Rapid affinity purification of erythropoietin from biological samples using disposable monoliths.

Identification of post-translational modifications of proteins in biological samples often requires access to preanalytical purification and concentration methods. In the purification step high or low molecular weight substances can be removed by size exclusion filters, and high abundant proteins can be removed, or low abundant proteins can be enriched, by specific capturing tools. In this paper is described the experience and results obtained with a recently emerged and easy-to-use affinity purification kit for enrichment of the low amounts of EPO found in urine and plasma specimens. The kit can be used as a pre-step in the EPO doping control procedure, as an alternative to the commonly used ultrafiltration, for detecting aberrantly glycosylated isoforms. The commercially available affinity purification kit contains small disposable anti-EPO monolith columns (6 ?L volume, Ø7 mm, length 0.15 mm) together with all required buffers. A 24-channel vacuum manifold was used for simultaneous processing of samples. The column concentrated EPO from 20 mL urine down to 55 ?L eluate with a concentration factor of 240 times, while roughly 99.7% of non-relevant urine proteins were removed. The recoveries of Neorecormon (epoetin beta), and the EPO analogues Aranesp and Mircera applied to buffer were high, 76%, 67% and 57%, respectively. The recovery of endogenous EPO from human urine was 65%. High recoveries were also obtained when purifying human, mouse and equine EPO from serum, and human EPO from cerebrospinal fluid. Evaluation with the accredited EPO doping control method based on isoelectric focusing (IEF) showed that the affinity purification procedure did not change the isoform distribution for rhEPO, Aranesp, Mircera or endogenous EPO. The kit should be particularly useful for applications in which it is essential to avoid carry-over effects, a problem commonly encountered with conventional particle-based affinity columns. The encouraging results with EPO propose that similar affinity monoliths, with the appropriate antibodies, should constitute useful tools for general applications in sample preparation, not only for doping control of EPO and other hormones such as growth hormone and insulin but also for the study of post-translational modifications of other low abundance proteins in biological and clinical research, and for sample preparation prior to in vitro diagnostics.

Atypical EPO profiles in anti-doping analyses

Résumé : Erythropoietin (EPO) is a glycoprotein hormone endogenously produced by the kidney, whose main physiological role is the stimulation of erythropoiesis. Since the beginning of the nineties, recombinant human EPO (rhEPO), a potent anti-anaemia treatment drug, has been manufactured by pharmaceutical industries. However, the erythropoiesis stimulating power of rhEPO was rapidly misused by unscrupulous athletes in order to improve their performances in endurance sports. Endogenous EPO has the same amino-acid backbone as most of recombinant forms; the molecules however differ through their respective glycosylation patterns. This difference constitutes the basis of the usual EPO screening test (IEF) developed in 2000 and still currently used in all anti-doping laboratories of the world. Nowadays, 3 EPO generations have been commercialized. The fight against EPO abuse is a continuous challenge for anti-doping laboratories. The diversity of recombinant EPO forms and the continuous development of new ones considerably confuse the identification of EPO doping. Several facets of this fight were investigated in this work. One of the limiting aspects of doping agents screening is the availability of positive samples. Therefore, 2nd and 3rd generation EPOS, namely NESP and C.E.R.A., were injected to healthy subjects in the frame of pilot clinical studies. These latter allowed to review the current EPO identification criteria defined by the World Anti-Doping Agency (WADA) in the case of NESP and to validate and implement a new assay targeting C.E.R.A. in human serum. Both studies resulted in the determination of the respective detection windows of NESP and C.E.R.A. in biological fluids. Following that, Dynepo, a 1st generation EPO presenting similarities with the endogenous form, was also in the centre of a similar clinical study. Our work aimed to overcome the actual identification criteria, which are not adapted to Dynpeo, and to propose an alternative pattern classification method based on the discriminant analysis of IEF EPO profiles. This method might be validated for other EPO forms in the future. The detection window of this molecule was also determined. Under particular conditions, confounding effects can complicate the identification of EPO in biological matrices. For example, athletes having performed a strenuous physical effort can excrete modified isoforms of endogenous EPO, making it very similar to some recombinant forms. Such phenomena, called effort urines, were reproduced under controlled conditions and, after characterization of effort EPO, an urinary biochemical marker was proposed to unequivocally identify effort urines. It also happens that EPO analyses fail to detect endogenous levels of EPO. Such profiles were thoroughly investigated and potential causes identified. Natural reasons relying on urine properties and test specificity were underlined, but the possible addition of adulterant agents in urine samples was also considered. Therefore, a simple biochemical assay targeting the suspected substances was set up. Our work was based on the characterization of atypical EPO profiles from different origins. Therefore, 3 EPO molecules representing the 3 generations of the drug and 2 confounding effects confusing the results interpretation were studied. These studies resulted in tangible applications for the laboratory, the best example of which being the C.E.R.A. assay, but also in scientific findings allowing to improve our comprehension of EPO doping in sport.

Erythropoietin (EPO) immunoaffinity columns--a powerful tool for purifying EPO and its recombinant analogues.

The sample preparation method preceding the urinary erythropoietin (EPO) doping test is based on several concentration and ultrafiltration steps. In order to improve the quality of isoelectric focusing (IEF) gel results and therefore, the sensitivity of the EPO test, new sample preparation methods relying on affinity purification were recently proposed. This article focuses on the evaluation and validation of disposable immunoaffinity columns targeting both endogenous and recombinant EPO molecules in two World Anti-Doping Agency (WADA) accredited anti-doping laboratories. The use of the columns improved the resolution of the IEF profiles considerably when compared with the classical ultrafiltration method, and the columns' ability to ensure the isoform integrity of the endogenous and exogenous EPO molecules was confirmed. Immunoaffinity columns constitute therefore a potent and reliable tool for the preparation of urine samples and their use will significantly improve the sensitivity and specificity of the actual urinary EPO test.

Accelerated digestion for high-throughput proteomics analysis of whole bacterial proteomes.

In bottom-up proteomics, rapid and efficient protein digestion is crucial for data reliability. However, sample preparation remains one of the rate-limiting steps in proteomics workflows. In this study, we compared the conventional trypsin digestion procedure with two accelerated digestion protocols based on shorter reaction times and microwave-assisted digestion for the preparation of membrane-enriched protein fractions of the human pathogenic bacterium Staphylococcus aureus. Produced peptides were analyzed by Shotgun IPG-IEF, a methodology relying on separation of peptides by IPG-IEF before the conventional LC-MS/MS steps of shotgun proteomics. Data obtained on two LC-MS/MS platforms showed that accelerated digestion protocols, especially the one relying on microwave irradiation, enhanced the cleavage specificity of trypsin and thus improved the digestion efficiency especially for hydrophobic and membrane proteins. The combination of high-throughput proteomics with accelerated and efficient sample preparation should enhance the practicability of proteomics by reducing the time from sample collection to obtaining the results.

Comparative genomics of emerging pathogens in the Candida glabrata clade

BACKGROUND: Candida glabrata follows C. albicans as the second or third most prevalent cause of candidemia worldwide. These two pathogenic yeasts are distantly related, C. glabrata being part of the Nakaseomyces, a group more closely related to Saccharomyces cerevisiae. Although C. glabrata was thought to be the only pathogenic Nakaseomyces, two new pathogens have recently been described within this group: C. nivariensis and C. bracarensis. To gain insight into the genomic changes underlying the emergence of virulence, we sequenced the genomes of these two, and three other non-pathogenic Nakaseomyces, and compared them to other sequenced yeasts. RESULTS: Our results indicate that the two new pathogens are more closely related to the non-pathogenic N. delphensis than to C. glabrata. We uncover duplications and accelerated evolution that specifically affected genes in the lineage preceding the group containing N. delphensis and the three pathogens, which may provide clues to the higher propensity of this group to infect humans. Finally, the number of Epa-like adhesins is specifically enriched in the pathogens, particularly in C. glabrata. CONCLUSIONS: Remarkably, some features thought to be the result of adaptation of C. glabrata to a pathogenic lifestyle, are present throughout the Nakaseomyces, indicating these are rather ancient adaptations to other environments. Phylogeny suggests that human pathogenesis evolved several times, independently within the clade. The expansion of the EPA gene family in pathogens establishes an evolutionary link between adhesion and virulence phenotypes. Our analyses thus shed light onto the relationships between virulence and the recent genomic changes that occurred within the Nakaseomyces.

Suunnitelma Ethernet-pohjaisen TCP/IP-verkon toteuttamiseksi prosessitietoliikenteen tarpeisiin Helsingin Energiassa

Prosessitietoliikenneverkko toimii mm. energian tuotannon, siirron ja jakelun käytönvalvontajärjestelmien tiedonsiirron alustana. Tietoliikenneverkkojen rooli osana yrityksen operatiivisia prosesseja kasvaa järjestelmäuusintojen myötä ja järjestelmien tehokas hyödyntäminen vaatii luotettavat sekä nopeat tietoverkot. Uusittavien järjestelmien tietoliikenneratkaisut perustuvat pääsääntöisesti Ethernetpohjaiseen TCP/IP-verkkotekniikkaan. Tällä hetkellä prosessijärjestelmien tiedonsiirtoyhteydet kenttälaitteiden ja valvomojärjestelmien välillä on pääsääntöisesti toteutettu PCM-siirtojärjestelmällä tai kiinteän yhteyden modeemeilla. Käytössä oleva PCM-tekniikka on elinkaarensa loppupuolella ja tiedonsiirtonopeudet sekä liityntärajapinnat eivät täytä nykyaikaisten prosessijärjestelmien tiedonsiirto¬vaatimuksia. Työssä käytiin läpikeskeisimmät Ethernet-tekniikkaan ja TCP/IP-protokollaperheeseen liittyvät standardit ja verkon aktiivilaitteet. Vaatimusmäärittelyissä selvitettiin Helsingin Energian eri liiketoimintayksiköiden tarpeita sekä energia-alan lainsäädännön vaatimuksia prosessitietoliikenneverkolle. Tarpeiden ja vaatimusten pohjalta verrattiin kolmea eri vaihtoehtoista verkkotekniikkaa: kytkintekniikkaa, reititintekniikkaa ja MPLS-tekniikkaa. Vertailun perusteella valitulla verkkotekniikalla tehtiin suunnitelma avoimesta, valmistajasta riippumattomasta standardeihin perustuvasta yritystason Ethernet-pohjaisesta TCP/IP prosessiverkkoratkaisusta. Johtopäätöksissä pohditaan valitun ratkaisun käytettävyyttä, tietoturvaa sekä toimenpiteitä niiden kehittämiseksi.

Numerical analysis of including phase change material in a domestic hot water cylinder

The domestic hot water cylinder incorporates encapsulated pcm placed in 57 vertical pipes. The use of PCM increases the thermal energy storage capacity of the cylinder and allows the use of low cost electricity during low peak periods. After experimental validation the numerical model developed in the project will be used to optimize the distribution of the pcm inside the water tank.

Los elementos personales del Impuesto sobre la Renta de no Residentes

Las peculiaridades del gravamen de las rentas obtenidas por no residentes determinan que su regulación contemple la mayor parte de los elementos personales actualmente previstos por nuestra legislación fiscal. El objeto de este artículo es proceder a un análisis sistemático del contribuyente, el sustituto, el responsable, los retenedores y el representante del Impuesto sobre la Renta de no Residentes, así como de las relaciones entre ellos, teniendo en cuenta las modificaciones que en la Ley 41/1998, de 9 de diciembre, del Impuesto sobre la Renta de no Residentes, ha introducido la Ley 46/2002, de 18 de diciembre, de reforma parcial del Impuesto sobre la Renta de las Personas Físicas y de modificación de los Impuestos sobre Sociedades y sobre la Renta de no Residentes.

Thermal energy storage with phase change materials in building envelopes

Els materials de canvi de fase (PCM) han estat considerats per a l’emmagatzematge tèrmic en edificis des de 1980. Amb la inclusió dels PCM en plaques de guix, guix, formigó o altres materials que s’utilitzen per a cobrir les parets, l’emmagatzematge tèrmic pot ser part de les estructures fins i tot en edificis lleugers. Les noves tècniques de microencapsulació han obert moltes possibilitats en aplicacions per a edificis. El treball que es presenta és el desenvolupament d’un formigó innovador mesclat amb PCM microencapsulat, amb un punt de fusió de 26 oC i una entalpia de canvi de fase de 110 kJ/kg. El primer experiment va ser la inclusió del PCM microencapsulat dins del formigó i la construcció d’una caseta amb aquest nou formigó-PCM. Es va construir una segona caseta al costat de la primera amb les mateixes característiques i orientació però amb formigó convencional que serveix com a referència. Durant els anys 2005 i 2006 es va analitzar el comportament d’ambdues casetes i més tard es va edificar un mur Trombe a la paret sud de totes dues per investigar la seva influència durant la tardor i l’hivern.

Impact of extracardiac findings during cardiac MR on patient management and outcome.

BACKGROUND: Cardiac magnetic resonance (CMR) is increasingly used to assess heart diseases. Relevant non-cardiac diseases may also be incidentally found on CMR images. The aim of this study was to determine the prevalence and nature of incidental extra-cardiac findings (IEF) and their clinical impact in non-selected patients referred for CMR. MATERIAL/METHODS: MR images of 762 consecutive patients (515 men, age: 56±18 years) referred for CMR were prospectively interpreted by 2 radiologists blinded for any previous imaging study. IEFs were classified as major when requiring treatment, follow-up, or further investigation. Clinical follow-up was performed by checking hospital information records and by calling referring physicians. The 2 endpoints were: 1) non-cardiac death and new treatment related to major IEFs, and 2) hospitalization related to major IEFs during follow-up. RESULTS: Major IEFs were proven in 129 patients (18.6% of the study population), 14% of those being unknown before CMR. During 15±6 month follow-up, treatment of confirmed major IEFs was initiated in 1.4%, and no non-cardiac deaths occurred. Hospitalization occurred in 8 patients (1.0% of the study population) with confirmed major IEFs and none occurred in the remaining 110 patients with unconfirmed/unexplored major IEFs (p<0.001). CONCLUSIONS: Screening for major IEFs in a population referred for routine CMR changed management in 1.4% of patients. Major IEFs unknown before CMR but without further exploration, however, carried a favorable prognosis over a follow-up period of 15 months.

Determinação teórica das conformações mais estáveis da trealose em solução aquosa

In this work the most abundant trehalose conformers for the isolated molecule as well as for the water solvated system are selected. The theoretical tecniques employed are ab initio calculations in the gas phase and in aqueous solution using the PCM model. A conformational map is built for the glycosidic angles (phi and psi) and the search for the most abundant structures is explained. The final structures are validated by the agreement found between experimental and theoretical values for ³J H,C along the glycosidic linkage.

Contribuição do fomento do órgão florestal de Minas Gerais na lucratividade e na redução de riscos para produtores rurais

O objetivo deste estudo foi realizar a análise financeira e a simulação de risco de investimento em projetos de reflorestamentos com eucalipto visando à produção de carvão e madeira para celulose, com e sem fomento florestal do Instituto Estadual de Florestas (IEF/MG). A análise financeira foi realizada mediante os métodos de avaliação de projetos florestais, e para a análise de risco utilizou-se a técnica de simulação de Monte Carlo, por meio do programa @RISK. Entre os projetos testados, aquele visando à produção de carvão com fomento do IEF obteve melhor desempenho financeiro. Verificou-se que os custos de colheita, transporte e carvoejamento são, juntos, responsáveis pela maior parcela do custo total dos projetos.A simulação da análise de risco indicou que as variáveis que afetaram o Valor Presente Líquido (VPL), nos projetos cuja produção final era o carvão, na sua ordem de importância (R), foram: preço dos produtos, produtividade da floresta, taxa de juros, custo de colheita e custo de implantação. Já para a produção de madeira a ordem de importância foi alterada quando se analisou o custo de colheita e de implantação, sendo este último mais influente, de forma negativa, sobre o VPL do Projeto sem fomento florestal.

Tolerance of eucalypt (Eucalyptus spp.) seedlings to imazapyr in nutritive solution

Imazapyr has presented excellent results in controlling coppices in stand reforms of eucalypt forests, despite differences in the efficacy levels. To find out whether these different responses are caused by the genetic variability of the cultivated materials, two experiments were carried out under greenhouse conditions with different imazapyr doses in a hydroponic system in plastic vases containing 2,500 mL solution. The clones IEF-1 (Eucalyptus grandis x Eucalyptus sp. hybrid), GE 463 (E. urophylla x E. grandis), and MN 445 (E. grandis x Eucalyptus sp. hybrid) were used in the first assay, and IEF-1, IEF2 (E. grandis x E. urophylla) x Eucalyptus sp. hybrid) and the clones 129 and 7182 (E. grandis x Eucalyptus sp. hybrids) in the second assay. Thirty days after transplanting the seedlings to a nutritive solution, imazapyr was applied to the solution at doses of 0.00, 0.05, 0.10, 0.20, 0.40, 0.80, 1.60 and 3.20 muL L-1. Clone GE 463 proved to be more tolerant to imazapyr than clones IEF-1 and MN 445 in the first assay; however, in the second, clone 7182 was the most tolerant. Thus, doses should also be differentiated when controlling coppices, according to the cultivated clone.

Cytokines produced by susceptible and resistant mice in the course of Paracoccidioides brasiliensis infection

Paracoccidioidomycosis (PCM) is the most prevalent deep mycosis in Latin America and presents a wide spectrum of clinical manifestations. We established a genetically controlled murine model of PCM, where A/Sn mice develop an infection which mimics the benign disease (immune responses which favor cellular immunity) and B10.A animals present the progressive disseminated form of PCM (preferential activation of B cells and impairment of cellular immune responses). To understand the immunoregulatory phenomena associated with resistance and susceptibility in experimental PCM, A/Sn and B10.A mice were studied regarding antigen-elicited secretion of monokines (TNF-a and TGF-ß) and type-1 (IL-2 and IFN-g) and type-2 (IL-4,5,10) cytokines. Total lymph node cells from resistant mice infected ip with P. brasiliensis produced early and sustained levels of IFN-g and IL-2; type-2 cytokines (IL-4 and IL-5) started to appear 8 weeks after infection. In contrast, susceptible mice produced low levels of IFN-g concomitant with significant levels of IL-5 and IL-10 early in the infection. In the chronic phase of the disease, susceptible animals presented a transitory secretion of IL-2, and IL-4. In the pulmonary infection IL-4, IL-5 and IL-10 were preferentially detected in the lung cells washings of susceptible animals. After in vitro challenge with fungal antigens, normal peritoneal macrophages from B10.A mice secreted high levels of TGF-ß and low levels of TNF-a. In contrast, macrophages from A/Sn animals released high levels of TNF-a associated with a small production of TGF-ß. The in vivo depletion of IFN-g not only abrogated the resistance of A/Sn mice but also diminished the relative resistance of B10.A animals. The in vivo depletion of IL-4 did not alter the disease outcome, whereas administration of rIL-12 significantly enhanced resistance in susceptible animals. Taken together, these results suggest that an early secretion of high levels of TNF-a and IFN-g followed by a sustained secretion of IL-2 and IFN-g plays a dominant role in the resistance mechanisms to P. brasiliensis infection. In contrast, an early and ephemeral secretion of low levels of TNF-a and IFN-g associated with production of IL-5, IL-10 and TGF-ß characterizes the progressive disease of susceptible animals.

Differential response to gepirone but not to chlordiazepoxide in malnourished rats subjected to learned helplessness

The learned helplessness (LH) paradigm is characterized by learning deficits resulting from inescapable events. The aims of the present study were to determine if protein-calorie malnutrition (PCM) alters learning deficits induced by LH and if the neurochemical changes induced by malnutrition alter the reactivity to treatment with GABA-ergic and serotonergic drugs during LH. Well-nourished (W) and PCM Wistar rats (61 days old) were exposed or not to inescapable shocks (IS) and treated with gepirone (GEP, 0.0-7.5 mg/kg, intraperitoneally, N = 128) or chlordiazepoxide (0.0-7.5 mg/kg, intraperitoneally, N = 128) 72 h later, 30 min before the test session (30 trials of escape learning). The results showed that rats exposed to IS had higher escape latency than non-exposed rats (12.6 ± 2.2 vs 4.4 ± 0.8 s) and that malnutrition increased learning impairment produced by LH. GEP increased the escape latency of W animals exposed or non-exposed to IS, but did not affect the response of PCM animals, while chlordiazepoxide reduced the escape deficit of both W and PCM rats. The data suggest that PCM animals were more sensitive to the impairment produced by LH and that PCM led to neurochemical changes in the serotonergic system, resulting in hyporeactivity to the anxiogenic effects of GEP in the LH paradigm.