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Gene expression orchestration is a key question in fundamental and applied research. Different models for transcription regulation were proposed, yet the dynamic regulation of RNA polymerase II (RNAP II) activity remains a matter of debate. To improve our knowledge of this topic, we investigated RNAP II motility in eukaryotic cells by combining single particle tracking (SPT) and fluorescence correlation spectroscopy (FCS) techniques, to take advantage of their different sensitivities in order to analyze together slow and fast molecular movements. Thanks to calibrated samples, we developed a benchmark for quantitative analysis of molecular dynamics, to eliminate the main potential instrumental biases. We applied this workflow to study the diffusion of RPB1, the catalytic subunit of RNAP II. By a cross-analysis of FCS and SPT, we could highlight different RPB1 motility states and identifyed a stationary state, a slow diffusion state, and two different modes of subdiffusion. Interestingly, our analysis also unveiled the oversampling by RPB1 of nuclear subdomains. Based on these data, we propose a novel model of spatio-temporal transcription regulation. Altogether, our results highlight the importance of combining microscopy approaches at different time scales to get a full insight into the real complexity of molecular kinetics in cells.
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ARN Polimerasa II , Imagen Individual de Molécula , Núcleo Celular , Transcripción Genética , MicroscopíaRESUMEN
Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique to probe the molecular environment of fluorophores. The analysis of FLIM images is usually performed with time consuming fitting methods. For accelerating this analysis, sophisticated deep learning architectures based on convolutional neural networks have been developed for restrained lifetime ranges but they require long training time. In this work, we present a simple neural network formed only with fully connected layers able to analyze fluorescence lifetime images. It is based on the reduction of high dimensional fluorescence intensity temporal decays into four parameters which are the phasor coordinates, the mean and amplitude-weighted lifetimes. This network called Phasor-Net has been applied for a time domain FLIM system excited with an 80 MHz laser repetition frequency, with negligible jitter and afterpulsing. Due to the restricted time interval of 12.5 ns, the training range of the lifetimes was limited between 0.2 and 3.0 ns; and the total photon number was lower than 106, as encountered in live cell imaging. From simulated biexponential decays, we demonstrate that Phasor-Net is more precise and less biased than standard fitting methods. We demonstrate also that this simple architecture gives almost comparable performance than those obtained from more sophisticated networks but with a faster training process (15 min instead of 30 min). We finally apply successfully our method to determine biexponential decays parameters for FLIM experiments in living cells expressing EGFP linked to mCherry and fused to a plasma membrane protein.
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ERK1/2 involvement in cell death remains unclear, although many studies have demonstrated the importance of ERK1/2 dynamics in determining cellular responses. To untangle how ERK1/2 contributes to two cell death programs, we investigated ERK1/2 signaling dynamics during hFasL-induced apoptosis and TNF-induced necroptosis in L929 cells. We observed that ERK1/2 inhibition sensitizes cells to apoptosis while delaying necroptosis. By monitoring ERK1/2 activity by live-cell imaging using an improved ERK1/2 biosensor (EKAR4.0), we reported differential ERK1/2 signaling dynamics between cell survival, apoptosis, and necroptosis. We also decrypted a temporally shifted amplitude- and frequency-modulated (AM/FM) ERK1/2 activity profile in necroptosis versus apoptosis. ERK1/2 inhibition, which disrupted ERK1/2 signaling dynamics, prevented TNF and IL-6 gene expression increase during TNF-induced necroptosis. Using an inducible cell line for activated MLKL, the final executioner of necroptosis, we showed ERK1/2 and its distinctive necroptotic ERK1/2 activity dynamics to be positioned downstream of MLKL.
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Control of cell death/survival balance is an important feature to maintain tissue homeostasis. Dependence receptors are able to induce either survival or cell death in presence or absence of their ligand, respectively. However, their precise mechanism of action and their physiological importance are still elusive for most of them including the MET receptor. We evidence that pro-apoptotic fragment generated by caspase cleavage of MET localizes to the mitochondria-associated membrane region. This fragment triggers a calcium transfer from endoplasmic reticulum to mitochondria, which is instrumental for the apoptotic action of the receptor. Knock-in mice bearing a mutation of MET caspase cleavage site highlighted that p40MET production is important for FAS-driven hepatocyte apoptosis, and demonstrate that MET acts as a dependence receptor in vivo. Our data shed light on new signaling mechanisms for dependence receptors' control of cell survival/death balance, which may offer new clues for the pathophysiology of epithelial structures.
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Muerte Celular/fisiología , Supervivencia Celular/fisiología , Proteínas Proto-Oncogénicas c-met/fisiología , Animales , Células Cultivadas , Retículo Endoplásmico/metabolismo , Humanos , Ratones , Ratones Transgénicos , Mitocondrias/metabolismo , Transporte de Proteínas , ProteolisisRESUMEN
How nuclear proteins diffuse and find their targets remains a key question in the transcription field. Dynamic proteins in the nucleus are classically subdiffusive and undergo anomalous diffusion, yet the underlying physical mechanisms are still debated. In this study, we explore the contribution of interactions to the generation of anomalous diffusion by the means of fluorescence spectroscopy and simulation. Using interaction-deficient mutants, our study indicates that HEXIM1 interactions with both 7SK RNA and positive transcription elongation factor b are critical for HEXIM1 subdiffusion and thus provides evidence of the effects of protein-RNA interaction on molecular diffusion. Numerical simulations allowed us to establish that the proportions of distinct oligomeric HEXIM1 subpopulations define the apparent anomaly parameter of the whole population. Slight changes in the proportions of these oligomers can lead to significant shifts in the diffusive features and recapitulate the modifications observed in cells with the various interaction-deficient mutants. By combining simulations and experiments, our work opens new prospects in which the anomaly α coefficient in diffusion becomes a helpful tool to infer alterations in molecular interactions.
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Núcleo Celular/metabolismo , Factor B de Elongación Transcripcional Positiva/metabolismo , ARN Largo no Codificante/metabolismo , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/metabolismo , Línea Celular Tumoral , Simulación por Computador , Difusión , Humanos , Modelos Moleculares , Unión Proteica , ARN Largo no Codificante/genética , Espectrometría de FluorescenciaRESUMEN
Ets-1 transcription factor overexpression in breast cancers is associated with invasive features and is associated with a poor prognosis. Beyond its role in driving carcinoma cell invasion, in this study, we wished to determine whether Ets-1 overexpression in cancer cells promotes angiogenesis by creating a paracrine pro-invasive environment for endothelial cells as well. To address this question, we set up different co-culture models of cancer cells with endothelial cells. Conditioned media from cancer cells induced endothelial cell proliferation, migration and morphogenesis in matrix models. Of note, co-culture assays in three-dimensional matrix models also revealed the reciprocal induction of cancer cell morphogenesis by endothelial cells, in support of an angiocrine action on tumor cells. Ets-1 emerged as a key regulator of the angiogenic potential of breast cancer cells, favoring their ability to induce, in a paracrine manner, the morphogenesis of endothelial cells and also to physically interact with the latter. Nevertheless, Ets-1 overexpression in cancer cells also restrained their chemoattractive potential for endothelial cells both in Boyden chambers and in ex vivo 3D co-cultures. Finally, Ets-1 modulation in breast cancer cells qualitatively altered the angiogenic pattern of experimental in vivo tumors, with a balance between vessel recruitment and intratumoral small capillaries sprouting. Taken together, our data highlight a critical and intriguing role for Ets-1 in the angiogenic potential of breast cancer cells, and reveal another facet of Ets-1 oncogenic activities.
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Neoplasias de la Mama/irrigación sanguínea , Células Endoteliales/citología , Neovascularización Patológica/genética , Proteína Proto-Oncogénica c-ets-1/genética , Proteína Proto-Oncogénica c-ets-1/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Proliferación Celular , Técnicas de Cocultivo , Medios de Cultivo Condicionados/química , Células Endoteliales/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Invasividad Neoplásica , Trasplante de Neoplasias , Neovascularización Patológica/metabolismo , Comunicación Paracrina , Regulación hacia ArribaRESUMEN
Most cell physiology events are dictated by the integration of perceived signals and the elaboration by cells of adapted answers via the execution of proper transcriptional programs. In order to ensure an optimal control of these answers, many regulation mechanisms have been selected throughout the evolution, thus allowing to fine-tune transcript expression. The transcriptional pause and its release by P-TEFb (Positive Transcription Elongation Factor) have been evidenced two decades ago. Since then, the importance of such mechanisms has been highlighted by the association between alterations of this machinery and the appearance of diseases. P-TEFb and Brd4 have thus recently emerged as potential therapeutical targets for cancers and AIDS notably. In this review, we present a brief case history and an up-to-date synthesis of models for transcriptional pause release. We later discuss on the pathophysiological processes associated with this mechanism and clinical trials targeting Brd4 and P-TEFb.
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Terapia Molecular Dirigida/métodos , Proteínas Nucleares/fisiología , Factor B de Elongación Transcripcional Positiva/fisiología , ARN Polimerasa II/metabolismo , Factores de Transcripción/fisiología , Transcripción Genética/fisiología , Proteínas de Ciclo Celular , HumanosRESUMEN
The Sda /Cad antigen reported on glycoconjugates of human tissues has an increasingly recognized wide impact on the physio-pathology of different biological systems. The last step of its biosynthesis relies on the enzymatic activity of the ß1,4-N-acetylgalactosaminyltransferase-II (B4GALNT2), which shows the highest expression level in healthy colon. Previous studies reported the occurrence in human colonic cells of two B4GALNT2 protein isoforms that differ in the length of their cytoplasmic tail, the long isoform showing an extended 66-amino acid tail. We examined here, the subcellular distribution of the two B4GALNT2 protein isoforms in stably transfected colonic LS174T cells and in transiently transfected HeLa cells using fluorescence microscopy. While a similar subcellular distribution at the trans-Golgi cisternae level was observed for the two isoforms, our study pointed to an atypical subcellular localization of the long B4GALNT2 isoform into dynamic vesicles. We demonstrated a critical role of its extended cytoplasmic tail for its Golgi targeting and post-Golgi sorting and highlighted the existence of a newly described post-Golgi sorting signal as well as a previously undescribed fate of a Golgi glycosyltransferase. DATABASE: The proteins ß1,4GalNAcT II, ß1,4-GalT1, FucT I, FucT VI and ST3Gal IV are noted B4GALNT2, B4GALT1, FUT1, FUT6 and ST3GAL4, whereas the corresponding human genes are noted B4GALNT2, B4GALT1, FUT1, FUT6 and ST3GAL4 according to the HUGO nomenclature.
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Neoplasias del Colon/metabolismo , Aparato de Golgi/metabolismo , N-Acetilgalactosaminiltransferasas/metabolismo , Fracciones Subcelulares/metabolismo , Secuencia de Aminoácidos , Neoplasias del Colon/patología , Células HeLa , Humanos , Isoformas de Proteínas , Transporte de Proteínas , Homología de Secuencia , Células Tumorales CultivadasRESUMEN
Calcium (Ca2+) release from the endoplasmic reticulum plays an important role in many cell-fate defining cellular processes. Traditionally, this Ca2+ release was associated with the ER Ca2+ release channels, inositol 1,4,5triphosphate receptor (IP3R) and ryanodine receptor (RyR). Lately, however, other calcium conductances have been found to be intracellularly localized and to participate in cell fate regulation. Nonetheless, molecular identity and functional properties of the ER Ca2+ release mechanisms associated with multiple diseases, e.g. prostate cancer, remain unknown. Here we identify a new family of transient receptor potential melastatine 8 (TRPM8) channel isoforms as functional ER Ca2+ release channels expressed in mitochondria-associated ER membranes (MAMs). These TRPM8 isoforms exhibit an unconventional structure with 4 transmembrane domains (TMs) instead of 6 TMs characteristic of the TRP channel archetype. We show that these 4TM-TRPM8 isoforms form functional channels in the ER and participate in regulation of the steady-state Ca2+ concentration ([Ca2+]) in mitochondria and the ER. Thus, our study identifies 4TM-TRPM8 isoforms as ER Ca2+ release mechanism distinct from classical Ca2+ release channels.
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Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Mitocondrias/metabolismo , Neoplasias de la Próstata/metabolismo , Canales Catiónicos TRPM/genética , Canales Catiónicos TRPM/metabolismo , Anciano , Empalme Alternativo , Línea Celular Tumoral , Células Epiteliales/citología , Células Epiteliales/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Próstata/citología , Próstata/metabolismo , Neoplasias de la Próstata/genética , Dominios Proteicos , Canales Catiónicos TRPM/químicaRESUMEN
Biochemical studies have revealed that the RNA Polymerase II (RNAPII) pause release is triggered by phosphorylation of the transcription machinery by the positive transcription elongation factor b (P-TEFb). However, there are no direct report that P-TEFb and RNA polymerase II interact in single living cells and the biophysical mechanisms mediating this association are still unclear. Förster resonance energy transfer (FRET) detects molecular interactions at the subcellular level. Time domain fluorescence lifetime imaging provides an accurate quantification of FRET efficiency, EFRET, because it is fluorochrome concentration-independent and insensitive to fluorescence bleed-through. However, the way FRET signal is usually analyzed does not provide information about the areas where protein-protein interactions take place. In this work, we developed a method, dubbed FRET image correlation spectroscopy (FICS), which relied on FRET fluorescence lifetime imaging image acquisition and image correlation spectroscopy of EFRET clusters to quantify the spatial distribution of interaction clusters in the nucleus. The combination of high content FRET microscopy with batch image analysis allowed a robust statistical analysis. By applying FICS, we characterized the area and density of interaction clusters between P-TEFb and RNAPII or histone H2A in single living cells. The FICS method applied to cells expressing genetically engineered mutated proteins confirmed that the histidine-rich domain of P-TEFb is required for its interaction with RNAPII. Furthermore, it demonstrated that P-TEFb was also located in close vicinity to histone H2A, independently of its interactions with RNAPII. These results support the hypothesis that P-TEFb dynamics on chromatin regulate its recruitment on RNAPII.
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Cromatina/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Histonas/metabolismo , Factor B de Elongación Transcripcional Positiva/metabolismo , ARN Polimerasa II/metabolismo , Transcripción Genética , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Cromatina/genética , Histonas/genética , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Osteosarcoma/genética , Osteosarcoma/metabolismo , Osteosarcoma/patología , Fosforilación , Factor B de Elongación Transcripcional Positiva/genética , Unión Proteica , ARN Polimerasa II/genética , Células Tumorales CultivadasRESUMEN
Photodynamic therapy (PDT) has developed into a new clinical and non-invasive treatment for cancer over the past 30 years. By the combination of three non-toxic partners, i.e. a photosensitizer (PS), molecular oxygen (O2) and light, cytotoxic reactive oxygen species (ROS) are locally produced leading to irreversible vascular and cellular damage. In the present study, we report for the first time that the combination of two photosensitizers (2 PSs: Protoporphyrin IX, PpIX and Hypericin, Hy) loaded in the same lipid nanocapsules (LNCs) leads to enhanced photodynamic therapy efficiency when compared with previously reported systems. The 2 PS-loaded LNCs are shown to increase the in vitro phototoxicity at the nanomolar range (IC50 = 274 and 278 nM on HeLa and MDA-MB-231 cell lines, respectively), whereas the corresponding single PS-loaded LNCs at the same concentration exhibit a phototoxicity two times lower. Intracellular localization in HeLa cells indicates a subcellular asymmetry of PpIX and Hy, in the plasma, ER membranes and round internal structures. The biodistribution of LNCs was studied upon different routes of injection into Swiss nude mice; based on the obtained data, LNCs were injected intratumorally and used to slow the growth of xenograft tumors in mice. The results obtained in this study suggest that the combination of two or more PSs may be a promising strategy to improve the efficacy of conventional photodynamic therapy as well as to reduce dark toxicity.
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Copepods are small crustaceans capable to survive in various aquatic environments. Their responses to changes in different external factors such as salinity and temperature can be observed at different integration levels from copepod genes to copepod communities. Until now, no thorough observation of the temperature or salinity effect stresses on copepods has been done by optical microscopy. In this study, we used autofluorescence to visualize these effects on the morphology of the calanoid copepod Pseudodiaptomus marinus maintained during several generations in the laboratory at favorable and stable conditions of salinity (30 psu) and temperature (18°C). Four different stress experiments were conducted: at a sharp decrease in temperature (18 to 4°C), a moderate decrease in salinity (from 30 to 15 psu), a major decrease in salinity (from 30 to 0 psu), and finally a combined stress with a decrease in both temperature and salinity (from 18°C and 30 psu to 4°C and 0 psu). After these stresses, images acquired by confocal laser scanning microscopy (CLSM) revealed changes in copepod cuticle and muscle structure. Low salinity and/or temperature stresses affected both the detection of fluorescence emitted by muscle sarcomeres and the distance between them. In the remaining paper we will use the term sarcomeres to describe the elements located within sarcomeres and emitted autofluorescence signals. Quantitative study showed an increase in the average distance between two consecutive sarcomeres from 2.06 +/- 0.11 µm to 2.44 +/- 0.42 µm and 2.88 +/- 0.45µm after the exposure to major haline stress (18°C, 0 psu) and the combined stress (4°C, 0 psu), respectively. These stresses also caused cuticle cracks which often occurred at the same location, suggesting the cuticle as a sensitive area for osmoregulation. Our results suggest the use of cuticular and muscle autofluorescence as new biomarkers of stress detectable in formalin-preserved P. marinus individuals. Our label-free method can be easily applied to a large number of other copepod species or invertebrates with striated musculature.
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Copépodos/fisiología , Miofibrillas/fisiología , Estrés Fisiológico/fisiología , Animales , Femenino , Salinidad , Sarcómeros/fisiología , TemperaturaRESUMEN
We have discovered and developed a series of molecules (thiazole benzenesulfonamides). HA15, the lead compound of this series, displayed anti-cancerous activity on all melanoma cells tested, including cells isolated from patients and cells that developed resistance to BRAF inhibitors. Our molecule displayed activity against other liquid and solid tumors. HA15 also exhibited strong efficacy in xenograft mouse models with melanoma cells either sensitive or resistant to BRAF inhibitors. Transcriptomic, proteomic, and biochemical studies identified the chaperone BiP/GRP78/HSPA5 as the specific target of HA15 and demonstrated that the interaction increases ER stress, leading to melanoma cell death by concomitant induction of autophagic and apoptotic mechanisms.
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Antineoplásicos/administración & dosificación , Resistencia a Antineoplásicos/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Melanoma/tratamiento farmacológico , Sulfonamidas/administración & dosificación , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Chaperón BiP del Retículo Endoplásmico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Melanoma/metabolismo , Ratones , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Sulfonamidas/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Since its cloning a decade ago, TRPM8 channel has emerged as a promising prognostic marker and a putative therapeutic target in prostate cancer (PCa). However, recent studies have brought to light the complexity of TRPM8 isoforms in PCa. Consequently, the respective role of each TRPM8 isoform needs to be deciphered prior to considering TRPM8 as an attractive therapeutic target. Full-length (6 transmembrane (TM)-domain) TRPM8 channel is overexpressed in early PCa and repressed in advanced prostate tumors whereas the localization of the truncated, 4TM-TRPM8 channel (4 transmembrane (TM)-domain), in the membranes of endoplasmic reticulum (ER) is independent of the pathogenic status of epithelial cells. In the same line, expression of non-channel cytoplasmic small TRPM8 isoforms (namely sM8) is conserved in cancer cells. In this study, we identify sM8s as putative regulator of PCa cell death. Indeed, suppression of sM8 isoforms was found to induce concomitantly ER stress, oxidative stress, p21 expression and apoptosis in human epithelial prostate cancer cells. We furthermore demonstrate that induction of such mechanisms required the activity of 4TM-TRPM8 channels at the ER-mitochondria junction. Our study thus suggests that targeting sM8 could be an appropriate strategy to fight prostate cancer.
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Neoplasias de la Próstata/patología , Canales Catiónicos TRPM/metabolismo , Animales , Apoptosis/fisiología , Línea Celular Tumoral , Femenino , Xenoinjertos , Humanos , Masculino , Ratones , Ratones Desnudos , Neoplasias de la Próstata/metabolismo , Isoformas de Proteínas/metabolismoRESUMEN
Particle-based photodynamic therapy (PPDT) holds great promise in theranostic applications. Herein, we demonstrate that PPDT based on gold nanorods coated with an indocyanine green (ICG)-loaded silica shell allows for the inactivation of the Crohn's disease-associated adherent-invasive Escherichia coli strain LF82 (E. coli LF82) under pulsed laser light irradiation at 810 nm. Fine-tuning of the plasmonic structures together with maximizing the photosensitizer loading onto the nanostructures allowed optimizing the singlet oxygen generation capability and the PPDT efficiency. Using a nanoparticle concentration low enough to suppress photothermal heating effects, 6 log10 reduction in E. coli LF82 viability could be achieved using gold nanostructures displaying a plasmonic band at 900 nm. An additional modality of nanoparticle-based photoinactivation of E. coli is partly observed, with 3 log10 reduction of bacterial viability using Au NRs@SiO2 without ICG, due to the two-photon induced formation of reactive oxygen species. Interaction of the particles with the bacterial surface, responsible for the disruption of the bacterial integrity, together with the generation of moderate quantities of singlet oxygen could account for this behavior.
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Uncoupling of ERK1/2 phosphorylation from subcellular localization is essential towards the understanding of molecular mechanisms that control ERK1/2-mediated cell-fate decision. ERK1/2 non-catalytic functions and discoveries of new specific anchors responsible of the subcellular compartmentalization of ERK1/2 signaling pathway have been proposed as regulation mechanisms for which dynamic monitoring of ERK1/2 localization is necessary. However, studying the spatiotemporal features of ERK2, for instance, in different cellular processes in living cells and tissues requires a tool that can faithfully report on its subcellular distribution. We developed a novel molecular tool, ERK2-LOC, based on the T2A-mediated coexpression of strictly equimolar levels of eGFP-ERK2 and MEK1, to faithfully visualize ERK2 localization patterns. MEK1 and eGFP-ERK2 were expressed reliably and functionally both in vitro and in single living cells. We then assessed the subcellular distribution and mobility of ERK2-LOC using fluorescence microscopy in non-stimulated conditions and after activation/inhibition of the MAPK/ERK1/2 signaling pathway. Finally, we used our coexpression system in Xenopus laevis embryos during the early stages of development. This is the first report on MEK1/ERK2 T2A-mediated coexpression in living embryos, and we show that there is a strong correlation between the spatiotemporal subcellular distribution of ERK2-LOC and the phosphorylation patterns of ERK1/2. Our approach can be used to study the spatiotemporal localization of ERK2 and its dynamics in a variety of processes in living cells and embryonic tissues.
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Genes Reporteros , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Análisis de la Célula Individual/métodos , Xenopus laevis/embriología , Animales , Diferenciación Celular , MAP Quinasa Quinasa 1/genética , MAP Quinasa Quinasa 1/metabolismo , Sistema de Señalización de MAP Quinasas , Ratones , Proteína Quinasa 1 Activada por Mitógenos/genética , Células 3T3 NIH , Fosforilación , Transducción de Señal , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismoRESUMEN
The potential of gold nanorods post-coated with a 20 nm silica shell loaded with verteporfin (Au NRs@SiO2-VP) as efficient near-infrared nanostructures for photodynamic therapy under continuous wave and pulsed-mode excitation to eradicate a virulent strain of E. coli associated with urinary tract infection is described.
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Antibacterianos/farmacología , Escherichia coli/efectos de los fármacos , Oro/farmacología , Nanotubos/química , Fármacos Fotosensibilizantes/farmacología , Porfirinas/farmacología , Dióxido de Silicio/farmacología , Antibacterianos/química , Oro/química , Oxígeno/química , Fotoquimioterapia , Fármacos Fotosensibilizantes/química , Porfirinas/química , Dióxido de Silicio/química , VerteporfinaRESUMEN
Biofilm formation of Pseudomonas aeruginosa and Staphylococcus aureus on food-contact-surfaces represents a significant risk for the public health. In this context, the present study investigates the relationship between the environmental conditions of biofilm formation and the resistance to disinfectants. Therefore, a static biofilm reactor, called NEC-Biofilm System, was established in order to study the effect of growth temperature (20, 30 and 37°C), and of the surface type (stainless steel and polycarbonate), on biofilm resistance to disinfectants. These conditions were selected to mimic the biofilm formation on abiotic surfaces of food processing industries. The antibiofilm assays were performed on biofilms grown during 24 h. The results showed that the growth temperature influenced significantly the biofilm resistance to disinfectants. These data also revealed that the growth temperature has a significant effect on the biofilm structure of both bacteria. Furthermore, the increase of the biofilm growth temperature increased significantly the algD transcript level in sessile P. aeruginosa cells, whereas the icaA one was not affected in S. aureus cells. Overall, our findings show that the biofilm structure and matrix cannot fully explain the biofilm resistance to disinfectant agents. Nevertheless, it underlines the intimate link between environmental conditions, commonly met in food sectors, and the biofilm resistance to disinfectants.