HspR mutations are naturally selected in Bifidobacterium longum when successive heat shock treatments are applied.

The development of molecular tools allowed light to be shed on several widespread genetic mechanisms aiming at limiting the effect of molecular damage on bacterial survival. For some bacterial taxa, there are limited tools in the genetic toolbox, which restricts the possibilities to investigate the molecular basis of their stress response. In that case, an alternative strategy is to study genetic variants of a strain under stress conditions. The comparative study of the genetic determinants responsible for their phenotypes, e.g., an improved tolerance to stress, offers precious clues on the molecular mechanisms effective in this bacterial taxon. We applied this approach and isolated two heat shock-tolerant strains derived from Bifidobacterium longum NCC2705. A global analysis of their transcriptomes revealed that the dnaK operon and the clpB gene were overexpressed in both heat shock-tolerant strains. We sequenced the hspR gene coding for the negative regulator of dnaK and clpB and found point mutations affecting protein domains likely responsible for the binding of the regulators to the promoter DNA. Complementation of the mutant strains by the wild-type regulator hspR restored its heat sensitivity and thus demonstrated that these mutations were responsible for the observed heat tolerance phenotype.

Six weeks' sebacic acid supplementation improves fasting plasma glucose, HbA1c and glucose tolerance in db/db mice.

AIM: To investigate the impact of chronic ingestion of sebacic acid (SA), a 10-carbon medium-chain dicarboxylic acid, on glycaemic control in a mouse model of type 2 diabetes (T2D). METHODS: Three groups of 15 db/db mice were fed for 6 weeks either a chow diet (Ctrl) or a chow diet supplemented with 1.5 or 15% (SA(1.5%) and SA(15%) , respectively) energy from SA. Fasting glycaemia was measured once a week and HbA1c before and after supplementation. An oral glucose tolerance test (OGTT) was performed at the end of the supplementation. Gene expression was determined by transcriptomic analysis on the liver of the Ctrl and SA(15%) groups. RESULTS: After 42 days of supplementation, fasting glycaemia and HbA1c were ∼70 and 25% lower in the SA(15%) group compared with the other groups showing a beneficial effect of SA on hyperglycaemia. During OGTT, plasma glucose area under the curve was reduced after SA(15%) compared with the other groups. This effect was associated with a tendency for an improved insulin response. In the liver, Pck1 and FBP mRNA were statistically decreased in the SA(15%) compared with Ctrl suggesting a reduced hepatic glucose output induced by SA. CONCLUSION: Dietary supplementation of SA largely improves glycaemic control in a mouse model of T2D. This beneficial effect may be due to (i) an improved glucose-induced insulin secretion and (ii) a reduced hepatic glucose output.

Contribution of mesothelial cells in the expression of inflammatory-related factors in omental adipose tissue of obese subjects.

OBJECTIVE: The objective of this study was to determine the contribution of mesothelial cells, present in human omental adipose tissue (OAT) but not in the subcutaneous depot (SAT), on the expression of inflammation-related factors. DESIGN: Comparison of the expression profiles of inflammation-related genes in mesothelial cells with those in the adipocyte-enriched (AEF) and stromal vascular fractions (SVF) and localization of interleukin-18 (IL-18) expression in adipose depots. SUBJECTS: Eleven obese Caucasian female subjects undergoing gastric bypass surgery (body mass index: 43.6+/-1.3 kg/m(2); age: 41.6+/-2.3 years). MEASUREMENTS: The expression profiles of cytokine and chemokine-related genes in mesothelial cells and in cell fractions prepared from OAT were assessed by the microarray technique. The differential expression of IL-18 was confirmed by real-time PCR and the protein was localized in adipose depots by immunohistochemistry. RESULTS: Microarray data analysis demonstrated that, of the 16 cytokine and chemokine-related genes that were upregulated in mesothelial cells compared with the AEF, IL-18 was the cytokine with the highest differential expression. IL-18 expression was similar in mesothelial cells and the SVF. In both SAT and OAT, IL-18 was immunolocalized in neutrophils and mast cells, but not in macrophages nor adipocytes. This cytokine was also detected in mesothelial cells in OAT. This additional source of expression may explain the higher IL-18 expression levels in OAT than SAT (+5.9-fold). CONCLUSION: By their capacity to express inflammatory-related factors, and in particular the proinflammatory cytokine IL-18 in OAT, mesothelial cells appear as a new player in the process of low-grade inflammation associated with obesity.

A toxicogenomics approach to identify new plausible epigenetic mechanisms of ochratoxin a carcinogenicity in rat.

Ochratoxin A (OTA) is a mycotoxin occurring naturally in a wide range of food commodities. In animals, it has been shown to cause a variety of adverse effects, nephrocarcinogenicity being the most prominent. Because of its high toxic potency and the continuous exposure of the human population, OTA has raised public health concerns. There is significant debate on how to use the rat carcinogenicity data to assess the potential risk to humans. In this context, the question of the mechanism of action of OTA appears of key importance and was studied through the application of a toxicogenomics approach. Male Fischer rats were fed OTA for up to 2 years. Renal tumors were discovered during the last 6 months of the study. The total tumor incidence reached 25% at the end of the study. Gene expression profile was analyzed in groups of animals taken in intervals from 7 days to 12 months. Tissue-specific responses were observed in kidney versus liver. For selected genes, microarray data were confirmed at both mRNA and protein levels. In kidney, several genes known as markers of kidney injury and cell regeneration were significantly modulated by OTA. The expression of genes known to be involved in DNA synthesis and repair, or genes induced as a result of DNA damage, was only marginally modulated. Very little or no effect was found amongst genes associated with apoptosis. Alterations of gene expression indicating effects on calcium homeostasis and a disruption of pathways regulated by the transcription factors hepatocyte nuclear factor 4 alpha (HNF4alpha) and nuclear factor-erythroid 2-related factor 2 (Nrf2) were observed in the kidney but not in the liver. Previous data have suggested that a reduction in HNF4alpha may be associated with nephrocarcinogenicity. Many Nrf2-regulated genes are involved in chemical detoxication and antioxidant defense. The depletion of these genes is likely to impair the defense potential of the cells, resulting in chronic elevation of oxidative stress in the kidney. The inhibition of defense mechanism appears as a highly plausible new mechanism, which could contribute to OTA carcinogenicity.

Zinc status of healthy elderly adults: response to supplementation.

The zinc status of 53 healthy elderly subjects was evaluated. The dietary Zn intake estimated by 24-h recall was 9.2 mg/d and 65% of subjects had intakes less than two-thirds of the RDA. Mean serum Zn concentration (13.0 mumol/L) and urinary Zn excretion (7.0 mumol/d) were normal. The Zn content of platelets, mononuclear cells, and polymorphonuclear cells was 5.8, 147, and 135 nmol/10(9) cells, respectively. Seventeen subjects were supplemented for 28 d with 30 mg Zn/d. The mean concentration of Zn in serum and urine increased 24% and 2.5-fold, respectively. Zn content of platelets and leukocytes did not change with Zn supplementation. The concentration of visceral proteins (ie, albumin, prealbumin, transferrin, and retinol-binding protein) and immunoglobulins (ie, IgG, IgA, and IgM) did not change with Zn supplementation. The data indicate that aging per se does not necessarily imply poor Zn status.

Zinc absorption in healthy elderly humans and the effect of diet.

Absorption of a zinc stable isotope was measured on two consecutive occasions in nine young and eight elderly healthy men aged 24-40 and 70-83 y, respectively. A zinc stable-isotope label (0.8 mg 70Zn) was added to a test meal of either high or low zinc bioavailability, depending mainly on phytic acid content. Zinc absorption from the high-bioavailability test meal was not significantly different (P > 0.05) in the young (38.9 +/- 9.8%, mean +/- SD) and elderly (35.0 +/- 10.9%) subjects. Zinc absorption from the low-bioavailability test meal was 40% and 43% lower, at 23.4 +/- 10.2% and 19.8 +/- 6.1% in these young and elderly men, respectively. Again, no significant effect of age was found. These results show that aging does not lead to nutritionally relevant changes in zinc absorption and in the effect of dietary inhibitors on zinc absorption. Thus, zinc absorption ability seems to be preserved in healthy elderly people, at least until the age of 80 y.

Cutaneous copper and zinc losses in burns.

To measure the exudative cutaneous copper (Cu) and zinc (Zn) losses in burns, 10 patients, aged 36 +/- 9 years (mean +/- s.d.) with burns covering 33 +/- 10 per cent of the total body surface area, were studied from the first postburn day (D1) until D7. All intakes and losses were analysed for Cu, Zn and nitrogen (N) content. Cutaneous losses were extracted from textiles surrounding the patients. Urinary excretions were 0.12 +/- 0.06mg/24h for Cu, 0.9 +/- 0.6mg/24h for Zn, and 14.1 +/- 4.4g/24h for N. Mean daily exudative losses through wound seepage from D1 to D7 were 4.7 +/- 2.1mg/24h for Cu, 27.1 +/- 14.4mg/24h for Zn, and 8.7 +/- 3.8g/24h for N. The cumulated mean losses over 7 days were 37mg for Cu, and 212mg for Zn, representing respectively 20-40 per cent and 5-10 per cent of normal body content. Serum Cu and Zn levels were strongly depressed. The urinary Cu/N ratios correlated with clinical improvement. We conclude that the exudative Cu and Zn losses during the first week postburn contribute significantly to the increased nutrient requirements in burns.

Effect of a mixture of bovine milk oligosaccharides, Lactobacillus rhamnosus NCC4007 and long-chain polyunsaturated fatty acids on catch-up growth of intra-uterine growth-restricted rats.

AIM: To investigate the effect of a nutritional mixture (bovine milk oligosaccharides, Lactobacillus rhamnosus NCC4007, arachidonic and docosahexaenoic acid) on growth of intrauterine growth-restricted (IUGR) rats. METHODS: IUGR was induced by maternal food restriction. The offspring (males and females) were assigned to: REF (non-IUGR, no mixture), IUGRc (IUGR, no mixture), or IUGRmx (IUGR, mixture). The mixture was given from day 7 to day 58, when tissues and plasma from half of the animals were collected for hormones, metabolites and microarray analysis. The rest received a high-fat diet (HFD) until day 100. Glucose tolerance was measured at 56 and 98 days, and body fat content at 21, 52 and 97 days. RESULTS: IUGRmx had the greatest growth during lactation, but from day 22 to day 54, both IUGR groups gained less body weight than the REF (P < 0.05). In the short-term (58 days), IUGRmx tended to be longer (P = 0.06) and had less body fat (P = 0.03) than IUGRc. These differences were not seen after HFD. Microarray analysis of hepatic mRNA expression at 58 and 100 days revealed a gender-dependent treatment effect, and expression of genes related to lipid metabolism was the most affected. Twelve of these genes were selected for studying differences in DNA methylation in the promoter region, for some, we observed age- and gender-related differences but none because of treatment. CONCLUSION: The nutritional intervention promoted catch-up growth and normalized excessive adiposity in IUGR animals at short-term. The benefits did not extend after a period of HFD. IUGR and early diet had gender-dependent effects on hepatic gene expression.

Murine intestinal cells expressing Trpm5 are mostly brush cells and express markers of neuronal and inflammatory cells.

To determine the role in chemosensation of intestinal solitary cells that express taste receptors and Trpm5, we carried out a microarray study of the transcriptome of FACS-sorted transgenic mouse intestinal cells expressing enhanced green fluorescent protein (eGFP) under the control of the Trpm5 promoter and compared it with that of intestinal cells that do not express eGFP. The findings of the study are: 1) Morphology and expression of markers show that most eGFP+ cells are brush cells. 2) The majority of proteins known to be involved in taste signal transduction are expressed in the eGFP+ cells, although the isoforms are not always the same. 3) eGFP+ cells express pre- and postsynaptic markers and nerves are often found in close proximity. 4) Several genes that play a role in inflammation are expressed specifically in eGFP+ cells. Furthermore, these cells express the entire biosynthesis pathway of leucotriene C4, an eicosanoid involved in modulation of intestinal smooth muscle contraction. 5) Angiotensinogen, renin, and succinate receptor genes are expressed in the eGFP+ cells, suggesting a role in the regulation of water and sodium transport, vasomotricity, and blood pressure. These data suggest that the Trpm5-expressing cells integrate many signals, including chemical signals from ingested food, and that they may regulate several physiological functions of the gastrointestinal tract.

The development of a high-density canine microarray.

DNA microarrays can give global transcriptional views of cellular responses to disease, development, nutrition, and other biological states. They can be used to elucidate biological networks, develop diagnostics, and identify genetic targets and molecular mechanisms. The technology is widely used and can be a valuable complement to more "disease-centric" focused arrays. For these reasons, Nestlé designed a custom canine Affymetrix microarray representing transcripts from multiple tissues for use in areas where a more focused microarray had not already been developed. Sufficient numbers of sequences representing messenger RNAs (mRNAs) or expressed sequence tags (ESTs) is integral for the design of a global microarray chip. This chip was designed using public domain sequences (GenBank) and sequences from a proprietary canine EST database. In order to enrich the chip with annotated transcripts, both of these sequence sets were BLASTed against the nonredundant protein database. The sequences on the microarray were isolated from more than 48 different tissues. The final compliment of sequences had sequences unique to GenBank (3160), unique to the proprietary EST database (17,620), and present in both sources (1996). In comparison with human sequences (RefSeq), 74% of the canine sequences matched a human sequence.

Effect of dietary phytic acid on zinc absorption in the healthy elderly, as assessed by serum concentration curve tests.

Zn absorption was investigated in healthy elderly subjects aged 71-78 years and in young subjects aged 23-43 years using serum concentration curve (SCC) tests. Both groups had similar Zn and protein status. The increase in serum Zn was monitored for 180 min after ingestion of 200 ml of soya milk enriched with 50 mg of Zn. Three levels of phytic acid were used: 0 g/200 ml (totally dephytinized soya milk), 0.13 g/200 ml (half dephytinized), and 0.26 g/200 ml (natural phytic acid content). In a first study the effect of 0 v. 0.26 g/200 ml phytic acid was compared in 10 elderly and 10 young subjects, each subject receiving both treatments. In a second study soya milks with 0 and 0.13 g/200 ml were tested in nine elderly and ten young subjects, again receiving both treatments. Mean areas under the curve of the SCC tests conducted with the 0 g/200 ml soya milk were found to be the same in both studies. Phytic acid strongly depressed Zn absorption in both studies (P < or = 0.05), but to a greater extent at the 0.26 g/200 ml level. No difference was found between the groups of young and elderly subjects. Therefore, no significant effect of aging on Zn absorption, as evaluated by the SCC test, or on the inhibitory effect of phytic acid was detected.

Microarray data analysis: a practical approach for selecting differentially expressed genes.

BACKGROUND: The biomedical community is rapidly developing new methods of data analysis for microarray experiments, with the goal of establishing new standards to objectively process the massive datasets produced from functional genomic experiments. Each microarray experiment measures thousands of genes simultaneously producing an unprecedented amount of biological information across increasingly numerous experiments; however, in general, only a very small percentage of the genes present on any given array are identified as differentially regulated. The challenge then is to process this information objectively and efficiently in order to obtain knowledge of the biological system under study and by which to compare information gained across multiple experiments. In this context, systematic and objective mathematical approaches, which are simple to apply across a large number of experimental designs, become fundamental to correctly handle the mass of data and to understand the true complexity of the biological systems under study. RESULTS: The present report develops a method of extracting differentially expressed genes across any number of experimental samples by first evaluating the maximum fold change (FC) across all experimental parameters and across the entire range of absolute expression levels. The model developed works by first evaluating the FC across the entire range of absolute expression levels in any number of experimental conditions. The selection of those genes within the top X% of highest FCs observed within absolute expression bins was evaluated both with and without the use of replicates. Lastly, the FC model was validated by both real time polymerase chain reaction (RT-PCR) and variance data. Semi-quantitative RT-PCR analysis demonstrated 73% concordance with the microarray data from Mu11K Affymetrix GeneChips. Furthermore, 94.1% of those genes selected by the 5% FC model were found to lie above measurement variability using a SDwithin confidence level of 99.9%. CONCLUSION: As evidenced by the high rate of validation, the FC model has the potential to minimize the number of required replicates in expensive microarray experiments by extracting information on gene expression patterns (e.g. characterizing biological and/or measurement variance) within an experiment. The simplicity of the overall process allows the analyst to easily select model limits which best describe the data. The genes selected by this process can be compared between experiments and are shown to objectively extract information which is biologically & statistically significant.

Protein-energy oral supplementation in malnourished nursing-home residents. A controlled trial.

OBJECTIVES: To validate a nutritional intervention programme for elderly people living in nursing homes. DESIGN: In a prospective, randomized, controlled study of 88 residents, we determined nutritional status at day 0 and day 60 using a record of dietary intake, anthropometry, hand-grip strength and mini-nutritional assessment. Dietary intake, grip strength and body weight were also recorded at day 30. We divided subjects into four groups according to their mini-nutritional assessment score. Those with a score 24 received no oral supplementation. Those at risk of malnutrition (with a score of 17-23.5) were randomized to oral supplementation. Those with a score <17 received oral supplementation. We recorded the amount of oral supplements consumed daily. RESULTS: Compliance with oral supplementation was good, and daily intake averaged about 400 kcal. The total energy intake on day 60 was significantly higher in both of the groups that received supplements. Following supplementation, most subjects at risk of malnutrition improved their mini-nutritional assessment score and increased their weight (by 1.4 +/- 0.5 kg). Neither the mini-nutritional assessment score nor weight improved in subjects at risk of malnutrition who did not receive supplements. Supplementation in the malnourished group resulted in a mean mini-nutritional assessment score increase (from 13.9 +/- 2.6 to 17.1 +/- 3.9) and a mean weight gain of 1.5 +/- 0.4 kg. CONCLUSION: Oral nutritional supplements are well accepted and result in increased daily protein and energy intake, body weight and nutritional status in most malnourished patients and in those at risk of malnutrition.