Soybean lunasin mediates colon carcinogenesis by inducing apoptosis and preventing outgrowth of metastasis

Colorectal cancer (CRC) is the third most common cancer worldwide. Various factors such as age, lifestyle and dietary patterns affect the risk of having CRC. Epidemiological studies showed a chemopreventive effect of soy consumption against CRC. However, which component(s) of soybean is associated with this reduced risk is not yet fully delineated. The objective of this research was to evaluate the anti-colon cancer potential of lunasin isolated from defatted soybean flour using in vitro and in vivo models of CRC. Lunasin was isolated from defatted soybean flour by a combination of different chromatographic and ultrafiltration techniques. The anti-colon cancer potential of lunasin was determined using different human colon cancer cell lines in vitro and a CRC liver metastasis model in vivo. Lunasin caused cytotoxicity to different human colon cancer cells with an IC50 value of 13.0, 21.6, 26.3 and 61.7 µM for KM12L4, RKO, HCT-116 and HT-29 human colon cancer cells, respectively. This cytotoxicity correlated with the expression of the α5 integrin on human colon cancer cells with a correlation coefficient of 0.78. The mechanism involved in the cytotoxic effect of lunasin was through cell cycle arrest and induction of the mitochondrial pathway of apoptosis. In KM12L4 human colon cancer cells, lunasin caused a G2/M phase arrest increasing the percentage of cells at G2/M phase from 12% (PBS-treated) to 24% (treated with 10 µM lunasin). This arrest was attributed to the capability of lunasin to increase the expression of cyclin dependent kinase inhibitors p21 and p27. At 10 µM, lunasin increased the expression of p21 and p27 in KM12L4 colon cancer cells by 2.2- and 2.3-fold, respectively. Flow cytometric analysis showed that lunasin at 10 µM increased the percentage of cells undergoing apoptosis from 13.6% to 24.7%. This is further supported by fluorescence microscopic analysis of KM12L4 cells treated with 10 µM lunasin showing chromatin condensation and DNA fragmentation. The mechanism involved is through modification of proteins involved in the mitochondrial pathway of apoptosis in KM12L4 cells as 10 µM lunasin reduced the expression of the anti-apoptotic Bcl-2 protein by 2-fold and increased the expression of the pro-apoptotic proteins Bax, cytochrome c and nuclear clusterin by 2.2-, 2.1- and 2.3- fold, respectively. This led to increased expression and activity of the executioner of apoptosis, caspase-3 by 1.8- and 2.3-fold, respectively. This pro-apoptotic property of lunasin can be attributed to its capability to internalize into the cytoplasm and nucleus of colon cancer cells 24 h and 72 h after treatment, respectively. In addition, lunasin mediated metastasis of colon cancer cells in vitro by inhibiting the focal adhesion kinase activation thereby reducing expression of extracellular regulated kinase and nuclear factor kappa B and finally inhibiting migration of colon cancer cells. In KM12L4 colon cancer cells, 10 µM lunasin resulted in the reduction of phosphorylation of focal adhesion kinase and extracellular regulated kinase by 2.5-fold, resulting in the reduced nuclear translocation of p50 and p65 NF-κB subunits by 3.8- and 1.4-fold, respectively. In an in vivo model of CRC liver metastasis, daily intraperitoneal administration of lunasin at 4 mg/kg body weight resulted in the inhibition of KM12L4 liver metastasis as shown by the reduction of the number of liver metastases from 28 (PBS-treated) to 14 (lunasin-treated, P = 0.047) and reduction in tumor burden as measured by liver weight/body weight from 0.13 (PBS-treated) to 0.10 (lunasin-treated, P = 0.039). Moreover, lunasin potentiated the anti-metastatic effect of the chemotherapeutic drug oxaliplatin given at 5 mg/kg body weight twice per week. Lunasin and oxaliplatin combination resulted in a more potent inhibition of outgrowth of KM12L4 cell metastases to the liver reducing the number of liver metastases by 6-fold and reducing the tumor burden in the liver by 3-fold when compared to PBS-treated group. This can be attributed by the capability of lunasin and oxaliplatin to reduce expression of proliferating cell nuclear antigen in liver-tumor tissue as measured by immunohistochemical staining. The results of this research for the first time demonstrated the anti-colon cancer potential of lunasin isolated from defatted soybean flour which might contribute to the chemopreventive effect of soybean in CRC as seen in different epidemiological studies. In conclusion, lunasin isolated from defatted soybean flour mediated colon carcinogenesis by inducing apoptosis and preventing outgrowth of metastasis. We suggest that the results of this research serve as a basis for further study on the chemopreventive effect of lunasin against CRC and a possible adjuvant role for lunasin in therapy of patients with metastatic CRC.

Efecto citotóxico de hipoclorito de sodio y EDTA sobre células madre de pulpa dental

Introducción. La presente investigación surge de la importancia que implica la irrigación en relación a la regeneración del complejo dentino-pulpar. En la actualidad, una gran cantidad de artículos han enfocado sus objetivos en el efecto antibacteriano de soluciones irrigantes, sin embargo, son pocas las investigaciones que evalúan el efecto de dichas soluciones sobre las células madre presentes en la pulpa dental. Objetivo. Evaluar el efecto citotóxico de hipoclorito de sodio y EDTA sobre células madre de pulpa dental in vitro. Materiales y métodos. Se utilizaron Células madre de pulpa dental cortesía de la Unidad de Odontología Integral y Especialidades del Centro de Investigación y Desarrollo en Ciencias de la Salud. Para la evaluación de la citotoxicidad, se utilizó la solución de EDTA a concentraciones de 1.7%, 1%, 0.5% y 0.1% y NaOCl al 0.25%, 0.15%, 0.1% y 0.5% por triplicado. Ambas moléculas (EDTA y NaOCl) se incubaron con las células a 37° C, 5% de CO2 en una atmosfera a 95% de humedad durante 3 y 24 horas. Los controles negativos fueron incubados con PBS. Resultados. En los resultados se observó una alta toxicidad del NaOCl frente a las DPSCs a 3 y 24 horas de exposición. Así mismo se observó que la combinación del NaOCl y el EDTA no disminuyó el efecto citotóxico sobre las células, a excepción de la combinación de EDTA .1% + NaOCl .05% la cual presentó un ligero aumento en la viabilidad celular. Por otra parte, todas las concentraciones evaluadas de la solución de EDTA fueron menos toxicas cuando se incubaron durante 3 y 24 horas frente a las células. Conclusiones. De acuerdo a los resultados observados en el presente estudio podemos concluir lo siguiente: el NaOCl presentó alta citotoxicidad en todas sus concentraciones a las 3 y 24 horas de exposición, el EDTA presentó una mayor viabilidad a las 3 y 24 horas cuando se utilizó al 0.5% y 0.10%, la combinación del EDTA y NaOCl resultó menos citotóxica cuando se utilizó al 0.1% y 0.5% respectivamente a las 3 horas de exposición.

Dipsacus asperoides (Xue Duan) inhibits spinal cord injury-induced inflammatory responses in rats

Purpose: To investigate the effect of Dipsacus asperoides (Xue Duan), a traditional Chinese medicine, on rats with spinal cord injury (SCI). Methods: In this study a total of 40 adult rats were used after inducing SCI where Xue Duan was applied on experimental group and phosphate-buffered saline (PBS) was administered in corresponding control groups. Intraperitoneal administration of both compounds for a period of four weeks (28 days) was carried out at a dose of 10 mg/kg/day. Bright field microscopy was performed on the tissues. Results: Bright Field microscopy of tissue sections showed significant reduction in cavity area that resulted from injury, that is from 0.19 ± 0.05 mm2 to 0.09 ± 0.03 mm2 (p < 0.01) in untreated and treated groups respectively. Similarly western blotting results showed a decrease in the expression of NF-kB p65 and I-kBα (p < 0.01). These two compounds are important in increasing secondary pathophysiology in SCI. The results for MPO activity also revealed significantly reduced infiltration of leukocytes to the injury site (p < 0.01). Conclusion: This study reveals the positive effect of the plant material in reducing inflammation in rats with traumatic SCI.

AGRIFIS - simulador de prospecção de cenários e avaliação de projetos de produção de sementes, óleo in natura e biodiesel de mamona.

Este trabalho propõe um modelo de prospecção de cenários e avaliação de projetos de atividades econômicas de produção agrícola de mamona caracterizada como agricultura familiar, extração de óleo vegetal in natura de sementes de mamona e produção de biodiesel (transesterificação), na forma de cadeia produtiva integrada. O modelo utiliza indicadores de avaliação econômico-flnanceiro, ambiental e social, como por exemplo, VPL, TIR, PBS, PBD, custo de produção, renda familiar, número de famílias assentadas e funcionários empregados, e outros, para identificar o desempenho de cada proposta de projeto. Para tanto, foi desenvolvido um ambiente computacional "AGRIFIS" para abrigar o modelo proposto nesse trabalho, que é uma ferramenta de apoio à tomada de decisão para investimentos em cadeia produtiva de mamona, pois se pode estimar o fluxo de caixa futuro de uma determinada proposta de projeto, incluindo as lucratividades, as despesas, a rentabilidade das famílias assentadas para determinadas condições pré-estabelecidas.

Effect of Aloe vera extract on the improvement of the respiratory activity of leukocytes of matrinxã during the transport stress

This study evaluated the effect of extract of Aloe vera in the transport water of matrinxã (Brycon amazonicus) fish on stress response and leukocyte respiratory activity. Fish was transported for 4 h in water containing Aloe at levels 0; 0.02; 0.2 and 2 mg/L, and sampled before transport 2, 4, 24 and 96 h after for determination of plasma glucose and respiratory activity of leukocytes. An additional in vitro assay was conducted with another fish species, pacu (Piaractus mesopotamicus), to test the respiratory burst of leukocytes exposed to Aloe extract (0.0, phosphate-buffered saline (PBS) only) at 0.1, 0.2, 0.5 and 1 mg/L). Plasma glucose increased after 2 and 4 h of transport and returned to control levels within 24 h, but the addition of Aloe in the transport water did not affect the level of blood glucose. However, at 2 h of transport, Aloe enhanced the respiratory activity of leukocytes in a dose-dependent way. The highest value of respiratory burst activity of leukocytes was observed in the fish transported in water containing Aloe at 2 mg/L. The enhancing effect of the plant extract on the production of oxygen radicals was confirmed in vitro in leukocytes of pacu incubated in Aloe at concentrations 0.1 and 0.2 mg/L. The results suggest that Aloe vera is a modulator of the immune system in fish improving the innate immune response tested.

Ocorrência da infecção por Toxoplasma gondii e avaliação da imunização em caprinos do sertão do cabugi, Rio Grande do Norte

Toxoplasmosis is one zoonosis caused by Toxoplasma gondii protozoan. Goats, amongst the production animals, are one of the species most susceptible to this parasite, being one them main involved agents in ovine and goat abortions, determining great economic losses and implications for public health, since the presence it parasite in the products of goat origin, consist in one of the main sources of infection for the man. In this study 244 blood samples in 8 farms situated in 4 cities from the Sertão do Cabugi region, Rio Grande do Norte State, northeast of Brazil and, tested by ELISA assay. The results had shown a prevalence of 47.13% for anti- T. gondii antibodies and a significant association between positivity and variable evaluated as age, locality and property. The IgG avidity assay evaluated in 115 positive samples was carried to discriminate acute and chronic infection. Twelve samples (10.4%) had presented antibodies of low avidity while 103 (89.6%) presented high avidity antibodies; indicating that most of the animals was precocious exposure to the parasite. Significant difference was verified only for the variable sex. We also evaluate the capacity of recombinant adenoviruses codifying SAG1, SAG2, SAG3 and CMV in inducing activation of specific immune response in goat. These 109 animals received 109 pfu of the AdSAG1, AdSAG2, AdSAG3, AdCMV or PBS in vaccine protocol with 3 immunizations. Serum samples of the each animal, before and after mmunization, had been submitted to the ELISA. The results demonstrate that the immunizations had induced the production of IgG antibodies specific against T. gondii proteins

Microspheres for Liver Radiomicrospheres Therapy and Planning

Liver cancer accounts for nearly 10% of all cancers in the US. Intrahepatic Arterial Radiomicrosphere Therapy (RMT), also known as Selective Internal Radiation Treatment (SIRT), is one of the evolving treatment modalities. Successful patient clinical outcomes require suitable treatment planning followed by delivery of the microspheres for therapy. The production and in vitro evaluation of various polymers (PGCD, CHS and CHSg) microspheres for a RMT and RMT planning are described. Microparticles with a 30±10 µm size distribution were prepared by emulsion method. The in vitro half-life of the particles was determined in PBS buffer and porcine plasma and their potential application (treatment or treatment planning) established. Further, the fast degrading microspheres (≤ 48 hours in vitro half-life) were labeled with 68Ga and/or 99mTc as they are suitable for the imaging component of treatment planning, which is the primary emphasis of this dissertation. Labeling kinetics demonstrated that 68Ga-PGCD, 68Ga-CHSg and 68Ga-NOTA-CHSg can be labeled with more than 95% yield in 15 minutes; 99mTc-PGCD and 99mTc-CHSg can also be labeled with high yield within 15-30 minutes. In vitro stability after four hours was more than 90% in saline and PBS buffer for all of them. Experiments in reconstituted hemoglobin lysate were also performed. Two successful imaging (RMT planning) agents were found: 99mTc-CHSg and 68Ga-NOTA-CHSg. For the 99mTc-PGCD a successful perfusion image was obtained after 10 minutes, however the in vivo degradation was very fast (half-life), releasing the 99mTc from the lungs. Slow degrading CHS microparticles (> 21 days half-life) were modified with p-SCN-b-DOTA and labeled with 90Y for production of 90Y-DOTA-CHS. Radiochemical purity was evaluated in vitro and in vivo showing more than 90% stability after 72 and 24 hours respectively. All agents were compared to their respective gold standards (99mTc-MAA for 68Ga-NOTA-CHSg and 99mTc-CHSg; 90Y-SirTEX for 90Y-DOTA-CHS) showing superior in vivo stability. RMT and RMT planning agents (Therapy, PET and SPECT imaging) were designed and successfully evaluated in vitro and in vivo.

PROTEIN-TRIGGERED SUSTAINED DRUG RELEASE CONTROLLED BY APTAMERS BOUND TO OLIGOMER-CROSSLINKED ALGINATE

Sustained drug release systems provide many advantages over traditional delivery methods such as extending the time in which the drug is found to be within an effective concentration within the therapeutic window, which decreases the frequency of administration of the drug, and increases patient compliance. Research using polyacrylamide crosslinked by oligomers containing an aptamer sequence, has demonstrated a pulsatile release over 50 minutes triggered by a 2 mM target adenosine concentration. This thesis aims to build off this concept by designing a system that delivers in a sustained manner when triggered by micromolar target concentrations reflective of disease in vivo, using macromolecular targets. For example, the disease wet age related macular degeneration (wet AMD) is associated with increased concentrations of the protein vascular endothelial growth factor (VEGF-A) – a macromolecule. Patients with wet AMD would benefit from the implantation of devices or microspheres that release drugs in a sustained manner in response to local VEGF concentrations. In this thesis, we hypothesize that the protein lysozyme, used to demonstrate proof-of-concept, could trigger the increased release of drugs from oligomer-crosslinked alginate. The objectives are to (i) demonstrate sustained release from alginate, (ii) design oligomer crosslinked alginate that degrades in response to lysozyme, and then (iii) use these systems to control the release of FITC-dextran with and without lysozyme. A series of control experiments and analyses were used to optimize the crosslinking of alginate by annealed oligomers. The cumulative release of FITC-dextran (MW 20,000) from oligomer crosslinked alginate increased by 3.4 μg when lysozyme (3 μM) was introduced at 48 hours, as opposed to controls which released only 0.2 μg. FITC-loaded alginate microspheres coated by oligomer-crosslinked alginate released 15% more FITC-dextran over 120 hours when placed into 3 μM of lysozyme than without lysozyme. Controls of alginate crosslinked with PEG or control oligomers (without a lysozyme aptamer sequence) had no changes in release with lysozyme. The incorporation of a lysozyme aptamer onto oligomers used to crosslink alginate disks or alginate coatings on microspheres resulted in different diffusion and release of FITC-dextran into PBS with or without lysozyme. This approach could be adapted for the delivery of drugs to diseases with specific protein profiles such as wet AMD.

MARS (Media Asset Retrieval System): Puget Sound’s Digital Archive

The MARS (Media Asset Retrieval System) Project is the collaborative effort of public broadcasters,libraries and schools in the Puget Sound region to create a digital online resource that provides access to content produced by public broadcasters via the public libraries. Convergence ConsortiumThe Convergence Consortium is a model for community collaboration, including organizations such as public broadcasters, libraries, museums, and schools in the Puget Sound region to assess the needs of their constituents and pool resources to develop solutions to meet those needs. Specifically, the archives of public broadcasters have been identified as significant resources for the local communities and nationally. These resources can be accessed on the broadcasters websites, and through libraries and used by schools, and integrated with text and photographic archives from other partners.MARS’ goalCreate an online resource that provides effective access to the content produced locally by KCTS (Seattle PBS affiliate) and KUOW (Seattle NPR affiliate). The broadcasts will be made searchable using the CPB Metadata Element Set (under development) and controlled vocabularies (to be developed). This will ensure a user friendly search and navigation mechanism and user satisfaction.Furthermore, the resource can search the local public library’s catalog concurrently and provide the user with relevant TV material, radio material, and books on a given subject.The ultimate goal is to produce a model that can be used in cities around the country.The current phase of the project assesses the community’s need, analyzes the current operational systems, and makes recommendations for the design of the resource.Deliverables• Literature review of the issues surrounding the organization, description and representation of media assets• Needs assessment report of internal and external stakeholders• Profile of the systems in the area of managing and organizing media assetsfor public broadcasting nationwideActivities• Analysis of information seeking behavior• Analysis of collaboration within the respective organizations• Analysis of the scope and context of the proposed system• Examining the availability of information resources and exchangeof resources among users

Alcances y limitaciones del tercer pilar de la doctrina de responsabilidad de proteger en la intervención militar de la crisis Libia (2011-2013)

El siguiente trabajo de investigación pretende analizar no solo la manera en la que la Intervención en la crisis Libia (2011-2013) se vio justificada a la luz de lo que establece la Doctrina de R2P, sino también investigar si logró cumplir con los objetivos de la misma. Con tal propósito, se evaluará si la intervención Militar Humanitaria por parte de la OTAN bajo la resolución 1973 del Consejo de Seguridad de la ONU, se justifica desde los objetivos que establece la Doctrina de Responsabilidad de Proteger y cumple parcialmente con los propósitos fundamentales de prevenir, reaccionar y reconstruir en la crisis Libia de 2011- 2013. El método que se utilizará para responder a la pregunta de este estudio de caso, será en su totalidad cualitativo, descriptivo y analítico. En lo que respecta a las fuentes, se usarán fuentes secundarias como artículos académicos y fuentes primarias provenientes de noticias de periódicos, revistas, resoluciones AG y del CS de la ONU.

Las empresas energéticas y la gran estrategia estadounidense hacia Irak : una reflexión sobre el rol de Exxon Mobil y Chevron como grupos económicos y de presión (2001- 2011)

La monografía pretende explicar el rol desempeñado por Exxon Mobil y Chevron en la formulación de la Gran Estrategia del gobierno Bush hacia Irak. Especialmente, se sostiene que las dos compañías multinacionales mencionadas lograron que la intervención militar en Irak, fuera pensada como un objetivo fundamental de la política energética del gobierno Bush. Para lograr este objetivo, Chevron y Exxon aprovecharon principalmente su posición en la economía nacional estadounidense. De hecho, lograron celebrar contratos a largo plazo para la extracción del crudo y de gas en Irak. Fundamentándose en un análisis documental, estas compañías son analizadas como grupos de presión empresarial y grupos económicos, cuyos beneficios derivados de la invasión en Irak pueden encontrarse incluso durante el gobierno Obama.