Browsing by Subject "FLOW-CYTOMETRY"

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  • Mora, Diego; Filardi, Rossella; Arioli, Stefania; Boeren, Sjef; Aalvink, Steven; de Vos, Willem M. (2019)
    The growing commercial interest in multi-strain formulations marketed as probiotics has not been accompanied by an equal increase in the evaluation of quality levels of these biotechnological products. The multi-strain product VSL#3 was used as a model to setup a microbiological characterization that could be extended to other formulations with high complexity. Shotgun metagenomics by deep Illumina sequencing was applied to DNA isolated from the commercial VSL#3 product to confirm strains identity safety and composition. Single-cell analysis was used to evaluate the cell viability, and beta-galactosidase and urease activity have been used as marker to monitor the reproducibility of the production process. Similarly, these lots were characterized in detail by a metaproteomics approach for which a robust protein extraction protocol was combined with advanced mass spectrometry. The results identified over 1600 protein groups belonging to all strains present in the VSL#3 formulation. Of interest, only 3.2 % proteins showed significant differences mainly related to small variations in strain abundance. The protocols developed in this study addressed several quality criteria that are relevant for marketed multi-strain products and these represent the first efforts to define the quality of complex probiotic formulations such as VSL#3.
  • Spilling, Kristian; Schulz, Kai G.; Paul, Allanah J.; Boxhammer, Tim; Achterberg, Eric P.; Hornick, Thomas; Lischka, Silke; Stuhr, Annegret; Bermudez, Rafael; Czerny, Jan; Crawfurd, Kate; Brussaard, Corina P. D.; Grossart, Hans-Peter; Riebesell, Ulf (2016)
    About a quarter of anthropogenic CO2 emissions are currently taken up by the oceans, decreasing seawater pH. We performed a mesocosm experiment in the Baltic Sea in order to investigate the consequences of increasing CO2 levels on pelagic carbon fluxes. A gradient of different CO2 scenarios, ranging from ambient (similar to 370 mu atm) to high (similar to 1200 mu atm), were set up in mesocosm bags (similar to 55m(3)). We determined standing stocks and temporal changes of total particulate carbon (TPC), dissolved organic carbon (DOC), dissolved inorganic carbon (DIC), and particulate organic carbon (POC) of specific plankton groups. We also measured carbon flux via CO2 exchange with the atmosphere and sedimentation (export), and biological rate measurements of primary production, bacterial production, and total respiration. The experiment lasted for 44 days and was divided into three different phases (I: t0-t16; II: t17-t30; III: t31-t43). Pools of TPC, DOC, and DIC were approximately 420, 7200, and 25 200 mmol Cm-2 at the start of the experiment, and the initial CO2 additions increased the DIC pool by similar to 7% in the highest CO2 treatment. Overall, there was a decrease in TPC and increase of DOC over the course of the experiment. The decrease in TPC was lower, and increase in DOC higher, in treatments with added CO2. During phase I the estimated gross primary production (GPP) was similar to 100 mmol C m(-2) day(-1), from which 75-95% was respired, similar to 1% ended up in the TPC (including export), and 5-25% was added to the DOC pool. During phase II, the respiration loss increased to similar to 100% of GPP at the ambient CO2 concentration, whereas respiration was lower (85-95% of GPP) in the highest CO2 treatment. Bacterial production was similar to 30% lower, on average, at the highest CO2 concentration than in the controls during phases II and III. This resulted in a higher accumulation of DOC and lower reduction in the TPC pool in the elevated CO2 treatments at the end of phase II extending throughout phase III. The "extra" organic carbon at high CO2 remained fixed in an increasing biomass of small-sized plankton and in the DOC pool, and did not transfer into large, sinking aggregates. Our results revealed a clear effect of increasing CO2 on the carbon budget and mineralization, in particular under nutrient limited conditions. Lower carbon loss processes (respiration and bacterial remineralization) at elevated CO2 levels resulted in higher TPC and DOC pools than ambient CO2 concentration. These results highlight the importance of addressing not only net changes in carbon standing stocks but also carbon fluxes and budgets to better disentangle the effects of ocean acidification.
  • Morani, Marco; Duc Mai, Thanh; Krupova, Zuzana; Defrenaix, Pierre; Multia, Evgen; Riekkola, Marja-Liisa; Taverna, Myriam (2020)
    This work reports on the development of the first capillary electrophoresis methodology for the elucidation of extracellular vesicles' (EVs) electrokinetic distributions. The approach is based on capillary electrophoresis coupled with laser-induced fluorescent (LIF) detection for the identification and quantification of EVs after their isolation. Sensitive detection of these nanometric entities was possible thanks to an 'inorganic-species-free' background electrolyte. This electrolyte was made up of weakly charged molecules at very high concentrations to stabilize EVs, and an intra-membrane labelling approach was used to prevent EV morphology modification. The limit of detection for EVs achieved using the developed CE-LIF method reached 8 x 10(9) EV/mL, whereas the calibration curve was acquired from 1.22 x 10(10) to 1.20 x 10(11) EV/mL. The CE-LIF approach was applied to provide the electrokinetic distributions of various EVs of animal and human origins, and visualize different EV subpopulations from our recently developed high-yield EV isolation method. (C) 2020 Elsevier B.V. All rights reserved.
  • Boonk, Stephanie E.; Zoutman, Willem H.; Marie-Cardine, Anne; van der Fits, Leslie; Out-Luiting, Jacoba J.; Mitchell, Tracey J.; Tosi, Isabella; Morris, Stephen L.; Moriarty, Blaithin; Booken, Nina; Felcht, Moritz; Quaglino, Pietro; Ponti, Renata; Barberio, Emanuela; Ram-Wolff, Caroline; Jantti, Kirsi; Ranki, Annamari; Bernengo, Maria Grazia; Klemke, Claus-Detlev; Bensussan, Armand; Michel, Laurence; Whittaker, Sean; Bagot, Martine; Tensen, Cornelis P.; Willemze, Rein; Vermeer, Maarten H. (2016)
    Differentiation between Sezary syndrome and erythrodermic inflammatory dermatoses can be challenging, and a number of studies have attempted to identify characteristic immunophenotypic changes and molecular biomarkers in Sezary cells that could be useful as additional diagnostic criteria. In this European multicenter study, the sensitivity and specificity of these immunophenotypic and recently proposed but unconfirmed molecular biomarkers in Sezary syndrome were investigated. Peripheral blood CD4(+) T cells from 59 patients with Sezary syndrome and 19 patients with erythrodermic inflammatory dermatoses were analyzed for cell surface proteins by flow cytometry and for copy number alterations and differential gene expression using custom-made quantitative PCR plates. Experiments were performed in duplicate in two independent centers using standard operating procedures with almost identical results. Sezary cells showed MYC gain (40%) and MNT loss (66%); up-regulation of DNM3 (75%), TWIST1 (69%), EPHA4 (66%), and PLS3 (66%); and down-regulation of STAT4 (91%). Loss of CD26 (>= 80% CD4(+) T cells) and/or CD7 (>= 40% CD4(+) T cells) and combination of altered expression of STAT4, TWIST1, and DNM3 or PLS3 could distinguish, respectively, 83% and 98% of patients with Sezary syndrome from patients with erythrodermic inflammatory dermatoses with 100% specificity. These additional diagnostic panels will be useful adjuncts in the differential diagnosis of Sezary syndrome versus erythrodermic inflammatory dermatoses.
  • Silva, Luis; Calleja, Maria Ll.; Ivetic, Snjezana; Huete-Stauffer, Tamara; Roth, Florian; Carvalho, Susana; Moran, Xose Anxelu G. (2021)
    In coral reefs, dissolved organic matter (DOM) cycling is a critical process for sustaining ecosystem functioning. However, global and local stressors have caused persistent shifts from coral- to algae-dominated benthic communities. The influence of such phase shifts on DOM nature and its utilization by heterotrophic bacterioplankton remains poorly studied. Every second month for one year, we retrieved seawater samples enriched in DOM produced by coral- and algae-dominated benthic communities in a central Red Sea reef during a full annual cycle. Seawater incubations were conducted in the laboratory under in situ temperature and light conditions by inoculating enriched DOM samples with bacterial assemblages collected in the surrounding waters. Dissolved organic carbon (DOC) concentrations were higher in the warmer months (May-September) in both communities, resulting in higher specific growth rates and bacterial growth efficiencies (BGE). However, these high summer values were significantly enhanced in algal-DOM relative to coral-DOM, suggesting the potential for bacterioplankton biomass increase in reefs with algae replacing healthy coral cover under warmer conditions. The potential exacerbation of heterotrophic bacterial activity in the ongoing widespread regime shift from coral- to algae-dominated communities may have detrimental consequences for the overall health of tropical coral reefs. (C) 2020 The Authors. Published by Elsevier B.V.
  • Giri, Anil K.; Ianevski, Aleksander (2022)
    Introduction The interactions between leukemic blasts and cells within the bone marrow environment affect oncogenesis, cancer stem cell survival, as well as drug resistance in hematological cancers. The importance of this interaction is increasingly being recognized as a potentially important target for future drug discoveries and developments. Recent innovations in the high throughput drug screening-related technologies, novel ex-vivo disease-models, and freely available machine-learning algorithms are advancing the drug discovery process by targeting earlier undruggable proteins, complex pathways, as well as physical interactions (e.g. leukemic cell-bone microenvironment interaction). Area covered In this review, the authors discuss the recent methodological advancements and existing challenges to target specialized hematopoietic niches within the bone marrow during leukemia and suggest how such methods can be used to identify drugs targeting leukemic cell-bone microenvironment interactions. Expert opinion The recent development in cell-cell communication scoring technology and culture conditions can speed up the drug discovery by targeting the cell-microenvironment interaction. However, to accelerate this process, collecting clinical-relevant patient tissues, developing culture model systems, and implementing computational algorithms, especially trained to predict drugs and their combination targeting the cancer cell-bone microenvironment interaction are needed.
  • Bach, Lennart T.; Taucher, Jan; Boxhammer, Tim; Ludwig, Andrea; Achterberg, Eric P.; Alguero-Muniz, Maria; Anderson, Leif G.; Bellworthy, Jessica; Buedenbender, Jan; Czerny, Jan; Ericson, Ylva; Esposito, Mario; Fischer, Matthias; Haunost, Mathias; Hellemann, Dana; Horn, Henriette G.; Hornick, Thomas; Meyer, Jana; Sswat, Michael; Zark, Maren; Riebesell, Ulf; Kristineberg KOSMOS Consortium (2016)
    Every year, the oceans absorb about 30% of anthropogenic carbon dioxide (CO2) leading to a re-equilibration of the marine carbonate system and decreasing seawater pH. Today, there is increasing awareness that these changes-summarized by the term ocean acidification (OA)-could differentially affect the competitive ability of marine organisms, thereby provoking a restructuring of marine ecosystems and biogeochemical element cycles. In winter 2013, we deployed ten pelagic mesocosms in the Gullmar Fjord at the Swedish west coast in order to study the effect of OA on plankton ecology and biogeochemistry under close to natural conditions. Five of the ten mesocosms were left unperturbed and served as controls (similar to 380 mu atm pCO(2)), whereas the others were enriched with CO2-saturated water to simulate realistic end-of-the-century carbonate chemistry conditions (mu 760 mu atm pCO(2)). We ran the experiment for 113 days which allowed us to study the influence of high CO2 on an entire winter-to-summer plankton succession and to investigate the potential of some plankton organisms for evolutionary adaptation to OA in their natural environment. This paper is the first in a PLOS collection and provides a detailed overview on the experimental design, important events, and the key complexities of such a "long-term mesocosm" approach. Furthermore, we analyzed whether simulated end-of-the-century carbonate chemistry conditions could lead to a significant restructuring of the plankton community in the course of the succession. At the level of detail analyzed in this overview paper we found that CO2-induced differences in plankton community composition were non-detectable during most of the succession except for a period where a phytoplankton bloom was fueled by remineralized nutrients. These results indicate: (1) Long-term studies with pelagic ecosystems are necessary to uncover OA-sensitive stages of succession. (2) Plankton communities fueled by regenerated nutrients may be more responsive to changing carbonate chemistry than those having access to high inorganic nutrient concentrations and may deserve particular attention in future studies.
  • Kuuliala, Krista; Kuuliala, Antti; Koivuniemi, Riitta; Kautiainen, Hannu; Repo, Heikki; Leirisalo-Repo, Marjatta (2016)
    Objective To find novel predictors of treatment response to disease-modifying antirheumatic drugs (DMARDs), we studied activation of STAT (signal transducers and activators of transcription) 6 and 1 in circulating leukocytes of patients with rheumatoid arthritis (RA). Methods 19 patients with untreated recent-onset RA, 16 patients with chronic RA irresponsive to synthetic DMARDs and 37 healthy volunteers provided blood samples for whole blood flow cytometric determination of intracellular STAT6 and STAT1 phosphorylation, expressed as relative fluorescence units, in response to IL-4 and IFN-gamma, respectively. Phosphorylation was restudied and treatment response (according to European League Against Rheumatism) determined after 1-year treatment with synthetic DMARDs in recent-onset RA and with biological DMARD in synthetic DMARD-irresponsive RA. Estimation-based exact logistic regression was used to investigate relation of baseline variables to treatment response. 95% confidence intervals of means were estimated by bias-corrected bootstrapping and the significance between baseline and follow-up values was calculated by permutation test. Results At baseline, levels of phosphorylated STAT6 (pSTAT6) induced by IL-4 in monocytes were higher in those who achieved good treatment response to synthetic DMARDs than in those who did not among patients with untreated RA (OR 2.74, 95% CI 1.05 to 9.47), and IFN-gamma-stimulated lymphocyte pSTAT1 levels were higher in those who achieved good treatment response to a biological drug than in those who did not among patients with chronic RA (OR 3.91, 95% CI 1.12 to 20.68). During follow-up, in recent-onset RA patients with good treatment response to synthetic DMARDS, the lymphocyte pSTAT6 levels decreased (p = 0.011), and, consequently, the ratio of pSTAT1/pSTAT6 in lymphocytes increased (p = 0.042). Conclusion Cytokine-stimulated STAT6 and STAT1 phosphorylation in circulating leukocytes was associated with treatment response to DMARDs in this pilot study. The result, if confirmed in larger studies, may aid in developing personalized medicine in RA.