578 research outputs found
Impact of Narrow Constraint on Single Cell Motion
Die extrazellulÀre Mikroumgebung spielt eine grundlegende Rolle bei der Entwicklung
von Metastasen und hat einen groĂen Einfluss auf die Wahl der Migrationsstrategien, die von Karzinomzellen wĂ€hrend der Invasion angewandt werden. In-vitro Anordnungen sind hilfreiche Instrumente fuÌr die Untersuchung von Zellmigration und -invasion, da sie grundlegende Merkmale von In-vivo-Geweben reproduzieren können. Ziel dieser Forschungsarbeit ist es, die FĂ€higkeit von mesenchymalen und epithelialen Brusttumorzellen zu untersuchen, sich zu verfluÌssigen und durch enge und starre Mikrostrukturen zu navigieren. Wir verwendeten eine mikrofluidische Vorrichtung mit trichterförmigen Mikroverengungen und verglichen das Verhalten von fuÌnf verschiedenen menschlichen Brustkrebszelllinien in der Mikrovorrichtung bei
Stimulation durch Chemoattraktoren. Wir fanden heraus, dass grundsÀtzlich verschiedene Zelllinien das gleiche invasive Potenzial haben, da normale Epithelzellen in der Lage waren, durch die stark komprimierenden Trichter zu wandern, Àhnlich wie die invasiveren mesenchymalen Zellen. Wir fanden auch heraus, dass die Migration der normalen Epithelzellen auch ohne einen chemo-attraktiven Stimulus stattfindet. Wir konzentrierten unsere Beobachtungen auf die Rolle des Aktin- und IntermediÀrfilament-Zytoskeletts wÀhrend der eingeschrÀnkten Migration und zeigten, dass das Aktin-Zytoskelett eine starke und langanhaltende Reorganisation erfÀhrt, damit die Zellen durch die engen Verengungen kriechen können. Wir sahen keinen Hinweis darauf, dass das Keratin- und Vimentin-Zwischenfilament- Zytoskelett wÀhrend der Invasion in die Mikroverengungen eine aktive mechanische Rolle
spielte. Insbesondere die Expression des Vimentin-Zwischenfilamentproteins korrelierte in unserem Versuchsaufbau nicht mit der InvasionsfÀhigkeit einzelner Zellen. Unter diesen Voraussetzungen wurden die passiven (ElastizitÀt und ViskositÀt) und aktiven (KontraktilitÀt) viskoelastischen Eigenschaften der Zellen weiter untersucht und quantifiziert. Wir fanden keinen signifikanten Unterschied in der passiven viskoelastischen Reaktion der Zellen, nachdem sie oszillierenden DruckkrÀften mittels AFM-Sondierung ausgesetzt waren, was darauf hindeutet, dass ElastizitÀt und ViskositÀt nicht zur Unterscheidung zwischen invasiven und nicht-invasiven Zellen verwendet werden können. Es wurde kein Hinweis darauf gefunden, dass die
Kompressionsversteifung die Invasion durch die Mikroverengungen entweder behindert oder fördert. SchlieĂlich haben wir bei der Betrachtung aktiver viskoelastischer Parameter die kontraktile Reaktion unserer Zelllinien verglichen, wenn sie mit dem mikrofluidischen optischen Strecker Laser-StreckkrĂ€ften ausgesetzt wurden. Hier fanden wir eine klare Korrelation zwischen den Zelllinien, die ein invasives Verhalten in den Mikroverengungen zeigten, und denjenigen, die eine aktive (substratunabhĂ€ngige) kontraktile Reaktion in der optischen Streckvorrichtung
zeigten. Wir kommen zu dem Schluss, dass ein entscheidender Faktor fuÌr eine erfolgreiche Migration durch hohe rĂ€umliche Enge die FĂ€higkeit der Zellen ist, aktiv Aktin-Stressfasern zu erzeugen und abzubauen, was sich in der FĂ€higkeit manifestiert, von einer substratabhĂ€ngigen und stressfaserbasierten KontraktilitĂ€t zu einer substratunabhĂ€ngigen kortikalen KontraktilitĂ€t zu wechseln
Creation and optimisation of a yeast whole-cell network
Il presente lavoro Ăš consistito nella creazione ed ottimizzazione di una rete virtuale di lievito, che possa predire i risultati di esperimenti su ceppi di lievito mutati; esso ha pertanto richiesto i seguenti passi:
1) Creazione di un modello simulabile di S. cerevisiae, impiegando il linguaggio delle Petri Nets; la rete definitiva si estende dai livelli generici schematizzati fino ai livelli specifici in cui i singoli processi sono descritti in dettaglio.
2) Creazione di scripts per simulare ed ottimizzare il modello; lo script di simulazione coniuga le regole delle Petri Nets e lâalgoritmo di Gillespie, mentre il âtraining e testingâ impiegano lo script di simulazione allâinterno di un metodo Monte Carlo.
3) Acquisizione dati per la fase di training: misure sperimentali della fluorescenza delle GFP citoplasmatica ed endoplasmatica in ceppi mutanti. Altri dati sulla fluorescenza della GFP, trascrizionalmente indotta dalla UPR, sono stati ottenuti da Jonikas et al (2009).
4) "Training e testingâ per ottimizzare la rete affinchĂ© mostri le proprietĂ cercate.
Ho inoltre contribuito alla progettazione di esyN (Bean et al, 2014), un sito per la costruzione e condivisione di Petri Nets, fornendo anche gli script necessari per la simulazione ed analisi delle sue reti in ambiente R.
This work has consisted in the creation and optimization of a yeast virtual cell, which should be able to predict the results of experiments performed on mutant yeast strains. Therefore, it has required the following steps:
1) Creation of a simulable model of S. Cerevisiae by employing the Petri Net language. The final network spans from a generic, schematic layer to specific layers, where cellular processes are described in details.
2) Creation of scripts for simulating and optimizing the model. The simulation script employs Petri Net firing rules and the Gillespie algorithm, whereas the âtraining and testingâ stage employ that simulation script inside a Monte Carlo method.
3) Acquisition of data for the training stage: experimental measures of cytoplasmic and endoplasmic GFP in yeast mutant strains. Jonikas et al. (2009) have provided other data on the GFP fluorescence, transcriptionally induced by the UPR.
4) âTraining and testingâ for optimizing the network so that it may show the required features.
I have also contributed to the creation of esyN (Bean et al, 2014), a web tool for building and sharing Petri Nets; I have also provided all the scripts required for simulating and analysing its output networks inside a R environment
Increased expression and activity of p75NTR are crucial events in azacitidine-induced cell death in prostate cancer
The high affinity nerve growth factor (NGF) NGF receptor, p75NTR, is a member of the tumor necrosis factor (TNF) receptor superfamily that shares a conserved intracellular death domain capable of inducing apoptosis and suppressing growth in prostate epithelial cells. Expression of this receptor is lost as prostate cancer progresses and is minimal in established prostate cancer cell lines. We aimed to verify the role of p75NTR in the azacitidine-mediated antitumor effects on 22Rv1 and PC3 androgen-independent prostate cancer cells. In the present study, we reported that the antiproliferative and pro-apoptotic effects of 5-azacytidine (azacitidine) were more marked in the presence of physiological concentrations of NGF and were reduced when a blocking p75NTR antibody or the selective p75NTR inhibitor, Ro 08-2750, were used. Azacitidine increased the expression of p75NTR without interfering with the expression of the low affinity NGF receptor TrkA and induced caspase 9-dependent caspase 3 activity. Taken together, our results suggest that the NGF network could be a candidate for future pharmacological manipulation in aggressive prostate cancer
The use of fentanyl buccal tablets for breakthrough pain by using doses proportional to opioid basal regimen in a home care setting.
Abstract
The dose of rapid onset opioids to be given for breakthrough cancer pain (BTcP) is controversial. Dose proportional to the basal opioid regimen seem to be safe and effective in hospital units. However, data in other less protected settings, like home care, are lacking. The aim of this open-label study was to assess the efficacy and safety in a group of patients with BTcP followed at home, after giving a dose of fentanyl buccal tablets (FBT) proportional to the opioid basal regimen, skipping the steps for dose titration. Consecutive patients admitted to a home care program presenting BTcP episodes and receiving stable doses of opioids for background pain were selected. Data from four consecutive episodes of BTcP were collected. For each episode, patients were instructed to routinely collect changes in pain intensity and severe adverse effects when pain got severe (T0) and to reassess the same items 15 min after FBT, given as a rescue medication in doses proportional to the daily opioid doses used for background pain (T15). One hundred twenty episodes of BTcP were recorded in 30 patients. One hundred eight episodes were defined as successfully treated, while 12 episodes required a further administration of opioids. Pain intensity significantly decreased at T15 (p < 0.001). In 95.5 and 90.8 % of episodes treated, there was a reduction in pain intensity of more than 33 and 50 %, respectively. No relevant adverse effects were recorded, even in older patients. This study suggests that FBT given in doses proportional to the basal opioid regimen for the management of BTcP is very effective and safe in clinical practice in the home care setting
Hypoxia sustains glioblastoma radioresistance through ERKs/DNA-PKcs/HIF-1α functional interplay
The molecular mechanisms by which glioblastoma multiforme (GBM) refracts and becomes resistant to radiotherapy treatment remains largely unknown. This radioresistance is partly due to the presence of hypoxic regions, which are frequently found in GBM tumors. We investigated the radiosensitizing effects of MEK/ERK inhibition on GBM cell lines under hypoxic conditions. Four human GBM cell lines, T98G, U87MG, U138MG and U251MG were treated with the MEK/ERK inhibitor U0126, the HIF-1α inhibitor FM19G11 or Îł-irradiation either alone or in combination under hypoxic conditions. Immunoblot analysis of specific proteins was performed in order to define their antiâoncogenic or radiosensitizing roles in the different experimental conditions. MEK/ERK inhibition by U0126 reverted the transformed phenotype and significantly enhanced the radiosensitivity of T98G, U87MG, U138MG cells but not of the U251MG cell line under hypoxic conditions. U0126 and ERK silencing by siRNA reduced the levels of DNA protein kinase catalytic subunit (DNA-PKcs), Ku70 and K80 proteins and clearly reduced HIF-1α activity and protein expression. Furthermore, DNA-PKcs siRNA-mediated silencing counteracted HIF-1α activity and downregulated protein expression suggesting that ERKs, DNA-PKcs and HIF-1α cooperate in radioprotection of GBM cells. Of note, HIF-1α inhibition under hypoxic conditions drastically radiosensitized all cell lines used. MEK/ERK signal transduction pathway, through the sustained expression of DNA-PKcs, positively regulates HIF-1α protein expression and activity, preserving GBM radioresistance in hypoxic condition
esyN: network building, sharing and publishing.
The construction and analysis of networks is increasingly widespread in biological research. We have developed esyN ("easy networks") as a free and open source tool to facilitate the exchange of biological network models between researchers. esyN acts as a searchable database of user-created networks from any field. We have developed a simple companion web tool that enables users to view and edit networks using data from publicly available databases. Both normal interaction networks (graphs) and Petri nets can be created. In addition to its basic tools, esyN contains a number of logical templates that can be used to create models more easily. The ability to use previously published models as building blocks makes esyN a powerful tool for the construction of models and network graphs. Users are able to save their own projects online and share them either publicly or with a list of collaborators. The latter can be given the ability to edit the network themselves, allowing online collaboration on network construction. esyN is designed to facilitate unrestricted exchange of this increasingly important type of biological information. Ultimately, the aim of esyN is to bring the advantages of Open Source software development to the construction of biological networks.This is the final published version of the paper. It's also available from PLOS at http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0106035
Rapid Prototyping of 3D Biochips for Cell Motility Studies Using Two-Photon Polymerization
The study of cellular migration dynamics and strategies plays a relevant role in the
understanding of both physiological and pathological processes. An important example
could be the link between cancer cell motility and tumor evolution into metastatic
stage. These strategies can be strongly influenced by the extracellular environment
and the consequent mechanical constrains. In this framework, the possibility to study
the behavior of single cells when subject to specific topological constraints could
be an important tool in the hands of biologists. Two-photon polymerization is a
sub-micrometric additive manufacturing technique that allows the fabrication of 3D
structures in biocompatible resins, enabling the realization of ad hoc biochips for cell
motility analyses, providing different types of mechanical stimuli. In our work, we present
a new strategy for the realization of multilayer microfluidic lab-on-a-chip constructs
for the study of cell motility which guarantees complete optical accessibility and the
possibility to freely shape the migration area, to tailor it to the requirements of the
specific cell type or experiment. The device includes a series of micro-constrictions
that induce different types of mechanical stress on the cells during their migration. We
show the realization of different possible geometries, in order to prove the versatility of
the technique. As a proof of concept, we present the use of one of these devices for the
study of the motility of murine neuronal cancer cells under high physical confinement,
highlighting their peculiar migration mechanisms
Intermediate filaments ensure resiliency of single carcinoma cells, while active contractility of the actin cortex determines their invasive potential
During the epithelial-to-mesenchymal transition, the intracellular cytoskeleton undergoes severe
reorganization which allows epithelial cells to transition into a motile mesenchymal phenotype.
Among the different cytoskeletal elements, the intermediate filaments keratin (in epithelial cells)
and vimentin (in mesenchymal cells) have been demonstrated to be useful and reliable histological
markers. In this study, we assess the potential invasiveness of six human breast carcinoma cell lines
and two mouse fibroblasts cells lines through single cell migration assays in confinement. We find
that the keratin and vimentin networks behave mechanically the same when cells crawl through
narrow channels and that vimentin protein expression does not strongly correlate to single cells
invasiveness. Instead, we find that what determines successful migration through confining spaces
is the ability of cells to mechanically switch from a substrate-dependent stress fibers based
contractility to a substrate-independent cortical contractility, which is not linked to their tumor
phenotype
Meta-analysis of phase III trials of docetaxel alone or in combination with chemotherapy in metastatic breast cancer
Opioid switching from and to tapentadol extended release in cancer patients: conversion ratio with other opioids
Objectives:
The aim of this exploratory study was to assess the conversion ratios between tapentadol and other opioids in patients requiring an opioid switching.
Methods:
A prospective study was carried out in a convenience sample of consecutive patients admitted to an acute palliative care unit and a home care unit for a period of 1 year. Patients who were switched from/to tapentadol were selected. The initial ratio between tapentadol and other opioids, expressed as oral morphine equivalents was 1:3.3. The subsequent doses were flexible and were changed to fit the patientsâ needs. Pain intensity and distress score were recorded until opioid doses were stable. In all, 37 patients were examined; 24 and 13 patients were switched from and to tapentadol, respectively.
Results:
The most frequent sequences were tapentadolâmorphine (18 patients) in one direction, and morphineâtapentadol (8 patients) in the other direction. In the sequence tapentadolâmorphine and morphineâtapentadol, the mean final tapentadolâmorphine ratios were 3.9:1 (SD 2.3), and 1:4.5 (SD 3.2), respectively, which did not differ significantly from the initial established conversion ratio. A minority of patients were switched from/to tapentadol to/from other opioids. Globally, the initial ratio did not change after switching took place.
Conclusion:
Data suggest that a conversion ratio between tapentadol and other opioids, expressed in oral morphine equivalents could be 1:3.3 in both direction, particularly in patients who are switched in conditions of equianalgesia. The limited number of patients prevents a definitive conclusion to be drawn, and data should be interpreted with caution, given the exploratory nature of the study and the question of the low number of patients should be addressed in future studie
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