Conditioning of desert sandy soil and investigation of the ameliorative effects of poultry manure and bentonite treatment rate on plant growth.

Soil is the base of any ecosystem since it conserves nutrients and water for plant roots including agriculture and plantations. In dry and semi-arid places across the world, including the UAE, sandy soils are common. Their fertility is extremely low, and production is hampered by a number of agronomic challenges. Soil conditioner sources like bentonite and chicken manure might be used to improve the poor sandy soil attributes and hence boost soil productivity. From November 2019 to March 2020, an experiment was conducted to investigate the growth rates of Bougainvillea following bentonite and chicken manure amendments to sandy soil taken from Lehbab, Dubai. Bougainvillea was evaluated for its plant height (cm), max length of primary branch (cm), the number of leaves per plant, number of secondary branches, shoot weight (g), root length (cm), root weight (g), root/shoot ratio, chlorophyll contents, and chlorophyll a* and b*. In this experiment, a complete randomized design (CRD) with five treatments was used (10 replications per treatment). According to the findings, bentonite and chicken manure additions considerably influence the productive properties of sandy soil, as indicated by Bougainvillea growth. Additionally, the research suggests that Bougainvillea may be efficiently planted with 10% bentonite and 15% chicken manure applied to sandy soil, resulting in the healthiest plants compared to other amendments.

Characterization of 3 different types of aquaporins in Carcinus maenas and their potential role in osmoregulation.

Intertidal crustaceans like Carcinus maenas shift between an osmoconforming and osmoregulating state when inhabiting full-strength seawater and dilute environments, respectively. While the bodily fluids and environment of marine osmoconformers are approximately isosmotic, osmoregulating crabs inhabiting dilute environments maintain their bodily fluid osmolality above that of their environment by actively absorbing and retaining osmolytes (e.g., Na+, Cl-, urea) while eliminating excess water. Few studies have investigated the role of aquaporins (AQPs) in the osmoregulatory organs of crustaceans, especially within brachyuran species. In the current study, three different aquaporins were identified within a transcriptome of C. maenas, including a classical AQP (CmAQP1), an aquaglyceroporin (CmGLP1), and a big-brain protein (CmBIB1), all of which are expressed in the gills and the antennal glands. Functional expression of these aquaporins confirmed water transport capabilities for CmAQP1, CmGLP1, but not for CmBIB1, while CmGLP1 also transported urea. Higher relative CmAQP1 mRNA expression within tissues of osmoconforming crabs suggests the apical/sub-apically localized channel attenuates osmotic gradients created by non-osmoregulatory processes while its downregulation in dilute media reduces the water permeability of tissues to facilitate osmoregulation. Although hemolymph urea concentrations rose upon exposure to brackish water, urea was not detected in the final urine. Due to its urea-transport capabilities, CmGLP1 is hypothesized to be involved in a urea retention mechanism believed to be involved in the production of diluted urine. Overall, these results suggest that AQPs are involved in osmoregulation and provide a basis for future mechanistic studies investigating the role of AQPs in volume regulation in crustaceans.

Efficacy of octreotide in bleeding recurrence from small bowel angioectasia: A comparative study.

INTRODUCTION: Fifty percent of small bowel bleeding is caused by angioectasia and the rebleeding rate due to small bowel angioectasia (SBA) is 80%. Its endoscopic treatment is difficult. Beneficial effects of octreotide on gastrointestinal angioectasia have been described, but no studies have reported its efficacy in SBA. AIM: Our aim was to investigate the effectiveness of octreotide in the prevention of rebleeding due to SBA. MATERIAL AND METHODS: Sixteen patients with bleeding caused by SBA were assigned to treatment with octreotide 100 µg/24 h SC, for at least 6 months, and compared with a non-treatment group of 36 patients. The primary outcome was the rebleeding rate, and the secondary outcomes were the number of hospital readmissions, bleeding-related death, and adverse effects. RESULTS: Octreotide was administered for 10.5 ± 8.4 months. Follow-up was 12.9 ± 17.3 months and 15.3 ± 17.7 months, in the treatment and non-treatment groups, respectively (p = 0.09). At the end of follow-up, 4 (25%) treatment group patients and 26 (72.2%) non-treatment group patients presented with rebleeding (p = 0.002). In the treatment group and non-treatment group, the cumulative probability of remaining rebleeding-free at one year was 79% vs 44.2%, and 79% vs 34.6% at 2 years, respectively (p = 0.05). Through the multiple logistic regression analysis, treatment was the protective variable. Six patients presented with adverse events. One of those patients (6.25%) had a major adverse event. CONCLUSIONS: Our results suggest that treatment with octreotide could be efficacious in the prevention of rebleeding due to SBA.

Quantifying citrate-enhanced phosphate root uptake using microdialysis.

AIMS: Organic acid exudation by plant roots is thought to promote phosphate (P) solubilisation and bioavailability in soils with poorly available nutrients. Here we describe a new combined experimental (microdialysis) and modelling approach to quantify citrate-enhanced P desorption and its importance for root P uptake. METHODS: To mimic the rhizosphere, microdialysis probes were placed in soil and perfused with citrate solutions (0.1, 1.0 and 10 mM) and the amount of P recovered from soil used to quantify rhizosphere P availability. Parameters in a mathematical model describing probe P uptake, citrate exudation, P movement and citrate-enhanced desorption were fit to the experimental data. These parameters were used in a model of a root which exuded citrate and absorbed P. The importance of soil citrate-P mobilisation for root P uptake was then quantified using this model. RESULTS: A plant needs to exude citrate at a rate of 0.73 µmol cm-1 of root h-1 to see a significant increase in P absorption. Microdialysis probes with citrate in the perfusate were shown to absorb similar quantities of P to an exuding root. CONCLUSION: A single root exuding citrate at a typical rate (4.3 × 10-5 µmol m-1 of root h-1) did not contribute significantly to P uptake. Microdialysis probes show promise for measuring rhizosphere processes when calibration experiments and mathematical modelling are used to decouple microdialysis and rhizosphere mechanisms.

Robotic-Assisted Partial Nephrectomy: Techniques to Improve Clinical Outcomes.

PURPOSE OF REVIEW: To summarize current options available for robot-assisted partial nephrectomy RECENT FINDINGS: Partial nephrectomy (PN) is a standard treatment option for management of cT1 renal masses. It may be carried out by multiple approaches. Robot-assisted (RA) PN is one such option. The goal of treatment is both correct oncological (negative surgical margins) and functional (preservation of sufficient amount of renal parenchyma of the operated kidney) outcome. Appropriate outcomes depend on multiple factors. There are many, but among others tumor characteristics (size, location, i.e., tumor complexity), patient baseline renal function, patient comorbidities, and performance status etc. Based on all these, the surgeon adapts the intervention for each mass/patient by preoperative planning, absence/use/duration of warm or cold ischemia, perioperative imaging, resection technique adapted to tumor location and depth of invasion, use of hemostatics, type and degree of renal parenchymal closure and others details. Nephroprotective agents have not shown efficacy so far. It should not be forgotten that surgeon's experience plays a key role in the achievement of good results. Although multiple factors have a role in the RA partial nephrectomy, surgeon experience and adaptation of technique of intervention have the crucial role in the achievement of both functional and oncological results.

Application of Bayesian statistics to estimate nitrous oxide emission factors of three nitrogen fertilisers on UK grasslands.

Trapezoidal integration by linear interpolation of data points is by far the most commonly used method of cumulative flux calculations of nitrous oxide (N2O) in studies that use flux chambers; however, this method is incapable of providing accurate uncertainty estimates. A Bayesian approach was used to calculate N2O emission factors (EFs) and their associated uncertainties from flux chamber measurements made after the application of nitrogen fertilisers, in the form of ammonium nitrate (AN), urea (Ur) and urea treated with Agrotain® urease inhibitor (UI) at four grassland sites in the UK. The comparison between the cumulative fluxes estimated using the Bayesian and linear interpolation methods were broadly similar (R2 = 0.79); however, the Bayesian method was capable of providing realistic uncertainties when a limited number of data points is available. The study reports mean EF values (and 95% confidence intervals) of 0.60 ±â€¯0.63, 0.29 ±â€¯0.22 and 0.26 ±â€¯0.17% of applied N emitted as N2O for the AN, Ur and UI treatments, respectively. There was no significant difference between N2O emissions from the Ur and UI treatments. In the case of the automatic chamber data collected at one site in this study, the data did not fit the log-normal model, implying that more complex models may be needed, particularly for measurement data with high temporal resolution.

Dinámica Pulmonar de Estructuras Anatómicas de Interés en Imágenes 4DCT / Pulmonary Dynamics of Anatomical Structures of Interest in 4DCT Images

Resumen: El presente trabajo muestra una aplicación del algoritmo Chan-Vese para la segmentación semi-automática de estructuras anatómicas de interés (pulmones y tumor pulmonar) en imágenes de 4DCT de tórax, así como su reconstrucción tridimensional. La segmentación y reconstrucción se realizó en 10 imágenes de TAC, las cuales conforman un ciclo inspiración-espiración. Se calculó el desplazamiento máximo para el caso del tumor pulmonar usando las reconstrucciones del inicio de la inspiración, el inicio de la espiración, y la información del voxel. El método propuesto logra segmentar de manera apropiada las estructuras estudiadas sin importar su tamaño y forma. La reconstrucción tridimensional nos permite visualizar la dinámica de las estructuras de interés a lo largo del ciclo respiratorio. En un futuro se espera poder contar con mayor evidencia del buen desempeño del método propuesto y contar con la retroalimentación del experto clínico, ya que el conocimiento de características de estructuras anatómicas, como su dimensión y posición espacial, ayuda en la planificación de tratamientos de Radioterapia (RT), logrando optimizar las dosis de radiación hacia las células cancerosas y minimizarla en órganos sanos. Por lo tanto, la información encontrada en este trabajo puede resultar de interés para la planificación de tratamientos de RT.

Abstract: This paper presents an application of the Chan-Vese algorithm for a semi-automatic segmentation of anatomical structures of interest (lungs and lung tumor) in thorax 4DCT images, as well as its threedimensional reconstruction. Segmentations and reconstructions were performed in 10 CT images, which conform an inspiration-expiration cycle. The maximum displacement of the lung tumor was calculated using the reconstructions of the beginning of inspiration, beginning of expiration, and the voxel size information. The proposed method was able to succesfully segment the studied structures regardless of their size and shape. The threedimensional reconstruction allow us to visualize the dynamics of the structures of interest throughout the respiratory cycle. In the near future, we are expecting to be able to have more evidence of the good performance of the proposed segmentation approach, and to have feedback from a clinical expert, giving the fact that the knowledge of anatomical structures characteristics, such as their size and spatial location, may help in the planning of radiotherapy treatments (RT), optimizing the radiation dose to cancer cells and minimizing it in healthy organs. Therefore, the information found in this work may be of interest for the planning of RT treatments.

Sistema de Apoyo para la Cuantificación Semiautomática de Fibrosis Pulmonar en Imágenes de TAC / Support System for Semiautomatic Quantification of Pulmonary Fibrosis in CT Images

Resumen: En este trabajo se presenta un método para calcular los niveles de fibrosis pulmonar en imágenes de tomografía axial computarizada. Se utilizó un algoritmo de segmentación semiautomática basado en el método de Chan-Vese. El método mostró similitudes de forma cualitativa en la región de la fibrosis con respecto al experto clínico. Sin embargo es necesario validar los resultados con una base de datos mayor. El método propuesto aproxima un porcentaje de fibrosis de forma fácil para apoyar su implementación en la práctica clínica minimizando la subjetividad del experto médico y generando una estimación cuantitativa de la región de fibrosis.

Abstract: A method to estimate the pulmonary fibrosis in computed tomography (CT) imaging is presented. A semi-automatic segmentation algorithm based on the Chan-Vese method was used. The proposed method shows a similar fibrosis región with respect to clinical expert. However, the results need to be validated in a bigger data base. The proposed method approximates a fibrosis percentage that allows to achieve this procedure easily in order to support its implementation in the clinical practice minimizing the clinical expert subjectivity and generating a quantitative estimation of fibrosis region.

[Ischemia is not an independent predictive factor of chronic renal failure after partial nephrectomy in a solitary kidney in patients without pre-operative renal insufficiency]. / L'ischémie a un impact limité sur la fonction rénale après néphrectomie partielle sur rein unique chez les patients sans insuffisance rénale préopératoire.

OBJECTIVE: To assess the influence of vascular clamping and ischemia time on long-term post-operative renal function following partial nephrectomy (PN) for cancer in a solitary kidney. PATIENTS AND METHODS: This is a retrospective study including 259 patients managed by PN between 1979 and 2010 in 13 centers. Clamping use, technique choice (pedicular or parenchymal clamping), ischemia time, and peri-operative data were collected. Pre-operative and last follow-up glomerular filtration rates were compared. A multivariate analysis using a Cox model was performed to assess the impact of ischemia on post-operative chronic renal failure risk. RESULTS: Mean tumor size was 4.0±2.3cm and mean pre-operative glomerular filtration rate was 60.8±18.9mL/min. One hundred and six patients were managed with warm ischemia (40.9%) and 53 patients with cold ischemia (20.5%). Thirty patients (11.6%) have had a chronic kidney disease. In multivariate analysis, neither vascular clamping (P=0.44) nor warm ischemia time (P=0.1) were associated with a pejorative evolution of renal function. Pre-operative glomerular filtration rate (P<0.0001) and blood loss volume (P=0.02) were significant independent predictive factors of long-term renal failure. CONCLUSION: Renal function following PN in a solitary kidney seems to depend on non-reversible factors such as pre-operative glomerular filtration rate. Our findings minimize the role of vascular clamping and ischemia time, which were not significantly associated with chronic renal failure risk in our study. LEVEL OF EVIDENCE: 5.

Predictive factors of chronic kidney disease stage V after partial nephrectomy in a solitary kidney: a multi-institutional study.

OBJECTIVE: Partial Nephrectomy (PN) in a solitary kidney is at risk of chronic kidney disease (CKD) stage V and/or haemodialysis (HD). Our objective was to determine predictive factors of CKD stage V in this population. MATERIAL & METHODS: Data from 300 patients were retrospectively collected from 16 tertiary centres. Clinical and operative parameters, tumor characteristics and renal function before surgery were analyzed. Patients with and without CKD stage V (defined as MDRD<1 5 ml/min) were compared using χ2 and Student-t tests for qualitative and quantitative variables, respectively. Predictive factors of CKD stage V were evaluated with a multivariable analysis using a Cox regression model. RESULTS: Median age and BMI were 63 years old and 26 kg/m², respectively. Most of the patients (65%) were male with an anatomic solitary kidney (88.3%). Median tumor size was 4 cm and 98% were malignant tumors. Median operative time, blood loss and clamping time were 180 min, 350 ml and 20 min respectively. Renal cooling was used in 19.3% and clamping of the pedicle was performed in 61.6%. Twenty five patients (8.5%) presented post operative CKD stage V at last follow-up and 18 underwent HD (6%) post-operatively because of acute renal insufficiency. There was no difference between CKD stage V and non CKD stage V patients concerning Charlson index, operative time (180 min vs 179 min, p = 0.39), blood loss (475 ml vs 350 ml, p = 0.51), use of renal cooling and type of clamping. Patients with CKD stage V were older (70 vs 63 years old, p = 0.005), had a lower baseline renal function (clearance MDRD 41 vs. 62 ml/min, p<0.0001) and an increased tumor size (p = 0.02). Complications occurred in 91 patients (30%) with 16% of minor (Clavien 1-2) and 14% of major (Clavien > 2) complications, respectively. In multivariable analysis, baseline MDRD, BMI, and the occurrence of a minor complication were independent predictive factors of post operative CKD stage V. CONCLUSION: PN in a solitary kidney is at risk of post-operative CKD stage V and HD. Pre-operative altered renal function and post operative complications are the main predictive factors of permanent CKD stage V.

Mast cell/IL-4 control of Francisella tularensis replication and host cell death is associated with increased ATP production and phagosomal acidification.

Mast cells are now recognized as effective modulators of innate immunity. We recently reported that mast cells and secreted interleukin-4 (IL-4) effectively control intramacrophage replication of Francisella tularensis Live Vaccine Strain (LVS), and that mice deficient in mast cells or IL-4 receptor (IL-4R(-/-)) exhibit greater susceptibility to pulmonary challenge. In this study, we further evaluated the mechanism(s) by which mast cells/IL-4 control intramacrophage bacterial replication and host cell death, and found that IL-4R(-/-) mice exhibited significantly greater induction of active caspase-3 within lung macrophages than wild-type animals following intranasal challenge with either LVS or the human virulent type A strain SCHU S4. Treatment of LVS-infected bone-marrow-derived macrophages with a pancaspase inhibitor (zVAD) did not alter bacterial replication, but minimized active caspase-3 and other markers (Annexin V and propidium iodide) of cell death, whereas treatment with both rIL-4 and zVAD resulted in concomitant reduction of both parameters, suggesting that inhibition of bacterial replication by IL-4 was independent of caspase activation. Interestingly, IL-4-treated infected macrophages exhibited significantly increased ATP production and phagolysosomal acidification, as well as enhanced mannose receptor upregulation and increased internalization with acidification, which correlated with observations in mast cell-macrophage co-cultures, with resultant decreases in F. tularensis replication.

Elastic registration based on particle filter in radiotherapy images with brain deformations.

This paper presents the evaluation of the accuracy of an elastic registration algorithm, based on the particle filter and an optical flow process. The algorithm is applied in brain CT and MRI simulated image datasets, and MRI images from a real clinical radiotherapy case. To validate registration accuracy, standard indices for registration accuracy assessment were calculated: the dice similarity coefficient (DICE), the average symmetric distance (ASD) and the maximal distance between pixels (Dmax). The results showed that this registration process has good accuracy, both qualitatively and quantitatively, suggesting that this method may be considered as a good new option for radiotherapy applications like patient's follow up treatment.

Determination of (fluoro)quinolone antibiotic residues in pig kidney using liquid chromatography-tandem mass spectrometry. I. Laboratory-validated method.

A new LC-MS/MS method has been developed for the multiresidue determination of 11 (fluoro)quinolone antibiotics (FQs), including acidic and amphoteric species, around their maximum residue level (MRL) in pig kidney. The procedure involves a common sample preparation by solid-phase extraction on disposable extraction cartridges followed by a fast reversed-phase liquid chromatography-tandem mass spectrometry analysis. The method was validated according to the Commission Decision 2002/657/CE. The accuracy of the method was satisfactory with recoveries included in the interval 80-100%. The precision results showed mean repeatability and reproducibility coefficients of 7.4% and 11.8%, respectively. Limits of quantification much lower than the MRLs could be obtained.

Determination of (fluoro)quinolone antibiotic residues in pig kidney using liquid chromatography-tandem mass spectrometry. Part II: intercomparison exercise.

A recently in-house validated method for the liquid chromatography-tandem mass spectrometry (LC-MS/MS) determination of eleven (fluoro)quinolone antibiotics (FQs) in pig kidney has been fully validated through an intercomparison exercise. This ring trial involved eight European laboratories and was based on the Commission Decision 2002/657/CE for validation of method and on the IUPAC protocol for method-performances studies. The laboratories data were submitted to a one-way analysis of variance. Satisfactory results were obtained for each FQ with regards to within- and between-laboratory reproducibility and accuracy. The method was validated for the simultaneous qualitative and quantitative determination of the eleven FQs in pig kidney around their maximum residue limit (MRL) as defined in the European Council Regulation 2377/90/EEC.

Study of the feasibility of using a pellicular anion-exchange column for separation of transferrin isoforms in human serum by HPLC with UV detection.

A method has been optimised for the separation of glycoforms of human serum transferrin, using a high-performance pellicular anion-exchange chromatographic column. The effect of the eluent pH and of the column temperature on the separation of transferrin glycoforms was studied using a standard solution of commercially available human serum transferrin. An HPLC system equipped with an ultraviolet detector was used for the analysis. No immunoassay was used after the anion-exchange chromatographic separation of the glycoforms, in contrast with most currently used methods. The method was applied to the separation and quantification of transferrin glycoforms in serum from a healthy, non-pregnant woman, after saturation of transferrin with iron and further precipitation of lipoproteins. The whole chromatographic run, including re-equilibration of the column, took 35 min.

Screening test to detect meat adulteration through the determination of hemoglobin by cation exchange chromatography with diode array detection.

A simple sample preparation procedure, consisting of an extraction step with Milli-Q water as extraction solvent for hemoglobins from meat samples, followed by a filtration step with a cellulose acetate filter, is applied. After cation exchange chromatographic separation and diode array detection, different peak patterns for extracted hemoglobins of cow, lamb or pork meat are obtained. Other heme-group containing proteins like myoglobin or cytochrome C, which could be also detected with diode array detection at 416 nm, are chromatographically separated from the hemoglobins. By the use of these characteristic peak patterns, the species of the meat can be specified permitting the qualitative assessment of meat adulteration with the proposed screening method.

Validation of a liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of 11 (fluoro)quinolone antibiotics in swine kidney.

A LC-MS-MS method has been validated for the simultaneous quantification of 11 (fluoro)quinolone antibiotics at the maximum residue level (MRL) in swine kidney. The studied compounds were danofloxacine, cinoxacine, ciprofloxacine, noxacine, enrofloxacine, flumequine, marbofloxacine, nalidixic acid, norfloxacine, ofloxacine and oxolinic acid. The method involves solid-phase extraction of these compounds followed by LC-MS-MS analysis using an electrospray ionisation interface. Limits of quantification < or = 50 microg/kg could be obtained in swine kidney, much lower than every MRL. The validation is discussed. This work was carried out in order to support the European Union policy on consumer health

Development of new analytical methods for selenium speciation in selenium-enriched yeast material.

A sequential extraction allowing the discrimination of water-soluble and non-soluble selenium fractions has been developed to evaluate the availability of selenium (Se) in an Se-enriched yeast candidate reference material. The fractionation of selenium-containing compounds in the extracts was achieved on preparative grade 200 Superdex 75 and columns. It showed that water-soluble selenium is present in several fractions with a large mass distribution. Low-molecular- (< or = 10,000) and high-molecular-mass selenocompounds (range 10,000-100,000) were considered separately for further experiments. The analytical approach for low-molecular-mass selenocompounds was based onanion-exchange HPLC with on-line inductively coupled plasma (ICP) MS for quantitative analysis. Selenocystine, selenomethionine, selenite and selenate were quantified in the fractions isolated in preparative chromatography. The study revealed the existence of various unidentified Se species in yeast material. The Se-containing proteins in the yeast material have been further separated and selenium quantified by the combination of gel electrophoresis and electrothermal vaporization-ICP-MS. This new approach allows the separation of the proteins with high resolution by sodium dodecylsulfate-polyacrylamide gel electrophoresis and the sensitive determination of selenium in the protein bands.

Identification, characterization and determination of metal-binding proteins by liquid chromatography. A review.

The use of liquid chromatography in the separation and determination of metal-binding proteins is reviewed. Advantages and drawbacks of different chromatographic techniques based on various principles: size exclusion, ion exchange (cationic and ionic), reversed phase and affinity, are presented and discussed. The topic "metal-binding proteins" is considered and presented from two different points of view. The first one regards metal speciation in biological samples (serum and blood). In metal speciation studies, the exact identity of the protein to which the metal is bound often remains unknown. The second point of view is that, frequently, the interest of analyzing metal-binding proteins is not related anymore to the metallic fraction of the protein, but to other chemical structures attached to the protein, such as carbohydrates, which indirectly determine how good the function of the protein is. In this review, special attention is paid to studies dealing with the glycosylation of transferrin, and with the glycated isoform of haemoglobin.

Identification and quantification of major bovine milk proteins by liquid chromatography.

In the field of food quality, bovine milk products are of particular interest due to the social and economic importance of the dairy products market. However, the risk of fraudulent manipulation is high in this area, for instance, replacing milk powder by whey is very interesting from an economic point of view. Therefore, there is a need to have suitable analytical methods available for the determination of all milk components, which is currently not the case, especially for the main proteins. The detection of potential manipulations requires then a clear analytical characterisation of each type of bovine milk, what constitutes the goal of this work. The separation of the major milk proteinic components has been carried out by ion-pair reversed-phase HPLC with photodiode array detection, using a C4 column. The overall optimisation has been achieved using a statistical experimental design procedure. The identification of each protein was ascertained using retention times, peak area ratios and second derivative UV spectra. Quantification was based on calibration curves drawn using purified proteins. Major sources of uncertainty were identified and the full uncertainty budget was established. The procedure was initially developed using the skimmed milk powder certified reference material CRM 063R and then applied to various types of commercial milks as well as to raw milk. The method is able to separate and quantify the seven major proteins (K-casein, alphas2-casein, alphas1-casein, beta-casein, alpha-lactalbumin, beta-lactoglobulin B and beta-lactoglobulin A) in one run and also to provide precise determinations of the total protein concentration. These are important results towards the further development of a reference method for major proteins in milk. In addition, the use of a certified material reference is suggested in order to make comparisons of method performances possible.