Fibrillar extracellular matrix produced by pericyte-like cells facilitates glioma cell dissemination.

Gliomagenesis induces profound changes in the composition of the extracellular matrix (ECM) of the brain. In this study, we identified a cellular population responsible for the increased deposition of collagen I and fibronectin in glioblastoma. Elevated levels of the fibrillar proteins collagen I and fibronectin were associated with the expression of fibroblast activation protein (FAP), which is predominantly found in pericyte-like cells in glioblastoma. FAP+ pericyte-like cells were present in regions rich in collagen I and fibronectin in biopsy material and produced substantially more collagen I and fibronectin in vitro compared to other cell types found in the GBM microenvironment. Using mass spectrometry, we demonstrated that 3D matrices produced by FAP+ pericyte-like cells are rich in collagen I and fibronectin and contain several basement membrane proteins. This expression pattern differed markedly from glioma cells. Finally, we have shown that ECM produced by FAP+ pericyte-like cells enhances the migration of glioma cells including glioma stem-like cells, promotes their adhesion, and activates focal adhesion kinase (FAK) signaling. Taken together, our findings establish FAP+ pericyte-like cells as crucial producers of a complex ECM rich in collagen I and fibronectin, facilitating the dissemination of glioma cells through FAK activation.

Recommendations for reporting equivalent black carbon (eBC) mass concentrations based on long-term pan-European in-situ observations.

A reliable determination of equivalent black carbon (eBC) mass concentrations derived from filter absorption photometers (FAPs) measurements depends on the appropriate quantification of the mass absorption cross-section (MAC) for converting the absorption coefficient (babs) to eBC. This study investigates the spatial-temporal variability of the MAC obtained from simultaneous elemental carbon (EC) and babs measurements performed at 22 sites. We compared different methodologies for retrieving eBC integrating different options for calculating MAC including: locally derived, median value calculated from 22 sites, and site-specific rolling MAC. The eBC concentrations that underwent correction using these methods were identified as LeBC (local MAC), MeBC (median MAC), and ReBC (Rolling MAC) respectively. Pronounced differences (up to more than 50 %) were observed between eBC as directly provided by FAPs (NeBC; Nominal instrumental MAC) and ReBC due to the differences observed between the experimental and nominal MAC values. The median MAC was 7.8 ± 3.4 m2 g-1 from 12 aethalometers at 880 nm, and 10.6 ± 4.7 m2 g-1 from 10 MAAPs at 637 nm. The experimental MAC showed significant site and seasonal dependencies, with heterogeneous patterns between summer and winter in different regions. In addition, long-term trend analysis revealed statistically significant (s.s.) decreasing trends in EC. Interestingly, we showed that the corresponding corrected eBC trends are not independent of the way eBC is calculated due to the variability of MAC. NeBC and EC decreasing trends were consistent at sites with no significant trend in experimental MAC. Conversely, where MAC showed s.s. trend, the NeBC and EC trends were not consistent while ReBC concentration followed the same pattern as EC. These results underscore the importance of accounting for MAC variations when deriving eBC measurements from FAPs and emphasize the necessity of incorporating EC observations to constrain the uncertainty associated with eBC.

Positive matrix factorization of seasonally resolved organic aerosol at three different central European background sites based on nuclear magnetic resonance Aerosolomics data.

Concentration data derived from 1H NMR analysis of the water-soluble organic compounds from fine aerosol (PM2.5) at three Central European background stations, Kosetice, Frýdlant (both in the Czech Republic), and Melpitz (Germany), were used for detailed source apportionment analysis. Two winter and two summer episodes (year 2021) with higher organic concentrations and similar wind directions were selected for NMR analyses. The concentration profiles of 61 water-soluble organic compounds were determined by NMR Aerosolomics and a principal component analysis (PCA) was performed on this dataset. Based on the PCA results, 23 compounds were selected for positive matrix factorization (PMF) analysis in order to identify dominant aerosol sources at rural background sites in Central Europe. Both the PCA and the subsequent PMF analyses clearly distinguished the characteristics of winter and summer aerosol particles. In summer, four factors were identified from PMF and were associated with biogenic aerosol (61-78 %), background aerosol (9-15 %), industrial biomass combustion (7-13 %), and residential heating (5-13 %). In winter, only 3 factors were identified - industrial biomass combustion (33-49 %), residential heating (37-45 %) and a background aerosol (8-30 %). The main difference was observed in the winter season with a stronger contribution of emissions from industrial biomass burning at the Czech stations Kosetice and Frýdlant (47-49 %) compared to the Melpitz station (33 %). However, in general, there were negligible differences in identified sources between stations in the given seasons, indicating a certain homogeneity in PM2.5 composition within Central Europe at least during the sampling periods.

Continuous short-term acclimation to moderate cold elicits cardioprotection in rats, and alters ß-adrenergic signaling and immune status.

Moderate cold acclimation (MCA) is a non-invasive intervention mitigating effects of various pathological conditions including myocardial infarction. We aim to determine the shortest cardioprotective regimen of MCA and the response of ß1/2/3-adrenoceptors (ß-AR), its downstream signaling, and inflammatory status, which play a role in cell-survival during myocardial infarction. Adult male Wistar rats were acclimated (9 °C, 1-3-10 days). Infarct size, echocardiography, western blotting, ELISA, mitochondrial respirometry, receptor binding assay, and quantitative immunofluorescence microscopy were carried out on left ventricular myocardium and brown adipose tissue (BAT). MultiPlex analysis of cytokines and chemokines in serum was accomplished. We found that short-term MCA reduced myocardial infarction, improved resistance of mitochondria to Ca2+-overload, and downregulated ß1-ARs. The ß2-ARs/protein kinase B/Akt were attenuated while ß3-ARs translocated on the T-tubular system suggesting its activation. Protein kinase G (PKG) translocated to sarcoplasmic reticulum and phosphorylation of AMPKThr172 increased after 10 days. Principal component analysis revealed a significant shift in cytokine/chemokine serum levels on day 10 of acclimation, which corresponds to maturation of BAT. In conclusion, short-term MCA increases heart resilience to ischemia without any negative side effects such as hypertension or hypertrophy. Cold-elicited cardioprotection is accompanied by ß1/2-AR desensitization, activation of the ß3-AR/PKG/AMPK pathways, and an immunomodulatory effect.

A year-round observation of δ13C of dicarboxylic acids and related compounds in fine aerosols: Implications from Central European background site.

Isotopic analysis of specific compounds in aerosols can be a useful tool when studying atmospheric processes. Here, we present the results of stable carbon isotope ratio (δ13C) measurements performed on a one-year set (n = 96, Sep. 2013-Aug. 2014) of dicarboxylic acids and related compounds in PM1 at a rural Central European background site, Kosetice (Czech Republic). The most 13C enriched acid was oxalic (C2, annual average = -16.6 ± 5.0‰) followed by malonic (C3, avg. = -19.9 ± 6.6‰) and succinic (C4, avg. = -21.3 ± 4.6‰) acids. Thus, δ13C values decreased with an increase in carbon numbers. Azelaic acid (C9, avg. = -27.2 ± 3.6‰) was found to be the least 13C enriched. A comparison of δ13C of dicarboxylic acids from other background sites, especially in Asia, shows similar values to those from the European site. This comparison also showed that C2 is more 13C enriched at background sites than at urban ones. In general, we did not observe significant seasonal differences in δ13C values of dicarboxylic acids at the Central European station. We observed statistically significant differences (p value < 0.05) between winter and summer δ13C values solely for C4, glyoxylic acid (ωC2), glutaric acid (C5) and suberic acid (C8). The only significant correlations between δ13C of C2 and δ13C of C3 were found in spring and summer, suggesting that the oxidation of C3 to C2 is significant in these months with a strong contribution from biogenic aerosols. The strongest season-independent annual correlation was observed in δ13C values between C2 and C4, the two dominant dicarboxylic acids. Therefore, C4 appears to be the main intermediate precursor of C2 throughout the whole year.

Different approaches to explore the impact of COVID-19 lockdowns on carbonaceous aerosols at a European rural background site.

To prevent the fast spread of COVID-19, worldwide restrictions have been put in place, leading to a reduction in emissions from most anthropogenic sources. In this study, the impact of COVID-19 lockdowns on elemental (EC) and organic (OC) carbon was explored at a European rural background site combining different approaches: - "Horizontal approach (HA)" consists of comparing concentrations of pollutants measured at 4 m a.g.l. during pre-COVID period (2017-2019) to those measured during COVID period (2020-2021); - "Vertical approach (VA)" consists of inspecting the relationship between OC and EC measured at 4 m and those on top (230 m) of a 250 m-tall tower in Czech Republic. The HA showed that the lockdowns did not systematically result in lower concentrations of both carbonaceous fractions unlike NO2 (25 to 36 % lower) and SO2 (10 to 45 % lower). EC was generally lower during the lockdowns (up to 35 %), likely attributed to the traffic restrictions whereas increased OC (up to 50 %) could be attributed to enhanced emissions from the domestic heating and biomass burning during this stay-home period, but also to the enhanced concentration of SOC (up to 98 %). EC and OC were generally higher at 4 m suggesting a greater influence of local sources near the surface. Interestingly, the VA revealed a significantly enhanced correlation between EC and OC measured at 4 m and those at 230 m (R values up to 0.88 and 0.70 during lockdown 1 and 2, respectively), suggesting a stronger influence of aged and long distance transported aerosols during the lockdowns. This study reveals that lockdowns did not necessarily affect aerosol absolute concentrations but it certainly influenced their vertical distribution. Therefore, analyzing the vertical distribution can allow a better characterization of aerosol properties and sources at rural background sites, especially during a period of significantly reduced human activities.

Huntingtin Co-Isolates with Small Extracellular Vesicles from Blood Plasma of TgHD and KI-HD Pig Models of Huntington's Disease and Human Blood Plasma.

(1) Background: Huntington's disease (HD) is rare incurable hereditary neurodegenerative disorder caused by CAG repeat expansion in the gene coding for the protein huntingtin (HTT). Mutated huntingtin (mHTT) undergoes fragmentation and accumulation, affecting cellular functions and leading to neuronal cell death. Porcine models of HD are used in preclinical testing of currently emerging disease modifying therapies. Such therapies are aimed at reducing mHTT expression, postpone the disease onset, slow down the progression, and point out the need of biomarkers to monitor disease development and therapy efficacy. Recently, extracellular vesicles (EVs), particularly exosomes, gained attention as possible carriers of disease biomarkers. We aimed to characterize HTT and mHTT forms/fragments in blood plasma derived EVs in transgenic (TgHD) and knock-in (KI-HD) porcine models, as well as in HD patients' plasma. (2) Methods: Small EVs were isolated by ultracentrifugation and HTT forms were visualized by western blotting. (3) Results: The full length 360 kDa HTT co-isolated with EVs from both the pig model and HD patient plasma. In addition, a ~70 kDa mutant HTT fragment was specific for TgHD pigs. Elevated total huntingtin levels in EVs from plasma of HD groups compared to controls were observed in both pig models and HD patients, however only in TgHD were they significant (p = 0.02). (4) Conclusions: Our study represents a valuable initial step towards the characterization of EV content in the search for HD biomarkers.

Dextran Sulphate Sodium Acute Colitis Rat Model: A Suitable Tool for Advancing Our Understanding of Immune and Microbial Mechanisms in the Pathogenesis of Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is a group of disorders causing inflammation in the digestive tract. Recent data suggest that dysbiosis may play a pivotal role in the IBD pathogenesis. As microbiome-based therapeutics that modulate the gut ecology have been proposed as a novel strategy for preventing IBD, the aim of presenting study was to evaluate the dextran sulphate sodium (DSS) rat model mainly in terms of microbial shifts to confirm its suitability for dysbiosis study in IBD. Acute colitis was induced using 5% DSS solution for seven days and rats were euthanized five days after DSS removal. The faecal/caecal microbiota was analyzed by next generation sequencing. Disease activity index (DAI) score was evaluated daily. Blood and colon tissue immunophenotyping was assessed by flow cytometry and histological, haematological, and biochemical parameters were also evaluated. The colitis induction was reflected in a significantly higher DAI score and changes in all parameters measured. This study demonstrated significant shifts in the colitis-related microbial species after colitis induction. The characteristic inflammation-associated microbiota could be detected even after a five day-recovery period. Moreover, the DSS-model might contribute to an understanding of the effect of different treatments on extraintestinal organ impairments. The observation that certain bacterial species in the gut microbiota are associated with colitis raises the question of whether these organisms are contributors to, or a consequence of the disease. Despite some limitations, we confirmed the suitability of DSS-induced colitis model to monitor microbial changes during acute colitis, in order to test attractive new microbiome-based therapies.

Proteomic Analysis of Human Neural Stem Cell Differentiation by SWATH-MS.

The unique properties of stem cells to self-renew and differentiate hold great promise in disease modelling and regenerative medicine. However, more information about basic stem cell biology and thorough characterization of available stem cell lines is needed. This is especially essential to ensure safety before any possible clinical use of stem cells or partially committed cell lines. As proteins are the key effector molecules in the cell, the proteomic characterization of cell lines, cell compartments or cell secretome and microenvironment is highly beneficial to answer above mentioned questions. Nowadays, method of choice for large-scale discovery-based proteomic analysis is mass spectrometry (MS) with data-independent acquisition (DIA). DIA is a robust, highly reproducible, high-throughput quantitative MS approach that enables relative quantification of thousands of proteins in one sample. In the current protocol, we describe a specific variant of DIA known as SWATH-MS for characterization of neural stem cell differentiation. The protocol covers the whole process from cell culture, sample preparation for MS analysis, the SWATH-MS data acquisition on TTOF 5600, the complete SWATH-MS data processing and quality control using Skyline software and the basic statistical analysis in R and MSstats package. The protocol for SWATH-MS data acquisition and analysis can be easily adapted to other samples amenable to MS-based proteomics.

Seasonal changes in stable carbon isotopic composition in the bulk aerosol and gas phases at a suburban site in Prague.

Isotope fractionation between the gas and aerosol phases is an important phenomenon for studying atmospheric processes. Here, for the first time, seasonally resolved stable carbon isotope ratio (δ13C) values are systematically used to study phase interactions in bulk aerosol and gaseous carbonaceous samples. Seasonal variations in the δ13C of total carbon (TC; δ13CTC) and water-soluble organic carbon (WSOC; δ13CWSOC) in fine aerosol particles (PM2.5) as well as in the total carbon of part of the gas phase (TCgas; δ13CTCgas) were studied at a suburban site in Prague, Czech Republic, Central Europe. Year-round samples were collected for the main and backup filters from 14 April 2016 to 1 May 2017 every 6 days with a 48 h sampling period (n = 66). During all seasons, the highest 13C enrichment was found in WSOC, followed by particulate TC, whereas the highest 13C depletion was found in gaseous TC. We observed a clear seasonal pattern for all δ13C, with the highest values in winter (avg. δ13CTC = -25.5 ± 0.8‰, δ13CWSOC = -25.0 ± 0.7‰, δ13CTCgas = -27.7 ± 0.5‰) and the lowest values in summer (avg. δ13CTC = -27.2 ± 0.5‰, δ13CWSOC = -26.4 ± 0.3‰, δ13CTCgas = -28.9 ± 0.3‰). This study supports the existence of different aerosol sources at the site during the year. Despite the different seasonal compositions of carbonaceous aerosols, the isotope difference (Δδ13C) between δ13CTC (aerosol) and δ13CTCgas (gas phase) was similar during the seasons (year avg. 1.97 ± 0.50‰). Moreover, Δδ13C between WSOC and TC in PM2.5 showed a difference between spring and winter, but in general, these values were also similar year-round (year avg. 0.71 ± 0.37‰). During the entire period, TCgas and WSOC were the most 13C-depleted and most 13C-enriched fractions, respectively, and although the resulting difference Δ(δ13CWSOC - Î´13CTCgas) was significant, it was almost invariant throughout the year (2.67 ± 0.44‰). The present study suggests that the stable carbon isotopic fractionation between the bulk aerosol and gas phases is probably not entirely dependent on the chemical composition of individual carbonaceous compounds from different sources.

Targeted mass spectrometry for monitoring of neural differentiation.

Human multipotent neural stem cells could effectively be used for the treatment of a variety of neurological disorders. However, a defining signature of neural stem cell lines that would be expandable, non-tumorigenic, and differentiate into desirable neuronal/glial phenotype after in vivo grafting is not yet defined. Employing a mass spectrometry approach, based on selected reaction monitoring, we tested a panel of well-described culture conditions, and measured levels of protein markers routinely used to probe neural differentiation, i.e. POU5F1 (OCT4), SOX2, NES, DCX, TUBB3, MAP2, S100B, GFAP, GALC, and OLIG1. Our multiplexed assay enabled us to simultaneously identify the presence of pluripotent, multipotent, and lineage-committed neural cells, thus representing a powerful tool to optimize novel and highly specific propagation and differentiation protocols. The multiplexing capacity of this method permits the addition of other newly identified cell type-specific markers to further increase the specificity and quantitative accuracy in detecting targeted cell populations. Such an expandable assay may gain the advantage over traditional antibody-based assays, and represents a method of choice for quality control of neural stem cell lines intended for clinical use.

Mass absorption cross-section and absorption enhancement from long term black and elemental carbon measurements: A rural background station in Central Europe.

Black carbon (BC) is a dominant aerosol light absorber, and its brown carbon (BrC) coating can enhance absorption and lead to uncertainties concerning the radiative forcing estimation. This study investigates the mass absorption cross-section of equivalent BC (MACeBC) during a long-term field measurement (2013-2017) at a rural Central European site. The MAC enhancement factor (Eabs) and the contribution of BrC coatings to the absorption coefficient (Babs) were estimated by combining different approaches. The annual mean Babs and MACeBC values decreased slightly over the measurement period associated with change in the submicron aerosol size distribution. Regardless of the wavelength, Babs exhibited clear seasonal and diurnal variations, with higher values in winter when a higher absorption Ångström exponent (1.4) was observed due to the local biomass burning (BB). In contrast, MACeBC did not have a distinct temporal trend at 600 nm (7.84 ± 2.79 m2 g-1), while it showed a seasonal trend at 370 nm with higher values in winter (15.64 ± 4.77 m2 g-1). During this season, Eabs_660 was 1.18 ± 0.27 and did not exhibit any clear wavelength dependence, despite the influence of BB. During the study period, BrC-attributed absorption was observed in 31% of the samples, with a contribution of up to 40% of total Babs. In summer, the Eabs_660 increased to 1.59 ± 0.60, when a larger BC coating could be formed by secondary aerosol fractions. During this season, MACeBC_660 and Eabs_660 showed comparable source profiles that were mainly associated with aged air masses over central Europe, thereby supporting the fact that characteristics of coating materials formed during atmospheric aging are a major factor driving the MACeBC_660 measured at the regional background site. Further field investigations of the composition of BC coatings would help to better understand and estimate uncertainties related to the radiative effect of aerosols.

Emerging Roles of Exosomes in Huntington's Disease.

Huntington's disease (HD) is a rare hereditary autosomal dominant neurodegenerative disorder, which is caused by expression of mutant huntingtin protein (mHTT) with an abnormal number of glutamine repeats in its N terminus, and characterized by intracellular mHTT aggregates (inclusions) in the brain. Exosomes are small extracellular vesicles that are secreted generally by all cell types and can be isolated from almost all body fluids such as blood, urine, saliva, and cerebrospinal fluid. Exosomes may participate in the spreading of toxic misfolded proteins across the central nervous system in neurodegenerative diseases. In HD, such propagation of mHTT was observed both in vitro and in vivo. On the other hand, exosomes might carry molecules with neuroprotective effects. In addition, due to their capability to cross blood-brain barrier, exosomes hold great potential as sources of biomarkers available from periphery or carriers of therapeutics into the central nervous system. In this review, we discuss the emerging roles of exosomes in HD pathogenesis, diagnosis, and therapy.

Distinctive Sources Govern Organic Aerosol Fractions with Different Degrees of Oxygenation in the Urban Atmosphere.

Understanding how the sources of an atmospheric organic aerosol (OA) govern its burden is crucial for assessing its impact on the environment and adopting proper control strategies. In this study, the sources of OA over Beijing were assessed year-around based on the combination of two separation approaches for OA, one from chemical fractionation into the high-polarity fraction of water-soluble organic matter (HP-WSOM), humic-like substances (HULIS), and water-insoluble organic matter (WISOM), and the other from statistical grouping using positive matrix factorization (PMF) of high-resolution aerosol mass spectra. Among the three OA fractions, HP-WSOM has the highest O/C ratio (1.36), followed by HULIS (0.56) and WISOM (0.17). The major sources of different OA fractions were distinct: HP-WSOM was dominated by more oxidized oxygenated OA (96%); HULIS by cooking-like OA (40%), less oxidized oxygenated OA (27%), and biomass burning OA (21%); and WISOM by fossil fuel OA (77%). In addition, our results provide evidence that mass spectral-based PMF factors are associated with specific substructures in molecules. These structures are further discussed in the context of the FT-IR results. This study presents an overall relationship of OA groups monitored by chemical and statistical approaches for the first time, providing insights for future source apportionment studies.

Spatial-temporal variability of aerosol sources based on chemical composition and particle number size distributions in an urban settlement influenced by metallurgical industry.

The Moravian-Silesian region of the Czech Republic with its capital city Ostrava is a European air pollution hot spot for airborne particulate matter (PM). Therefore, the spatiotemporal variability assessment of source contributions to aerosol particles is essential for the successful abatement strategies implementation. Positive Matrix Factorization (PMF) was applied to highly-time resolved PM0.15-1.15 chemical composition (1 h resolution) and particle number size distribution (PNSD, 14 nm - 10 µm) data measured at the suburban (Ostrava-Plesná) and urban (Ostrava-Radvanice) residential receptor sites in parallel during an intensive winter campaign. Diel patterns, meteorological variables, inorganic and organic markers, and associations between the chemical composition factors and PNSD factors were used to identify the pollution sources and their origins (local, urban agglomeration and regional). The source apportionment analysis resolved six and four PM0.15-1.15 sources in Plesná and Radvanice, respectively. In Plesná, local residential combustion sources (coal and biomass combustion) followed by regional combustion sources (residential heating, metallurgical industry) were the main contributors to PM0.15-1.15. In Radvanice, local residential combustion and the metallurgical industry were the most important PM0.15-1.15 sources. Aitken and accumulation mode particles emitted by local residential combustion sources along with common urban sources (residential heating, industry and traffic) were the main contributors to the particle number concentration (PNC) in Plesná. Additionally, accumulation mode particles from local residential combustion sources and regional pollution dominated the particle volume concentration (PVC). In Radvanice, local industrial sources were the major contributors to PNC and local coal combustion was the main contributor to PVC. The source apportionment results from the complementary datasets elucidated the relevance of highly time-resolved parallel measurements at both receptor sites given the specific meteorological conditions produced by the regional orography. These results are in agreement with our previous studies conducted at this site. Graphical abstract.

Protein changes in synaptosomes of Huntington's disease knock-in mice are dependent on age and brain region.

Molecular changes at synapses are thought to underly the deficits in motor and cognitive dysfunction seen in Huntington's disease (HD). Previously we showed in synaptosome preparations age dependent changes in levels of selected proteins examined by western blot assay in the striatum of Q140/Q140 HD mice. To assess if CAG repeat length influenced protein changes at the synapse, we examined synaptosomes from 6-month old heterozygote HD mice with CAG repeat lengths ranging from 50 to 175. Analysis of 19 selected proteins showed that increasing CAG repeat length in huntingtin (HTT) increased the number of affected proteins in HD striatal synaptosomes. Moreover, SDS-soluble total HTT (WT plus mutant HTT) and pThr3 HTT were reduced with increasing CAG repeat length, and there was no pSer421 mutant HTT detected in any HD mice. A LC-MS/MS and bioinfomatics study of synaptosomes from 2 and 6-month old striatum and cortex of Q140/Q7 HD mice showed enrichment of synaptic proteins and an influence of age, gender and brain region on the number of protein changes. HD striatum at 6 months had the most protein changes that included many HTT protein interactors, followed by 2-month old HD striatum, 2-month old HD cortex and 6-month HD cortex. SDS-insoluble mutant HTT was detected in HD striatal synaptosomes consistent with the presence of aggregates. Proteins changed in cortex differed from those in striatum. Pathways affected in HD striatal synaptosomes that were not identified in whole striatal lysates of the same HD mouse model included axon guidance, focal adhesion, neurotrophin signaling, regulation of actin cytoskeleton, endocytosis, and synaptic vesicle cycle. Results suggest that synaptosomes prepared from HD mice are highly informative for monitoring protein changes at the synapse and may be preferred for assessing the effects of experimental therapies on synaptic function in HD.

Expression of lamin C2 in mammalian oocytes.

Lamin C2 (LMN C2) is a short product of the lamin a gene. It is a germ cell-specific lamin and has been extensively studied in male germ cells. In this study, we focussed on the expression and localization of LMN C2 in fully-grown germinal vesicle (GV) oocytes. We detected LMN C2 in the fully-grown germinal vesicle oocytes of various mammalian species with confirmation done by immunoblotting the wild type and Lmnc2 gene deleted testes. Expression of LMN C2 tagged with GFP showed localization of LMN C2 to the nuclear membrane of the oocyte. Moreover, the LMN C2 protein notably disappeared after nuclear envelope breakdown (NEBD) and the expression of LMN C2 was significantly reduced in the oocytes from aged females and ceased altogether during meiotic maturation. These results provide new insights regarding LMN C2 expression in the oocytes of various mammalian species.

A Comparative Analysis of Multipotent Mesenchymal Stromal Cells derived from Different Sources, with a Focus on Neuroregenerative Potential.

Multipotent mesenchymal stromal cells (MSCs) can be considered an accessible therapeutic tool for regenerative medicine. Here, we compared the growth kinetics, immunophenotypic and immunomodulatory properties, gene expression and secretome profile of MSCs derived from human adult bone marrow (BM-MSCs), adipose tissue (AT-MSCs) and Wharton's jelly (WJ-MSCs) cultured in clinically-relevant conditions, with the focus on the neuroregenerative potential. All the cell types were positive for CD10/CD29/CD44/CD73/CD90/CD105/HLA-ABC and negative for CD14/CD45/CD235a/CD271/HLA-DR/VEGFR2 markers, but they differed in the expression of CD34/CD133/CD146/SSEA-4/MSCA-1/CD271/HLA-DR markers. BM-MSCs displayed the highest immunomodulatory activity compared to AT- and WJ-MSCs. On the other hand, BM-MSCs secreted the lower content and had the lower gene expression of neurotrophic growth factors compared to other cell lines, which may be caused by the higher sensitivity of BM-MSCs to nutrient limitations. Despite the differences in growth factor secretion, the MSC secretome derived from all cell sources had a pronounced neurotrophic potential to stimulate the neurite outgrowth of DRG-neurons and reduce the cell death of neural stem/progenitor cells after H2O2 treatment. Overall, our study provides important information for the transfer of basic MSC research towards clinical-grade manufacturing and therapeutic applications.

Luminex xMAP Assay to Quantify Cytokines in Cancer Patient Serum.

Cytokines, chemokines, and growth factors are key mediators of cell proliferation, migration, and immune response, and in tumor microenvironment, such factors contribute to regulation of tumor growth, immune cell recruitment, angiogenesis, and metastasis. In body fluids, levels of inflammatory mediators reflect the patient immune response to the disease and may predict the effects of targeted therapies. Significant improvements in cytokine detection techniques have been made during last 10 years leading to sensitive quantification of such potent molecules present in low pg/mL levels. Among the techniques, Luminex xMAP® multiplex assays allow for simultaneous quantification of up to 100 analytes with high sensitivity, broad dynamic range of quantification, high throughput, and minimal sample requirements. In this chapter we describe a detailed protocol for the application of xMAP assays using Luminex® 200™ analyzer with xPonent® acquisition software to quantify cytokines, chemokines, and growth factors secreted to blood serum and plasma of cancer patients. We also discuss how sample preparation, instrument settings, and standard curve fitting algorithms can influence validity of obtained results. Special attention is paid to data analysis using open source R statistical environment and we provide an example dataset of cytokine levels measured in serum and corresponding R script for standard curve fitting and concentration estimates.