2
S434 Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576 Fig. 1. Encapsulation procedure for IB3-1 cells (left) and DoE analysis (right). of the interleukin family, chemokines, growth factors and soluble forms of adhesion molecules. The experiments demonstrated that most of the analyzed pro- teins, were secreted both by the free and encapsulated cells, even if in a different extent. In order to determine the biotechnological applications of this procedure, we determined whether encapsu- lated IB3-1 cells, could be induced to pro-inflammatory responses, after treatment with TNF-alpha. As expected, TNF-alpha induced a sharp increase in the secretion of interleukins, chemokines and growth factors. Of great interest was the evidence that induction of IL-6 and IL-8 occurs also by encapsulated IB3-1 cells. Fig. 1. doi:10.1016/j.jbiotec.2010.09.612 [P-M.25] Variant Morphologies of Bacterial Inclusion Bodies Improve Substrate Material Properties that Support Mammalian Cell Pro- liferation Elena García-Fruitós 1,2 , Joaquín Seras 1,2 , Esther Vazquez 2,3,, Antonio Villaverde 2,3 1 CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Bellaterra08193 Barcelona, Spain 2 Institute for Biotechnology and Biomedicine, Universitat Autònoma de Barcelona, Bellaterra08193 Barcelona, Spain 3 Department of Genetics and Microbiology, Universitat Autònoma de Barcelona, Bellaterra08193 Barcelona, Spain Keywords: Bacterial inclusion bodies; Cell proliferation; Tissue engineering; Nanoparticle geometry A diversity of materials for biomedical applications is produced in bacteria, and some of them, such as metals, are straightfor- ward obtained as particulate entities. We have here explored the biofabrication process of bacterial inclusion bodies, particulate proteinaceous particles (ranging from 50 to 500 nm in diame- ter) that have been recently described as suitable materials for topographical modification of surfaces suitable for tissue engi- neering (Garcia-Fruitos et al., 2009). In general, inclusion bodies have been observed as spherical or pseudo-spherical particles with only minor morphological variability, mostly restricted to their size (Margreiter et al., 2008; Carrio et al., 1998; Peternel et al., 2008). However, we have identified a cellular gene in the producing bacte- ria Escherichia coli (clpP) that controls the in vivo fabrication process of inclusion bodies. A ClpP deficiency alters the natural dynamics of protein incorporation into the in-reconstruction protein particles. Then, when producing inclusion bodies formed by an aggregation- prone GFP, they show unusual tear-shaped forms with enhanced surface-volume ratios. These inclusion bodies promote mammalian cell attachment, growth and differentiation upon surface decora- tion significantly better that those produced in wild type cells and in other mutants in heat shock genes, as observed in both epithelial (BHK21) but especially in nervous cells (PC12). Implications of the genetic control of nanoparticle geometry are discussed in the con- text of the biological fabrication of protein-based nanoparticles and regarding the biomedical potential of bacterial inclusion bodies in regenerative medicine. References Garcia-Fruitos, E., Rodriguez-Carmona, E., Diez-Gil, C., Ferraz, R.M., Vazquez, E., Corchero, J.L., et al., 2009. Surface Cell Growth Engineering Assisted by a Novel Bacterial Nanomaterial. Advanced Materials 21, 4249–4253. Margreiter, G., Messner, P., Caldwell, K.D., Bayer, K., 2008. Size characterization of inclusion bodies by sedimentation field-flow fractionation. J Biotechnol 138, 67–73. Carrio, M.M., Corchero, J.L., Villaverde, A., 1998. Dynamics of in vivo protein aggre- gation: building inclusion bodies in recombinant bacteria. FEMS Microbiol Lett 169, 9–15. Peternel, S., Jevsevar, S., Bele, M., Gaberc-Porekar, V., Menart, V., 2008. New prop- erties of inclusion bodies with implications for biotechnology. Biotechnol Appl Biochem 49, 239–246. doi:10.1016/j.jbiotec.2010.09.613 [P-M.26] Bacterially produced inclusion bodies as biocompatible materi- als for substrate-dependent mammalian cell proliferation J. Seras 1,2 , C. Díez-Gil 4,1 , E. Vazquez 2,1 , S. Krabbenborg 4,1 , E. Rodríguez-Carmona 2,3 , J.L. Corchero 1,2 , R.M. Ferraz 2,3 , M. Cano- Sarabia 1,4 , I. Ratera 1,4 , N. Ventosa 1,4 , O. Cano 1,2 , N. Ferrer- Miralles 1,2 , E. García-Fruitós 1,2 , J. Veciana 1,4 , A. Villaverde 1,2,1 CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Spain 2 Institute for Biotechnology and Biomedicine, Universitat Autònoma de Barcelona, Spain 3 Department of Genetics and Microbiology, Universitat Autònoma de Barcelona, Spain 4 Department of Molecular Nanoscience and Organic Materials, Insti- tut de Ciencia de Materials de Barcelona (CSIC), Spain Keywords: Inclusion bodies; Tissue engineering; Biomaterials; Pro- tein engineering Bacterial inclusion bodies are particulate proteinaceous par- ticles (in the nano and microscales) commonly observed as by- products of recombinant protein production processes. Recently, we have described that these protein clusters, being mechanically stable and biocompatible, can be used as cost-effective substrate materials for the topographical modification of surfaces in the context of tissue engineering, as when decorating polystyrene culture plates, the growth of mammalian cells is significantly stim- ulated (Garcia-Fruitos et al., 2009). Interestingly, different types of mammalian cells respond differentially (regarding attachment and proliferation) to inclusion body variants of the same model protein obtained in different bacterial mutants, in which specific functions of the quality control have been genetically suppressed. This indicates that the nanoscale properties conferred to inclusion bodies by the aggregated protein, as embedded in alternative con- formational states, have a macroscopic eco in biological interfaces. This differential mechano-sensing cell reaction to the cell nano- environment can be progressively understood on the basis of the relevant nanoscale properties of protein particles that can be tuned, in a finest way, than in conventional materials resulting from chem- ical synthesis.

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Page 1: Bacterially produced inclusion bodies as biocompatible materials for substrate-dependent mammalian cell proliferation

S434 Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576

Fig. 1. Encapsulation procedure for IB3-1 cells (left) and DoE analysis (right).

of the interleukin family, chemokines, growth factors and solubleforms of adhesion molecules.

The experiments demonstrated that most of the analyzed pro-teins, were secreted both by the free and encapsulated cells, evenif in a different extent. In order to determine the biotechnologicalapplications of this procedure, we determined whether encapsu-lated IB3-1 cells, could be induced to pro-inflammatory responses,after treatment with TNF-alpha. As expected, TNF-alpha induceda sharp increase in the secretion of interleukins, chemokines andgrowth factors. Of great interest was the evidence that induction ofIL-6 and IL-8 occurs also by encapsulated IB3-1 cells.

Fig. 1.

doi:10.1016/j.jbiotec.2010.09.612

[P-M.25]

Variant Morphologies of Bacterial Inclusion Bodies ImproveSubstrate Material Properties that Support Mammalian Cell Pro-liferation

Elena García-Fruitós 1,2, Joaquín Seras 1,2, Esther Vazquez 2,3,∗,Antonio Villaverde 2,3

1 CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN),Bellaterra08193 Barcelona, Spain2 Institute for Biotechnology and Biomedicine, Universitat Autònomade Barcelona, Bellaterra08193 Barcelona, Spain3 Department of Genetics and Microbiology, Universitat Autònoma deBarcelona, Bellaterra08193 Barcelona, SpainKeywords: Bacterial inclusion bodies; Cell proliferation; Tissueengineering; Nanoparticle geometry

A diversity of materials for biomedical applications is producedin bacteria, and some of them, such as metals, are straightfor-ward obtained as particulate entities. We have here explored thebiofabrication process of bacterial inclusion bodies, particulateproteinaceous particles (ranging from 50 to 500 nm in diame-ter) that have been recently described as suitable materials fortopographical modification of surfaces suitable for tissue engi-neering (Garcia-Fruitos et al., 2009). In general, inclusion bodieshave been observed as spherical or pseudo-spherical particles withonly minor morphological variability, mostly restricted to their size(Margreiter et al., 2008; Carrio et al., 1998; Peternel et al., 2008).However, we have identified a cellular gene in the producing bacte-ria Escherichia coli (clpP) that controls the in vivo fabrication processof inclusion bodies. A ClpP deficiency alters the natural dynamics ofprotein incorporation into the in-reconstruction protein particles.Then, when producing inclusion bodies formed by an aggregation-prone GFP, they show unusual tear-shaped forms with enhancedsurface-volume ratios. These inclusion bodies promote mammaliancell attachment, growth and differentiation upon surface decora-

tion significantly better that those produced in wild type cells andin other mutants in heat shock genes, as observed in both epithelial(BHK21) but especially in nervous cells (PC12). Implications of thegenetic control of nanoparticle geometry are discussed in the con-text of the biological fabrication of protein-based nanoparticles andregarding the biomedical potential of bacterial inclusion bodies inregenerative medicine.

References

Garcia-Fruitos, E., Rodriguez-Carmona, E., Diez-Gil, C., Ferraz, R.M., Vazquez, E.,Corchero, J.L., et al., 2009. Surface Cell Growth Engineering Assisted by a NovelBacterial Nanomaterial. Advanced Materials 21, 4249–4253.

Margreiter, G., Messner, P., Caldwell, K.D., Bayer, K., 2008. Size characterization ofinclusion bodies by sedimentation field-flow fractionation. J Biotechnol 138,67–73.

Carrio, M.M., Corchero, J.L., Villaverde, A., 1998. Dynamics of in vivo protein aggre-gation: building inclusion bodies in recombinant bacteria. FEMS Microbiol Lett169, 9–15.

Peternel, S., Jevsevar, S., Bele, M., Gaberc-Porekar, V., Menart, V., 2008. New prop-erties of inclusion bodies with implications for biotechnology. Biotechnol ApplBiochem 49, 239–246.

doi:10.1016/j.jbiotec.2010.09.613

[P-M.26]

Bacterially produced inclusion bodies as biocompatible materi-als for substrate-dependent mammalian cell proliferation

J. Seras 1,2, C. Díez-Gil 4,1, E. Vazquez 2,1, S. Krabbenborg 4,1, E.Rodríguez-Carmona 2,3, J.L. Corchero 1,2, R.M. Ferraz 2,3, M. Cano-Sarabia 1,4, I. Ratera 1,4, N. Ventosa 1,4, O. Cano 1,2, N. Ferrer-Miralles 1,2, E. García-Fruitós 1,2, J. Veciana 1,4, A. Villaverde 1,2,∗

1 CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN),Spain2 Institute for Biotechnology and Biomedicine, Universitat Autònomade Barcelona, Spain3 Department of Genetics and Microbiology, Universitat Autònoma deBarcelona, Spain4 Department of Molecular Nanoscience and Organic Materials, Insti-tut de Ciencia de Materials de Barcelona (CSIC), SpainKeywords: Inclusion bodies; Tissue engineering; Biomaterials; Pro-tein engineering

Bacterial inclusion bodies are particulate proteinaceous par-ticles (in the nano and microscales) commonly observed as by-products of recombinant protein production processes. Recently,we have described that these protein clusters, being mechanicallystable and biocompatible, can be used as cost-effective substratematerials for the topographical modification of surfaces in thecontext of tissue engineering, as when decorating polystyreneculture plates, the growth of mammalian cells is significantly stim-ulated (Garcia-Fruitos et al., 2009). Interestingly, different typesof mammalian cells respond differentially (regarding attachmentand proliferation) to inclusion body variants of the same modelprotein obtained in different bacterial mutants, in which specificfunctions of the quality control have been genetically suppressed.This indicates that the nanoscale properties conferred to inclusionbodies by the aggregated protein, as embedded in alternative con-formational states, have a macroscopic eco in biological interfaces.This differential mechano-sensing cell reaction to the cell nano-environment can be progressively understood on the basis of therelevant nanoscale properties of protein particles that can be tuned,in a finest way, than in conventional materials resulting from chem-ical synthesis.

Page 2: Bacterially produced inclusion bodies as biocompatible materials for substrate-dependent mammalian cell proliferation

Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576 S435

Reference

Garcia-Fruitos, E., Rodriguez-Carmona, E., Diez-Gil, C., Ferraz, R.M., Vazquez, E.,Corchero, J.L., et al., 2009. Surface Cell Growth Engineering Assisted by a NovelBacterial Nanomaterial. Advanced Materials 21, 4249–4253.

doi:10.1016/j.jbiotec.2010.09.614

[P-M.27]

Structural Analysis of Toxic Waste -Induced Chromosome Aber-rations by Atomic Force Microscopy

Tarek Y.S. Kapiel 1,2,3, Narguess Hossameldin Marei 1,2,3,∗, KareemDorri Zaki 1,2,3, Ahmed M. Osman 1,2,3, Eman Galal Zakaria 1,2,3,Menattallah Elserafy 1,2,3

1 Biotechnology students faculty of sciences Cairo University, Egypt2 Botany Department, Faculty of Science, Cairo University, Egypt3 Biotechnology student at GUC (german university in cairo), EgyptKeywords: Chromosome aberration; Atomic force microscope(AFM)

Light microscopy has been used to analyse chromosome damagethat occurs following exposure of cells to chemical pollution. Thevarious types of chromosome aberration, i.e. chromosome breaks,chromosome exchange, chromatid gaps, chromatid breaks, etc.,have been classified by light microscopy. Complicated chromosomeaberrations are produced as a result of chemical toxic waste. Theatomic force microscope has become a useful tool to visualize bio-logical molecules at nanometer resolution. We adapt a new methodemploying Atomic Force Microscope AFM for nanometer-levelstructural analysis of chromosome damage induced by chemicaltoxic waste. Damaged metaphase chromosomes induced by chem-ical pollution were marked under a light microscope. Then thedetailed structure of chromosomes was visualized by the AFM.The height data of chromosomes obtained by AFM provided usefulinformation to distinguish chromatid gaps and breaks and muchother chromosome aberration. The structure of the various typesof chromosome aberration such as break point regions induced bychemical toxic waste was imaged by AFM. Many types of biologi-cal molecules including DNA and protein have been observed in airand/or water by AFM. Here, we report a new method using AFMto analyse the chromosome structure in the region of DNA damageinduced by heavy chemical pollution to further analyse the struc-tural changes in chromatin. These observations indicated that AFMis a useful tool for analysis of chromosome aberrations induced bychemical pollution.

doi:10.1016/j.jbiotec.2010.09.615

[P-M.28]

Applying Random Graphs for Proteomic Data Analysis of RCCPatients

M. Antoniotti 1, M. Borsani 1, E. Gianazza 2, F. Magni 2, G. Mauri 1, I.Zoppis 1,∗

1 Department of Informatics, Systems and Communication. Universityof Milano-Bicocca., Italy2 Department of Experimental Medicine. University of Milano-Bicocca.,ItalyKeywords: Proteomics; Random Graphs

Among the several “omics” techniques (Kolker, 2009), pro-teomics represents a promising area to define new biomarkers in

Table 1H0 VS HA. Observed average values for different periods of the follow-up

Statistics Time Av. Obs. Value Av. Crit. Value Power

1 0 – 1 23 ≥ 17 0.77791 1 – 2 28 ≥ 17 0.73471 2 – 3 24 ≥ 18 0.75771 3 – 4 20 ≥ 15 0.82542 0 – 1 2 ≥ 2 ≈ 12 1 – 2 4 ≥ 2 ≈ 12 2 – 3 9 ≥ 2 ≈ 12 3 – 4 9 ≥ 3 ≈ 13 0 – 1 247 ≤ 247 ≈ 13 1 – 2 245 ≤ 247 ≈ 13 2 – 3 240 ≤ 247 ≈ 13 3 – 4 239 ≤ 246 ≈ 14 0 – 1 10 ≥ 4 0.86344 1 – 2 8 ≥ 4 0.84424 2 – 3 46 ≥ 4 0.9114 3 – 4 12 ≥ 2 0.8505

biological fluids which can characterize and predict multi-factorialdiseases. Therefore, it is important also in this context to supportthe search with robust analysis which offers suitable models foravailable data. In this regard, we analyzed correlation structuresamong proteomic spectra of protein profiles during the follow-upperiod of Renal Cell Carcinoma (RCC) patients. The study of thesestructures can offer information about the protein expressions, andtheir structured presence can be useful in future studies of thebiomarker discovery problem. Specifically, these dependencies canrefer to “how information about a region of the spectra helps to inferthe modification of other regions over time”.

This analysis was performed first by adapting magnetic bead-based purification (ClinProt system) followed by matrix-assistedlaser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS) to profile human urine proteins (Bosso et al.,2008). Then, by applying random graph models (Bolloba, 1985):i.e. by assuming predefined families of (random graph) models, weanalyzed whether structured correlations between spectra regions(these regions whose intensity signals are subsequently correlatedin time) emerge from the real patient data. This evidence canemerge by evaluating different graph characteristics when used astests in a Neyman Pearson framework.

The main target of our tests is to show some evidence for realdata representation to be provided by models assessing struc-tured correlation between parts of the spectra (White et al., 1976).Following the Neyman-Pearson approach we want this evidenceagainst the hypothesis for these dependencies to be distributedsimply by chance (null hypothesis H0) (Gilbert and Random Graphs,1959; Erdos and Renyi, 1957). Numerical results permits to rejectthe null hypothesis in favour of the proposed alternative (Table 1).

We claim that it will be worthwhile to extend the work indifferent ways. We are immediately interested in the followingopportunities.

Exploring hypotheses with more suitable statistics and consid-ering different modular structures for the alternative (hypothesisHA) E.g., we could run experiments with indexes taken from the richliterature on the social network analysis (Wasserman and Faust,1994).

We analysed subsequent spectra over time even if the existenceof structures can also emerge when considering wider periods ofthe follow-up.

Finally, we used correlation as measure for analysing dependen-cies even if more suitable measures can be considered as well (forinstance mutual information).

References

Kolker, E., 2009. OMICS: A Journal of Integrative Biology 13 (6), 451.