Flow cytometer results of the live/dead assay. We found that both CBNs reduced the concentration of media proteins. After 2Â h of exposure, fibroblasts exposed to GFs exhibited a fast-mechanical regulation, a 10% decrease in modulus and after 24Â h, the modulus had decreased by 20%. A wide range of test methods is available for measuring modulus, but there is currently some uncertainty within parts of the user community about the reliability where Fts is the tip-sample indentation force (N), E is the elastic Youngâs modulus (Pa) of the sample, Î´ is the sample mean indentation, \(\nu\) is the Poissonâs ratio (defined as 0.5 for soft live cells), and R is the microsphere radius. Nat Nanotechnol. Light fâ¦ https://doi.org/10.1016/j.jtemb.2017.01.005. https://doi.org/10.1007/s10616-010-9263-3. https://doi.org/10.1111/j.1539-6924.2010.01446.x. Explicitly state where your pillow is behaving elastically? Molecular dynamics analysis, as well as experimental studies, had shown that 2D carbon nanomaterials have enhanced protein adsorption from culture media [30, 31]. The excess of ROS results in additional glutathione residues to the actin monomers during the polymerization process of F-actin . However, the introduced analysis of the length modifications in the actin networks once exposed to CBNs and their estimation of cellâs Young modulus indicates a deleterious effect on the mechanical stability of living cells that was opposite to the results of the viability assays. In the experiments, the viability assays lead to infer that both CBNs are innocuous. Cells were seeded in culture media overnight on 5Â cm diameter Petri dishes at a concentration of 2.5âÃâ106Â cell/mL. Disaggregation of actin filaments has resulted in reducing the cellâs Youngâs modulus, and it has been used as a correlation between the F-actin network and the cellâs mechanical stability . No significant differences were observed between the two nanomaterials by t test. Result Precautions. Yue H, Wei W, Yue Z, Wang B, Luo N, Gao Y, et al. Gavara N, Chadwick RS. 2016;110:2528â39. Method for quantitative measurements of the elastic modulus of biological cells in AFM indentation experiments. https://doi.org/10.1021/nl060177c. The lack of F-actin filaments results in lower Youngâs modulus values [20,21,22,23,24]. https://doi.org/10.1016/j.carbon.2007.03.033. De Nicola M, Bellucci S, Traversa E, De Bellis G, Micciulla F, Ghibelli L. Carbon nanotubes on Jurkat cells: effects on cell viability and plasma membrane potential. A-Level-Physics-HQ---Experiment-Worksheet---Young's-Modulus-v.3. There are some other numbers exists which provide us a measure of elastic properties of a material. Martens JC, Radmacher M. Softening of the actin cytoskeleton by inhibition of myosin II. Youngâs Modulus(Experimental) : 8465.746 Ksi 2. Results are expressed as the meanâÂ±âstandard deviation (SD). Integrating physiological regulation with stem cell and tissue homeostasis. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Meyer G, Amer NM. https://doi.org/10.1039/C2IB20117H. Subramanian V, Semenzin E, Hristozov D, Zabeo A, Malsch I, McAlea E, et al. 2017;5:41. https://doi.org/10.3389/fcell.2017.00041. Variation of mechanical property of single-walled carbon nanotubes-treated cells explored by atomic force microscopy. Graphene-based nanomaterials: biological and medical applications and toxicity. 2 Results. All authors read and approved the final manuscript. Physicochemical characteristics of nanomaterials that affect pulmonary inflammation. Carbon nanotubes disrupt iron homeostasis and induce anemia of inflammation through inflammatory pathway as a secondary effect distant to their portal-of-entry. c GFs outside the cell near the cellâs membrane. PubMed CentralÂ Then, cells were incubated for 2 and 24Â h at 37Â Â°C under 5% CO2 atmosphere. These interference behaviors raise concerns about the reliability of toxicity assays that may lead to uncertainty and contradictory results [6,7,8,9]. Neuron. University. The planar sheet shape and larger size of the GFs than the MWCNTs are likely responsible for modifications to the cellsâ environment, such as media protein adsorption . We also acknowledge Dr. John Gonzalez for his cooperation using the flow cytometer at the Faculty of Medicine, Universidad de Los Andes. J Nanobiotechnol 17, 32 (2019). https://doi.org/10.1016/j.injury.2007.02.007. The SSA is a critical parameter in in vitro assays that evaluate the response of cells to nanomaterial exposure, as detected on the basis of catalytic activity, acidity/alkalinity, absorption, fluorescence signaling, adsorption capacity, and metabolic dynamics . Cookies policy. 2010;267:89â105. Therefore, shape and dimensionality are becoming relevant parameters to define the potential toxicity of NMs [42, 43]. The data collected in the video can be used to complete a table and graph. An important assumption in the analysis using this model is that the microsphere indentation is much smaller than the finite cell height (10â20% of the cell thickness) to minimize artifacts in extracted elasticity maps originating from contributions of the hard substrate due to the finite cell thickness . Three replicates per CBN were tested, each group has a complete field image of the well, see Additional file 1. Crist RM, Grossman JH, Patri AK, Stern ST, Dobrovolskaia MA, Adiseshaiah PP, et al. The percentage of cells expressing ROS production was two times higher on cells exposed to GF than in those exposed to MWCNT, Fig.Â 3. TableÂ 1 summarizes the characterization results carried out in phosphate buffer solution (PBS) and culture media (DMEM) as well as the information provided by the manufacturer. 2010;5:e9445. 2003;37:1â17. 1984;119:58â64. Rev Policy Res. Springer Nature. 2014;276:12â24. Youngâs Modulus (Y) =stress/strain Youngâs modulus can be used in the following equation: F = ð(âð³ ð³ð)ð¨ In this equation, F is equal to the force applied to the structure, Y is the Youngâs modulus for the material, ÎL is the change in length of the material when the force is applied to it, L 0 is the initial length, and A is https://doi.org/10.1002/anie.201209099. Burgess RJ, Agathocleous M, Morrison SJ. To estimate the elastic Youngâs modulus of the fibroblast cells, the forceâvolume (FâV) mode was used. The relative trigger force used was 2Â nN to minimize the cell deformation and the damage to the cells. Sanchez VC, Jachak A, Hurt RH, Kane AB. J Phys Chem B. 2014;811:73â91. YOUNGâS MODULUS PRACTICAL OBJECTIVE â¢ To determine the Youngâs Modulus of a steel wire using a graphical method. 2008;456:95â100. Freestone B, Krishnamoorthy S, Lip GY. Sci Rep. 2015;5:10886. https://doi.org/10.1038/srep10886. Today's classroom version of the same experiment is typically performed using a laser beam as the source. Graphene oxide flakes (XG Sciences Inc, Lansing, MI, USA) were suspended in 1Ã PBS at 1Â mg/mL. Elastic Youngâ modulus maps and three-dimensional topographic reconstruction of live NIH3T3 in culture media before and after exposure to CBNs: a unexposed cells, b cells exposed 2Â h to MWCNTs, c cells exposed 24Â h to MWCNTs, d cells exposed 2Â h to GFs and e cells exposed 24Â h to GFs. Sokolov I, Dokukin ME, Guz NV. Dimitriadis EK, Horkay F, Maresca J, Kachar B, Chadwick RS. In addition, the introduced force/volume technique permit to simultaneously quantify a single cells volume variation at different periods that has provided information of the process of cell death. The FâZ curve rate was set to 1Â Hz (1Â s) to minimize viscoelastic contributions . 2013;18:2473â507. In this study, the images were obtained with a resolution of 32âÃâ32 pixels, with an approximate acquisition time of 35Â min per image. Previous works have concentrated on studying the toxicity effects based on the SSA . The results demonstrated that MWCNTs and GF induced cytotoxicity in fibroblast at different timescales. 10Â Âµg of each sample was placed on an XploRA confocal Raman spectrometer (Horiba Scientific, Kyoto, Japan; located at Universidad de Los Andes). The protein quantification was carried out using the Pierce bicinchoninic acid assay (BCA) (ThermoFisher, Cat 23225, MD, USA). The samples were then centrifuged at 12,000Â rpm for 10Â min. The Young Modulus is named after Thomas Young (1773 to 1829) who was a British polymath, contributing to our understanding of optics, physiology, and Egyptology, among other fields. According to various experimental observations and results, the magnitude of the strain produced in a given material is the same irrespective of the fact whether the stress is tensile or compressive. J R Soc Interface. Schmidt DR, Waldeck H, Kao WJ. https://doi.org/10.1016/j.bpj.2013.12.037. This website and its content is subject to our Terms and The photodetector optical sensitivity was obtained by extracting the slope of the FâZ curve. London WC1R 4HQ. 2008. https://doi.org/10.1038/nprot.2008.23. NIH3T3 fibroblasts were exposed for 24Â h to 10 and 30Â Âµg/mL CBNs. The medium was then removed, and the wells were washed twice with PBS 1Ã containing Mg2+. ArticleÂ 'Young's Modulus' or modulus of elasticity is a measurement of the rate of change of strain as a function of stress. https://doi.org/10.1111/risa.12394. By comparing the effects of uniaxial tensile load on the change of the deflection curve with gravity, the relation between the actual and directly measured tensile Young's modulus is obtained. Our study is focused on the cellular mechanical response, which mainly depends on cytoskeletal integrity . Course. Nature. After 24Â h of exposure to MWCNTs adsorbed 21.5Â Âµg/mL of albumin, in contrast, the exposure to GFs had albumin adsorption of 127Â Âµg/mL, see Fig.Â 7. 2010;62:1â16. Carbon. Our Objective. Cartagena-Rivera AX, Logue JS, Waterman CM, Chadwick RS. In: Puleo D, Bizios R, editors. 2010;30:1634â44. In light of these problems, international organizations such as the National Institute for Occupational Safety and Health (NIOSH), the Organization for Economic Cooperation and Development (OECD), the European Community (EC), and various non-governmental organizations (NGOs) are urging the development of label-free in vitro methods that avoid interference with NMs and improve assaysâ reliability [10, 11]. ArticleÂ Chai Hao. 2006;6:1261â8. 2002;13:33â7. Multimedia University. 1Â Î¼L of the supernatant of each sample was diluted to 1:100, 1:1000 and 1:10,000 with deionized water. The slide with its slits is most commonly purchased from a manufacturer who provides a measured value for the slit separation distance - the d value in Young's equation. The assessment of CBNâs cytotoxicity has been highly debated recently. These assays are strongly dependent on the properties of the nanomaterials, for example; specific surface area (SSA), size, surface defects, and surface charge, and the host response. Biomaterials. Gourlay CW, Ayscough KR. This AFM mode is based on the acquisition of low-speed, quasi-static FâZ curves predefined by the user grid of points, resulting in the simultaneous acquisition of topography and material properties maps . The control cells exceeded, up to eight times, the length of the fibers of the cells exposed to both CBNs (MWCNT and GF). It is reported that 2D nanomaterials as GF are more likely to provide a better surface for protein residues anchorage where the proteins do not require as much deformation as with more rigid 1D CNTs. Albumin and mammalian cell culture: implications for biotechnology applications. Finally, calcein-AM green dye (Life Technologies, Carlsbad, CA, USA) for cell viability, DHE dye (Life Technologies, Carlsbad, CA, USA) for ROS production, and Hoechst 33342 dye (Life Technologies, Carlsbad, CA, USA) for DNA staining were added. Journal of Nanobiotechnology Pastrana, H.F., Cartagena-Rivera, A.X., Raman, A. et al. Cells were incubated for an additional 2Â h and 24Â h. The cells were then washed twice with warm culture medium (37Â Â°C), and 2Â mL of fresh medium was added to each dish. The PBS has removed washing with distilled water twice. J Am Chem Soc. Cells were maintained at 37Â Â°C during the AFM experiments using a Petri dish holder and a heater on the MFP-3D scanner XâY stage (Asylum Research, Santa Barbara, CA, USA). The length of the actin fibers in cells exposed to GF was 50% shorter than the fibers of the cells exposed to MWCNT. Discher DE, Janmey P, Wang Y-L. Tissue cells feel and respond to the stiffness of their substrate. Hilal N, Bowen WR, Alkhatib L, Ogunbiyi O. The length of the actin fibers can be used as a descriptor for the cytoskeleton disruption that explains the changes on cells Youngâs modulus induced by the exposure of CBNs, at a concentration of 50Â Âµg/mL. J Phys Chem B. It has been previously demonstrated that rather than introduce toxicity by the presence of iron, MWCNTs promotes iron metabolism sequestration inducing cellular inflammation [45, 47]. Cytotechnology. Ou L, Song B, Liang H, Liu J, Feng X, Deng B, et al. To Dr. Helena Groot and Diana Narvaez for access to the cell culture facilities at Genetics Laboratory Universidad de Los Andes. Kroll A, Dierker C, Rommel C, Hahn D, Wohlleben W, Schulze-Isfort C, et al. Inherent characterization of nanomaterials, as well as the host response and metabolic conditions, is required to identify the relevant properties related to nanomaterials toxicity; otherwise, the results are meaningless [38, 39]. https://doi.org/10.1007/978-0-387-98161-1_1. Youngâs modulus, also known as the tensile modulus, elastic modulus or traction modulus (measured in Pa, which is a pressure unit(N.m^-2; 10MPa is equivalent to 1Kg force per square millimeter) is a mechanical property of linear elastic materials. The Young Modulus is also known as Young's Modulus or the elastic modulus or tensile modulus.. The damage on actin fibers provides evidence of cellular stress due to ROS production. 2015;10:2423â50. Injury. Ma J, Li R, Liu Y, Qu G, Liu J, Guo W, et al. Local viscoelastic properties of live cells investigated using dynamic and quasi-static atomic force microscopy methods. Sci Rep. 2016;6:28443. https://doi.org/10.1038/srep28443. Lab Report Young Modulus Matriculation â A lab report is basically how you describe what youâve carried out in a lab experiment, what you discovered, and the outcomes. J Funct Biomater. Metabolic regulation of stem cell function. Surface curvature relation to protein adsorption for carbon-based nanomaterials. Kroll A, Pillukat MH, Hahn D, Schnekenburger J. After 24Â h, 100Â ÂµL of MWCNTs or GFs at 1Â mg/mL were added to each well to a final concentration of 50Â Âµg/mL. It is a measure of the stiffness of a material that is independent of the particular sample of a substance. 2 Apparatus required PubMedÂ The more detailed results are provided below. There is more protein adsorption, ten times, in GF regarding their SSA. Toxicol Appl Pharmacol. https://doi.org/10.1016/j.neuron.2011.05.011. 2006;84:282â92. By contrast, at longer exposure times (24Â h), the cellsâ metabolic stress was induced by ROS production, and cytoskeleton damage, which led to an increase in cell volume, probably as result of sodium/potassium pumps dysfunction . Significant at pâ<â0.05, n: 1000 points per group, t-test. Part Fibre Toxicol. Results 1. Our results of physicochemical characterization (size, SSA, and impurities) of the CBNs differ from the ones provided by manufacturers. Elastic Modulus Measurement J D Lord and R Morrell Abstract: Elastic modulus is an intrinsic material property and a key parameter in engineering design and materials development. Some statics formulae and data plots led to finding the latter data during the experiment. It is more evident when the adsorbed proteins are calculated considering the SSA; in the case of MWCNT, its protein adsorption regarding the SSA is 3.04Â Âµg/m2 while the GF had protein adsorption of 33.5Â Âµg/m2. Front Cell Dev Biol. Mammalian iron homeostasis in health and disease: uptake, storage, transport, and molecular mechanisms of action. 2013;52:4986â97. Toxicol Lett. Young's modulus, denoted by the symbol 'Y' is defined or expressed as the ratio of tensile or compressive stress (Ï) to the longitudinal strain (Îµ). However, other characteristics related to shape and dimensionality can be determinant on NMs behavior into the organisms. 2008;20:474204. https://doi.org/10.1088/0953-8984/20/47/474204. The incorporation of carbon-based nanomaterials in novel and everyday technologies requires to overcome its potential toxic effects by its beneficial properties; it is particularly true for those technologies in intimate contact with biological structures . The values of Youngâs Modulusfor Elasticity (E) for 0.3% carbon (mild) steel, 60/40 brass, Mahogany (hardwood) and Parana Pine (softwood) have been successfully determined from laboratory experiments as 31.4, 16.2, 2.84, 1.41 in (x106psi) respectively and compared with some published results showing good correlation. 2011;8:78â84. AR responsible for document preparation, substantial contribution on experimental design, results analysis and technical support during experiment. They found a correlation between the filamentous actin cytoskeleton disruption and the cellsâ Youngâs modulus decrease on cells that had phagocytose the nanoparticles [58, 59]. CASÂ Nanda SS, Yi DK, Kim K. Study of antibacterial mechanism of graphene oxide using Raman spectroscopy. Youngâs modulus Experiment Youngâs modulus Experiment. In summary, our results suggest that toxicity assessments need to consider additional techniques apart from the traditional, here demonstrated with the integration of the single the cellsâ mechanical properties. HFP was responsible for document preparation, biological experimental set-up, AFM analysis, cytometry measurements, nanomaterials characterization, LEAP systems experiment and protein adsorption. Kay AR. The effective uptake of CBNs into the cells depends on nanomaterial properties (size, shape, surface charge, protein corona) and the cell line (fibroblast, macrophage, neuron) . The MFP3D-Bio AFM system (Asylum Research, Santa Barbara, CA, USA) located in the Birck Nanotechnology Center at Purdue University was used in this work. 2007;45:1425â32. PubMed CentralÂ Nevertheless, after 24Â h of exposure to MWCNT the Youngâs modulus dramatically decrease by 13.6% compared with 2Â h results. AC was responsible for AFM experimentation, AFM analysis, document preparation, biological experimentation. Our study shows the dependence of the Youngâs modulus on exposure time. Biophys J. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Here are some of the subjects to search for on the cover of a lab report: Risk Anal. 3. NIH3T3 cells were seeded overnight in a specific 96-well plate (Cyntellect, San Diego, CA, USA) for laser-enabled analysis and processing (LEAPâ¢) (Cyntellect, San Diego, CA, USA; located at Bindley, Purdue University) at 5âÃâ105 cell/mL concentration in culture media. Cytoskeleton damage is a sign of cellular homeostasis dysfunction, due to cytotoxic products such as high concentration of radical oxygen species (ROS) . The Young modulus is often regarded as the quintessential material property, and students can learn to measure it. The results from live/dead assay confirms a slightly higher cytotoxic effect expected from MWCNT probably secondary to a larger SSA, nickel impurities, and facilitated cell uptake. Atomic force microscopy is a powerful tool for the measurement of mechanical properties of cells in their physiological environment [56,57,58,59,60]. Recently, excessive ROS production has been demonstrated to be exogenously induced by CBNs [25,26,27,28,29]. The yellow arrow indicates a GF. Visalli G, FacciolÃ A, Iannazzo D, Piperno A, Pistone A, Di Pietro A. However, the quantitative connections related to the ROS production and the cell mechanical properties with the CBNsâ dimensionality, such as the cylindrical MWCNT with one dimension out the nanoscale against planar GF with two dimensions, remains poorly understood. The role of the iron catalyst in the toxicity of multi-walled carbon nanotubes (MWCNTs). J Biomed Nanotechnol. This behavior has been previously demonstrated via molecular dynamics simulations, where ÏâÏ stacking with the aromatic residues of proteins showed to be reduced in cylindrical nanostructures, limiting the adsorption [30, 31, 36, 65]. We cultured the NIH3T3 in DMEM (Gibco, Waltham, Massachusetts, USA) containing a low concentration of d-glucose (1000Â mgÂ Lâ1), 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA), 1% penicillinâstreptomycin (Invitrogen, Carlsbad, CA), and 0.1% amphotericin B (Sigma-Aldrich, St. Louis, MO). Of fibroblast cells, no major variation on the cell near the nucleus modulus constant of 6063,... Group of cells in AFM indentation experiments assessment: limitations and challenges the GFs exhibited a planar shape to! Is designed to determine Youngâs modulus and shear modulus â¦ Youngâs modulus on time! Viability assays lead to uncertainty and contradictory results [ 6,7,8,9 ] nanotube-induced:! Liu YX, Karsai a, Pillukat MH, Hahn D, Scherthan Ionizing... To any trainee whoâs working in the toxicity effects based on the force applied but! Cells exposed to both CBNs normal NMs exposure in the live/dead assays Frennesson CI the gated group of labeled. Show an increased ROS production that does not only depend on the force volume using... Lead to uncertainty and contradictory results [ 6,7,8,9 ] nanotubes cell interaction Wang W-H Geahlen! The commonly used DCF test for oxidative stress really show determine Youngâs was... Pietro a mechanical properties responses at various times allowed understanding the effects of surface curvature and surface characteristics carbon-based! Cytotoxicity in fibroblast at different timescales [ 53 ] and ex vivo and microtubules is how easily is... Bowen WR, Alkhatib L, Ogunbiyi O Adiseshaiah PP, et al Additional file 1 ( live/dead cell ). On cells toxicity from both CBNs, at a concentration of 50Â Âµg/mL Hattori h, Wei W, Z... Before starting experiment characterization of Multiwall carbon nanotubes cell young's modulus experiment results cell exposed to MWCNTs for h..., multi-frequency, and the wells were washed thoroughly with PBS 1Ã containing Mg2+ is how easily it bended. Interactions: are current in vitro methods in nanoparticle risk assessment: limitations and challenges h... Mondal S, Broggi F, et al an experiment and recording your results determination of lateral... Nitrogen was used as the adsorbate cell viability was observed by biochemical (. Was removed, excessive ROS production has been highly debated recently of 23 engineered nanomaterials a! Method of Young 's modulus experiment as dimensionality relation to uncertainty and contradictory results [ 6,7,8,9 ] characterization... The source out only in PBS, centrifuged, and impurities ) of cellsâ! Operating principle and applications of AFM are well documented in the supernatant after to. Complete a table and graph, Mondal S, Rape AD, Wang B Luo. Stress by ROS production [ 26 ] characteristics related to shape and dimensionality shape of CBN affects... Into the instrument ; nitrogen young's modulus experiment results used as the meanâÂ±âstandard deviation ( SD ) A.X. Raman... Zimmer CC, Liu Y, Qu G, Wang W-H, Geahlen RL Raman! Dhe ) as a secondary effect distant to their dimensionality, Pietroiusti a, Dierker C et!, biophysics and biomechanics of cells exposed to both CBNs some of these are Bulk modulus and shear â¦! Substantial contribution on experimental design, results analysis, data were analyzed up indentations... Image of the supernatant after the exposure to them CBNs are innocuous, Weber JK, DR... Epithelial cells in AFM indentation experiments length of actin monomers during the polymerization process of F-actin 19... And nanomedicine: concepts of nanotoxicology to satisfy this assumption, data interpretation, critical and... By calcein marker reactive oxygen species-induced actin glutathionylation controls actin dynamics in neutrophils in cells exposed 50Â! Sd ) evidence of cellular responses a Young 's modulus constant of MPa... Mwcnts seems to exert more intracellular effects interfering with cellular structures and breaking lysosomal increasing. The medium was then removed, and quantitative nanomechanical mapping of live cells using conical atomic force microscope tips also! ( Y ) after dehydration, samples were not exposed to CNBs cartagena-rivera,... Acute toxicity but induce intracellular reactive oxygen species-induced actin glutathionylation controls actin dynamics in.., harvested, centrifuged, and the wells were washed thoroughly with PBS with... Seeded in culture medium thin specimens biological and medical applications and toxicity GQ, Mu GG, al. The Youngâs modulus of a material that is independent of the graph use... Of two metal frames F1 and F2 10Â Âµg/mL, cellsâ viability had a marginal reduction of 2 for! Risk assessment: limitations and challenges 488 conjugated-phalloidin marker evidenced significant shortening on cell to. ) ( Fig.Â 4b ) 2020| Updated: Sep 8, 2020 was diluted to 1:100, 1:1000 1:10,000... To GF was 50 % shorter than the fibers in cells exposed to MWCNTs and GF induced cytotoxicity fibroblast., Eldawud R, Liu Y, et al Yi DK, K.. Which may be enhanced on planar nanomaterials such as graphene flakes 2020| Updated: Sep 8 2020. In vitro biochemical assays cytotoxicity than their SSA we evidenced that after 2Â h results before move. Subject to our Terms and Conditions NIH3T3 fibroblasts were exposed to both CBNs are triggered is registered England! Than the fibers of the CBNs differ from the Colciencias Colombia Scholarship Francisco Jose de Caldas 2012 were out...
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