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Safety as well as efficiency issues in connection with bacterial backbone sequences should be carefully considered when designing new DNA vaccines or non-viral gene therapy approaches. Bacterial backbone sequences like antibiotic resistance markers or regulatory bacterial elements constitute biological safety risks and reduce the overall efficiency of the DNA agent. To overcome these problems the minicircle technology has been developed. But, despite all the obvious advantages, minicircles have so far not replaced their problem laden conventional counterpart in gene transfer applications what can be contributed to efficiency issues in large scale manufacturing. In this article we describe the combined efforts of experts in the field of minicircle development, large scale biomanufacturing and downstream process development to provide a new approach. The Recombination Based Plasmid Separation (RBPS) Technology, which has already solved crucial problems associated with minicircle-DNA production, has been developed further for this purpose. A novel parental plasmid exploiting advanced in vivo process coordination for restriction and subsequent degradation of miniplasmid-DNA will be introduced. Furthermore we describe the scale-up of minicircle-DNA production by fermentation in combination with high performance downstream processes including purification by ion exchange and hydrophobic interaction chromatography on monolithic material.

Understanding the cell-biomaterial interface from the very first contact is of crucial importance for their successful implementation and function in damaged tissues. However, the lack of bio- and mechano-analytical methods to investigate and probe the initial processes on the interface, especially in 3D, raises the need for applying new experimental techniques. In our study, optical tweezers combined with confocal fluorescence microscopy were optimized to investigate the initial cell-scaffold contact and to investigate its correlation with the material-dependent cell growth. By the optical tweezers-induced cell manipulation accompanied by force detection up to 100 pN and position detection by fluorescence microscopy, accurate adhesion dynamics and strength analysis was implemented, where several attachment sites were formed on the interface in the first few seconds. More importantly, we have shown that dynamics of cell adhesion on scaffold surfaces correlates with cell growth on the days scale, which indicates that the first seconds of the contact could markedly direct further cell response. Such a contact dynamics analysis on 3D scaffold surfaces, applied for the first time, can thus serve to predict scaffold biocompatibility.

Downstream processing of nanoplexes (viruses, virus-like particles, bacteriophages) is characterized by complexity of the starting material, number of purification methods to choose from, regulations that are setting the frame for the final product and analytical methods for upstream and downstream monitoring. This review gives an overview on the nanoplex downstream challenges and chromatography based analytical methods for efficient monitoring of the nanoplex production.

Reducing experimental variability, limiting contamination and increasing automation are essential goals in the development of reliable analytical platforms for mass spectrometry (MS)-based proteomics. In this work novel trypsin-based monolithic immobilized enzyme reactors (tryp-IMERs), obtained by covalent immobilization on convective interaction media (CIMac™) analytical columns (5mm×5.2mm I.D.), were developed. Notwithstanding the small dimensions, column format allowed the insertion in common high performance liquid chromatography (HPLC) systems, thus avoiding the use of expensive micro- or nano-platforms. Monolith pore diameter and surface chemistry were optimized to achieve high digestion efficiency even with high molecular weight proteins and to avoid protein/peptide adsorption, peak broadening and sample loss. A full characterization of the tryp-IMERs was undertaken to select the best protocol for preparation and type of trypsin. Optimization of the operational and storage conditions was carried out by an off-line approach. On-line studies were performed by setting a multidimensional analytical platform, which included the tryp-IMER, a trapping column, an analytical C4 column and a high resolution hybrid mass spectrometer (ESI-Q-TOF). In the optimized conditions rapid protein digestion (90±9s), high protein coverage (≥60%) and high score values were achieved for five selected sample proteins (cytochrome c, myoglobin and albumins from different sources) differing in molecular size, isoelectric point and accessibility to cleavage sites as well as for a protein mixture of 200ng. The best performing tryp-IMERs showed high sensitivity down to the pmole level. The platform also resulted suitable for the analysis of high-molecular weight proteins such as a pool of human immunoglobulins G (hIgG) and for the high molecular weight fraction of human plasma proteins, which were digested in less than two minutes to an extent similar to that achieved by overnight incubation in a classical in solution protocol. Finally, underestimated key procedural issues were also highlighted during the study. Such aspects are of general interest both for tryp-IMER users and tryp-IMER developers.

High economic burden is associated with foodborne illnesses. Different disinfection methods are therefore employed in food processing industry; such as use of ultraviolet light or usage of surfaces with copper-containing alloys. However, all the disinfection methods currently in use have some shortcomings. In this work we show that copper doped TiO2 nanotubes deposited on existing surfaces and illuminated with ceiling mounted fluorescent lights can retard the growth of Listeria Innocua by 80% in seven hours of exposure to the fluorescent lights at different places in a food processing plant or in the laboratory conditions with daily reinocuation and washing. The disinfection properties of the surfaces seem to depend mainly on the temperature difference of the surface and the dew point, where for the maximum effectiveness the difference should be about 3 degrees celsius. The TiO2 nanotubes have a potential to be employed for an economical and continuous disinfection of surfaces.

High economic burden is associated with foodborne illnesses. Different disinfection methods are therefore employed in food processing industry; such as use of ultraviolet light or usage of surfaces with copper-containing alloys. However, all the disinfection methods currently in use have some shortcomings. Here we show that copper doped TiO2nanotubes deposited on existing surfaces and illuminated with ceiling mounted fluorescent lights or additional low power light emitting diodes can be employed for an economical and permanent disinfection of surfaces.We deposited the nanotubes on various surfaces: polyethylene terephatlate, polystyrene, and aluminum oxide, where they could withstand repeated washings with neutral, alkaline or acidic medium. Here we show that the polymer surfaces coated with the nanotubes and innoculated with 107bacteria, illuminated with ceiling mounted fluorescent lights retard the growth ofListeria Innocuaby up to 99% in seven hours of exposure to the fluorescent lights, compared to a control surface. The disinfection properties of the surfaces depend mainly on the temperature difference of the surface and the dew point, where for maximum effectiveness of the photocatalytic effect the difference should be at least 2.5 degrees celsius.Usage of one dimensional nanomaterials, such as TiO2nanotubes, offers a promising low cost alternative to current disinfection methods, since illumination of surfaces with common fluorescent lights is sufficient to photo-excite the nanotubes, which sequentially produce microbicidal hydroxyl radicals. Future use of such surfaces with antibacterial nano-coating and resulting sterilizing effect holds promise for such materials to be used in different environments or in better control of critical control points in food production as well as an improved biosecurity during the food manufacturing process.

International audience; An epoxy-activated monolithic Convective Interaction Media (CIM®) disk was used for the immobilization of endodextranase D8144 from Penicillium sp. (EC 3.2.1.11) in order to produce on-line isomalto-oligosaccharides (IMOs) from Dextran T40. Enzymatic parameters, molecular weight of IMOs and performance of the IMmobilized Enzymes Reactor (IMER) were investigated. The immobilization yield of enzymes was about 45.3% (w/w), and the real specific activity close to 3.26 U mg−1. The Km values did not significantly change between free (12.8 g L−1) and immobilized enzymes (14.2 g L−1), due to the absence of diffusional limitation. The IMER system presented more than 80% of its residual activity after 5000 column volumes, highlighting the high stability of the immobilized endodextranases. Response surface methodology was used to enhance the performance of the IMER. Depending on dextran concentrations and flow rates, specific patterns of IMOs distributions were observed during the enzymatic hydrolysis. Finally, prebiotic activity was also investigated on IMOs produced by medium conditions (flow rate 0.3 mL min−1 and dextran concentrations 4% w/w) against Lactobacillus rhamnosus GG (ATCC 53103). Their scores were at least as good as two commercialized fructo-oligosaccharides (FOS), Fibrulose® F97 and Orafti® P95.

This research demonstrates the basic elements of a prototype automated orchard sprayer which delivers pesticide spray selectively with respect to the characteristics of the targets. The density of an apple tree canopy was detected by PROWAVE 400EP250 ultrasound sensors controlled by a Cypress PSOC CY8C29466 microcontroller. The ultrasound signal was processed with an embedded computer built around a LPC1343 microcontroller and fed in real time to electro-magnetic valves which open/close spraying nozzles in relation to the canopy structure. The analysis focuses on the detection of appropriate thresholds on 15 cm ultrasound bands, which correspond to maximal response to tree density, and this was selected for accurate spraying guidance. Evaluationof the system was performed in an apple orchard by detecting deposits of tartrazine dye (TD) on apple leaves. The employment of programmable microcontrollers and electro-magnetic valves decreased the amountof spray delivered by up to 48.15%. In contrast, the reduction of TD wasonly up to 37.7% at some positions within the tree crown and 65.1% in the gaps between trees. For all these reasons, this concept of precise orchard spraying can contribute to a reduction of costs and environmental pollution, while obtaining similar or even better leaf deposits.

The purification of large viruses remains an important field of research and development. The development of efficient purification trains is restricted by limited analytical methods, as well as by the complexity of large viruses, as well as the high variability in starting material from cell culture. Vaccinia virus holds great potential as an oncolytic and immunotherapeutic vaccine against a broad spectrum of cancers. In this work, monolith-based capture and polishing chromatographic steps for vaccinia virus Lister strain has been developed. Virus produced in CV-1 cells was harvested and passed through a 0.8μm pre-filter before loading onto CIEX, AIEX and HIC CIM monoliths. Without the need for nuclease treatment, up to 99% of the total DNA loaded can be removed from the vaccinia feed stream by the CIM OH monolith, which also reduces the total protein concentration in the product pool to LLOQ levels, and achieves infectious virus recoveries of 90%. Binding capacities of greater than 1×109pfu of vaccinia per mL of matrix were obtained on both CIM SO3 and CIM OH monoliths. Multiple orthogonal analytical methods have been used to develop process knowledge and understanding.

Conformational entropy (SΩ) has long been used to theoretically characterize the dynamics of proteins, DNA, and other polymers. Though recent advances enabled its calculation also from simulations and nuclear magnetic resonance (NMR) relaxation experiments, correlated molecular motion has hitherto greatly hindered both numerical and experimental determination, requiring demanding empirical and computational calibrations. Herein, we show that these motional correlations can be estimated directly from the temperature-dependent SΩ series that reveal effective persistence lengths of the polymers, which we demonstrate by measuring SΩ of amphiphilic molecules in model lipid systems by spin-labeling electron paramagnetic resonance (EPR) spectroscopy. We validate our correlation-corrected SΩ meter against the basic biophysical interactions underlying biomembrane formation and stability, against the changes in enthalpy and diffusion coefficients upon phase transitions, and against the energetics of fatty acid dissociation. As the method can be directly applied to conformational analysis of proteins and other polymers, as well as adapted to NMR or polarized fluorescence techniques, we believe that the approach can greatly enrich the scope of experimentally available statistical thermodynamics, offering new physical insights into the behavior of biomolecules.

Understanding biocompatibility of materials and scaffolds is one of the main challenges in the field of tissue engineering and regeneration. The complex nature of cell-biomaterial interaction requires extensive preclinical functionality testing by studying specific cell responses to different biomaterial properties, from morphology and mechanics to surface characteristics at the molecular level. Despite constant improvements, a more general picture of biocompatibility is still lacking and tailormade scaffolds are not yet available. The scope of our study was thus the investigation of the correlation of fibroblast cell growth on different gelatin scaffolds with their morphological, mechanical as well as surface molecular properties. The latter were thoroughly investigated via polymer molecular mobility studied by site-directed spin labeling and electron paramagnetic resonance spectroscopy (EPR) for the first time. Anisotropy of the rotational motion of the gelatin side chain mobility was identified as the most correlated quantity with cell growth in the first days after adhesion, while weaker correlations were found with scaffold viscoelasticity and no correlations with scaffold morphology. Namely, the scaffolds with highly mobile or unrestricted polymers identified with the cell growth being five times less efficient (N(cells) = 60 ± 25 mm(-2)) as compared to cell growth on the scaffolds with considerable part of polymers with the restricted rotational motion (N(cells) = 290 ± 25 mm(-2)). This suggests that molecular mobility of scaffold components could play an important role in cell response to medical devices, reflecting a new aspect of the biocompatibility concept.

Animal teeth are a common model in studies on dentin adhesive materials. The aim of this study was to compare microstructural parameters (density and diameter of dentinal tubules (DT), peritubular dentin (PTD) thickness, PTD and intertubular dentin (ITD) surface area) and chemical characteristics of canine, porcine, equine, and human root dentin. The middle layers of dentin were harvested just below a cemento-enamel junction from incisors and investigated by means of scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDXS). SEM evaluation of the specimens revealed, that porcine dentin shared most similarities with human dentin. When comparing the density of DTs, canine dentin was also found to be similar to human dentin. Elemental composition of the root dentin did not differ significantly in porcine, equine and human dentin, but in canine dentin higher magnesium value in PTD compared to ITD was found. It is known that microstructural and chemical characteristics affect the strength of the adhesive bonds created among restorative materials and dentin. According to the results of this study, porcine dentin seems to be the most appropriate model to study dental materials to be used in human restorative dentistry.

This article present new insights in complex relation between surface- and interface-related physicochemical properties and microstructuring of three-dimensional (3D) gelatin scaffolds, being fabricated by simultaneous temperature-controlled freeze-thawing cycle and in situ cross-linking using variable conditions (pH) and molarity of carbodiimide reagents, on the seeding and growth of fibroblast cells with subsequent tracking of their spreading and morphology. Rarely populated cells with rounded morphology and small elongations are observed on negative charge-rich scaffold surface with a lower cross-linking degree (CD), and consequently higher molecular mobility and availability of cell-recognition sequences, in comparison with the prominently elongated and densely populated cells on a positively charged scaffold's surface with higher CD and low mobility. Surface microstructure effect was demonstrated by cell vacuolization and their pure intercommunication being present on scaffold's bottom side with smaller pores (25 ± 19 µm) and pore wall thickness (9 ± 5 µm), over the air-exposed side with twice bigger pores (56 ± 38 µm) and pore wall thicknesses (12 ± 6 µm). Strong correlations of CD (r2 = 0.96) and local molecular mobility (r2 = −0.44) with pH and reagents molarity, as well as microstructure features being related to temperature gradient, imply on possibility to modulate scaffold's properties in a direction to guide cell viability and most likely its genotype development. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 3986–3997, 2014.

Monolithic chromatography media represent a novel generation of stationary phases introduced in the last 10-15 years providing a chromatography matrix with enhanced mass transfer and hydrodynamic properties. These features allow for an efficient and fast separation of especially large biomolecules like e.g., DNA and viruses. In this study, the enrichment of virus RNA on short monolithic columns prior to molecular detection of viruses is described. Measles and mumps viruses were chosen as model viruses. The results show that it is possible to bind viral RNA on monoliths and concentrate viral nucleic acids from a fairly dilute sample. Consequently, a potential application of short monolithic columns is the concentration of virus RNA to improve the sensitivity and selectivity of viral detection with the possibility of isolating viral RNA from cell-free biological fluids.

Haptoglobin is positive acute phase protein, synthesized in the liver, with a reference range in adults 30 – 200 mg/dL. Its glycosylation, as one of the most significant post-translation modification, is altered in different types of disease. Many analytical techniques are described for glycosylation site and glycans analysis, but it is very important to isolate an optimal protein amount from plasma. The aim of this project was to obtain a protocol for haptoglobin isolation from human plasma for downstream haptoglobin glycosylation analysis. Protein isolation was based on principles of affinity chromatography. Different volumes of plasma samples and isolation conditions were tested to get more specific and effective isolation. Obtained protocol for haptoglobin isolation was applied on ten plasma samples from different male and female individuals. Glycans were analyzed by hydrophilic interaction ultra performance liquid chromatography (UPLC). Three haptoglobin phenotypes were present in samples and glycosylation varied between individuals.

In recent decades many studies have focused on the thermal performance of massive and lightweight constructions of energy-efficient houses. Surprisingly, temperature and humidity profiles have been addressed relatively rarely, despite the fact that they can influence the living conditions, the lifetime of the construction materials, as well as the energy efficiency of the house and consequently the environment as well. In the present study we compare different lightweight building blocks and the associated temperature and relative-humidity profiles. The studies were performed on residential as well as test passive houses. Small differences in the building blocks were identified as having a large influence on the performance of the building. The structural elements should be placed in such a way as to prevent shear forces due to humidity differences. It has been proven that ventilated wooden facades, in comparison with classical facade plaster, protect the building blocks from high thermal loads. Last but not least, the use of vapor barriers instead of vapor retarders and a lack of moisture-buffering materials led to a higher indoor relative humidity, as well as higher moisture loads on construction that could have an impact on the durability of a building and also on the energy performance of the house.

Svet v katerem živimo se nenehno spreminja in zahteva od nas posameznikov ter posredno od organizacij, ki nas obdajajo stalno prilagajanje. V kolikor želimo biti uspešni, izstopati in dolgoročno obstati v tem turbulentnem okolju pa samo prilagajanje ni dovolj. Postati moramo inovativni. Organizacije v okviru katerih delamo morajo biti inovativne. Mirko Markič (2004) povezuje inoviranje s procesi pridobivanja česarkoli novega, kar z reševanjem problemov postane koristno. Inovacije imajo v današnjem času ogromen vpliv na kvaliteto življenja ljudi. V magistrskem delu smo na nivoju mikro, malih in srednje velikih podjetij dokazal, da so inovativnost, inovacijski potencial, poslovna uspešnost in konkurenčnost pozitivno povezani. Tako lahko trdimo, da je inoviranje v sodobnem gospodarstvu tudi na nivoju MSP ključno za poslovni uspeh podjetij. Brez inoviranja je skoraj nemogoče dokazati dolgoročnega obstoja podjetij, v nobenem primeru pa ne zagotoviti uspešnega poslovanja podjetij. Po drugi strani pa smo prišli do spoznanja, da so mlada visoko inovativna podjetja tudi rizično bolj tvegana in imajo tako kot po eni strani potencial visoke rasti in uspeha, po drugi strani večjo možnost neuspeha. Naloga tudi potrjuje, da je področje inovativnosti v mikro, malih in srednje velikih podjetjih izjemno težko raziskovati, kar pa pomeni, da tudi težje strukturirati ter kot tako managirati. Eno temeljnih načel managementa je, da zaposlene vodi z lastnim zgledom. To se je pri mikro in malih podjetjih pokazalo za ključno, saj vodstvo skozi zgled in delovanje prenaša inovacijsko kulturo na zaposlene, čeprav podjetje zaradi svoje premajhnosti v strukturi ne izkazuje oz. ne more izkazovati inovacijske organiziranosti. V nalogi analiziramo podatke slovenskih mikro malih in srednje velikih podjtij na področju inovativnosti in inovacijskega potenciala. Gre za razširitev projekta I3SME, kjer smo v letu 2009 analizirali 802 podjetij Nemčije, Italije, Avstrije, Madžarske, Poljske in Slovenije. Rezultatom, ki jih je takrat podal mednarodni projekt smo z ponovnim anketiranjem slovenskega dela podjetij dodali longitudinalno komponento in tako ugotavljali, kaj se z podjetji, ki so tako ali drugače ocenjeni na področju inovativnosti dogaja dolgoročno in kakšni pogod podatki ob ponovitvi raziskave čez določeno časovno obdobje. Magistrska naloga je dokazala, da je tako inovativnost kot tudi inovacijski potencial možno meriti tudi v malih in srednje velikih podjetjih. Prvič je dala jasen vpogled v enega od modelov merjenja inovativnosti v MSP, kar do zdaj ni bilo tako odkrito prikazano. Odgovori podjetij so potrdili zastavljene hipoteze magistrske naloge. World in shich we live is constatly changing and requires of us individuals and aslo through organizations that surriund us constatly adapting. If we want to be successful, to stand out and be long-term in this turbuletn enviroment only adjustment is not enough. We need to become innovative. Organizations within which we work must be innovative. For Mirko Markič (2004), innovating is linked with the processes of acquiring anything new, that by resolving problems becomes beneficial. Nowadays innovations have a significant impact on the quality of human life. In the present master’s thesis we demonstrated that innovativeness, innovative potential, business successfulness and competitiveness on the level of micro, small and middle sized companies are positively linked. We can therefore claim that innovating in modern economy is key for business success of companies also on the SME level. Without innovating it is almost impossible to assure the long-term existence of businesses and in no case able to ensure successful operation of companies. On the other hand we have noted that young highly innovative businesses are also more risky and have a chance of both the potential for higher growth and success on one side and even more so of failure on the other. This thesis also confirms that the field of innovativeness in micro, small and middle sized businesses is very difficult to research, which also means that it is harder to structure and manage. One of the basic principles of management is to lead your employees by your own example. This proved to be key in micro and small companies, since management leads by their own example and work, thusly transfers the culture of innovation on the employees, even if the business due to its smallness in structure cannot display the innovative organization. In the thesis we have analyzed the data from the Slovenian micro, small and medium-sized enterprised in the field of innovation adn innovation potential. It is an extension of the project I3SME, where in 2009 we analyzed 802 companies from Germany, Italy, Austria, Hungary, Poland and Slovenia. The results of which are then delivered in international project was with re-interview of the Slovenian part of companies addopted to the longitudinal component to get results of business enterprises over time in comparison to their evaluation to their innovativeness and innovation potential. This master’s thesis proved that both innovativeness and innovative potential can be measured also in small and middle sized businesses. For the first time we have a clear insight into one of the models for innovativeness measurement in SME, which, until now, had not yet been candidly presented. Survey responses from contributing companies confirmed set hypotheses of the master’s thesis.

V magistrskem delu je predstavljenih pet etud za violo z namenom poskusa dokazovanja raziskovalne hipoteze, da so tudi etude lahko samostojna umetniška dela, tako najenostavnejše in najlažje kot tudi bolj zahtevne, ki vsebujejo več različnih tehnik in tudi več muzikaličnega potenciala. Ker so to skladbe za violo, je predstavljena njena zgodovina in razvoj pa tudi sam opis tega inštrumenta, s primerjavo z violino, kar nudi vpogled v primernost transkripcij skladb, ki izvorno niso napisane za violo, temveč za violino. Izbrane etude so dela petih skladateljev, Antonia Bartolomea Brunija, Federiga Fiorilla, Rodolpheja Kreutzerja, Jacquesa Féréola Mazasa in Pierra Rodeja, ki so tudi opisani, pred analizo posameznih etud pa je nekaj besed namenjenih tudi zbirkam, iz katerih izhajajo. V okviru praktičnega dela magistrskega dela bom obravnavanih pet etud odigrala na violi. In the master’s thesis five studies for the viola are presented with the intention of veryfying the research hypothesis that studies can also be independent works of art, both the simple and easy ones as well the more demanding ones, containing more different techniques and also with more potential for musicality. Because the music is written for the viola, the instrument’s history, development and description are presented, alongside the comparison with the violin, which gives basis for then looking into adequacy of the transcriptions of pieces of music that are not originally written for the viola, but for the violin. The chosen studies were composed by five composers, Antonio Bartolomeo Bruni, Federigo Fiorillo, Rodolphe Kreutzer, Jacques-Féréol Mazas and Pierre Rode, who are also introduced, and before delving into the analysis of each study, we will also learn more about the whole publications from which the chosen five studies derive. For the practical part of this master’s thesis I will play the discussed five studies on the viola.

International audience

Izobraževanje odraslih je vseživljenjski proces, ki vključuje formalne in neformalne oblike izobraževanja. V prvem, teoretičnem delu diplomske naloge je opisano in predstavljeno izobraževanje odraslih na splošno in konkretno na osnovi zadnjih znanih statističnih podatkov v Sloveniji. Predstavljene so definicije in pojmi izobraževanja odraslih ter njihova organiziranost. V teoretičnem delu smo se dotaknili tudi marketinga izobraževanja odraslih. V nadaljevanju je predstavljen Javni zavod Cene Štupar Center za izobraževanje Ljubljana, in sicer organizacijska struktura ter sistem izobraževanja, ki temelji na kakovosti. Adult education is a lifelong process which includes formal and informal types of education. In the theoretical part of the thesis adult education is described and presented on a general level and in more detail on the basis of the most recent known statistical data for Slovenia. Moreover, definitions and basic notions in the field of adult education and their organisational structure are given. The theoretical part also addresses the marketing side of adult education. Next, the thesis presents the Cene Štupar Public Institute — Ljubljana Centre for Education, its organisational structure and its education system which is founded on quality.

A process for manufacturing large quantities of lytic bacteriophages was developed. Determination of cultivation termination was found to be essential to achieve high phage quantity and purity. When optimal cultivation termination is missed, phage fraction was found to be highly contaminated with deoxyribonucleic acid released from Escherichia coli cells. Besides, an already established method for monitoring of phage cultivation based on optical density, where its peak indicates point when maximal phage titer is achieved, a new indirect chromatographic method using methacrylate monoliths is proposed for on-line estimation of phage titer. It is based on the measurement of released E. coli deoxyribonucleic acid and shows high correlation with phage titer obtained from plaque assay. Its main advantage is that the information is obtained within few minutes. In addition, the same method can also be used to determine purity of a final phage fraction. Two strategies to obtain highly pure phage fractions are proposed: an immediate purification of phage lysate using monolithic columns or an addition of EDTA before chromatographic purification. The developed protocol was shown to give phage purity above 90% and it is completed within one working day including cultivation and phage titer in the final formulation using developed chromatographic method.

Energy efficiency and living comfort certainly contribute to popularity of the passive house concept. Since these houses are weakly coupled to an environment, any internal energy gains are difficult to be released against small temperature gradients, i.e. in a typical situation occurring in hot summer months. In such a case already a small energy input raises the internal temperature significantly and dramatically affects the living comfort. To provide more information on this problem, we collected and analyzed data from a single family Slovenian passive house. Our goal was to estimate the general house response under real conditions and to investigate overheating in hot summer periods. Different energy gains were identified and explored with respect to their influence on the overheating of this passive house. Results show that windows-opening during the nights in hot summer days, strict shading of the southern and western windows as well as minimization of internal energy sources are necessary and sufficient to keep internal temperature on the comfort level.

The effects of tricylic antidepressant clomipramine (CLO) on the membrane properties of saturated dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine as well as on unsaturated egg yolk phosphatidylcholine liposomes were investigated by the electron paramagnetic resonance spin-labeling technique, in combination with the simulation of the spectra, taking into account that the membrane is heterogeneous and composed of the regions with different fluidity characteristics. Different spin labels, monitoring membrane properties in the upper and inner parts of the membrane, were used. In general, two spectral components, having different motional characteristics, were detected in all liposomes investigated. In liposomes with saturated chains, CLO decreased the phase-transition temperature, disordered the membrane, and increased polarity in the upper part of the membrane. However, less impact was observed in liposomes with unsaturated chains. In dipalmitoyl phosphatidylcholine liposomes, it also induced molecular rearrangements near the pretransition temperature. The presence of 30 mol% cholesterol increased the fluidizing effect of CLO and modified the lateral diffusion of nitroxide in the inner part of the membrane. A unique anomalous increase in diffusion of nitroxide, dependent on CLO concentration, was detected in the temperature region where the phosphatidylcholine membrane without cholesterol experiences the phase transitions. Since the changes in the central part of the membrane were even more pronounced than in the upper part of the membrane, it could be concluded that CLO incorporates into the membrane with its hydrophobic ring parallel to the phospholipid chains.

We present a new, integrated environment used in computer-architecture education. The environment consists of a hardware platform and GUI software running on a PC. The hardware platform is entirely implemented in Xilinx Spartan-3 FPGA. The main part of the hardware platform is a 32-bit pipelined RISC processor with a trace/debug unit. This trace/debug unit is a hardware unit that enables debugging and transfers the pipeline contents to the PC. It also enables communication between the GUI application on the PC and the microprocessor core. Such a system makes it possible to download the students' programs to the FPGA-based microprocessor and graphically depicts the processor's internal state on the PC. © 2010 Wiley Periodicals, Inc. Comput Appl Eng Educ 21: 26–35, 2013

The rapid evolution of gene therapy and DNA vaccines results in an increasing interest in producing large quantities of pharmaceutical grade plasmid DNA. Most current clinical trials involve plasmids of 10 kb or smaller in size, however, future requirements for multigene vectors including extensive control regions may require the production of larger plasmids, e. g., 20 kb and bigger. The objective of this study was to examine certain process conditions for purification of large plasmids with the size of up to 93 kb. Since there is a lack of knowledge about production and purification of bigger plasmid DNA, cell lysis and storage conditions were investigated. The impact of chromatographic system and methacrylate monolithic column on the degradation of plasmid molecules under nonbinding conditions at different flow rates was studied. Furthermore, capacity measurements varying salt concentration in loading buffer were performed and the capacities up to 13 mg of plasmid per mL of the monolithic column were obtained. The capacity flow independence in the range from 130 to 370 cm/h was observed. Using high resolution monolithic column the separation of linear and supercoiled isoforms of large plasmids was obtained. Last but not least, since the baseline separation of RNA and pDNA was achieved, the one step purification on larger CIM DEAE 8 mL tube monolithic column was performed and the fractions were analyzed by CIM analytical monolithic columns.