A 1D High Performance Thin Layer Chromatography Method Validated to Quantify Phospholipids Including Cardiolipin and Monolysocardiolipin from Biological Samples

The aim of this study is to identify high performance thin layer chromatography (HPTLC) conditions allowing the separation and quantification of mammalian cellular phospholipids (PLs) (sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and especially phosphatidic acid, cardiolipin, and monolysocardiolipin, these latter two being specifically located in mitochondria membranes). In order to make this method faster and easier, a 1D HPTLC method is chosen, testing several eluents as well as several staining methods. A pre‐conditioning of HPTLC plates with boric acid and a copper staining reagent followed by carbonization are selected for the quality of PL separation and homogeneity of staining. The selected conditions are discussed and the method validation is performed according to the International Conference on Harmonization guidelines. Linearity is effective between 1 and 8 µg and limit of quantification is between 0.5 and 2.3 µg depending on PL classes. Precision measurements show coefficients of variation <6%, and when amounts are close to the detection limit, <12%. Lipid extracts of tumor cell lines or isolated mitochondria are used to assess PL profiles. This shows that the HPTLC method can be used routinely to follow level variations of PLs. Practical Applications: The changes in PL composition play a crucial role in tumor processes and regulate cellular functions modulating cellular signaling or mitochondrial metabolism. The simple and cost‐effective 1D HPTLC method that is developed is applied to lipid extracts of whole tumor cells or hepatocyte‐isolated mitochondria. It is sensitive as well as precise to detect variations of phosphatidic acid or cardiolipin levels linked to physio‐pathological conditions. It can also be used to investigate the composition changes of other membrane PLs. Moreover, with a simultaneous analysis of 14 samples/standards on the same plate (six plates per day), this method is adapted for large series of samples.     

Biosynthetic Plasticity Enables Production of Fluorinated Aurachins

Enzyme promiscuity has important implications in the field of biocatalysis. In some cases, structural analogues of simple metabolic building blocks can be processed through entire pathways to give natural product derivatives that are not readily accessible by chemical means. In this study, we explored the plasticity of the aurachin biosynthesis pathway with regard to using fluoro- and chloroanthranilic acids, which are not abundant in the bacterial producers of these quinolone antibiotics. The incorporation rates of the tested precursor molecules disclosed a regiopreference for halogen substitution as well as steric limitations of enzymatic substrate tolerance. Three previously undescribed fluorinated aurachin derivatives were produced in preparative amounts by fermentation and structurally characterized. Furthermore, their antibacterial activities were evaluated in comparison to their natural congener aurachin D.     

Microbiological, epidemiological, and food safety aspects of Enterobacter sakazakii

Enterobacter sakazakii is considered to be an opportunistic pathogen and has been implicated in foodborne diseases causing meningitis or enteritis, especially in neonates and infants. The U.S FoodNet 2002 survey rate of invasive infections with this organism in infants under 1 year of age was 1 per 100,000 infants. Severity of the disease is a matter of concern. In a recent study on the occurrence of E. sakazakii in production environments from food (milk powder, chocolate, cereal, potato, and pasta) factories and households, this organism was isolated with varying frequency from nearly all environments examined, strongly indicating that it is widespread. Stationary phase E. sakazakii cells are remarkably resistant to osmotic and drying stresses compared with other species of the Enterobacteriacae. In this article, we review the literature on this organism with special respect to the information relevant for food safety.     

Germin-like protein Cit s 1 and profilin Cit s 2 are major allergens in orange (Citrus sinensis) fruits

Oranges are clinically relevant allergenic foods. To date, orange allergens have not been characterized in detail. The study is aimed at analyzing the sensitization profile in orange-sensitized subjects with and without clinical allergy, and to identify orange allergens. Fifty-six sensitized subjects with self-reported reactions to orange were grouped into reactors (anaphylaxis or multiple episodes of immediate reactions and/or positive challenge tests) and non-reactors (negative open food challenge tests). Allergens were characterized by IgE immunoblotting, N-terminal sequencing, IgE-inhibition assays, and mediator release assays were performed to determine the allergenic potency of orange profilin. Of 56 subjects, 23 were classified as orange allergic showing mainly an oral allergy syndrome. Of 23 subjects classified as orange allergic, 22 were sensitized to profilin, Cit s 2. In patients with mono-sensitization to profilin in vitro histamine releases up to 75% from basophils were induced using orange extract and purified plant profilins. Of the allergic patients 78% were sensitized to germin-like protein, Cit s 1. Both allergens showed retained IgE reactivity in heat-processed orange juice. Interestingly, subjects with and without clinical allergy showed a comparable sensitization profile. Profilin and germin-like proteins are major orange allergens. The potential clinical relevance of orange profilin was indicated by its strong capacity to release histamine from basophils. However, a predominant sensitization to both allergens in subjects without symptoms also indicates a high frequency of clinically insignificant sensitization.     

Electrolyzed water and its application in the food industry

Electrolyzed water (EW) is gaining popularity as a sanitizer in the food industries of many countries. By electrolysis, a dilute sodium chloride solution dissociates into acidic electrolyzed water (AEW), which has a pH of 2 to 3, an oxidation-reduction potential of >1,100 mV, and an active chlorine content of 10 to 90 ppm, and basic electrolyzed water (BEW), which has a pH of 10 to 13 and an oxidation-reduction potential of -800 to -900 mV. Vegetative cells of various bacteria in suspension were generally reduced by > 6.0 log CFU/ml when AEW was used. However, AEW is a less effective bactericide on utensils, surfaces, and food products because of factors such as surface type and the presence of organic matter. Reductions of bacteria on surfaces and utensils or vegetables and fruits mainly ranged from about 2.0 to 6.0 or 1.0 to 3.5 orders of magnitude, respectively. Higher reductions were obtained for tomatoes. For chicken carcasses, pork, and fish, reductions ranged from about 0.8 to 3.0, 1.0 to 1.8, and 0.4 to 2.8 orders of magnitude, respectively. Considerable reductions were achieved with AEW on eggs. On some food commodities, treatment with BEW followed by AEW produced higher reductions than did treatment with AEW only. EW technology deserves consideration when discussing industrial sanitization of equipment and decontamination of food products. Nevertheless, decontamination treatments for food products always should be considered part of an integral food safety system. Such treatments cannot replace strict adherence to good manufacturing and hygiene practices.     

Escherichia coli O157 and non-O157 Shiga toxin-producing Escherichia coli in fecal samples of finished pigs at slaughter in Switzerland

Fecal samples from 630 slaughtered finisher pigs were examined by PCR to assess the shedding of Escherichia coli O157 (rfbE) and Shiga toxin-producing E. coli (STEC, stx). The proportion of positive samples was 7.5% for rfbE and 22% for stx. By colony hybridization, 31 E. coli O157 and 45 STEC strains were isolated, and these strains were further characterized by phenotypic and genotypic traits. Among E. coli O157 strains, 30 were sorbitol positive, 30 had an H type other than H7, and none harbored stx genes. Intimin (eae), enterohemolysin (ehxA), EAST1 (astA), and porcine A/E-associated protein (paa) were present in 10, 3, 26, and 6% of strains. Among them, one eae-gamma1-positive O157:H7 strain testing positive for ehxA and astA and two eae-alpha1-positive O157:H45 strains were classified as enteropathogenic E. coli (EPEC). The O157:H45 EPEC harbored the EAF plasmid and the bfpA gene, factors characteristic for typical EPEC. The isolated STEC strains (43 sorbitol positive) belonged to 11 O:H serotypes, including three previously reported in human STEC causing hemolytic uremic syndrome (O9:H-, O26:H-, and O103:H2). All but one strain harbored stx2e. The eae and ehxA genes, which are strongly correlated with human disease, were present in only one O103:H2 strain positive for stx1 and paa, whereas the astA gene was found more frequently (14 strains). High prevalence of STEC was found among finisher pigs, but according to the virulence factors the majority of these strains seem to be of low virulence.     

Lipoxygenase Products in the Urine Correlate with Renal Function and Body Temperature but not with Acute Transplant Rejection

Acute transplant rejection is the leading cause of graft loss in the first months after kidney transplantation. Lipoxygenase products mediate pro- and anti-inflammatory actions and thus we aimed to correlate the histological reports of renal transplant biopsies with urinary lipoxygenase products concentrations to evaluate their role as a diagnostic marker. This study included a total of 34 kidney transplant recipients: 17 with an acute transplant rejection and 17 controls. LTE4, LTB4, 12-HETE and 15-HETE concentrations were measured by enzyme immunoassay. Urinary lipoxygenase product concentrations were not significantly changed during an acute allograft rejection. Nevertheless, LTB4 concentrations correlated significantly with the body temperature (P ≤ 0.05) 3 months after transplantation, and 12- and 15-HETE concentrations correlated significantly with renal function (P ≤ 0.05) 2 weeks after transplantation. In conclusion, our data show a correlation for LTB4 with the body temperature 3 months after transplantation and urinary 12- and 15-HETE concentrations correlate positively with elevated serum creatinine concentrations but do not predict acute allograft rejection.     

Adventitial Tertiary Lymphoid Organs as Potential Source of MicroRNA Biomarkers for Abdominal Aortic Aneurysm

Abdominal aortic aneurysm (AAA) is an inflammatory disease associated with marked changes in the cellular composition of the aortic wall. This study aims to identify microRNA (miRNA) expression in aneurysmal inflammatory cells isolated by laser microdissection from human tissue samples. The distribution of inflammatory cells (neutrophils, B and T lymphocytes, mast cells) was evaluated in human AAA biopsies. We observed in half of the samples that adventitial tertiary lymphoid organs (ATLOs) with a thickness from 0.5 to 2 mm were located exclusively in the adventitia. Out of the 850 miRNA that were screened by microarray in isolated ATLOs (n = 2), 164 miRNAs were detected in ATLOs. The three miRNAs (miR-15a-3p, miR-30a-5p and miR-489-3p) with the highest expression levels were chosen and their expression quantified by RT-PCR in isolated ATLOs (n = 4), M1 (n = 2) and M2 macrophages (n = 2) and entire aneurysmal biopsies (n = 3). Except for the miR-30a-5p, a similar modulation was found in ATLOs and the two subtypes of macrophages. The modulated miRNAs were then evaluated in the plasma of AAA patients for their potential as AAA biomarkers. Our data emphasize the potential of miR-15a-3p and miR-30a-5p as biomarkers of AAA but also as triggers of ATLO evolution. Further investigations will be required to evaluate their targets in order to better understand AAA pathophysiology.     

Arsenic removal with iron(II) and iron(III) in waters with high silicate and phosphate concentrations

Arsenic removal by passive treatment, in which naturally present Fe(II) is oxidized by aeration and the forming iron(III) (hydr)oxides precipitate with adsorbed arsenic, is the simplest conceivable water treatment option. However, competing anions and low iron concentrations often require additional iron. Application of Fe(II) instead of the usually applied Fe(III) is shown to be advantageous, as oxidation of Fe(II) by dissolved oxygen causes partial oxidation of As(III) and iron(III) (hydr)oxides formed from Fe(II) have higher sorption capacities. In simulated groundwater (8.2 mM HCO3(-), 2.5 mM Ca2+, 1.6 mM Mg2+, 30 mg/L Si, 3 mg/L P, 500 ppb As(III), or As(V), pH 7.0 +/- 0.1), addition of Fe(II) clearly leads to better As removal than Fe(III). Multiple additions of Fe(II) further improved the removal of As(II). A competitive coprecipitation model that considers As(III) oxidation explains the observed results and allows the estimation of arsenic removal under different conditions. Lowering 500 microg/L As(III) to below 50 microg/L As(tot) in filtered water required > 80 mg/L Fe(III), 50-55 mg/L Fe(II) in one single addition, and 20-25 mg/L in multiple additions. With As(V), 10-12 mg/L Fe(II) and 15-18 mg/L Fe(III) was required. In the absence of Si and P, removal efficiencies for Fe(II) and Fe(III) were similar: 30-40 mg/L was required for As(II), and 2.0-2.5 mg/L was required for As(V). In a field study with 22 tubewells in Bangladesh, passive treatment efficiently removed phosphate, but iron contents were generally too low for efficient arsenic removal.     

Relevant aspects of Arcobacter spp. as potential foodborne pathogen

Arcobacter species are Gram-negative spiral-shaped organisms belonging to the family Campylobacteraceae that can grow microaerobically or aerobically. The Arcobacter organisms also have the ability to grow at 15 degrees C, which is a distinctive feature that differentiates Arcobacter species from Campylobacter species. Cultural detection of Arcobacter is generally performed by an enrichment step and takes 4 to 5 days. In the last few years, several studies comparing different culture-based protocols have been published. Furthermore, DNA-based assays have also been established for rapid and specific identification of Arcobacter spp. Recent evidence suggests that Arcobacter, especially A. Butzleri, may be involved in human enteric diseases. Moreover, A. butzleri has also occasionally been found in cases of human extraintestinal diseases. However, up to now, little is known about the mechanisms of pathogenicity or potential virulence factors of Arcobacter spp. There is evidence that livestock animals may be a significant reservoir of Arcobacter spp. and over the last few years, the presence of these organisms in raw meat products as well as in surface and ground water has received increasing attention. In view of control measures to be used to prevent or to eliminate the hazard of Arcobacter spp. in food, several treatments have been evaluated for their effectiveness. While the role of Arcobacter spp. in human disease awaits further evaluation, a precautionary approach is advisable. Measures aimed at reduction or eradication of Arcobacter from the human food chain should be encouraged. With this article, we review the recent literature on this organism with a special emphasis on the information relevant to food safety.