A next generation sequencing gene panel for use in the diagnosis of anorexia nervosa.

PURPOSE: The aim of this study was to increase knowledge of genes associated with anorexia nervosa (AN) and their diagnostic offer, using a next generation sequencing (NGS) panel for the identification of genetic variants. The rationale underlying this test is that we first analyze the genes associated with syndromic forms of AN, then genes that were found to carry rare variants in AN patients who had undergone segregation analysis, and finally candidate genes intervening in the same molecular pathways or identified by GWAS or in mouse models. METHODS: We developed an NGS gene panel and used it to screen 68 Italian AN patients (63 females, 5 males). The panel included 162 genes. Family segregation study was conducted on available relatives of probands who reported significant genetic variants. RESULTS: In our analysis, we found potentially deleterious variants in 2 genes (PDE11A and SLC25A13) associated with syndromic forms of anorexia and predicted deleterious variants in the following 12 genes: CD36, CACNA1C, DRD4, EPHX2, ESR1, GRIN2A, GRIN3B, LRP2, NPY4R, PTGS2, PTPN22 and SGPP2. Furthermore, by Sanger sequencing of the promoter region of NNAT, we confirmed the involvement of this gene in the pathogenesis of AN. Family segregation studies further strengthened the possible causative role of CACNA1C, DRD4, GRIN2A, PTGS2, SGPP2, SLC25A13 and NNAT genes in AN etiology. CONCLUSION: The major finding of our study is the confirmation of the involvement of the NNAT gene in the pathogenesis of AN; furthermore, this study suggests that NGS-based testing can play an important role in the diagnostic evaluation of AN, excluding syndromic forms and increasing knowledge of the genetic etiology of AN. LEVEL OF EVIDENCE: Level I, experimental study.

Development of Salmon Sperm DNA/Regenerated Silk Bio-Based Films for Biomedical Studies on Human Keratinocyte HaCaT Cells under Solar Spectrum.

In this study, we fabricated adhesive patches from silkworm-regenerated silk and DNA to safeguard human skin from the sun's rays. The patches are realized by exploiting the dissolution of silk fibers (e.g., silk fibroin (SF)) and salmon sperm DNA in formic acid and CaCl2 solutions. Infrared spectroscopy is used to investigate the conformational transition of SF when combined with DNA; the results indicated that the addition of DNA provides an increase in the SF crystallinity. UV-Visible absorption and circular dichroism spectroscopy showed strong absorption in the UV region and the presence of B-form of DNA once dispersed in the SF matrix, respectively. Water absorption measurements as well as thermal dependence of water sorption and thermal analysis, suggested the stability of the fabricated patches. Biological results on cellular viability (MTT assay) of keratinocyte HaCaT cells after exposures to the solar spectrum showed that both SF and SF/DNA patches are photo-protective by increasing the cellular viability of keratinocytes after UV component exposure. Overall, these SF/DNA patches promise applications in wound dressing for practical biomedical purposes.

Dietary supplements for intestinal inflammation.

Intestinal inflammation leads to various chronic diseases, collectively known as inflammatory bowel disease (IBD). IBD mainly affects the large intestine, but it can also affect the gastrointestinal tract as a whole. Its major symptoms are pain, diarrhea, and weight loss, and it is usually associated with deficiencies of both macro- and micronutrients. Unluckily, after some time the body develops resistance against the already available drugs: thus, many patients fail to maintain remission, which is achieved in less than 50% of cases. Diet is a major determinant of gut inflammation. An unbalanced diet can affect the gut microbiota and cause dysbiosis, which is related to a dysregulated host immune response. The Mediterranean Diet its renowned for its anti-inflammatory effects and for preventing dysbiosis. In order to improve management and treatment of intestinal inflammatory diseases, it should become common practice to integrate the patient's diet with dietary supplements with anti-inflammatory effects (probiotics, butyrate, phosphatidylcholine, lactoferrin, palmitoylethanolamide, silymarin, and omega 3), which maintain the stability of the intestinal microbial cohort and strengthen the mucosal barrier, thus preventing or soothing IBD symptoms. Dietary supplements may help fight the high costs, the adverse side effects, and the recurrent relapses typical of drug use.

Dietary supplements for polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is one of the most prevalent female endocrine reproductive disorders, affecting between 4 to 18% of the women in their reproductive age. It is generally characterized by several clinical aspects, among which anovulation, inflammation and infertility. Moreover, PCOS has several health implications, including increased metabolic, reproductive, and psychological risks. Previously, metformin and to some extent thiazolidinediones were considered as drug of choice for PCOS management, but they had several side-effects, and controversial results were obtained about their efficiency, especially in non-insulin-resistant non-obese patients. Thus, alternative treatment options are now being studied for PCOS, including different natural molecules and complementary medicines (CM) for the improvement of their health, wellbeing and fertility. Recently, treatment of PCOS patients with different natural molecules, coming from nutritional supplements and herbal medicines, has attained satisfactory results with the absence of any side effects. In this review, four natural molecules, curcumin, vitamin D, inositol and CoQ10 are discussed for their therapeutic ability. These molecules proved to decrease insulin sensitivity and inflammation, to improve the restoration of ovarian function, and they could restore hormonal balance and regulate the menstrual cycle, all of which are the main features and major concerns for women suffering from PCOS.

From Achille Bertelli onward: more than 100 years of research and production of dietary supplements based on natural molecules typical of the Mediterranean diet.

Achille Bertelli was an aeronautics pioneer and an innovative entrepreneur of the pharmaceutical industry. After graduating in Chemistry in Italy, he moved to the United States of America where he opened a chemical-pharmaceutical laboratory in San Francisco in 1879, and later moved back to Italy where he opened a chemical and pharmaceutical industry in Milan (1886). The "A. Bertelli" pharmaceutical company developed the famous cough pills "Catramina Bertelli", as well as new cosmetics and perfumes. Apart from his chemical experience, Achille Bertelli was a passionate aeronautics expert. He wrote many essays on this topic and devoted himself to aeronautical experiments by designing the apparatus "Autovol", "Aerocurvo", "Autovol no. 2", "Autovol no. 3", and "Aerostave", which are considered the prototypes of the helicopter. Achille Bertelli was also the president of the Electric Company of Salò, which installed an electrical system that served the lighting in many cities on Lake Garda (Italy). Finally, Achille Bertelli also participated in the Italian revival after the First World War, especially by supporting the agricultural revival. Throughout his life, Achille Bertelli teamed with several famous people from all over Italy, such as Gabriele D'Annunzio, Cesare Lombroso and Cordero di Montezemolo. Today, Achille Bertelli's interest for natural molecules, his ideas, and his entrepreneurial approach are carried forward by his descendant, Matteo Bertelli.

Ion mobility mass spectrometry with surface activated chemical ionisation as a method for studying the domain of water clusters.

Water holds great relevance in various biological and biochemical systems. Water behaves as an excellent solvent, a reactant, a product and a catalyst of the reaction. The organisation of the water molecules, synergised by hydrogen bonds, builds up the structure of the water clusters. These water clusters significantly influence biological functions. To study the domain of water clusters using Ion mobility mass spectrometry with surface activated chemical ionisation. The experimental analysis was aimed to determine the water behaviour in terms of cluster formation before and after the application of a physical effect, namely low-frequency irradiation. A sanist platform-based spectrometer, manufactured by ISB srl with SACI version for protein analysis, was used as the equipment. Furthermore, for samples, we used pure de-ionised water, a part of which was used virgin, and another part was irradiated. Ion-mobility mass spectrometry (IM-MS) procedure was adopted as the experimental method. An electromagnetic frequency fields generator was used to subject the test samples to electromagnetic radiations between 7 Hz to 80 Hz. The presence of neutral water species was confirmed in the water samples. For the same m/z, water ion clusters in the untreated water were found to have a much higher intensity than the electromagnetically treated water. The presence of a water cluster near the (M+H)+ in electromagnetically treated dilute arginine solution was also confirmed. It is possible to detect water ion clusters by using Ion mobility mass spectrometry and SACI with low surface potential (47 V). The water cluster formation and its characteristics were found to be different in the treated and non-treated water. The electromagnetic radiations of low frequency seem to affect the hydrogen bonds of the water molecules.

Foods of the Mediterranean diet: citrus, cucumber and grape.

Fruit and vegetables are excellent sources of health-promoting bioactive compounds and nutraceuticals. Regular consumption of fruit and vegetables helps prevent the onset and progression of many non-communicable diseases. The Mediterranean diet envisages consumption of healthy vegetables and fruit on a daily basis for maximum health benefits. Traditional use envisages vegetable-based and fruit-based diets, and many studies scientifically proved the beneficial effects of Mediterranean vegetables and fruits. Rich in bioactive phytochemicals, citrus, cucumbers and grapes have antioxidant, anti-inflammatory, antimicrobial, cardioprotective, anti-ageing and anti-cancer properties. Studies indicate that intake of citrus, cucumbers and grapes reduces hypertension, hyperlipidemia, skin problems and infections and improves the health of the cardiovascular and nervous systems. These beneficial effects are mediated by several bioactive molecules present in Mediterranean diet vegetables and fruits, such as citrus, cucumbers and grapes. Indeed, they contains flavones, isoflavones, tannins, polyphenols and many beneficial natural molecules. This review focuses on the bioactive ingredients in citrus fruit, cucumbers and grapes, all components of the Mediterranean diet, and their health effects. A deep understanding of Mediterranean diet's components, as well as clinical trials to test natural molecules beneficial effects, will permit to further explore the therapeutic potential of the Mediterranean diet in several pathological conditions.

Foods of the Mediterranean diet: tomato, olives, chili pepper, wheat flour and wheat germ.

Mediterranean people, which follows a diet rich in minimally-processed plant-based foods, are believed to live longer and healthier lives than many other populations in the Western world. Epidemiological and clinical data suggest that the Mediterranean diet has beneficial effects for several chronic diseases, such as cardiovascular diseases, obesity, cancer and diabetes. Although the mechanisms of action of the Mediterranean diet are not completely clear, the synergistic effects of a number of its components and their bioactive phytochemicals exert antioxidant, anti-inflammatory, anti-microbial and anti-cancer effects. The Mediterranean diet includes daily consumption of whole cereals, fruit, vegetables and legumes in moderate proportions, weekly consumption of white meat in low to moderate proportions and occasionally sweets and chocolates in small amounts. Since olive oil is the main lipids source, it has special significance for health. Healthy fruit and vegetables, rich in phytochemicals, are a major proportion of this diet and contribute to the overall nutritional value and bioactivity of its components. Here we review the nutritional and health benefits of wheat germ, tomatoes, olives and chili pepper, items at the base of Mediterranean diet food pyramid that provides beneficial molecules, such as polyphenols, vitamins and flavonoids, and exert anti-inflammatory, anti-microbial and anti-oxidative actions.

Dietary supplements for obesity.

Obesity and associated complications including diabetes, cardiometabolic dysfunction, disability, malignancy and premature mortality are considered epidemic. Research on obesity is therefore of worldwide importance. The development of obesity is a multifactorial phenomenon with contributions from biological, behavioral, genetic and environmental factors. Obesity and its associated issues require various lifestyle modifications and treatment options such medication, exercise, diet, surgery, pharmacological therapy and dietary supplements. Dietary supplements are considered an attractive alternative to traditional therapy due to their low toxicity profile and their accessibility to the general population. Dietary supplements may include one or more dietary ingredients. In this narrative review, we analyze the effects on obesity and obesity-related issues of various natural components. For example, there are a myriad of supplements that have been used as dietary supplements for weight loss such as minerals, vitamins, amino acids, metabolites, herbs, and plant extracts. This narrative review aims to present the benefits and side-effects of several ingredients of dietary supplements for weight loss and treatment of obesity. In particular, the mechanism of action, results of clinical trials, and possible side effects will be presented for the following ingredients: ß-Glucans, bitter orange, calcium, vitamin D, chitosan, chromium, cocoa, coleus forskohlii, conjugate linoleic acid, ephedra sinica, fucoxanthin, garcinia cambogia, glucomannan, green coffee, green tea, guar gum, raspberry, hoodia gordonii, irvingia gabonensis, phenylpropylamine, pyruvate, white kidney bean.

Polyphenols and Lactobacillus reuteri in oral health.

Oral health is one of the necessary preludes to the overall quality of life. Several medical procedures and therapies are available to treat oral diseases in general and periodontal diseases in particular, yet caries, periodontitis, oral cancer, and oral infections remain a global concern. Natural molecules, with their anti-oxidant, anti-inflammatory, and anti-microbic properties, are one of the main sources of oral health and dental health care, and should be supplemented to exploit their beneficial effects. A possible way to improve the intake of these molecules is adhering to a diet that is rich in fruits, vegetables, and probiotics, which has many beneficial properties and can improve overall health and wellbeing. The Mediterranean diet, in particular, provides several beneficial natural molecules, mainly because of the precious nutrients contained in its typical ingredients, mainly plant-based (olives, wine, citrus fruits, and many more). Its beneficial effects on several diseases and in increasing the overall wellbeing of the population are currently being studied by physicians. Among its nutrients, polyphenols (including, among other molecules, lignans, tannins, and flavonoids) seem to be of outmost importance: several studies showed their anticariogenic properties, as well as their effects in decreasing the incidence of non-communicable diseases. Therefore, plant-derived molecules - such as polyphenols - and probiotics - such as Lactobacillus reuteri - have shown a significant potential in treating and curing oral diseases, either alone or in combination, owing to their antioxidant and antimicrobial properties, respectively.

Polymorphisms, diet and nutrigenomics.

Every human being possesses an exclusive nutritional blueprint inside their genes. Bioactive food components and nutrients affect the expression of such genes. Nutrigenomics is the science that analyzes gene-nutrient interactions (nutrigenetics), which can lead to the development of personalized nutritional recommendations to maintain optimal health and prevent disease. Genomic diversity among various ethnic groups might affect nutrients bioavailability as well as their metabolism. Nutrigenomics combines different branches of science including nutrition, bioinformatics, genomics, molecular biology, molecular medicine, and epidemiology. Genes regulate intake and metabolism of different nutrients, while nutrients positively or negatively influence the expression of a number of genes; testing of specific genetic polymorphisms may therefore become a useful tool to manage weight loss and to fully understand gene-nutrient interactions. Indeed, several approaches are used to study gene-nutrient interactions: epigenetics, the study of genome modification not related to changes in nucleotide sequence; transcriptomics, the study of tissue-specific and time-specific RNA transcripts; proteomics, the study of proteins involved in biological processes; and metabolomics, the study of changes of primary and secondary metabolites in body fluids and tissues. Hence, the use of nutrigenomics to improve and optimize a healthy, balanced diet in clinical settings could be an effective approach for long-term lifestyle changes that might lead to consistent weight loss and improve quality of life.

Clinical assessment for diet prescription.

Accurate nutritional assessment based on dietary intake, physical activity, genetic makeup, and metabolites is required to prevent from developing and/or to treat people suffering from malnutrition as well as other nutrition related health issues. Nutritional screening ought to be considered as an essential part of clinical assessment for every patient on admission to healthcare setups, as well as on change in clinical conditions. Therefore, a detailed nutritional assessment must be performed every time nutritional imbalances are observed or suspected. In this review we have explored different techniques used for nutritional and physical activity assessment. Dietary Intake (DI) assessment is a multidimensional and complex process. Traditionally, dietary intake is assessed through self-report techniques, but due to limitations like biases, random errors, misestimations, and nutrient databases-linked errors, questions arise about the adequacy of self-reporting dietary intake procedures. Despite the limitations in assessing dietary intake (DI) and physical activity (PA), new methods and improved technologies such as biomarkers analysis, blood tests, genetic assessments, metabolomic analysis, DEXA (Dual-energy X-ray absorptiometry), MRI (Magnetic resonance imaging), and CT (computed tomography) scanning procedures have made much progress in the improvement of these measures. Genes also plays a crucial role in dietary intake and physical activity. Similarly, metabolites are also involved in different nutritional pathways. This is why integrating knowledge about the genetic and metabolic markers along with the latest technologies for dietary intake (DI) and physical activity (PA) assessment holds the key for accurately assessing one's nutritional status and prevent malnutrition and its related complications.

Gene variants in eating disorders. Focus on anorexia nervosa, bulimia nervosa, and binge-eating disorder.

Eating disorders such as anorexia nervosa, bulimia nervosa and binge-eating disorder, have a deep social impact, concluding with death in cases of severe disease. Eating disorders affect up to 5% of the population in the industrialized countries, but probably the phenomenon is under-detection and under-diagnosis. Eating disorders are multifactorial disorders, resulting from the interaction between environmental triggers, psychological factors, but there is also a strong genetic component. In fact, genetic factors predispose for approximately 33-84% to anorexia nervosa, 28-83% to bulimia nervosa, and 41-57% to binge eating disorder. Twins and family studies have provided an unassailable proof on the heritability of these disorders. Other types of genetic studies, including genome-wide association studies, whole genome sequencing and linkage analysis, allowed to identify the genes and their variants associated with eating disorders and moreover global collaborative efforts have led to delineate the etiology of these disorders. Next Generation Sequencing technologies can be considered as an ideal diagnostic approach to identify not only the common variants, such as single nucleotide polymorphism, but also rare variants. Here we summarize the present knowledge on the molecular etiology and genetic determinants of eating disorders including serotonergic genes, dopaminergic genes, opioid genes, appetite regulation genes, endocannabinoid genes and vitamin D3.

Main nutritional deficiencies.

Nutrition is the source of energy that is required to carry out all the processes of human body. A balanced diet is a combination of both macro- and micronutrients. "Nutritional inadequacy" involves an intake of nutrients that is lower than the estimated average requirement, whereas "nutritional deficiency" consists of severely reduced levels of one or more nutrients, making the body unable to normally perform its functions and thus leading to an increased risk of several diseases like cancer, diabetes, and heart disease. Malnutrition could be caused by environmental factors, like food scarcity, as well as disease conditions, like anorexia nervosa, fasting, swallowing inability, persistent vomiting, impaired digestion, intestinal malabsorption, or other chronic diseases. Nutritional biomarkers - like serum or plasma levels of nutrients such as folate, vitamin C, B vitamins, vitamin D, selenium, copper, zinc - could be used for the evaluation of nutrient intake and dietary exposure. Macronutrients deficiencies could cause kwashiorkor, marasmus, ketosis, growth retardation, wound healing, and increased infection susceptibility, whereas micronutrient - like iron, folate, zinc, iodine, and vitamin A - deficiencies lead to intellectual impairment, poor growth, perinatal complications, degenerative diseases associated with aging and higher morbidity and mortality. Preventing macro- and micronutrient deficiency is crucial and this could be achieved through supplementation and food-based approaches.

Metabolomics application for the design of an optimal diet.

Precision nutrition is an emerging branch of nutrition science that aims to use modern omics technologies (genomics, proteomics, and metabolomics) to assess an individual's response to specific foods or dietary patterns and thereby determine the most effective diet or lifestyle interventions to prevent or treat specific diseases. Metabolomics is vital to nearly every aspect of precision nutrition. It can be targeted or untargeted, and it has many applications. Indeed, it can be used to comprehensively characterize the thousands of chemicals in foods, identify food by-products in human biofluids or tissues, characterize nutrient deficiencies or excesses, monitor biochemical responses to dietary interventions, track long- or short-term dietary habits, and guide the development of nutritional therapies. Indeed, metabolomics can be coupled with genomics and proteomics to study and advance the field of precision nutrition. Integrating omics with epidemiological and clinical data will begin to define the beneficial effects of human food metabolites. In this review, we present the metabolome and its relationship to precision nutrition. Moreover, we describe the different techniques used in metabolomics and present how metabolomics has been applied to advance the field of precision nutrition by providing notable examples and cases.

Methodology for clinical research.

A clinical research requires a systematic approach with diligent planning, execution and sampling in order to obtain reliable and validated results, as well as an understanding of each research methodology is essential for researchers. Indeed, selecting an inappropriate study type, an error that cannot be corrected after the beginning of a study, results in flawed methodology. The results of clinical research studies enhance the repertoire of knowledge regarding a disease pathogenicity, an existing or newly discovered medication, surgical or diagnostic procedure or medical device. Medical research can be divided into primary and secondary research, where primary research involves conducting studies and collecting raw data, which is then analysed and evaluated in secondary research. The successful deployment of clinical research methodology depends upon several factors. These include the type of study, the objectives, the population, study design, methodology/techniques and the sampling and statistical procedures used. Among the different types of clinical studies, we can recognize descriptive or analytical studies, which can be further categorized in observational and experimental. Finally, also pre-clinical studies are of outmost importance, representing the steppingstone of clinical trials. It is therefore important to understand the types of method for clinical research. Thus, this review focused on various aspects of the methodology and describes the crucial steps of the conceptual and executive stages.

Ethical considerations regarding animal experimentation.

Animal experimentation is widely used around the world for the identification of the root causes of various diseases in humans and animals and for exploring treatment options. Among the several animal species, rats, mice and purpose-bred birds comprise almost 90% of the animals that are used for research purpose. However, growing awareness of the sentience of animals and their experience of pain and suffering has led to strong opposition to animal research among many scientists and the general public. In addition, the usefulness of extrapolating animal data to humans has been questioned. This has led to Ethical Committees' adoption of the 'four Rs' principles (Reduction, Refinement, Replacement and Responsibility) as a guide when making decisions regarding animal experimentation. Some of the essential considerations for humane animal experimentation are presented in this review along with the requirement for investigator training. Due to the ethical issues surrounding the use of animals in experimentation, their use is declining in those research areas where alternative in vitro or in silico methods are available. However, so far it has not been possible to dispense with experimental animals completely and further research is needed to provide a road map to robust alternatives before their use can be fully discontinued.

Lactobacillus rhamnosus GG attenuates interferon-{gamma} and tumour necrosis factor-alpha-induced barrier dysfunction and pro-inflammatory signalling.

The intestinal epithelium forms a protective barrier against luminal contents and the external environment, mediated via intercellular tight junctions (TJs). The TJ can be disrupted via cell signalling induced by either enteric pathogens or pro-inflammatory cytokines, thereby contributing to various intestinal disorders ranging from acute infectious diarrhoea to chronic inflammatory bowel diseases. Probiotics, such as Lactobacillus rhamnosus GG (LGG), are reported to confer beneficial effects on epithelial cells, including antagonizing infections and reducing overt pro-inflammatory responses, but the underlying mechanisms of these observed effects require further characterization. We hypothesized that probiotics preserve barrier function by interfering with pro-inflammatory cytokine signalling. Caco-2bbe cells were seeded into Transwells to attain polarized monolayers with intercellular TJs. Monolayers were inoculated apically with the probiotic LGG 3 h prior to the addition of IFN-γ (100 ng ml(-1)) to the basolateral medium overnight. The monolayers were then placed in fresh basal medium±TNF-α (10 ng ml(-1)) and transepithelial electrical resistance (TER) measurements were taken over the time-course of TNF-α stimulation. To complement the TER findings, cells were processed for zona occludens-1 (ZO-1) immunofluorescence staining. As a measure of TNF-α downstream signalling, cells were immunofluorescently stained for NF-κB p65 subunit and CXCL-8 mRNA was quantified by qRT-PCR. Basal cell culture medium was collected after overnight TNF-α stimulation to measure secreted chemokines, including CXCL-8 (interleukin-8) and CCL-11 (eotaxin). Following LGG inoculation, IFN-γ priming and 24 h TNF-α stimulation, epithelial cells maintained TER and ZO-1 distribution. LGG diminished the nuclear translocation of p65, demonstrated by both immunofluorescence and CXCL-8 mRNA expression. CXCL-8 and CCL-11 protein levels were decreased in LGG-inoculated, cytokine-challenged cells. These findings indicate that LGG alleviates the effects of pro-inflammatory cytokines on epithelial barrier integrity and inflammation, mediated, at least in part, through inhibition of NF-κB signalling.

Enterohemorrhagic Escherichia coli O157:H7 gene expression profiling in response to growth in the presence of host epithelia.

BACKGROUND: The pathogenesis of enterohemorrhagic Escherichia coli (EHEC) O157:H7 infection is attributed to virulence factors encoded on multiple pathogenicity islands. Previous studies have shown that EHEC O157:H7 modulates host cell signal transduction cascades, independent of toxins and rearrangement of the cytoskeleton. However, the virulence factors and mechanisms responsible for EHEC-mediated subversion of signal transduction remain to be determined. Therefore, the purpose of this study was to first identify differentially regulated genes in response to EHEC O157:H7 grown in the presence of epithelial cells, compared to growth in the absence of epithelial cells (that is, growth in minimal essential tissue culture medium alone, minimal essential tissue culture medium in the presence of 5% CO(2), and Penassay broth alone) and, second, to identify EHEC virulence factors responsible for pathogen modulation of host cell signal transduction. METHODOLOGY/PRINCIPAL FINDINGS: Overnight cultures of EHEC O157:H7 were incubated for 6 hr at 37 degrees C in the presence or absence of confluent epithelial (HEp-2) cells. Total RNA was then extracted and used for microarray analyses (Affymetrix E. coli Genome 2.0 gene chips). Relative to bacteria grown in each of the other conditions, EHEC O157:H7 cultured in the presence of cultured epithelial cells displayed a distinct gene-expression profile. A 2.0-fold increase in the expression of 71 genes and a 2.0-fold decrease in expression of 60 other genes were identified in EHEC O157:H7 grown in the presence of epithelial cells, compared to bacteria grown in media alone. CONCLUSION/SIGNIFICANCE: Microarray analyses and gene deletion identified a protease on O-island 50, gene Z1787, as a potential virulence factor responsible for mediating EHEC inhibition of the interferon (IFN)-gamma-Jak1,2-STAT-1 signal transduction cascade. Up-regulated genes provide novel targets for use in developing strategies to interrupt the infectious process.

Escherichia albertii and Hafnia alvei are candidate enteric pathogens with divergent effects on intercellular tight junctions.

Attaching-effacing lesion-inducing Escherichia albertii and the related, but non-attaching-effacing organism, Hafnia alvei, are both implicated as enteric pathogens in humans. However, effects of these bacteria on epithelial cells are not well-characterized. Related enteropathogens, including enterohemorrhagic Escherichia coli O157:H7, decrease epithelial barrier function by disrupting intercellular tight junctions in polarized epithelia. Therefore, this study assessed epithelial barrier function and tight junction protein distribution in polarized epithelia following bacterial infections. Polarized epithelial (MDCK-I and T84) cells grown on filter supports were infected apically with E. coli O157:H7, E. albertii, and H. alvei for 16h at 37 degrees C. All strains decreased transepithelial electrical resistance and increased permeability to a dextran probe in a host cell-dependent manner. Immunofluorescence microscopy showed that both E. coli O157:H7 and E. albertii, but not H. alvei, caused a redistribution of the tight junction protein zona occludens-1. In contrast to E. coli O157:H7, E. albertii and H. alvei did not redistribute claudin-1. Western blotting of whole cell protein extracts demonstrated that each bacterium caused differential changes in tight junction protein expression, dependent on the host cell. These findings demonstrate that E. albertii and H. alvei are candidate enteric pathogens that have both strain-specific and host epithelial cell-dependent effects.