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HPLC is established as a fast convenient analytical technology for characterizing the content of empty and full capsids in purified samples containing adeno-associated virus (AAV). UV-based monitoring unfortunately over-estimates the proportion of full capsids and offers little value for characterizing unpurified samples. The present study combines dual-wavelength UV monitoring with intrinsic fluorescence, extrinsic fluorescence, and light-scattering to extend the utility of HPLC for supporting development of therapeutic AAV-based drugs. Applications with anion exchange (AEC), cation exchange (CEC), and size exclusion chromatography (SEC) are presented. Intrinsic fluorescence increases sensitivity of AAV detection over UV and enables more objective estimation of empty and full capsid ratios by comparison of their respective peak areas. Light scattering enables identification of AAV capsids in complex samples, plus semiquantitative estimation of empty and full capsid ratios from relative peak areas of empty and full capsids. Extrinsic Picogreen fluorescence enables semiquantitative tracking of DNA with all HPLC methods at all stages of purification. It does not detect encapsidated DNA but reveals DNA associated principally with the exteriors of empty capsids. It also enables monitoring of host DNA contamination across chromatograms. These enhancements support many opportunities to improve characterization of raw materials and process intermediates, to accelerate process development, provide rapid in-process monitoring, and support process validation.

Za uspešno uravnavanje vnetnih reakcij pri poškodbah ali infekcijah je pravilno delovanje vnetnih in protivnetnih mehanizmov imunskega sistema ključnega pomena. Vnetje je pri zdravem človeku uravnavano s pravilnim razmerjem v količini vnetnih in protivnetnih mediatorjev, saj tako ne pride do prekomernega sistemskega vnetja ali sepse. Izločanje vnetnih in protivnetnih molekul nadzorujeta humoralni sistem (kortizol) preko hipotalamo-hipofizo-suprarenalne osi in živčni sistem preko živca vagusa. V zadnjih dvajsetih letih so znanstveniki iz različnih laboratorijev po svetu poročali o najrazličnejših odkritjih, ki so privedle do razumevanja mehanizma in pomena novo odkritega holinergičnega protivnetnega odziva, ki ga nadzira živec vagus. Holinergični protivnetni odziv temelji na aktivaciji živca vagusa ter prenosu signala na vranični živec, pri čemer se iz perifernih živčnih končičev slednjega v vranici sprošča noradrenalin, ta pa stimulira podvrsto limfocitov T, da v vranici začnejo sintetizirati in izločati acetilholin. Acetilholin se veže na α7 nikotinske receptorje na makrofagih in preko različnih mehanizmov preprečuje izločanje vnetnih citokinov. Določanje izražanja mRNA alfa-7 nikotinskih receptorjev v periferni krvi bi bila lahko dokaj enostavna metoda, s katero bi pri človeku lahko določali aktivnost holinergične protivnetne poti v različnih bolezenskih stanjih. V metodologiji smo predlagali protokol dela za vzpostavitev rutinskega merjenja izražanja mRNA α7 nikotinskih receptorjev, ki zajema izolacijo PBMC iz človeške krvi, izolacijo RNA in dvostopenjski RT-PCR. To bi lahko postalo uporabno metodološko orodje za klinične raziskave, s časom pa potencialno tudi kot rutinski diagnostični biomarker stanja holinergičnega protivnetnega odziva. Ugotovitve glede postopkov izolacije PBMC in RT-PCR smo primerjali z metodologijo izolacije, ki so jo opisali v raziskavah, kjer so raven izražanja mRNA nikotinskih acetilholinskih receptorjev v celicah iz periferne krvi pri bolnikih že določali. Nova in do sedaj redko uporabljena je metoda imunomagnetne negativne selekcije z EasySep, ki temelji na označevanju krvnih celic z monoklonskimi protitelesi in njihovo izolacijo s pomočjo magnetnih delcev. Ta metoda se zdi še posebej uporabna, saj hitro in v zadostni količini omogoča izolacijo specifičnih podvrst limfocitov kot tudi monocitov. S predlagano metodologijo qPCR želimo ugotavljati razlike v izražanju gena za α7 nikotinski receptor pri bolnikih z različnimi oblikami in jakostjo vnetja v primerjavi z zdravimi. Normal activity of inflammatory and anti-inflammatory mechanism of immune system is crucial for successful regulation of inflammation caused by infections and injuries. Inflammation is normally regulated by the right proportion of inflammatory and anti-inflammatory cytokines, so excessive systemic inflammation and sepsis are excluded. Secretion of inflammatory and anti-inflammatory cytokines is regulated by humoral system (cortisol) through hypothalamic-pituitary-adrenal axis and nervous system through vagus nerve. In the last 20 years it has been reported about many different discoveries by laboratories around the world, which lead to understanding of the mechanism and meaning of the newly discovered cholinergic anti-inflammatory pathway, which is supervised by vagus nerve. Cholinergic anti-inflammatory pathway is based on activation of vagus nerve which transducts the signal to splenic nerve, which secretes noradrenaline, the activator of specific subset of T lymphocytes in spleen. T lymphocytes then start secreting acetylcholine, which binds to α7 nicotinic receptors on macrophages and prevents the secretion of inflammatory cytokines through different molecular mechanisms. Determination of alpha-7 nicotinic receptor mRNA expression in human peripheral blood could present a simple method to determine the activity of human cholinergic anti-inflammatory response during different diseases. We have suggested methodological protocol to establish routine measurement of alpha-7 nicotinic receptor mRNA expression, which includes isolation of PBMC from human peripheral blood, isolation of whole RNA and two-level RT-PCR. This could become a useful methodological tool for clinical researches and in time also a routine diagnostic biomarker for determining the condition of cholinergic anti-inflammatory response. Findings about protocols for isolation of PBMC and RT-PCR were compared to the methodology of isolation of PMBC, which was used for routine measurement of alpha-7 nicotinic receptor mRNA expression in peripheral blood of different patients, described in different researches. Newly discovered and rarely used method for isolation of specific cells of PBMC is immunomagnetic negative selection with EasySep, which is based on labelling blood cells with monoclonal antibodies and suspension of magnetic particles. This method seems to be very useful, because it sufficiently and quickly enables isolating of specific subsets of lymphocytes and monocytes. With the suggested method and qPCR the differences between alpha-7 nicotinic receptor mRNA expression in patients with different forms and intensities of inflammation and healthy people would like to be found.

Purification of high-quality plasmid DNA in large quantities is a crucial step in its production for therapeutic use and is usually conducted by different chromatographic techniques. Large-scale preparations require the optimization of yield and homogeneity, while maximizing removal of contaminants and preserving molecular integrity. The advantages of Convective Interaction Media

Purification of high-quality plasmid DNA in large quantities is a crucial step in its production for therapeutic use and is usually conducted by different chromatographic techniques. Large-scale preparations require the optimization of yield and homogeneity, while maximizing removal of contaminants and preserving molecular integrity. The advantages of Convective Interaction Media® (CIM®) monolith stationary phases, including low backpressure, fast separation of macromolecules, and flow-rate-independent resolution qualified them to be used effectively in separation of plasmid DNA on laboratory as well as on large scale. A development and scale-up of plasmid DNA downstream process based on chromatographic monoliths is described and discussed below. Special emphasis is put on the introduction of process analytical technology principles and tools for optimization and control of a downstream process.

Blood coagulation mechanisms forming a blood clot and preventing hemorrhage have been extensively studied in the last decades. Knowing the mechanisms behind becomes very important particularly in the case of blood vessel diseases. Real-time and accurate diagnostics accompanied by the therapy are particularly needed, for example, in diseases related to retinal vasculature. In our study, we employ for the first time fluorescence hyperspectral imaging (fHSI) combined with the spectral analysis algorithm concept to assess physical as well as functional information of blood coagulation in real-time. By laser-induced local disruption of retinal vessels to mimic blood leaking and subsequent coagulation and a proper fitting algorithm, we were able to reveal and quantify the extent of local blood coagulation through direct identification of the change of oxyhemoglobin concentration within few minutes. We confirmed and illuminated the spatio-temporal evolution of the essential role of erythrocytes in the coagulation cascade as the suppliers of oxygenated hemoglobin. By additional optical tweezers force manipulation, we showed immediate aggregation of erythrocytes at the coagulation site. The presented fluorescence-based imaging concept could become a valuable tool in various blood coagulation diagnostics as well as theranostic systems if coupled with the laser therapy.

Many chronic diseases manifest themselves in prolonged inflammation and often ignored dysregulated lipid metabolism, both also associated with inhalation of certain nanomaterials. Limited knowledge of involved molecular events and their causal connections prevents reliable prediction of outcomes by efficient testing strategies. To unravel how acute nanomaterial exposure leads to chronic conditions, we employed advanced microscopy and omics in vitro , in vivo and in silico . For selected metal-oxide nanomaterials, we show that epithelial cells survive the exposure by excreting internalized nanomaterials and passivating them on the surface, employing elevated lipid synthesis. Macrophages, on the contrary, attack the defending epithelium but die degrading passivized complexes, releasing nanomaterial, which is reuptaken by epithelial cells. Constant proinflammatory signalling recruits new phagocytes that feed the vicious cycle of events resulting in a long-lasting response to a single exposure. The discovered mechanism predicts the nanomaterial-associated in vivo chronic outcomes based on simple in vitro measurements and potentially enlightens other chronic diseases. Graphical abstract

Fluorescent carbon dots (CDs) are potential tools for the labeling of cells with many advantages such as photostability, multicolor emission, small size, rapid uptake, biocompatibility, and easy preparation. Affinity towards organelles can be influenced by the surface properties of CDs which affect the interaction with the cell and cytoplasmic distribution. Organelle targeting by carbon dots is promising for anticancer treatment; thus, intracellular trafficking and cytotoxicity of cationic CDs was investigated. Based on our previous study, we used quaternized carbon dots (QCDs) for treatment and monitoring the behavior of two human cancer cell MCF-7 and HeLa lines. We found similarities between human cancer cells and mouse fibroblasts in the case of QCDs uptake. Time lapse microscopy of QCDs-labeled MCF-7 cells showed that cells are dying during the first two hours, faster at lower doses than at higher ones. QCDs at a concentration of 100 µg/mL entered into the nucleus before cellular death; however, at a dose of 200 µg/mL, blebbing of the cellular membrane occurred, with a subsequent penetration of QCDs into the nuclear area. In the case of HeLa cells, the dose-depended effect did not happen; however, the labeled cells were also dying in mitosis and genotoxicity occurred nearly at all doses. Moreover, contrasted intracellular compartments, probably mitochondria, were obvious after 24 h incubation with 100 µg/mL of QCDs. The levels of reactive oxygen species (ROS) slightly increased after 24 h, depending on the concentration, thus the genotoxicity was likely evoked by the nanomaterial. A decrease in viability did not reach IC 50 as the DNA damage was probably partly repaired in the prolonged G0/G1 phase of the cell cycle. Thus, the defects in the G2/M phase may have allowed a damaged cell to enter mitosis and undergo apoptosis. The anticancer effect in both cell lines was manifested mainly through genotoxicity.

Tryptic hydrolysis of β-Lactoglobulin (β-Lg) is attracting more and more attention due to the reduced allergenicity and the functionality of resulting hydrolysates. To produce hydrolysates in an economically viable way, immobilized trypsin reactors (IMTRs), based on polymethacrylate monolith with pore size 2.1 μm (N1) and 6 μm (N2), were developed and used in a flow-through system. IMTRs were characterized in terms of permeability and enzymatic activity during extensive usage. N1 showed twice the activity compared with N2, correlating well with its almost two times higher amount of immobilized trypsin. N2 showed high stability over 18 cycles, as well as over more than 30 weeks during storage. The efficiency of IMTRs on hydrolyzing β-Lg was compared with free trypsin, and the resulting hydrolysates were analyzed by MALDI-TOF/MS. The final hydrolysis degree by N1 reached 9.68% (86.58% cleavage sites) within 4 h, while only around 6% (53.67% cleavage sites) by 1.5 mg of free trypsin. Peptides analysis showed the different preference between immobilized trypsin and free trypsin. Under the experimental conditions used in this study, the potential cleavage site Lys135-Phe136 was resistant against the immobilized trypsin in N1. This article is protected by copyright. All rights reserved

Fibrinogen (FIB) is a secretory glycoprotein synthesized by hepatocytes that has a key role in blood clotting. Its glycosylation has not been studied in detail and little is known about the biological variability of FIB N-glycosylation, mainly due to the lack of fast, simple, and robust approaches to purify FIB from blood plasma samples. In recent years, customised chromatographic monoliths have been used for a variety of biological applications due to their unique characteristics. Here we describe development and optimisation of monolithic supports bearing monoclonal anti-human fibrinogen antibodies in a single column as well as in multi-well plate formats with high FIB specificity and binding capacity for fast immunoaffinity purification of FIB from human blood samples. The developed semi-high-throughput workflow has been successfully applied for FIB immunoaffinity isolation and subsequent ultra performance liquid chromatography N-glycosylation analysis in ten healthy human individuals, demonstrating the potential of monolithic supports in glycomics studies.

Key properties of monolithic chromatographic supports, make them suitable for separation and/or concentration of large biomolecules, especially virus particles and viral genomes. One by one, the studies that have been completed so far, contributed to the knowledge that monolith chromatography has hardly any limitation to be applied in virus research. Viruses of different sizes, possessing icosahedral structure and symmetrical morphology, as well as rod-shaped or filamentous viruses with helical structure, even enveloped ones, all of them could be successfully managed by means of monolith chromatography. Same is true for viral genomes, primarily when being distinct from other nucleic acid forms present in a host cell. This review is exclusively focussed on viruses. It describes the application of monolith chromatography to different problematics within the virus research field. The reviewed achievements offer new possibilities and trigger new aspects in virology. This article is protected by copyright. All rights reserved