Occurrence of ochratoxin A-producing fungi in grapes grown in Italy

A study was carried out to investigate fungi present on grapes grown in Italy. Aspergillus and Penicillium spp. isolates were identified and studied in vitro, and their ability to produce ochratoxin A (OA) was investigated. The survey involved nine vineyards, three located in northern Italy and six located in southern Italy. In 1999 and 2000, bunches of grapes at different growth stages were collected from all nine vineyards, and berry samples were placed in moist chambers and incubated. The resultant fungal colonies were then transferred to petri dishes containing Czapek yeast agar and incubated at 25 degrees C for 7 days; the fungal isolates were identified and then cultivated in liquid Czapek yeast medium and evaluated for their ability to produce OA. During the survey, 508 isolates were collected, with 477 belonging to Aspergillus spp. and 31 belonging to Penicillium spp. Among the aspergilli, species of the Fumigati, Circumdati, and Nigri sections were identified, with species of the Nigri section (464 isolates) largely predominating; for species of the Nigri section, 108 isolates were uniseriate, 270 were biseriate, and 86 were identified as Aspergillus carbonarius. Black aspergilli isolated over the 2 years of the study showed a very similar pattern. On average, the biseriates represented about 60% of the isolates collected in both years and were followed by uniseriates (21%) and A. carbonarius (19%). The most toxigenic strains proved to be those of A. carbonarius; about 60% of these isolates were OA producers and produced the highest levels of OA. A. carbonarius was more frequent in the south, but in both areas the percentages of OA-producing isolates remained the same.     

Soybean allergen detection methods--a comparison study

Soybean containing products are widely consumed, thus reliable methods for detection of soy in foods are needed in order to make appropriate risk assessment studies to adequately protect soy allergic patients. Six methods were compared using eight food products with a declared content of soy: a direct sandwich ELISA based on polyclonal rabbit antibody (ab) to raw soy flakes, a commercial and an in-house competitive ELISA both based on ab to denatured, 'renatured' soy protein, an enzyme-allergosorbent test (EAST) inhibition based on two sera from soy allergic patients, histamine release (HR) using basophils passively sensitized with patient serum and a PCR method detecting soy DNA. Eight food products were selected as model foods to test the performance of the methods. There was an overall good agreement between the methods in terms of ranks of soy content but not the quantity. The sandwich ELISA aimed at native soy proteins had the lowest detection limit of 0.05 ppm, but only identified soy in 5/8 products, and generally in lower amounts compared to other methods. The competitive ELISA had a higher detection limit of 21 ppm, but seemed more successful in detecting processed soy. Only HR, EAST inhibition and PCR detected soy in all eight products. In spite of a general good correlation in terms of ranks of soy content, more than a single method may be necessary to confirm the presence of soy in foods.     

High level expression, purification and physico- and immunochemical characterisation of recombinant Pen a 1: a major allergen of shrimp

Well-characterised and immunologically active recombinant allergens are of eminent importance for improvement of diagnostic tools and immunotherapy of allergic diseases. The use of recombinant allergens has several advantages such as the more precise quantification of the active substance compared to allergen extracts and the reduced risk of contamination with other allergenic proteins compared to purified natural allergens. Optimised standard protocols for expression and purification and a detailed physico-chemical characterisation of such recombinant allergens are necessary to ensure consistent quality and comparability of results obtained with recombinant material. In this study the major allergen Pen a 1 of brown shrimp (Penaeus aztecus) was expressed in E. coli and purified in two steps by immobilised metal chelate-affinity chromatography (IMAC) and size-exclusion chromatography. Identity and purity were verified with N-terminal sequencing and peptide mass fingerprinting. Circular dichroism and NMR-spectroscopy indicated an alpha-helical flexible structure of rPen a 1 which is in accordance with the known structure of tropomyosins. Finally, the recombinant allergen proved to be immunologically reactive in IgE Western blot analysis and ELISA. This study provides a protocol for the preparation of recombinant shrimp tropomyosin in standardised quality.     

Nonalcoholic Fatty Liver Disease and Chronic Kidney Disease: Epidemiology,Pathogenesis, and Clinical and Research Implications

Nonalcoholic fatty liver disease (NAFLD) has become the most common cause of chronic liver disease worldwide, affecting up to ~30% of adult populations. NAFLD defines a spectrum of progressive liver conditions ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma, which often occur in close and bidirectional associations with metabolic disorders. Chronic kidney disease (CKD) is characterized by anatomic and/or functional renal damage, ultimately resulting in a reduced glomerular filtration rate. The physiological axis linking the liver and kidneys often passes unnoticed until clinically significant portal hypertension, as a major complication of cirrhosis, becomes apparent in the form of ascites, refractory ascites, or hepatorenal syndrome. However, the extensive evidence accumulated since 2008 indicates that noncirrhotic NAFLD is associated with a higher risk of incident CKD, independent of obesity, type 2 diabetes, and other common renal risk factors. In addition, subclinical portal hypertension has been demonstrated to occur in noncirrhotic NAFLD, with a potential adverse impact on renal vasoregulation. However, the mechanisms underlying this association remain unexplored to a substantial extent. With this background, in this review we discuss the current evidence showing a strong association between NAFLD and the risk of CKD, and the putative biological mechanisms underpinning this association. We also discuss in depth the potential pathogenic role of the hepatorenal reflex, which may be triggered by subclinical portal hypertension and is a poorly investigated but promising research topic. Finally, we address emerging pharmacotherapies for NAFLD that may also beneficially affect the risk of developing CKD in individuals with NAFLD.     

Role of Citric Acid in Delaying Gypsum Precipitation

The comprehension of the mechanisms of nucleation and growth that regulate the gypsum (calcium sulfate dihydrate) precipitation is fundamental to prevent scaling problems. The present paper reports measurements of the induction period for gypsum nucleation when citric acid is added in solution as an additive to retard scale formation. The supersaturation ratio was varied in the interval 2.3‐6.0, the concentration of citric acid ranged from 0.01 to 0.30 g/L, while in all experiments the temperature was fixed to 25°C. The induction period values were compared to those previously obtained, by using the same experimental technique, in the absence of foreign ions in the mother liquor, showing that the citric acid has a strong retarding effect towards gypsum nucleation.     

Conjugated linoleic acid and oxidative stress

At the present time, conjugated linoleic acid (CLA) is the subject of a growing number of studies since it has been demonstrated to possess anticarcinogenic and antiatherogenic activities in experimental animal models and to increase in some pathological states in humans. In both situations, CLA has been claimed to be involved in oxidative stress, as an antioxidant in the first case and as a primary product of a free-radical attack on polyunsaturated fatty acids (PUFA) in the other. The controversial results are due mostly to a lack of a suitable methodology because the presence of conjugated dienes (CD) in lipid moiety has been taken for years as evidence of lipid peroxidation due to the occurrence of this structure in fatty acid hydroperoxides. We have recently developed a new methodology that consists of the extraction of fatty acids, including CD fatty acid hydroperoxides, by mild saponification and their separation and identification by high-performance liquid chromatography with diode array detector. Fatty acid analyses of liver homogenate, oxidized in vitro either with Fe-ADP or t-butyl hydroperoxide (t-ButylHP), of lamb and rats fed CLA at levels known to prevent carcinogenesis, showed that CLA and its metabolites steadily decreased during oxidative stress and that they are more prone to oxidation than their corresponding methylene-interrupted fatty acids. No significant antioxidant effect of CLA was detected in any model tested. However, CD fatty acid hydroperoxides increased in the t-ButylHP model but not in the Fe-ADP model, owing probably to the degradation of CD fatty acid hydroperoxides induced by this oxidative agent. In conclusion, CLA and its metabolites seem to behave, under oxidative stress, as regular PUFA. Thus, it is highly unlikely that the peculiar effects of CLA are directly related to interference in lipoperoxidative processes.     

Three months of Western diet induces small intestinal mucosa alteration in TLR KO mice

Several studies support the role of Western‐style diet (WD) in inflammatory bowel disease (IBD). Toll‐like receptors/NOD‐like receptors (TLRs/NLRs) are important to maintain a healthy epithelium as well as inducing inflammation. Given that dietary factors influence IBD development, that epithelial dysfunction is thought to be involved in initiating intestinal inflammation and that TLR‐NLR are involved in maintenance of the functionality of intestinal epithelium as well as in regulating inflammation, we decided to examine the role of TLR signals in the triggering events that lead to alteration of the small intestinal epithelium associated to consumption of WD. C57BL/6J mice deficient for TLR2, 4, 9, or NOD2 and wild‐type (WT) were fed a WD or a standard diet for 3 months. The effects of WD on small intestinal samples were evaluated by histological and immunohistochemical analysis. After 3 months, WD modifies the morphology and the organization of the small intestine in TLR9 KO mice compared with WT mice and the others TLRs. The most interesting change involved the expression of proliferative and differentiation markers of WNT signaling, Ki67 and FzD5. Mice deficient in TLR2, 4, and NOD2 have a significant reduction in the proliferative cell numbers but do not show any signs of histological alterations. Our results suggest that TLR9 is an important protective factor in intestinal epithelial homeostasis and provide new insights into an unrecognized role of TLR9 signaling in the small intestinal mucosa dysfunction associated with WD.     

Zero‐Depth Interfacial Nanopore Capillaries

High‐fidelity analysis of translocating biomolecules through nanopores demands shortening the nanocapillary length to a minimal value. Existing nanopores and capillaries, however, inherit a finite length from the parent membranes. Here, nanocapillaries of zero depth are formed by dissolving two superimposed and crossing metallic nanorods, molded in polymeric slabs. In an electrolyte, the interface shared by the crossing fluidic channels is mathematically of zero thickness and defines the narrowest constriction in the stream of ions through the nanopore device. This novel architecture provides the possibility to design nanopore fluidic channels, particularly with a robust 3D architecture maintaining the ultimate zero thickness geometry independently of the thickness of the fluidic channels. With orders of magnitude reduced biomolecule translocation speed, and lowered electronic and ionic noise compared to nanopores in 2D materials, the findings establish interfacial nanopores as a scalable platform for realizing nanofluidic systems, capable of single‐molecule detection.     

The Galois lattice as a hierarchical structure for topological relations

This paper presents the construction and the comparison of Galois lattices of topological relations for qualitative spatial representation and reasoning. The lattices rely on a correspondence between computational operations working on quantitative data, on the one hand, and topological relations working on qualitative knowledge units, on the other hand. After introducing the context of the present research work, i.e. the RCC-8 model of topological relations, we present computational operations for checking topological relations on spatial regions. From these operations are derived two sets of computational conditions that can be associated to topological relations through a Galois connection. The associated Galois lattices are presented and compared. Elements on the practical use of the lattices for representing spatial knowledge and for reasoning are also introduced and discussed.