361 research outputs found

    Progenitor cells are responsible for formation of human prostate epithelium primary cultures

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    To analyze cell viability and morphology of primary cell cultures from CD133 immunolabeled and sorted cells from epithelium of patients suffering from benign prostate hyperplasia (BPH). Methods: Cells obtained from 5 patients were divided in two fractions. First fraction (CD133+/CD133–) was cultivated in DMEM with 10% FBS. Second fraction was mixed with CD133 microbeads and immunomagnetically divided into CD133+ and CD133– fractions. These cells were cultivated and followed-up for 2 weeks. Cells were stained for Annexin V FITC/propidium iodide. Results: Seventy CD133+/CD133– cultures, thirty-one of CD133+ and thirty-one of CD133– cells were established. There were 5-fold and 3-fold increase of CD133+/CD133- and CD133+ cell number after 2 weeks, respectively. CD133+/CD133– and CD133+ monolayers displayed epithelial-like morphology and cytokeratine expression. CD133– cultures collapsed. Cell viability within CD133+ and CD133– populations was 90.1 ± 6.3% and 24.3 ± 6.2%, respectively. Apoptotic index was 9.0 ± 6.1% and 28.5 ± 23.8% within CD133+ and CD133– cultures, respectively. Conclusions: CD133 separated human primary epithelial cell cultures displayed differences in morphology, viability and apoptosis occurrence. Immunomagnetic sorting can be recommended in each in vitro experiments with primary cell cultures in order to provide more objective results.Цель: оценить жизнеспособность и морфологию клеток первичных клеточных культур, полученных из меченных по CD133 и полученных с помощью клеточной сортировки клеток эпителия пациентов с доброкачественной гиперплазией предстательной железы (BPH). Методы: клетки, полученные от 5 пациентов, были разделены на 2 фракции. Первую фракцию (CD133+/CD133–) выращивали в DMEM с 10% FBS али с CD133 магнитными гранулами и с помощью магнита разделили клетки на CD133+- и CD133–-фракции. Далее клетки культивировали в течении 2 нед. Клетки окрашивали аннексиномV FITC/пропидий йодидом. Результаты: получено 70 CD133+/CD133- -культур клеток, 31 CD133+ и 31 CD133–. Через 2 нед культивирования отмечали 5-кратное и 3-кратное увеличение количества CD133+/CD133– и CD133+ клеток соответственно. CD133+/CD133- -и CD133+-клетки росли в монослое и имели морфологию эпителиальных клеток, экспрессировали цитокератин. CD133–-клетки не выжили. Выживаемость клеток в популяциях CD133+ и CD133– была 90,1 ± 6,3% и 24,3 ± 6,2% соответственно. Показатель апоптического индекса для культур CD133+ и CD133– был 9,0 ± 6,1% и 28,5 ± 23,8% соответственно. Выводы: показаны различия в морфологии, выживаемости клеток и частоте апоптоза для эпителиальных клеток, разделенных в зависимости от экспрессии CD133. Сортировка клеток с помощью иммуномагнитного разделения рекомендована для каждого in vitro эксперимента с использованием первичных клеточных культур для получения более объективных результатов

    Droplet-based digital antibiotic susceptibility screen reveals single-cell clonal heteroresistance in an isogenic bacterial population

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    Since antibiotic resistance is a major threat to global health, recent observations that the traditional test of minimum inhibitory concentration (MIC) is not informative enough to guide effective antibiotic treatment are alarming. Bacterial heteroresistance, in which seemingly susceptible isogenic bacterial populations contain resistant sub-populations, underlies much of this challenge. To close this gap, here we developed a droplet-based digital MIC screen that constitutes a practical analytical platform for quantifying the single-cell distribution of phenotypic responses to antibiotics, as well as for measuring inoculum effect with high accuracy. We found that antibiotic efficacy is determined by the amount of antibiotic used per bacterial colony forming unit (CFU), not by the absolute antibiotic concentration, as shown by the treatment of beta-lactamase-carrying Escherichia coli with cefotaxime. We also noted that cells exhibited a pronounced clustering phenotype when exposed to near-inhibitory amounts of cefotaxime. Overall, our method facilitates research into the interplay between heteroresistance and antibiotic efficacy, as well as research into the origin and stimulation of heterogeneity by exposure to antibiotics. Due to the absolute bacteria quantification in this digital assay, our method provides a platform for developing reference MIC assays that are robust against inoculum-density variations

    Droplet-based digital antibiotic susceptibility screen reveals single-cell clonal heteroresistance in an isogenic bacterial population

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    Since antibiotic resistance is a major threat to global health, recent observations that the traditional test of minimum inhibitory concentration (MIC) is not informative enough to guide effective antibiotic treatment are alarming. Bacterial heteroresistance, in which seemingly susceptible isogenic bacterial populations contain resistant sub-populations, underlies much of this challenge. To close this gap, here we developed a droplet-based digital MIC screen that constitutes a practical analytical platform for quantifying the single-cell distribution of phenotypic responses to antibiotics, as well as for measuring inoculum effect with high accuracy. We found that antibiotic efficacy is determined by the amount of antibiotic used per bacterial colony forming unit (CFU), not by the absolute antibiotic concentration, as shown by the treatment of beta-lactamase-carrying Escherichia coli with cefotaxime. We also noted that cells exhibited a pronounced clustering phenotype when exposed to near-inhibitory amounts of cefotaxime. Overall, our method facilitates research into the interplay between heteroresistance and antibiotic efficacy, as well as research into the origin and stimulation of heterogeneity by exposure to antibiotics. Due to the absolute bacteria quantification in this digital assay, our method provides a platform for developing reference MIC assays that are robust against inoculum-density variations

    Towards an Architecture Proposal for Federation of Distributed DES Simulators

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    The simulation of large and complex Discrete Event Systems (DESs) increasingly imposes more demanding and urgent requirements on two aspects accepted as critical: (1) Intensive use of models of the simulated system that can be exploited in all phases of its life cycle where simulation can be used, and methodologies for these purposes; (2) Adaptation of simulation techniques to HPC infrastructures, as a method to improve simulation efficiency and to have scalable simulation environments. This paper proposes a Model Driven Engineering approach (MDE) based on Petri Nets (PNs) as formal model. This approach proposes a domain specific language based on modular PNs from which efficient distributed simulation code is generated in an automatic way. The distributed simulator is constructed over generic simulation engines of PNs, each one containing a data structure representing a piece of net and its simulation state. The simulation engine is called simbot and versions of it are available for different platforms. The proposed architecture allows, in an efficient way, a dynamic load balancing of the simulation work because the moving of PN pieces can be realized by moving a small number of integers representing the subnet and its state

    The pentameric nucleoplasmin fold is present in Drosophila FKBP39 and a large number of chromatin-related proteins.

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    Nucleoplasmin is a histone chaperone that consists of a pentameric N-terminal domain and an unstructured C-terminal tail. The pentameric core domain, a doughnut-like structure with a central pore, is only found in the nucleoplasmin family. Here, we report the first structure of a nucleoplasmin-like domain (NPL) from the unrelated Drosophila protein, FKBP39, and we present evidence that this protein associates with chromatin. Furthermore, we show that two other chromatin proteins, Arabidopsis thaliana histone deacetylase type 2 (HD2) and Saccharomyces cerevisiae Fpr4, share the NPL fold and form pentamers, or a dimer of pentamers in the case of HD2. Thus, we propose a new family of proteins that share the pentameric nucleoplasmin-like NPL domain and are found in protists, fungi, plants and animals.We are grateful to Gunter Stier for providing the vector; Michael Nilges, Oleg Fedorov, Benjamin Bardiaux, Stefanie Hartmann and Wolfgang Rieping for helpful discussions; and Daniel Nietlispach for NMR expertise. We thank Renato Paro for generously providing us with an anti-FKBP39 antibody. We would like to thank the Wellcome Trust for financial support (grant 082010/Z/07/Z). V.T.F. and E.D.L. acknowledge support from Engineering and Physical Sciences Research Council under grants GR/R99393/01 and EP/C015452/1 for the creation of the Deuteration Laboratory platform operating within the Grenoble Partnership for Structural Biology. V.T.F. also acknowledges support from the European Union under contract RII3-CT-2003-505925. J.B.A. acknowledges the provision of a postdoctoral fellowship held at Keele University. M.R.P. and D.M.G. were supported by the Medical Research Council and Cancer Research UK grants to D.M.G. A.A.W. is a recipient of a Wellcome Trust Fellowship092441/Z/10/Z. J.D. and M.D. were supported by the Harmonia 5 Grant 2013/10/M/NZ2/00298 from the Polish National Science Center. The authors would like to thank the Institut Laue-Langevin (ILL), the European Synchrotron Radiation Facility (ESRF) and the European Molecular Biology Laboratory Hamburg outstation (EMBL-HH) for the provision of beamtime and access to the experimental facilities of D22, ID14eh3 and X33 respectively. We would also like to thank the local contacts at all the facilities for providing assistance in using the beam lines.This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.jmb.2015.03.01

    The Pentameric Nucleoplasmin Fold Is Present in Drosophila FKBP39 and a Large Number of Chromatin-Related Proteins

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    Nucleoplasmin is a histone chaperone that consists of a pentameric N-terminal domain and an unstructured C-terminal tail. The pentameric core domain, a doughnut-like structure with a central pore, is only found in the nucleoplasmin family. Here, we report the first structure of a nucleoplasmin-like domain (NPL) from the unrelated Drosophila protein, FKBP39, and we present evidence that this protein associates with chromatin. Furthermore, we show that two other chromatin proteins, Arabidopsis thaliana histone deacetylase type 2 (HD2) and Saccharomyces cerevisiae Fpr4, share the NPL fold and form pentamers, or a dimer of pentamers in the case of HD2. Thus, we propose a new family of proteins that share the pentameric nucleoplasmin-like NPL domain and are found in protists, fungi, plants and animals

    The Enzymatic Activity of Type 1 Iodothyronine Deiodinase (D1) is Low in Liver Hemangioma: A Preliminary Study

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    Type 1 iodothyronine deiodinase (D1) is a crucial enzyme which converts the prohormone thyroxine (T4) into active tri-iodothyronine (T3). There has been strong evidence that the metabolism of thyroid hormones is disturbed in some neoplastic tissues such as thyroid, renal, and breast cancer. However, there are few available data about D1 enzyme activity in benign tumors such as hemangioma, which is the most common primary liver tumor. Hence this study aimed to determine the enzymatic activity of D1 in hemangiomas in relation to healthy liver tissue. Seven tumors and healthy control tissues were obtained from patients who had liver resection due to hemangioma. The activity was assessed by measurement of radioactive iodine released by deiodination catalyzed by D1. It was found that D1 activity was significantly lower in the hemagiomas than in the healthy surrounding tissue (p = 0.0017). The results indicated that thyroid hormones play important roles not only in the regulation of cell metabolism, but also in cell growth, division, and apoptosis. The active form T3 acts through its nuclear receptors and influences the up- and down-regulation of target genes. Healthy liver tissue expresses a high level of D1, but disturbed D1 activity may result in changes in the local concentration of T3 which may impair gene transcription. These finding demonstrate a low enzymatic activity of D1 in liver hemangioma and suggest an as yet unknown role of thyroid hormones in this type of benign liver tumor

    The quest for companions to post-common envelope binaries. II. NSVS14256825 and HS0705+6700

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    We report new mid-eclipse times of the two close binaries NSVS14256825 and HS0705+6700, harboring an sdB primary and a low-mass main-sequence secondary. Both objects display clear variations in the measured orbital period, which can be explained by the action of a third object orbiting the binary. If this interpretation is correct, the third object in NSVS14256825 is a giant planet with a mass of roughly 12 M_Jup. For HS0705+6700, we provide evidence that strengthens the case for the suggested periodic nature of the eclipse time variation and reduces the uncertainties in the parameters of the brown dwarf implied by that model. The derived period is 8.4 yr and the mass is 31 M_Jup, if the orbit is coplanar with the binary. This research is part of the PlanetFinders project, an ongoing collaboration between professional astronomers and student groups at high schools.Comment: Accepted by Astron. and Astrophy
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