Cytopathology smears from autopsies: A viable storage method for molecular analysis.

INTRODUCTION: Cytology appears to be a viable option to histological samples for proper storage and maintenance of autopsy material for DNA extraction and analysis. In the present study, we tested the feasibility of using archived air-dried smears produced at the time of the autopsy for simple molecular analysis, comparing quantity and quality of the DNA extracted from the smears to that of correspondent histological specimens. METHODS: Air-dried cytological smears were obtained from scrapings of exactly the same areas collected for histological study. DNA was extracted using a commercially available protocol from all samples, with calculation of purity ratio and overall concentration. The integrity of the extracted DNA was also verified through conventional polymerase chain reaction (PCR). RESULTS: Five cases of lung tumours (2 small cell carcinomas and 3 adenocarcinomas) were collected. Percentage of tumour cells and necrosis ranged from 30% to 90% and from 10% to 40%, respectively, in the cytological preparations, and from 50% to 90% and from 10% to 80%, respectively, in the histological preparations. Purity ratio (260/280) had a median of 1.87 in cytology vs 1.94 in histology. Mean DNA concentration among the cytological preparations was 2653 ng/mL (range 1684-3980 ng/mL) vs 757.2 ng/mL among the histological preparations (range 456-1829 ng/mL. DNA from all five cases of cytology was successfully amplified by conventional PCR, in contrast to none from the histology specimens. CONCLUSIONS: Archived air-dried smears scraped from tumoural lesions in autopsies have proven to yield a good concentration of quality DNA for conventional PCR, with better results than formalin-fixed paraffin embedded material.

Redox Activation of Nox1 (NADPH Oxidase 1) Involves an Intermolecular Disulfide Bond Between Protein Disulfide Isomerase and p47phox in Vascular Smooth Muscle Cells.

Objective- PDI (protein disulfide isomerase A1) was reported to support Nox1 (NADPH oxidase) activation mediated by growth factors in vascular smooth muscle cells. Our aim was to investigate the molecular mechanism by which PDI activates Nox1 and the functional implications of PDI in Nox1 activation in vascular disease. Approach and Results- Using recombinant proteins, we identified a redox interaction between PDI and the cytosolic subunit p47phox in vitro. Mass spectrometry of crosslinked peptides confirmed redox-dependent disulfide bonds between cysteines of p47phox and PDI and an intramolecular bond between Cys 196 and 378 in p47phox. PDI catalytic Cys 400 and p47phox Cys 196 were essential for the activation of Nox1 by PDI in vascular smooth muscle cells. Transfection of PDI resulted in the rapid oxidation of a redox-sensitive protein linked to p47phox, whereas PDI mutant did not promote this effect. Mutation of p47phox Cys 196, or the redox active cysteines of PDI, prevented Nox1 complex assembly and vascular smooth muscle cell migration. Proximity ligation assay confirmed the interaction of PDI and p47phox in murine carotid arteries after wire injury. Moreover, in human atheroma plaques, a positive correlation between the expression of PDI and p47phox occurred only in PDI family members with the a' redox active site. Conclusions- PDI redox cysteines facilitate Nox1 complex assembly, thus identifying a new mechanism through which PDI regulates Nox activity in vascular disease.

Nox1 in cardiovascular diseases: regulation and pathophysiology.

Since its discovery in 1999, a number of studies have evaluated the role of Nox1 NADPH oxidase in the cardiovascular system. Nox1 is activated in vascular cells in response to several different agonists, with its activity regulated at the transcriptional level as well as by NADPH oxidase complex formation, protein stabilization and post-translational modification. Nox1 has been shown to decrease the bioavailability of nitric oxide, transactivate the epidermal growth factor receptor, induce pro-inflammatory signalling, and promote cell migration and proliferation. Enhanced expression and activity of Nox1 under pathologic conditions results in excessive production of reactive oxygen species and dysregulated cellular function. Indeed, studies using genetic models of Nox1 deficiency or overexpression have revealed roles for Nox1 in the pathogenesis of cardiovascular diseases ranging from atherosclerosis to hypertension, restenosis and ischaemia/reperfusion injury. These data suggest that Nox1 is a potential therapeutic target for vascular disease, and drug development efforts are ongoing to identify a specific bioavailable inhibitor of Nox1.

Stathmin involvement in the maternal embryonic leucine zipper kinase pathway in glioblastoma.

BACKGROUND: Maternal Embryonic Leucine Zipper Kinase (MELK) is a serine/threonine kinase involved in cell cycle, differentiation, proliferation, and apoptosis. These multiple features are consistent with it being a potential anticancer target. Nevertheless, the MELK pathway in tumorigenesis is not yet completely understood. This study aims to identify proteins associated with MELK pathway in astrocytomas. To this end, proteomic data of the human glioma cell line U87MG transfected with siRNA for MELK were compared with non-target transfected control cells and compared with oligonucleotide microarray data. RESULTS: In both assays, we identified stathmin/oncoprotein 18 (STMN1), involved in cell cycle. STMN1 gene expression was further assessed in a series of 154 astrocytomas and 22 non-neoplastic brain samples by qRT-PCR. STMN1 expression was significantly increased in malignant diffusely infiltrative astrocytomas compared with pilocytic astrocytoma (p < 0.0001). A strong correlation between MELK and STMN1 expressions was observed (r = 0.741, p < 0.0001) in glioblastoma (GBM) samples. However, no difference on survival times was found when compared GBM cases with upregulated and downregulated STMN1 (Breslow = 0.092, median survival time: 11 and 13 months, respectively). Functional assays knocking down MELK by siRNA in GBM cell line showed that gene and protein expression of both MELK and stathmin were diminished. On the other hand, when the same analysis was performed for STMN1, only stathmin gene and protein was silenced. CONCLUSIONS: The results presented herein point stahtmin as a downstream target in the MELK pathway that plays a role in malignant progression of astrocytomas.

Quantitative proteomic analysis shows differentially expressed HSPB1 in glioblastoma as a discriminating short from long survival factor and NOVA1 as a differentiation factor between low-grade astrocytoma and oligodendroglioma.

BACKGROUND: Gliomas account for more than 60 % of all primary central nervous system neoplasms. Low-grade gliomas display a tendency to progress to more malignant phenotypes and the most frequent and malignant gliomas are glioblastomas (GBM). Another type of glioma, oligodendroglioma originates from oligodendrocytes and glial precursor cells and represents 2-5 % of gliomas. The discrimination between these two types of glioma is actually controversial, thus, a molecular distinction is necessary for better diagnosis. METHODS: iTRAQ-based quantitative proteomic analysis was performed on non-neoplastic brain tissue, on astrocytoma grade II, glioblastoma with short and long survival and oligodendrogliomas. RESULTS: We found that expression of nucleophosmin (NPM1), glucose regulated protein 78 kDa (GRP78), nucleolin (NCL) and heat shock protein 90 kDa (HSP90B1) were increased, Raf kinase inhibitor protein (RKIP/PEBP1) was decreased in glioblastoma and they were associated with a network related to tumor progression. Expression level of heat shock protein 27 (HSPB1/HSP27) discriminated glioblastoma presenting short (6 ± 4 months, n = 4) and long survival (43 ± 15 months, n = 4) (p = 0.00045). Expression level of RNA binding protein nova 1 (NOVA1) differentiated low-grade oligodendroglioma and astrocytoma grade II (p = 0.0082). Validation were done by Western blot, qRT-PCR and immunohistochemistry in a larger casuistry. CONCLUSION: Taken together, our quantitative proteomic analysis detected the molecular triad, NPM1, GRP78 and RKIP participating together with NCL and HSP27/HSPB1 in a network related to tumor progression. Additionally, two new important targets were uncovered: NOVA1 useful for diagnostic refinement differentiating astrocytoma from oligodendroglioma, and HSPB1/HSP27, as a predictive factor of poor prognosis for GBM.

Quantitative proteomic analysis and functional studies reveal that nucleophosmin is involved in cell death in glioblastoma cell line transfected with siRNA.

Previously, we reported that nucleophosmin (NPM) was increased in glioblastoma multiforme (GBM). NPM is a phosphoprotein related to apoptosis, ribosome biogenesis, mitosis, and DNA repair, but details about its function remain unclear. We treated U87MG and A172 cells with small interference RNA (siRNA) and obtained a reduction of 80% in NPM1 expression. Knockdown at the protein level was evident after the 4th day and was maintained until the 7th day of transfection that was investigated by quantitative proteomic analysis using isobaric tags. The comparison of proteomic analysis of NPM1-siRNA against controls allowed the identification of 14 proteins, two proteins showed increase and 12 presented a reduction of expression levels. Gene ontology assigned most of the hypoexpressed proteins to apoptosis regulation, including GRP78. NPM1 silencing did not impair cell proliferation until the 7th day after transfection, but sensitized U87MG cells to temozolomide (TMZ), culminating with an increase in cell death and provoking at a later period a reduction of colony formation. In a large data set of GBM patients, both GRP78 and NPM1 genes were upregulated and presented a tendency to shorter overall survival time. In conclusion, NPM proved to participate in the apoptotic process, sensitizing TMZ-treated U87MG and A172 cells to cell death, and in association with upregulation of GRP78 may be helpful as a predictive factor of poor prognosis in GBM patients.

Changes in the expression of proteins associated with aerobic glycolysis and cell migration are involved in tumorigenic ability of two glioma cell lines.

BACKGROUND: The most frequent and malignant brain cancer is glioblastoma multiforme (GBM). In gliomas, tumor progression and poor prognosis are associated with the tumorigenic ability of the cells. U87MG cells (wild-type p53) are known to be tumorigenic in nude mice, but T98G cells (mutant p53) are not tumorigenic. We investigated the proteomic profiling of these two cell lines in order to gain new insights into the mechanisms that may be involved in tumorigenesis. RESULTS: We found 24 differentially expressed proteins between T98G and U87MG cells. Gene Ontology supports the notion that over-representation of differentially expressed proteins is involved in glycolysis, cell migration and stress oxidative response. Among those associated with the glycolysis pathway, TPIS and LDHB are up-regulated in U87MG cells. Measurement of glucose consumption and lactate production suggests that glycolysis is more effective in U87MG cells. On the other hand, G6PD expression was 3-fold higher in T98G cells and this may indicate a shift to the pentose-phosphate pathway. Moreover, GRP78 expression was also three-fold higher in T98G than in U87MG cells. Under thapsigargin treatment both cell lines showed increased GRP78 expression and the effect of this agent was inversely correlated to cell migration. Quantitative RT-PCR and immunohistochemistry of GRP78 in patient samples indicated a higher level of expression of GRP78 in grade IV tumors compared to grade I and non-neoplastic tissues, respectively. CONCLUSIONS: Taken together, these results suggest an important role of proteins involved in key functions such as glycolysis and cell migration that may explain the difference in tumorigenic ability between these two glioma cell lines and that may be extrapolated to the differential aggressiveness of glioma tumors.

Proteomic analysis of low- to high-grade astrocytomas reveals an alteration of the expression level of raf kinase inhibitor protein and nucleophosmin.

Proteomic approaches have been useful for the identification of aberrantly expressed proteins in complex diseases such as cancer. These proteins are not only potential disease biomarkers, but also targets for therapy. The aim of this study was to identify differentially expressed proteins in diffuse astrocytoma grade II, anaplastic astrocytoma grade III and glioblastoma multiforme grade IV in human tumor samples and in non-neoplastic brain tissue as control using 2-DE and MS. Tumor and control brain tissue dissection was guided by histological hematoxylin/eosin tissue sections to provide more than 90% of tumor cells and astrocytes. Six proteins were detected as up-regulated in higher grade astrocytomas and the most important finding was nucleophosmin (NPM) (p<0.05), whereas four proteins were down-regulated, among them raf kinase inhibitor protein (RKIP) (p<0.05). We report here for the first time the alteration of NPM and RKIP expression in brain cancer. Our focus on these proteins was due to the fact that they are involved in the PI3K/AKT/mTOR and RAS/RAF/MAPK pathways, known for their contribution to the development and progression of gliomas. The proteomic data for NPM and RKIP were confirmed by Western blot, quantitative real-time PCR and immunohistochemistry. Due to the participation of NPM and RKIP in uncontrolled proliferation and evasion of apoptosis, these proteins are likely targets for drug development.

Imbalance between nitric oxide and superoxide anion induced by uncoupled nitric oxide synthase contributes to human melanoma development.

Melanoma is the most aggressive type of cutaneous tumors due to its metastatic potential and high mortality. Increased levels of reactive oxygen species, including superoxide anion (O2-), and the consequent installation of a pro-oxidant environment are associated with melanoma development. The enzyme nitric oxide synthase (NOS), responsible for the production of nitric oxide (NO), when uncoupled is as a source of O2-, for example by the absence of its cofactor tetrahydrobiopterin (BH4). Western blot analysis showed increased expression of endothelial and inducible NOS in human melanoma cells, altering the stoichiometry between NOS levels and BH4 concentration and together with decreased BH4:BH2 ratio are contributing to NOS uncoupling. The treatment of melanoma cells with exogenous BH4 increased NO concentration and decreased O2- levels, leading to NOS coupling, which in turn reduced cell viability, cell proliferation and the ability of melanoma cells to form melanoma spheroids. Moreover, BH4 level restoration rendered melanoma cells more sensitive to apoptosis, demonstrating the role of dysfunctional NOS in melanoma genesis.

Efeito do ultra-som na prevenção da hérnia incisional mediana em ratos / Effect of ultrasonic therapy on prevention of midline incisional Hernia in rats

OBJETIVO: Investigar se o ultra-som é capaz de evitar o aparecimento da hérnia incisional (HI), uma vez que o seu tratamento ainda é um problema não resolvido. MÉTODO: Induziu-se a HI, por secção da linha alba, em 20 ratos Wistar, distribuídos em dois grupos: um recebeu a aplicação do ultra-som, na área operada, por 14 dias e o outro por 28 dias, cada qual com seu subgrupo controle (não tratado). Utilizou-se aparelho com cabeçote reduzido, modo pulsado (1:5), freqüência de 3,0MHz e intensidade de 0,5W/cm², durante 5min/dia, iniciando-se as aplicações no primeiro dia pós-operatório. Ao final, mediu-se o diâmetro transverso do anel herniário ou da largura da cicatriz na linha alba, definindo-se a HI quando esses valores ultrapassassem 2mm. A densidade de vasos sangüíneos, de fibroblastos e de fibras colágenas, nessas estruturas, foi estudada à microscopia óptica. As médias e o erro padrão foram submetidos à análise de variância (ANOVA) e ao teste de comparação múltipla de Tukey-Kramer. RESULTADOS: Dos animais tratados com o ultra-som, 60 por cento não desenvolveram HI e, nos restantes, o anel herniário foi menor do que os não tratados (p<0,0001). A densidade de vasos sangüíneos (p=0,0031), de fibroblastos (p<0,0001) e de fibras colágenas (p=0,0015) também foi maior, em relação aos controles. Não houve diferença significante nesses parâmetros, comparando-se os dois períodos de tratamento. CONCLUSÃO: O ultra-som evitou o aparecimento da HI ou reduziu o seu tamanho, quando aplicado por 14 dias pós-operatórios, nas condições deste experimento.

BACKGROUND: To investigate if ultrasonic therapy is effective in avoidance of incisional hernia (IH), once the ideal technique for repair of IH is still a non-solved problem, in spite of prostheses usage. METHODS: IH was created by linea alba opening over 20 male Wistar rats, distributed in two groups: one group received ultrasound over the wound, during 14 days (5 animals) and the other for 28 days (5 animals), each one with the same number of animals as controls. The ultrasound was applied using a small probe, 1:5 pulse mode, 3.0MHz frequency and 0.5W/cm² intensity, for 5 minutes daily, from the first postoperative day. The width of the linea alba scar or the hernia ring was measured at the end of every period. The IH was defined when these openings were over 2mm wide. Some specimens from hernia sac or linea alba scar were obtained for the analysis of vessels, fibroblasts, and colagen fibers density. Standard deviation and medians were obtained by variation analysis and Tukey-Kramer multi-comparison test. RESULTS: The ultrasound group showed a linea alba scar in 60 percent. The other 40 percent presented the hernia ring smaller than the non treated group (p<0.0001). The ultrasound increased density of blood vessels (p=0,0031), fibroblasts (p<0,0001), and collagen fibers (p=0,0015). There was no difference between the two treated groups. CONCLUSION: The ultrasound avoided the IH to appear or reduced its width. Fourteen days of treatment were sufficient for this purpose.

Crecimiento de Clostridium botulinum en medios con ajo (Allium sativum) / Growth of Clostridium botulinum in media with garlic (Allium sativum)

El efecto del ajo sobre el crecimiento y toxinogénesis de Clostridium botilinum fue estudiado utilizando como sustrato el jugo obtenido por previsión y filtración de dientes de las variedades "blanco" y "colorado", y dientes cortados en trozos. El pH de ambos varió, según los lotes, entre 5,7 y 6,0. Los diluyentes de los medios con ajo fueron caldo peptona-glucosa-extracto de levadura, o agua destilada. C. botulinum a 110 fue sembrado en diluciones del jugo y en los medios con trozos e incubado en atmósferas aireada y anaeróbica, 15 d a 37-C. botulinum creció y produjo toxina en los diferents sustratos, en concentraciones variables (desde vestigios hasta 5.000 DL50/ml) pero menores que el control (10.000 DL50/ml). Mientras no se amplíen los resultados preliminares obtenido en este trabajo, la industria conservera deberá tener en cuenta que para su preparación como conserva acuosa en envase hermético o aireado, el ajo deberá ser considerado un producto de acidez baja apto para el crecimiento y producción de toxina de C. botulinum, no esterilizable por el calor, por lo que el contenido deberá ser acidificado hasta un pH equilibrado de 4,5 o ,menos, de acuerdo a las regulamentaciones generales vigentes para el tratamiento de alimentos conservados de acidez baja