Bovine mammary epithelial cells can grow and express milk protein synthesis genes at reduced fetal bovine serum concentration.

Milk proteins produced by lactating cells isolated from bovine mammary tissue can offer a sustainable solution to the high protein demand of a global growing population. Serum is commonly added to culture systems to provide compounds necessary for optimal growth and function of the cells. However, in a cellular agricultural context, its usage is desired to be decreased. This study aims at examining the minimum level of fetal bovine serum (FBS) required for the growth and functionality of bovine mammary epithelial cells (MECs). The cells were isolated from dairy cows in early and mid-lactation and cultured in reduced concentrations of FBS (10%, 5%, 1.25%, and 0%). Real-time cell analysis showed a significant effect of lactation stage on growth rate and 5% FBS resulted in similar growth rate as 10% while 0% resulted in the lowest. The effect of reducing FBS on cell functionality was examined by studying the expressions of selected marker genes involved in milk protein and fat synthesis, following differentiation. The gene expressions were not affected by the level of FBS. A reduction of FBS in the culture system of MEC, at least down to 5%, does not assert any negative effect on the growth and expression levels of studied genes. As the first attempt in developing an in-vitro model for milk component production using MEC, our results demonstrate the potential of MEC to endure FBS-reduced conditions.

Obesity Development in a Miniature Yucatan Pig Model: A Multi-compartmental Metabolomics Study on Cloned and Normal Pigs Fed Restricted or Ad Libitum High-Energy Diets.

Miniature-pig models for human metabolic disorders such as obesity and metabolic syndrome are gaining popularity. However, in-depth knowledge on the phenotypic and metabolic effects of metabolic dysregulation is lacking, and ad libitum feeding is not well-characterized in these pig breeds. Therefore, an investigation was performed into the metabolome of Yucatan minipigs fed ad libitum or restricted diets. Furthermore, we used cloned and conventional minipigs to assess if cloning reflects a presumably lowered variation between subjects. For 5 months, 17 female Yucatan minipigs were fed either ad libitum or restricted Western-style diets. Serum, urine, and liver tissues were collected and analyzed by non-targeted liquid chromatography-mass spectrometry metabolomics and by biochemical analyses. Several metabolic pathways were deregulated as a result of obesity and increased energy-dense feed intake, particularly the hepatic glutathione pathway and the pantothenic acid and tryptophan metabolic pathways in serum and urine. Although cloned minipigs were phenotypically similar to wild-type minipigs, the metabolomics analysis of serum and liver tissues showed several altered pathways, such as amino acid and purine metabolism. These changes, as an effect of cloning, could limit the use of cloned models in dietary intervention studies and provides no evidence of decreased variability between subjects.

Bioactive Whey Protein Concentrate and Lactose Stimulate Gut Function in Formula-fed Preterm Pigs.

OBJECTIVE: Formula feeding is associated with compromised intestinal health in preterm neonates compared with maternal milk, but the mechanisms behind this are unclear. We hypothesized that the use of maltodextrin and whey protein concentrates (WPCs) with reduced bioactivity owing to thermal processing are important factors. METHOD: Ninety-two cesarean-delivered preterm pigs were fed increasing doses of formulas for 5 days (24-120 mL ·â€Škg ·â€Šday). In experiment 1, 4 groups of pigs (n = 15-16) were fed lactose- or maltodextrin-dominant formulas (lactose/maltodextrin ratios 3:1 or 1:3, respectively), containing WPC with either high or low levels of IgG (WPC1 or WPC2, respectively). In experiment 2, 2 groups of pigs (n = 15-16) were fed lactose-dominant formulas with either a bioactive WPC (BioWPC, produced by reduced thermal-processing) or a conventional WPC (ConWPC). RESULTS: In experiment 1, pigs fed formula with WPC1 had higher villi, hexose absorption, and lactase activity in small intestine, relative to WPC2, but predominantly with the lactose-dominant formula (all P < 0.05). In experiment 2, the BioWPC product had higher bioactivity, as indicated by higher IgG, lactoferrin, and TGF-ß2 levels, and better enterocyte proliferation in vitro. Pigs fed the BioWPC formula showed better feeding tolerance and higher intestinal villi and lactase activity (all P < 0.05). The BioWPC formula-fed pigs also had greater physical activity (P < 0.05 on day 4) and tended to show improved hexose absorption and decreased gut permeability (both P ≤ 0.09). CONCLUSIONS: Infant formulas containing lactose as the main carbohydrate, and WPC with reduced thermal processing, may support gut maturation and health in sensitive, preterm neonates.

Spray Dried, Pasteurised Bovine Colostrum Protects Against Gut Dysfunction and Inflammation in Preterm Pigs.

OBJECTIVE: Feeding bovine colostrum (BC) improves gut maturation and function and protects against necrotizing enterocolitis, relative to formula in newborn preterm pigs. Before BC can be used for preterm infants, it is important to test if the milk processing, required to reduce bacterial load and increase shelf life, may affect bioactivity and efficacy of a BC product. METHODS: We investigated if spray dried, pasteurised BC had protective effects on gut function in preterm pigs, relative to formula. After a 2-day total parenteral nutrition period, preterm pigs were fed formula for a few hours (to induce a proinflammatory state) followed by 2 days of formula (FORM, n = 14), BC (colostrum [COLOS], n = 14), spray-dried BC (POW, n = 8), or pasteurised, spray-dried BC (POWPAS, n = 9). RESULTS: Spray drying and pasteurisation of BC decreased the concentration of transforming growth factor-ß1, -ß2 and increased protein aggregation. All of the 3 BC groups had reduced necrotizing enterocolitis severity, small intestinal levels of IL-1ß, -8, and colonic lactic acid levels, and increased intestinal villus height, hexose absorption, and digestive enzyme activities, relative to the FORM group (all P < 0.05). All of the 3 BC diets stimulated epithelial cell migration in a wound-healing model with IEC-6 cells. CONCLUSIONS: Spray drying and pasteurisation affect BC proteins, but do not reduce the trophic and anti-inflammatory effects of BC on the immature intestine. It remains to be studied if BC products will benefit preterm infants just after birth when human milk is often not available.

Effect of growth factors, estradiol 17-ß, and short chain fatty acids on the intestinal HT29-MTX cells : Growth factors and SCFAs effects on intestinal E12 cells.

Peptides growth factors, hormones, and short chain fatty acids (SCFAs) are constantly in contact with the human bowel when secreted by gland or ingested by food, as milk and colostrum, or, as in the case of SCFAs, produced by fermentation processes. This study considers the effect of growth factors, estradiol 17-ß, and SCFAs on the metabolic activity and proliferation of undifferentiated HT29-MTX-E12 (E12) cells. In particular, the aim of the present study was the characterization of the human intestinal cell line E12 for its suitability as an in vitro intestinal model for cell-nutrient interaction studies. The effect of insulin-like growth factors (IGF)-I, epidermal growth factors (EGF), transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-ß), estradiol 17-ß and butyrate, propionate, and acetate was assessed on metabolic activity and proliferation of E12 cells using AlamarBlue(TM) assay and PicoGreen® assay, respectively. IGF-I and estradiol 17-ß significantly (P < 0.05; P < 0.001) increased both metabolic activity and proliferation in a concentration-dependent manner, whereas TGF-α, at the concentration of 1 ng/mL, significantly (P < 0.05) reduced the metabolic activity of the cells. Further, a dose-dependent inhibition of cell metabolic activity was detected in the presence of all SCFAs tested. Butyrate showed to be the most active in the inhibition of E12 metabolic activity and its effect was enhanced by the presence of propionate and acetate. E12 cells, in undifferentiated state, showed to be a suitable in vitro model for cell-nutrient interaction studies, providing an opportunity to examine the potential role of growth factors, hormones and SCFAs in the regulation of the intestinal cell viability.

Guaianolides and a seco-Eudesmane from the Resinous Exudates of Cushion Bush (Leucophyta brownii) and Evaluation of Their Cytostatic and Anti-inflammatory Activity.

A detailed phytochemical investigation of a dichloromethane extract of the resinous exudates of the cushion bush plant (Leucophyta brownii) resulted in the isolation of the new 8,12-guaianolides leucophytalins A (5) and B (6), the new 1,10-seco-eudesmane leucophytalin C (10), six rare 8,12-guaianolides (1-4, 7, and 8), and the xanthanolide tomentosin (9). The structures of all isolated compounds were elucidated on the basis of spectroscopic and spectrometric analyses. The structures of compounds isolated in crystalline form, including leucophytalins A and C, were further confirmed by X-ray crystallography. The crude extract exhibited moderate cytostatic activity against a breast cancer (MCF-7) and human colon cancer (HT-29) cell line with IC50 values of 9.3 and 18 µg/mL, respectively, and anti-inflammatory activity against the macrophage-like cell line RAW 264.7 with IC50 values of 3.9 and 6.1 µg/mL for thromboxane B2 and prostaglandin E2 production, respectively. The isolated compounds were evaluated for their cytostatic activity against MCF-7 and HT-29 cells (1, 3-10) and their anti-inflammatory activity against RAW 264.7 cells (1-10). All isolated compounds are most likely derived from (+)-germacrene A, and a biosynthetic pathway is proposed for these sesquiterpenoids.

Developmental potential and kinetics of pig embryos with different cytoplasmic volume.

The effects of cytoplasmic volumes on development and developmental kinetics of in vitro produced porcine embryos were investigated. During hand-made cloning (HMC), selected cytoplasts were separated into two groups according to their size in relation to the initial oocyte: ~75% or ~50%. Following two fusion steps and activation (day 0), reconstructed embryos were cultured in vitro for 6 days. Cleavage rates on day 2 as well as blastocyst rates and cell numbers on day 6 were recorded. Results showed that embryo development was no different for ~50% versus ~75% cytoplasm at first fusion. This result was used in the following experiments, where the effect of varying cytoplasm volume in second fusion to obtain a final cytoplasm volume of ~75% to ~200% was tested. The results showed that the lowest quality was obtained when the final cytoplasm volume was ~75% and the highest quality at ~200% of the original oocyte. Similar results were observed in parthenogenetic (PA) embryos activated with different cytoplasmic volumes. A common pattern for the developmental kinetics of HMC and PA embryos was observed: the smaller group tended to have a longer time for the first two cell cycles, but subsequently a shorter time to form morula and blastocyst. In conclusion, the developmental kinetics of in vitro produced embryos was affected by the cytoplasm volume of the initial oocyte, and this further accounted for the developmental ability of the reconstructed embryos.

Long-term effect on in vitro cloning efficiency after treatment of somatic cells with Xenopus egg extract in the pig.

In somatic cell nuclear transfer (SCNT), donor cell reprogramming is considered as a biologically important and vulnerable event. Various donor cell pre-treatments with Xenopus egg extracts can promote reprogramming. Here we investigated if the reprogramming effect of one treatment with Xenopus egg extract on donor cells was maintained for several cell passages. The extract treatment resulted in increased cell-colony formation from early passages in treated porcine fibroblasts (ExTES), and increased development of cloned embryos. Partial dedifferentiation was observed in ExTES cells, shown as a tendency towards upregulation of NANOG, c-MYC and KLF-4 and downregulation of DESMIM compared with ExTES at Passage 2. Compared with our routine SCNT, continuously increased development of cloned embryos was observed in the ExTES group, and ExTES cloned blastocysts displayed hypermethylated DNA patterns and hypermethylation of H3K4me3 and H3K27me3 in ICM compared with TE. All seven recipients became pregnant after transferral of ExTES cloned embryos and gave birth to 7-22 piglets per litter (average 12). In conclusion, our results demonstrate that one treatment of porcine fibroblasts with Xenopus egg extract can result in long-term increased ability of the cells to promote their in vitro function in subsequent SCNT. Finally these cells can also result in successful development of cloned embryos to term.

Developmental potential of pig embryos reconstructed by use of sow versus pre-pubertal gilt oocytes after somatic cell nuclear transfer.

In this study, the developmental ability of cloned embryos using gilt versus sow oocytes was evaluated under the hypothesis that the efficiency of nuclear transfer using gilt oocytes was lower than that of sow oocytes, but that it could be optimized. Five experiments were performed with routine production of cloned embryos with sow oocytes serving as the control. Results showed that: Experiment 1: Blastocyst rates of cloned embryos with gilt oocytes was about half compared with control. Experiment 2: An extended maturation time of 48 h used for gilt oocytes resulted in lower blastocyst rates after cloning. Experiment 3: Development of cloned embryos with gilt oocytes was improved by co-culture with sow oocytes. Experiment 4: After maturation of gilt oocytes using follicular fluid from gilt instead of sow, the oocytes were sorted into large and small oocytes, and after cloning, blastocyst rates were higher using large gilt oocytes compared with small oocytes; however, the rate remained lower compared with control. Experiment 5: Six sow recipients received a total of 503 morulae and blastocysts cloned from gilt oocytes (four recipients) and 190 cloned from sow oocytes (two recipients). All recipients became pregnant and went to term, resulting in 26 (gilt oocytes) and six (sow oocytes) piglets. In conclusion, results confirmed that nuclear transfer efficiency was higher using sow versus gilt oocytes, but the use of gilt oocytes can be optimized by sorting after ooplasm size following maturation and by maturing gilt and sow oocytes together.

Postnatal amniotic fluid intake reduces gut inflammatory responses and necrotizing enterocolitis in preterm neonates.

Preterm neonates are susceptible to gastrointestinal disorders such as necrotizing enterocolitis (NEC). Maternal milk and colostrum protects against NEC via growth promoting, immunomodulatory, and antimicrobial factors. The fetal enteral diet amniotic fluid (AF), contains similar components, and we hypothesized that postnatal AF administration reduces inflammatory responses and NEC in preterm neonates. Preterm pigs (92% gestation) were delivered by caesarean section and fed parental nutrition (2 days) followed by enteral (2 days) porcine colostrum (COLOS, n = 7), infant formula (FORM, n = 13), or AF supplied before and after introduction of formula (AF, n = 10) in experiment 1, and supplied only during the enteral feeding period in experiment 2 (FORM, n = 16; AF, n = 14). The NEC score was reduced in both AF and COLOS pigs, relative to FORM, when AF was provided prior to full enteral feeding (9.9 and 7.7 compared with 17.3, P < 0.05). There was no effect of AF when provided only during enteral feeding. AF pigs showed decreased bacterial abundance in colon and intestinal inflammation-related genes (e.g., TNF-α, IL-1α, IL-6, NOS) were downregulated, relative to FORM pigs with NEC. Anti-inflammatory properties of AF were supported by delayed maturation and decreased TNF-α production in murine dendritic cells, as well as increased proliferation and migration, and downregulation of IL-6 expression in intestinal cells (IEC-6, IPEC-J2). Like colostrum, AF may reduce NEC development in preterm neonates by suppressing the proinflammatory responses to enteral formula feeding and gut colonization when provided before the onset of NEC.

Corrigendum: A westernized diet changed the colonic bacterial composition and metabolite concentration in a dextran sulfate sodium pig model for ulcerative colitis.

[This corrects the article DOI: 10.3389/fmicb.2023.1018242.].

A westernized diet changed the colonic bacterial composition and metabolite concentration in a dextran sulfate sodium pig model for ulcerative colitis.

Introduction: Ulcerative colitis (UC) is characterized by chronic inflammation in the colonic epithelium and has a blurred etiology. A western diet and microbial dysbiosis in the colon were reported to play a role in UC development. In this study, we investigated the effect of a westernized diet, i.e., increasing fat and protein content by including ground beef, on the colonic bacterial composition in a dextran sulfate sodium (DexSS) challenged pig study. Methods: The experiment was carried out in three complete blocks following a 2×2 factorial design including 24 six-week old pigs, fed either a standard diet (CT) or the standard diet substituted with 15% ground beef to simulate a typical westernized diet (WD). Colitis was induced in half of the pigs on each dietary treatment by oral administration of DexSS (DSS and WD+DSS, respectively). Samples from proximal and distal colon and feces were collected. Results and discussion: Bacterial alpha diversity was unaffected by experimental block, and sample type. In proximal colon, WD group had similar alpha diversity to CT group and the WD+DSS group showed the lowest alpha diversity compared to the other treatment groups. There was a significant interaction between western diet and DexSS for beta diversity, based on Bray-Curtis dissimilarly. The westernized diet and DexSS resulted in three and seven differentially abundant phyla, 21 and 65 species, respectively, mainly associated with the Firmicutes and Bacteroidota phyla followed by Spirochaetota, Desulfobacterota, and Proteobacteria. The concentration of short-chain fatty acids (SCFA) was lowest in the distal colon. Treatment had a slight effect on the estimates for microbial metabolites that might have valuable biological relevance for future studies. The concentration of putrescine in the colon and feces and that of total biogenic amines was highest in the WD+DSS group. We conclude that a westernized diet could be a potential risk factor and an exacerbating agent for UC by reducing the abundance of SCFA-producing bacteria, increasing the abundance of pathogens such as Helicobacter trogontum, and by increasing the concentration of microbial proteolytic-derived metabolites in the colon.

Production of novel fusarielins by ectopic activation of the polyketide synthase 9 cluster in Fusarium graminearum.

Like many other filamentous fungi, Fusarium graminearum has the genetic potential to produce a vast array of unknown secondary metabolites. A promising approach to determine the nature of these is to activate silent secondary metabolite gene clusters through constitutive expression of cluster specific transcription factors. We have developed a system in which an expression cassette containing the transcription factor from the targeted PKS cluster disrupts the production of the red mycelium pigment aurofusarin. This aids with identification of mutants as they appear as white colonies and metabolite analyses where aurofusarin and its intermediates are normally among the most abundant compounds. The system was used for constitutive expression of the local transcription factor from the PKS9 cluster (renamed FSL) leading to production of three novel fusarielins, F, G and H. This group of compounds has not previously been reported from F. graminearum or linked to a biosynthetic gene in any fungal species. The toxicity of the three novel fusarielins was examined against colorectal cancer cell lines where fusarielin H was more potent than fusarielin F and G.

Comparative analysis of inflamed and non-inflamed colon biopsies reveals strong proteomic inflammation profile in patients with ulcerative colitis.

BACKGROUND: Accurate diagnostic and monitoring tools for ulcerative colitis (UC) are missing. Our aim was to describe the proteomic profile of UC and search for markers associated with disease exacerbation. Therefore, we aimed to characterize specific proteins associated with inflamed colon mucosa from patients with acute UC using mass spectrometry-based proteomic analysis. METHODS: Biopsies were sampled from rectum, sigmoid colon and left colonic flexure from twenty patients with active proctosigmoiditis and from four healthy controls for proteomics and histology. Proteomic profiles of whole colonic biopsies were characterized using 2D-gel electrophoresis, and peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was applied for identification of differently expressed protein spots. RESULTS: A total of 597 spots were annotated by image analysis and 222 of these had a statistically different protein level between inflamed and non-inflamed tissue in the patient group. Principal component analysis clearly grouped non-inflamed samples separately from the inflamed samples indicating that the proteomic signature of colon mucosa with acute UC is strong. Totally, 43 individual protein spots were identified, including proteins involved in energy metabolism (triosephosphate isomerase, glycerol-3-phosphate-dehydrogenase, alpha enolase and L-lactate dehydrogenase B-chain) and in oxidative stress (superoxide dismutase, thioredoxins and selenium binding protein). CONCLUSIONS: A distinct proteomic profile of inflamed tissue in UC patients was found. Specific proteins involved in energy metabolism and oxidative stress were identified as potential candidate markers for UC.

Increased blastocyst formation of cloned porcine embryos produced with donor cells pre-treated with Xenopus egg extract and/or digitonin.

Pre-treating donor cells before somatic cell nuclear transfer (SCNT, 'cloning') may improve the efficiency of the technology. The aim of this study was to evaluate the early development of cloned embryos produced with porcine fibroblasts pre-treated with a permeabilizing agent and extract from Xenopus laevis eggs. In Experiment 1, fetal fibroblasts were permeabilized by digitonin, incubated in egg extract and, after re-sealing of cell membranes, cultured for 3 or 5 days before use as donor cells in handmade cloning (HMC). Controls were produced by HMC with non-treated donor cells. The blastocyst rate for reconstructed embryos increased significantly when digitonin-permeabilized, extract-treated cells were used after 5 days of culture after re-sealing. In Experiment 2, fetal and adult fibroblasts were treated with digitonin alone before re-sealing the cell membranes, then cultured for 3 or 5 days and used as donor cells in HMC. Treatment with digitonin alone increased the blastocyst rate, but only when fetal, and not adult fibroblasts, were used as donor cells, and only after 3 days of culture. In conclusion, we find a time window for increased efficiency of porcine SCNT using donor cells after pre-treatment with permeabilization/re-sealing and Xenopus egg extract. Interestingly, we observe a similar increase in cloning efficiency by permeabilization/re-sealing of donor cells without extract treatment that seems to depend on choice of donor cell type. Thus, pre-treatment of donor cells using permeabilizing treatment followed by re-sealing and in vitro culture for few days could be a simple way to improve the efficiency of porcine cloning.

Proliferative effect of whey from cows' milk obtained at two different stages of pregnancy measured in MCF-7 cells.

Dietary estrogens may play a role in the etiology of hormone-dependent cancers like breast cancer. Cow's milk contains various endogenous estrogens and feed derived phytoestrogens that potentially contribute to an estrogenic effect of milk in consumers, and therefore we evaluated the effect of milk (whey) in a proliferation assay with estrogen-sensitive MCF-7 human breast cancer cells. Milk samples were obtained from 22 cows representing different stages of pregnancy (first and second half) and whey was produced from the milk. 0·1, 0·25 or 0·5% whey was included in the cell culture medium and after 6 days of treatment cell proliferation was assessed by a colorimetric method with a fluorometer. Whey induced significant (P<0·05) proliferative effects compared with control cells with no added whey at all concentrations tested. There was no difference in the proliferative effect of whey depending on the stage of pregnancy from which the milk was obtained. We did not observe anti-proliferative effects when whey was tested in the presence of 10 pm estradiol in the medium. In conclusion, these results indicate that whey, irrespective of the pregnancy stage from which the milk was obtained induced a significant proliferative response in MCF-7 cells and no anti-proliferative effect, which may be caused, at least in part, by estrogens present in milk. The implications of our findings in relation to for example breast cancer will have to be studied further in other model systems preferentially in vivo.

Proliferative effect of whey from cows' milk varying in phyto-oestrogens in human breast and prostate cancer cells.

Intake of dietary phyto-oestrogens has received a great deal of attention owing to their potential influence on hormone-sensitive cancers such as breast and prostate cancer. Cows' milk contains phyto-oestrogens and the content varies according to the composition of the feed and the type and amount of legumes used. In this study we evaluated the proliferative effect of milk (whey) with different phyto-oestrogen content in human breast (MCF-7) and prostate cancer cells (PC-3). Milk was obtained from cows fed either a birdsfoot trefoil-timothy silage based ration (B1) or two different red clover silage based diets (R1 and R2) resulting in total phyto-oestrogen contents of 403, 1659 and 1434 ng/ml for the B1, R1 and R2 diets, respectively. Whey was produced from the milk and added to cell culture medium in concentrations up to 10% for MCF-7 cells and 5% for PC-3 cells. Cell proliferation was measured fluorometrically after 7 d for MCF-7 cells and 5 d for PC-3 cells. There was no significant difference in the proliferative effect of whey from the different dietary treatments at any of the whey concentrations tested. An anti-proliferative effect (P<0·01) of 5 and 10% whey was seen when tested in the presence of 10 pM oestradiol in the medium. This effect was independent of dietary treatment of cows. Whey induced a significant (P<0·01) proliferative response in PC-3 cells independent of dietary treatment. Purified equol in concentrations similar to equol concentrations in milk decreased PC-3 cell proliferation, and therefore the stimulatory effect of whey in PC-3 cells is believed to be mediated by other bioactives than equol. In conclusion, our results suggest that using whey in these proliferation assays, it was not possible to discriminate between milk with high or low levels of phyto-oestrogens.

Role of Short Chain Fatty Acids to Counteract Inflammatory Stress and Mucus Production in Human Intestinal HT29-MTX-E12 Cells.

Short chain fatty acids (SCFAs), especially butyrate (BUT), are known to promote intestinal health, but their role in the protection of intestinal barrier integrity is poorly characterized. The aim of the study was to set up an in vitro model of human colon epithelium using HT29-MTX-E12 cells to delineate the potential role of SCFAs under stress conditions. Accordingly, the HT29-MTX-E12 cells were differentiated for 42 days and subsequently exposed to dextran sulphate sodium (DSS). Further, the effects of BUT or its mixture with acetate and propionate (SCFAs-MIX) were tested to study proliferation, epithelial integrity and mucus production. The results showed that the concentration of 10% DSS for 24 h decreased the TEER about 50% compared to the control in HT29-MTX-E12 cells. The pre-treatment on HT29-MTX-E12 cells with BUT or SCFAs-MIX at specific concentrations significantly (p < 0.05) reduced the DSS-induced damage on epithelial cell integrity and permeability. Further, the treatment with specific concentrations of BUT and SCFAs-MIX for 24 h significantly promoted ZO-1, MUC2 and MUC5AC mRNA expression (p < 0.005). The present study demonstrated the suitability of HT29-MTX-E12 cells treated with DSS as an in vitro stress model of inflammatory bowel disease, which enabled us to understand the effect of bioactive SCFAs on the intestinal barrier.

Metabolomic phenotyping of a cloned pig model.

BACKGROUND: Pigs are widely used as models for human physiological changes in intervention studies, because of the close resemblance between human and porcine physiology and the high degree of experimental control when using an animal model. Cloned animals have, in principle, identical genotypes and possibly also phenotypes and this offer an extra level of experimental control which could possibly make them a desirable tool for intervention studies. Therefore, in the present study, we address how phenotype and phenotypic variation is affected by cloning, through comparison of cloned pigs and normal outbred pigs. RESULTS: The metabolic phenotype of cloned pigs (n = 5) was for the first time elucidated by nuclear magnetic resonance (NMR)-based metabolomic analysis of multiple bio-fluids including plasma, bile and urine. The metabolic phenotype of the cloned pigs was compared with normal outbred pigs (n = 6) by multivariate data analysis, which revealed differences in the metabolic phenotypes. Plasma lactate was higher for cloned vs control pigs, while multiple metabolites were altered in the bile. However a lower inter-individual variability for cloned pigs compared with control pigs could not be established. CONCLUSIONS: From the present study we conclude that cloned and normal outbred pigs are phenotypically different. However, it cannot be concluded that the use of cloned animals will reduce the inter-individual variation in intervention studies, though this is based on a limited number of animals.

Establishing Cell Lines from Fresh or Cryopreserved Tissue from the Great Crested Newt (Triturus cristatus):A Preliminary Protocol.

This study describes a successful protocol for establishing cell lines from the threatened Triturus cristatus in terms of collection, preparing, establishing, cryopreserving, thawing and quality checking. Different parameters such as media, media change, fresh vs. cryopreserved tissue and seeding density were tested to optimize culture conditions for this species. With fresh tissue, no considerable differences in the use of two different media were found, but with cryopreserved tissue, a combination of ITS (insulin/transferrin/selenite) and 2-mercaptoethanol had a positive effect on growth. Real-time measurements on the cell lines were used, for the first time in amphibian cells, to investigate the effect of different treatments such as media change with or without washing. Media change had a positive impact on the cells, whereas the effect was negative when combined with washing. It is concluded that establishment of cell lines is possible from the great crested newt, especially when using fresh tissue, but much more challenging if the tissue has been cryopreserved. Real-time measurement during cell culture is a useful tool to visualize the sensitivity of amphibian cells during different culture treatments.