Ana Paula Benaduce

Boron nitride nanotube reinforced hydroxyapatite composite: mechanical and tribological performance and in-vitro biocompatibility to osteoblasts.

This study proposes boron nitride nanotube (BNNT) reinforced hydroxyapatite (HA) as a novel composite material for orthopedic implant applications. The spark plasma sintered (SPS) composite structure shows higher density compared to HA. Minimal lattice mismatch between HA and BNNT leads to coherent bonding and strong interface. HA-4 wt% BNNT composite offers excellent mechanical properties-120% increment in elastic modulus, 129% higher hardness and 86% more fracture toughness, as compared to HA. Improvements in the hardness and fracture toughness are related to grain refinement and crack bridging by BNNTs. HA-BNNT composite also shows 75% improvement in the wear resistance. The wear morphology suggests localized plastic deformation supported by the sliding of outer walls of BNNT. Osteoblast proliferation and cell viability show no adverse effect of BNNT addition. HA-BNNT composite is, thus, envisioned as a potential material for stronger orthopedic implants.

Wear behavior and in vitro cytotoxicity of wear debris generated from hydroxyapatite-carbon nanotube composite coating.

This work evaluates the effect of carbon nanotube (CNT) addition to plasma-sprayed hydroxyapatite (HA) coating on its tribological behavior, biocompatibility of the coating, and cytotoxicity of CNT-containing wear debris. Biological response of the CNT-containing wear debris is critical for osteoblasts, the bone-forming cells, and macrophages, the cells that clear up wear debris from blood stream. The addition of 4 wt % CNTs to HA coating reduces the volume of wear debris generation by 80% because of the improved elastic modulus and fracture toughness. CNT reinforcement has a pronounced effect on the particle size in the wear debris and subsequent biological response. There was a slight increase in the numbers and viability of osteoblasts grown on HA-CNT compared with HA alone. The cytotoxic effect of HA and HA-CNT debris to macrophages and osteoblasts was similar, demonstrating that loose CNT does not pose a problem to these cells.

Quantification of carbon nanotube induced adhesion of osteoblast on hydroxyapatite using nano-scratch technique

This paper explores the nano-scratch technique for measuring the adhesion strength of a single osteoblast cell on a hydroxyapatite (HA) surface reinforced with carbon nanotubes (CNTs). This technique efficiently separates out the contribution of the environment (culture medium and substrate) from the measured adhesion force of the cell, which is a major limitation of the existing techniques. Nano-scratches were performed on plasma sprayed hydroxyapatite (HA) and HA-CNT coatings to quantify the adhesion of the osteoblast. The presence of CNTs in HA coating promotes an increase in the adhesion of osteoblasts. The adhesion force and energy of an osteoblast on a HA-CNT surface are 17 ± 2 µN/cell and 78 ± 14 pJ/cell respectively, as compared to 11 ± 2 µN/cell and 45 ± 10 pJ/cell on a HA surface after 1 day of incubation. The adhesion force and energy of the osteoblasts increase on both the surfaces with culture periods of up to 5 days. This increase is more pronounced for osteoblasts cultured on HA-CNT. Staining of actin filaments revealed a higher spreading and attachment of osteoblasts on a surface containing CNTs. The affinity of CNTs to conjugate with integrin and other proteins is responsible for the enhanced attachment of osteoblasts. Our results suggest that the addition of CNTs to surfaces used in medical applications may be beneficial when stronger adhesion of osteoblasts is desired.

A mathematical model for growth in weight of silver catfish (Rhamdia quelen) (Heptapteridae, Siluriformes, Teleostei)

The use of a mathematical model applied to biological science helps to predict the specific data. Based on biological data (weight and age) of silver catfish, Rhamdia quelen, a mathematical model was elaborated based on a nonlinear difference equation to demonstrate the relationship between age and growth in weight. Silver catfish growth was described following the Beverton-Holt model Pt+1 = (r Pt) / (1+ a Pt ), where r > 0 is the maximum growth rate and a > 0 is a constant of growth inhibition. The solution of this equation is Pt= 1 /{[1/P0 - a / (r-1)] 1/rt + a/ (r-1)}, were P0 is the initial weight of the fish. Through this model it was observed that the female reaches the theoretical maximum weight approximately at the age of 18 years and the male at the age of 12 years in a natural environment.

High waterborne Mg does not attenuate the toxic effects of Fe, Mn, and Ba on Na+ regulation of Amazonian armored catfish tamoatá (Hoplosternum litoralle)

Formation water (FoW) is a by-product from oil and gas production and usually has high concentrations of soluble salts and metals. Calcium (Ca) and magnesium (Mg) have been shown to reduce the toxicity of metals to aquatic animals, and previous study showed that high waterborne Ca exerts mild effect against disturbances on Na+ regulation in Amazonian armored catfish tamoatá (Hoplosternum littorale) acutely exposed to high Fe, Mn, and Ba levels. Here, we hypothesized that high Mg levels might also reduce the toxic effects of these metals on Na+ regulation of tamoatá. The exposure to 5% FoW promoted an increase in Na+ uptake and a rapid accumulation of Na+ in all tissues analyzed (kidney<plasma<gills<carcass<liver), besides increasing the branchial activity of both NKA and v-type H+-ATPase in fish. High waterborne Mg lowered Na+ efflux rates and markedly inhibited Na+ uptake, and also reduced both NKA activity and newly Na+ accumulation in gills of fish. High Fe levels increased Na+ net losses and inhibited Na+ uptake in tamoatá. The diffusive Na+ losses and the newly accumulated Na+ in gills were reduced in fish exposed to high Mn and Ba. High waterborne Ba also inhibited NKA in gills, while both high Mn and Ba inhibited v-type H+-ATPase in kidney of tamoatá. High Mg did not lessen the toxic effect of Fe on Na+ net fluxes, and reduced even more Na+ uptake and the newly Na+ accumulation in gills and plasma, and did not prevent the inhibition of both NKA and v-type H+-ATPases in kidney. Furthermore, Mg did not attenuate the effect of Mn on inhibition Na+ uptake, keeping the activity of v-type H+-ATPase in kidney significantly lowered. High Mg levels mildly attenuated the effects of Ba in Na+ balance by increasing the new accumulation of Na+ in liver, and restore the activity of both NKA and v-type H+-ATPase in gills of tamoatá. Overall, high waterborne Mg does not have a strong contribution to, or have only minor effects, in protecting tamoatá against disruptions in Na+ regulation mediated by high Fe, Mn, and Ba levels.

Abstract 4992: Development of a novel radiation-induced targeted immunotherapy strategy through oligonucleotide aptamer conjugation

Monoclonal antibodies (mAbs) produce dramatic anti-tumor responses in multiple solid tumors. Unfortunately, systemic administration can result in end organ retention and dose-limiting toxicities. Radiotherapy (RT) is a standard-of-care treatment modality that induces direct tumor cell kill through DNA damage. While studies assessing the synergy of RT-induced tumor cell kill with mAbs are ongoing, an additional unexploited effect of RT is the induction of stress response products in the tumor microenvironment, such as vascular endothelial growth factor (VEGF). Oligonucleotide aptamers are short pieces of nucleic acid that exhibit little to no immunogenicity compared to mAbs, making them ideal delivery vehicles for therapeutic mAbs. The purpose of this study was to assess the feasibility of a novel strategy for targeted tumor delivery of existing immunotherapeutic mAbs through conjugation to ligands that bind to radiation induced products. An antibody recognizing the costimulatory molecule 41BB was covalently modified with a 21-mer ssDNA oligonucleotide linker sequence that serves as an anchor site for the VEGF aptamer synthesized with a 3’ extension. Annealing of complementary strands formed a 41BB mAb-VEGF aptamer conjugate. First, VEGF aptamer conjugation to modified 41BB mAb was verified by polyacrylamide gel electrophoresis analysis which revealed the expected size shift of the 41BB mAb-VEGF aptamer conjugate versus the unannealed controls. Conjugation was then verified using flow cytometry. Recombinant mouse 41BB-Fc was immobilized on Protein A Dynabeads and 41BB mAb conjugated with 5’ biotinylated VEGF aptamer. 41BB mAb-VEGF aptamer conjugate retention on Dynabeads was confirmed by using streptavidin-PE 5’ biotin of aptamer attached to mAb in conjugate binding epitope target. Functionality of 41BB mAb-VEGF aptamer conjugate was confirmed in vitro using a CD8+ T cell costimulation assay. CD8+ T cells were isolated from 8 week old female C57BL6/J splenocytes, and exposed to sub-optimal levels of CD3 mAb. 41BB mAb-VEGF aptamer conjugate, unconjugated 41BB mAb and isotype control were added to activated cells. Elevated levels of interferon gamma were observed and equivalent in conjugated and unconjugated 41BB mAbs. These results indicate that conjugation of 41BB mAb-VEGF aptamer is feasible and that conjugation does not affect 41BB mAb functionality. Use of this novel targeting approach to improve the therapeutic index of immunotherapeutic mAbs requires validation in murine tumor models. Citation Format: Ana Paula Benaduce, Randall Brenneman, Diana Cardero, Brett Schrand, Eli Gilboa, Adrian Ishkanian. Development of a novel radiation-induced targeted immunotherapy strategy through oligonucleotide aptamer conjugation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4992.

Abstract 5352: Ultraviolet radiation induces carcinogenesis in a novel transgenic mouse model of melanoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Melanomagenesis is caused by environmental factors, such as ultraviolet (UV) radiation, as well as genetic factors. The Nucleotide Excision Repair (NER) pathway repairs photoproducts induced by UV radiation. In humans, NER pathway disruption leads to the development of skin cancers, including melanoma. Upon UV exposure, mice deficient in Xpc, an important protein in the NER pathway, develop skin cancer; but not melanoma. The Endothelin 3 (Edn3) signaling pathway is essential for melanocyte precursor cell development. In humans, this pathway has also been associated with melanoma progression and its metastatic potential. The purpose of this study is the development of a UV-dependent melanoma mouse model that combines Xpc deficiency with the over-activation of the Edn3 pathway. Transgenic mice over-expressing Edn3 under the control of the keratin 5 promoter (K5-Edn3) and carrying a targeted mutation in Xpc were exposed to a single suberythemal neonatal dose of UV radiation. Immunostaining and histomorphology were used to confirm the melanocytic origin of primary skin tumors. Melanomas were only found in animals with the K5-Edn3 transgene. High penetrance was observed in Xpc null (67%, n=3) and Xpc heterozygous (67%, n=3) mice in comparison to Xpc wild type (33%, n=3) mice. RNA extracted from melanomas of Xpc null (n=2), Xpc heterozygous (n=1) and Xpc wild type (n=1) mice were screened for UV signature mutations in Hras, Kras and Nras. No mutations were found in any of these genes. These results indicate that UV radiation exposure, in conjunction with over-activation of the Edn3 pathway is sufficient to lead to melanomagenesis in mice independently of mutations in Hras, Kras or Nras. Citation Format: Ana Paula Benaduce, Deannys Batista, Gabriel Grilo, Karen Jorge, Diana Cardero, Clara Milikowski, Lidia Kos. Ultraviolet radiation induces carcinogenesis in a novel transgenic mouse model of melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5352. doi:10.1158/1538-7445.AM2014-5352

Abstract 1276: UV radiation interacts with Endothelin 3 and loss of nucleotide excision repair pathway to promote melanomagenesis.

Melanomagenesis is influenced by the interaction of environmental (such as UV radiation) and genetic factors. The Xpa gene is important in the Nucleotide Excision Repair (NER) pathway. Similar to humans afflicted with Xeroderma Pigmentosum, which results from mutations in genes that play a role in NER, Xpa deficient mice show high sensitivity to UV light, leading to skin cancer development. However, Xpa deficient mice do not develop melanoma even upon UV exposure. The Endothelin 3 (Edn3) signaling pathway is essential for proliferation, survival and migration of melanocyte precursor cells. In humans, this pathway has also been implicated in melanoma progression and its metastatic potential. The purpose of this study is to develop a UV induced melanoma mouse model that combines Xpa deficiency with the over-activation of the Edn3 pathway. To this end, transgenic mice over-expressing Edn3 under the control of the keratin 5 promoter (K5-Edn3) and carrying a targeted mutation in Xpa were exposed to a single suberythemal neonatal dose of UV radiation. A subgroup of mice was also exposed to a second dose of UV radiation at 6 weeks of age. Histomorphology and immunostaining were used to confirm the melanocytic origin of primary skin tumors and metastases. Melanoma was only found in animals with the K5-Edn3 transgene. High penetrance was observed in animals exposed to one dose of UV radiation that were Xpa null (100%, n=3) when compared to Xpa heterozygous (73%, n=11) or Xpa wild type (43%, n=7). Enlarged and hyperpigmented lymph nodes were present in animals with melanoma skin lesions and these were diagnosed as local metastases. These results indicate that UV radiation exposure, in conjunction with over-activation of the Edn3 pathway is sufficient to lead to melanomagenesis in mice. Citation Format: Ana Paula Benaduce, Deannys Batista, Gabriel Grilo, Karen Jorge, Lidia Kos. UV radiation interacts with Endothelin 3 and loss of nucleotide excision repair pathway to promote melanomagenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1276. doi:10.1158/1538-7445.AM2013-1276