HPLC-FLD methods to quantify chloroaluminum phthalocyanine in nanoparticles, plasma and tissue: application in pharmacokinetic and biodistribution studies

Analytical and bioanalytical methods of high-performance liquid chromatography with fluorescence detection (HPLC-FLD) were developed and validated for the determination of chloroaluminum phthalocyanine in different formulations of polymeric nanocapsules, plasma and livers of mice. Plasma and homogenized liver samples were extracted with ethyl acetate, and zinc phthalocyanine was used as internal standard. The results indicated that the methods were linear and selective for all matrices studied. Analysis of accuracy and precision showed adequate values, with variations lower than 10% in biological samples and lower than 2% in analytical samples. The recoveries were as high as 96% and 99% in the plasma and livers, respectively. The quantification limit of the analytical method was 1.12 ng/ml, and the limits of quantification of the bioanalytical method were 15 ng/ml and 75 ng/g for plasma and liver samples, respectively. The bioanalytical method developed was sensitive in the ranges of 15-100 ng/ml in plasma and 75-500 ng/g in liver samples and was applied to studies of biodistribution and pharmacokinetics of AlClPc. (C) 2011 Elsevier B.V. All rights reserved.

Föld és ég. Az ember és a természeti környezet közötti kapcsolat jellemzése az adatnégyzetek módszerével

E cikk végső soron két tudományos állítás igazolását célozza: (1) létrehozható egy olyan, kétdimenziós struktúra, amely képes információveszteség nélkül négydimenziós humán–környezet problémákat megjeleníteni (az adatnégyzetek módszere); valamint (2) a boldogság/környezetterhelés hányados (az égi lábnyom) stratégiai jelentőségű a fenntarthatóságot célzó vitákban. A tanulmány szerkezete a következő: A második részben bemutatjuk az adatnégyzetek módszerének általános struktúráját és matematikai hátterét, valamint ismertetjük azt a négy mutatót, amelyek rendszerünkben az ember és a környezet közötti kapcsolatot mérni hivatottak. A harmadik rész az adatnégyzetek között lehetséges nem-szimmetrikus kapcsolatokat mutatja be, ami esetünkben különféle népességnagyság, illetve a boldogság monetizáltságának eltérő mértéke esetén fordulhat elő. A negyedik részben bevezetjük és jellemezzük az égi lábnyom fogalmát. Az ötödik részben e fogalom további jelentőségre tesz szert, ahol a boldogság elérésének modelljét mutatjuk be (a boldogság útjai). A hatodik rész a materiális (illetve nem materiális), valamint a monetáris (illetve nem monetáris) átváltások lehetőségét ismerteti. Végül esettanulmányunk Magyarország 2005-ös adatnégyzetét mutatja be, összevetve azt néhány hasonló ország adatnégyzetével.

Földrajzi információs rendszerek és a Digitális Föld

Napjainkra a társadalmi–gazdasági fejlődés elérte azt a szintet, amikor a rendelkezésre álló információ tömege olyan hatalmassá vált, melyet csak rendszerezett formában tud az ember hasznosítani. A társadalom és a tudomány fejlődésének információ igénye és az egyén tudásszomja is csak akkor elégülhet ki, ha a határtalannak tűnő adattömeget földrajzilag határokhoz kötjük, és így vizsgáljuk az adattartalmakat egyéb dimenzióit. Ez azt jelenti, hogy digitalizálni kell a Földet. E témakör legfontosabb elemeiről szól ez a fejezet, melyet szakmegkötés nélkül ajánlhatunk minden hallgatónak, hisz a földrajzi információs rendszerek nemcsak a környezettudományhoz, nemcsak regionális társadalmi–gazdasági kutatásokhoz kapcsolódnak, hanem mindenki számára hasznosak lehetnek.

Highly Toxic Microcystis aeruginosa Strain, Isolated from So Paulo-Brazil, Produce Hepatotoxins and Paralytic Shellfish Poison Neurotoxins

While evaluating several laboratory-cultured cyanobacteria strains for the presence of paralytic shellfish poison neurotoxins, the hydrophilic extract of Microcystis aeruginosa strain SPC777-isolated from Billings`s reservoir, So Paulo, Brazil-was found to exhibit lethal neurotoxic effect in mouse bioassay. The in vivo test showed symptoms that unambiguously were those produced by PSP. In order to identify the presence of neurotoxins, cells were lyophilized, and the extracts were analyzed by HPLC-FLD and HPLC-MS. HPLC-FLD analysis revealed four main Gonyautoxins: GTX4(47.6%), GTX2(29.5%), GTX1(21.9%), and GTX3(1.0%). HPLC-MS analysis, on other hand, confirmed both epimers, with positive Zwitterions M(+) 395.9 m/z for GTX3/GTX2 and M(+) 411 m/z for GTX4/GTX1 epimers. The hepatotoxins (Microcystins) were also evaluated by ELISA and HPLC-MS analyses. Positive immunoreaction was observed by ELISA assay. Alongside, the HPLC-MS analyses revealed the presence of [l-ser(7)] MCYST-RR. The N-methyltransferase (NMT) domain of the microcystin synthetase gene mcyA was chosen as the target sequence to detect the presence of the mcy gene cluster. PCR amplification of the NMT domain, using the genomic DNA of the SPC777 strain and the MSF/MSR primer set, resulted in the expected 1,369 bp product. The phylogenetic analyses grouped the NMT sequence with the NMT sequences of other known Microcystis with high bootstrap support. The taxonomical position of M. aeruginosa SPC777 was confirmed by a detailed morphological description and a phylogenetic analysis of 16S rRNA gene sequence. Therefore, co-production of PSP neurotoxins and microcystins by an isolated M. aeruginosa strain is hereby reported for the first time.

Verification of a mixed high-order accurate DNS code for laminar turbulent transition by the method of manufactured solutions

This paper presents results on a verification test of a Direct Numerical Simulation code of mixed high-order of accuracy using the method of manufactured solutions (MMS). This test is based on the formulation of an analytical solution for the Navier-Stokes equations modified by the addition of a source term. The present numerical code was aimed at simulating the temporal evolution of instability waves in a plane Poiseuille flow. The governing equations were solved in a vorticity-velocity formulation for a two-dimensional incompressible flow. The code employed two different numerical schemes. One used mixed high-order compact and non-compact finite-differences from fourth-order to sixth-order of accuracy. The other scheme used spectral methods instead of finite-difference methods for the streamwise direction, which was periodic. In the present test, particular attention was paid to the boundary conditions of the physical problem of interest. Indeed, the verification procedure using MMS can be more demanding than the often used comparison with Linear Stability Theory. That is particularly because in the latter test no attention is paid to the nonlinear terms. For the present verification test, it was possible to manufacture an analytical solution that reproduced some aspects of an instability wave in a nonlinear stage. Although the results of the verification by MMS for this mixed-order numerical scheme had to be interpreted with care, the test was very useful as it gave confidence that the code was free of programming errors. Copyright (C) 2009 John Wiley & Sons, Ltd.

Numerical solution of the two-phase expansion of a metastable flashing liquid jet using the dispersion-controlled dissipative scheme

This paper presents a study of the stationary phenomenon of superheated or metastable liquid jets, flashing into a two-dimensional axisymmetric domain, while in the two-phase region. In general, the phenomenon starts off when a high-pressure, high-temperature liquid jet emerges from a small nozzle or orifice expanding into a low-pressure chamber, below its saturation pressure taken at the injection temperature. As the process evolves, crossing the saturation curve, one observes that the fluid remains in the liquid phase reaching a superheated condition. Then, the liquid undergoes an abrupt phase change by means of an oblique evaporation wave. Across this phase change the superheated liquid becomes a two-phase high-speed mixture in various directions, expanding to supersonic velocities. In order to reach the downstream pressure, the supersonic fluid continues to expand, crossing a complex bow shock wave. The balance equations that govern the phenomenon are mass conservation, momentum conservation, and energy conservation, plus an equation-of-state for the substance. A false-transient model is implemented using the shock capturing scheme: dispersion-controlled dissipative (DCD), which was used to calculate the flow conditions as the steady-state condition is reached. Numerical results with computational code DCD-2D vI have been analyzed. Copyright (C) 2009 John Wiley & Sons, Ltd.

Drosophila melanogaster lipins are tissue-regulated and developmentally regulated and present specific subcellular distributions

Lipins constitute a novel family of Mg2+-dependent phosphatidate phosphatases that catalyze the dephosphorylation of phosphatidic acid to yield diacylglycerol, an important intermediate in lipid metabolism and cell signaling. Whereas a single lipin is detected in less complex organisms, in mammals there are distinct lipin isoforms and paralogs that are differentially expressed among tissues. Compatible with organism tissue complexity, we show that the single Drosophila Lpin1 ortholog (CG8709, here named DmLpin) expresses at least three isoforms (DmLpinA, DmLpinK and DmLpinJ) in a temporal and spatially regulated manner. The highest levels of lipin in the fat body, where DmLpinA and DmLpinK are expressed, correlate with the highest levels of triacylglycerol (TAG) measured in this tissue. DmLpinK is the most abundant isoform in the central nervous system, where TAG levels are significantly lower than in the fat body. In the testis, where TAG levels are even lower, DmLpinJ is the predominant isoform. Together, these data suggest that DmLpinA might be the isoform that is mainly involved in TAG production, and that DmLpinK and DmLpinJ could perform other cellular functions. In addition, we demonstrate by immunofluorescence that lipins are most strongly labeled in the perinuclear region of the fat body and ventral ganglion cells. In visceral muscles of the larval midgut and adult testis, lipins present a sarcomeric distribution. In the ovary chamber, the lipin signal is concentrated in the internal rim of the ring canal. These specific subcellular localizations of the Drosophila lipins provide the basis for future investigations on putative novel cellular functions of this protein family.

Determinação do momento crítico de flambagem lateral com distorção em vigas mistas contínuas de aço e concreto com perfis de alma senoidal

As vigas mistas de aço e concreto estão sendo largamente utilizadas em construções de edifícios e pontes. Ao se combinar o aço com o concreto obtêm-se estruturas mais econômicas, uma vez que se tira proveito das melhores características de cada material. Nas regiões de momento negativo de uma viga mista contínua, a mesa inferior e parte da alma estão comprimidas, se a alma do perfil não tiver rigidez suficiente para evitar a flexão lateral, ela distorcerá gerando um deslocamento lateral e um giro na mesa comprimida, caracterizando um modo de flambagem denominado flambagem lateral com distorção (FLD). O procedimento de verificação à FLD da EN 1994-1-1:2004 originou o método de cálculo da ABNT NBR 8800:2008, entretanto a EN 1994-1-1:2004 não fornece expressão para o cálculo do momento crítico elástico, enquanto a ABNT NBR 8800:2008 prescreve uma formulação proposta por Roik, Hanswille e Kina (1990) desenvolvida para vigas mistas com perfis de alma plana. Embora as normas prescrevam um método de verificação à FLD para vigas mistas com perfis de alma plana, poucos estudos têm sido feitos sobre esse estado-limite. Além disso, tanto a ABNT NBR 8800:2008 quanto as normas internacionais não abordam perfis de alma senoidal. Neste trabalho, foram implementadas análises de flambagem elástica, com auxílio do software ANSYS 14.0 (2011), em modelos de elementos finitos que retratem o comportamento à FLD de vigas mistas de aço e concreto com perfis de alma plana e senoidal. Os modelos numéricos foram constituídos pelo perfil de aço, por uma mola rotacional que restringe parcialmente o giro da mesa superior e uma restrição ao deslocamento lateral, ao longo de todo o comprimento da viga. Os resultados numéricos são comparados com os obtidos pelas formulações de Roik, Hanswille e Kina (1990) e de Hanswille (2002), adaptadas para levar em consideração a corrugação da alma do perfil de aço. Para avaliação das formulações supracitadas e da consistência da modelagem numérica adotada, o momento crítico elástico foi determinado para vigas mistas com perfis de aço de alma plana. Como resultado, um método para o cálculo do momento crítico elástico de vigas mistas de alma senoidal é proposto.

Data mining framework for fatty liver disease classification in ultrasound: a hybrid feature extraction paradigm

PURPOSE: Fatty liver disease (FLD) is an increasing prevalent disease that can be reversed if detected early. Ultrasound is the safest and ubiquitous method for identifying FLD. Since expert sonographers are required to accurately interpret the liver ultrasound images, lack of the same will result in interobserver variability. For more objective interpretation, high accuracy, and quick second opinions, computer aided diagnostic (CAD) techniques may be exploited. The purpose of this work is to develop one such CAD technique for accurate classification of normal livers and abnormal livers affected by FLD. METHODS: In this paper, the authors present a CAD technique (called Symtosis) that uses a novel combination of significant features based on the texture, wavelet transform, and higher order spectra of the liver ultrasound images in various supervised learning-based classifiers in order to determine parameters that classify normal and FLD-affected abnormal livers. RESULTS: On evaluating the proposed technique on a database of 58 abnormal and 42 normal liver ultrasound images, the authors were able to achieve a high classification accuracy of 93.3% using the decision tree classifier. CONCLUSIONS: This high accuracy added to the completely automated classification procedure makes the authors' proposed technique highly suitable for clinical deployment and usage.

Analysis of polycyclic aromatic hydrocarbons in atmospheric particulate samples by microwaveassisted extraction and liquid chromatography

A methodology based on microwave-assisted extraction (MAE) and LC with fluorescence detection (FLD) was investigated for the efficient determination of 15 polycyclic aromatic hydrocarbons (PAHs) regarded as priority pollutants by the US Environmental Protection Agency and dibenzo(a,l)pyrene in atmospheric particulate samples. PAHs were successfully extracted from real outdoor particulate matter (PM) samples with recoveries ranging from 81.4±8.8 to 112.0±1.1%, for all the compounds except for naphthalene (62.3±18.0%) and anthracene (67.3±5.7%), under the optimum MAE conditions (30.0 mL of ACN for 20 min at 110ºC). No clean-up steps were necessary prior to LC analysis. LOQs ranging from 0.0054 ng/m3 for benzo( a)anthracene to 0.089 ng/m3 for naphthalene were reached. The validated MAE methodology was applied to the determination of PAHs from a set of real world PM samples collected in Oporto (north of Portugal). The sum of particulate-bound PAHs in outdoor PM ranged from 2.5 and 28 ng/m3.

A numerical analysis of generalized Boussinesq-type equation using continuos/discontinuous FEM

An improved class of Boussinesq systems of an arbitrary order using a wave surface elevation and velocity potential formulation is derived. Dissipative effects and wave generation due to a time-dependent varying seabed are included. Thus, high-order source functions are considered. For the reduction of the system order and maintenance of some dispersive characteristics of the higher-order models, an extra O(mu 2n+2) term (n ??? N) is included in the velocity potential expansion. We introduce a nonlocal continuous/discontinuous Galerkin FEM with inner penalty terms to calculate the numerical solutions of the improved fourth-order models. The discretization of the spatial variables is made using continuous P2 Lagrange elements. A predictor-corrector scheme with an initialization given by an explicit RungeKutta method is also used for the time-variable integration. Moreover, a CFL-type condition is deduced for the linear problem with a constant bathymetry. To demonstrate the applicability of the model, we considered several test cases. Improved stability is achieved.

O citocromo b5 no complexo enzimático do citocromo P450

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Genética Molecular e Biomedicina

Implementação de método em HPLC para a determinação de PAH´s em óleos vegetais

Os Hidrocarbonetos Policíclicos Aromáticos (HPA’s), doravante designados PAH’s são considerados poluentes prioritários, devido às suas características tóxicas, cancerígenas e mutagénicas. No entanto, apenas 16 destes compostos são referidos na lista de compostos orgânicos prioritários, na qual estão inseridos os 4 PAH’s estudados neste trabalho - Benzo[a]antraceno, Benzo[a]Pireno, Benzo[b]fluoranteno e o criseno. A origem dos PAH’s no ambiente provém de fontes naturais e antropogénicas. Vários processos têm sido desenvolvidos para remover e/ou reduzir estes contaminantes no ambiente. A sua maioria passa por reduzir a poluição causada por fontes antropogénicas. No entanto, devido à dificuldade de remover totalmente estes contaminantes é necessário controlar a concentração destes compostos durante o processo de fabrico de alimentos. Os PAH’s podem ser encontrados em diferentes géneros alimentícios tais como óleos vegetais. Sendo a Sovena produtora destes surge como objectivo deste trabalho implementar e validar um método analítico para a quantificação dos 4 PAH’s nos óleos crus e refinados de forma a garantir que o contaminante é eliminado ou reduzido até níveis aceitáveis. Esta quantificação foi feita em HPLC com detector de fluorescência (FLD), utilizando-se o método da adição de padrão interno, neste caso o Benzo[b]Criseno. Para avaliar a linearidade do método procedeu-se à representação gráfica da função Área=f (concentração), obtendo – se uma função linear na gama de 1 a 50μg/kg. A média da precisão medida através do coeficiente de variação variou entre 1,63 e 7,49%, a exactidão obtida pela média da percentagem de recuperação variou entre 65,0 e 107,5% sem matriz e entre 59,2 e 87,8% com matriz (óleo alimentar). O limite de detecção obtido foi de 0,5 μg/kg e o limite de quantificação 1 μg/kg. Através da comparação de resultados com laboratórios externos foi possível um constante desenvolvimento no sentido de melhorar os resultados.

Immunoreactive proteins of Saccharopolyspora rectivirgula for farmer's lung serodiagnosis.

PURPOSE: Saccharopolyspora rectivirgula is the principal cause of farmer's lung disease (FLD). Serodiagnosis is based on immunoprecipitation techniques or enzyme immunoassays with homemade crude antigens and is not standardized. We aimed to produce specific recombinant antigens for the development of a standardized ELISA. EXPERIMENTAL DESIGN: We recruited 41 patients and 43 healthy exposed controls from five university hospital pneumology departments in France and Switzerland. S. rectivirgula proteins were extracted, separated by 2D electrophoresis, and subjected to Western blotting, with sera from FLD patients or controls. FLD-specific proteins were identified by MS and were produced as recombinant antigens. The diagnostic performance of ELISA tests using the recombinant antigens was assessed with all the sera from FLD patients and controls. RESULTS: We identified 25 FLD-specific proteins, some of which play important roles in transport, nutrition, or virulence. We produced 17 of these proteins as recombinant antigens and assessed their suitability for inclusion in the ELISA test. A combination of three of these proteins (SR1FA, SR17, and SR22) proved remarkably effective at discriminating between patients and controls, with a sensitivity of 83% and a specificity of 77%. CONCLUSIONS AND CLINICAL RELEVANCE: The recombinant antigens produced in this study constitute a major step toward the improvement of diagnostic performance and the standardization of FLD serodiagnosis.

A hypomorphic mutation in lpin1 induces progressively improving neuropathy and lipodystrophy in the rat.

The Lpin1 gene encodes the phosphatidate phosphatase (PAP1) enzyme Lipin 1, which plays a critical role in lipid metabolism. In this study we describe the identification and characterization of a rat model with a mutated Lpin1 gene (Lpin1(1Hubr)), generated by N-ethyl-N-nitrosourea mutagenesis. Lpin1(1Hubr) rats are characterized by hindlimb paralysis and mild lipodystrophy that are detectable from the second postnatal week. Sequencing of Lpin1 identified a point mutation in the 5'-end splice site of intron 18 resulting in mis-splicing, a reading frameshift, and a premature stop codon. As this mutation does not induce nonsense-mediated decay, it allows the production of a truncated Lipin 1 protein lacking PAP1 activity. Lpin1(1Hubr) rats developed hypomyelination and mild lipodystrophy rather than the pronounced demyelination and adipocyte defects characteristic of Lpin1(fld/fld) mice, which carry a null allele for Lpin1. Furthermore, biochemical, histological, and molecular analyses revealed that these lesions improve in older Lpin1(1Hubr) rats as compared with young Lpin1(1Hubr) rats and Lpin1(fld/fld) mice. We observed activation of compensatory biochemical pathways substituting for missing PAP1 activity that, in combination with a possible non-enzymatic Lipin 1 function residing outside of its PAP1 domain, may contribute to the less severe phenotypes observed in Lpin1(1Hubr) rats as compared with Lpin1(fld/fld) mice. Although we are cautious in making a direct parallel between the presented rodent model and human disease, our data may provide new insight into the pathogenicity of recently identified human LPIN1 mutations.