Dynamic molecular events associated to Plasmodium berghei gametogenesis through proteomic approach.

Plasmodium mature sexual cycle occurs in the vector mosquitoes and ensures the transmission to a new host. Gametogenesis takes place within minutes in the vector midgut. Gametocytes have to complete a deep nuclear reorganization, quick differentiation, and in the case of male gametocytes, intracytoplasmic flagellum assembly that results in free-swimming microgametes required for macrogamete fertilization. In efforts to improve our knowledge of molecular mechanisms involved in gamete morphogenesis, we carried out a nanoLC/MSMS based quantitative proteomic analysis throughout the xanthurenic acid-induced gametogenesis of the rodent parasite P. berghei. Time-course analyses were performed 7 and 15min after gametogenesis induction. From 2617 iTRAQ-labelled peptides, 49 proteins were found differentially abundant. Proteins related to RNA translation, DNA, and protein biosynthesis were most prominent and strongly regulated. The energetic metabolic pathway, glycolysis, environmental stress response, RNA/protein biosynthesis, mitosis and axoneme formation, both related to tubulin-associated cytoskeleton dynamic, were predominant regulated cell processes at protein level during the differentiation. Our results also include 26 phosphoproteins in gametocytes/gametes. This first iTRAQ-based proteomic time course analysis of Plasmodium gamete development sheds light on the biological protein orchestration within gametogenesis. SIGNIFICANCE: Malaria is one of the most serious and widespread parasitic diseases that affected humans in medicine history. The increasing emergence of resistance of parasites from Plasmodium genus to the available antimalarial drugs and the absence of efficient vaccines require an urgent need of development of new therapeutic strategies to fight against that disease. The sexual reproduction is a key step of Plasmodium life cycle and constitutes an attractive target for the development of new therapeutic approaches since it would control malaria based on an inhibition of the parasite transmission to Anopheles, and then to humans. Male and female gamete formation (gametogenesis) is thus a biological event that is determinant for the perpetuation of the parasite in which drastic morphological and metabolic changes occur in a short time interval, resulting in the production of 8 male gametes from a male gametocyte, and fertilization of the female gamete. Development of such transmission-blocking strategies required in deep understanding of the molecular and cellular events associated to gametogenesis. Despite several studies, our understanding on gametogenesis is still incomplete and requires further investigations. This work is the first large-scale quantitative proteomic insight into the P. berghei gamete morphogenesis providing valuable time course data. Plasmodium gametogenesis clearly requires regulation of expression and phosphorylation of proteins belonging to different metabolic pathways and functions, in order to produce mature male and female gametes.

Desarrollo y validación de un método verde para la extracion y cuantificación de residuos de cipermetrina en tejido animal / Fevelopment and validation of green method for the extraction and quantification of cypermethrin residues in animal tissue / Desenvolvimento e validação do método verde para a extração e quantificação dos resíduos da cipermetrina no tecido animal

En este trabajo se desarrolló y validó un método simple, ambientalmente amigable y efectivo, para la extracción y cuantificación de cipermetrina en muestras de bovino (hígado, grasa perirrenal y músculo) utilizando dispersión de matriz en fase sólida (MSPD) y cromatografía de gases con detector de ionización de llama (GC-FID). Diferentes parámetros del método se evaluaron, tales como el dispersante, solvente de extracción y volumen de solvente. Los mejores resultados para la extracción de cipermetrina por MSPD fueron: 0,20 g de muestra macerados con 0,80 g de silica gel y extraídos con 5,00 mL de acetona. El procedimiento propuesto fue validado mostrando comportamiento lineal en el intervalo de 10,20-400,40 µg/L (R²=0,9988). Los límites de detección y cuantificación fueron 2,00 y 5,70 µg/L respectivamente, con una desviación estándar relativa de 0,0875 (n=5). Este método permite determinar cipermetrina hasta niveles traza en muestras de tejido animal, con una recuperación de 98,96%.

In the present study a simple, effective and environmentally friendly method was developed and validated for the extraction and quantification of cypermethrin in animal tissue (meat, fat and liver) based on matrix solid-phase dispersion (MSPD) and gas chromatography with flame ionization detector (GC-FID). Different parameters of the method were evaluated, such as dispersant, extractive solvent and solvent volume. The best results for the extraction of cypermethrin by MSPD were 0.20 g of sample with 0.80 g of silica gel and 5.00 mL of acetone as eluting solvent. The proposed procedure was validated showing linear behavior in the interval of 10.20 to 400.40 µg/L (R²= 0.9988). Detection and quantification limits ranged from 2.00 and 5.70 µg/L respectively, with a standard deviation of 0.0875 (n=5). This method enables to determine cypermethrin at trace level in animal tissue samples, with a recovery of 98.96%.

Neste trabalho foi desenvolvido e validado um método verde para extração e quantificação de cipermetrina em amostras de fígado, gordura perirrenal e músculo em gado bovino, por meio da técnica de dispersão de matriz em fase solida (MSPD) e cromatografia de gases com detecção de ionização de chama (GC-FID). Foram avaliados diferentes parâmetros do método, como dispersante, extração com solvente e volume de solvente. Os melhores resultados para a extracção da cipermetrina por MSPD foram 0.20 g da amostra macerada com 0.80 g de gel de sílica e extraiu-se com 5.00 mL de acetona. O método proposto foi validada mostrando um comportamento linear na gama testada (10.20-400.40 µg/L) com R² de 0.9988. Os limites de detecção e de quantificação foram 2.00 e 5.70 µg/L, respectivamente, com um desvio padrão de 0.0875 (n = 5). O método proposto neste trabalho permitiu determinar níveis traça da cipermetrina em amostras de tecidos animai, com uma recuperação de 98.96%.

Comparison of the neutrophil proteome in trauma patients and normal controls.

Neutrophils have an impressive array of microbicidal weapons, and in the presence of a pathogen, progress from a quiescent state in the bloodstream to a completely activated state. Failure to regulate this activation, for example, when the blood is flooded with cytokines after severe trauma, causes inappropriate neutrophil activation that paradoxically, is associated with tissue and organ damage. Acidic proteomic maps of quiescent human neutrophils were analyzed and compared to those of activated neutrophils from severe trauma patients. The analysis revealed 114 spots whose measured volumes differed between activated and quiescent neutrophils, with 27 upregulated and 87 downregulated in trauma conditions. Among the identified proteins, grancalcin, S100-A9 and CACNB2 reinforce observed correlations between motility and ion flux, ANXA3, SNAP, FGD1 and Zfyve19 are involved in vesicular transport and exocytosis, and GSTP1, HSPA1 HSPA1L, MAOB, UCH-L5, and PPA1 presented evidence that activated neutrophils may have diminished protection against oxidative damage and are prone to apoptosis. These are discussed, along with proteins involved in cytoskeleton reorganization, reactive oxygen species production, and ion flux. Proteins such as Zfyve19, MAOB and albumin- like protein were described for the first time in the neutrophil. In this work we achieved the identification of several proteins potentially involved in inflammatory signaling after trauma, as well as proteins described for the first time in neutrophils.

Estandarizacióndde un método analítico para extacción y cuantificación de cipeermetrina en pastos / Standardizatio oof analytical method for extraction and quantiication of cypeermetrhin in grass / Padronização demmétodo analítico para extração e quantificação de cipermetrina em pattagens

En el presente trabajo se estandarizó un método analítico para la extracción y determinación de cipermetrina en muestras de pastos utilizando microextracción en fase sólida (MEFS) en modo de inmersión con posterior desorción y determinación por cromatografía de gases con detector de ionización de llama (CG-DILL). Las condiciones óptimas para la extracción de cipermetrina en pastos por ID-MEFS fueron: 5 g de muestra se calentaron con 10 mL de solución acuosa de acetona al 1% v/v durante 10 minutos; de esta solución se tomaron 4 mL para llevar a cabo el proceso de extracción por MEFS utilizando una fibra de PDMS con exposición de 30 min a 500 rpm y 60 °C. La desorción del analito se llevó a cabo a 270 °C durante 6 minutos. El procedimiento propuesto mostró comportamiento lineal en el rango probado (5-300 µg/L) con R² de 0,999. Los límites de detección y cuantificación fueron 1,53 y 4,97 ng/mL, respectivamente, con una desviación estándar relativa de 8,57% (n = 6). El método propuesto en este trabajo permite determinar cipermetrina en muestras de pastos hasta niveles de trazas con una recuperación de 99,08%.

In this study, an analytical method has been developed to determine cypermethrin in grass matrices using direct immersion solid-phase microextraction (DI-SPME), coupled to gas chromatography with flame ionization detector (GC-FID). The optimized DI-MEFS experimental procedures to extract cypermethrin in grass matrix were: 5 g of the sample were heated with 10 mL of acetone 1% v/v in water during 10 min. 4 mL of this solution were used for extracting with a polydimethylsiloxane (PDMS)-coated fiber at 60 °C and 500 rpm during 30 min. The analyte desorption was performed at 270 °C for 6 min. The proposed procedure showed linear behavior in the range tested (5-300 mg/L) with R² of 0.999. The detection and quantification limits were 1.53 and 4.97 ng/ mL respectively with a standard deviation of 8.57% (n = 6). The method proposed in this paper allows determining cypermethrin in samples of grass to trace levels with a recovery of 99.08%.

No presente estudo, se padronizou um método analítico para a extração e determinação de cipermetrina em amostras de grama usando microextração em fase sólida (MEFS) no modo de imersão com dessorção subseqüentes e determinação por cromatografia gasosa com detector de ionização de chama (CG-DIC). As condições ótimas para a extração de cipermetrina em pastagens por ID-MEFS foram: 5 g de amostra foram aquecidos com 10 mL de solução aquosa de acetona a 1% v/v por 10 minutos. Desta solução, 4 mL foram tomadas para executar o processo de extração MEFS utilizando uma fibra PDMS submetida por 30 min a 500 rpm e 60 °C. A dessorção do analito foi realizada a 270 °C por 6 minutos. O procedimento proposto apresentou um comportamento linear no intervalo testado (500-300 mg/L) com R² de 0,999. Os limites de detecção e quantificação foram 1,53 e 4,97 ng / mL, respectivamente, com um desvio padrão relativo de 8,57% (n = 6). O método proposto neste trabalho possibilita determinar cipermetrina em amostras de pastagens com uma recuperação de 99,08% nos traços.

Proteomic analysis of honey bee brain upon ontogenetic and behavioral development.

The honey bee (Apis mellifera) is a social insect that shows complex and integrated behaviors. Its ability to read and respond to several sets of extrinsic and intrinsic signals is fundamental for the modulation of individual activities and social systems. For instance, A. mellifera behavior changes upon the ontogenetic differentiation from nurse to forager worker subcastes. In this work, brain proteomes of nurses and foragers were compared by two-dimensional gel electrophoresis within pH range of 4-7 in order to find proteins related to such an ontogenetic and behavioral development. Twenty differentially expressed proteins were detected by gel image computational analysis, and identified by peptide mass fingerprinting using MALDI-TOF mass spectrometry. Nurse brain showed increased expression of major royal jelly proteins (MRJP1, MRJP2 and MRJP7), which are related to determination of castes during the honey bee larvae differentiation. Immunocytochemistry and electron microscopy showed that MRJP1 was localized in the cytoplasm of brain cells, seemingly along filaments of the cytoskeleton, in the antennal lobe, optical lobe and mushroom body. Also, MRJP1 was deposited on the rhabdom, a structure of the retinular cells, composed of numerous tubules. Such evidence suggests that MRJP1 could be associated to proteins of filamentous structures. MRJP1 was also found in intercellular spaces between cells in mushrooms bodies, indicating that it is a secreted protein. Other proteins implicated in protein synthesis and putative functions in the olfactory system were also up-regulated in the nurse brain. Experienced foragers overexpressed proteins possibly involved in energy production, iron binding, metabolic signaling and neurotransmitter metabolism. Such differential expression of proteins may be related to ontogenetic and behavior changes in A. mellifera.