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Background: Fluorescent proteins (FPs) are pivotal reagents for flow cytometry analysis or fluorescent microscopy. A new generation of immunoreagents (fluobodies/chromobodies) has been developed by fusing recombinant nanobodies to FPs. Methods: We analyzed the quality of such biomolecules by a combination of gel filtration and SDS-PAGE to identify artefacts due to aggregation or material degradation. Results: In the SDS-PAGE run, unexpected bands corresponding to separate fluobodies were evidenced and characterized as either degradation products or artefacts that systematically resulted in the presence of specific FPs and some experimental conditions. The elimination of N-terminal methionine from FPs did not impair the appearance of FP fragments, whereas the stability and migration characteristics of some FP constructs were strongly affected by heating in loading buffer, which is a step samples undergo before electrophoretic separation. Conclusions: In this work, we provide explanations for some odd results observed during the quality control of fluobodies and summarize practical suggestions for the choice of the most convenient FPs to fuse to antibody fragments. [ABSTRACT FROM AUTHOR]

Abstract: Phenolic acids are known to possess antioxidant activities whilst chitosan is a biocompatible polymer with antibacterial activity against a broad spectrum of bacteria. Merging both types of molecules could therefore provide several potential applications. In this work, antioxidant properties of phenolic acid–functionalized-chitosan were investigated after being prepared from structurally-different phenolic acids (caffeic and gallic acids) and chitosan using the laccase from Trametes versicolor as the reaction initiator. A laccase-mediated oxidation kinetic of phenolic acids was monitored by UV–vis spectroscopy and cyclic voltammetry, as well as spin-trapping electron paramagnetic resonance spectroscopy (ST-EPR). The pH was shown to have a significant effect on the degree of phenolic acid self-polymerization, indicating the involvement of phenolate anions within the formations of coupled polyphenol products, and their functionalities, i.e. antioxidant activity. All the phenolic acid-functionalized-chitosans displayed greatly improved ABTS radical cation scavenging capacities, compared with the untreated chitosan. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

As proteins are key molecules in living cells, knowledge about their structure can provide important insights and applications in science, biotechnology, and medicine. However, many protein structures are still a big challenge for existing high-resolution structure-determination methods, as can be seen in the number of protein structures published in the Protein Data Bank. This is especially the case for less-ordered, more hydrophobic and more flexible protein systems. The lack of efficient methods for structure determination calls for urgent development of a new class of biophysical techniques. This work attempts to address this problem with a novel combination of site-directed spin labelling electron spin resonance spectroscopy (SDSL-ESR) and protein structure modelling, which is coupled by restriction of the conformational spaces of the amino acid side chains. Comparison of the application to four different protein systems enables us to generalize the new method and to establish a general procedure for determination of protein structure. [ABSTRACT FROM AUTHOR]

Abstract: Site-directed spin-labeling electron spin resonance (SDSL-ESR) is a promising tool for membrane protein structure determination. Here we propose a novel way to translate the local structural constraints gained by SDSL-ESR data into a low-resolution structure of a protein by simulating the restrictions of the local conformational spaces of the spin label attached at different protein sites along the primary structure of the membrane-embedded protein. We test the sensitivity of this approach for membrane-embedded M13 major coat protein decorated with a limited number of strategically placed spin labels employing high-throughput site-directed mutagenesis. We find a reasonably good agreement of the simulated and the experimental data taking a protein conformation close to the one determined by fluorescence resonance energy transfer analysis [P.V. Nazarov, R.B.M. Koehorst, W.L. Vos, V.V. Apanasovich, M.A. Hemminga, FRET study of membrane proteins: determination of the tilt and orientation of the N-terminal domain of M13 major coat protein, Biophys. J. 92 (2007) 1296–1305]. [Copyright &y& Elsevier]

Abstract: Small-Angle X-ray Scattering (SAXS) curves of pullulan oligomers and gellan gum were modeled with a new string of beads model. The model enables one to simulate polysaccharide single helices with different pitch values, number of monomers per pitch, and cross-sectional radius, as well as different random coils with excluded volume taken into account. The vast conformational space of possible polysaccharide structures is systematically scanned and, for each selected polysaccharide 3D structure, the SAXS curve is calculated and compared to the SAXS experimental data. All structures with RMSSD≤1 are retained as the solutions. By allowing the distribution of equally good solutions to the given experimental SAXS curve, the new model enables one to get a phase space of possible polymer structures in the aqueous environment. Most importantly it avoids using pre-assumed secondary structure elements to fit the experimental data. [Copyright &y& Elsevier]

Abstract: A series of new phosphinate compounds were designed and synthesized as inhibitors of the d-glutamic acid-adding enzyme (MurD) involved in peptidoglycan biosynthesis. They were tested against the MurD enzyme from Escherichia coli, allowing initial structure–activity relationships to be deduced. Two compounds had IC50 values near 100μM and constitute a promising starting point for further development. [Copyright &y& Elsevier]

Standard methods of characterization of electron paramagnetic resonance (EPR) spectra of spin-labeled biomembranes limit the resolution of lateral heterogeneity to only two or three domain types. This disables examination of the structure—function relationship in complex membranes, which might be composed of a larger number of different domain types. To enable exploration of this kind, a new approach based on analysis of EPR spectra with multi-run, hybrid evolutionary optimization is proposed here. From the multiple runs a quasi-continuous distribution of membrane spectral parameters (order parameter, proportion of spectral component, polarity correction factor, rotational correlation time and broadening constant) can be constructed and presented by a new presentation technique CODE (colored distribution of EPR spectral parameters). Through this the concept of a “soft” picture of membrane heterogeneity is introduced, in contrast to the standard “discrete” domain picture. The “soft” characterization method, established on synthetic spectra, was used to examine the lateral heterogeneity of liposome membranes as well as of membranes of neutrophils from healthy and asthmatic horses. In liposome membranes the determined number of domain types was the same as already established by standard procedures of EPR spectra line-shape interpretation. In membranes of neutrophils a quasi-continuous distribution of membrane domain properties was detected by the new method. [ABSTRACT FROM AUTHOR]

The only free thiol group of bovine serum albumin (BSA) was coupled in a high yield with some novel thiol-reactive clenbuterol analogues. The unreacted cysteines were probed with maleimide spin label to determine the yield of the coupling reaction. A novel approach to determining free thiol groups of BSA quantitatively by electron paramagnetic resonance spectroscopy and spectral decomposition without the usual gel-filtration step or extensive dialysis is presented. [Copyright &y& Elsevier]

Infectious haematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are widely distributed fish pathogens in Europe. A reverse transcriptase–polymerase chain reaction (RT–PCR) assay was developed for the detection of both viruses as an alternative method to virus assay in cell culture. Oligonucleotide primers corresponding to highly conserved regions of glycoprotein G-gene sequences were used for IHNV. For the detection of IPNV the VP2-coding region was selected for RT–PCR amplification. Products of the expected size were amplified from total ribonucleic acid (RNA) extracts of infected cells. The optimized RT–PCR methods successfully detected viral RNA from ovarian and seminal fluids and other organs. To enhance the sensitivity and specificity of RT–PCR, a semi-nested PCR assay was tested using additional specific inner primers for reamplification of products obtained by RT–PCR. Because of the possibility of template carry-over contamination, a closed one step RT–PCR method was tested. This technically simplified approach was then combined with the PCR–enzyme linked immunosorbent assay (ELISA) method for the detection of amplification products and verification using specific biotinylated probes. The test provides an additional tool for the detection of IHNV and IPNV which is rapidly and easily performed and is highly sensitive, especially for the detection of IHNV in fish samples coinfected with IPNV. The PCR–ELISA method for the detection of RT–PCR products enables the screening of large numbers of samples and offers the possibility for automatisation of diagnostic work. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is a pathogenic bacterium that can form biofilms in food processing plants, allowing the bacteria to survive despite the control measures applied. As the surface of the bacteria is covered with versatile polysaccharides and proteins, these influence the interactions of the bacterium with any surface. The unique properties and high stability of fungal proteins make them good candidates for the control of bacteria by targeting surface structures. We screened a group of fungal lectins and protease inhibitors from different fungal species, protein folds and known targets for their antibacterial and antibiofilm activity against model strains of Listeria innocua and Listeria monocytogenes. Several of them significantly decreased the viability of biofilm bacteria, but had no effect on bacterial growth parameters at 37°C and thus had no antibacterial activity. Fungal lectins significantly impaired biofilm development even at room temperature, which was attributed to exposure to lectins during adhesion. The tested fungal proteins also reduced biofilm development on biological model surfaces. The observed antibiofilm activity of fungal proteins suggests that they have the potential to modulate interactions between bacteria and/or between bacteria and surfaces, which could be used in the future to reduce surface contamination by Listeria. [ABSTRACT FROM AUTHOR]

Immobilized enzyme reactors (IMERs) are critical tools for developing novel oligosaccharides based on the enzymatic catalysis of polysaccharides. In this paper, a novel glucuronan lyase from Peteryoungia rosettiformans was produced, purified, and then immobilized on a CIM® CDI disk for cleaving glucuronan. The results showed that around 63.6% of glycuronan lyases (800.9 μg) were immobilized on the disk. The Vmax values of immobilized glucuronan lyases did not significantly change (56.9 ± 4.7 μM∙min−1), while the Km values (0.310 ± 0.075 g∙L−1) increased by 2.5 times. It is worth noting that immobilized glucuronan lyases overcame the catalytic inhibition of free enzymes observed under high glucuronan concentrations (0.5–2 g∙L−1). circumscribed central composite design (CCCD) and response surface methodology (RSM) showed that glucuronan concentration, flow rate, and reaction time significantly affected the yield of oligoglucuronans. The degree of polymerization (DP) of degraded glucuronan ranged from DP 2–8 according to the results obtained by high performance anion exchange chromatography coupled with a pulsed amperometric detector (HPAEC-PAD). The IMER retained 50.9% activity after running 2373 column volumes of glucuronan. Finally, this glucuronan lyase reactor was tentatively connected to an immobilized laccase reactor to depolymerize, and gallic acid (GA) was added to glucuronan. Approximately 8.5 mg of GA was added onto 1 g of initial glucuronan, and the GA–oligoglucuronan conjugates showed notable antioxidant activity. [ABSTRACT FROM AUTHOR]

Enzymatic modification of polysaccharides is an efficient and environmentally friendly method to enhance their bioactivities. Immobilized enzymes represent a technology that enables the reuse of enzymes and increase their stability. In this study, laccases from Trametes versicolor were covalently immobilized on CIMmultus® CDI monolithic column, which was employed to graft gallic acid (GA) onto the backbone of T40 dextran. 1.43 mg laccases were successfully immobilized, yielding a mass conversion rate of 32.17 %. The kinetic parameters of free and immobilized laccases were respectively 0.0056 and 0.1770 U for Vmax and 0.0121 and 0.7510 mmol·l−1 for Km. Remarkably, 91.29 % of the enzymatic activity of immobilized laccases was preserved after catalyzing 1200 ml of reactant. A central composite design was carried out to investigate the influence of T40 dextran concentration and flow rate on the phenolization. Response surface methodology was used for predicting the optimal conditions, showed that a 2.07 % mass ratio of phenolization could be achieved with 2 % dextran and a flow rate of 0.89 ml·min−1. The GA-dextran conjugate, produced under the best conditions, revealed a DPPH radical inhibition activity of 63.97 % and an IC 50 of 73.39 µg·ml−1. This work describes a new IMER for ecofriendly producing phenolized polysaccharides. [Display omitted] • Laccases were covalently immobilized on CDI CIMmultus® monolithic column. • Immobilized laccases reactor was used to add phenol groups to T40 dextran. • The effects of flow rate and substrate concentration on phenolization were explored. • Response surface methodology (RSM) was used to predict optimal operation parameters. • The GA-dextran conjugate exhibited antioxidant activity. [ABSTRACT FROM AUTHOR]

Air pollution is an environmental factor associated with an increased risk of neurodegenerative diseases, such as Alzheimer's and Parkinson's, characterized by decreased cognitive abilities and memory. The limited models of sporadic Alzheimer's disease fail to replicate all pathological hallmarks of the disease, making it challenging to uncover potential environmental causes. Environmentally driven models of Alzheimer's disease are thus timely and necessary. We used live-cell confocal fluorescent imaging combined with high-resolution stimulated emission depletion (STED) microscopy to follow the response of retinoic acid-differentiated human neuroblastoma SH-SY5Y cells to nanomaterial exposure. Here, we report that exposure of the cells to some particulate matter constituents reproduces a neurodegenerative phenotype, including extracellular amyloid beta-containing plaques and decreased neurite length. Consistent with the existing in vivo research, we observed detrimental effects, specifically a substantial reduction in neurite length and formation of amyloid beta plaques, after exposure to iron oxide and diesel exhaust particles. Conversely, after exposure to engineered cerium oxide nanoparticles, the lengths of neurites were maintained, and almost no extracellular amyloid beta plaques were formed. Although the exact mechanism behind this effect remains to be explained, the retinoic acid differentiated SH-SY5Y cell in vitro model could serve as an alternative, environmentally driven model of neurodegenerative diseases, including Alzheimer's disease. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The recent clinical success of messenger RNA (mRNA) technology in managing the Covid pandemic has triggered an unprecedented innovation in production and analytical technologies for this therapeutic modality. mRNA is produced by enzymatic transcription of plasmid DNA (pDNA) using polymerase in a cell-free process of in vitro transcription. After transcription, the pDNA is considered a process-related impurity and is removed from the mRNA product enzymatically, chromatographically, or by precipitation. Regulatory requirements are currently set at 10 ng of template pDNA per single human dose, which typically ranges between 30 and 100 µg. Here, we report the development of a generic procedure based on enzymatic digestion and chromatographic separation for the determination of residual pDNA in mRNA samples, with a limit of quantification of 2.3 ng and a limit of detection of less than 0.1 ng. The procedure is based on enzymatic degradation of mRNA and anion exchange HPLC separation, followed by quantification of residual pDNA with a chromatographic method that is already widely adopted for pDNA quality analytics. The procedure has been successfully applied for in-process monitoring of three model mRNAs and a self-amplifying RNA (saRNA) and can be considered a generic substitution for qPCR in mRNA in-process control analytical strategy, with added benefits that it is more cost-efficient, faster, and sequence agnostic. [ABSTRACT FROM AUTHOR]

Biological functions of cell membranes and their correlation to the heterogeneity of the latter's lipid composition are still poorly understood. Fluorescence provides one of the most versatile tools for studying biological membranes. However, few bright and photostable fluorescent probes for labeling plasma membranes are available. We have designed and synthesized two such probes, 8 and 9, that are based on the thiazole-coumarin scaffold. Both are environment sensitive and exhibit similar shifts of emission spectra in a variety of solvents as probes based on 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD). In particular, the second, positively charged probe 9 labels the plasma membrane selectively with limited redistribution to other membranes of the cell. Unfortunately, compared to the other two probes tested, 8 and 6-NBD-PC, it exhibits the highest rate of photobleaching. Nevertheless, these new thiazole-coumarin based membrane probes provide a viable approach to the design of novel membrane probes. [ABSTRACT FROM AUTHOR]

Generating activatable probes that report about molecular vicinity through contact-based mechanisms such as aggregation can be very convenient. Specifically, such probes change a particular spectral property only at the intended biologically relevant target. Xanthene derivatives, for example rhodamines, are able to form aggregates. It is typical to examine aggregation by absorption spectroscopy but for microscopy applications utilizing fluorescent probes it is very important to perform characterization by measuring fluorescence spectra. First we show that excitation spectra of aqueous solutions of rhodamine 6G can be very informative about the aggregation features. Next we establish the dependence of the fluorescence emission spectral maximum shift on the dimer concentration. The obtained information helped us confirm the possibility of aggregation of a recently designed and synthesized rhodamine 6G-based pH-activatable fluorescent probe and to study its pH and concentration dependence. The size of the aggregation-induced emission spectral shift at specific position on the sample can be measured by fluorescence microspectroscopy, which at particular pH allows estimation of the local concentration of the observed probe at microscopic level. Therefore, we show that besides aggregation-caused quenching and aggregation-induced emission also aggregation-induced emission spectral shift can be a useful photophysical phenomenon. [ABSTRACT FROM AUTHOR]

Messenger RNA (mRNA) is becoming an increasingly important therapeutic modality due to its potential for fast development and platform production. New emerging RNA modalities, such as circular RNA, drive the need for the development of non-affinity purification approaches. Recently, the highly efficient chromatographic purification of mRNA was demonstrated with multimodal monolithic chromatography media (CIM® PrimaS), where efficient mRNA elution was achieved with an ascending pH gradient approach at pH 10.5. Here, we report that a newly developed chromatographic material enables the elution of mRNA at neutral pH and room temperature. This material demonstrates weak anion-exchanging properties and an isoelectric point of 5.3. It enables the baseline separation of mRNA (at least up to 10,000 nucleotides (nt) in size) from parental plasmid DNA (regardless of isoform composition) with both a NaCl gradient and ascending pH gradient approach, while mRNA elution is achieved in a pH range of 5–7. In addition, the basic structure of the novel material is a chromatographic monolith, enabling convection-assisted mass transfer of large RNA molecules to and from the active surface. This facilitates the elution of mRNA in 3–7 column volumes with more than 80% elution recovery and uncompromised integrity. This is demonstrated by the purification of a model mRNA (size 995 nt) from an in vitro transcription reaction mixture. The purified mRNA is stable for at least 34 days, stored in purified H2O at room temperature. [ABSTRACT FROM AUTHOR]

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 (5 mm×5.2 mm 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±9 s), 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 200 ng. 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. [ABSTRACT FROM AUTHOR]

Human plasma transferrin (Tf) N-glycosylation has been mostly studied as a marker for congenital disorders of glycosylation, alcohol abuse, and hepatocellular carcinoma. However, inter-individual variability of Tf N-glycosylation is not known, mainly due to technical limitations of Tf isolation in large-scale studies. Here, we present a highly specific robust high-throughput approach for Tf purification from human blood plasma and detailed characterization of Tf N-glycosylation on the level of released glycans by ultra-high-performance liquid chromatography based on hydrophilic interactions and fluorescence detection (HILIC-UHPLC-FLD), exoglycosidase sequencing, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). We perform a large-scale comparative study of Tf and immunoglobulin G (IgG) N-glycosylation analysis in two human populations and demonstrate that Tf N-glycosylation is associated with age and sex, along with multiple biochemical and physiological traits. Observed association patterns differ compared to the IgG N-glycome corroborating tissue-specific N-glycosylation and specific N-glycans' role in their distinct physiological functions. A large-scale comparative study in two human populations analyzes human plasma transferrin and immunoglobulin G (IgG) N-glycosylation profiles and their association with age, sex, and various biochemical and physiological traits. [ABSTRACT FROM AUTHOR]

• Development of reproducible and scalable grafting procedure for chromatographic monoliths. • Up to 72 mg lysozyme /mL dynamic binding capacity in CEX mode using grafted monoliths. • Grafted monoliths result in up to 15 mg pDNA /mL dynamic binding capacity in AEX mode. • Long and dense grafted layer is optimal for pDNA capture in terms of elution recovery. • pDNA isolation on 1 mL scale with 3.4 g/L·h productivity and complete RNA removal. Increased requirements for plasmid DNA (pDNA) in gene therapy and vaccination efforts bring the need for efficient large-scale production processes with high purity and productivity. Chromatographic purification of pDNA is often a bottleneck due to low dynamic binding capacities (DBC), pDNA recovery and/or low selectivity of columns. Ion exchangers (IEX) with charged groups on surface extenders (polyions formed by a grafting reaction and subsequents modification) have attracted attention due to their greatly enhanced productivity compared with traditional IEX. The ultimate goal of our investigation was to increase the DBC for pDNA on a monolith stationary phase by grafting the surface with linear polymethacrylate brushes (grafted chains), while retaining high pDNA recovery and chromatographic selectivity between pDNA and RNA impurities. The inner walls of the Convective Interaction Media® monolith channels were successfully coated with polyglycidyl methacrylate linear chains of varying densities and lengths by employing a controlled radical polymerization reaction. Glycidyl groups throughout the length of the formed grafted chains were converted to cation- (CEX) or anion-exchanging (AEX) moieties. Preliminary chromatographic evaluation of novel grafted CEX columns for different model proteins exhibited 10-times higher DBC with high elution recovery (99 %). Similar DBC improvement was proven for 7.3 kilobase pair pDNA, where the DBC 10 increased from 1.8 mg pDNA per mL of non-grafted chromatographic support up to 15.2 mg per the most densely distributed and the longest grafted chains, modified with weak AEX groups. However, the DBC increase was partially at the expense of pDNA elution recovery, which presumably decreased due to entanglement of pDNA molecules inside the dense grafted layer. To confirm the hypothesis, we have prepared a series of columns differing in length and density of grafted chains and tested them for pDNA capture. A satisfactory compromise between high pDNA DBC and elution recovery was found with relatively long and low density grafted chains. The optimal grafted AEX column with 1 mL bed volume was evaluated for pDNA capture from neutralized E. coli lysate. With a capacity of 13.5 mg pDNA per mL support, ≥95 % elution recovery, and complete RNA clearance, the pDNA was successfully purified at loading flow rate of at least 15 column volumes per min. According to our knowledge and literature search, these process characteristics would enable one of the highest pDNA isolation productivities to be achieved with currently tested IEX. [ABSTRACT FROM AUTHOR]

The COVID‐19 pandemic triggered an unprecedented surge in development of mRNA‐based vaccines. Despite the need to increase process productivity and thus decrease the cost of mRNA vaccines, limited scientific literature is available on strategies to increase the yield of in vitro transcription (IVT) reaction, the unit operation with highest cost of goods, which has traditionally been performed as a batch reaction. Single‐use bioreactors are traditionally used for cell‐based production of biopharmaceuticals, but some core functionalities, such as controlled and automated feed addition, are potentially useful for cell‐free mRNA processes. We report the production of 2 g mRNA in an Ambr® 250 Modular bioreactor system with a starting volume of 100 mL, reaching a maximum mRNA concentration of 12 g L−1 by a fed‐batch IVT approach, and demonstrate the feasibility of continuous fed‐batch production, paving the way towards continuous manufacturing of mRNA. [ABSTRACT FROM AUTHOR]

A comprehensive understanding of the transient heat- and mass-transfer processes on the submicron scale requires the development of novel, non-invasive, temperature-measurement techniques. Here, we present a fluorescence-based method for the non-invasive characterization of transient temperature fields on a submicron scale using a temperature-sensitive erbium/ytterbium co-doped transparent glass–ceramic. This inorganic material is more stable against degradation and photobleaching than traditional organic dyes. Transient heat-conduction experiments were performed on an Er:GPF1Yb0.5Er glass–ceramic sample, which was simultaneously used as a temperature sensor. The temperatures were determined by measuring the fluorescence spectrum and by measuring the intensity variations of the emission spectrum in the temperature range between 25 and 150 °C. A spatial resolution of 400 nm was achieved across an approximately 200 μm × 200 μm field of view at an imaging frequency of 7 Hz. The conducted experiments demonstrate that Er-doped glass–ceramics permit non-invasive and transient thermal imaging and can be adopted for the analysis of heat-transfer processes on submicron scales. [ABSTRACT FROM AUTHOR]

The use of plasmid DNA (pDNA) as a pharmaceutical tool has increased since it represents a safer vector for gene transfer compared to viral vectors. Different pDNA extraction methods have been described; among them is alkaline lysis, currently the most commonly used. Although alkaline lysis represents an established method for isolation of pDNA, some drawbacks are recognized, such as entrapment of pDNA in cell debris, leading to lower pDNA recovery; the time-consuming process; and increase of the volume due to the buffers used, all leading to increased cost of production. We compared the concentration of extracted pDNA when two methods for extracting pDNA from Escherichia coli were used: alkaline lysis and a method based on membrane electroporation, electroextraction. At the same time, we also studied the effect of different pulse protocols on bacterial inactivation. The concentration of pDNA was assayed with anion exchange chromatography. When alkaline lysis was used, two incubations of lysis time (5 and 10 min) were compared in terms of the amount of isolated pDNA. We did not observe any difference in pDNA concentration regardless of incubation time used. In electroextraction, different pulse protocols were used in order to exceed the pDNA concentration obtained by alkaline lysis. We show that electroextraction gives a higher concentration of extracted pDNA than alkaline lysis, suggesting the use of electroporation as a potentially superior method for extracting pDNA from E. coli. In addition, electroextraction represents a quicker alternative to alkaline lysis for extracting pDNA. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Genistein (5,7,4′-trihydroxyisoflavone) the common soy beans isoflavone has attracted scientific interest due to its antioxidant, estrogenic, antiangiogenic and aniticancer activities. The aim of the present study was to investigate the interaction of genistein with biological (erythrocyte) and model membranes (dimyristoyl- and dipalmitoylphosphatidylcholine). Using Laurdan and Prodan as fluorescent probes, we demonstrated phase behavior and membrane fluidity changes induced by genistein. ESR spectroscopy revealed alterations caused by genistein in membrane domains structure and mobility of spin probes with free radicals located at different depths of membrane. The method of ESR spectra decomposition and computer simulation of the recorded spectra were used in order to visualize domain coexistence by GHOST condensation method. Fluorescence and ESR spectroscopy experiments performed at different temperatures enabled us to observe the effect of isoflavone on phospholipid bilayers in either gel or liquid crystalline phase. It was concluded that genistein preferentially intercalated into lipid headgroup region, to some extent into polar–apolar interface and only in minimal degree into hydrophobic core of the membrane. According to our best knowledge this is the first study on modification of domain structure of membranes by genistein. [Copyright &y& Elsevier]

Abstract: 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 7.1mg/mL at 0.4M NaCl for 21kbp, 12.0mg/mL at 0.4M NaCl for 39.4kbp and 8.4mg/mL at 0.5M NaCl for 62.1kbp. 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. [Copyright &y& Elsevier]

Abstract: 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 4.5×1013 pfu/mL while T7 and lambda showed capacity of 1×1013 pfu/mL, all corresponding to values of around 1mg/mL. Interestingly, capacity for lambda phage was increased 5-fold by increasing NaCl concentration in a loaded sample from 0 to 0.2M 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 50nm for T7 phage and 60nm for lambda phage matching closely capside size thus indicating monolayer adsorption while 80nm layer thickness was estimated for M13 phage showing its orientation along the pore. [Copyright &y& Elsevier]

Abstract: Resveratrol and piceatannol are plant-derived polyphenols possessing extremely wide range of biological activities such as cancer chemopreventive, cardio- and neuroprotective, antioxidant, anti-inflammatory, anticancer and lifespan extending properties. Despite great interest in these stilbenes, their interactions with lipid bilayers have not been extensively studied. In the present work, the interaction of both resveratrol and piceatannol with model membranes composed of phosphatidylcholine (DMPC and DPPC) was investigated by means of fluorescence spectroscopy, differential scanning calorimetry (DSC) and electron spin resonance spectroscopy (ESR). Generalized polarization of two fluorescent probes Laurdan and Prodan measured in pure lipid and lipid:stilbene mixtures revealed that resveratrol and piceatannol changed bilayer properties in both gel-like and liquid crystalline phase and interacted with lipid headgroup region of the membrane. These findings were corroborated by DSC experiments in which the stilbene-induced decrease of lipid melting temperature and transition cooperativity were recorded. Resveratrol and piceatannol restricted also the ESR-measured mobility of spin probes GluSIN18, 5DSA and 16DSA with nitroxide group localized at different depths. Since the most pronounced effect was exerted on the spin probe located near membrane surface, we concluded that also ESR results pointed to the preferential interaction of resveratrol and piceatannol with headgroup region of lipid bilayer. [Copyright &y& Elsevier]

Abstract: The membrane lipid composition of living cells generally adjusts to the prevailing environmental and physiological conditions. In this study, membrane activity and lipid composition of the Gram-negative bacterium Vibrio sp. DSM14379, grown aerobically in a peptone-yeast extract medium supplemented with 0.5, 1.76, 3, 5 or 10% (w/v) NaCl, was determined. The ability of the membrane to reduce a spin label was studied by EPR spectroscopy under different salt concentrations in cell suspensions labeled with TEMPON. For lipid composition studies, cells were harvested in a late exponential phase and lipids were extracted with chloroform–methanol–water, 1:2:0.8 (v/v). The lipid polar head group and acyl chain compositions were determined by thin-layer and gas–liquid chromatographies. 31P-NMR spectroscopy was used to study the phase behaviour of the cell lipid extracts with 20 wt.% water contents in a temperature range from −10 to 50 °C. The results indicate that the ability of the membrane to reduce the spin label was highest at optimal salt concentrations. The composition of both polar head groups and acyl chains changed markedly with increasing salinity. The fractions of 16:0, 16:1 and 18:0 acyl chains increased while the fraction of 18:1 acyl chains decreased with increasing salinity. The phosphatidylethanolamine fraction correlated inversely with the lysophosphatidylethanolamine fraction, with phosphatidylethanolamine exhibiting a minimum, and lysophosphatidylethanolamine a maximum, at the optimum growth rate. The fraction of lysophosphatidylethanolamine was surprisingly high in the lipid extracts. This lipid can form normal micellar and hexagonal phases and it was found that all lipid extracts form a mixture of lamellar and normal isotropic liquid crystalline phases. This is an anomalous behaviour since the nonlamellar phases formed by total lipid extracts are generally of the reversed type. [Copyright &y& Elsevier]

Abstract: The influence of glass surface modification in order to determine strength of the monolith attachment was studied. Modification consists of pre-treatment of the glass with chemicals or boiling in deionized water, silanization and drying has been investigated on different types of glass. Amount of silane groups was determined by measurement of the contact angle between the glass surface and water drop. The highest values were found for soda–lime glass. Strength of the monolith attachment was established by pumping ethanol through the monolithic capillaries and measuring the pressure drop at which monolith was dislodged. Surprisingly, it was found that the critical part of the glass surface modification procedure is glass pre-treatment. Good results were obtained with glass boiled in water for 2.5h or more. [Copyright &y& Elsevier]

Abstract: A deoxyribonuclease bioreactor was prepared by immobilization of deoxyribonuclease I through epoxy groups inherently present on poly (glycidyl methacrylate-co-ethylene dimethacrylate) monoliths. Columns with various levels of DNase activity were prepared varying immobilization temperature, pH, time and method. The apparent Michaelis–Menten constant, , and turnover number, , for immobilized DNase determined by on-line frontal analysis method were, respectively, 0.28g of DNAl-1 and 16dA260nmmin-1mg-1 of immobilized DNase. The highest activity of immobilized DNase was detected at 1mM calcium ions concentration and mirrored properties of free enzyme; however, reaction temperature in the range from 25 to 37°C has no significant effect on activity of immobilized DNase in contrary to free enzyme. The CIM DNase bioreactor was used for elimination of DNA contaminants in RNA samples prior to reverse transcription followed by PCR. [Copyright &y& Elsevier]

Nanoparticle toxicity assessments have moved closer to physiological conditions while trying to avoid the use of animal models. An example of new in vitro exposure techniques developed is the exposure of cultured cells at the air–liquid interface (ALI), particularly in the case of respiratory airways. While the commercially available VITROCELL® Cloud System has been applied for the delivery of aerosolized substances to adherent cells under ALI conditions, it has not yet been tested on lung surfactant and semi-adherent cells such as alveolar macrophages, which are playing a pivotal role in the nanoparticle-induced immune response. Objectives: In this work, we developed a comprehensive methodology for coating semi-adherent lung cells cultured at the ALI with aerosolized surfactant and subsequent dose-controlled exposure to nanoparticles (NPs). This protocol is optimized for subsequent transcriptomic studies. Methods: Semi-adherent rat alveolar macrophages NR8383 were grown at the ALI and coated with lung surfactant through nebulization using the VITROCELL® Cloud 6 System before being exposed to TiO 2 NM105 NPs. After NP exposures, RNA was extracted and its quantity and quality were measured. Results: The VITROCELL® Cloud system allowed for uniform and ultrathin coating of cells with aerosolized surfactant mimicking physiological conditions in the lung. While nebulization of 57 μ L of 30 mg/mL TiO 2 and 114 μ L of 15 mg/mL TiO 2 nanoparticles yielded identical cell delivered dose, the reproducibility of dose as well as the quality of RNA extracted were better for 114 μ L. [ABSTRACT FROM AUTHOR]

A new chromatographic medium, Convective Interaction Media® (CIM) disk monolithic columns, was applied to plant virus concentration. The ability of the columns to concentrate highly diluted plant viruses was tested on a model plant virus, rod-shaped tomato mosaic virus (ToMV). Enzyme-linked immunosorbent assay (ELISA) was used for the quantitative analysis. The virus was concentrated using a strong anion exchanger, CIM quaternary amine (QA) disk monolithic column. A high salt concentration was used to elute the concentrated virus from the columns. It has been demonstrated that ToMV, which had been diluted considerably below the sensitivity of ELISA, was concentrated by several orders of magnitude in the one-step procedure. Concentrated virus preparations could be used directly for ELISA testing. In comparison with methods described for concentrating plant viruses from irrigation water, the above procedure may provide a much faster and more efficient way to concentrate highly diluted plant viruses. The procedure could be applied to the testing of other highly diluted plant viruses, and to concentrating viruses for antiserum production. [Copyright &y& Elsevier]

Using EPR spectroscopy a typical lateral domain structure was detected in the membranes of spin-labeled bovine erythrocyte ghosts. The spectral parameters were determined by decomposing the EPR spectrum into three spectral components and tuned by a hybrid-evolutionary-optimization method. In our experiments the lateral domain structure and its properties were influenced by the variation in the temperature and by the addition of n-butanol. The specific responses of the particular domain types were detected. For the most-ordered domain type a break was seen in the temperature dependence of its order parameter, while the order parameters of the two less-ordered domain types exhibited a continuous decrease. Below the break-point temperature the alcohol-induced membrane fluidity variation is mainly a consequence of the change in the proportions of the least- and the most-ordered domain type and not the change of the domain-type ordering or dynamics (with n-butanol concentration). On the other hand, the fluidity variation above the break-point temperature arises from both types of changes. Interestingly, the proportion of the domain type that has its order parameter between that of the least- and the most-ordered domain type remains almost constant with concentration as well as with temperature, which implies its stability. Such characterization of the lateral membrane domain structure could be beneficial when considering the lipid–protein interactions, because it can be assumed that the activity of the membrane-bound enzyme depends on the properties of the particular domain type. [Copyright &y& Elsevier]

Nanotechnologies hold great promise for various applications. To predict and guarantee the safety of novel nanomaterials, it is essential to understand their mechanism of action in an organism, causally connecting adverse outcomes with early molecular events. This is best investigated using noninvasive advanced optical methods, such as high-resolution live-cell fluorescence microscopy, which require stable labeling of nanoparticles with fluorescent dyes. However, as shown here, when the labeling is performed inadequately, unbound fluorescent dyes and inadvertently altered chemical and physical properties of the nanoparticles can result in experimental artefacts and erroneous conclusions. To prevent such unintentional errors, we introduce a tested minimal combination of experimental methods to enable artefact-free fluorescent labeling of metal-oxide nanoparticles—the largest subpopulation of nanoparticles by industrial production and applications—and demonstrate its application in the case of TiO2 nanotubes. We (1) characterize potential changes of the nanoparticles' surface charge and morphology that might occur during labeling by using zeta potential measurements and transmission electron microscopy, respectively, and (2) assess stable binding of the fluorescent dye to the nanoparticles with either fluorescence intensity measurements or fluorescence correlation spectroscopy, which ensures correct nanoparticle localization. Together, these steps warrant the reliability and reproducibility of advanced optical tracking, which is necessary to explore nanomaterials' mechanism of action and will foster widespread and safe use of new nanomaterials. [ABSTRACT FROM AUTHOR]

Separation of empty and full AAV8 capsids was achieved during their elution from a weak anion exchanger with an ascending pH gradient at low conductivity. Experimental data suggest elution was mediated by loss of positive charge from the exchanger. The method produced a full capsid peak with fewer empty capsids than elution of a strong anion exchanger with a salt gradient. Elution of the weak exchanger by sodium chloride gradients or by pH gradients in the presence of sodium chloride gave inferior separation performance. Pre-elution of empty capsids with a pH step allowed full capsids to be eluted by salt without compromising separation. Loading at intermediate pH prevented empty capsid binding and enabled step elution of full capsids in a physiological buffer environment. [ABSTRACT FROM AUTHOR]

To avoid a devastating effect of eye vision impairment on the information flow from the eye to our brain, enormous effort is being put during the last decades into the development of more sensitive diagnostics and more efficient therapies of retinal tissue. While morphology can be impressively imaged by optical coherence tomography, molecular-associated pathology information can be provided almost exclusively by auto-fluorescence-based methods. Among the latter, the recently developed fluorescence lifetime imaging ophthalmoscopy (FLIO) has the potential to provide both structural information and interacting pictures at the same time. The requirements for FLIO laser sources are almost orthogonal to the laser sources used in phototherapy that is expected to follow up the FLIO diagnostics. To make theranostics more effective and cheaper, the complete system would need to couple at least the modalities of low-power high-repetition-rate FLIO and precision high-pulse energy-adjustable repetition rate phototherapy. In addition, the intermediate-power high repetition rate for two-photon excitation would also be desired to increase the depth resolution. In our work, compact fiber-laser based on high-speed gain-switched laser diode has been shown to achieve adaptable/independently tunable repetition rate and energy per pulse allowing coupled fluorescence lifetime diagnostics via two-photon excitation and phototherapy via laser-induced photodisruption on a local molecular environment in a complex ex vivo retinal tissue. [ABSTRACT FROM AUTHOR]

Nanomaterial (NM) characteristics may affect the pulmonary toxicity and inflammatory response, including specific surface area, size, shape, crystal phase or other surface characteristics. Grouping of TiO 2 in hazard assessment might be challenging because of variation in physicochemical properties. We exposed C57BL/6 J mice to a single dose of four anatase TiO 2 NMs with various sizes and shapes by intratracheal instillation and assessed the pulmonary toxicity 1, 3, 28, 90 or 180 days post-exposure. The quartz DQ12 was included as benchmark particle. Pulmonary responses were evaluated by histopathology, electron microscopy, bronchoalveolar lavage (BAL) fluid cell composition and acute phase response. Genotoxicity was evaluated by DNA strand break levels in BAL cells, lung and liver in the comet assay. Multiple regression analyses were applied to identify specific TiO 2 NMs properties important for the pulmonary inflammation and acute phase response. The TiO 2 NMs induced similar inflammatory responses when surface area was used as dose metrics, although inflammatory and acute phase response was greatest and more persistent for the TiO 2 tube. Similar histopathological changes were observed for the TiO 2 tube and DQ12 including pulmonary alveolar proteinosis indicating profound effects related to the tube shape. Comparison with previously published data on rutile TiO 2 NMs indicated that rutile TiO 2 NMs were more inflammogenic in terms of neutrophil influx than anatase TiO 2 NMs when normalized to total deposited surface area. Overall, the results suggest that specific surface area, crystal phase and shape of TiO 2 NMs are important predictors for the observed pulmonary effects of TiO 2 NMs. • Inflammation and acute phase response was highest and more persistent for TiO 2 tube. • TiO 2 tube and DQ12 caused pulmonary alveolar proteinosis. • Rutile TiO 2 NMs were more inflammogenic in terms of neutrophil influx than anatase. • BET surface area strongly correlated with neutrophil influx for both crystal phases. • BET surface area, crystal phase and shape of TiO 2 NMs are predictors for the effects. [ABSTRACT FROM AUTHOR]

Legionella pneumophila can cause a potentially fatal form of humane pneumonia (Legionnaires' disease), which is most problematic in immunocompromised and in elderly people. Legionella species is present at low concentrations in soil, natural and artificial aquatic systems and is therefore constantly entering man-made water systems. The environment temperature for it's ideal growth range is between 32 and 42°C, thus hot water pipes represent ideal environment for spread of Legionella. The bacteria are dormant below 20°C and do not survive above 60°C. The primary method used to control the risk from Legionella is therefore water temperature control. There are several other effective treatments to prevent growth of Legionella in water systems, however current disinfection methods can be applied only intermittently thus allowing Legionella to grow in between treatments. Here we present an alternative disinfection method based on antibacterial coatings with Cu-TiO2 nanotubes deposited on preformed surfaces. In the experiment the microbiocidal efficiency of submicron coatings on polystyrene to the bacterium of the genus Legionella pneumophila with a potential use in a water supply system was tested. The treatment thus constantly prevents growth of Legionella pneumophila in presence of water at room temperature. Here we show that 24-hour illumination with low power UVA light source (15 W/m2 UVA illumination) of copper doped TiO2 nanotube coated surfaces is effective in preventing growth of Legionella pneumophila. Microbiocidal effects of Cu-TiO2 nanotube coatings were dependent on the flow of the medium and the intensity of UV-A light. It was determined that tested submicron coatings have microbiocidal effects specially in a non-flow or low-flow conditions, as in higher flow rates, probably to a greater possibility of Legionella pneumophila sedimentation on the coated polystyrene surfaces, meanwhile no significant differences among bacteria reduction was noted regarding to non or low flow of medium. [ABSTRACT FROM AUTHOR]

The potential of live-cell stimulated emission depletion (STED) nanoscopy has not yet been fully exploited. Currently, the main limitation is the small number of fluorophores and probes that can sustain high light intensity/high dose employed in STED. Namely, fluorophores suitable for STED nanoscopy must be bright and highly photostable and exhibit a large Stokes shift. To expand the list of available probes, we synthesized and evaluated several new membrane probes for live-cell STED nanoscopy. Of the tested probes, probes MePyr500, ThiaCN545 and NB640 not only allow high-resolution STED images, but also partition into the intracellular membranes relatively quickly, thus lacking the selectivity of labelling solely the plasma membrane. During experiments, cytotoxicity was observed merely with the probe ThiaCN545, which blebs the plasma membrane. In comparison with commercially available CellMask Orange and STAR RED (KK114) DPPE, all our tested probes exhibited better photostability with the exception of NB640, which had the fastest bleaching rate of all tested probes. The best overall results can be assigned to the probe MePyr500, providing high-resolution STED images as well as high photostability with no noticeable cytotoxicity, making it an excellent candidate for further development. [ABSTRACT FROM AUTHOR]

Bacterial infections acquired in healthcare facilities including hospitals, the so called healthcare acquired or nosocomial infections, are still of great concern worldwide and represent a significant economical burden. One of the major causes of morbidity is infection with Methicillin Resistant Staphylococcus aureus (MRSA), which has been reported to survive on surfaces for several months. Bactericidal activity of copper-TiO2 thin films, which release copper ions and are deposited on glass surfaces and heated to high temperatures, is well known even when illuminated with very weak UVA light of about 10 μW/cm2. Lately, there is an increased intrerest for one-dimensional TiO2 nanomaterials, due to their unique properties, low cost, and high thermal and photochemical stability. Here we show that copper doped TiO2 nanotubes produce about five times more ·OH radicals as compared to undoped TiO2 nanotubes and that effective surface disinfection, determined by a modified ISO 22196:2011 test, can be achieved even at low intensity UVA light of 30 μW/cm2. The nanotubes can be deposited on a preformed surface at room temperature, resulting in a stable deposition resistant to multiple washings. Up to 103 microorganisms per cm2 can be inactivated in 24 hours, including resistant strains such as Methicillin-resistant Staphylococcus aureus (MRSA) and Extended-spectrum beta-lactamase Escherichia coli (E. coli ESBL). This disinfection method could provide a valuable alternative to the current surface disinfection methods. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Bead matrices have been used in affinity chromatography to purify molecules in multiple applications. For instance, the hepatitis B surface antigen (HBsAg) is one of the molecules purified by this technique for human vaccine development programs. However, the use of monolithic supports have emerged as the advantageous choice for affinity chromatography based on convective mass transfer, a high number of channels, and low backpressures at high flow rates. For this reason, several experiments were conducted to determine the suitability of CB.Hep-1 monoclonal antibody (mAb) immunosorbent developed on carboxyimidazole (CDI)-monolithic supports (ligand concentrations: 0.5, 1.0, and 7.0 mg/mL) for HBsAg particle purification. Key results from this study show the highest amounts of HBsAg adsorbed (3059.31 ± 865.71 μg HBsAg/mL immunosorbent, n = 2), and HBsAg eluted (2884.50 ± 541.01 μg HBsAg/mL immunosorbent, n = 2), were estimated in the 1.0 mg/mL-CDI-CB. Hep-1 mAb monolithic support immunosorbents. In addition, the ligand leakage was always < 3 ng mAb/μg HBsAg (approved limit) in the 1.0 mg/mL-CDI-CB.Hep-1 mAb immunosorbents. Experiments also evidenced the high purity and molecular homogeneity of purified HBsAg particles (< 95 %) across 20 purification cycles. Therefore, the ligand concentration could be reduced up to 1.0 mg/mL, which would enable a notable decrease in the mAb amount required for vaccine manufacturing, as compared to bead matrices (4.0 mg/mL). This study demonstrated that CDI-CB.Hep-1 mAb monolithic support immunosorbents are best suited for assessing the large-scale purification performance of HBsAg particles for human vaccine development programs at low ligand concentration and high flow rates. [ABSTRACT FROM AUTHOR]

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 0.8 μm 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 × 10 9 pfu 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. [ABSTRACT FROM AUTHOR]

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 K m 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. [ABSTRACT FROM AUTHOR]

• OH column removes majority of DNA, protein and media with 100% phage recovery. • PrimaS and H-Bond column substantially reduce endotoxin levels (7 log reduction). • Fast AEX-MALS analytical method can be used for bacteriophage quantification. Bacteriophages represent immense potential as therapeutic agents. Many of the most compelling applications of bacteriophages involve human therapy, some pertinent to gene therapy, others involving antibiotic replacement. Phages themselves are considered safe for humans. However, phage lysates may contain many kinds of harmful by-products, especially endotoxins of gram-negative bacteria and protein toxins produced by many pathogenic bacterial species. In bacteriophage research and therapy, most applications ask for highly purified phage suspensions, as such it is crucial to reduce proteins, endotoxins, DNA and other contaminants. In this article we present an efficient two-step chromatographic purification method for P. aeruginosa bacteriophage PP-01, using Convective Interaction Media (CIM®) monoliths, that is cGMP compliant and easy to scale-up for most stringent production of the therapeutic phage. First chromatographic step on CIMmultus OH resulted in 100% bacteriophage recovery with a reduction of 98 % protein and more than 99 % DNA content. Polishing was conducted using three different column options, CIMmultus with QA, H-Bond and PrimaS ligands. For PP-01 bacteriophage all three different options worked, but multimodal ligands H-Bond and PrimaS outperformed traditional QA in endotoxin removal (7 log step reduction). Additionally, an HPLC analytical method was developed to estimate phage concentration and impurity profile in different in-process samples. The HPLC method shows good correlation with drop assay titration, provides useful insights and can be run very fast with just 20 min per sample analysis. [ABSTRACT FROM AUTHOR]