Validation by a Collaborative Interlaboratory Study of an ELISA Method for the Detection of Caseinate Used as a Fining Agent in Wine

The clarification or fining of wine removes undesirable substances such as proteins, phenols, and tannin compounds that would cloud the wine and cause bitterness and astringency. Caseinates have useful fining properties, but their residues could present a risk for allergic subjects. A commercial kit that was developed to detect caseinates in food has been examined for its applicability to a wine matrix; it is sensitive to caseinates at concentrations as low as 1 ppm. The general characteristics of the caseinate assay, described below in detail, are as follows. It is a sandwich enzyme-linked immunosorbent assay (ELISA): the microplate is first coated with a specific anti-casein antibody; and after incubation with the wine sample, a secondary anti-casein antibody conjugated with horseradish peroxidase is added to form a sandwich. The antibodies have been tested for their immunoreactivity and the reproducibility of antigen recognition has been measured. An interlaboratory collaborative trial was organized to evaluate the performance of this ELISA method and its ease of use by laboratories routinely dealing with wine/food analyses. The results satisfy the criteria established by the International Organisation of Vine and Wine in the Compendium of International Methods of Analysis MA-EAS1-07-ETCOL.     

HS-SPME/GC-MS Method to Characterise the Flavour of Italian Pasta: Potential Application to Assess the Quality of the Products

Food flavour, consisting of a large number of volatile compounds, has great influence on consumer acceptability and, when safety and nutritional value are guaranteed, sensory parameters become the discriminating factor in the product quality assessment which determines the differentiation on the market. The present work combines head space-solid phase micro-extraction and gas chromatography mass spectrometry techniques to analyse the volatile fraction of Italian durum wheat pasta. The aim was the optimization of an analytical method suitable to show possible differences in pasta flavour richness and identify compounds that could be used as food quality markers. Twenty-three odorants were identified and confirmed by comparing their mass spectra with those of 27 standard substances. The optimum conditions for extraction temperature and time, respectively, resulted from 70 °C and 90 min; the best recoveries were achieved with 10.5 g of sample. The developed method was applied for the relative quantitative analysis of different priced pasta to verify the technique capability to highlight possible quality differences.     

Phenotypic and genotypic characterization of Oenococcus oeni strains isolated from Italian wines

A phenotypic and genotypic characterization of 84 Oenococcus oeni isolates from Italian wines of different oenological areas was carried out. Numerical analysis of fatty acid profiles grouped the isolates into two clusters at low level of similarity (63%), the minor cluster containing seven isolates besides the type and the reference strains. Forthy-eight O. oeni isolates, representative of the two clusters, showed no differences in their metabolic properties (heterolactic fermentation pattern, citrate degradation capability and formation of some secondary metabolites). Moreover, the analysis of species-specific randomly amplified polymorphic DNA and 16S-23S rDNA intergenic spacer region polymorphism as well as the sequence-specific separation of V3 region from 16S rDNA by denaturing gradient gel electrophoresis demonstrated a substantial homogeneity among the isolates. On the basis of ApaI Pulsed Field Gel Electrophoresis (PFGE) restriction patterns, the 84 isolates were grouped into five different clusters at 70% similarity, but no correlation with the phenotypic groups could be demonstrated. However, by combining phenotypic and genotypic data, the 84 O. oeni isolates grouped into eight phenotypic-genotypic combined profiles and a relationship between the origin of the isolates and their combined profile became evident, so that a sort of strain specificity can be envisaged for each wine-producing area.     

Why Our Patients Like Daily Hemodialysis

The option of daily hemodialysis (HD) was discussed in November 1998 with a group of 35 HD patients on home or self‐care/limited‐care HD in a single, freestanding unit. After the meeting, 3 patients on home HD chose to switch to daily HD. The clinical success of the first patient and the immediate followers was one of the main reasons for further extension of this experience. At the time of this writing (February 2000), 10 patients were on a daily HD program (8 at home and 2 in a self‐care/limited‐care center) and one was in training for home daily HD. One further patient who tried 1 month of daily HD dropped out for logistic reasons. On daily HD, patients are dialyzed 2 – 3 hours/day, 6 days/ week, with blood flow of 270 – 300 mL/min, on bicarbonate dialysate with individually determined levels of Na and K. The schedule is flexible and a switch to 3 – 4 dialyses/week is occasionally allowed for working needs or for vacation. In addition to the well‐known clinical advantages (better well‐being, blood pressure control, nutrition, etc.), some patients preferred daily HD because of easier organization of daily activities, including work schedule. Patients initially feared frequent needle punctures and excessive burden on partners, but those concerns proved to be less a problem than anticipated. All current patients are willing to continue daily HD; only a nursing shortage limits further extension of the program in the self‐care/limited‐care center.     

Advances in anodic alumina membranes thin film fuel cell: CsH2PO4 pore-filler as proton conductor at room temperature

Anodic alumina membranes (AAM) filled with cesium hydrogen phosphate proton conductor have been tested as inorganic composite electrolyte for hydrogen–oxygen thin film (≤50 μm) fuel cell (TFFC) working at low temperatures (25 °C), low humidity (T_gas = 25 °C) and low Pt loading (1 mg cm⁻²). Single module TFFC delivering a peak power of around 15–27 mW cm⁻², with open circuit voltage (OCV) of about 0.9 V and short circuit current density in the range 80–160 mA cm⁻² have been fabricated. At variance with pure solid acid electrolytes showing reproducibility problems due to the scarce mechanical resistance, the presence of porous alumina support allowed to replicate similar fuel cell performances over numerous AAM/CsH₂PO₄ assemblies. A scale-up process of the electrodic area has been optimized in order to increase the delivered peak power of AAM thin film fuel cell. Morphological, chemical and electrochemical studies on the alumina composite electrolyte have been carried out by means of scanning electron microscopy, X-ray diffractometry, Micro-Raman spectroscopy, DTA/DTG analysis, ac impedance spectroscopy and single fuel cell tests.     

The effect of butter grains on physical properties of butter-like emulsions

Milk fat exists as globules in its natural state in milk. The potential of using globular fat to modulate the rheological properties and crystallization behavior in butter-like emulsions was studied in the present work. We conducted a comparative study of butter-like emulsions, with a fat phase consisting of 0, 10, 25, 50, or 100% anhydrous milk fat (AMF), the remaining fat being butter grains, and all samples containing 20% water, to obtain systematic variation in the ratio of globular fat. All emulsions were studied over 4 wk of storage at 5°C. By combining small and large deformation rheology, we conducted a detailed characterization of the rheological behavior of butter-like emulsions. We applied differential scanning calorimetry to monitor thermal behavior, confocal laser scanning microscopy for microstructural analysis, and low-field pulsed nuclear magnetic resonance spectrometry to measure solid fat content. By combining these techniques, we determined that increasing the fraction of globular fat (by mixing with butter grains) decreases the hardness of butter-like emulsions up to an order of magnitude at d 1. However, no difference was observed in thermal behavior as a function of butter grain content, as all emulsions containing butter grains revealed 2 endothermal peaks corresponding to the high (32.7°C ± 0.6) and medium (14.6°C ± 0.1) melting fractions of fatty acids. In terms of microstructure, decreasing the amount of butter grains in the emulsions resulted in formation of a denser fat crystal network, corresponding to increased hardness. Moreover, microstructural analysis revealed that the presence of butter grains resulted in faster formation of a continuous fat crystal network compared with the 100% AMF sample, which was dominated by crystal clusters surrounded by liquid oil. During storage, hardness remained stable and no changes in thermal behavior were observed, despite an increase in solid fat content of up to 5%. After 28 d of storage, we observed no difference in either microstructural or rheological properties, indicating that formation of primary bonds occurs primarily within the first day of storage. The rheological behavior of butter-like emulsions is not determined solely by hardness, but also by stiffness related to secondary bonds within the fat crystal network. The complex rheological behavior of milk fat-based emulsions is better characterized using multiple parameters.     

Genomic Analysis Made It Possible to Identify Gene-Driver Alterations Covering the Time Window between Diagnosis of Neuroblastoma 4S and the Progression to Stage 4

Neuroblastoma (NB) is a tumor of the developing sympathetic nervous system. Despite recent advances in understanding the complexity of NB, the mechanisms that determine its regression or progression are still largely unknown. Stage 4S NB is characterized by a favorable course of disease and often by spontaneous regression, while progression to true stage 4 is a very rare event. Here, we focused on genomic analysis of an NB case that progressed from stage 4S to stage 4 with a very poor outcome. Array-comparative genomic hybridization (a-CGH) on tumor-tissue DNA, and whole-exome sequencing (WES) on exosomes DNA derived from plasma collected at the onset and at the tumor progression, pointed out relevant genetic changes that can explain this clinical worsening. The combination of a-CGH and WES data allowed for the identification iof somatic copy number aberrations and single-nucleotide variants in genes known to be responsible for aggressive NB. KLRB1, MAPK3 and FANCA genes, which were lost at the time of progression, were studied for their possible role in this event by analyzing in silico the impact of their expression on the outcome of 786 NB patients.     

Lipidomic Approaches to Study HDL Metabolism in Patients with Central Obesity Diagnosed with Metabolic Syndrome

The metabolic syndrome (MetS) is a cluster of cardiovascular risk factors characterised by central obesity, atherogenic dyslipidaemia, and changes in the circulating lipidome; the underlying mechanisms that lead to this lipid remodelling have only been partially elucidated. This study used an integrated “omics” approach (untargeted whole serum lipidomics, targeted proteomics, and lipoprotein lipidomics) to study lipoprotein remodelling and HDL composition in subjects with central obesity diagnosed with MetS (vs. controls). Compared with healthy subjects, MetS patients showed higher free fatty acids, diglycerides, phosphatidylcholines, and triglycerides, particularly those enriched in products of de novo lipogenesis. On the other hand, the “lysophosphatidylcholines to phosphatidylcholines” and “cholesteryl ester to free cholesterol” ratios were reduced, pointing to a lower activity of lecithin cholesterol acyltransferase (LCAT) in MetS; LCAT activity (directly measured and predicted by lipidomic ratios) was positively correlated with high-density lipoprotein cholesterol (HDL-C) and negatively correlated with body mass index (BMI) and insulin resistance. Moreover, many phosphatidylcholines and sphingomyelins were significantly lower in the HDL of MetS patients and strongly correlated with BMI and clinical metabolic parameters. These results suggest that MetS is associated with an impairment of phospholipid metabolism in HDL, partially led by LCAT, and associated with obesity and underlying insulin resistance. This study proposes a candidate strategy to use integrated “omics” approaches to gain mechanistic insights into lipoprotein remodelling, thus deepening the knowledge regarding the molecular basis of the association between MetS and atherosclerosis.     

Assessing T-Cell Immunity in Kidney Transplant Recipients with Absent Antibody Production after a 3rd Dose of the mRNA-1273 Vaccine

The vulnerable population of kidney transplant recipients (KTRs) are low responders to COVID-19 vaccines, so specific immune surveillance is needed. The interferon-gamma (IFN-γ) release assay (IGRA) is effective in assessing T cell-mediated immunity. We assessed SARS-CoV-2-directed T cell responses in KTRs with absent antibody production after a third dose of the mRNA-1273 vaccine, using two different IGRAs. A cohort of 57 KTRs, who were actively followed up, received a third dose of the mRNA-1273 vaccine. After the evaluation of humoral immunity to SARS-CoV-2, 14 seronegative patients were tested with two commercial IGRAs (SD Biosensor and Euroimmun). Out of 14 patients, one and three samples were positive by IGRAs with Euroimmun and SD Biosensor, respectively. The overall agreement between the two assays was 85.7% (κ = 0.444). In addition, multivariate linear regression analysis showed no statistically significant association between the IFN-γ concentration, and the independent variables analyzed (age, gender, years since transplant, total lymphocytes cells/mcl, CD3+ cells/mcl, CD3+ CD4+ cells/mcl, CD3+ CD8+ cells/mcl, CD19+ cells/mcl, CD3-CD16+CD56+ cells/mcl) (p > 0.01). In a vulnerable setting, assessing cellular immune response to complement the humoral response may be advantageous. Since the two commercial IGRAs showed a good agreement on negative samples, the three discordant samples highlight the need for further investigations.