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We have built a quantitative systems toxicology modeling framework focused on the early prediction of oncotherapeutic‐induced clinical intestinal adverse effects. The model describes stem and progenitor cell dynamics in the small intestinal epithelium and integrates heterogeneous epithelial‐related processes, such as transcriptional profiles, citrulline kinetics, and probability of diarrhea. We fitted a mouse‐specific version of the model to quantify doxorubicin and 5‐fluorouracil (5‐FU)‐induced toxicity, which included pharmacokinetics and 5‐FU metabolism and assumed that both drugs led to cell cycle arrest and apoptosis in stem cells and proliferative progenitors. The model successfully recapitulated observations in mice regarding dose‐dependent disruption of proliferation which could lead to villus shortening, decrease of circulating citrulline, increased diarrhea risk, and transcriptional induction of the p53 pathway. Using a human‐specific epithelial model, we translated the cytotoxic activity of doxorubicin and 5‐FU quantified in mice into human intestinal injury and predicted with accuracy clinical diarrhea incidence. However, for gefitinib, a specific‐molecularly targeted therapy, the mice failed to reproduce epithelial toxicity at exposures much higher than those associated with clinical diarrhea. This indicates that, regardless of the translational modeling approach, preclinical experimental settings have to be suitable to quantify drug‐induced clinical toxicity with precision at the structural scale of the model. Our work demonstrates the usefulness of translational models at early stages of the drug development pipeline to predict clinical toxicity and highlights the importance of understanding cross‐settings differences in toxicity when building these approaches. [ABSTRACT FROM AUTHOR]

The effect of NaCl concentration during sporulation and subsequent growth on the distributions of times associated with different stages of the lag phase of individual spores of nonproteolytic Clostridium botulinum strain Eklund 17B is determined. The results on distribution of lag times for individual spores have helped in understanding the mechanisms that can be used in improving the predictions of growth in food and refining risk assessments.

The successive generation times for single cells of Escherichia coli K-12 were measured and the histograms they generated were used as empirical distributions to simulate growth of the population as the result of the multiplication of its single cells. The results show that the differences in the lag time duration was reported for greater inoculum sizes, but those cells were severely stressed by starvation or heating or growth conditions were very close to the growth-no growth boundary.

Campylobacter jejuni is a leading cause of bacterial gastroenteritis in the developed world. The role of a homologue of the negative transcriptional regulatory protein HspR, which in other organisms participates in the control of the heat-shock response, was investigated. Following inactivation of hspR in C. jejuni, members of the HspR regulon were identified by DNA microarray transcript profiling. In agreement with the predicted role of HspR as a negative regulator of genes involved in the heat-shock response, it was observed that the transcript amounts of 13 genes were increased in the hspR mutant, including the chaperone genes dnaK, grpE and clpB, and a gene encoding the heat-shock regulator HrcA. Proteomic analysis also revealed increased synthesis of the heat-shock proteins DnaK, GrpE, GroEL and GroES in the absence of HspR. The altered expression of chaperones was accompanied by heat sensitivity, as the hspR mutant was unable to form colonies at 44 [degrees]C. Surprisingly, transcriptome analysis also revealed a group of 17 genes with lower transcript levels in the hspR mutant. Of these, eight were predicted to be involved in the formation of the flagella apparatus, and the decreased expression is likely to be responsible for the reduced motility and ability to autoagglutinate that was observed for hspR mutant cells. Electron micrographs showed that mutant cells were spiral-shaped and carried intact flagella, but were elongated compared to wild-type cells. The inactivation of hspR also reduced the ability of Campylobacter to adhere to and invade human epithelial INT-407 cells in vitro, possibly as a consequence of the reduced motility or lower expression of the flagellar export apparatus in hspR mutant cells. It was concluded that, in C. jejuni, HspR influences the expression of several genes that are likely to have an impact on the ability of the bacterium to successfully survive in food products and subsequently infect the consumer.

Campylobacter jejuni is a zoonotic pathogen and the most common cause of bacterial foodborne diarrhoeal illness worldwide. To establish intestinal colonization prior to either a commensal or pathogenic interaction with the host, C. jejuni will encounter iron-limited niches where there is likely to be intense competition from the host and normal microbiota for iron. To gain a better understanding of iron homeostasis and the role of ferric uptake regulator (Fur) in iron acquisition in C. jejuni, a proteomic and transcriptome analysis of wild-type and fur mutant strains in iron-rich and iron-limited growth conditions was carried out. All of the proposed iron-transport systems for haemin, ferric iron and enterochelin, as well as the putative iron-transport genes p19, Cj1658, Cj0177, Cj0178 and cfrA, were expressed at higher levels in the wild-type strain under iron limitation and in the fur mutant in iron-rich conditions, suggesting that they were regulated by Fur. Genes encoding a previously uncharacterized ABC transport system (Cj1660-Cj1663) also appeared to be Fur regulated, supporting a role for these genes in iron uptake. Several promoters containing consensus Fur boxes that were identified in a previous bioinformatics search appeared not to be regulated by iron or Fur, indicating that the Fur box consensus needs experimental refinement. Binding of purified Fur to the promoters upstream of the p19, CfrA and CeuB operons was verified using an electrophoretic mobility shift assay (EMSA). These results also implicated Fur as having a role in the regulation of several genes, including fumarate hydratase, that showed decreased expression in response to iron limitation. The known PerR promoters were also derepressed in the C. jejuni Fur mutant, suggesting that they might be co-regulated in response to iron and peroxide stress. These results provide new insights into the effects of iron on metabolism and oxidative stress response as well as the regulatory role of Fur. Present address: Swammerdam Institute for Life Sciences, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands. Abbreviation: EMSA, electrophoretic mobility shift assay. The raw microarray data are available as a supplement to the online version of this paper (at http://mic.sgmjournals.org), which also has links to Supplementary Tables A-C (available with the online version of the paper only).

Byline: Roberto Carcoba (1), Carmen Pin (2), Ana Rodriguez (1) Keywords: Lactococcus lactis; Chemostat cultures; Lactose metabolism; Dilution rate; pH Abstract: Lactococcus lactis subsp. lactis IPLA 947 is a starter strain isolated from a Spanish farmhouse acid-coagulated cheese. To use this strain as starter culture in Afuega'l Pitu cheese manufacture, anaerobic lactose-limited chemostat cultures were used to optimise the active biomass production in a simple medium (BRFS). Growth, lactose consumption and metabolite production were measured at different pH values (5.5, 6.0 and 6.5) and dilution rates (0.06 to 0.96 h.sup.-1). The influence of both variables on the quoted parameters was described by means of mathematical models. These provided a satisfactory representation of the experimental data. The bacterial response surface showed a significant increase in the biomass when the pH increased and the dilution rate decreased. As dilution rate increased, biomass yield increased but only at the lowest pH. The lactose consumption and the ratio of metabolic end products were also influenced by both variables. As dilution rate decreased, the residual lactose decreased and increased amounts of metabolites other than lactate (acetate, formate, ethanol) were produced. The minimum lactose consumption was obtained at pH 5.5 and 0.96 h.sup.-1 dilution rate. A significant rise of acetate and formate levels occurred as pH increased. A maximum lactate level, along with an acetate, formate and ethanol production close to the minimum, was predicted by mathematical modelling at 0.5173 h.sup.-1 dilution rate and pH 6.054. Author Affiliation: (1) Instituto de Productos Lacteos de Asturias (IPLA-CSIC), Apdo. 85, 33300, Villaviciosa, Asturias, Spain (2) Institute of Food Research, Norwich Research Park, Norwich, NR4 7UA, UK Article History: Registration Date: 01/06/2004 Received Date: 28/01/2004 Online Date: 02/07/2004

Research has been conducted on Brochothrix thermosphacta glucose metabolism. The role of atmosphere composition in this metabolism has been investigated and the decision whether the metabolism is dominantly anaerobic or anaerobic for a given atmosphere composition has been made.

Research was conducted to examine a novel numerical approach in estimating bacterial growth parameters using detection times generated by different initial counts. Detection times were subjected to a transformation involving the maximum specific growth rate and the ratios between the different inoculum sizes and the constant detectable level of counts. The more accurate the cell number at the detection level, the better the estimate will be for the variance of the lag times of the individual cells.

Abstract Background Microarrays are widely used for the study of gene expression; however deciding on whether observed differences in expression are significant remains a challenge. Results A computing tool (ArrayLeaRNA) has been developed for gene expression analysis. It implements a Bayesian approach which is based on the Gumbel distribution and uses printed genomic DNA control features for normalization and for estimation of the parameters of the Bayesian model and prior knowledge from predicted operon structure. The method is compared with two other approaches: the classical LOWESS normalization followed by a two fold cut-off criterion and the OpWise method (Price, et al. 2006. BMC Bioinformatics. 7, 19), a published Bayesian approach also using predicted operon structure. The three methods were compared on experimental datasets with prior knowledge of gene expression. With ArrayLeaRNA, data normalization is carried out according to the genomic features which reflect the results of equally transcribed genes; also the statistical significance of the difference in expression is based on the variability of the equally transcribed genes. The operon information helps the classification of genes with low confidence measurements. ArrayLeaRNA is implemented in Visual Basic and freely available as an Excel add-in at http://www.ifr.ac.uk/safety/ArrayLeaRNA/ Conclusion We have introduced a novel Bayesian model and demonstrated that it is a robust method for analysing microarray expression profiles. ArrayLeaRNA showed a considerable improvement in data normalization, in the estimation of the experimental variability intrinsic to each hybridization and in the establishment of a clear boundary between non-changing and differentially expressed genes. The method is applicable to data derived from hybridizations of labelled cDNA samples as well as from hybridizations of labelled cDNA with genomic DNA and can be used for the analysis of datasets where differentially regulated genes predominate.

Stability analysis, often overlooked in pharmacometrics, is essential to explore dynamical systems. The model developed by Friberg et al.1 to describe drug‐induced hematotoxicity is widely used to support decisions across drug development, and parameter values are often identified from observed blood counts. We use stability analysis to study the parametric dependence of stable and unstable solutions of several Friberg‐type models and highlight the risks associated with system instability in the context of nonlinear mixed effects modeling. We emphasize the consequences of unstable solutions on prediction performance by demonstrating nonbiological system behaviors in a real case study of drug‐induced thrombocytopenia. Ultimately, we provide simple criteria for identifying parameters associated with stable solutions of Friberg‐type models. For instance, in the original Friberg model, we find that stability depends only on the parameter that governs the feedback from peripheral cells to progenitors and provide the exact range of values that results in stable solutions. [ABSTRACT FROM AUTHOR]

Drug-induced gastrointestinal toxicities (DI-GITs) are among the most common adverse events in clinical trials. High prevalence of DI-GIT has persisted among new drugs due in part to the lack of robust experimental tools to allow early detection or to guide optimization of safer molecules. Developing in vitro assays for the leading GI toxicities (nausea, vomiting, diarrhoea, constipation, and abdominal pain) will likely involve recapitulating complex physiological properties that require contributions from diverse cell/tissue types including epithelial, immune, microbiome, nerve, and muscle. While this stipulation may be beyond traditional 2D monocultures of intestinal cell lines, emerging 3D GI microtissues capture interactions between diverse cell and tissue types. These interactions give rise to microphysiologies fundamental to gut biology. For GI microtissues, organoid technology was the breakthrough that introduced intestinal stem cells with the capability of differentiating into each of the epithelial cell types and that self-organize into a multi-cellular tissue proxy with villus- and crypt-like domains. Recently, GI microtissues generated using miniaturized devices with microfluidic flow and cyclic peristaltic strain were shown to induce Caco2 cells to spontaneously differentiate into each of the principle intestinal epithelial cell types. Second generation models comprised of epithelial organoids or microtissues co-cultured with non-epithelial cell types can successfully reproduce cross-'tissue' functional interactions broadening the potential of these models to accurately study drug-induced toxicities. A new paradigm in which in vitro assays become an early part of GI safety assessment could be realized if microphysiological systems (MPS) are developed in alignment with drug-discovery needs. Herein, approaches for assessing GI toxicity of pharmaceuticals are reviewed and gaps are compared with capabilities of emerging GI microtissues (e.g., organoids, organ-on-a-chip, transwell systems) in order to provide perspective on the assay features needed for MPS models to be adopted for DI-GIT assessment. [ABSTRACT FROM AUTHOR]

The dynamic model for the growth of a bacterial population was applied to model the lag period and exponential growth of Listeria monocytogenes under conditions of fluctuating temperature and water activity values. Observations for several scenarios in which exponential growth was halted by a sudden change in the temperature and/or water activity values were in good agreement with predictions.

The effects of C[O.sub.2] on gene expression and neurotoxin formation by proteolytic Clostridium botulinum type A1 strain ATCC 3502 are analyzed during the growth cycle. Microarray analysis has enabled the identification of coding sequences whose expression profiles have matched with those of the neurotoxin cluster and a DNA microarray is used for studying the transcriptome.

A study was conducted to assess the suitability of real-time PCR for the quantification of Listeria monocytogenes under different stress conditions and no-stress conditions in a culture model. The differences observed between the data obtained from low-stress conditions plate count and real-time PCR data are related to the stress imposed on L. monocytogenes and changed during growth.

The development of a stochastic model is reported to simulate the growth of the population resulting from the single-cell division times in Escherichia coli. Results suggest that greater the variability of the single-cell interdivision intervals, the shorter are both the lag time and the doubling time of the population.

A novel attempt is made to determine the distributions of times associated with the various stages of germination and subsequent growth from spores and the relationships between these stages. As the variability in postgermination stages is relatively large, the first spore to germinate will not necessarily be the first spore to produce actively dividing cells and start neurotoxin production.

The distinct growth and death toll of Aeromonas hydrophilia were calculated in laboratory environment with a mixture of different temperatures, pH along with the percentage of CO2 and O2 in the environment. The predictive models were formed from the information available and also were updated with the help of the results procured from the scrutiny made of the seafood experiments for this purpose and the combase information.

Haematological toxicity associated with cancer therapeutics is monitored by changes in blood cell count, and their primary effect is on proliferative progenitors in the bone marrow. Using observations in rat bone marrow and blood, we characterize a mathematical model that comprises cell proliferation and differentiation of the full haematopoietic phylogeny, with interacting feedback loops between lineages in homeostasis as well as following carboplatin exposure. We accurately predicted the temporal dynamics of several mature cell types related to carboplatin‐induced bone marrow toxicity and identified novel insights into haematopoiesis. Our model confirms a significant degree of plasticity within bone marrow cells, with the number and type of both early progenitors and circulating cells affecting cell balance, via feedback mechanisms, through fate decisions of the multipotent progenitors. We also demonstrated cross‐species translation of our predictions to patients, applying the same core model structure and considering differences in drug‐dependent and physiology‐dependent parameters. [ABSTRACT FROM AUTHOR]

Drug-induced gastrointestinal toxicities (GITs) rank among the most common clinical side effects. Preclinical efforts to reduce incidence are limited by inadequate predictivity of in vitro assays. Recent breakthroughs in in vitro culture methods support intestinal stem cell maintenance and continual differentiation into the epithelial cell types resident in the intestine. These diverse cells self-assemble into microtissues with in vivo -like architecture. Here, we evaluate human GI microtissues grown in transwell plates that allow apical and/or basolateral drug treatment and 96-well throughput. Evaluation of assay utility focused on predictivity for diarrhea because this adverse effect correlates with intestinal barrier dysfunction which can be measured in GI microtissues using transepithelial electrical resistance (TEER). A validation set of widely prescribed drugs was assembled and tested for effects on TEER. When the resulting TEER inhibition potencies were adjusted for clinical exposure, a threshold was identified that distinguished drugs that induced clinical diarrhea from those that lack this liability. Microtissue TEER assay predictivity was further challenged with a smaller set of drugs whose clinical development was limited by diarrhea that was unexpected based on 1-month animal studies. Microtissue TEER accurately predicted diarrhea for each of these drugs. The label-free nature of TEER enabled repeated quantitation with sufficient precision to develop a mathematical model describing the temporal dynamics of barrier damage and recovery. This human 3D GI microtissue is the first in vitro assay with validated predictivity for diarrhea-inducing drugs. It should provide a platform for lead optimization and offers potential for dose schedule exploration. [ABSTRACT FROM AUTHOR]

Summary: The gastrointestinal tract is a highly complex organ in which multiple dynamic physiological processes are tightly coordinated while interacting with a dense and extremely diverse microbial population. From establishment in early life, through to host‐microbe symbiosis in adulthood, the gut microbiota plays a vital role in our development and health. The effect of the microbiota on gut development and physiology is highlighted by anatomical and functional changes in germ‐free mice, affecting the gut epithelium, immune system and enteric nervous system. Microbial colonisation promotes competent innate and acquired mucosal immune systems, epithelial renewal, barrier integrity, and mucosal vascularisation and innervation. Interacting or shared signalling pathways across different physiological systems of the gut could explain how all these changes are coordinated during postnatal colonisation, or after the introduction of microbiota into germ‐free models. The application of cell‐based in‐vitro experimental systems and mathematical modelling can shed light on the molecular and signalling pathways which regulate the development and maintenance of homeostasis in the gut and beyond. [ABSTRACT FROM AUTHOR]

Our work addresses two key challenges, one biological and one methodological. First, we aim to understand how proliferation and cell migration rates in the intestinal epithelium are related under healthy, damaged (Ara-C treated) and recovering conditions, and how these relations can be used to identify mechanisms of repair and regeneration. We analyse new data, presented in more detail in a companion paper, in which BrdU/IdU cell-labelling experiments were performed under these respective conditions. Second, in considering how to more rigorously process these data and interpret them using mathematical models, we use a probabilistic, hierarchical approach. This provides a best-practice approach for systematically modelling and understanding the uncertainties that can otherwise undermine the generation of reliable conclusionsÐuncertainties in experimental measurement and treatment, difficult-to-compare mathematical models of underlying mechanisms, and unknown or unobserved parameters. Both spatially discrete and continuous mechanistic models are considered and related via hierarchical conditional probability assumptions. We perform model checks on both in-sample and out-of-sample datasets and use them to show how to test possible model improvements and assess the robustness of our conclusions. We conclude, for the present set of experiments, that a primarily proliferation-driven model suffices to predict labelled cell dynamics over most time-scales. [ABSTRACT FROM AUTHOR]

Intestinal homeostasis is maintained by a hierarchy of immune defenses acting in concert to minimize contact between luminal microorganisms and the intestinal epithelial cell surface. The intestinal mucus layer, covering the gastrointestinal tract epithelial cells, contributes to mucosal homeostasis by limiting bacterial invasion. In this study, we used ?d T-cell-deficient (TCRd-/-) mice to examine whether and how ?d T-cells modulate the properties of the intestinal mucus layer. Increased susceptibility of TCRd-/- mice to dextran sodium sulfate (DSS)-induced colitis is associated with a reduced number of goblet cells. Alterations in the number of goblet cells and crypt lengths were observed in the small intestine and colon of TCRd-/- mice compared with C57BL/6 wild-type (WT) mice. Addition of keratinocyte growth factor to small intestinal organoid cultures from TCRd-/- mice showed a marked increase in crypt growth and in both goblet cell number and redistribution along the crypts. There was no apparent difference in the thickness or organization of the mucus layer between TCRd-/- and WT mice, as measured in vivo. However, ?d T-cell deficiency led to reduced sialylated mucins in association with increased gene expression of gel-secreting Muc2 and membrane-bound mucins, including Muc13 and Muc17. Collectively, these data provide evidence that ?d T cells play an important role in the maintenance of mucosal homeostasis by regulating mucin expression and promoting goblet cell function in the small intestine. [ABSTRACT FROM AUTHOR]

Mucins are the main components of the gastrointestinal mucus layer. Mucin glycosylation is critical to most intermolecular and intercellular interactions. However, due to the highly complex and heterogeneous mucin glycan structures, the encoded biological information remains largely encrypted. Here we have developed a methodology based on force spectroscopy to identify biologically accessible glycoepitopes in purified porcine gastric mucin (pPGM) and purified porcine jejunal mucin (pPJM). The binding specificity of lectins Ricinus communis agglutinin I (RCA), peanut (Arachis hypogaea) agglutinin (PNA), Maackia amurensis lectin II (MALII), and U/ex europaeus agglufinin I (UEA) was utilized in force spectroscopy measurements to quantify the affinity and spatial distribution of their cognate sugars at the molecular scale. Binding energy of 4, 1.6, and 26 aJ was determined on pPGM for RCA, PNA, and UEA. Binding was abolished by competition with free ligands, demonstrating the validity of the affinity data. The distributions of the nearest binding site separations estimated the number of binding sites in a 200-nm mucin segment to be 4 for RCA, PNA, and UEA, and 1.8 for MALII. Binding site separations were affected by partial defucosylation of pPGM. Furthermore, we showed that this new approach can resolve differences between gastric and jejunum mucins. [ABSTRACT FROM AUTHOR]

Loss of culturability of Salmonella enterica subsp. enterica serovar Typhimurium has been observed in mixed cultures with anaerobic fecal bacteria under conditions that allow local interaction between cells, such as cell contact. A reduction of a population of culturable S. Typhimurium on the order of ~104 to 105 CFU/ml was observed in batch anaerobic mixed cultures with fecal samples from different human donors. Culturability was not affected either in supernatants collected at several times from fecal cultures, when separated from fecal bacteria by a membrane of 0.45-&8764;mpore size, or when in contact with inactivated fecal bacterial cells. Loss of culturability kinetics was characterized by a sharp reduction of several logarithmic units followed by a pronounced tail. A mathematical model was developed to describe the rate of loss of culturability as a function of the frequency of encounters between populations and the probability of inactivation after encounter. The model term F(S . F)1/2 quantifies the effect of the concentration of both populations, fecal bacteria (F) and S. Typhimurium (S), on the loss of culturability of S. Typhimurium by cell contact with fecal bacteria. When the value of F(S . F)1/2 decreased below ca. 1015 (CFU/ml)2, the frequency of encounters sharply decreased, leading to the deceleration of the inactivation rate and to the tailing off of the S. Typhimurium population. The probability of inactivation after encounter, P, was constant, with an estimated value of∼10-5 for all data sets. P might be characteristic of the mechanism of growth inhibition after a cell encounter. [ABSTRACT FROM AUTHOR]

Background Salmonella Typhimurium is an important pathogen of human and animals. It shows a broad growth range and survives in harsh conditions. The aim of this study was to analyze transcriptional responses to a number of growth and stress conditions as well as the relationship of metabolic pathways and/or cell functions at the genome-scale-level by network analysis, and further to explore whether highly connected genes (hubs) in these networks were essential for growth, stress adaptation and virulence. Results De novo generated as well as published transcriptional data for 425 selected genes under a number of growth and stress conditions were used to construct a bipartite network connecting culture conditions and significantly regulated genes (transcriptional network). Also, a genome scale network was constructed for strain LT2. The latter connected genes with metabolic pathways and cellular functions. Both networks were shown to belong to the family of scalefree networks characterized by the presence of highly connected nodes or hubs which are genes whose transcription is regulated when responding to many of the assayed culture conditions or genes encoding products involved in a high number of metabolic pathways and cell functions. The five genes with most connections in the transcriptional network (wraB, ygaU, uspA, cbpA and osmC) and in the genome scale network (ychN, siiF (STM4262), yajD, ybeB and dcoC) were selected for mutations, however mutagenesis of ygaU and ybeB proved unsuccessful. No difference between mutants and the wild type strain was observed during growth at unfavorable temperatures, pH values, NaCl concentrations and in the presence of H2O2. Eight mutants were evaluated for virulence in C57/BL6 mice and none differed from the wild type strain. Notably, however, deviations of phenotypes with respect to the wild type were observed when combinations of these genes were deleted. Conclusion Network analysis revealed the presence of hubs in both transcriptional and functional networks of S. Typhimurium. Hubs theoretically confer higher resistance to random mutation but a greater susceptibility to directed attacks, however, we found that genes that formed hubs were dispensable for growth, stress adaptation and virulence, suggesting that evolution favors non-essential genes as main connectors in cellular networks. [ABSTRACT FROM AUTHOR]

We investigated if the transcriptional response of Salmonella Typhimurium to temperature and acid variations was hysteretic, i.e. whether the transcriptional regulation caused by environmental stimuli showed memory and remained after the stimuli ceased. The transcriptional activity of non-replicating stationary phase cells of S. Typhimurium caused by the exposure to 45°C and to pH 5 for 30 min was monitored by microarray hybridizations at the end of the treatment period as well as immediately and 30 minutes after conditions were set back to their initial values, 25°C and pH 7. One hundred and two out of 120 up-regulated genes during the heat shock remained up-regulated 30 minutes after the temperature was set back to 25°C, while only 86 out of 293 down regulated genes remained down regulated 30 minutes after the heat shock ceased. Thus, the majority of the induced genes exhibited hysteresis, i.e., they remained up-regulated after the environmental stress ceased. At 25°C the transcriptional regulation of genes encoding for heat shock proteins was determined by the previous environment. Gene networks constructed with up-regulated genes were significantly more modular than those of down-regulated genes, implying that down-regulation was significantly less synchronized than upregulation. The hysteretic transcriptional response to heat shock was accompanied by higher resistance to inactivation at 50°C as well as cross-resistance to inactivation at pH 3; however, growth rates and lag times at 43°C and at pH 4.5 were not affected. The exposure to pH 5 only caused up-regulation of 12 genes and this response was neither hysteretic nor accompanied of increased resistance to inactivation conditions. Cellular memory at the transcriptional level may represent a mechanism of adaptation to the environment and a deterministic source of variability in gene regulation. [ABSTRACT FROM AUTHOR]

We developed a slow structural relaxation model to describe cellular dynamics in the crypt of the mouse small intestine. Cells are arranged in a three dimensional spiral the size of which dynamically changes according to cell production demands of adjacent villi. Cell differentiation and proliferation is regulated through Wnt and Notch signals, the strength of which depends on the local cell composition. The highest level of Wnt activity is associated with maintaining equipotent stem cells (SC), Paneth cells and common goblet-Paneth cell progenitors (CGPCPs) intermingling at the crypt bottom. Low levels of Wnt signalling area are associated with stem cells giving rise to secretory cells (CGPCPs, enteroendocrine or Tuft cells) and proliferative absorptive progenitors. Deciding between these two fates, secretory and stem/absorptive cells, depends on Notch signalling. Our model predicts that Notch signalling inhibits secretory fate if more than 50% of cells they are in contact with belong to the secretory lineage. CGPCPs under high Wnt signalling will differentiate into Paneth cells while those migrating out from the crypt bottom differentiate into goblet cells. We have assumed that mature Paneth cells migrating upwards undergo anoikis. Structural relaxation explains the localisation of Paneth cells to the crypt bottom in the absence of active forces. The predicted crypt generation time from one SC is 4-5 days with 10-12 days needed to reach a structural steady state. Our predictions are consistent with experimental observations made under altered Wnt and Notch signalling. Mutations affecting stem cells located at the crypt floor have a 50% chance of being propagated throughout the crypt while mutations in cells above are rarely propagated. The predicted recovery time of an injured crypt losing half of its cells is approximately 2 days. [ABSTRACT FROM AUTHOR]

Specific molecular-receptor interactions with gut epithelium cells are important in understanding bioactivity of food components and drugs, binding of commensal microflora, attachment and initiation of defense mechanisms against pathogenic bacteria and for development of targeted delivery systems to the gut. However, methods for probing such interactions are lacking. Methodology has been developed and validated to measure specific molecular-receptor interactions on living human colorectal cancer cells as in vitro models for the gut epithelium. Atomic force microscopy (AFM) was used to measure ligand-receptor interactions and to map receptor locations on cell surfaces. Measurements were made using silica beads attached to the AFM tip-cantilever assembly, which were functionalized by coupling of ligands to the bead surface. Wheat germ agglutinin (WGA) binds to the glycosylated extracellular domain III of the epidermal growth factor receptor. Methodology was tested by measuring binding of WGA to the surface of confluent monolayers of living Caco-2 human intestinal epithelial cells. The measured modal detachment force of 125 pN is typical of values expected for single molecule interactions. Adhesive events were used to map the location of binding sites on the cell surface revealing heterogeneity in their distribution within and between cells within the monolayer.--Gunning, A. P., Chambers, S., Pin, C., Man, A. L., Morris, V. J., Nicoletti, C. Mapping specific adhesive interactions on living human intestinal epithelial cells with atomic force microscopy. [ABSTRACT FROM AUTHOR]

Abstract: Data from a database on microbial responses to the food environment (ComBase, see www.combase .cc) were used to study the boundary of growth several pathogens (Aeromonas hydrophila, Escherichia coli, Listeria monocytogenes, Yersinia enterocolitica). Two methods were used to evaluate the growth/no growth interface. The first one is an application of the Minimum Convex Polyhedron (MCP) introduced by Baranyi et al. [Baranyi, J., Ross, T., McMeekin, T., Roberts, T.A., 1996. The effect of parameterisation on the performance of empirical models used in Predictive Microbiology. Food Microbiol. 13, 83–91.]. The second method applies logistic regression to define the boundary of growth. The combination of these two different techniques can be a useful tool to handle the problem of extrapolation of predictive models at the growth limits. [Copyright &y& Elsevier]

A generalised z-value concept has been applied to analyse the relationship between the lag and the growth rate of Yersinia enterocolitica at a range of temperature, atmospheric carbon dioxide and oxygen percentages. The product of the specific growth rate and the lag (the “work to be done” during the lag phase) is found to be independent of temperature. However, it does depend on the CO2 and O2 concentrations, though the effect of oxygen was less noticeable than the effect of carbon dioxide. [Copyright &y& Elsevier]

Abstract: We aim to predict the population density of Salmonella spp. through the pork supply chain under dynamic environmental conditions (pH, a w and temperature) that fluctuate from growth to survival/slow inactivation. To do this, the dependence of the probability of growth, and of the growth and inactivation rate on the temperature, pH and a w were modelled. Probabilistic and kinetic measurements, i.e. growth and survival curves, were collected from the ComBase database (www.combase.cc). Conditions at which selected data used to fit the models were generated covered wide ranges that are relevant to the pork supply chain. Probabilistic and kinetic models were combined to give predictions on the concentration of Salmonella spp. at any stage of the pork supply chain under fluctuating pH, a w and/or temperature. Models were implemented in a user-friendly computing tool freely available from http://www.ifr.ac.uk/safety/SalmonellaPredictions/. This program provides estimates on the population dynamics of Salmonella spp. at any stage of the pork supply chain and its predictive performance has been validated in several pork products. [Copyright &y& Elsevier]

Abstract: We have investigated the effect of storage temperature (0–20 °C) and carbon dioxide concentration (0–100% v/v, balance nitrogen) on the growth of Carnobacterium maltaromaticum, Serratia proteamaculans, Yersinia intermedia and Shewanella baltica, as well as on the growth of a mixed culture of the four species. These species were identified as the organisms responsible for spoilage in mackerel fillets packed under modified atmospheres in preliminary studies. The growth rates of all those groups were measured at several temperatures between 0 and 20 °C in cultures stored under air. Models describing the dependence of the maximum specific growth rate on temperature, CO2 and O2 were developed for each organism. C. maltaromaticum was the organism that showed the highest resistance to CO2 and to the lack of O2, while under aerobic condition at 0 °C. S. baltica showed the fastest growth rate. Model predictions were compared with observations on naturally contaminated horse mackerel fillets packed under modified atmospheres as well as inoculated with the bacteria strains used to generate the data for model development. These validation studies showed a good performance of models under constant and fluctuating temperature conditions. Models were implemented in a user-friendly computing tool called “Fishmap” (freely available at http://www.azti.es/en/fish-map-software.html). This program predicts the growth of spoilage bacteria in horse mackerel fillets stored at constant and fluctuating temperature conditions under modified atmospheres and under air. [Copyright &y& Elsevier]