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The conditions of the gastrointestinal tract may change the physicochemical properties of nanoparticles (NPs) and therewith the bioavailability of orally taken NPs. Therefore, we assessed the impact ofin vitrogastrointestinal digestion on the protein corona of polystyrene NPs (PS-NPs) and their subsequent translocation across anin vitrointestinal barrier. A co-culture of intestinal Caco-2 and HT29-MTX cells was exposed to 50 nm PS-NPs of different charges (positive and negative) in two forms: pristine and digested in anin vitrogastrointestinal digestion model.In vitrodigestion significantly increased the translocation of all, except the “neutral”, PS-NPs. Uponin vitrodigestion, translocation was 4-fold higher for positively charged NPs and 80- and 1.7-fold higher for two types of negatively charged NPs. Digestion significantly reduced the amount of protein in the corona of three out of four types of NPs. This reduction of proteins was 4.8-fold for “neutral”, 3.5-fold for positively charged and 1.8-fold for one type of negatively charged PS-NPs.In vitrodigestion also affected the composition of the protein corona of PS-NPs by decreasing the presence of higher molecular weight proteins and shifting the protein content of the corona to low molecular weight proteins. These findings are the first to report thatin vitrogastrointestinal digestion significantly affects the protein corona and significantly increases thein vitrotranslocation of differently charged PS-NPs. These findings stress the importance of including thein vitrodigestion in futurein vitrointestinal translocation screening studies for risk assessment of orally taken NPs. [ABSTRACT FROM PUBLISHER]

Intestinal translocation is a key factor for determining bioavailability of nanoparticles (NPs) after oral uptake. Therefore, we evaluated threein vitrointestinal cell models of increasing complexity which might affect the translocation of NPs: a mono-culture (Caco-2 cells), a co-culture with mucus secreting HT29-MTX cells and a tri-culture with M-cells. Cell models were exposed to well characterized differently sized (50 and 100 nm) and charged (neutral, positively and negatively) polystyrene NPs. In addition, two types of negatively charged NPs with different surface chemistries were used. Size strongly affected the translocation of NPs, ranging up to 7.8% for the 50 nm NPs and 0.8% for the 100 nm NPs. Surface charge of NPs affected the translocation, however, surface chemistry seems more important, as the two types of negatively charged 50 nm NPs had an over 30-fold difference in translocation. Compared with the Caco-2 mono-culture, presence of mucus significantly reduced the translocation of neutral 50 nm NPs, but significantly increased the translocation of one type of negatively charged NPs. Incorporation of M-cells shifted the translocation rates for both NPs closer to those in the mono-culture model. The relative pattern of NP translocation in all three models was similar, but the absolute amounts of translocated NPs differed per model. We conclude that for comparing the relative translocation of different NPs, using one intestinal model is sufficient. To choose the most representative model for risk assessment,in vivoexperiments are now needed to determine thein vivotranslocation rates of the used NPs. [ABSTRACT FROM PUBLISHER]

Intestinal translocation is a key factor for determining bioavailability of nanoparticles (NPs) after oral uptake. Therefore, we evaluated three in vitro intestinal cell models of increasing complexity which might affect the translocation of NPs: a mono-culture (Caco-2 cells), a co-culture with mucus secreting HT29-MTX cells and a tri-culture with M-cells. Cell models were exposed to well characterized differently sized (50 and 100 nm) and charged (neutral, positively and negatively) polystyrene NPs. In addition, two types of negatively charged NPs with different surface chemistries were used. Size strongly affected the translocation of NPs, ranging up to 7.8% for the 50 nm NPs and 0.8% for the 100 nm NPs. Surface charge of NPs affected the translocation, however, surface chemistry seems more important, as the two types of negatively charged 50 nm NPs had an over 30-fold difference in translocation. Compared with the Caco-2 mono-culture, presence of mucus significantly reduced the translocation of neutral 50 nm NPs, but significantly increased the translocation of one type of negatively charged NPs. Incorporation of M-cells shifted the translocation rates for both NPs closer to those in the mono-culture model. The relative pattern of NP translocation in all three models was similar, but the absolute amounts of translocated NPs differed per model. We conclude that for comparing the relative translocation of different NPs, using one intestinal model is sufficient. To choose the most representative model for risk assessment, in vivo experiments are now needed to determine the in vivo translocation rates of the used NPs. [ABSTRACT FROM AUTHOR]

The mode of action of silver nanoparticles (AgNPs) is suggested to be exerted through both Ag+and AgNP dependent mechanisms. Ingestion is one of the major NP exposure routes, and potential effects are often studied using Caco-2 cells, a well-established model for the gut epithelium. MCF-7 cells are epithelial breast cancer cells with extensive well-characterized toxicogenomics profiles. In the present study, we aimed to gain a deeper understanding of the cellular molecular responses in Caco-2 and MCF-7 cells after AgNP exposure in order to evaluate whether epithelial cells derived from different tissues demonstrated similar responses. These insights could possibly reduce the size of cell panels for NP hazard identification screening purposes. AgNPs of 20, 30, 60, and 110 nm, and AgNO3were exposed for 6 h and 24 h. AgNPs were shown to be taken up and dissolve intracellularly. Compared with MCF-7 cells, Caco-2 cells showed a higher sensitivity to AgNPs, slower gene expression kinetics and absence of NP size-dependent responses. However, on a molecular level, no significant differences were observed between the two cell types. Transcriptomic analysis showed that Ag(NP) exposure caused (oxidative) stress responses, possibly leading to cell death in both cell lines. There was no indication for effects specifically induced by AgNPs. Responses to AgNPs appeared to be induced by silver ions released from the AgNPs. In conclusion, differences in mRNA responses to AgNPs between Caco-2 and MCF-7 cells were mainly related to timing and magnitude, but not to a different underlying mechanism. [ABSTRACT FROM AUTHOR]

Summary: Transgene expression in maize cells changed from intron-independent to intron-dependent by an exact exchange of the bar coding region for that of cat. By deletion mapping an approximately 100 nucleotide sequence element at the 5′ end of the cat coding region was identified that, when inserted at the translation start site of the bar gene, impaired expression. Successive inclusion of the salT intron in the 5′ untranslated region (UTR) restored expression near to wild-type bar expression levels. A chimeric gfp gene, but not nptII gene, behaved similarly. These observations are in agreement with the view that intron-mediated enhancement of transgene expression does not enhance, but rather restores expression of an impaired gene. [ABSTRACT FROM AUTHOR]

Beta- carotene 15,15′- monooxygenase 1 knockout ( Bcmo1−/−) mice accumulate beta-carotene (BC) similarly to humans, whereas wild-type ( Bcmo1+/+) mice efficiently cleave BC. Bcmo1− /− mice are therefore suitable to investigate BC-induced alterations in gene expression in lung, assessed by microarray analysis. Bcmo1− /− mice receiving control diet had increased expression of inflammatory genes as compared to BC-supplemented Bcmo1− /− mice and Bcmo1+/+ mice that received either control or BC-supplemented diets. Differential gene expression in Bcmo1− /− mice was confirmed by real-time quantitative PCR. Histochemical analysis indeed showed an increase in inflammatory cells in lungs of control Bcmo1− /− mice. Supported by metabolite and gene-expression data, we hypothesize that the increased inflammatory response is due to an altered BC metabolism, resulting in an increased vitamin A requirement in Bcmo1−/− mice. This suggests that effects of BC may depend on inter-individual variations in BC-metabolizing enzymes, such as the frequently occurring human polymorphisms in BCMO1. [ABSTRACT FROM AUTHOR]

Background: Dietary non-digestible carbohydrates stimulate the gut microflora and are therefore presumed to improve host resistance to intestinal infections. However, several strictly controlled rat infection studies showed that non-digestible fructo-oligosaccharides (FOS) increase, rather than decrease, translocation of Salmonella towards extra-intestinal sites. In addition, it was shown that FOS increases intestinal permeability already before infection. The mechanism responsible for this adverse effect of FOS is unclear. Possible explanations are altered mucosal integrity due to changes in tight junctions or changes in expression of defense molecules such as antimicrobials and mucins. To examine the mechanisms underlying weakening of the intestinal barrier by FOS, a controlled dietary intervention study was performed. Two groups of 12 rats were adapted to a diet with or without FOS. mRNA was collected from colonic mucosa and changes in gene expression were assessed for each individual rat using Agilent rat whole genome microarrays. Results: Among the 997 FOS induced genes we observed less mucosal integrity related genes than expected with the clear permeability changes. FOS did not induce changes in tight junction genes and only 8 genes related to mucosal defense were induced by FOS. These small effects are unlikely the cause for the clear increase in intestinal permeability that is observed. FOS significantly increased expression of 177 mitochondria-related genes. More specifically, induced expression of genes involved in all five OXPHOS complexes and the TCA cycle was observed. These results indicate that dietary FOS influences intestinal mucosal energy metabolism. Furthermore, increased expression of 113 genes related to protein turnover, including proteasome genes, ribosomal genes and protein maturation related genes, was seen. FOS upregulated expression of the peptide hormone proglucagon gene, in agreement with previous studies, as well as three other peptide hormone genes; peptide YY, pancreatic polypeptide and cholecystokinin. Conclusion: We conclude that altered energy metabolism may underly colonic barrier function disruption due to FOS feeding in rats. [ABSTRACT FROM AUTHOR]

Data on the molecular response of the intestine to the food-borne pathogen Salmonella are derived from in vitro studies, whereas in vivo data are lacking. We performed an oral S. enteritidis infection study in Wistar rats to obtain insight in the in vivo response in time. Expression profiles of ileal mucosa (IM) and Peyer's patches (PP) were generated using DNA microarrays at days 1, 3, and 6 postinfection. An overview of Salmonella-regulated processes was obtained and confirmed by quantitative real-time PCR on pooled and individual samples. Salmonella-induced gene expression responses in vivo are fewer and smaller than observed in vitro, and the response develops over a longer period of time. Few effects are seen at day 1 and mainly occur in IM, suggesting the mucosa as the primary site of invasion. Later, a bigger response is observed, especially in PP. Decreased expression of anti-microbial peptides genes (in IM at day 1) suggests inhibition of this process by Salmonella. Newly identified target processes are carbohydrate transport (increased expression in IM at day 1) and phase I and phase II detoxification (decreased expression at days 3 and 6). Increase of cytokine and chemokine expression occurs at later time points, both in PP and IM. Pancreatitis-associated protein, lipocalin 2, and calprotectin, potential inflammatory marker proteins, showed induced expression from day 3 onward. We conclude that the in vivo gene expression response of the ileum to Salmonella differs to a large extent from the response seen in vitro. [ABSTRACT FROM AUTHOR]

cDNA microarray technology is becoming the technique of choice for studying gene expression and gene expression patterns. Although experimental protocols are available, only limited methodological information on microarray manufacture, hybridization, and signal interpretation has been published. The aim of this paper is to provide more insight into the practical aspects of microarray construction and hybridization. The influence of the size, composition, and concentration of the spotted DNA fragments on the final hybridization signal and the effect of hybridization volume, sample concentration, and sample depletion have been tested and are discussed. [Copyright &y& Elsevier]

Reports the discovery of a missing lithotroph and its identification as an autotrophic member of the order Planctomycetales. Unculturability of the organism by conventional microbiological techniques; How the bacterium is responsible for anaerobic oxidation of ammonia; Implication for the removal of ammonia nitrogen from waste water.

Evidence from cell culture studies indicates that &bgr;-carotene-(BC)-derived apocarotenoid signaling molecules can modulate the activities of nuclear receptors that regulate many aspects of adipocyte physiology. Two BC metabolizing enzymes, the BC- 15,159-oxygenase (Bcmo1) and the BC-99,109-oxygenase (Bcdo2) are expressed in adipocytes. Bcmo1 catalyzes the conversion of BC into retinaldehyde and Bcdo2 into &bgr;-109-apocarotenal and &ggr;-ionone. Here we analyzed the impact of BC on body adiposity of mice. To genetically dissect the roles of Bcmo1 and Bcdo2 in this process, we used wild-type and Bcmo1-/- mice for this study. In wild-type mice, BC was converted into retinoids. In contrast, Bcmo1-/- mice showed increased expression of Bcdo2 in adipocytes and &bgr;-10'-apocarotenol accumulated as the major BC derivative. In wild-type mice, BC significantly reduced body adiposity (by 28%), leptinemia and adipocyte size. Genome wide microarray analysis of inguinal white adipose tissue revealed a generalized decrease of mRNA expression of peroxisome proliferator-activated receptor &ggr;(PPAR&ggr;) target genes. Consistently, the expression of this key transcription factor for lipogenesis was significantly reduced both on the mRNA and protein levels. Despite &bgr;-10'-apocarotenoid production, this effect of BC was absent in Bcmo1-/- mice, demonstrating that it was dependent on the Bcmo1-mediated production of retinoids. Our study evidences an important role of BC for the control of body adiposity in mice and identifies Bcmo1 as critical molecular player for the regulation of PPAR&ggr; activity in adipocytes [ABSTRACT FROM AUTHOR]

Background: Microbial infections induce ileal pancreatitis-associated protein/regenerating gene III (PAP/RegIII) mRNA expression. Despite increasing interest, little is known about the PAP/RegIII protein. Therefore, ileal mucosal PAP/RegIII protein expression, localization, and fecal excretion were studied in rats upon Salmonella infection.Results: Salmonella infection increased ileal mucosal PAP/RegIII protein levels in enterocytes located at the crypt-villus junction. Increased colonization and translocation of Salmonella was associated with higher ileal mucosal PAP/RegIII levels and secretion of this protein in feces.Conclusions: PAP/RegIII protein is increased in enterocytes of the ileal mucosa during Salmonella infection and is associated with infection severity. PAP/RegIII is excreted in feces and might be used as a new and non-invasive infection marker. [ABSTRACT FROM AUTHOR]

We have shown in several controlled rat and human infection studies that dietary calcium improves intestinal resistance and strengthens the mucosal barrier. Reinforcement of gut barrier function may alleviate inflammatory bowel disease (IBD). Therefore, we investigated the effect of supplemental calcium on spontaneous colitis development in an experimental rat model of IBD. HLA-B27 transgenic rats were fed a purified high-fat diet containing either a low or high calcium concentration 130 and 120 mmol CaHPO4/kg diet, respectively) for almost 7 wk. Inert chromium EDTA (CrEDTA) was added to the diets to quantify intestinal permeability by measuring urinary CrEDTA excretion. Relative fecal wet weight was determined to quantify diarrhea. Colonic inflammation was determined histologically and by measuring mucosal interleukin (IL)-1β. In addition, colonic mucosal gene expression of individual rats was analyzed using whole- genome microarrays. The calcium diet significantly inhibited the increase in intestinal permeability and diarrhea with time in HLA-B27 rats developing colitis compared with the control transgenic rats. Mucosal IL-1β levels were lower in calcium-fed rats and histological colitis scores tended to be lower (P = 0.08). Supplemental calcium prevented the colitis-induced increase in the expression of extracellular matrix remodeling genes (e.g. matrix metalloproteinases, procollagens, and fibronectin), which was confirmed by quantitative real-time PCR and gelatin zymography. In conclusion, dietary calcium ameliorates several important aspects of colitis severity in HLA-B27 transgenic rats. Reduction of mucosal irritation by luminal components might be part of the mechanism. These results show promise for supplemental calcium as effective adjunct therapy for IBD. [ABSTRACT FROM AUTHOR]

Summary: Microarray technology makes it feasible to analyse the expression of thousands of different gene elements in a single experiment. Most informative are ‘whole genome’ arrays, where all gene expression products of a single species or variety are represented. Such arrays are now available for a limited number of model species. However, for other, less well-documented species other routes are still necessary to obtain informative arrays. This includes the use of cDNA libraries. To enhance the amount of information that can be obtained from cDNA libraries, redundancy needs to be minimised, and the number of cDNAs relevant for the conditions of interest needs to be increased. Here, we used representational difference analysis (RDA), a mRNA subtraction procedure, as a tool to enhance the efficiency of cDNA libraries to be used to generate microarrays. Tomato was chosen as a model system for a less well-documented species. cDNA libraries for two distinct physiological conditions of tomato fruits, red and green, were made. The libraries were characterized by sequencing and hybridisation analysis. The RDA procedure was shown to be effective in selecting for genes of relevance for the physiological conditions under investigation, and against constitutively expressed genes. At the same time, redundancy was reduced, but complete normalisation was not obtained, and subsequent sequence analysis will be required to obtain non-redundant arrays. Further, known and putative ripening-related cDNAs were identified in hybridisation experiments on the basis of RNA populations as isolated from the green and red stage of ripening. [Copyright &y& Elsevier]