclude that the gut microbiota is required for heme-induced epithelial hyperproliferation and hyperplasia because of the capacity to reduce mucus barrier function.
Deze zomer veel tekenbeten verwacht
Vliet, A.J.H. van; Wijngaard, K. van den - \ 2015
Nature Today (2015)9 juli.
tekenbeten - ziekten overgebracht door teken - lyme-ziekte - muizen - borrelia burgdorferi - tekenbesmettingen - registratie - tick bites - tickborne diseases - lyme disease - mice - tick infestations - registration
Naar verwachting zullen komende maand een half miljoen mensen door een teek gebeten worden. Afgelopen maand werden aanzienlijk meer tekenbeten gemeld via Tekenradar.nl dan in eerdere jaren. Teken kunnen met een beet de ziekte van Lyme overbrengen. Het is daarom belangrijk dat mensen zich na een bezoek ‘in het groen’ goed controleren op tekenbeten. Mensen die toch de ziekte van Lyme oplopen, kunnen meedoen aan het onderzoek naar langdurige klachten na Lyme. De gezondheid van de deelnemers wordt een jaar lang gevolgd. Dit jaar kunnen nog 500 mensen meedoen aan het onderzoek.
Molecular and physiological assessment of metabolic health : adipose tissue, transcriptome analysis and challenge tests
Duivenvoorde, L.P.M. - \ 2015
Wageningen University. Promotor(en): Jaap Keijer, co-promotor(en): Evert van Schothorst. - Wageningen : Wageningen University - ISBN 9789462573017 - 186
muizen - metabolisme - gezondheid - vetweefsel - transcriptomen - stofwisselingsstoornissen - fysiologie - laboratoriumdieren - mice - metabolism - health - adipose tissue - transcriptomes - metabolic disorders - physiology - laboratory animals

Summary of main findings

Maintenance of metabolic health not only ensures that energy is made available in times of need and stored in times of excess, but also prevents resistance to nutritional cues, ectopic lipid accumulation and dysfunction of metabolic organs. The proportion of humans that is at risk for reduced metabolic health increases worldwide due to the current epidemic of obesity and the increase in both mean and maximum life span. Better understanding of the various factors that influence metabolic health may offer opportunities to fight this threat to human health. This thesis aims to assess metabolic health using transcriptome analysis and non-invasive challenge tests. Special focus is on the development and validation of InCa-based non-invasive challenge tests. In most chapters of this thesis white adipose tissue (WAT) formed the major organ of interest because of its key role in whole-body energy homeostasis. WAT function was, among others, studied with whole-genome gene expression analysis, which, compared to single parameter analysis, extends the scale and depth of understanding biological processes.

Metabolic health was also quantified as metabolic flexibility, with the use of non-invasive, indirect calorimetry (InCa) based challenge tests. One of the InCa based challenge tests described in this thesis, the oxygen restriction (OxR) challenge, is a novel approach to investigate metabolic flexibility in mice. In each study, OxR was applied acute ([O2] reduction within 30 minutes) and for a short period of 6 hours in fasted mice. The other two InCa-based challenge tests: fasting and re-feeding and fasting and glucose consumption are nutrient-based and were described previously, although in different formats and settings.

In chapter 2 we demonstrate that dietary restriction on a high-fat diet (HF-DR) improves metabolic health of mice compared with mice receiving the same diet on an ad libitum basis (HF-AL). Already after five weeks of restriction, the serum levels of cholesterol and leptin were significantly decreased in HF-DR mice, whereas their glucose tolerance and serum adiponectin levels were increased. The body weight and measured serum parameters remained stable in the following 7 weeks of restriction, implying metabolic adaptation. To understand the molecular events associated with this adaptation, we analysed gene expression in WAT with whole genome microarrays. HF-DR strongly influenced gene expression in WAT; in total, 8643 genes were differentially expressed between both groups of mice, with a major role for genes involved in lipid metabolism and mitochondrial functioning. DR also increases mitochondrial density in WAT. These results show that WAT, indeed, has an important role in the improvement of metabolic health of dietary restricted mice and suggest that the development of substrate efficiency plays an important role in the observed changes in health status. Finally, mitochondrial density might be used as a marker for WAT health status.

Chapter 3 shows how indirect calorimetry can be used to noninvasively assess metabolic and age-related flexibility in mice. In this study, we tested the sensitivity and response stability over time of three InCa-based treatments in old versus adult mice. For the first treatment, diurnal patterns of respiratory exchange ratio were followed for 24 hours under standard conditions. For the second and third treatment, which were both based on a challenge approach, mice were fasted and either received a glucose bolus to test switch-effectiveness from fat to glucose oxidation (Treatment 2), or were exposed to oxygen restriction (OxR, Treatment 3) in the InCa system, which was introduced as a novel approach to asses metabolic flexibility. Opposite to the mice that were dietary restricted (chapter 2), aging appeared to increase adiposity and decrease WAT mitochondrial density, which further suggests that WAT mitochondrial density might be used as a marker for WAT health. We observed that the test results of the first treatment were not stable between test periods, possibly because of behavioural differences within the group of old mice between both measurements. For the second treatment, no differences between groups were observed. With Treatment 3, however, stable significant differences could be detected: old mice did not maintain reduced oxygen consumption under OxR during both measurements, whereas adult mice did. Further biochemical and gene expression analyses showed that OxR affected glucose and lactate homeostasis in liver and WAT of adult mice, supporting the observed differences in oxygen consumption. This was the first study to show that InCa analysis of the response to OxR is a sensitive and reproducible treatment to noninvasively measure age-impaired metabolic health in mice. Evaluation of metabolic health under non-challenged conditions may be confounded by behavioural-induced variation between animals

The study described in chapter 4 followed up on the promising results that were obtained with the OxR challenge in chapter 3. In this study we tested whether OxR can also be used to reveal diet-induced health effects in an early stage. Early detection of diet-induced health effects might shorten animal experiments and reduce costs and age-related variation. Timely identification may increase options for reversal. Mice were exposed to a low-fat (LF) or high-fat (HF) diet for only 5 days, after which they were exposed to OxR or remained under normoxic conditions. The response to OxR was assessed by calorimetric measurements, followed by analysis of gene expression in liver and WAT. A novelty described in this chapter was the analysis of serum markers for protein glycation and oxidation, to detect differences in the response to OxR between LF and HF mice. Although HF feeding increased body weight, HF and LF mice did not differ in indirect calorimetric values under normoxic conditions and in a fasting state. Exposure to OxR however, increased oxygen consumption and lipid oxidation in HF mice versus LF mice. Furthermore, OxR induced gluconeogenesis and an antioxidant response in the liver of HF mice, whereas it induced de novo lipogenesis and an antioxidant response in eWAT of LF mice, indicating that HF and LF mice differed in their adaptation to OxR. OxR also increased serum markers of protein glycation and oxidation in HF mice, whereas these changes were absent in LF mice. From this study we concluded that OxR is a promising new method to test food products on potential beneficial effects for metabolic health.

The study described in chapter 5 aimed to assess differences in metabolic health of mice on iso-caloric diets differing in fatty acid composition using the OxR challenge. We also implemented a fasting and re-feeding challenge. One diet, the HFpu diet, predominantly contained poly-unsaturated fatty acids (PUFAs), which are considered to be healthier than saturated fatty acids (SFAs) that mainly made up the fat component of the second diet, the HFs diet. Since health effects of fatty acids also depend on the ratio of dietary omega-6 to omega-3 PUFAs (n6/n3 ratio), this ratio was kept similar between both diets. Mice received the isocaloric high-fat diets for six months, during and after which several biomarkers for health were measured. We found that HFpu and HFs diets only induce minor differences in static health markers: HFpu and HFs mice did not differ in body weight, total adiposity, adipose tissue health, serum adipokines, whole body energy balance, or circadian rhythm. HFpu and HFs mice also had a similar glucose tolerance, even though HFs mice had more triglycerides in liver and skeletal muscle and larger adipocytes in the eWAT depot. Interestingly, HFs mice were less flexible in their response to both fasting and re-feeding and OxR, which shows the relevance and sensitivity of InCa-based challenge tests. We concluded that InCa-based challenge tests are a valuable contribution to the analysis of metabolic health in mice. Challenge tests in the InCa system may, furthermore, reveal relevant consequences of small changes in metabolic health status, such as adipocyte hypertrophy or ectopic lipid storage.

Chapter 6 describes an in-depth study to the response to OxR both at whole body level using InCa and serum metabolomics (amino acids and (acyl)carnitines) and at WAT level using transcriptomics and the analysis of amino acid and (acyl)carnitine levels. Serum and tissue amino acids levels indicate the level of protein catabolism and certain amino acids are, typically, increased in obese individuals. Serum and tissue (acyl)carnitine levels indicate the rate and completeness of mitochondrial fatty acid oxidation; serum acylcarnitine levels are significantly increased in individuals that suffer from ambient oxygen restriction. The metabolic adaptation to OxR was studied in diet-induced moderately obese mice that received a high-fat diet (HFpu diet, as in chapter 5) for 6 weeks, which is expected to lead to WAT expansion and possibly to reduce oxygen availability in WAT. We found that OxR reduced mitochondrial oxidation at whole-body level, as shown by a reduction in whole-body oxygen consumption and an increase in serum long-chain acylcarnitine levels. WAT did not seem to contribute to this serum profile, since only short-chain acylcarnitines were increased in WAT and gene expression analysis indicated an increase in mitochondrial oxidation, based on coordinate down-regulation of Sirt4, Gpam and Chchd3/Minos3. In addition, OxR did not induce oxidative stress in WAT, but increased molecular pathways involved in cell growth and proliferation. OxR increased levels of tyrosine, lysine and ornithine in serum and of leucine/isoleucine in WAT. This study shows that OxR limits oxidative phosphorylation at whole-body level, but in WAT compensatory mechanisms seem to operate. The down-regulation of the mitochondria-related genes Sirt4, Gpam, and Chchd3 may be considered as a biomarker profile for WAT mitochondrial reprogramming in response to acute exposure to limited oxygen availability.

To conclude, the work presented in this thesis provides more insight in the analysis of metabolic health in mice with the use of transcriptome analysis and InCa-based challenge tests. We show that non-invasive tests using the InCa-system are more likely to reveal differences in metabolic flexibility than invasive challenge tests, such as the oral glucose tolerance test. Furthermore, we show that the challenge approach is more sensitive than analysis of metabolic health under non-challenged (free-feeding) conditions. Transcriptome analysis proved to be very valuable to provide in-depth molecular understanding of the mechanisms underlying reduced or improved metabolic health. Ideally, transciptomic or metabolomic approaches should be integrated with InCa-based challenge tests to further extent physiological understanding of diet-induced health effects.

p21 Ablation in Liver Enhances DNA Damage, Cholestasis, and Carcinogenesis
Ehedego, H. ; Boekschoten, M.V. ; Hu, W. ; Doler, C. ; Haybaeck, J. ; Gassler, N. ; Muller, M.R. ; Liedtke, C. ; Trautwein, C. - \ 2015
Cancer Research 75 (2015)6. - ISSN 0008-5472 - p. 1144 - 1155.
kinase inhibitor p21 - human hepatocellular-carcinoma - cell-cycle progression - rad51 overexpression - expression - p21(waf1/cip1) - mice - inflammation - regeneration - repair
Genetic mouse studies suggest that the NF-¿B pathway regulator NEMO (also known as IKK¿) controls chronic inflammation and carcinogenesis in the liver. However, the molecular mechanisms explaining the function of NEMO are not well defined. Here, we report that overexpression of the cell-cycle regulator p21 is a critical feature of liver inflammation and carcinogenesis caused by the loss of NEMO. NEMO¿hepa mice develop chronic hepatitis characterized by increased hepatocyte apoptosis and proliferation that causes the development of fibrosis and hepatocellular carcinoma (HCC), similar to the situation in human liver disease. Having identified p21 overexpression in this model, we evaluated its role in disease progression and LPS-mediated liver injury in double mutant NEMO¿hepa/p21-/- mice. Eight-week-old NEMO¿hepa/p21-/- animals displayed accelerated liver damage that was not associated with alterations in cell-cycle progression or the inflammatory response. However, livers from NEMO¿hepa/p21-/- mice displayed more severe DNA damage that was further characterized by LPS administration correlating with higher lethality of the animals. This phenotype was attenuated by genetic ablation of the TNF receptor TNF-R1 in NEMO¿hepa/p21-/- mice, demonstrating that DNA damage is induced via TNF. One-year-old NEMO¿hepa/p21-/- mice displayed greater numbers of HCC and severe cholestasis compared with NEMO¿hepa animals. Therefore, p21 overexpression in NEMO¿hepa animals protects against DNA damage, acceleration of hepatocarcinogenesis, and cholestasis. Taken together, our findings illustrate how loss of NEMO promotes chronic liver inflammation and carcinogenesis, and they identify a novel protective role for p21 against the generation of DNA damage.
Vreterij in consumptie-aardappelen : inventarisatie van vreterij op aardappelpercelen en in de bewaring
Rozen, K. van; Huiting, H.F. - \ 2015
Lelystad : Praktijkonderzoek Plant & Omgeving, onderdeel van Wageningen UR, Business Unit PPO-agv - 49
akkerbouw - aardappelen - diagnose - gewasbescherming - plagen - agriotes - athous - melolontha melolontha - phyllopertha horticola - naaktslakken - deroceras reticulatum - arion - agrotis - diplopoda - muizen - arable farming - potatoes - diagnosis - plant protection - pests - slugs - mice
In 2012 en 2013 is onderzoek verricht naar de symptomen en de impact van gaten in knollen. Percelen zijn bezocht om de mate van vraat aan de knollen te beoordelen en de oorzaak vast te stellen. Dit leverde schade op door het hele land. Oost-Nederland is de regio waar het meest onderzoek heeft plaatsgevonden. Daar zijn de meeste percelen bezocht en bemonsterd. De meeste schade werd veroorzaakt door ritnaalden. De symptomen zijn beschreven en afgebeeld. De symptomen zijn vergeleken met drycore, een aantasting wat ook gaatjes tot gevolg heeft. Enkele percelen met engerlingen en slakken zijn aangetroffen. Van enkele andere aantasters zijn de symptomen beschreven.
Unravelling mechanisms of dietary flavonoid-mediated health effects: effects on lipid metabolism and genotoxicity
Hoek-van den Hil, E.F. - \ 2015
Wageningen University. Promotor(en): Ivonne Rietjens; Jaap Keijer, co-promotor(en): Peter Hollman. - Wageningen : Wageningen University - ISBN 9789462573031 - 157
flavanoïden - flavonoïden - vetzuren - quercetine - flavonolen - lichaamsgewicht - lipidenmetabolisme - hart- en vaatziekten - lever - vetweefsel - gezondheid - genotoxiciteit - voeding - muizen - flavanoids - flavonoids - fatty acids - quercetin - flavonols - body weight - lipid metabolism - cardiovascular diseases - liver - adipose tissue - health - genotoxicity - nutrition - mice

Summary

Consumption of foods containing flavonoids is associated with a reduced risk of cardiovascular diseases (CVD), possibly by lipid-lowering effects. On the other hand, for one of these flavonoids, quercetin, also genotoxicity was shown especially in in vitro bioassays. Therefore, the first aim of this thesis was to identify mechanisms underlying potential beneficial health effects of flavonoids. The focus was on hepatic lipid metabolism and circulating lipids and a molecular and physiological approach was used. Secondly, we aimed to study the potential in vivo genotoxic effects of quercetin by transcriptome analyses in liver and small intestine, since these represent the tissues of first contact exposed to relatively high levels upon oral intake of flavonoids.

Circulating lipids are important CVD-related risk markers, which are in general determined with commercially available enzyme-based assays. However, the usual enzyme in these assays, peroxidase, has previously been reported to be inhibited by flavonoids. Therefore, we have studied in chapter 2 whether these assays can adequately be used in flavonoid research. We observed that various flavonoid aglycones interfere with peroxidase used in triglycerides (TG) and free fatty acids (FFA) enzymatic assays, reporting incorrect lower TG and FFA levels than actually present. Furthermore, addition of metabolites such as isorhamnetin or quercetin-3-O-glucuronide, the major metabolite of quercetin in human and rat plasma, to murine serum also resulted in a significant reduction of the detected TG levels, while a trend was seen towards reduced FFA levels. It can be concluded that when applying these biochemical assays, vigilance is needed and alternative analytical methods assessing FFA or TG levels should preferably be applied for studying the biological effects of flavonoids on TG and FFA levels.

In chapter 3 mechanistic and physiological effects of quercetin on hepatic lipid metabolism were studied. C57BL/6JOlaHsd male adult mice received a mild high-fat (30 en%) diet without or with supplementation of 0.33% (w/w) quercetin for 12 weeks. Gas chromatography and 1H-NMR were used to quantitatively measure serum lipid profiles. Whole genome microarray analysis of liver tissue was used to identify potential mechanisms underlying altered circulating lipid levels by quercetin supplementation. Body weight, energy intake and hepatic lipid accumulation did not differ significantly between the quercetin and the control group. In serum of quercetin-fed mice, TG levels were decreased by 14% (p<0.001) and total poly unsaturated fatty acids (PUFA) levels were increased by 13% (p<0.01). Levels of palmitic acid, oleic acid, and linoleic acid were all decreased by 9-15% (p<0.05) in quercetin-fed mice. Both palmitic acid and oleic acid can be oxidized by omega-oxidation. Gene expression profiling showed indeed that quercetin increased hepatic lipid metabolism, especially omega-oxidation. At the gene level, this was reflected by the up-regulation of cytochrome P450 (Cyp) 4a10, Cyp4a14, Cyp4a31 and Acyl-CoA thioesterase 3 (Acot3). Two relevant regulators, cytochrome P450 oxidoreductase (Por, rate limiting for cytochrome P450 activities) and the transcription factor constitutive androstane receptor (Car; official symbol Nr1i3) were also up- regulated in the quercetin-fed mice. We concluded that quercetin intake increased hepatic lipid omega-oxidation and lowered corresponding circulating lipid levels, which may contribute to potential beneficial effects of quercetin on CVD.

Subsequently, in chapter 4 effects of quercetin supplementation were studied in mice given a high-fat (40 en%) background diet. The set-up of the experiment was the same as in chapter 3, with the exception of the background diet that was used, which was different in fat content and composition. This high-fat diet-induced body weight gain, and serum and hepatic lipid accumulation, which are all known risk factors for CVD. The aim of this study was to investigate the effects and underlying molecular mechanisms of the effects of the flavonoid quercetin on hepatic lipid metabolism in mice given this high-fat diet background. C57BL/6JOlaHsd male adult mice received the high-fat diet without or with supplementation of 0.33% (w/w) quercetin for 12 weeks. Body weight gain was 29% lower in quercetin fed mice versus control mice (p<0.01), while the energy intake was not significantly different. Quercetin supplementation lowered high-fat diet-induced hepatic lipid accumulation to 29% of the amount present in the control mice (p<0.01). 1H-NMR serum lipid profiling revealed that the supplementation also significantly lowered high-fat diet-induced increases in serum lipid levels. Global gene expression profiling of liver showed that cytochrome P450 2b (Cyp2b) genes, key target genes of the transcription factor Car, were down-regulated. However, the induction of omega-oxidation observed by quercetin supplementation to a mild high-fat (30en%) diet (chapter 3), was not observed this time with the high-fat (40en%) diet. Cumulatively, quercetin decreased high-fat diet-induced body weight gain, hepatic lipid accumulation and serum lipid levels. This was accompanied by regulation of cytochrome P450 2b genes in liver, which are considered to be under transcriptional control of CAR. The quercetin effects are likely dependent on the fat content and composition of the diet.

In chapter 5 we investigated whether flavonoids from other flavonoid subclasses can exert the same effects as we observed for quercetin. Effects of quercetin, hesperetin, epicatechin, apigenin and anthocyanins, in C57BL/6JOlaHsd male adult mice fed a high-fat diet for 12 weeks were compared, relative to a normal-fat diet. High-fat diet-induced body weight gain was significantly lowered by all flavonoids (17-29%), but most by quercetin. Quercetin significantly lowered high-fat diet-induced hepatic lipid accumulation (by 71%). High-fat diet-induced increases of mesenteric adipose tissue weight and serum leptin levels were significantly lowered by quercetin, hesperetin, and anthocyanins. Adipocyte cell size and adipose tissue inflammation were not affected.

The effects on body weight and adiposity could not be explained by individual significant differences in energy intake, energy expenditure, nor by differences in activity. Lipid metabolism was not changed as measured by indirect calorimetry or expression of known lipid metabolic genes in liver and white adipose tissue. Hepatic expression of Cyp2b9 was strongly down-regulated by all flavonoids. Overall, all five flavonoids lowered parameters of high-fat diet-induced adiposity, with quercetin being most effective.

Next to the beneficial health effects of flavonoids, the safety of flavonoids is under discussion, mainly because of potential genotoxic effects found for quercetin in vitro. Therefore, in chapter 6 the in vivo genotoxicity of this flavonoid was studied by transcriptome analyses in two tissues, small intestine and liver, where the highest exposure to quercetin is expected. This is especially of interest in view of high intake by widely available food supplements. Quercetin (0.33%) supplemented to a high-fat diet was administered to C57BL/6JOlaHsd male adult mice during 12 weeks. Serum alanine aminotransferase and aspartate aminotransferase levels revealed no indications for hepatotoxicity. General microarray pathway analysis of liver and small intestinal tissue samples showed no regulation of genotoxicity related pathways. In addition, analysis of DNA damage pathways in these tissues did also not point at genotoxicity. Furthermore, comparison with a published classifier set of transcripts for identifying genotoxic compounds did not reveal any similarities in the regulation of these classifier set by quercetin. Available microarray datasets of known genotoxic liver carcinogens, 2-acetylaminofluorene and aflatoxin B1 in mice were taken along as positive controls for comparison, and indeed showed genotoxic properties (regulation of genotoxic related genes) in the analyses. This transcriptomic analysis showed that supplementation with quercetin at ~350 mg/kg bw/day for 12 weeks did not induce genotoxicity in liver and small intestine.

In conclusion, we have shown in vivo efficacy of flavonoids reflected by effects on metabolic health parameters, including hepatic lipid metabolism. These effects on hepatic lipid metabolism seemed to be related or influenced by the transcription factor CAR. The dietary contexts appeared to modify the health effects. The five studied flavonoids in general showed the same effects, with quercetin being the most effective. No genotoxicity of quercetin was found by transcriptome analyses in liver and small intestine. Overall, we have obtained indications for beneficial health effects of flavonoids in mice, which makes it interesting to study if these effects can be extrapolated to humans to further explore their potential as functional compounds of dietary flavonoid intake.

Muizenschade in Friesland
Roerink, G.J. - \ 2015
Wageningen : Wageningen UR Alterra
graslanden - vegetatiemonitoring - remote sensing - schade - beweidingsschade - monitoring - muizen - friesland - grasslands - vegetation monitoring - damage - browsing damage - mice
Friesland is getroffen door een muizenplaag. Al meer dan 12 duizend ha grasland heeft zodanige schade opgelopen dat het waarneembaar wordt op satellietbeelden. Door de zachte winter en de warme droge zomer van afgelopen jaar is de muizenpopulatie in de loop van 2014 in Friesland geëxplodeerd. De muizenkolonies graven gangen onder de graslandzode en vreten de wortels ervan op zodat het gras afsterft. Alterra brengt via satellietbeelden de schade in kaart.
Dietary Linoleic and a-Linolenic Acid Affect Anxiety-Related Responses and Exploratory Activity in Growing Pigs
Clouard, C.M. ; Gerrits, W.J.J. ; Kerkhof, I. van; Smink, W. ; Bolhuis, J.E. - \ 2015
The Journal of Nutrition 145 (2015)2. - ISSN 0022-3166 - p. 358 - 364.
polyunsaturated fatty-acids - docosahexaenoic acid - inflammatory processes - open-field - behavior - brain - rats - mice - deficiency - piglets
Background: Growing evidence suggests that the dietary ratio of linoleic acid (LA) to a-linolenic acid (ALA), the precursors of arachidonic acid (AA) and docosahexaenoic acid (DHA), respectively, may affect behavior in mammals. Objective: This study aimed at evaluating the impact of dietary LA and ALA intake on behaviors of growing pigs, a pertinent model for human nutrition. Methods: At 7 wk of age, 32 pigs were allocated to 4 dietary treatments varying in daily intake of LA (1.3 and 2.6 g · kg body weight-0.75 · d-1 for low- and high-LA groups, respectively) and ALA (0.15 and 1.5 g · kg body weight-0.75 · d-1 for low- and high-ALA groups, respectively) for 4 wk. Between days 12 and 18, general behavior in the home pen was observed and pigs were subjected to an open field and novel object test. At 11 wk of age, brain fatty acid composition was analyzed. Results: Compared with high LA intake, low LA intake increased the time spent on exploration, particularly nosing in the home pen (P <0.05) and the open field (P <0.05), and tended to reduce the time spent lying with eyes open in the home pen (P = 0.09). Time spent lying with eyes open also tended to be affected by LA × ALA interaction (P = 0.08). A high-LA/high-ALA intake (ratio of 2; P <0.05) and a low-LA/high-ALA intake (ratio of 1; P = 0.06) decreased the latency to approach the novel object compared with a low-LA/low-ALA intake (ratio of 9). DHA in the frontal cortex was positively correlated with exploratory behaviors in the home pen (rs = 0.56, P <0.01), whereas AA was negatively correlated with time spent lying with eyes closed (rs = –0.48, P <0.01). Conclusions: Low LA intake and a low dietary LA:ALA ratio increased exploration and decreased anxiety-related behaviors in pigs. It is suggested that changes in brain DHA and AA induced by dietary LA and ALA intake mediate these behavioral changes.
Oxygen restriction as challenge test reveals early high-fat-diet-induced changes in glucose and lipid metabolism
Duivenvoorde, L.P.M. ; Schothorst, E.M. van; Derous, D. ; Stelt, I. van der; Masania, J. ; Rabbani, N. ; Thornalley, P.J. ; Keijer, J. - \ 2015
Pflugers Archiv-European Journal of Physiology 467 (2015)6. - ISSN 0031-6768 - p. 1179 - 1193.
adipose-tissue - gene-expression - intermittent hypoxia - energy-expenditure - insulin-resistance - transcriptional regulation - mass-spectrometry - mice - obesity - oxidation
Challenge tests stress homeostasis and may reveal deviations in health that remain masked under unchallenged conditions. Ideally, challenge tests are non-invasive and applicable in an early phase of an animal experiment. Oxygen restriction (OxR; based on ambient, mild normobaric hypoxia) is a non-invasive challenge test that measures the flexibility to adapt metabolism. Metabolic inflexibility is one of the hallmarks of the metabolic syndrome. To test whether OxR can be used to reveal early diet-induced health effects, we exposed mice to a low-fat (LF) or high-fat (HF) diet for only 5 days. The response to OxR was assessed by calorimetric measurements, followed by analysis of gene expression in liver and epididymal white adipose tissue (eWAT) and serum markers for e.g. protein glycation and oxidation. Although HF feeding increased body weight, HF and LF mice did not differ in indirect calorimetric values under normoxic conditions and in a fasting state. Exposure to OxR; however, increased oxygen consumption and lipid oxidation in HF mice versus LF mice. Furthermore, OxR induced gluconeogenesis and an antioxidant response in the liver of HF mice, whereas it induced de novo lipogenesis and an antioxidant response in eWAT of LF mice, indicating that HF and LF mice differed in their adaptation to OxR. OxR also increased serum markers of protein glycation and oxidation in HF mice, whereas these changes were absent in LF mice. Cumulatively, OxR is a promising new method to test food products on potential beneficial effects for human health.
Inulin-type fructans modulate intestinal Bifidobacterium species populations and decrease fecal short-chain fatty acids in obese women
Salazar, N. ; Dewulf, E.M. ; Neyrinck, A.M. ; Bindels, L.B. ; Cani, P.D. ; Mahillon, J. ; Vos, W.M. de; Thissen, J.P. ; Gueimonde, M. ; Reyes-Gavilán, C.G. de los; Delzenne, N.M. - \ 2015
Clinical Nutrition 34 (2015)3. - ISSN 0261-5614 - p. 501 - 507.
gradient gel-electrophoresis - protein-coupled receptor - gut microbiota - mice - prebiotics - increases - glucose - diet - pcr - fermentation
Background & aims : Inulin-type fructans (ITF) prebiotics promote changes in the composition and activity of the gut microbiota. The aim of this study was to determine variations on fecal short chain fatty acids (SCFA) concentration in obese women treated with ITF and to explore associations between Bifidobacterium species, SCFA and host biological markers of metabolism. Methods Samples were obtained in a randomized, double blind, parallel, placebo-controlled trial, with 30 obese women randomly assigned to groups that received either 16 g/day ITF (n = 15) or maltodextrin (n = 15) for 3 months. The qualitative and quantitative analysis of Bifidobacterium spp. was performed in feces by PCR-DGGE and q-PCR, and SCFA profile was analyzed by gas chromatography. Spearman correlation analysis was performed between the different variables analyzed. Results The species Bifidobacterium longum, Bifidobacterium pseudocatenulatum and Bifidobacterium adolescentis were significantly increased at the end of the treatment in the prebiotic group (p <0.01) with being B. longum negatively correlated with serum lipopolysaccharide (LPS) endotoxin (p <0.01). Total SCFA, acetate and propionate, that positively correlated with BMI, fasting insulinemia and homeostasis model assessment (HOMA) (p <0.05), were significantly lower in prebiotic than in placebo group after the treatment period. Conclusions ITF consumption selectively modulates Bifidobacterium spp. and decreases fecal SCFA concentration in obese women. ITF could lessen metabolic risk factors associated with higher fecal SCFA concentration in obese individuals.
Mechanisms of amiodarone and valproic acid induced liver steatosis in mouse in vivo act as a template for other hepatotoxicity models
Vitins, A.P. ; Kienhuis, A.S. ; Speksnijder, E.N. ; Roodbergen, M. ; Luijten, M. ; Ven, L.T.M. van der - \ 2014
Archives of Toxicology 88 (2014)8. - ISSN 0340-5761 - p. 1573 - 1588.
gene-expression profiles - hepatic steatosis - lipid homeostasis - drug discovery - mice - metabolism - toxicogenomics - receptor - disruption - resistance
Liver injury is the leading cause of drug-induced toxicity. For the evaluation of a chemical compound to induce toxicity, in this case steatosis or fatty liver, it is imperative to identify markers reflective of mechanisms and processes induced upon exposure, as these will be the earliest changes reflective of disease. Therefore, an in vivo mouse toxicogenomics study was completed to identify common pathways, nuclear receptor (NR) binding sites, and genes regulated by three known human steatosis-inducing compounds, amiodarone (AMD), valproic acid (VPA), and tetracycline (TET). Over 1, 4, and 11 days of treatment, AMD induced changes in clinical chemistry parameters and histopathology consistent with steatosis. Common processes and NR binding sites involved in lipid, retinol, and drug metabolism were found for AMD and VPA, but not for TET, which showed no response. Interestingly, the pattern of enrichment of these common pathways and NR binding sites over time was unique to each compound. Eleven biomarkers of steatosis were identified as dose responsive and time sensitive to toxicity for AMD and VPA. Finally, this in vivo mouse study was compared to an AMD rat in vivo, an AMD mouse primary hepatocyte, and a VPA human primary hepatocyte study to identify concordance for steatosis. We conclude that concordance is found on the process level independent of species, model or dose*time point.
Growth regulation, imprinting, and epigenetic transcription-related gene expression differs in lung of deceased transgenic cloned and normal goats
Meng, L. ; Jia, R.X. ; Sun, Y. ; Wang, Z.Y. ; Wan, Y.J. ; Zhang, Y.L. ; Zhong, B.S. ; Wang, F. - \ 2014
Theriogenology 81 (2014)3. - ISSN 0093-691X - p. 459 - 466.
cell nuclear transfer - dna methylation - nationwide survey - tumor-suppressor - bovine clones - aberrant - calves - mice - cdkn1c - cattle
Somatic cell nuclear transfer (SCNT) is a promising technique to produce mammalian transgenic clones. Only a small proportion of manipulated embryos, however, can develop into viable offspring. The abnormal growth and development of cloned animals, furthermore, are accompanied by aberrant lung development. Our objective was to investigate molecular background of lung developmental problems in transgenic (random insertion of exogenous DNA) cloned goats. We examined expression of 15 genes involved in growth regulation, imprinting, and epigenetic transcription in lung tissue of deceased transgenic cloned and normal goats of various ages. Compared with normal goats of the same age from conventional reproduction, expression of 13 genes (BMP4, FGF10, GHR, HGFR, PDGFR, RABP, VEGF, H19, CDKNIC, PCAF, MeCP2, HDAC1, and Dnmt3b) decreased in transgenic cloned goats that died at or shortly after birth; Expression of eight genes (FGF10, PDGFR, RABP, VEGF, PCAF, HDAC1, MeCP2, and Dnmt3b) decreased in fetal death of transgenic cloned goats. Expression of two epigenetic transcription genes (PCAF and Dnmt3b) decreased in disease death of transgenic cloned goats (1-4 months old). Disruptions in gene expression might be associated with the high neonatal mortality in transgenic cloned animals. These findings have implications in understanding the low efficiency of transgenic cloning. (C) 2014 Elsevier Inc. All rights reserved.
Circulating angiopoietin-like 4 links proteinuria with hypertriglyceridemia in nephrotic syndrome
Clement, L.C. ; Mace, C. ; Avila-Casado, C. ; Joles, J.A. ; Kersten, A.H. ; Chugh, S.S. - \ 2014
Nature Medicine 20 (2014)1. - ISSN 1078-8956 - p. 37 - 46.
lipoprotein-lipase - fatty-acids - gene-expression - adipose-tissue - angptl4 - disease - serum - rats - mice - glomeruli
The molecular link between proteinuria and hyperlipidemia in nephrotic syndrome is not known. We show in the present study that plasma angiopoietin-like 4 (Angptl4) links proteinuria with hypertriglyceridemia through two negative feedback loops. In previous studies in a rat model that mimics human minimal change disease, we observed localized secretion by podocytes of hyposialylated Angptl4, a pro-proteinuric form of the protein. But in this study we noted high serum levels of Angptl4 (presumably normosialylated based on a neutral isoelectric point) in other glomerular diseases as well. Circulating Angptl4 was secreted by extrarenal organs in response to an elevated plasma ratio of free fatty acids (FFAs) to albumin when proteinuria reached nephrotic range. In a systemic feedback loop, these circulating pools of Angptl4 reduced proteinuria by interacting with glomerular endothelial alpha(v)beta(5) integrin. Blocking the Angptl4-beta(5) integrin interaction or global knockout of Angptl4 or beta(5) integrin delayed recovery from peak proteinuria in animal models. But at the same time, in a local feedback loop, the elevated extrarenal pools of Angptl4 reduced tissue FFA uptake in skeletal muscle, heart and adipose tissue, subsequently resulting in hypertriglyceridemia, by inhibiting lipoprotein lipase (LPL)-mediated hydrolysis of plasma triglycerides to FFAs. Injecting recombinant human ANGPTL4 modified at a key LPL interacting site into nephrotic Buffalo Mna and Zucker Diabetic Fatty rats reduced proteinuria through the systemic loop but, by bypassing the local loop, without increasing plasma triglyceride levels. These data show that increases in circulating Angptl4 in response to nephrotic-range proteinuria reduces the degree of this pathology, but at the cost of inducing hypertriglyceridemia, while also suggesting a possible therapy to treat these linked pathologies.
Development of the recombinase-based in vivo expression technology in Streptococcus thermophilus and validation using the lactose operon promoter
Junjua, M. ; Galia, W. ; Gaci, N. ; Uriot, O. ; Genay, M. ; Bachmann, H. ; Kleerebezem, M. ; Dary, A. ; Roussel, Y. - \ 2014
Journal of Applied Microbiology 116 (2014)3. - ISSN 1364-5072 - p. 620 - 631.
lactic-acid bacteria - gene-expression - lactococcus-lactis - human gut - yogurt - system - mice - identification - transformation - proteinase

Aims

To construct and validate the recombinase-based in vivo expression technology (R-IVET) tool in Streptococcus thermophilus (ST).

Methods and Results

The R-IVET system we constructed in the LMD-9 strain includes the plasmid pULNcreB allowing transcriptional fusion with the gene of the site-specific recombinase Cre and the chromosomal cassette containing a spectinomycin resistance gene flanked by two loxP sites. When tested in M17 medium, promoters of the genes encoding the protease PrtS, the heat-shock protein Hsp16 and of the lactose operon triggered deletion of the cassette, indicating promoter activity in these conditions. The lactose operon promoter was also found to be activated during the transit in the murine gastrointestinal tract.

Conclusions

The R-IVET system developed in ST is relatively stable, functional, very sensitive and can be used to assay activity of promoters, which are specifically active in in vivo conditions.

Significance and Impact of the Study

This first adaptation of R-IVET to ST provides a highly valuable tool allowing an exploration of the physiological state of ST in the GIT of mammals, fermentation processes or dairy products.
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