36 research outputs found
Mechanical strain induces involution-associated events in mammary epithelial cells
<p>Abstract</p> <p>Background</p> <p>Shortly after weaning, a complex multi-step process that leads to massive epithelial apoptosis is triggered by tissue local factors in the mouse mammary gland. Several reports have demonstrated the relevance of mechanical stress to induce adaptive responses in different cell types. Interestingly, these signaling pathways also participate in mammary gland involution. Then, it has been suggested that cell stretching caused by milk accumulation after weaning might be the first stimulus that initiates the complete remodeling of the mammary gland. However, no previous report has demonstrated the impact of mechanical stress on mammary cell physiology. To address this issue, we have designed a new practical device that allowed us to evaluate the effects of radial stretching on mammary epithelial cells in culture.</p> <p>Results</p> <p>We have designed and built a new device to analyze the biological consequences of applying mechanical stress to cells cultured on flexible silicone membranes. Subsequently, a geometrical model that predicted the percentage of radial strain applied to the elastic substrate was developed. By microscopic image analysis, the adjustment of these calculations to the actual strain exerted on the attached cells was verified. The studies described herein were all performed in the HC11 non-tumorigenic mammary epithelial cell line, which was originated from a pregnant BALB/c mouse. In these cells, as previously observed in other tissue types, mechanical stress induced ERK1/2 phosphorylation and c-Fos mRNA and protein expression. In addition, we found that mammary cell stretching triggered involution associated cellular events as Leukemia Inhibitory Factor (LIF) expression induction, STAT3 activation and AKT phosphorylation inhibition.</p> <p>Conclusion</p> <p>Here, we show for the first time, that mechanical strain is able to induce weaning-associated events in cultured mammary epithelial cells. These results were obtained using a new practical and affordable device specifically designed for such a purpose. We believe that our results indicate the relevance of mechanical stress among the early post-lactation events that lead to mammary gland involution.</p
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RuBi-Glutamate: two-photon and visible-light photoactivation of neurons and dendritic spines
We describe neurobiological applications of RuBi-Glutamate, a novel caged-glutamate compound based on ruthenium photochemistry. RuBi-Glutamate can be excited with visible wavelengths and releases glutamate after one- or two-photon excitation. It has high quantum efficiency and can be used at low concentrations, partly avoiding the blockade of GABAergic transmission present with other caged compounds. Two-photon uncaging of RuBi-glutamate has a high spatial resolution and generates excitatory responses in individual dendritic spines with physiological kinetics. With laser beam multiplexing, RuBi-Glutamate uncaging can also be used to depolarize and fire pyramidal neurons with single-cell resolution. RuBi-Glutamate therefore enables the photo-activation of neuronal dendrites and circuits with visible or two-photon light sources, achieving single spine, or single cell, precision
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Bifunctional poly(acrylamide) hydrogels through orthogonal coupling chemistries
Biomaterials for cell culture allowing simple and quantitative presentation of instructive cues enable rationalization of the interplay between cells and their surrounding microenvironment. Poly(acrylamide) (PAAm) hydrogels are popular 2D-model substrates for this purpose. However,
quantitative and reproducible biofunctionalization of PAAm hydrogels with multiple ligands in a trustable, controlled, and independent fashion is not trivial. Here, we describe a method for bifunctional modification of PAAm hydrogels with thioland amine- containing biomolecules with controlled densities in an independent, orthogonal manner. We developed copolymer networks of AAm with 9% acrylic acid and 2% N-(4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)phenyl)acrylamide. The covalent binding of thiol- and amine- containing chromophores at tunable concentrations was demonstrated and quantified by UV spectroscopy. The morphology, mechanical properties, and homogeneity of the copolymerized hydrogels were characterized by scanning electron microscopy, dynamic mechanical analysis, and confocal microscopy studies. Our copolymer hydrogels were bifunctionalized with polylysine and a laminin-mimetic peptide using the specific chemistries. We analyzed the effect of binding protocol of the two components in the maturation of cultured postmitotic cortical neurons. Our substrates supported neuronal attachment, proliferation, and neuronal differentiation. We found that neurons cultured on our hydrogels bifunctionalized with ligand-specific chemistries in a sequential fashion exhibited higher maturation at comparable culture times than using a simultaneous bifunctionalization strategy, displaying a higher number of neurites, branches, and dendritic filopodia. These results demonstrate the relevance of quantitative and optimized coupling chemistries for the performance of simple biomaterials and with sensitive cell types
Synchronized cell attachment triggered by photo-activatable adhesive ligands allows QCM-based detection of early integrin binding
The Quartz Crystal Microbalance with dissipation (QCM-D) technique was applied to monitor and quantify integrin-RGD recognition during the early stages of cell adhesion. Using QCM-D crystals modified with a photo-activatable RGD peptide, the time point of presentation of adhesive ligand at the surface of the QCM-D crystal could be accurately controlled. This allowed temporal resolution of early integrin-RGD binding and the subsequent cell spreading process, and their separate detection by QCM-D. The specificity of the integrin-RGD binding event was corroborated by performing the experiments in the presence of soluble cyclicRGD as a competitor, and cytochalasin D as inhibitor of cell spreading. Larger frequency change in the QCM-D signal was observed for cells with larger spread area, and for cells overexpressing integrin avb3 upon stable transfection. This strategy enables quantification of integrin activity which, in turn, may allow discrimination among different cell types displaying distinct integrin subtypes and expression levels thereof. On the basis of these findings, we believe the strategy can be extended to other photoactivatable ligands to characterize cell membrane receptors activity, a relevant issue for cancer diagnosis (and prognosis) as other several pathologies.Fil: Iturri, Jagoba. Max Planck Institute for Polymer Research; AlemaniaFil: GarcĂa FernĂĄndez, Luis. Max Planck Institute for Polymer Research; AlemaniaFil: Reuning, Ute. Technische Universitat Munchen; AlemaniaFil: GarcĂa, AndrĂ©s J.. Georgia Institute Of Techology; Estados UnidosFil: del Campo, ArĂĄnzazu. Max Planck Institute for Polymer Research; AlemaniaFil: Salierno, Marcelo Javier. Max Planck Institute for Polymer Research; Alemania. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas; Argentin
Tuba8 Drives Differentiation of Cortical Radial Glia into Apical Intermediate Progenitors by Tuning Modifications of Tubulin C Termini
Most adult neurons and glia originate from radial glial progenitors (RGs), a type of stem cell typically extending from the apical to the basal side of the developing cortex. Precise regulation of the choice between RG self-renewal and differentiation is critical for normal development, but the mechanisms underlying this transition remain elusive. We show that the non-canonical tubulin Tuba8, transiently expressed in cortical progenitors, drives differentiation of RGs into apical intermediate progenitors, a more restricted progenitor type lacking attachment to the basal lamina. This effect depends on the unique C-terminal sequence of Tuba8 that antagonizes tubulin tyrosination and Î2 cleavage, two post-translational modifications (PTMs) essential for RG fiber maintenance and the switch between direct and indirect neurogenesis and ultimately distinct neuronal lineage outcomes. Our work uncovers an instructive role of a developmentally regulated tubulin isotype in progenitor differentiation and provides new insights into biological functions of the cellular tubulin PTM âcode.â Radial glial progenitors of the developing mouse cortex differentiate into a more restricted progenitor type, apical intermediate progenitors. Ramos et al. find that Tuba8 drives this differentiation by tuning tyrosination and Î2 cleavage, post-translational modifications of tubulin C termini, highlighting the functional significance of the âtubulin codeâ in cortical progenitor differentiation.Fil: Ramos, Susana I.. King's College London; Reino UnidoFil: Makeyev, Eugene V.. King's College London; Reino UnidoFil: Salierno, Marcelo Javier. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Patagonia Norte. Instituto de Investigaciones en Biodiversidad y Medioambiente. Universidad Nacional del Comahue. Centro Regional Universidad Bariloche. Instituto de Investigaciones en Biodiversidad y Medioambiente; ArgentinaFil: Kodama, Takashi. University Johns Hopkins; Estados UnidosFil: Kawakami, Yasuhiko. University Johns Hopkins; Estados UnidosFil: Sahara, Setsuko. King's College London; Reino Unid
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Bifunctional hydrogels containing the laminin motif IKVAV promote neurogenesis
Engineering of biomaterials with specific biological properties has gained momentum as a means to control stem cell behavior. Here, we address the effect of bifunctionalized hydrogels comprising polylysine (PL) and a 19-mer peptide containing the laminin motif IKVAV (IKVAV) on embryonic and adult neuronal progenitor cells under different stiffness regimes. Neuronal differentiation of embryonic and adult neural progenitors was accelerated by adjusting the gel stiffness to 2 kPa and 20 kPa, respectively.While gels containing IKVAV or PL alone failed to support long-term cell adhesion, in bifunctional gels, IKVAV synergized with PL to promote differentiation and formation of focal adhesions containing b1-integrin in embryonic cortical neurons. Furthermore, in adult neural stem cell culture, bifunctionalized gels promoted neurogenesis via the expansion of neurogenic clones. These data highlight the potential of synthetic matrices to steer stem and progenitor cell behavior via defined mechano-adhesive properties
Exploring the response of Marchantia polymorpha: Growth, morphology and chlorophyll content in the presence of anthracene
Polycyclic aromatic hydrocarbons (PAHs) were identified as hazardous contaminants that are ubiquitous and persistent in aquatic environments, where bryophytes sensu lato (mosses, liverworts and hornworts) are frequently present. Marchantia polymorpha (Class Hepaticae; thalloid liverwort) is known to respond fast to changes in the environment; it accumulates toxic substances in its tissues due to the lack of vascular and radicular systems and a reduced or absent cuticle. The objective of the present study was to quantify the effects of increasing concentrations of anthracene (0, 50 100, 280 ÎŒM) on the germination of propagules, plant morphology and chlorophyll content index (CCI) in M. polymorpha under in vitro cultures. The results show that anthracene had no statistical effect on germination or propagula formation. However, plants exposed to anthracene for 30 days showed significantly lowered the content of chlorophyll (measured as CCI), irregular growth patterns and the induction of thalli asexual reproduction as evidenced by the production of multicellular viable propagules in gemmae cups. Results of epifluorescence microscopy also showed concomitant accumulation of anthracene in the cell walls. All of these distinctive morphological and physiological adaptive responses indicators, clearly suggest that M. polymorpha are capable of resisting high (coal tar) anthracene concentrations.Fil: Spinedi, Nahuel. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Patagonia Norte. Instituto de Investigaciones en Biodiversidad y Medioambiente. Universidad Nacional del Comahue. Centro Regional Universidad Bariloche. Instituto de Investigaciones en Biodiversidad y Medioambiente; ArgentinaFil: Rojas, Nadia Gimena. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Patagonia Norte. Instituto de Investigaciones en Biodiversidad y Medioambiente. Universidad Nacional del Comahue. Centro Regional Universidad Bariloche. Instituto de Investigaciones en Biodiversidad y Medioambiente; ArgentinaFil: Storb Guzman, Romina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Patagonia Norte. Instituto de Investigaciones en Biodiversidad y Medioambiente. Universidad Nacional del Comahue. Centro Regional Universidad Bariloche. Instituto de Investigaciones en Biodiversidad y Medioambiente; ArgentinaFil: Cabrera, Juan Manuel. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Patagonia Norte. Instituto de Investigaciones en Biodiversidad y Medioambiente. Universidad Nacional del Comahue. Centro Regional Universidad Bariloche. Instituto de Investigaciones en Biodiversidad y Medioambiente; ArgentinaFil: Aranda, Elisabet. Universidad de Granada; EspañaFil: Salierno, Marcelo Javier. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Patagonia Norte. Instituto de Investigaciones en Biodiversidad y Medioambiente. Universidad Nacional del Comahue. Centro Regional Universidad Bariloche. Instituto de Investigaciones en Biodiversidad y Medioambiente; ArgentinaFil: Svriz, Maya. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Patagonia Norte; Argentina. Universidad Nacional de RĂo Negro; ArgentinaFil: Scervino, Jose Martin. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Patagonia Norte. Instituto de Investigaciones en Biodiversidad y Medioambiente. Universidad Nacional del Comahue. Centro Regional Universidad Bariloche. Instituto de Investigaciones en Biodiversidad y Medioambiente; Argentin
Heme oxygenase-1 in the forefront of a multi-molecular network that governs cellâcell contacts and filopodia-induced zippering in prostate cancer
Prostate cancer (PCa) cells display abnormal expression of cytoskeletal proteins resulting in an augmented capacity to resist chemotherapy and colonize distant organs. We have previously shown that heme oxygenase 1 (HO-1) is implicated in cell morphology regulation in PCa. Here, through a multi 'omics' approach we define the HO-1 interactome in PCa, identifying HO-1 molecular partners associated with the integrity of the cellular cytoskeleton. The bioinformatics screening for these cytoskeletal-related partners reveal that they are highly misregulated in prostate adenocarcinoma compared with normal prostate tissue. Under HO-1 induction, PCa cells present reduced frequency in migration events, trajectory and cell velocity and, a significant higher proportion of filopodia-like protrusions favoring zippering among neighboring cells. Moreover forced expression of HO-1 was also capable of altering cell protrusions in transwell co-culture systems of PCa cells with MC3T3 cells (pre-osteoblastic cell line). Accordingly, these effects were reversed under siHO. Transcriptomics profiling evidenced significant modulation of key markers related to cell adhesion and cellâcell communication under HO-1 induction. The integration from our omics-based research provides a four molecular pathway foundation (ANXA2/HMGA1/POU3F1; NFRSF13/GSN; TMOD3/RAI14/VWF; and PLAT/PLAU) behind HO-1 regulation of tumor cytoskeletal cell compartments. The complementary proteomics and transcriptomics approaches presented here promise to move us closer to unravel the molecular framework underpinning HO-1 involvement in the modulation of cytoskeleton pathways, pushing toward a less aggressive phenotype in PCa
EphA3 Expressed in the Chicken Tectum Stimulates Nasal Retinal Ganglion Cell Axon Growth and Is Required for Retinotectal Topographic Map Formation
BACKGROUND: Retinotopic projection onto the tectum/colliculus constitutes the most studied model of topographic mapping and Eph receptors and their ligands, the ephrins, are the best characterized molecular system involved in this process. Ephrin-As, expressed in an increasing rostro-caudal gradient in the tectum/colliculus, repel temporal retinal ganglion cell (RGC) axons from the caudal tectum and inhibit their branching posterior to their termination zones. However, there are conflicting data regarding the nature of the second force that guides nasal axons to invade and branch only in the caudal tectum/colliculus. The predominant model postulates that this second force is produced by a decreasing rostro-caudal gradient of EphA7 which repels nasal optic fibers and prevents their branching in the rostral tectum/colliculus. However, as optic fibers invade the tectum/colliculus growing throughout this gradient, this model cannot explain how the axons grow throughout this repellent molecule. METHODOLOGY/PRINCIPAL FINDINGS: By using chicken retinal cultures we showed that EphA3 ectodomain stimulates nasal RGC axon growth in a concentration dependent way. Moreover, we showed that nasal axons choose growing on EphA3-expressing cells and that EphA3 diminishes the density of interstitial filopodia in nasal RGC axons. Accordingly, in vivo EphA3 ectodomain misexpression directs nasal optic fibers toward the caudal tectum preventing their branching in the rostral tectum. CONCLUSIONS: We demonstrated in vitro and in vivo that EphA3 ectodomain (which is expressed in a decreasing rostro-caudal gradient in the tectum) is necessary for topographic mapping by stimulating the nasal axon growth toward the caudal tectum and inhibiting their branching in the rostral tectum. Furthermore, the ability of EphA3 of stimulating axon growth allows understanding how optic fibers invade the tectum growing throughout this molecular gradient. Therefore, opposing tectal gradients of repellent ephrin-As and of axon growth stimulating EphA3 complement each other to map optic fibers along the rostro-caudal tectal axis
A caged nicotine with nanosecond range kinetics and visible light sensitivity
We report the synthesis, characterization and applications of a ruthenium-bipyridine based caged nicotine. The complex [Ru(bpy)2(nic)2]2+ (where bpy=2,2' bipyridine and nic=nicotine (3-[(2S)-1-methylpyrrolidin-2-yl] pyridine)) releases nicotine with a quantum yield Ί=0.23 upon irradiation with biologically harmless, blue (473nm) or green (532nm) light. The photolysis reaction is clean and very fast, with a time constant of 17ns. The synthesis is simple and the obtained compound is characterized by NMR, UV-Vis spectroscopy and cyclic voltametry. We find that this compound is active in biological systems, being able to elicit action potentials in leech neurons. © 2010 Elsevier Inc.Fil: Filevich, Oscar. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Ciudad Universitaria. Instituto de QuĂmica, FĂsica de los Materiales, Medioambiente y EnergĂa. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de QuĂmica, FĂsica de los Materiales, Medioambiente y EnergĂa; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de QuĂmica InorgĂĄnica, AnalĂtica y QuĂmica FĂsica; ArgentinaFil: Salierno, Marcelo Javier. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Ciudad Universitaria. Instituto de QuĂmica, FĂsica de los Materiales, Medioambiente y EnergĂa. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de QuĂmica, FĂsica de los Materiales, Medioambiente y EnergĂa; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de QuĂmica InorgĂĄnica, AnalĂtica y QuĂmica FĂsica; ArgentinaFil: Etchenique, Roberto Argentino. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Ciudad Universitaria. Instituto de QuĂmica, FĂsica de los Materiales, Medioambiente y EnergĂa. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de QuĂmica, FĂsica de los Materiales, Medioambiente y EnergĂa; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de QuĂmica InorgĂĄnica, AnalĂtica y QuĂmica FĂsica; Argentin