[Implementation of the Lübeck model of themed movement activities in nursing. Cooperation at the local level to strengthen the prevention and health promotion in Berlin-Pankow]. / Umsetzung des Lübecker Modells Bewegungswelten in der Pflege. Kooperation auf kommunaler Ebene zur Stärkung von Prävention und Gesundheitsförderung in Berlin-Pankow.

In accordance with Prevention Act law and the associated new prevention mandate of the nursing care funds, more preventive and health promoting activities have been taking place in inpatient care facilities since 2016. The Lübeck model of themed movement activities as a part of the program "Grow older in Balance" of the Federal Center for Health Education was developed as a physical, mental, and socially activating prevention program for elderly people with physical and cognitive impairments.The regional implementation of the Lübeck model in inpatient care facilities, facilities for age-appropriate living, as well as in day-care, serves the Berlin project "Adaptation and implementation of the Lübeck model of themed movement activities in the model region of Pankow - Low threshold exercise offers in nursing facilities for the elderly." This model project of the regional network Qualitätsverbund Netzwerk im Alter - Pankow e. V. (QVNIA e. V.) is presented in the current article.The relevant local structures and cooperation as well as the resulting and program-related requirements for participating actors are highlighted. With the aim of sustainable implementation of physical activity promotion in the nursing setting, a possible implementation path and quality assurance measures are presented.The transfer has shown that the concept of the Lübeck model can also be implemented in metropolitan structures. However, the implementation requires the consideration of the regional framework conditions. A centralized local control with the corresponding structural and procedural expertise as well as quality assurance assets is necessary. Positive experiences in the implementation of the program were acquired during the model phase and recommendations for the continuation and further development can be derived.

Simultaneously tracing the geographical origin and presence of bovine milk in Italian water buffalo Mozzarella cheese using MALDI-TOF data of casein signature peptides.

Water buffalo (WB) casein (CN) and curd samples from indigenous Italian and international breeds were examined with the objective of identifying signature peptides that could function as an indicator to determine the origin of their milk products. CN in complex mixtures were digested with trypsin, and peptide fragments were subsequently identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). The unique presence of a ß-CN A variant and an internally deleted αs1-CN (f35-42) variant in international WB milk samples was ascertained by identifying signature tryptic peptides from either dephosphorylated or native CN. Four signature unphosphorylated peptides derived from ß-CN A, i.e. (f49-68) Asn(68) (2223.6 Da), (f1-28) Ser(10) (3169.4 Da), (f1-29) Ser(10) (3297.4 Da) and (f33-48) Thr(41) (1982 Da) and two from αs1-CN (f35-42) deleted fragments, i.e. (f23-34) Met(31) (1415.7 Da) and (f43-58) Val(44) (1752.7 Da), were identified. Two signature casein phosphopeptides (CPPs), i.e. ß-CN (f1-28) 4P (3489.1 Da) and ß-CN (f33-48) 1P (2062.0 Da), were identified in the tryptic hydrolysate of native casein or curd and cheese samples using in-batch hydroxyapatite (HA) chromatography. All these fragments functioned as analytical surrogates of two αs1- and ß-casein variants that specifically occur in the milk of international WB breeds. Furthermore, the bovine peptide ß-CN (f1-28) 4P had a distinct and lower molecular mass compared with the WB counterpart and functioned as a species-specific marker for all breeds of WB. Advantages of this analytical approach are that (i) peptides are easier to separate than proteins, (ii) signature peptide probes originating from specific casein variants allow for the targeting of all international WB milk, curd and cheese samples and (iii) bovine and WB casein in mixtures can be simultaneously determined in protected designation of origin (PDO) "Mozzarella di Bufala Campana" cheese. This analytical method enabled the specific detection of international WB and bovine casein with a sensitivity threshold of 2 and 0.78 %, respectively. Graphical Abstract Monitoring of prototypic tryptic CPPs by MALDI-TOF analysis in Mediterranean (A), Romanian (B), Indian (C), Polish (D) and Canadian (E) curd samples to guarantee the authenticity of the PDO "Mozzarella di Bufala Campana" cheese.

Comparison study of in vivo dose response to laser-driven versus conventional electron beam.

The long-term goal to integrate laser-based particle accelerators into radiotherapy clinics not only requires technological development of high-intensity lasers and new techniques for beam detection and dose delivery, but also characterization of the biological consequences of this new particle beam quality, i.e. ultra-short, ultra-intense pulses. In the present work, we describe successful in vivo experiments with laser-driven electron pulses by utilization of a small tumour model on the mouse ear for the human squamous cell carcinoma model FaDu. The already established in vitro irradiation technology at the laser system JETI was further enhanced for 3D tumour irradiation in vivo in terms of beam transport, beam monitoring, dose delivery and dosimetry in order to precisely apply a prescribed dose to each tumour in full-scale radiobiological experiments. Tumour growth delay was determined after irradiation with doses of 3 and 6 Gy by laser-accelerated electrons. Reference irradiation was performed with continuous electron beams at a clinical linear accelerator in order to both validate the dedicated dosimetry employed for laser-accelerated JETI electrons and above all review the biological results. No significant difference in radiation-induced tumour growth delay was revealed for the two investigated electron beams. These data provide evidence that the ultra-high dose rate generated by laser acceleration does not impact the biological effectiveness of the particles.

Identification of allergen encoding sequences in a novel food ingredient from Moringa oleifera leaves.

Alternative sources of edible proteins are required to feed the world's growing population, such as Moringa oleifera leaves, a protein source with a balanced amino acid composition. Since Moringa leaf proteins is a novel food in the EU and UK, an assessment of their potential allergenicity of is required. Proteins from Moringa leaf powder were characterised using traditional proteomic approaches. The proteins identified were evaluated for their allergenic potential using in-silico tools. The main proteins identified belonged to photosynthetic and metabolic pathways. In-silico analysis of the leaf proteome identified moritides as potential allergens by homology with a latex allergen implicated in fruit-latex syndrome. This analysis also identified a nsLTP, a major panallergen in food. The presence of these putative allergens was confirmed by de-novo sequencing. Our study allowed identification of putative allergens, Morintides and nsLTP. Further in-vitro and in-vivo investigations are required to confirm their allergenic potential.

The occurrence of genetic polymorphism and related non-allelic proteins increases the compositional complexity of goat α((s1)) -CN.

A genetic survey on three autochthonous goat breeds reared in Italy was carried out by a proteomic approach. This methodology, further to providing the phenotypic frequency of identified α(s1) genetic variants, allowed to determine (i) the additional constitutive presence of a non-allelic 'α(s1) -casein (CN) F like' protein in goat 'strong' α(s1) variants; (ii) an α(s1) -CN B(2) like protein, expressed at very low quantitative level, in goat 'weak' α(s1) -CN variants, and, as main focus; (iii) the occurrence of a new α(s1) -CN D(1) variant characterised by the lack of α(s1) (f59-69) sequence otherwise encoded by exon 9 in goat α(s1) B(2) reference. The same exon skipping event had been identified since 1990, as responsible of the 'weak quantitative class' of α(s1) -CN D variant (0.6 g/L), while the new α(s1) -CN D(1,) has been 'quantitatively' classified as an 'intermediate' variant, since 1.8 g/L per allele was assessed in the milk.

Deducing the electron-beam diameter in a laser-plasma accelerator using x-ray betatron radiation.

We investigate the properties of a laser-plasma electron accelerator as a bright source of keV x-ray radiation. During the interaction, the electrons undergo betatron oscillations and from the carefully measured x-ray spectrum the oscillation amplitude of the electrons can be deduced which decreases with increasing electron energies. From the oscillation amplitude and the independently measured x-ray source size of (1.8±0.3) µm we are able to estimate the electron bunch diameter to be (1.6±0.3) µm.

Lactosylated casein phosphopeptides as specific indicators of heated milks.

Casein phosphopeptides (CPP) were identified in small amounts in milks heated at various intensities by using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. CPP selectively concentrated on hydroxyapatite (HA) were regenerated using phosphoric acid mixed in the matrix. Unphosphorylated peptides not retained by HA were removed by buffer washing. This procedure enhanced the MALDI signals of CPP that are ordinarily suppressed by the co-occurrence of unphosphorylated peptides. CPP, belonging to the ß-casein (CN) family, i.e., (f1-29) 4P, (f1-28) 4P, and (f1-27) 4P, and the α(s2)-CN family, i.e., (f1-21) 4P and (f1-24) 4P, were observed in liquid and powder milk. The lactosylated counterparts were specific to intensely heated milks, but absent in raw and thermized/pasteurized milk. Most CPP with C-terminal lysines probably arose from the activity of plasmin; an enzyme most active in casein hydrolysis. A CPP analogue was used as the internal standard. The raw milk signature peptide ß-CN (f1-28) 4P constituted ~4.3% of the total ß-CN. Small amounts of lactosylated peptides, which varied with heat treatment intensity, were detected in the milk samples. The limit of detection of ultra-high-temperature milk adjunction in raw or pasteurized milk was ~10%.

Effect of sprouting on the proteome of chickpea flour and on its digestibility by ex vivo gastro-duodenal digestion complemented with jejunal brush border membrane enzymes.

The demand for sustainably produced proteins is increasing with the world population and is prompting a dietary shift toward plant sourced proteins. Vegetable proteins have lower digestibility and biological value compared to animal derived counterparts. We explored sprouting of chickpea seeds as a strategy for improving digestibility. Protein evolution associated with by the sprouting process was assessed by proteomics. The sprouting induced breakdown of seed storage proteins and doubled the release of free alpha-amino nitrogen in sprouted chickpea flour. During sprouting, several enzymes involved in plant development were newly expressed. An ex vivo model of gastroduodenal and jejunal digestion was applied to assess the bioaccessibility of the protein digests. Proteins from chickpea sprouts showed a greater susceptibility to digestion with a 10% increase in alpha amino nitrogen. Peptides with potential immunoreactivity or bioactivity were catalogued in both digested chickpea sprouts and seeds using an in-silico approach. Peptides belonging to the non-specific transfer proteins, which are allergens in pulses, and peptides belonging to an IgE-binding hemagglutinin protein could only be identified in the digested chickpea sprouts. The observation collected paved the way to immune-based evaluations to assess the effect of germination on the allergenic potential.

The effect of nitrogen fertilization on the expression of protein in wheat and tritordeum varieties using a proteomic approach.

Wheat, an essential ingredient for several bakery preparations, is also responsible for gluten-related diseases in sensitive subjects. The effect of the N fertilization rate (80 vs 160 kg N ha-1) on gluten protein expression profile has been evaluated considering two soft wheats (landrace and modern) and one tritordeum cultivar (cv), grown in the same experimental field in North Italy. The proteins of refined flour were characterized through advanced proteomic approaches, including chromatography (RP-HPLC) and electrophoresis. A static model system was used to simulate in vitro digestion and the digestome peptides were examined by mass spectrometry and in silico approaches, to investigate the celiac and allergenic sequences. The CD-toxic epitopes in the digested samples were quantified by means of a R5 ELISA assay. The N fertilization rate increased the grain protein content, but it did not lead to any difference in gluten composition, with exception of glu/glia ratio in the modern wheat cv. Moreover, the gluten composition and the occurrence of toxic/allergenic epitopes varied to a great extent, according mostly to the genotype. A lower immunoreactivity, determined using R5 ELISA, was detected for the digested tritordeum flours than for the landrace (-51%) or modern (-58%) cvs, while no significant difference was observed for the N rates between each genotype. In silico analysis showed that tritordeum has fewer CD epitopes belonging to the ω-gliadins and a lower LMW-GS than the landrace or modern cv. Tritordeum presented fewer α-gliadin allergenic epitopes than the modern wheat cv. The lower frequency of celiac epitopes in tritordeum, compared to the old and the modern wheat, is probably due to the absence of a D genome.

Multiparametric magnetic resonance imaging and clinical variables: Which is the best combination to predict reclassification in active surveillance patients?

INTRODUCTION & OBJECTIVES: We tested the role of multiparametric magnetic resonance imaging (mpMRI) in disease reclassification and whether the combination of mpMRI and clinicopathological variables could represent the most accurate approach to predict the risk of reclassification during active surveillance. MATERIALS & METHODS: Three-hundred eighty-nine patients (pts) underwent mpMRI and subsequent confirmatory or follow-up biopsy according to the Prostate Cancer Research International Active Surveillance (PRIAS) protocol. Pts with negative (-) mpMRI underwent systematic random biopsy. Pts with positive (+) mpMRI [Prostate Imaging Reporting and Data System, version 2 (PI-RADS-V2) score ≥3] underwent targeted + systematic random biopsies. Multivariate analyses were used to create three models predicting the probability of reclassification [International Society of Urological Pathology ≥ Grade Group 2 (GG2)]: a basic model including only clinical variables (age, prostate-specific antigen density, and number of positive cores at baseline), an Magnetic resonance imaging (MRI) model including only the PI-RADS score, and a full model including both the previous ones. The predictive accuracy (PA) of each model was quantified using the area under the curve. RESULTS: mpMRI negative (-) was recorded in 127 (32.6%) pts; mpMRI positive (+) was recorded in 262 pts: 72 (18.5%) had PI-RADS 3, 150 (38.6%) PI-RADS 4, and 40 (10.3%) PI-RADS 5 lesions. At a median follow-up of 12 months, 125 pts (32%) were reclassified to GG2 prostate cancer. The rate of reclassification to GG2 prostate cancer was 17%, 35%, 38%, and 52% for mpMRI (-), PI-RADS 3, 4, and 5, respectively (P < 0.001). The PA was 69% and 64% in the basic and MRI models, respectively. The full model had the best PA of 74%: older age (P = 0.023; Odds ratio (OR) = 1.040), prostate-specific antigen density (P = 0.037; OR = 1.324), number of positive cores at baseline (P = 0.001; OR = 1.441), and PI-RADS 3, 4, and 5 (overall P = 0.001; OR = 2.458, 3.007, and 3.898, respectively) were independent predictors of reclassification. CONCLUSIONS: Disease reclassification increased according to the PI-RADS score increase, at confirmatory or follow-up biopsy. However, a no-negligible rate of reclassification was found also in cases of mpMRI (-). The combination of mpMRI and clinicopathological variables still represents the most accurate approach to pts on active surveillance.

The Use of Multiparametric Magnetic Resonance Imaging for Follow-up of Patients Included in Active Surveillance Protocol. Can PSA Density Discriminate Patients at Different Risk of Reclassification?

INTRODUCTION: The objective of this study was to test Prostate Imaging Reporting and Data System (PI-RADS) classification on multiparametric magnetic resonance imaging (mpMRI) and MRI-derived prostate-specific antigen density (PSAD) in predicting the risk of reclassification in men in active surveillance (AS), who underwent confirmatory or per-protocol follow-up biopsy. MATERIALS AND METHODS: Three hundred eighty-nine patients in AS underwent mpMRI before confirmatory or follow-up biopsy. Patients with negative (-) mpMRI underwent systematic random biopsy. Patients with positive (+) mpMRI underwent targeted fusion prostate biopsies + systematic random biopsies. Different PSAD cutoff values were tested (< 0.10, 0.10-0.20, ≥ 0.20). Multivariable analyses assessed the risk of reclassification, defined as clinically significant prostate cancer of grade group 2 or more, during follow-up according to PSAD, after adjusting for covariates. RESULTS: One hundred twenty-seven (32.6%) patients had mpMRI(-); 72 (18.5%) had PI-RADS 3, 150 (38.6%) PI-RADS 4, and 40 (10.3%) PI-RADS 5 lesions. The rate of reclassification to grade group 2 PCa was 16%, 22%, 31%, and 39% for mpMRI(-) and PI-RADS 3, 4, and 5, respectively, in case of PSAD < 0.10 ng/mL2; 16%, 25%, 36%, and 44%, in case of PSAD 0.10 to 0.19 ng/mL2; and 25%, 42%, 55%, and 67% in case of PSAD ≥ 0.20 ng/mL2. PSAD ≥ 0.20 ng/mL2 (odds ratio [OR], 2.45; P = .007), PI-RADS 3 (OR, 2.47; P = .013), PI-RADS 4 (OR, 2.94; P < .001), and PI-RADS 5 (OR, 3.41; P = .004) were associated with a higher risk of reclassification. CONCLUSION: PSAD ≥ 0.20 ng/mL2 may improve predictive accuracy of mpMRI results for reclassification of patients in AS, whereas PSAD < 0.10 ng/mL2 may help selection of patients at lower risk of harboring clinically significant prostate cancer. However, the risk of reclassification is not negligible at any PSAD cutoff value, also in the case of mpMRI(-).

A non-canonical phosphorylation site in ß-casein A from non-Mediterranean water buffalo makes quantifiable the adulteration of Italian milk with foreign material by combined isoelectrofocusing-immunoblotting procedures.

The need of controlling illegal addition of water buffalo (WB) milk from foreign countries to the Italian counterpart devoted to the production of Protected Denomination of Origin (PDO) Mozzarella di Bufala Campana (MBC) cheese has promoted the development of simple, fast and cheap isoelectrofocusing (IEF) methods for evaluating the nature of the raw material to be used according to a high-throughput sample multiplexing format, avoiding the use of dedicated mass spectrometry-based procedures. Thus, combined proteomic methods were here integrated with optimized western blotting protocols in solving the complex IEF pattern of casein (CN) mixtures observed when Italian and foreign WB milk are mixed together. Identification of internally deleted αs1-CN hepta-phosphorylated species as well as of still unknown ß-CN A hexa-phosphorylated and N-terminally-nicked ß-CN A phosphorylated forms present uniquely in foreign WB milk samples, allowed recognizing these molecules as adulteration markers to be assayed in combined IEF-immunoblotting procedures; the latter ones showing optimal migration characteristics to be used in routine assays. A linear relationship between detected area of specific immunorecognized gel bands and percentage of international WB milk added to the Italian counterpart was verified, demonstrating that this method has an adulteration detection limit close to 3% v/v. Based on these results, this analytical procedure is here proposed as optimal one for evaluating the authenticity of PDO MBC cheese products.

Production, digestibility and allergenicity of hemp (Cannabis sativa L.) protein isolates.

Hemp (Cannabis sativa L.), traditionally cultivated for industrial use and harvested for fibers and seeds, has raised much interest as a sustainable crop in the last years. Recently, hemp seeds and derived oil have started to be used in a variety of food products. Hemp-based food products are considered less allergenic than those from other edible seeds, although this statement has never been experimentally verified. In this study high purity grade hemp flour (HF) and hemp protein isolate (HPI) were obtained through a fast and cheap process starting from defatted hemp cakes, a residue of hempseed oil extraction. HPI resulted enriched at nearly 86% protein, mainly constituted by the storage protein edestin (accounting for 70% total protein). In vitro protein digestibility was determined using a static model of gastrointestinal digestion (GID), which included a final step with purified brush border membrane (BBM) enzyme preparations. HF and HPI showed a high degree of digestibility. The survival of potential bioactive and/or allergenic peptide sequences in digests was investigated by peptidomic analysis. Only a limited number of sequences survived GID. Among them, fragments from 12 seed proteins. These fragments were precursors of sequences with potential bioactive peptides, which might justify the bioactivity of HPI hydrolysates, reported in previous studies. More importantly, all known hemp allergens, including the major thaumatin-like protein and LTP, were entirely eliminated by the HPI production process, neither fragments of the proteins were present after GID. These data support the use of HPI as an ingredient for hypoallergenic foods.

Comprehensive analysis of the peanut allergome combining 2-DE gel-based and gel-free proteomics.

In this work, we explored the "deep" seed peanut proteome by using both two dimensional electrophoresis (2-DE)-based analysis run under reducing and non-reducing condition (protein-centric) and LC-MS/MS gel-free proteomic (peptide-centric). The former approach allowed to identify high molecular weight disulfide-linked Ara h 1 and Ara h 3 heteroligomers and Ara h 1 homoligomers linked through covalent bonds other than disulfides. The occurrence of these protein complexes revealed natural interactions between Ara(s) subunits with a possible involvement in the allergenic potential of peanut. The second approach, also referred to as shot-gun proteomics, allowed the identification of 149 gene products, including low-abundance proteins escaped the 2-DE detection. Interestingly, we identified 60 proteins never catalogued previously. The complementary exploitation of two proteomic approaches enabled the access to new relevant information about the complexity of the peanut proteome, with special emphasis to the complement of allergens (allergome).

Extended pelvic lymph node dissection during radical prostatectomy: comparison between initial robotic experience of a high-volume open surgeon and his contemporary open series.

BACKGROUND: The aim of this study was to evaluate intra- and perioperative outcomes of a single high volume open radical prostatectomy (ORP) surgeon, during his learning curve period for robot-assisted radical prostatectomy (RARP) and extended pelvic lymph node dissection (ePLND). METHODS: The study included 264 intermediate-high risk prostate cancer patients, treated by ORP + ePLND or RARP + ePLND, prospectively collected. Descriptive statistics compared clinical and pathological variables between groups. Bivariate (Pearson) correlation analysis assessed the relationship between the number of lymph node (LN) removed, positive surgical margins (PSM), surgical time and the number of procedures performed per group. RESULTS: pT stage and Gleason score (GS) were lower in RARP than in ORP group (both P=0.04), while PSM were more frequent in the RARP group (40% vs. 25%; P=0.02). However, PSM decreased with the increase of RARP procedures. The number of LNs removed was 25 and 22, in RARP and ORP group (P=0.03). However, LN+ rate did not differ between groups (11% vs. 16%; P=0.216). In the RARP group, overall surgical time and ePLND time decreased with the increase of surgical procedures (all P<0.001). CONCLUSIONS: RARP requires significant learning curve to reduce operative room time and obtain PSM comparable to those of an ORP high-volume surgeon. On the contrary, the quality of ePLND during RARP seems to be not related to the number of procedures performed, allowing removal of a number of LNs that is clinically comparable to ORP.

Monitoring molecular composition and digestibility of ripened bresaola through a combined foodomics approach.

In this work, the effects of maturation time and simulated gastrointestinal digestion on the molecular and peptide profiles of "Bresaola Valtellina" were assessed through the foodomics approach, in this case food proteomics and peptidomics combined to other analytical and biological assays, aiming at depicting a holistic food quality. Human digestion of this Italian cured meat product was simulated using an in vitro static protocol and the degree of proteolysis and the in vitro bioactivity of the soluble free compounds in the digestates were evaluated by biochemical assays, e.g. SDS-PAGE, size exclusion HPLC, HPLC/MS, 1H NMR, enzymatic and antioxidant activities. The obtained results demonstrated that in vitro gastrointestinal digestion contributed to a considerable release of myofibrillar proteins by the muscle tissue. Data from SDS-PAGE, peptidomic and size exclusion HPLC assays showed that the in vitro digestion largely degraded proteins of muscle tissue to peptides smaller than 250 Da. The released peptides were likely responsible for the inhibitory activity on amylolytic enzymes and for the antioxidant properties elicited by the gastric digestates of Bresaola. Overall, the results demonstrated the negligible role of ripening in making meat proteins more bioaccessible, whereas they confirmed the highly in vitro digestibility of meat proteins from Bresaola. This study represents a new approach merging proteomics and foodomics to evaluate the effect of ripening and in vitro digestion on the bioactivity and bioaccessibility of proteins and peptides of meat products.

Difference in Frequency and Distribution of Nodal Metastases Between Intermediate and High Risk Prostate Cancer Patients: Results of a Superextended Pelvic Lymph Node Dissection.

Objectives: To evaluate the frequency and distribution of pelvic nodes metastases, in intermediate-high risk prostate cancer (PCa) patients (pts), who underwent open radical prostatectomy (ORP) and superextended pelvic lymph node dissection (sePLND). Patients and Methods: We retrospectively evaluated 630 consecutive pts with clinically localized, intermediate-high risk PCa, treated with ORP and sePLND from 2009 to 2016 at a single institution. The sePLND always removed all nodal/fibro-fatty tissue of the internal iliac, external iliac, obturator, common iliac, and presacral regions. Results: Positive lymph nodes (LN+) were found in 133 pts (21.1%). The median number of removed nodes and LN+ was 25 and 1, respectively. LN+ were found in 64 (48.1%), 58 (43.6%), 53 (39.8%), 16 (12%), and 20 (15%) pts and were present as a single site in 27 (20.3%), 22 (16.5%), 20 (15%), 0, and 6 (4.5%) cases in the internal iliac, external iliac, obturator, common iliac, and presacral chain, respectively. An ePLND would have correctly staged 127 (95%) pts but removed all LN+ in only 97 (73%) pts. Presacral nodes harbored LN+ in 20 patients. Among them, 18 were high-risk patients. Moreover, all but 1 pts with common iliac LN+ were in high risk group. Conclusions: These results suggest that removal of presacral and common iliac nodes could be omitted in intermediate risk pts. However, a PLND limited to external iliac, obturator, and internal iliac region may be adequate for nodal staging purpose, but not enough accurate if we aim to remove all possible site of LN+ in high risk pts.

Eventual limits of the current EU official method for evaluating milk adulteration of water buffalo dairy products and potential proteomic solutions.

The European reference method (ERM) recognises the fraudulent addition of bovine (B) milk in water buffalo (WB) milk/dairy products based on concomitant isoelectric focusing (IEF) detection of B γ2- and γ3-CN fragments after corresponding plasminolysis. We here used proteomics to characterise false positive results occurring in the ERM as being due to WB ß-CN(f100-209), which is also formed after plasminolysis of genuine WB milk/dairy products and comigrates in IEF with B γ2-CN. These ERM limitations were overcome by a dedicated proteomic procedure based on loading of B/WB milk/cheese CN extracts on a hydroxyapatite column, in situ trypsinolysis and elution of B ß-CN(f1-25)4P and WB ß-CN(f1-28)4P proteotypic peptides. Based on their similar ionisation properties and resolution in MALDI-TOF-MS, these phosphopeptides were identified as suitable markers for detection of B material in WB milk/dairy products to a detection limit of 0.8% v/v. This proteomic procedure is here proposed as integrative/alternative to the ERM.

Human IgE binding to the glycosidic moiety of bovine kappa-casein.

IgE ability for recognizing milk proteins was assayed in the serum of an adult atopic patient who outgrew cow milk allergy in early childhood. A number of protein species included in casein from bovine milk were detected by human IgE in immunoblotting experiments. Comparing these results with those obtained from an analysis using antibody preparations specifically directed toward the different casein fractions, IgE-reactive bands were identified as isoforms of kappa-casein. IgE-reactive protein was not present in neither bovine cheese, regardless of cheese-making technology and time ripening, nor milk from any other dairy animal, such as ewe, goat, and water buffalo. Chemical deglycosylation of protein bands immobilized onto nitrocellulose proved that the glycosidic moiety of bovine kappa-casein was principally involved in IgE recognition.

Immunochemical detection of formylated gamma(2)-casein in cheese.

An immunochemical approach has been developed to detect the use of formaldehyde as a bacteriostatic agent in dairy products. A synthetic peptide, reproducing the first five amino acid residues of the gamma(2)-casein sequence, was formylated to generate the novel haptenic structure, already well-recognized in formaldehyde-treated milk and arising out of molecular rearrangement after the addition of formaldehyde to the alpha-amino group of the histidine residue at the N terminus of gamma(2)-casein. A polyclonal antibodies preparation produced against the formylated peptide adduct proved to be a highly specific analytical tool for detecting the formylated adduct of gamma(2)-casein in formaldehyde-treated milk. Polyclonal antibodies obtained against the unmodified peptide were able to detect selectively residual native gamma(2)-casein in ripened cheese.