Synthesis of silver nanoparticles using DL-alanine for ESR dosimetry applications

The potential use of alanine for the production of nanoparticles is presented here for the first time. Silver nanoparticles were synthesized using a simple green method, namely the thermal treatment of silver nitrate aqueous solutions with in-alanine. The latter compound was employed both as a reducing and a capping agent. Particles with average size equal to 7.5 nm, face-centered cubic crystalline structure, narrow size distribution, and spherical shape were obtained. Interaction between the silver ions present on the surface of the nanoparticles and the amine group of the DL-alanine molecule seems to be responsible for reduction of the silver ions and for the stability of the colloid. The bio-hybrid nanocomposite was used as an ESR dosimeter. The amount of silver nanoparticles in the nanocomposite was not sufficient to cause considerable loss of tissue equivalency. Moreover, the samples containing nanoparticles presented increased sensitivity and reduced energetic dependence as compared with pure DL-alanine, contributing to the construction of small-sized dosimeters. (C) 2011 Elsevier Ltd. All rights reserved.

BJcuL, a lectin purified from Bothrops jararacussu venom, induces apoptosis in human gastric carcinoma cells accompanied by inhibition of cell adhesion and actin cytoskeleton disassembly

We show that BJcuL, a lectin purified from Bothrops jararacussu venom, exerts cytotoxic effects to gastric carcinoma cells MKN45 and AGS. This effect was due to the direct interaction with specific glycans on the cells surface and was observed by cell viability decrease, disorganization of actin filaments and apoptosis. In addition, BJcuL was able to reduce tumor cell adhesion to matrigel, what was inhibited by specific carbohydrate or partially inhibited when cells were pre-incubated with matrigel. Our results suggest that BJcuL was able to promote apoptosis in both tumor cells lines and therefore has a prospect for potential use in cancer therapy. (C) 2011 Elsevier Ltd. All rights reserved.

Genetic vaccine for tuberculosis (pVAXhsp65) primes neonate mice for a strong immune response at the adult stage

Abstract Background Vaccination of neonates is generally difficult due to the immaturity of the immune system and consequent higher susceptibility to tolerance induction. Genetic immunization has been described as an alternative to trigger a stronger immune response in neonates, including significant Th1 polarization. In this investigation we analysed the potential use of a genetic vaccine containing the heat shock protein (hsp65) from Mycobacterium leprae (pVAXhsp65) against tuberculosis (TB) in neonate mice. Aspects as antigen production, genomic integration and immunogenicity were evaluated. Methods Hsp65 message and genomic integration were evaluated by RT-PCR and Southern blot, respectively. Immunogenicity of pVAXhsp65 alone or combined with BCG was analysed by specific induction of antibodies and cytokines, both quantified by ELISA. Results This DNA vaccine was transcribed by muscular cells of neonate mice without integration into the cellular genome. Even though this vaccine was not strongly immunogenic when entirely administered (three doses) during early animal's life, it was not tolerogenic. In addition, pVAXhsp65 and BCG were equally able to prime newborn mice for a strong and mixed immune response (Th1 + Th2) to pVAXhsp65 boosters administered later, at the adult life. Conclusion These results suggest that pVAXhsp65 can be safely used as a priming stimulus in neonate animals in prime-boost similar strategies to control TB. However, priming with BCG or pVAXhsp65, directed the ensuing immune response triggered by an heterologous or homologous booster, to a mixed Th1/Th2 pattern of response. Measures as introduction of IL-12 or GM-CSF genes in the vaccine construct or even IL-4 neutralization, are probably required to increase the priming towards Th1 polarization to ensure control of tuberculosis infection.

Tissue distribution of DNA-Hsp65/TDM-loaded PLGA microspheres and uptake by phagocytic cells

This study aimed to demonstrate that microspheres, used as delivery vehicle of DNA-Hsp65/TDM [plasmid DNA encoding heat shock protein 65 (Hsp65) coencapsulated with trehalose dimycolate (TDM) into PLGA microspheres], are widely spread among several organs after intramuscular administration in BALB/c mice. In general, we showed that these particles were phagocytosed by antigen presenting cells, such as macrophages and dendritic cells. Besides, it was demonstrated herein that draining lymph node cells presented a significant increase in the number of cells expressing costimulatory molecules (CD80 and CD86) and MHC class II, and also that the administration of the DNA-Hsp65/TDM and vector/TDM formulations resulted in the up-regulation of CD80, CD86 and MHC class II expression when compared to control formulations (vector/TDM and empty). Regarding the intracellular trafficking we observed that following phagocytosis, the microspheres were not found in the late endosomes and/or lysosomes, until 15 days after internalization, and we suggest that these constructions were hydrolysed in early compartments. Overall, these data expand our knowledge on PLGA [poly (lactic-co- glycolic acid)] microspheres as gene carriers in vaccination strategies, as well as open perspectives for their potential use in clinical practice.

Topical anti-inflammatory activity of yacon leaf extracts

Smallanthus sonchifolius (Poepp.) H. Rob. , Asteraceae, known as yacon, is an herb that is traditionally used for the treatment of diabetes in folk medicine. However, recent studies have demonstrated that this plant has other interesting properties such as anti-microbial and anti-inflammatory actions. Thus, the purpose of this study was to evaluate the topical anti-inflammatory property of different extracts prepared from yacon leaves and analyze the role of different chemical classes in this activity. Three yacon leaf extracts were obtained: aqueous extract, where chlorogenic acid derivatives and sesquiterpene lactones were detected; leaf rinse extract, rich in sesquiterpene lactones; and polar extract, rich in chlorogenic acid derivatives. All the extracts exhibited anti-edematogenic activity in vivo (aqueous extract: 25.9% edema inhibition at 0.50 mg/ear; polar extract: 42.7% inhibition at 0.25 mg/ear; and leaf rinse extract: 44.1% inhibition at 0.25 mg/ear). The leaf rinse extract furnished the best results regarding neutrophil migration inhibition, and NO, TNF-? and PGE2 inhibition. These data indicate that both sesquiterpene lactones and chlorogenic acid derivatives contribute to the anti-inflammatory action, although sesquiterpene lactones seem to have more pronounced effects. In conclusion, yacon leaf extracts, particularly the sesquiterpene lactone-rich extract, has potential use as topical anti-inflammatory agent.

Expression of human protein S100A7 (psoriasin), preparation of antibody and application to human larynx squamous cell carcinoma

Background Up-regulation of S100A7 (Psoriasin), a small calcium-binding protein, is associated with the development of several types of carcinomas, but its function and possibility to serve as a diagnostic or prognostic marker have not been fully defined. In order to prepare antibodies to the protein for immunohistochemical studies we produced the recombinant S100A7 protein in E. coli. mRNA extracted from human tracheal tumor tissue which was amplified by RT-PCR to provide the region coding for the S100A7 gene. The amplified fragment was cloned in the vector pCR2.1-TOPO and sub-cloned in the expression vector pAE. The protein rS100A7 (His-tag) was expressed in E. coli BL21::DE3, purified by affinity chromatography on an Ni-NTA column, recovered in the 2.0 to 3.5 mg/mL range in culture medium, and used to produce a rabbit polyclonal antibody anti-rS100A7 protein. The profile of this polyclonal antibody was evaluated in a tissue microarray. Results The rS100A7 (His-tag) protein was homogeneous by SDS-PAGE and mass spectrometry and was used to produce an anti-recombinant S100A7 (His-tag) rabbit serum (polyclonal antibody anti-rS100A7). The molecular weight of rS100A7 (His-tag) protein determined by linear MALDI-TOF-MS was 12,655.91 Da. The theoretical mass calculated for the nonapeptide attached to the amino terminus is 12,653.26 Da (delta 2.65 Da). Immunostaining with the polyclonal anti-rS100A7 protein generated showed reactivity with little or no background staining in head and neck squamous cell carcinoma cells, detecting S100A7 both in nucleus and cytoplasm. Lower levels of S100A7 were detected in non-neoplastic tissue. Conclusions The polyclonal anti-rS100A7 antibody generated here yielded a good signal-to-noise contrast and should be useful for immunohistochemical detection of S100A7 protein. Its potential use for other epithelial lesions besides human larynx squamous cell carcinoma and non-neoplastic larynx should be explored in future.

ENDNOTE: A EXPERIÊNCIA DA FMVZ/USP NA CAPACITAÇÃO E NORMALIZAÇÃO DE TRABALHOS

O presente trabalho relata a experiência da Biblioteca Virginie Buff D’Ápice da FMVZ/USP na capacitação do uso do software EndNote e sua aplicabilidade por seus usuários; objetiva ainda demonstrar o potencial de uso dessa ferramenta na normalização de trabalhos científicos e acadêmicos. Com dados preliminares obtidos entre os anos de 2009 e 2010, além da experiência vivenciada nesse período, foi possível constatar que o uso do EndNote pelos estudantes que participaram da capacitação oferecida pela biblioteca promoveu um índice de acerto na normalização de seus trabalhos muito acima daqueles que não foram capacitados e/ou não o utilizaram o programa. Dessa forma, pode-se considerar que uma capacitação efetiva e o uso adequado do EndNote facilitam o trabalho do pesquisador que, ao utilizá-lo, pode focar mais seus esforços na escrita do trabalho, bem como o da biblioteca responsável pela normalização de trabalhos científicos/acadêmicos

MicroRNA expression profile in head and neck cancer: HOX-cluster embedded microRNA-196a and microRNA-10b dysregulation implicated in cell proliferation

Abstract Background Current evidence implicates aberrant microRNA expression patterns in human malignancies; measurement of microRNA expression may have diagnostic and prognostic applications. Roles for microRNAs in head and neck squamous cell carcinomas (HNSCC) are largely unknown. HNSCC, a smoking-related cancer, is one of the most common malignancies worldwide but reliable diagnostic and prognostic markers have not been discovered so far. Some studies have evaluated the potential use of microRNA as biomarkers with clinical application in HNSCC. Methods MicroRNA expression profile of oral squamous cell carcinoma samples was determined by means of DNA microarrays. We also performed gain-of-function assays for two differentially expressed microRNA using two squamous cell carcinoma cell lines and normal oral keratinocytes. The effect of the over-expression of these molecules was evaluated by means of global gene expression profiling and cell proliferation assessment. Results Altered microRNA expression was detected for a total of 72 microRNAs. Among these we found well studied molecules, such as the miR-17-92 cluster, comprising potent oncogenic microRNA, and miR-34, recently found to interact with p53. HOX-cluster embedded miR-196a/b and miR-10b were up- and down-regulated, respectively, in tumor samples. Since validated HOX gene targets for these microRNAs are not consistently deregulated in HNSCC, we performed gain-of-function experiments, in an attempt to outline their possible role. Our results suggest that both molecules interfere in cell proliferation through distinct processes, possibly targeting a small set of genes involved in cell cycle progression. Conclusions Functional data on miRNAs in HNSCC is still scarce. Our data corroborate current literature and brings new insights into the role of microRNAs in HNSCC. We also show that miR-196a and miR-10b, not previously associated with HNSCC, may play an oncogenic role in this disease through the deregulation of cell proliferation. The study of microRNA alterations in HNSCC is an essential step to the mechanistic understanding of tumor formation and could lead to the discovery of clinically relevant biomarkers.

Response of Saccharomyces cerevisiae to Cadmium and Nickel Stress: The Use of the Sugar Cane Vinasse as a Potential Mitigator

Most of the metals released from industrial activity, among them are cadmium (Cd) and nickel (Ni), inhibit the productivity of cultures and affect microbial metabolism. In this context, the aim of this work was to investigate the capacity of sugar cane vinasse to mitigate the adverse effects of Cd and Ni on cell growth, viability, budding rate and trehalose content of Saccharomyces cerevisiae, likely because of adsorption and chelating action. For this purpose, the yeast was grown batch-wise in YED medium supplemented with selected amounts of vinasse and Cd or Ni. The negative effects of Cd and Ni on S. cerevisiae growth and the mitigating one of sugar cane vinasse were quantified by an exponential model. Without vinasse, the addition of increasing levels of Cd and Ni reduced the specific growth rate, whereas in its presence no reduction was observed. Consistently with the well-proved toxicity of both metals, cell viability and budding rate progressively decreased with increasing their concentration, but in the presence of vinasse the situation was remarkably improved. The trehalose content of S. cerevisiae cells followed the same qualitative behavior as cell viability, even though the negative effect of both metals on this parameter was stronger. These results demonstrate the ability of sugar cane vinasse to mitigate the toxic effects of Cd and Ni.