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  • 1
    Language: English
    In: The Journal of Chemical Physics, 28 February 2014, Vol.140(8)
    Description: All atom molecular dynamics (MD) models provide valuable insight into the dynamics of biophysical systems, but are limited in size or length by the high computational demands. The latter can be reduced by simulating long term diffusive dynamics (also known as Langevin dynamics or Brownian motion) of the most interesting and important user-defined parts of the studied system, termed collective variables (colvars). A few hundred nanosecond-long biased MD trajectory can therefore be extended to millisecond lengths in the colvars subspace at a very small additional computational cost. In this work, we develop a method for determining multidimensional anisotropic position- and timescale-dependent diffusion coefficients ( D ) by analysing the changes of colvars in an existing MD trajectory. As a test case, we obtained D for dihedral angles of the alanine dipeptide. An open source Mathematica ® package, capable of determining and visualizing D in one or two dimensions, is available at https://github.com/lbf-ijs/DiffusiveDynamics . Given known free energy and D , the package can also generate diffusive trajectories.
    Keywords: Articles
    ISSN: 0021-9606
    E-ISSN: 1089-7690
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  • 2
    In: PLoS ONE, 2015, Vol.10(6)
    Description: Background & Aim TiO 2 nanoparticles have generally low toxicity in the in vitro systems although some toxicity is expected to originate in the TiO 2 -associated photo-generated radical production, which can however be modulated by the radical trapping ability of the serum proteins. To explore the role of serum proteins in the phototoxicity of the TiO 2 nanoparticles we measure viability of the exposed cells depending on the nanoparticle and serum protein concentrations. Methods & Results Fluorescence and spin trapping EPR spectroscopy reveal that the ratio between the nanoparticle and protein concentrations determines the amount of the nanoparticles’ surface which is not covered by the serum proteins and is proportional to the amount of photo-induced radicals. Phototoxicity thus becomes substantial only at the protein concentration being too low to completely coat the nanotubes’ surface. Conclusion These results imply that TiO 2 nanoparticles should be applied with ligands such as proteins when phototoxic effects are not desired - for example in cosmetics industry. On the other hand, the nanoparticles should be used in serum free medium or any other ligand free medium, when phototoxic effects are desired – as for efficient photodynamic cancer therapy.
    Keywords: Research Article
    E-ISSN: 1932-6203
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  • 3
    Language: English
    In: Journal of Chromatography A, 11 January 2013, Vol.1272, pp.50-55
    Description: ► Estimation for dynamic binding capacity from pressure drop data. ► Correlation between pore size and dynamic binding capacity. ► Estimation of dynamic binding capacity from a pressure drop for different thickness of grafted layer. ► Correlation of structural and chromatographic properties via pressure drop. Convective chromatographic media comprising of membranes and monoliths represent an important group of chromatographic supports due to their flow-unaffected chromatographic properties and consequently fast separation and purification even of large biological macromolecules. Consisting of a single piece of material, common characterization procedures based on analysis of a small sample assuming to be representative for the entire batch, cannot be applied. Because of that, non-invasive characterization methods are preferred. In this work pressure drop was investigated for an estimation of dynamic binding capacity (DBC) of proteins and plasmid DNA for monoliths with different pore sizes. It was demonstrated that methacrylate monolith surface area is reciprocally proportional to pore diameter and that pressure drop on monolith is reciprocally proportional to square pore size demonstrating that methacrylate monolith microstructure is preserved by changing pore size. Based on these facts mathematical formalism has been derived predicting that DBC is in linear correlation with the square root of pressure drop. This was experimentally confirmed for ion-exchange and hydrophobic interactions for proteins and plasmid DNA. Furthermore, pressure drop was also applied for an estimation of DBC in grafted layers of different thicknesses as estimated from the pressure drop data. It was demonstrated that the capacity is proportional to the estimated grafted layer thickness.
    Keywords: Convective Chromatographic Media ; Monoliths ; Pressure Drop ; Dynamic Binding Capacity ; Grafting ; Cim ; Chemistry
    ISSN: 0021-9673
    E-ISSN: 18733778
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  • 4
    Language: English
    In: Journal of Chromatography A, 11 October 2013, Vol.1311, pp.106-114
    Description: HPLC based analytical assay is a powerful technique that can be used to efficiently monitor plasmid DNA (pDNA) purity and quantity throughout the entire purification process. Anion exchange monolithic and non-porous particle based stationary phases were used to study the recovery of the different pDNA isoforms from the analytical column. Three differently sized pDNA molecules of 3.0 kbp, 5.2 kbp and 14.0 kbp were used. Plasmid DNA was injected onto columns under the binding conditions and the separation of the isoforms took place by increasing the ionic strength of the elution buffer. While there was no substantial decrease of the recovered supercoiled and linear isoforms of the pDNA with the increase of the plasmid size and with the increase of the flow rate (recoveries in all cases larger than 75%), a pronounced decrease of the oc isoform recovery was observed. The entrapment of the oc pDNA isoform occurred under non-binding conditions as well. The partial oc isoform elution from the column could be achieved by decreasing the flow rate of the elution mobile phase. The results suggested a reversible entrapment of the oc isoform in the restrictions within the pores of the monolithic material as well as within the intra-particle space of the non-porous particles. This phenomenon was observed on both types of the stationary phase morphologies and could only be connected to the size of a void space through which the pDNA needs to migrate. A prediction of reversible pDNA entrapment was successfully estimated with the calculation of Peclet numbers, , which defines the ratio between a convective and diffusive mass transport.
    Keywords: Plasmid DNA ; Isoform Separation ; Aex Chromatography ; Monolith ; Recovery ; Chemistry
    ISSN: 0021-9673
    E-ISSN: 18733778
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  • 5
    Language: English
    In: Journal of Chromatography A, 2011, Vol.1218(17), pp.2413-2424
    Description: The objective of this study was to investigate the behavior of large plasmids on the monolithic columns under binding and nonbinding conditions. The pressure drop measurements under nonbinding conditions demonstrated that the flow velocities under which plasmid passing monolith became hindered by the monolithic pore structure depended on the plasmid size as well as on the average monolith pore size; however, they were all very high exceeding the values encountered when applying CIM monolithic columns at their maximal flow rate. The impact of the ligand density and the salt concentration in loading buffer on binding capacity of the monolith for different sized plasmids was examined. For all plasmids the increase of dynamic binding capacity with the increase of salt concentration in the loading solution was observed reaching maximum of 7.1 mg/mL at 0.4 M NaCl for 21 kbp, 12.0 mg/mL at 0.4 M NaCl for 39.4 kbp and 8.4 mg/mL at 0.5 M NaCl for 62.1 kbp. Analysis of the pressure drop data measured on the monolithic column during plasmid loading revealed different patterns of plasmid binding to the surface, showing “car-parking problem” phenomena under certain conditions. In addition, layer thickness of adsorbed plasmid was estimated and at maximal dynamic binding capacity it matched calculated plasmid radius of gyration. Finally, it was found that the adsorbed plasmid layer acts similarly as the grafted layer responding to changes in solution's ionic strength as well as mobile phase flow rate and that the density of plasmid layer depends on the plasmid size and also loading conditions.
    Keywords: Plasmids ; Monoliths ; Convective Interaction Media (Cim) ; Dynamic Binding Capacity ; Purification ; Pressure Drop ; Chemistry
    ISSN: 0021-9673
    E-ISSN: 18733778
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  • 6
    Language: English
    In: Journal of chromatography, 2013, Vol.1276, pp.58-64
    Description: Purification of high quantities of human grade plasmid DNA is one of the most intensive production steps. Because of that several methods have been proposed, among them also chromatographic purification using methacrylate monoliths. Recently, a process comprising the combination of hydrophobic interaction (HIC) monolith and ion-exchange monolith was developed. In this work both chemistries were tried to be introduced on a single monolith. Methacrylate monoliths bearing octylamine groups, combination of butyl (C4) grafted methacrylate groups and diethylaminoethyl (DEAE) groups as well as grafted chains bearing both C4 and DEAE groups were prepared. All monoliths were investigated for their ionic and protein capacity and compared to conventional epoxy, C4, and DEAE methacrylate monoliths. Octylamine monolith and monolith bearing combination of C4 grafted methacrylate groups and DEAE groups were found to be the most promising candidates and were further tested for plasmid DNA (pDNA) dynamic binding capacity under ion-exchange (IEX) and HIC binding conditions and ability to separate open circular (OC) from supercoiled (SC) pDNA forms and RNA from pDNA. Since monolith bearing combination of grafted C4 methacrylate groups and DEAE groups was superior in all three tested features, exhibiting pDNA dynamic binding capacity of 4.7mg/ml under IEX conditions and 2.1mg/ml under HIC conditions, it was used for the development of a single step purification method and tested with pure pDNA as well as with cell lysate. Developed method removed over 99% of RNA, host cell proteins (HCP) and genomic DNA (gDNA) demonstrating capacity to purify around 1.5mg ofpDNA/ml of monolith from cell lysate. ; p. 58-64.
    Keywords: Hydrophobic Bonding ; Chromatography ; Purification Methods ; Rna ; Epoxides ; Plasmids ; Ion Exchange ; Hydrophobicity ; Humans ; Proteins ; Binding Capacity
    ISSN: 0021-9673
    Source: AGRIS (Food and Agriculture Organization of the United Nations)
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  • 7
    Language: English
    In: Journal of chromatography, 2013, Vol.1272, pp.50-55
    Description: Convective chromatographic media comprising of membranes and monoliths represent an important group of chromatographic supports due to their flow-unaffected chromatographic properties and consequently fast separation and purification even of large biological macromolecules. Consisting of a single piece of material, common characterization procedures based on analysis of a small sample assuming to be representative for the entire batch, cannot be applied. Because of that, non-invasive characterization methods are preferred. In this work pressure drop was investigated for an estimation of dynamic binding capacity (DBC) of proteins and plasmid DNA for monoliths with different pore sizes. It was demonstrated that methacrylate monolith surface area is reciprocally proportional to pore diameter and that pressure drop on monolith is reciprocally proportional to square pore size demonstrating that methacrylate monolith microstructure is preserved by changing pore size. Based on these facts mathematical formalism has been derived predicting that DBC is in linear correlation with the square root of pressure drop. This was experimentally confirmed for ion-exchange and hydrophobic interactions for proteins and plasmid DNA. Furthermore, pressure drop was also applied for an estimation of DBC in grafted layers of different thicknesses as estimated from the pressure drop data. It was demonstrated that the capacity is proportional to the estimated grafted layer thickness. ; p. 50-55.
    Keywords: Hydrophobic Bonding ; Chromatography ; Microstructure ; Prediction ; Plasmids ; Ion Exchange ; Proteins ; Binding Capacity ; Surface Area
    ISSN: 0021-9673
    Source: AGRIS (Food and Agriculture Organization of the United Nations)
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  • 8
    Language: English
    In: Journal of chromatography, 2013, Vol.1311, pp.106-114
    Description: HPLC based analytical assay is a powerful technique that can be used to efficiently monitor plasmid DNA (pDNA) purity and quantity throughout the entire purification process. Anion exchange monolithic and non-porous particle based stationary phases were used to study the recovery of the different pDNA isoforms from the analytical column. Three differently sized pDNA molecules of 3.0kbp, 5.2kbp and 14.0kbp were used. Plasmid DNA was injected onto columns under the binding conditions and the separation of the isoforms took place by increasing the ionic strength of the elution buffer. While there was no substantial decrease of the recovered supercoiled and linear isoforms of the pDNA with the increase of the plasmid size and with the increase of the flow rate (recoveries in all cases larger than 75%), a pronounced decrease of the oc isoform recovery was observed. The entrapment of the oc pDNA isoform occurred under non-binding conditions as well. The partial oc isoform elution from the column could be achieved by decreasing the flow rate of the elution mobile phase. The results suggested a reversible entrapment of the oc isoform in the restrictions within the pores of the monolithic material as well as within the intra-particle space of the non-porous particles. This phenomenon was observed on both types of the stationary phase morphologies and could only be connected to the size of a void space through which the pDNA needs to migrate. A prediction of reversible pDNA entrapment was successfully estimated with the calculation of Peclet numbers, Pe, which defines the ratio between a convective and diffusive mass transport. ; p. 106-114.
    Keywords: Purification Methods ; Anion Exchange ; Prediction ; Plasmids ; Mass Transfer ; High Performance Liquid Chromatography ; Ionic Strength
    ISSN: 0021-9673
    Source: AGRIS (Food and Agriculture Organization of the United Nations)
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  • 9
    Language: English
    In: Journal of Chromatography A, 08 February 2013, Vol.1276, pp.58-64
    Description: ► Single step purification of pDNA on miligram scale. ► Characterization of grafted HIC groups. ► Multidimensional purification of pDNA. ► Very fast separation of pDNA using monoliths. Purification of high quantities of human grade plasmid DNA is one of the most intensive production steps. Because of that several methods have been proposed, among them also chromatographic purification using methacrylate monoliths. Recently, a process comprising the combination of hydrophobic interaction (HIC) monolith and ion-exchange monolith was developed. In this work both chemistries were tried to be introduced on a single monolith. Methacrylate monoliths bearing octylamine groups, combination of butyl (C4) grafted methacrylate groups and diethylaminoethyl (DEAE) groups as well as grafted chains bearing both C4 and DEAE groups were prepared. All monoliths were investigated for their ionic and protein capacity and compared to conventional epoxy, C4, and DEAE methacrylate monoliths. Octylamine monolith and monolith bearing combination of C4 grafted methacrylate groups and DEAE groups were found to be the most promising candidates and were further tested for plasmid DNA (pDNA) dynamic binding capacity under ion-exchange (IEX) and HIC binding conditions and ability to separate open circular (OC) from supercoiled (SC) pDNA forms and RNA from pDNA. Since monolith bearing combination of grafted C4 methacrylate groups and DEAE groups was superior in all three tested features, exhibiting pDNA dynamic binding capacity of 4.7 mg/ml under IEX conditions and 2.1 mg/ml under HIC conditions, it was used for the development of a single step purification method and tested with pure pDNA as well as with cell lysate. Developed method removed over 99% of RNA, host cell proteins (HCP) and genomic DNA (gDNA) demonstrating capacity to purify around 1.5 mg of pDNA/ml of monolith from cell lysate.
    Keywords: Monolith ; Pdna ; Hic ; Iex ; Mix-Mode ; Purification ; Cim ; Chemistry
    ISSN: 0021-9673
    E-ISSN: 18733778
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  • 10
    Language: English
    In: Journal of Chromatography A, 2011, Vol.1218(17), pp.2438-2444
    Description: Binding of three different bacteriophages (phages), namely T7, lambda and M13 on methacrylate monoliths was investigated. Phage M13 exhibited the highest dynamic binding capacity of 4.5 × 10 pfu/mL while T7 and lambda showed capacity of 1 × 10 pfu/mL, all corresponding to values of around 1 mg/mL. Interestingly, capacity for lambda phage was increased 5-fold by increasing NaCl concentration in a loaded sample from 0 to 0.2 M while there was a constant capacity decrease for T7 and M13 phages. Under optimal conditions, recovery for all three phages approached 100%. Measurement of a pressure drop increase during loading enabled estimation of adsorbed phage layer thickness. At a maximal capacity it was calculated to be around 50 nm for T7 phage and 60 nm for lambda phage matching closely capside size thus indicating monolayer adsorption while 80 nm layer thickness was estimated for M13 phage showing its orientation along the pore.
    Keywords: Bacteriophage ; Purification ; Monoliths ; Layer Thickness ; Capacity ; Cim ; Chemistry
    ISSN: 0021-9673
    E-ISSN: 18733778
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