Department of Food Processing Technology and Management

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Start date: 2010End date: 2019

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    THE INFLUENCE OF SWELLING ON LOCAL ELASTIC PROPERTIES OF POLYACRYLAMIDE HYDROGELS
    (Open Science Framework, 2017) Ramesh, Subramani; Alicia, Izquierdo-Alvarez; Pinaki, Bhattacharya; Mathieu, Meerts; Paula, Moldenaers; Herman, Ramon; Hans Van, Oosterwyck
    Polyacrylamide (PAM)hydrogelsarecommonly usedas soft cell culture substrates for cell mechanical and mechanobiological studiesbecause oftheir tunable stiffness,easeof handling, transparent natureand surface functionalization to promote cell adhesion. The dependence of bulk rheological and local elastic properties (for example, as assessed by means of Atomic Force Microscopyor AFM) of PAM hydrogels onmonomer and cross-linkerconcentrationsand on polymerization temperature havebeen extensively investigated.PAM hydrogels are known to swell, which may affect their elastic properties and therefore may complicate the interpretation ofcell culturingexperiments because of a lack of control of substrate stiffness. Direct measurements of the effect of swelling on PAM elastic properties are scarce. We report here, for the first time, the direct observation of swelling (by measuring the volumetric swelling ratio) and its influence onlocal elastic properties, as measuredby AFM. Bulk rheological measurements were performed as well to enable the comparison between local and global elastic properties during and after hydrogel polymerization. Four PAM hydrogel compositions were considered,with corresponding storage shear moduliof 4530 Pa(termed stiffest), 2900 Pa(stiff), 538 Pa(soft)and 260 Pa(softest) as measured immediately after polymerization. These values as well as values obtained during hydrogel polymerization were found to be in good agreement with the local elastic moduli derived from AFM and assuming hydrogel incompressibility. After polymerization, the hydrogels were subjected to swelling conditions over six days in phosphate buffered salineand swelling ratios and local elastic moduli were measured each day.Additional short term measurements (at 0, 3, 6, 9, 12 and 24 hours) were performed for the soft and softest gelsto study their swelling kinetics in more detail. Swelling ratio and local elastic modulus did not change with time for the stiffest and stiff gels, while for the soft and softest gels substantialchanges between Day 0 and Day 1were found for both swelling ratio (21.6%and 133% increase for soft and softest gel respectively) and local elastic modulus (33.7%and 33.3% decrease for soft and softest gel respectively). Experimental data werefurther analysed theoretically by combining models of ideal elastomeric gels with a poroelastic swelling kinetics model, which confirmed the validity of observed trends with respect to literature data. The results demonstrate that swelling can have an important effect on PAM elastic properties and must be taken into account when using PAM as a cell culture substrate, particularly for PAM gels with low monomer and cross-linker concentrations
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    IMMERSED BOUNDARY MODELS FOR QUANTIFYING FLOW-INDUCED MECHANICAL STIMULI ON STEM CELLS SEEDED ON 3D SCAFFOLDS IN PERFUSION BIOREACTORS
    (PLOS Computational Biology, 2016-09-22) Yann, Guyot; Bart, Smeets; Tim, Odenthal; Ramesh, Subramani; Frank P, Luyten; Herman, Ramon; Ioannis, Papantoniou; Liesbet, Geris
    Perfusion bioreactors regulate flow conditions in order to provide cells with oxygen, nutrients and flow-associated mechanical stimuli. Locally, these flow conditions can vary depending on the scaffold geometry, cellular confluency and amount of extra cellular matrix deposition. In this study, a novel application of the immersed boundary method was introduced in order to represent a detailed deformable cell attached to a 3D scaffold inside a perfusion bioreactor and exposed to microscopic flow. The immersed boundary model permits the prediction of mechanical effects of the local flow conditions on the cell. Incorporating stiffness values measured with atomic force microscopy and micro-flow boundary conditions obtained from computational fluid dynamics simulations on the entire scaffold, we compared cell deformation, cortical tension, normal and shear pressure between different cell shapes and locations. We observed a large effect of the precise cell location on the local shear stress and we predicted flow-induced cortical tensions in the order of 5 pN/μm, at the lower end of the range reported in literature. The proposed method provides an interesting tool to study perfusion bioreactors processes down to the level of the individual cell’s micro-environment, which can further aid in the achievement of robust bioprocess control for regenerative medicine applications.
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    SPATIOTEMPORAL ANALYSES OF CELLULAR TRACTIONS DESCRIBE SUBCELLULAR EFFECT OF SUBSTRATE STIFFNESS AND COATING
    (Springer Link, 2019) Alicia, Izquierdo-Álvarez; Diego A, Vargas; Álvaro, Jorge-Peñas; Ramesh, Subramani; Marie-Mo, Vaeyens; Hans Van, Oosterwyck
    Cells interplay with their environment through mechanical and chemical interactions. To characterize this interplay, endothelial cells were cultured on polyacrylamide hydrogels of varying stiffness, coated with either fibronectin or collagen. We developed a novel analysis technique, complementary to traction force microscopy, to characterize the spatiotemporal evolution of cellular tractions: We identified subpopulations of tractions, termed traction foci, and tracked their magnitude and lifetime. Each focus consists of tractions associated with a local single peak of maximal traction. Individual foci were spread over a larger area in cells cultured on collagen relative to those on fibronectin and exerted higher tractions on stiffer hydrogels. We found that the trends with which forces increased with increasing hydrogel stiffness were different for foci and whole-cell measurements. These differences were explained by the number of foci and their average strength. While on fibronectin multiple short-lived weak foci contributed up to 30% to the total traction on hydrogels with intermediate stiffness, short-lived foci in such a number were not observed on collagen despite the higher tractions. Our approach allows for the use of existing traction force microscopy data to gain insight at the subcellular scale without molecular probes or spatial constraining of cellular tractions.
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    TWO-DIMENSIONAL NETWORK STABILITY OF NUCLEOBASES AND AMINO ACIDS ON GRAPHITE UNDER AMBIENT CONDITIONS: ADENINE, L-SERINE AND L-TYROSINE
    (Royal Society of Chemistry, 2010-03-09) Ilko, Bald; Sigrid, Weigelt; Xiaojing, Ma; Pengyang, Xie; Ramesh, Subramani; Mingdong, Dong; Chen, Wang; Wael, Mamdouh; Jianguo, Wang; Flemming, Besenbacher
    We have investigated the stability of two-dimensional self-assembled molecular networks formed upon co-adsorption of the DNA base, adenine, with each of the amino acids, L-serine and L-tyrosine, on a highly oriented pyrolytic graphite (HOPG) surface by drop-casting from a water solution. L-serine and L-tyrosine were chosen as model systems due to their different interaction with the solvent molecules and the graphite substrate, which is reflected in a high and low solubility in water, respectively, compared with adenine. Combined scanning tunneling microscopy (STM) measurements and density functional theory (DFT) calculations show that the self-assembly process is mainly driven by the formation of strong adenine–adenine hydrogen bonds. We find that pure adenine networks are energetically more stable than networks built up of either pure L-serine, pure L-tyrosine or combinations of adenine with L-serine or L-tyrosine, and that only pure adenine networks are stable enough to be observable by STM under ambient conditions.
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    BUILDING LAYER-BY-LAYER 3D SUPRAMOLECULAR NANOSTRUCTURES AT THE TEREPHTHALIC ACID/STEARIC ACID INTERFACE
    (Royal Society of Chemistry, 2011-07-14) Yinli, Li; Lei, Liu; Ramesh, Subramani; Yunxiang, Pan; Bo, Liu; Yanlian, Yang; Chen, Wang; Wael, Mamdouh; Flemming, Besenbacher; Mingdong, Dong
    By using the layer-by-layer deposition method, we build three dimensional (3D) supramolecular nanostructures by stacking small molecular species on top of the first buffer layer, which can be utilized to fabricate novel 3D supramolecular functional nanostructures.
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    GREEN SYNTHESIZED SILVER NANOPARTICLES: TOXICITY AGAINST POECILIA RETICULATA FISHES AND CERIODAPHNIA CORNUTA CRUSTACEANS
    (Springer Link, 2016-11-30) Ramachandran, Ishwarya; Baskaralingam, Vaseeharan; Sathappan, Shanthi; Subramani, Ramesh; Pitchaimani, Manogari,; Kannan, Dhanalakshmi; Sekar, Vijayakumar; Giovanni, Benelli
    Recently, the green synthesis of silver nanoparticles gained increasing attention due to interesting properties for optical, antimicrobial and pest control applications. However, their toxicity against micro-crustaceans and fishes has been scarcely explored, while most of the research efforts focused on mosquito control with the green-synthesized nanocomposites. In this study, we investigated the toxic effects of AgNO3, Cissus quadrangularis (Cq)-synthesized AgNPs and Cq extract in two different study models, the larvae of Poecilia reticulata fishes and adults of the micro-crustacean Ceriodaphnia cornuta. In both species, AgNO3 and Cq-AgNPs showed high mortality rates even if tested at very low doses. Molecular analysis revealed high DNA damages induced by Cq-AgNPs on both aquatic organisms. Furthermore, light microscopy studies evidenced lesions in the gills and vacuolization in the gills and in the abdomen of P. reticulata larvae. Overall, our research pointed out that the exposure of aquatic organisms to AgNO3 or green-fabricated AgNPs can damage fishes and crustaceans, posing noteworthy risks to the aquatic ecosystems.
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    SPATIOTEMPORAL ANALYSES OF CELLULAR TRACTIONS DESCRIBE SUBCELLULAR EFFECT OF SUBSTRATE STIFFNESS AND COATING
    (Springer Link, 2018-11-08) Alicia, Izquierdo-Álvarez; Diego A, Vargas; Álvaro, Jorge-Peñas; Ramesh, Subramani; Marie-Mo, Vaeyens; Hans, Van Oosterwyck
    Cells interplay with their environment through mechanical and chemical interactions. To characterize this interplay, endothelial cells were cultured on polyacrylamide hydrogels of varying stiffness, coated with either fibronectin or collagen. We developed a novel analysis technique, complementary to traction force microscopy, to characterize the spatiotemporal evolution of cellular tractions: We identified subpopulations of tractions, termed traction foci, and tracked their magnitude and lifetime. Each focus consists of tractions associated with a local single peak of maximal traction. Individual foci were spread over a larger area in cells cultured on collagen relative to those on fibronectin and exerted higher tractions on stiffer hydrogels. We found that the trends with which forces increased with increasing hydrogel stiffness were different for foci and whole-cell measurements. These differences were explained by the number of foci and their average strength. While on fibronectin multiple short-lived weak foci contributed up to 30% to the total traction on hydrogels with intermediate stiffness, short-lived foci in such a number were not observed on collagen despite the higher tractions. Our approach allows for the use of existing traction force microscopy data to gain insight at the subcellular scale without molecular probes or spatial constraining of cellular tractions.
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    PLASMODIUM FALCIPARUM-INFECTED ERYTHROCYTE KNOB DENSITY IS LINKED TO THE PFEMP1 VARIANT EXPRESSED
    (ASM Journals, 2015-10-06) Ramesh, Subramani; Katharina, Quadt; Anine E, Jeppesen; Casper, Hempel; Jens, Emil Vang Petersen; Tue, Hassenkam; Lars, Hviid; Lea, Barfod
    Members of the clonally variant Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family mediate adhesion of infected erythrocytes (IEs) to vascular receptors. PfEMP1 expression is normally confined to nanoscale knob protrusions on the IE surface membrane. To investigate the relationship between the densities of these IE surface knobs and the PfEMP1 variant expressed, we used specific antibody panning to generate three sublines of the P. falciparum clone IT4, which expresses the PfEMP1 variants IT4VAR04, IT4VAR32b, and IT4VAR60. The knob density in each subline was then determined by atomic force microscopy (AFM) and scanning electron microscopy (SEM) and compared to PfEMP1 and knob-associated histidine-rich protein (KAHRP) expression. Selection for uniform expression of IT4VAR04 produced little change in knob density, compared to unselected IEs. In contrast, selection for IT4VAR32b expression increased knob density approximately 3-fold, whereas IEs selected for IT4VAR60 expression were essentially knobless. When IT4VAR60+ IEs were subsequently selected to express IT4VAR04 or IT4VAR32b, they again displayed low and high knob densities, respectively. All sublines expressed KAHRP regardless of the PfEMP1 expressed. Our study documents for the first time that knob density is related to the PfEMP1 variant expressed. This may reflect topological requirements to ensure optimal adhesive properties of the IEs.
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    ROBUST SCALABLE SYNTHESIS OF A BIS-UREA DERIVATIVE FORMING THIXOTROPIC AND CYTOCOMPATIBLE SUPRAMOLECULAR HYDROGELS
    (Royal Society of Chemistry, 2019-06-06) Laurens A J, Rutgeerts; Al, Halifa Soulta; Ramesh, Subramani; Burak, Toprakhisar; Herman, Ramon; Monissa C, Paderes; Wim M, De Borggraeve; Jennifer, Patterson
    Synthetic hydrogels address a need for affordable, industrially scalable scaffolds for tissue engineering. Herein, a novel low molecular weight gelator is reported that forms self-healing supramolecular hydrogels. Its robust synthesis can be performed in a solvent-free manner using ball milling. Strikingly, encapsulated cells spread and proliferate without specific cell adhesion ligands in the nanofibrous material.
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    IMMERSED BOUNDARY MODELSFOR QUANTIFYING FLOW-INDUCED MECHANICAL STIMULI ON STEM CELLS SEEDED ON 3D SCAFFOLDS IN PERFUSION BIOREACTORS
    (PLOS Computational Biology, 2016-09-22) Yann, Guyot; Bart, Smeets; Tim, Odentha; Ramesh, Subramani; Frank P, Luyten; Herman, Ramon; Ioannis, Papantoniou; Liesbet, Geris
    Perfusion bioreactors regulate flow conditions in order to provide cells with oxygen, nutrients and flow-associated mechanical stimuli. Locally, these flow conditions can vary depending on the scaffold geometry, cellular confluency and amount of extra cellular matrix deposition. In this study, a novel application of the immersed boundary method was introduced in order to represent a detailed deformable cell attached to a 3D scaffold inside a perfusion bioreactor and exposed to microscopic flow. The immersed boundary model permits the prediction of mechanical effects of the local flow conditions on the cell. Incorporating stiffness values measured with atomic force microscopy and micro-flow boundary conditions obtained from computational fluid dynamics simulations on the entire scaffold, we compared cell deformation, cortical tension, normal and shear pressure between different cell shapes and locations. We observed a large effect of the precise cell location on the local shear stress and we predicted flow-induced cortical tensions in the order of 5 pN/μm, at the lower end of the range reportedin literature. The proposed method provides an interesting tool to study perfusion bioreactors processes down to the level of the individual cell’s micro-environment, which can furtheraid in the achievement of robust bioprocess control for regenerative medicine applications.