Characterization of the microbial community in ripened Pecorino Toscano cheese affected by pink discoloration.

Pink discoloration defect can cause economic losses for cheese producers due to the impossibility to sell the defected cheese, but few knowledge is currently available on the causes of this defect. To gain more insight on the causes that lead to the formation of pink discoloration in Pecorino Toscano cheese with the Protected Designation of Origin (PDO) status, the bacterial community in defected and not defected cheese was characterized by high-throughput sequencing of bacterial 16S rRNA gene. The bacterial community in the defected cheese significantly differed compared to the control. The relative abundance of the genera Acidipropionibacterium, Enterococcus, Escherichia/Shigella, Lactobacillus, Lentilactobacillus and Propionibacterium was higher in the cheese with pink discoloration defect. The concentration of short chain fatty acids and of lactic acid in cheese was measured and a shift towards the production of propionate in the cheese with pink discoloration defect was observed. Furthermore, the possible involvement of microbially produced vitamin B12 in the formation of pink discoloration was not supported by the data, since a tendency to a lower concentration of vitamin B12 was measured in the defected cheese compared to the control.

Exposure to perfluoroalkyl substances through human milk in preterm infants.

Perfluoroalkyl substances (PFASs) are environmental contaminants that have been shown to exert toxic effects, which are dependent upon concentration, in animals and humans. No specific data on the exposure of preterm infants to PFASs are available. We aimed to quantify the potential exposure of preterm infants to PFASs through human milk (HM), to be compared to the exposure data recently reported for infants by EFSA. The amount of PFASs in ten preterm (PHM) and ten donor HM (DHM) samples was evaluated, and the expected daily intake (EDI) at full enteral feeding was calculated. This EDI was compared to the mean and the 95th centile dietary exposure ranges at the lower bound for infants issued by EFSA. The calculated median EDI for total PFASs was 20.72 ng/kg/day (range 10.72-107.84) for PHM and 17.92 ng/kg/day (range 6.4-28.96) for DHM, which were both higher than mean exposure ranges reported for infants (2.4-12.2 ng/kg/day). The calculated EDI for DHM was far more similar to the 95th centile (4.5-27.9 ng/kg/day) dietary exposure ranges. For PHM samples, higher EDI values were obtained, with 4 out of 10 samples exceeding the upper limit of the 95th centile range.Conclusion: The exposure of preterm infants to PFASs through HM feeding might exceed reference values reported for older and healthier infants. Given the immunological and developmental vulnerability of preterm infants, the risks related to their exposure to PFASs should be further investigated, also focusing on how maternal exposure and subsequent transfer through HM feeding can be reduced. What is Known: • Perfluoroalkyl substances (PFASs) are environmental contaminants that have been shown to exert toxic effects, which are dependent upon concentration, in animals and humans. The EFSA has recently issued reference values for PFASs exposure for different age groups. • Infants might be exposed to PFASs prenatally, as these substances can cross the placenta, and postnatally, through breastfeeding. No specific data about exposure of preterm infants through human milk (HM) feeding are currently available. What is New: • The exposure of preterm infants to PFASs through HM feeding might exceed reference values reported for older and healthier infants. • Given the immunological and developmental vulnerability of preterm infants, the risks related to their exposure to PFASs deserve further investigation. As HM represents the optimal feeding for preterm infants, it will be fundamental to focus on how maternal exposure and subsequent transfer through HM feeding can be reduced.

Short communication: Monitoring the presence of perfluoroalkyl substances in Italian cow milk.

Perfluoroalkyl substances (PFAS) are fully fluorinated compounds widely used during the last 60 yr in the production of multiple industrial and consumer applications, such as food packaging, nonstick cookware, cleaning agents, and many more. These emerging contaminants have recently become of concern for human health because of their potential negative effects. The risk of exposure to PFAS for humans is mainly related to diet, and the increasing interest in food safety has led the European Commission to call Member States to monitor these contaminants in food matrices. The purpose of the present work was to perform the first monitoring on the presence of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), the 2 main and most widely investigated molecules of this family, in cow milk commercially available in Italy. We used an analytical protocol consisting of liquid-liquid extraction followed by 2 purification steps through solid-phase extraction cartridges and injection on an ultra-performance liquid chromatography-tandem mass spectroscopy system. The analysis of 67 samples of different types of cow milk from Italy demonstrated that contamination by PFOS was often present, although at relatively low concentrations (up to 97 ng/L), whereas PFOA was rarely found. On the basis of these results and data reported in the literature on this matrix, milk does not seem to be a major source of PFAS compared with other food categories such as fish and seafood. However, variability among different types of milk must be taken into account, and surveys of milk-derived products would be helpful to better define the risk for consumers.

Preliminary data on glyphosate, glufosinate, and metabolite contamination in Italian honey samples.

Glyphosate and glufosinate are among the most widely used pesticides in agriculture worldwide. Their extensive use leads to the presence of their residues on crops and in the surrounding environment. Beehives, bees, and apiculture products can represent potential sources for the accumulation of these substances and their metabolites, and the consequences for bee health, as well as the level of risk to human health from consuming contaminated food, are still unclear. Furthermore, information on the contamination levels of honey and other beehive products by these compounds remains poorly documented. This study is part of a broader research effort aimed at developing specific analytical methods for monitoring the level of these contaminants in bee products. The methodology employed enabled the acquisition of preliminary information concerning the levels of glyphosate and glufosinate contamination in honey samples obtained from various retailers in Italy to assess compliance with the limits established by Regulation 293/2013. The liquid chromatography tandem mass spectrometry analysis of the 30 honey samples revealed quantifiable levels of glyphosate in eight samples, with contamination ranging from 5.4 to 138.5 ng/g. Notably, one sample of the wild-flower type showed residue levels nearly three times the maximum residue limit. Additionally, trace levels of glyphosate contamination were detected in another ten samples. It is noteworthy that glufosinate and its metabolites were not detected in any of the analyzed samples within the established method's detection ranges.

Glyphosate and Glufosinate Residues in Honey and Other Hive Products.

Hive products have numerous beneficial properties; however, the hive's health is affected by the surrounding environment. The widespread use of herbicides in agriculture, such as glyphosate and glufosinate, has raised alarm among consumers, beekeepers, and environmentalists due to their potential to harm bees and humans through the consumption of bee products. This review aims to provide a comprehensive overview of the presence of glyphosate, glufosinate, and their metabolites in hive products, collecting and comparing available data from peer-reviewed research and surveys conducted across several countries. Moreover, it analyzes and discusses the potential impacts of these substances on human and bee health, analytical aspects, and recent regulatory developments. The data has revealed that these substances can be present in the different matrices tested, but the concentrations found are usually lower than the maximum residue limits set. However, the use of different methodologies with non-uniform analytical performances, together with an incomplete search for regulated analytes, leads to heterogeneity and makes comparisons challenging. In addition to the completion of studies on the toxicology of herbicide active ingredients, further monitoring actions are necessary, harmonizing analytical methodologies and data management procedures.

Analysis of Cobalamin (Vit B12) in Ripened Cheese by Ultra-High-Performance Liquid Chromatography Coupled with Mass Spectrometry.

The analysis of natural cobalamins in dairy products still represents an analytical challenge. The matrix's complexity, low concentration level, light sensitivity, and binding to proteins are just some of the aspects that make their quantification a difficult goal to achieve. Vitamin B12 plays a fundamental role in human nutrition, and its intake is closely linked to a diet that includes the consumption of food of animal origin. In the current literature, few studies have been carried out on the quantitation of cobalamin in ripened cheeses. A sensitive, selective, and robust ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) method was developed, validated, and applied on ripened cheeses from different species (cow, sheep, and goat) purchased from local Italian markets, highlighting species-dependent differences in vitamin B12 concentrations. The vitamin B12 extraction procedure was performed by converting all cobalamins to the cyanocobalamin form. Furthermore, solid-phase extraction was used for matrix clean-up and analyte preconcentration. The proposed method showed good performance in terms of linearity, sensitivity, reproducibility, and repeatability. The mean vitamin B12 content ranged from

Validation of a single liquid chromatography-tandem mass spectrometry approach for oxytetracycline determination in bull plasma, seminal plasma and urine.

Oxytetracycline is a broad-spectrum antibiotic, which inhibits protein synthesis and is generally used for the treatment of pneumonia, shipping fever, leptospirosis and wound infections in cattle and swine. The present work proposes a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for oxytetracycline quantification in bull plasma, seminal plasma and urine, requiring limited sample treatment before analysis. Extraction with trichloroacetic acid followed by dilution of the supernatant in mobile phase proved to be effective in all three matrices, allowing to rapidly process large batches of samples. Sharp and symmetrical peak shape was obtained using a BEH C18 reversed-phase column in a chromatographic run of just 3.5 min. The mass spectrometer operated in positive electrospray ionization mode and monitored specific transitions for oxytetracycline (461.1 → 425.8) and the internal standard demeclocycline (465.0 → 447.6). The method was validated over concentration ranges suitable for field concentrations of oxytetracycline found in each matrix, showing good linearity during each day of testing (R2 always >0.99), as also confirmed by analysis of variance (ANOVA) and lack-of-fit tests. Excellent accuracy and precision were demonstrated by calculated bias always within ±15% and CV% below 10% at all quality control (QC) levels in the three matrices. Matrix effect and recovery were investigated for both analytes, which showed consistent and comparable behaviour in each matrix. To our knowledge, this is the first validated approach for mass spectrometric determination of oxytetracycline in seminal plasma and urine. The method was successfully applied to samples collected during a pharmacokinetic study in bulls, allowing to assess the oxytetracycline concentration-time profile in plasma, seminal plasma and urine.

A single LC-MS/MS validated method for tulathromycin quantification in plasma, seminal plasma, and urine to be applied in a pharmacokinetic study in bull.

Tulathromycin is a macrolide antibiotic generally used for the treatment of respiratory diseases in cattle and swine. This work proposes an improvement of a previously published LC-MS/MS method for tulathromycin determination in pig serum, here validated in three different bull matrices: plasma, seminal plasma, and urine. The approach is based on a quick protein precipitation with acetonitrile, filtration, and sample dilution before injection, allowing to rapidly process large batches of samples. Analytes separation was obtained using a BEH C18 (50 × 2.1 mm, 1.7 µm) column, maintained at 40°C with a chromatographic run of 5 min. The method was fully validated over concentration ranges suitable for field levels of tulathromycin found in each matrix (0.01-1 µg/ml for plasma, 0.05-5 µg/ml for seminal plasma, and 0.1-10 µg/ml for urine), showing good linearity during each day of testing (R2 always >0.99). Accuracy and precision were within ±15% at all QC concentrations in all the three matrices. Furthermore, the use of tulathromycine-d7 as internal standard mitigated the potential impacts of matrix effect. The validated technique was successfully applied to samples collected during a pharmacokinetic study in bulls, allowing to monitor tulathromycin concentrations over time in the three matrices. To our knowledge, this is the first validated approach for LC-MS/MS quantification of tulathromycin in seminal plasma and urine.

Pharmacokinetics of oxytetracycline long-acting on plasma and semen of beef bulls.

The objectives of this investigation were to evaluate the pharmacokinetic parameters of oxytetracycline long-acting in plasma and seminal plasma after a single administration through either subcutaneous or intramuscular route at 10 mg/kg or 20 mg/kg dose. Four Simmental bulls, healthy and satisfactory potential breeders, were used. The route of administration either subcutaneous or intramuscular did not affect the mean values for 10 mg/kg dose in plasma (1,470 ng/mL vs. 1,330 ng/mL; P = 0.82) or seminal plasma (5,710 ng/mL vs. 5,390 ng/mL; P = 0.88), or for 20 mg/kg dose in plasma (2,540 ng/mL vs. 2,590 ng/mL; P = 0.96) or seminal plasma (25,600 ng/mL vs. 19,400 ng/mL; P = 0.58), respectively. Comparison between the 10 mg/kg and 20 mg/kg doses showed a difference in terms of mean plasma levels (1400 ng/mL vs. 2570 ng/mL; P = 0.07) and mean seminal plasma levels (6,480 ng/mL vs. 26,200 ng/mL; P = 0.004), respectively. After the dose of 10 mg/kg, plasma Cmax was 2,841 ng/mL at 12 h (Tmax) with a half-life of 20.1 h; seminal plasma Cmax was 11,515 ng/mL at 24 h (Tmax) with a half-life of 23.7 h. After the dose of 20 mg/kg, plasma Cmax was 5,269 ng/mL at 12 h (Tmax) with a half-life of 18.1 h; seminal plasma Cmax was 55,040 ng/mL at 24 h (Tmax) with a half-life of 15.7 h. Oxytetracycline long-acting may be an appropriate antibiotic, owing to its pharmacokinetic properties, that could be used for treating bulls' genital infections when its usage is indicated.

Pharmacokinetics of tulathromycin on plasma and semen of beef bulls.

The objective of this investigation was to evaluate the pharmacokinetic parameters of tulathromycin in plasma and semen of beef bulls after administering a single sc dose at two different sites in the neck. Four Simmental bulls with excellent temperament received a comprehensive physical exam that included breeding soundness examination. In addition, blood was collected and analyzed for CBC and chemical panel in order to rule out any subclinical liver or kidney disease. All bulls were diagnosed as healthy and satisfactory potential breeders. The mean plasma levels of tulathromycin for the two neck sites of sc administration were not different between posterior aspect of the ear where it attaches to the head (RP; regio parotidea; 77.9 ± 43.3 ng/mL; X ± SD) and to the middle of the neck (RC; regio collis lateralis; 73.7 ± 39.7 ng/mL; P = 0.84). The mean seminal plasma levels of tulathromycin after administration in the RP was 608 ± 374 ng/mL and for RC was 867 ± 599 ng/mL without differences between both sites (P = 0.29). The mean level of tulathromycin in plasma was 75.8 ± 40.2 ng/mL, which was lower than mean seminal plasma levels of 781 ± 482 ng/mL (P = 0.001). The plasma peak tulathromycin concentration (Cmax) was 160 ± 27 ng/mL at 21 ± 6 h (Tmax) post-administration. The seminal plasma Cmax was 1539 ± 44.4 ng/mL at 33.00 ± 18.00 h (Tmax) post-administration. The Cmax between plasma and seminal plasma were different (P = 0.008) without any differences in Tmax between plasma and seminal plasma (P = 0.35). The terminal half-life for plasma tulathromycin (81.4 ± 27.6 h) showed a tendency to be shorter than in seminal plasma (114.7 ± 21.7; P = 0.10). The plasma area under the curve concentration time from the first to the last sample (AUC0-last) was 15,440 ± 1717 ng/mL/h, which was significatively smaller compared with 171,071 ± 58,556 ng/mL/h for seminal plasma AUC0-last (P = 0.01). The plasma means residence time from the first to the last sample (MRT0-last) was 89.3 ± 5.1 h and it was shorter than for seminal plasma of 96.6 ± 5.0 h (P = 0.05). From the present investigation, it was concluded that tulathromycin is a suitable antibiotic based in its pharmacokinetic properties that could be used for treatment of bull genital infections when its application is indicated.

A critical point in chiral chromatography-mass spectrometry analysis of ketamine metabolites.

Ketamine is a widely used dissociative drug, whose quantification in plasma and urine can be of pharmacological, toxicological, and clinical interest. Although tandem mass spectrometry allows the reliable determination of ketamine and its metabolites in biological matrices, the structural similarity between norketamine (main active metabolite) and dehydronorketamine (a less relevant metabolite) can represent a critical aspect. These compounds differ exclusively in two hydrogen atoms, but the consequent two-unit difference in their mass/charge ratio is partially nullified by the isotopic abundance of the chlorine atom present in their structure. This, along with their similar fragmentation pattern, can result in the incorrect identification of the enantiomers of these ketamine metabolites even with triple quadrupole instruments, if shared transitions are monitored after chiral chromatography. The key to prevent norketamine overestimation is therefore observing analyte-specific MS/MS transitions. Here, we describe in detail how we investigated this issue, during the development of an analytical method for ketamine and norketamine enantiomer determination in plasma.

Dechlorane Plus and Related Compounds in Food-A Review.

Dechlorane Plus is a polychlorinated compound which has exclusively anthropic origin. This compound has been manufactured for close to 60 years for various applications, but mainly as flame retardant. Dechlorane Plus and other Dechlorane-related compounds (DRCs) are currently marketed as a replacement for Dechlorane, also known as Mirex, banned in 1978. These compounds share comparable properties to persistent organic pollutants (POPs), such as persistence in the environment, high lipophilicity, bioaccumulation through the food web and adverse effects on the environment and human health. Despite their long production history, they have been only recently reported in various environmental compartments, such as air, soil, and foodstuff. The aim of this review is to provide a picture of the current state of knowledge on worldwide DRC levels in food, in order to highlight gaps and research needs. The review compares the data on DRC contamination available in literature, considering different food categories and sampling country. In addition, it is specified whether the data were obtained from studies on foodstuff to estimate dietary intake, to evaluate the contamination near the e-waste treatment area or for environmental monitoring purposes.

Perfluoroalkyl contaminants in eggs from backyard chickens reared in Italy.

Per- and poly-fluoroalkyl substances (PFASs) are persistent and bioaccumulative compounds with adverse impacts on the environment and human health. Diet is one of the main sources of exposure to PFASs. Recently, the EFSA established a tolerable weekly intake (TWI) limit (4.4 ng/kg b.w.) for a mixture of the four major PFASs. Eggs and egg products can contribute to this intake, with their contamination possibly dependent on the husbandry system. Monitoring Italian eggs from backyard chickens revealed a relatively uniform PFAS contamination, with perfluoro-1-octanesulfonate being the most abundant. Contamination was detected to be significantly higher in eggs from backyard chickens than in eggs from commercial laying hens, consistent with a previous Italian study. According to the recently set TWI value, the consumption of eggs from backyard chickens could contribute significantly to dietary intake of PFASs (up to 29% of the TWI in children, considering the lower bound approach).

Design and In Vitro Study of a Dual Drug-Loaded Delivery System Produced by Electrospinning for the Treatment of Acute Injuries of the Central Nervous System.

Vascular and traumatic injuries of the central nervous system are recognized as global health priorities. A polypharmacology approach that is able to simultaneously target several injury factors by the combination of agents having synergistic effects appears to be promising. Herein, we designed a polymeric delivery system loaded with two drugs, ibuprofen (Ibu) and thyroid hormone triiodothyronine (T3) to in vitro release the suitable amount of the anti-inflammation and the remyelination drug. As a production method, electrospinning technology was used. First, Ibu-loaded micro (diameter circa 0.95-1.20 µm) and nano (diameter circa 0.70 µm) fibers were produced using poly(l-lactide) PLLA and PLGA with different lactide/glycolide ratios (50:50, 75:25, and 85:15) to select the most suitable polymer and fiber diameter. Based on the in vitro release results and in-house knowledge, PLLA nanofibers (mean diameter = 580 ± 120 nm) loaded with both Ibu and T3 were then successfully produced by a co-axial electrospinning technique. The in vitro release studies demonstrated that the final Ibu/T3 PLLA system extended the release of both drugs for 14 days, providing the target sustained release. Finally, studies in cell cultures (RAW macrophages and neural stem cell-derived oligodendrocyte precursor cells-OPCs) demonstrated the anti-inflammatory and promyelinating efficacy of the dual drug-loaded delivery platform.

Determination of 15 organophosphorus pesticides in Italian raw milk.

A study was conducted on raw cow's milk to measure the residues of 15 organophosphorus pesticides used as dairy cattle ectoparasiticides or as insecticides in crops used for animal feed. For this purpose a previously devised method was improved and validated. The samples were collected directly from tank trucks during delivery of 3,974 tonnes of raw milk at nine Italian dairy plants. Approximately 4.4% of the 298 samples analyzed contained residues only in traces. The main pollutant was chlorpyriphos.

Preliminary monitoring of the presence of perfluoroalkyl substances in Italian eggs from different breeding systems.

Perfluoroalkylated substances (PFASs) are a wide cluster of fluorinated molecules largely engaged industrially and commercially for many purposes. Because of the strength of the fluorine-carbon bond, PFASs show a firm tenacity against thermal degradation, hydrolysis, photolysis and biodegradation. On the other hand, such chemical stability gives them persistent environmental pollutant feature. In 2012, EFSA published a scientific report on PFASs in food, mentioning their adverse effects on health. Based on observational studies evidences, EFSA has recommended a tolerable daily intake (TDI) for the two most known PFASs, i.e. PFOS 150 ng/kg b.w./day and PFOA 1500 ng/kg b.w./day. The aim of this study was to monitor, for the first time, the level of contamination of PFASs in chicken eggs laid in Northern Italy. The eggs were collected from different rearing systems, in order to search a correlation between this variable and the contamination of PFASs. In this study four PFASs [perfluoro-nnonanoic acid (PFNA), perfluoro-noctanoic- acid (PFOA), sodium perfluoro-1- hexanesulfonate (PFHxS) and sodium perfluoro- 1-octanesulfonate (PFOS)] were analyzed by liquid chromatography-tandem mass spectrometer (LC-MS/MS). 132 eggs were analyzed, split up in 11 groups according to the geographical origin and rearing system. Results accord with literature data available for chicken eggs: almost all the samples show a PFASs contamination level under the limit of quantification (LOQ) of 0.25 ng/mL. No significant difference results from the rearing system, attesting an equal distribution and a concentration of PFASs detectable under the limit of quantification.

Adverse Effects, Transformation and Channeling of Aflatoxins Into Food Raw Materials in Livestock.

Aflatoxins are wide-spread harmful carcinogenic secondary metabolites produced by Aspergillus species, which cause serious feed and food contaminations and affect farm animals deleteriously with acute or chronic manifestations of mycotoxicoses. On farm, both pre-harvest and post-harvest strategies are applied to minimize the risk of aflatoxin contaminations in feeds. The great economic losses attributable to mycotoxin contaminations have initiated a plethora of research projects to develop new, effective technologies to prevent the highly toxic effects of these secondary metabolites on domestic animals and also to block the carry-over of these mycotoxins to humans through the food chain. Among other areas, this review summarizes the latest findings on the effects of silage production technologies and silage microbiota on aflatoxins, and it also discusses the current applications of probiotic organisms and microbial products in feeding technologies. After ingesting contaminated foodstuffs, aflatoxins are metabolized and biotransformed differently in various animals depending on their inherent and acquired physiological properties. These mycotoxins may cause primary aflatoxicoses with versatile, species-specific adverse effects, which are also dependent on the susceptibility of individual animals within a species, and will be a function of the dose and duration of aflatoxin exposures. The transfer of these undesired compounds from contaminated feed into food of animal origin and the aflatoxin residues present in foods become an additional risk to human health, leading to secondary aflatoxicoses. Considering the biological transformation of aflatoxins in livestock, this review summarizes (i) the metabolism of aflatoxins in different animal species, (ii) the deleterious effects of the mycotoxins and their derivatives on the animals, and (iii) the major risks to animal health in terms of the symptoms and consequences of acute or chronic aflatoxicoses, animal welfare and productivity. Furthermore, we traced the transformation and channeling of Aspergillus-derived mycotoxins into food raw materials, particularly in the case of aflatoxin contaminated milk, which represents the major route of human exposure among animal-derived foods. The early and reliable detection of aflatoxins in feed, forage and primary commodities is an increasingly important issue and, therefore, the newly developed, easy-to-use qualitative and quantitative aflatoxin analytical methods are also summarized in the review.

Occurrence of Mycotoxins in Extruded Commercial Cat Food.

The occurrence of the most important mycotoxins (deoxynivalenol, fumonisin B1 and B2, aflatoxins B1, B2, G1, and G2, ochratoxin A, zearalenone, T-2, and HT-2 toxins) was determined in 64 extruded cat foods purchased in Italy through ultra-performance liquid chromatography coupled with tandem mass spectrometry. Deoxynivalenol and fumonisins were the most common contaminants (quantified in 80 and 95% of the samples, respectively). Conversely, aflatoxins B2, G1, and G2 were not identified in any sample. Some cat foods exceeded the regulatory limit for aflatoxin B1 (n = 3) or the guidance values for zearalenone (n = 3), fumonisins (n = 2), ochratoxin A (n = 1), and T-2 (n = 1) recently established for pets in the European Union. A widespread co-occurrence of mycotoxins was observed (28, 42, and 8% of the samples contained quantifiable amounts of two, three, and four mycotoxins, respectively). This study describes criticisms regarding the mycotoxin issue in pet food and suggests an improvement of the monitoring of the pet food chain.

Expression of CLAVATA3 fusions indicates rapid intracellular processing and a role of ERAD.

The 12 amino acid peptide derived from the Arabidopsis soluble secretory protein CLAVATA3 (CLV3) acts at the cell surface in a signalling system that regulates the size of apical meristems. The subcellular pathway involved in releasing the peptide from its precursor is unknown. We show that a CLV3-GFP fusion expressed in transfected tobacco protoplasts or transgenic tobacco plants has very short intracellular half-life that cannot be extended by the secretory traffic inhibitors brefeldin A and wortmannin. The fusion is biologically active, since the incubation medium of protoplasts from CLV3-GFP-expressing tobacco contains the CLV3 peptide and inhibits root growth. The rapid disappearance of intact CLV3-GFP requires the signal peptide and is inhibited by the proteasome inhibitor MG132 or coexpression with a mutated CDC48 that inhibits endoplasmic reticulum-associated protein degradation (ERAD). The synthesis of CLV3-GFP is specifically supported by the endoplasmic reticulum chaperone endoplasmin in an in vivo assay. Our results indicate that processing of CLV3 starts intracellularly in an early compartment of the secretory pathway and that ERAD could play a regulatory or direct role in the active peptide synthesis.

Vitamin B12 determination in milk, whey and different by-products of ricotta cheese production by ultra performance liquid chromatography coupled with tandem mass spectrometry.

Vitamin B12 (cobalamin) is a metal complex composed of a central cobalt ion bonded to six ligands. It is essential for major biological functions such as protein, fat and carbohydrate metabolism, the maintenance of the central nervous system, and the formation of red blood cells. Since mammals cannot synthesize cobalamin, dietary intake represents the only natural source for humans. Dairy products can provide significant levels of cobalamin; moreover, the European Food Safety Authority (EFSA) panel has set the recommended intake at 4 µg/day for adults. Vitamin B12 content was determined in milk and several matrices related to the process of transformation of the residual whey from Parmigiano Reggiano cheese-making to obtain ricotta cheese. In addition, vitamin B12 degradation during ricotta cheese shelf-life was studied. The analyses were performed using an ultra performance liquid chromatography-tandem mass spectrometry method. Results show that vitamin B12 amount in ricotta from dairy and experimental cheese-making brings respectively 1/8 to 1/4 of the adequate intake in adults established by EFSA. In addition, shelf-life experiment shows that cobalamine is fairly rapidly degraded in ricotta: light effect seems to be significant, even if the light exposure is short. The use of photoprotective packaging material increases B12 shelf-life in the early stage of storage.