Военная служба бояр-шляхты и организация вооруженных формирований в Великом княжестве Литовском в первой половине XVI в.

В XVI в. социальное общество Великого княжества Литовского представляло собой сложную систему, основанную на феодальной иерархии. Все важные должности в государственном аппарате занимали люди знатного происхождения. В этот период одним из привилегированных сословий являлись представители военного сословия бояре-шляхта. За отправление ратной службы они получали от господаря участки земли. Нормы отправления земской службы были обусловлены количеством зависимого населения

Proteome analysis of bronchoalveolar lavage from calves infected with bovine respiratory syncytial virus-Insights in pathogenesis and perspectives for new treatments

Human and bovine respiratory syncytial viruses (HRSV/BRSV) are major causes of severe lower respiratory tract infections in children and calves, respectively. Shared epidemiological, clinical, pathological and genetic characteristics of these viruses make comparative research highly relevant. To characterise the host response against BRSV infection, bronchoalveolar lavage supernatant (BAL) from i) non-vaccinated, BRSV-infected ii) vaccinated, BRSV-infected and iii) non-infected calves was analysed by tandem mass spectrometry. Proteins were semi-quantified and protein expression was validated by immunoblotting. Correlations between selected proteins and pathology, clinical signs and virus shedding were investigated. Calves with BRSV-induced disease had increased total protein concentrations and a decreased number of proteins identified in BAL. The protein profile was characterised by neutrophil activation and a reduction in identified antioxidant enzymes. The presence of neutrophils in alveolar septa, the expression level of neutrophil-related or antioxidant proteins and LZTFL1 correlated significantly with disease. Citrullinated histone 3, an indicator of extracellular traps (ETs), was only detected in non-vaccinated, BRSV-infected animals. By bringing disequilibrium in the release and detoxification of reactive oxygen species, generating ETs and causing elastine degradation, exaggerated neutrophil responses might exacerbate RSV-induced disease. Neutrophil-mitigating or antioxidant treatments should be further explored

MS for investigation of time-dependent protein adsorption on surfaces in complex biological samples

Aim: This study aims at developing a nondestructive way for investigating proteinadsorption on surfaces such as biomaterials using mass spectrometry. Methods: Ventricular cerebrospinal fluid in contact with poly carbonate membranes were usedas adsorption templates and on-surface enzymatic digestion was applied to desorbproteins and cleave them into peptides. Mass spectrometric analysis provided bothprotein identification and determination of protein specific adsorption behavior. Results: In general, the adsorption increased with incubation time but also proteinspecifictime-resolved adsorption patterns from the complex protein solutionwere discovered. Conclusion: The method developed is a promising tool for thecharacterization of biofouling, which sometimes causes rejection and encapsulationof implants and can be used as complement to other surface analytical techniques. One problem associated with artificial materials in the body is that proteins in thebody interact with the surface, which sometimes causes rejection of the implant.In this study, a method for investigating the time-dependent protein adsorptionon a surface originating from a complex biological protein solution was developed.Compared with other surface analyses, this method can identify what proteins thatadsorbs on the surface. In addition, determination of protein-specific adsorptionbehavior in relation to incubation was possibl

Time-resolved proteomics of adenovirus infected cells

Viral infections cause large problems in the world and deeper understanding of the disease mechanisms is needed. Here we present an analytical strategy to investigate the host cell protein changes during human adenovirus type 2 (HAdV-C2 or Ad2) infection of lung fibro-blasts by stable isotope labelling of amino acids in cell culture (SILAC) and nanoLC-MS/MS. This work focuses on early phase of infection (6 and 12 h post-infection (hpi)) but the data is combined with previously published late phase (24 and 36 hpi) proteomics data to produce a time series covering the complete infection. As many as 2169 proteins were quantitatively monitored from 6 to 36 hpi, while some proteins were time-specific. After applying different filter criteria, 2027 and 2150 proteins were quantified at 6 and 12 hpi and among them, 431 and 544 were significantly altered at the two time points. Pathway analysis showed that the De novo purine and pyrimidine biosynthesis, Glycolysis and Cytoskeletal regulation by Rho GTPase pathways were activated early during infection while inactivation of the Integrin signalling pathway started between 6 and 12 hpi. Moreover, upstream regulator analysis predicted MYC to be activated with time of infection and protein and RNA data for genes controlled by this transcription factor showed good correlation, which validated the use of protein data for this prediction. Among the identified phosphorylation sites, a group related to glycolysis and cytoskeletal reorganization were up-regulated during infection. The results show specific aspects on how the host cell proteins, the final products in the genetic information flow, are influenced by Ad2 infection, which would be overlooked if only knowledge derived from mRNA data is considered

Data on the expression of cellular lncRNAs in human adenovirus infected cells

Expression of cellular long non-coding RNAs (lncRNAs) in human primary lung fibroblasts (IMR-90) during the course of adenovirus type 2 (Ad2) infection was studied by strand-specific whole transcriptome sequencing. In total, 645 cellular lncRNAs were expressed at a significant level and 398 of them were changed more than 2-fold. The changes in expression followed a distinct temporal pattern. Significantly, 80% of the changes occurred at the late phase and 80% of the de-regulated lncRNAs were up-regulated. The three largest groups of deregulated lncRNAs were 125 antisense RNAs, 111 pseudogenes and 85 long intergenic non-coding RNAs (lincRNAs). Lastly, more than 36% of lncRNAs have been shown to interact with RNA binding proteins. Keywords: Adenovirus, Long noncoding RNA, IncRNA, IMR-9

Mass Spectrometric Determination of the Effect of Surface Deactivation on Membranes Used for In-Situ Sampling of Cerebrospinal Fluid (CSF)

In this paper, a strategy for structured monitoring of surface modifications to control protein adsorption to membrane structures is presented. The already established on-surface enzymatic digestion (oSED) method combined with nano-liquid chromatography and tandem mass spectrometry (LC-MS/MS) analysis was employed for the analysis of proteins in ventricular cerebrospinal fluid (vCSF) from neurointensive care patients. Protein adsorption was studied by in-situ sampling in a temporally resolved manner on both immobilized native and Pluronic-deactivated membranes. Deactivation was significantly reducing the protein adsorption but it also induced novel selective properties of the surface. The proposed versatile strategy will facilitate protein-biomaterial, protein-polymer, protein-protein interaction studies in the future

Inhibition properties of free and conjugated leupeptin analogues

Leupeptin is a naturally occurring inhibitor of various proteases, in particular serine proteases. Following its discovery, the inhibitory properties of several other peptidyl argininals have been studied. The specificity of leupeptin is most likely due to the Leu-Leu-Argininal sequence, and its C-terminal aldehyde group has been suggested to enhance the binding efficiency and to be essential for function. The terminal aldehyde group makes the structure less vulnerable to carboxypeptidases. Here, we investigated whether the inhibitory function of leupeptin toward serine proteases is retained after oxidation or reduction of the aldehyde group. The oxidized form, which corresponds to the natural precursor, was shown to be superior to the reduced form in terms of inhibitory properties. However, the original leupeptin possessed enhanced inhibitory properties as compared with the oxidized form. Based on these results, new synthetic leupeptin analogues, 6-aminohexanoic acid (Ahx)-Phe-Leu-Arg-COOH and Ahx-Leu-Leu-Arg-COOH, were prepared by solid-phase peptide synthesis using the Fmoc strategy. In these analogues, the N-terminal capping acetyl group was replaced with a 6-aminohexanoyl group to allow conjugation. The structures of the modified leupeptin and the synthetic peptides were confirmed by mass spectrometry. Determination of the inhibitory properties against trypsin (IEC 3.4.21.4, Chymotrypsin IEC 3.4.21.1) revealed that these further modified tripeptides were tight binding inhibitors to their target enzyme, similar to the naturally occurring leupeptin, with Ki values generally in the micromolar range. The Ahx-Phe-Leu-Arg-COOH analogue was selected for conjugation to inorganic oxide nanoparticles and agarose gel beads. All conjugates exhibited inhibitory activity in the same range as for the free peptides.

Influence of surface modification and static pressure on microdialysis protein extraction efficiency

There is growing interest in using microdialysis (MD) for monitoring larger and more complexmolecules such as neuropeptides and proteins. This promotes the use of MD membranes withmolecular weight cut off (MWCO) of 100 kDa or above. The hydrodynamic property of themembrane goes to ultrafiltration or beyond, making the MD catheters more sensitive to pressure.In the meantime, despite the large pore size, studies have shown that membrane biofouling stilllead to unstable catheter performance. The objective is to study in vitro how 500 kDa dextranand Poloxamer 407 surface modification affect the fluid recovery (FR) and extraction efficiency(EE) of 100 kDa MWCO MD catheters. A pressure chamber was designed to facilitate the tests,using as MD sample a protein standard with similar concentrations as in human cerebral spinalfluid, comparing native and Poloxamer 407 modified MD catheters. The collected dialysatefractions were examined for FR and protein EE, employing Dot-it Spot-it Protein Assay for totalprotein EE and targeted mass spectrometry (MS) for EE of individual proteins and peptides. TheFR results suggested that the surface modified catheters were less sensitive to the pressure andprovide higher precision, and provided a FR closer to 100%. The surface modification did notshow a significant effect on the protein EE. The average total protein EE of surface modifiedcatheters was slightly higher than that of the native ones. The MS EE data of individual proteinsshowed a clear trend of complex response in EE with pressure

Plasma-derived extracellular vesicles from myocardial infarction patients inhibits tumor necrosis factor-alpha induced cardiac cell death.

RATIONALE: Extracellular vesicles (EVs) derived exogenously from pluripotent stem cells or endogenously from healthy human serum exert cardioprotective effects after injury. However role of endogenous EVs from myocardial infarction (MI) patients not well understood in this settings. METHODS AND RESULTS: The EVs from plasma of MI patients with preserved or reduced left ventricular ejection fraction (LVEF) and healthy controls (HC) were purified and characterized by flow cytometry, mass spectrometry (MS) and transmission electron microscopy (TEM). HCM and human cardiac microvascular endothelial cells (hCMVECs), under individual culture or co-culture, were used to study functional effects of EVs upon TNFα stimulation. These effects of EVs on HCM and hCMVECs were observed using cell death assays, western blots and confocal microscopy. Higher concentrations of platelet-, leukocyte-, endothelial- and erythrocyte-derived EVs were found in MI patients, both with preserved and reduced LVEF, compared to HC, and MS data on MI EVs proteome displayed alteration in several proteins. MI EVs protected HCM and hCMVECs against staurosporine-induced apoptosis. Furthermore, MI EVs were observed to abrogate TNFα-triggered HCM and hCMVECs death under both individually cultured and co-cultured conditions. MI EVs failed to inhibit TNFα induced hCMVECs and HCM activation when cultured individually, however co-cultured hCMVECs with HCM supported MI EVs capacity to attenuate TNFα induced cells activation. MI CD41+ EVs but not HC EVs were found to be internalized by HCM directly or migrated through hCMVECs to HCM. MI EVs indirectly restores TNFα mediated drop in mitochondrial membrane potential. CONCLUSIONS: Endogenous EVs from MI patients, regardless of severity of the MI exert cardioprotective potential upon TNFα-induced cell death. Patient-derived EVs needs to be further explored to elucidate their potential cardioprotective role during MI

Investigating the Impact of Sample Preparation on Mass Spectrometry-Based Drug-To-Antibody Ratio Determination for Cysteine- and Lysine-Linked Antibody–Drug Conjugates

Antibody–drug conjugates (ADCs) are heterogeneous biotherapeutics and differ vastly in their physicochemical properties depending on their design. The number of small drug molecules covalently attached to each antibody molecule is commonly referred to as the drug-to-antibody ratio (DAR). Established analytical protocols for mass spectrometry (MS)-investigation of antibodies and ADCs often require sample treatment such as desalting or interchain disulfide bond reduction prior to analysis. Herein, the impact of the desalting and reduction steps—as well as the sample concentration and elapsed time between synthesis and analysis of DAR-values (as acquired by reversed phase liquid chromatography MS (RPLC–MS))—was investigated. It was found that the apparent DAR-values could fluctuate by up to 0.6 DAR units due to changes in the sample preparation workflow. For methods involving disulfide reduction by means of dithiothreitol (DTT), an acidic quench is recommended in order to increase DAR reliability. Furthermore, the addition of a desalting step was shown to benefit the ionization efficiencies in RPLC–MS. Finally, in the case of delayed analyses, samples can be stored at four degrees Celsius for up to one week but are better stored at −20 °C for longer periods of time. In conclusion, the results demonstrate that commonly used sample preparation procedures and storage conditions themselves may impact MS-derived DAR-values, which should be taken into account when evaluating analytical procedures