text:
Dóna suport
PER A INVESTIGADORS
PER A JOVES CIENTÍFICS
PER A INDÚSTRIA I HOSPITALS
PER A PÚBLIC I PREMSA
Nanoscopy for nanomedicine
The main goal of our group is to use Super Resolution Microscopy (nanoscopy) to visualize and track in living cells and tissues self-assembled nanomaterials with therapeutic potential (nanomedicine).
TEM image of novel self-assembled nanofibers synthesized in the group.
The understanding of materials-cell interactions is the key towards the development of novel nanotechnology-based therapies for treatment of cancer and infectious diseases.
Our group aims to use a multidisciplinary approach, at the interface of chemistry, physics and biology, to develop novel nanomaterials for the treatment of cancer and infectious diseases.
We aim at the development of novel nanocarriers for drug delivery based on self-assembly, i.e. able to build themselves. Molecular self-organization is ubiquitous in the biological world and represents for us a source of inspiration for the design of nanostructures with biomedical potential. In particular we focus on the development of self-assembled nanoparticles and nanofibers able to selectively target diseased cells and deliver locally therapeutic moieties such as drugs and genetic material (e.g. DNA, siRNA, mRNA).
Right: Nanoparticles interactions with blood components imaged with conventional optical microscopy (left) and super resolution STORM microscopy (right).
A key point towards the development of novel nanotechnology-based therapies is the understanding of the behavior of nanomaterials in the complex biological environment. Here we use super resolution microscopy to track nanomaterials during their voyage in the biological environment and to visualize the interactions with blood components, immune system and target cells. We make use of a variety of super resolution techniques based on single molecule detection such a stochastic optical reconstruction microscopy (STORM), photoactivated localization microscopy (PALM), point accumulation for imaging in nanoscale topography (PAINT), and single particle tracking (SPT). These methods allow to achieve a resolution down to few nanometers and are therefore ideal to visualize nanosized synthetic objects in the biological environment. Super resolution microscopy provides a molecular picture of structure-activity relations and represent a guide towards the design of innovative materials for nanomedicine.
Lorenzo Albertazzi | Group Leader
Silvia Pujals Riatós | Senior Researcher
Pietro Delcanale | Postdoctoral Researcher
Teodora Andrian | PhD Student
Maria Arista Romero | PhD Student
Natàlia Feiner Gracia | PhD Student
Edgar Fuentes Fuentes | PhD Student
Adrianna Glinkowska Mares | PhD Student
Madhura Vijay Murar | PhD Student
Rodica Alis Olea | PhD Student
Roger Riera Brillas | PhD Student
Eleonora Giro | Masters Student
News/Jobs
IBEC and TU/e plant the seed of a promising partnership
Eight IBECers were in the Netherlands on 13th and 14th September for the first ever IBEC-ICMS Symposium, 'NanoSens&Med'.
Super-resolution imaging guides the design of biocompatible microswimmers
Two IBEC groups have clubbed together to combine their expertise and reveal new knowledge that could advance the design of micro- and nanomotors for applications in health.
BIST grant success for IBEC mothers
Three of IBEC's women researchers have been successful in BIST's recent 'To the Mothers of Science' call.
Synthetic enzymes for controlled drug delivery in cells
The Nanoscopy for Nanomedicine group has studied Single-Chain Polymeric Nanoparticles (SCPNs) mimicking enzymes as possible drug activators in biological environments, like the living cell.
"ERC: Getting into the lab with the latest winners"
An article by BIOCAT profiles the three winners in Catalonia of the last round of ERC Starting Grants, including IBEC's Lorenzo Albertazzi.
"STORMing their way to Nanoparticle–Protein Interaction Understanding"
A paper published in Small last month by Lorenzo Albertazzi's group is featured in Advanced Science News, Wiley publishing company's in-house news website. This platform presents advances in various fields of research for a general audience.
ERC success for Lorenzo Albertazzi
The Nanoscopy for Nanomedicine junior group leader was successful in the European Research Council's 2017 call for Starting Grants, of which just 17 out of the total of 406 have been awarded to scientists working in Spain.
IBEC research features in ChemComm's "Emerging Investigators" issue
IBEC junior group leader Lorenzo Albertazzi is a contributor to the 2017 edition of ChemComm Emerging Investigators, which is published annually by the UK's Royal Society of Chemistry.
IBEC project among 44 funded by AXA
The AXA Research Fund, the international scientific philanthropy initiative of global insurer AXA, officially announced last week that it will devote €15.6m in 2016 to 44 new research projects with leading academic institutions in 16 countries.
Playing with molecular Lego to build the next generation of drug delivery vectors
New IBEC junior group leader Lorenzo Albertazzi and his former colleagues at the Eindhoven University of Technology, working together with industry partner Novartis, have made a leap in drug delivery vectors by developing a new type of carrier with some groundbreaking improvements.
"Wrapping Up Pandemics in New Nanomaterials"
Lorenzo Albertazzi's research project funded by AXA, Novel approaches for Pandemic Virus Targeting Using Adaptive Polymers, is featured on the Granted Projects section of their website.
'Submarinos' en la sangre para llevar el medicamento a las células dañadas
New IBEC junior group leader Lorenzo Albertazzi is profiled in El Mundo's "Personajes Únicos" section this week.
Expert in novel drug delivery systems joins IBEC
Dr Lorenzo Albertazzi, a nanoscientist whose research focuses on creating smart self-assembling materials for therapeutic applications, is joining IBEC this September.
Novel approaches for Pandemic Virus Targeting Using Adaptive Polymers AXA Research Fund Lorenzo Albertazzi
NANOSTORM Design of Nanomaterials for Targeted Therapies Guided by Super Resolution Imaging ERC Lorenzo Albertazzi
TARGETSTORM Nanomateriales para terapias dirigidas contra el cáncer visualizados con microscopia de súper resolución STORM (2016-2019) MINECO Retos investigación: Proyectos I+D Lorenzo Albertazzi
NANOVAX Nanovacunas diseñadas para inmunoterapia antitumoral MINECO Acciones de Programación Conjunta Internacional Lorenzo Albertazzi/Josep Samitier
Ayudas para contratos Ramón y Cajal MINECO Ramon y Cajal Lorenzo Albertazzi
Understanding and measuring mechanical tumor properties to improve cancer diagnosis, treatment, and survival: Application to liquid biopsies
(2017-2020) Obra Social La Caixa Lorenzo Albertazzi
(See full publication list in ORCID)
Post, R. A. J., van der Zwaag, D., Bet, G., Wijnands, S. P. W., Albertazzi, L., Meijer, E. W., van der Hofstad, R. W., (2019). A stochastic view on surface inhomogeneity of nanoparticles Nature Communications 10, (1), 1663
The interactions between and with nanostructures can only be fully understood when the functional group distribution on their surfaces can be quantified accurately. Here we apply a combination of direct stochastic optical reconstruction microscopy (dSTORM) imaging and probabilistic modelling to analyse molecular distributions on spherical nanoparticles. The properties of individual fluorophores are assessed and incorporated into a model for the dSTORM imaging process. Using this tailored model, overcounting artefacts are greatly reduced and the locations of dye labels can be accurately estimated, revealing their spatial distribution. We show that standard chemical protocols for dye attachment lead to inhomogeneous functionalization in the case of ubiquitous polystyrene nanoparticles. Moreover, we demonstrate that stochastic fluctuations result in large variability of the local group density between particles. These results cast doubt on the uniform surface coverage commonly assumed in the creation of amorphous functional nanoparticles and expose a striking difference between the average population and individual nanoparticle coverage.
Feiner-Gracia, N., Olea, R. A., Fitzner, R., El Boujnouni, N., Van Asbeck, A. H., Brock, R., Albertazzi, L., (2019). Super-resolution imaging of structure, molecular composition, and stability of single oligonucleotide polyplexes Nano Letters 19, (5), 2784-2792
The successful application of gene therapy relies on the development of safe and efficient delivery vectors. Cationic polymers such as cell-penetrating peptides (CPPs) can condense genetic material into nanoscale particles, called polyplexes, and induce cellular uptake. With respect to this point, several aspects of the nanoscale structure of polyplexes have remained elusive because of the difficulty in visualizing the molecular arrangement of the two components with nanometer resolution. This limitation has hampered the rational design of polyplexes based on direct structural information. Here, we used super-resolution imaging to study the structure and molecular composition of individual CPP-mRNA polyplexes with nanometer accuracy. We use two-color direct stochastic optical reconstruction microscopy (dSTORM) to unveil the impact of peptide stoichiometry on polyplex structure and composition and to assess their destabilization in blood serum. Our method provides information about the size and composition of individual polyplexes, allowing the study of such properties on a single polyplex basis. Furthermore, the differences in stoichiometry readily explain the differences in cellular uptake behavior. Thus, quantitative dSTORM of polyplexes is complementary to the currently used characterization techniques for understanding the determinants of polyplex activity in vitro and inside cells.
Keywords: dSTORM, Gene delivery, Polyplexes, Stability, Super-resolution microscopy
Pujals, S., Feiner-Gracia, N., Delcanale, P., Voets, I., Albertazzi, L., (2019). Super-resolution microscopy as a powerful tool to study complex synthetic materials Nature Reviews Chemistry 3, (2), 68-84
Understanding the relations between the formation, structure, dynamics and functionality of complex synthetic materials is one of the great challenges in chemistry and nanotechnology and represents the foundation for the rational design of novel materials for a variety of applications. Initially conceived to study biology below the diffraction limit, super-resolution microscopy (SRM) is emerging as a powerful tool for studying synthetic materials owing to its nanometric resolution, multicolour ability and minimal invasiveness. In this Review, we provide an overview of the pioneering studies that use SRM to visualize materials, highlighting exciting recent developments such as experiments in operando, wherein materials, such as biomaterials in a biological environment, are imaged in action. Moreover, the potential and the challenges of the different SRM methods for application in nanotechnology and (bio)materials science are discussed, aiming to guide researchers to select the best SRM approach for their specific purpose.
Keywords: Bioinspired materials, Imaging techniques
Pujals, S., Albertazzi, L., (2019). Super-resolution microscopy for nanomedicine research ACS Nano 13, (9), 9707-9712
Super-resolution microscopy, or nanoscopy, revolutionized the field of cell biology, enabling researchers to visualize cellular structures with nanometric resolution, single-molecule sensitivity, and in multiple colors. However, the impact of these techniques goes beyond biology as the fields of nanotechnology and nanomedicine can greatly benefit from them, as well. Nanoscopy can visualize nanostructures in vitro and in cells and can contribute to the characterization of their structures and nano-bio interactions. In this Perspective, we discuss the potential of super-resolution imaging for nanomedicine research, its technical challenges, and the future developments we envision for this technology.
Kolpe, A., Arista-Romero, M., Schepens, B., Pujals, S., Saelens, X., Albertazzi, L., (2019). Super-resolution microscopy reveals significant impact of M2e-specific monoclonal antibodies on influenza A virus filament formation at the host cell surface Scientific Reports 9, (1), 4450
Influenza A virions are highly pleomorphic, exhibiting either spherical or filamentous morphology. The influenza A virus strain A/Udorn/72 (H3N2) produces copious amounts of long filaments on the surface of infected cells where matrix protein 1 (M1) and 2 (M2) play a key role in virus filament formation. Previously, it was shown that an anti-M2 ectodomain (M2e) antibody could inhibit A/Udorn/72 virus filament formation. However, the study of these structures is limited by their small size and complex structure. Here, we show that M2e-specific IgG1 and IgG2a mouse monoclonal antibodies can reduce influenza A/Udorn/72 virus plaque growth and infectivity in vitro. Using Immuno-staining combined with super-resolution microscopy that allows us to study structures beyond the diffraction limit, we report that M2 is localized at the base of viral filaments that emerge from the membrane of infected cells. Filament formation was inhibited by treatment of A/Udorn/72 infected cells with M2e-specific IgG2a and IgG1 monoclonal antibodies and resulted in fragmentation of pre-existing filaments. We conclude that M2e-specific IgGs can reduce filamentous influenza A virus replication in vitro and suggest that in vitro inhibition of A/Udorn/72 virus replication by M2e-specific antibodies correlates with the inhibition of filament formation on the surface of infected cells.
Riera, R., Feiner-Gracia, N., Fornaguera, C., Cascante, A., Borrós, S., Albertazzi, L., (2019). Tracking the DNA complexation state of pBAE polyplexes in cells with super resolution microscopy Nanoscale 11, (38), 17869-17877
The future of gene therapy relies on the development of efficient and safe delivery vectors. Poly(β-amino ester)s are promising cationic polymers capable of condensing oligonucleotides into nanoparticles – polyplexes – and deliver them into the cell nucleus, where the gene material would be expressed. The complexation state during the crossing of biological barriers is crucial: polymers should tightly complex DNA before internalization and then release to allow free DNA to reach the nucleus. However, measuring the complexation state in cells is challenging due to the nanometric size of polyplexes and the difficulties to study the two components (polymer and DNA) independently. Here we propose a method to visualize and quantify the two components of a polyplex inside cells, with nanometre scale resolution, using two-colour direct stochastic reconstruction super-resolution microscopy (dSTORM). With our approach, we tracked the complexation state of pBAE polyplexes from cell binding to DNA release and nuclear entry revealing time evolution and the final fate of DNA and pBAE polymers in mammalian cells.
Uroz, Marina, Garcia-Puig, Anna, Tekeli, Isil, Elosegui-Artola, Alberto, Abenza, Juan F., Marín-Llauradó, Ariadna, Pujals, Silvia, Conte, Vito, Albertazzi, Lorenzo, Roca-Cusachs, Pere, Raya, Ángel, Trepat, Xavier, (2019). Traction forces at the cytokinetic ring regulate cell division and polyploidy in the migrating zebrafish epicardium Nature Materials 18, 1015-1023
Epithelial repair and regeneration are driven by collective cell migration and division. Both cellular functions involve tightly controlled mechanical events, but how physical forces regulate cell division in migrating epithelia is largely unknown. Here we show that cells dividing in the migrating zebrafish epicardium exert large cell–extracellular matrix (ECM) forces during cytokinesis. These forces point towards the division axis and are exerted through focal adhesions that connect the cytokinetic ring to the underlying ECM. When subjected to high loading rates, these cytokinetic focal adhesions prevent closure of the contractile ring, leading to multi-nucleation through cytokinetic failure. By combining a clutch model with experiments on substrates of different rigidity, ECM composition and ligand density, we show that failed cytokinesis is triggered by adhesion reinforcement downstream of increased myosin density. The mechanical interaction between the cytokinetic ring and the ECM thus provides a mechanism for the regulation of cell division and polyploidy that may have implications in regeneration and cancer.
Liu, Yiliu, Pujals, Sílvia, Stals, Patrick J. M., Paulöhrl, Thomas, Presolski, Stanislav I., Meijer, E. W., Albertazzi, Lorenzo, Palmans, Anja R. A., (2018). Catalytically active single-chain polymeric nanoparticles: Exploring their functions in complex biological media Journal of the American Chemical Society 140, (9), 3423-3433
Dynamic single-chain polymeric nanoparticles (SCPNs) are intriguing, bioinspired architectures that result from the collapse or folding of an individual polymer chain into a nanometer-sized particle. Here we present a detailed biophysical study on the behavior of dynamic SCPNs in living cells and an evaluation of their catalytic functionality in such a complex medium. We first developed a number of delivery strategies that allowed the selective localization of SCPNs in different cellular compartments. Live/dead tests showed that the SCPNs were not toxic to cells while spectral imaging revealed that SCPNs provide a structural shielding and reduced the influence from the outer biological media. The ability of SCPNs to act as catalysts in biological media was first assessed by investigating their potential for reactive oxygen species generation. With porphyrins covalently attached to the SCPNs, singlet oxygen was generated upon irradiation with light, inducing spatially controlled cell death. In addition, Cu(I)- and Pd(II)-based SCPNs were prepared and these catalysts were screened in vitro and studied in cellular environments for the carbamate cleavage reaction of rhodamine-based substrates. This is a model reaction for the uncaging of bioactive compounds such as cytotoxic drugs for catalysis-based cancer therapy. We observed that the rate of the deprotection depends on both the organometallic catalysts and the nature of the protective group. The rate reduces from in vitro to the biological environment, indicating a strong influence of biomolecules on catalyst performance. The Cu(I)-based SCPNs in combination with the dimethylpropargyloxycarbonyl protective group showed the best performances both in vitro and in biological environment, making this group promising in biomedical applications.
Patiño, Tania, Feiner-Gracia, Natalia, Arqué, Xavier, Miguel-López, Albert, Jannasch, Anita, Stumpp, Tom, Schäffer, Erik, Albertazzi, Lorenzo, Sánchez, Samuel, (2018). Influence of enzyme quantity and distribution on the self-propulsion of non-Janus urease-powered micromotors Journal of the American Chemical Society 140, (25), 7896-7903
The use of enzyme catalysis to power micro- and nanomachines offers unique features such as biocompatibility, versatility, and fuel bioavailability. Yet, the key parameters underlying the motion behavior of enzyme-powered motors are not completely understood. Here, we investigate the role of enzyme distribution and quantity on the generation of active motion. Two different micromotor architectures based on either polystyrene (PS) or polystyrene coated with a rough silicon dioxide shell (PS@SiO2) were explored. A directional propulsion with higher speed was observed for PS@SiO2 motors when compared to their PS counterparts. We made use of stochastically optical reconstruction microscopy (STORM) to precisely detect single urease molecules conjugated to the micromotors surface with a high spatial resolution. An asymmetric distribution of enzymes around the micromotor surface was observed for both PS and PS@SiO2 architectures, indicating that the enzyme distribution was not the only parameter affecting the motion behavior. We quantified the number of enzymes present on the micromotor surface and observed a 10-fold increase in the number of urease molecules for PS@SiO2 motors compared to PS-based micromotors. To further investigate the number of enzymes required to generate a self-propulsion, PS@SiO2 particles were functionalized with varying amounts of urease molecules and the resulting speed and propulsive force were measured by optical tracking and optical tweezers, respectively. Surprisingly, both speed and force depended in a nonlinear fashion on the enzyme coverage. To break symmetry for active propulsion, we found that a certain threshold number of enzymes molecules per micromotor was necessary, indicating that activity may be due to a critical phenomenon. Taken together, these results provide new insights into the design features of micro/nanomotors to ensure an efficient development.
Delcanale, Pietro, Miret-Ontiveros, Bernat, Arista-Romero, Maria, Pujals, Silvia, Albertazzi, Lorenzo, (2018). Nanoscale mapping functional sites on nanoparticles by Points Accumulation for Imaging in Nanoscale Topography (PAINT) ACS Nano 12, (8), 7629-7637
The ability of nanoparticles to selectively recognize a molecular target constitutes the key toward nanomedicine applications such as drug delivery and diagnostics. The activity of such devices is mediated by the presence of multiple copies of functional molecules on the nanostructure surface. Therefore, understanding the number and the distribution of nanoparticle functional groups is of utmost importance for the rational design of effective materials. Analytical methods are available, but to obtain quantitative information at the level of single particles and single functional sites, i.e., going beyond the ensemble, remains highly challenging. Here we introduce the use of an optical nanoscopy technique, DNA points accumulation for imaging in nanoscale topography (DNA-PAINT), to address this issue. Combining subdiffraction spatial resolution with molecular selectivity and sensitivity, DNA-PAINT provides both geometrical and functional information at the level of a single nanostructure. We show how DNA-PAINT can be used to image and quantify relevant functional proteins such as antibodies and streptavidin on nanoparticles and microparticles with nanometric accuracy in 3D and multiple colors. The generality and the applicability of our method without the need for fluorescent labeling hold great promise for the robust quantitative nanocharacterization of functional nanomaterials.
Ardizzone, Antonio, Kurhuzenkau, Siarhei, Illa-Tuset, Sílvia, Faraudo, Jordi, Bondar, Mykhailo, Hagan, David, Van Stryland, Eric W., Painelli, Anna, Sissa, Cristina, Feiner, Natalia, Albertazzi, Lorenzo, Veciana, Jaume, Ventosa, Nora, (2018). Nanostructuring lipophilic dyes in water using stable vesicles, quatsomes, as scaffolds and their use as probes for bioimaging Small 14, (16), 1703851
Abstract A new kind of fluorescent organic nanoparticles (FONs) is obtained using quatsomes (QSs), a family of nanovesicles proposed as scaffolds for the nanostructuration of commercial lipophilic carbocyanines (1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI), 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indodicarbocyanine perchlorate (DiD), and 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indotricarbocyanine iodide (DiR)) in aqueous media. The obtained FONs, prepared by a CO2-based technology, show excellent colloidal- and photostability, outperforming other nanoformulations of the dyes, and improve the optical properties of the fluorophores in water. Molecular dynamics simulations provide an atomistic picture of the disposition of the dyes within the membrane. The potential of QSs for biological imaging is demonstrated by performing superresolution microscopy of the DiI-loaded vesicles in vitro and in cells. Therefore, fluorescent QSs constitute an appealing nanomaterial for bioimaging applications.
Casellas, Nicolas M., Pujals, Sílvia, Bochicchio, Davide, Pavan, Giovanni M., Torres, Tomás, Albertazzi, Lorenzo, García-Iglesias, Miguel, (2018). From isodesmic to highly cooperative: Reverting the supramolecular polymerization mechanism in water by fine monomer design Chemical Communications 54, (33), 4112-4115
Two structurally-similar discotic molecules able to self-assemble in water, forming supramolecular fibers, are reported. While both self-assembled polymers are indistinguishable from a morphological point-of-view, a dramatic change in their polymerization mechanism is observed (i.e., one self-assemble via an isodesmic mechanism, while the other shows one of the highest cooperativity values).
Krivitsky, Adva, Polyak, Dina, Scomparin, Anna, Eliyahu, Shay, Ofek, Paula, Tiram, Galia, Kalinski, Hagar, Avkin-Nachum, Sharon, Feiner Gracia, N., Albertazzi, Lorenzo, Satchi-Fainaro, Ronit, (2018). Amphiphilic poly(α)glutamate polymeric micelles for systemic administration of siRNA to tumors Nanomedicine: Nanotechnology, Biology, and Medicine 14, (2), 303-315
RNAi therapeutics carried a great promise to the area of personalized medicine: the ability to target "undruggable" oncogenic pathways. Nevertheless, their efficient tumor targeting via systemic administration had not been resolved yet. Amphiphilic alkylated poly(α)glutamate amine (APA) can serve as a cationic carrier to the negatively-charged oligonucleotides. APA polymers complexed with siRNA to form round-shaped, homogenous and reproducible nano-sized polyplexes bearing ~50 nm size and slightly negative charge. In addition, APA:siRNA polyplexes were shown to be potent gene regulators in vitro. In light of these preferred physico-chemical characteristics, their performance as systemically-administered siRNA nanocarriers was investigated. Intravenously-injected APA:siRNA polyplexes accumulated selectively in tumors and did not accumulate in the lungs, heart, liver or spleen. Nevertheless, the polyplexes failed to induce specific mRNA degradation, hence neither reduction in tumor volume nor prolonged mice survival was seen.
van Elsland, Daphne M., Pujals, Sílvia, Bakkum, Thomas, Bos, Erik, Oikonomeas-Koppasis, Nikolaos, Berlin, Ilana, Neefjes, Jacques, Meijer, Annemarie H., Koster, Abraham J., Albertazzi, Lorenzo, van Kasteren, Sander I., (2018). Ultrastructural imaging of salmonella-host interactions using super-resolution correlative light-electron microscopy of bioorthogonal pathogens ChemBioChem 19, (16), 1766-1770
The imaging of intracellular pathogens inside host cells is complicated by the low resolution and sensitivity of fluorescence microscopy and by the lack of ultrastructural information to visualize the pathogens. Herein, we present a new method to visualize these pathogens during infection that circumvents these problems: by using a metabolic hijacking approach to bioorthogonally label the intracellular pathogen Salmonella Typhimurium and by using these bioorthogonal groups to introduce fluorophores compatible with stochastic optical reconstruction microscopy (STORM) and placing this in a correlative light electron microscopy (CLEM) workflow, the pathogen can be imaged within its host cell context Typhimurium with a resolution of 20 nm. This STORM-CLEM approach thus presents a new approach to understand these pathogens during infection.
Oria, Roger, Wiegand, Tina, Escribano, Jorge, Elosegui-Artola, Alberto, Uriarte, Juan Jose, Moreno-Pulido, Cristian, Platzman, Ilia, Delcanale, Pietro, Albertazzi, Lorenzo, Navajas, Daniel, Trepat, Xavier, García-Aznar, José Manuel, Cavalcanti-Adam, Elisabetta Ada, Roca-Cusachs, Pere, (2017). Force loading explains spatial sensing of ligands by cells Nature 552, 219-224
Cells can sense the density and distribution of extracellular matrix (ECM) molecules by means of individual integrin proteins and larger, integrin-containing adhesion complexes within the cell membrane. This spatial sensing drives cellular activity in a variety of normal and pathological contexts1,2. Previous studies of cells on rigid glass surfaces have shown that spatial sensing of ECM ligands takes place at the nanometre scale, with integrin clustering and subsequent formation of focal adhesions impaired when single integrin–ligand bonds are separated by more than a few tens of nanometres3,4,5,6. It has thus been suggested that a crosslinking 'adaptor' protein of this size might connect integrins to the actin cytoskeleton, acting as a molecular ruler that senses ligand spacing directly3,7,8,9. Here, we develop gels whose rigidity and nanometre-scale distribution of ECM ligands can be controlled and altered. We find that increasing the spacing between ligands promotes the growth of focal adhesions on low-rigidity substrates, but leads to adhesion collapse on more-rigid substrates. Furthermore, disordering the ligand distribution drastically increases adhesion growth, but reduces the rigidity threshold for adhesion collapse. The growth and collapse of focal adhesions are mirrored by, respectively, the nuclear or cytosolic localization of the transcriptional regulator protein YAP. We explain these findings not through direct sensing of ligand spacing, but by using an expanded computational molecular-clutch model10,11, in which individual integrin–ECM bonds—the molecular clutches—respond to force loading by recruiting extra integrins, up to a maximum value. This generates more clutches, redistributing the overall force among them, and reducing the force loading per clutch. At high rigidity and high ligand spacing, maximum recruitment is reached, preventing further force redistribution and leading to adhesion collapse. Measurements of cellular traction forces and actin flow speeds support our model. Our results provide a general framework for how cells sense spatial and physical information at the nanoscale, precisely tuning the range of conditions at which they form adhesions and activate transcriptional regulation.
Duro-Castano, Aroa, Nebot, Vicent J., Niño-Pariente, Amaya, Armiñán, Ana, Arroyo-Crespo, Juan J., Paul, Alison, Feiner-Gracia, Natalia, Albertazzi, Lorenzo, Vicent, María J., (2017). Capturing "extraordinary" soft-assembled charge-like polypeptides as a strategy for nanocarrier design Advanced Materials , 29, (39), 1702888
The rational design of nanomedicines is a challenging task given the complex architectures required for the construction of nanosized carriers with embedded therapeutic properties and the complex interface of these materials with the biological environment. Herein, an unexpected charge-like attraction mechanism of self-assembly for star-shaped polyglutamates in nonsalty aqueous solutions is identified, which matches the ubiquitous "ordinary–extraordinary" phenomenon previously described by physicists. For the first time, a bottom-up methodology for the stabilization of these nanosized soft-assembled star-shaped polyglutamates is also described, enabling the translation of theoretical research into nanomaterials with applicability within the drug-delivery field. Covalent capture of these labile assemblies provides access to unprecedented architectures to be used as nanocarriers. The enhanced in vitro and in vivo properties of these novel nanoconstructs as drug-delivery systems highlight the potential of this approach for tumor-localized as well as lymphotropic delivery.
Keywords: Charge-like, Drug delivery, Polymer therapeutics, Polypeptides, Self-assembly
Labernadie, A., Kato, T., Brugués, A., Serra-Picamal, X., Derzsi, S., Arwert, E., Weston, A., González-Tarragó, V., Elosegui-Artola, A., Albertazzi, L., Alcaraz, J., Roca-Cusachs, P., Sahai, E., Trepat, X., (2017). A mechanically active heterotypic E-cadherin/N-cadherin adhesion enables fibroblasts to drive cancer cell invasion Nature Cell Biology 19, (3), 224-237
Cancer-associated fibroblasts (CAFs) promote tumour invasion and metastasis. We show that CAFs exert a physical force on cancer cells that enables their collective invasion. Force transmission is mediated by a heterophilic adhesion involving N-cadherin at the CAF membrane and E-cadherin at the cancer cell membrane. This adhesion is mechanically active; when subjected to force it triggers β-catenin recruitment and adhesion reinforcement dependent on α-catenin/vinculin interaction. Impairment of E-cadherin/N-cadherin adhesion abrogates the ability of CAFs to guide collective cell migration and blocks cancer cell invasion. N-cadherin also mediates repolarization of the CAFs away from the cancer cells. In parallel, nectins and afadin are recruited to the cancer cell/CAF interface and CAF repolarization is afadin dependent. Heterotypic junctions between CAFs and cancer cells are observed in patient-derived material. Together, our findings show that a mechanically active heterophilic adhesion between CAFs and cancer cells enables cooperative tumour invasion.
Feiner-Gracia, Natalia, Buzhor, Marina, Fuentes, Edgar, Pujals, S., Amir, Roey J., Albertazzi, Lorenzo, (2017). Micellar stability in biological media dictates internalization in living cells Journal of the American Chemical Society 139, (46), 16677-16687
The dynamic nature of polymeric assemblies makes their stability in biological media a crucial parameter for their potential use as drug delivery systems in vivo. Therefore, it is essential to study and understand the behavior of self-assembled nanocarriers under conditions that will be encountered in vivo such as extreme dilutions and interactions with blood proteins and cells. Herein, using a combination of fluorescence spectroscopy and microscopy, we studied four amphiphilic PEG–dendron hybrids and their self-assembled micelles in order to determine their structure–stability relations. The high molecular precision of the dendritic block enabled us to systematically tune the hydrophobicity and stability of the assembled micelles. Using micelles that change their fluorescent properties upon disassembly, we observed that serum proteins bind to and interact with the polymeric amphiphiles in both their assembled and monomeric states. These interactions strongly affected the stability and enzymatic degradation of the micelles. Finally, using spectrally resolved confocal imaging, we determined the relations between the stability of the polymeric assemblies in biological media and their cell entry. Our results highlight the important interplay between molecular structure, micellar stability, and cell internalization pathways, pinpointing the high sensitivity of stability–activity relations to minor structural changes and the crucial role that these relations play in designing effective polymeric nanostructures for biomedical applications.
Feiner-Gracia, Natalia, Beck, Michaela, Pujals, Sílvia, Tosi, Sébastien, Mandal, Tamoghna, Buske, Christian, Linden, Mika, Albertazzi, Lorenzo, (2017). Super-resolution microscopy unveils dynamic heterogeneities in nanoparticle protein corona Small 13, (41), 1701631
The adsorption of serum proteins, leading to the formation of a biomolecular corona, is a key determinant of the biological identity of nanoparticles in vivo. Therefore, gaining knowledge on the formation, composition, and temporal evolution of the corona is of utmost importance for the development of nanoparticle-based therapies. Here, it is shown that the use of super-resolution optical microscopy enables the imaging of the protein corona on mesoporous silica nanoparticles with single protein sensitivity. Particle-by-particle quantification reveals a significant heterogeneity in protein absorption under native conditions. Moreover, the diversity of the corona evolves over time depending on the surface chemistry and degradability of the particles. This paper investigates the consequences of protein adsorption for specific cell targeting by antibody-functionalized nanoparticles providing a detailed understanding of corona-activity relations. The methodology is widely applicable to a variety of nanostructures and complements the existing ensemble approaches for protein corona study.
Keywords: Heterogeneity, Mesoporous silica nanoparticles, Protein corona, Super-resolution imaging, Targeting
Van Onzen, A. H. A. M., Albertazzi, L., Schenning, A. P. H. J., Milroy, L. G., Brunsveld, L., (2017). Hydrophobicity determines the fate of self-assembled fluorescent nanoparticles in cells Chemical Communications 53, (10), 1626-1629
The fate of small molecule nanoparticles (SMNPs) composed of self-assembling intrinsically fluorescent π-conjugated oligomers was studied in cells as a function of side-chain hydrophobicity. While the hydrophobic SMNPs remained intact upon cellular uptake, the more hydrophilic SMNPs disassembled and dispersed throughout the cytosol.
Pujals, S., Tao, K., Terradellas, A., Gazit, E., Albertazzi, L., (2017). Studying structure and dynamics of self-Assembled peptide nanostructures using fluorescence and super resolution microscopy Chemical Communications 53, (53), 7294-7297
Understanding the formation and properties of self-Assembled peptide nanostructures is the basis for the design of new architectures for various applications. Here we show the potential of fluorescence and super resolution imaging to unveil the structural and dynamic features of peptide nanofibers with high spatiotemporal resolution.
Caballero, David, Blackburn, Sophie M., de Pablo, Mar, Samitier, Josep, Albertazzi, Lorenzo, (2017). Tumour-vessel-on-a-chip models for drug delivery Lab on a Chip 17, 3760-3771
Nanocarriers for drug delivery have great potential to revolutionize cancer treatment, due to their enhanced selectivity and efficacy. Despite this great promise, researchers have had limited success in the clinical translation of this approach. One of the main causes of these difficulties is that standard in vitro models, typically used to understand nanocarriers' behaviour and screen their efficiency, do not provide the complexity typically encountered in living systems. In contrast, in vivo models, despite being highly physiological, display serious bottlenecks which threaten the relevancy of the obtained data. Microfluidics and nanofabrication can dramatically contribute to solving this issue, providing 3D high-throughput models with improved resemblance to in vivo systems. In particular, microfluidic models of tumour blood vessels can be used to better elucidate how new nanocarriers behave in the microcirculation of healthy and cancerous tissues. Several key steps of the drug delivery process such as extravasation, immune response and endothelial targeting happen under flow in capillaries and can be accurately modelled using microfluidics. In this review, we will present how tumour-vessel-on-a-chip systems can be used to investigate targeted drug delivery and which key factors need to be considered for the rational design of these materials. Future applications of this approach and its role in driving forward the next generation of targeted drug delivery methods will be discussed.
Bakker, Maarten H., Lee, Cameron C., Meijer, E. W., Dankers, Patricia Y. W., Albertazzi, Lorenzo, (2016). Multicomponent supramolecular polymers as a modular platform for intracellular delivery ACS Nano 10, (2), 1845-1852
Supramolecular polymers are an emerging family of nanosized structures with potential use in materials chemistry and medicine. Surprisingly, application of supramolecular polymers in the field of drug delivery has received only limited attention. Here, we explore the potential of PEGylated 1,3,5-benzenetricarboxamide (BTA) supramolecular polymers for intracellular delivery. Exploiting the unique modular approach of supramolecular chemistry, we can coassemble neutral and cationic BTAs and control the overall properties of the polymer by simple monomer mixing. Moreover, this platform offers a versatile approach toward functionalization. The core can be efficiently loaded with a hydrophobic guest molecule, while the exterior can be electrostatically complexed with siRNA. It is demonstrated that both compounds can be delivered in living cells, and that they can be combined to enable a dual delivery strategy. These results show the advantages of employing a modular system and pave the way for application of supramolecular polymers in intracellular delivery.
Beun, L. H., Albertazzi, L., Van Der Zwaag, D., De Vries, R., Cohen Stuart, M. A., (2016). Unidirectional living growth of self-assembled protein nanofibrils revealed by super-resolution microscopy ACS Nano 10, (5), 4973-4980
Protein-based nanofibrils are emerging as a promising class of materials that provide unique properties for applications such as biomedical and food engineering. Here, we use atomic force microscopy and stochastic optical reconstruction microscopy imaging to elucidate the growth dynamics, exchange kinetics, and polymerization mechanism for fibrils composed of a de novo designed recombinant triblock protein polymer. This macromolecule features a silk-inspired self-assembling central block composed of GAGAGAGH repeats, which are known to fold into a β roll with turns at each histidine and, once folded, to stack, forming a long, ribbon-like structure. We find several properties that allow the growth of patterned protein nanofibrils: the self-assembly takes place on only one side of the growing fibrils by the essentially irreversible addition of protein polymer subunits, and these fibril ends remain reactive indefinitely in the absence of monomer ("living ends"). Exploiting these characteristics, we can grow stable diblock protein nanofibrils by the sequential addition of differently labeled proteins. We establish control over the block length ratio by simply varying monomer feed conditions. Our results demonstrate the use of engineered protein polymers in creating precisely patterned protein nanofibrils and open perspectives for the hierarchical self-assembly of functional biomaterials.
Keywords: Nanofibrils, Protein polymers, Self-assembly, STORM microscopy
Garzoni, M., Baker, M. B., Leenders, C. M. A., Voets, I. K., Albertazzi, L., Palmans, A. R. A., Meijer, E. W., Pavan, G. M., (2016). Effect of H-bonding on order amplification in the growth of a supramolecular polymer in water Journal of the American Chemical Society 138, (42), 13985-13995
While a great deal of knowledge on the roles of hydrogen bonding and hydrophobicity in proteins has resulted in the creation of rationally designed and functional peptidic structures, the roles of these forces on purely synthetic supramolecular architectures in water have proven difficult to ascertain. Focusing on a 1,3,5-benzenetricarboxamide (BTA)-based supramolecular polymer, we have designed a molecular modeling strategy to dissect the energetic contributions involved in the self-assembly (electrostatic, hydrophobic, etc.) upon growth of both ordered BTA stacks and random BTA aggregates. Utilizing this set of simulations, we have unraveled the cooperative mechanism for polymer growth, where a critical size must be reached in the aggregates before emergence and amplification of order into the experimentally observed fibers. Furthermore, we have found that the formation of ordered fibers is favored over disordered aggregates solely on the basis of electrostatic interactions. Detailed analysis of the simulation data suggests that H-bonding is a major source of this stabilization energy. Experimental and computational comparison with a newly synthesized 1,3,5-benzenetricarboxyester (BTE) derivative, lacking the ability to form the H-bonding network, demonstrated that this BTE variant is also capable of fiber formation, albeit at a reduced persistence length. This work provides unambiguous evidence for the key 1D driving force of hydrogen bonding in enhancing the persistency of monomer stacking and amplifying the level of order into the growing supramolecular polymer in water. Our computational approach provides an important relationship directly linking the structure of the monomer to the structure and properties of the supramolecular polymer.
Aloi, Antonio, Vargas Jentzsch, Andreas, Vilanova, Neus, Albertazzi, Lorenzo, Meijer, E. W., Voets, Ilja K., (2016). Imaging nanostructures by single-molecule localization microscopy in organic solvents Journal of the American Chemical Society 138, (9), 2953-2956
The introduction of super-resolution fluorescence microscopy (SRM) opened an unprecedented vista into nanoscopic length scales, unveiling a new degree of complexity in biological systems in aqueous environments. Regrettably, supramolecular chemistry and material science benefited far less from these recent developments. Here we expand the scope of SRM to photoactivated localization microscopy (PALM) imaging of synthetic nanostructures that are highly dynamic in organic solvents. Furthermore, we characterize the photophysical properties of commonly used photoactivatable dyes in a wide range of solvents, which is made possible by the addition of a tiny amount of an alcohol. As proof-of-principle, we use PALM to image silica beads with radii close to Abbe's diffraction limit. Individual nanoparticles are readily identified and reliably sized in multicolor mixtures of large and small beads. We further use SRM to visualize nm-thin yet μm-long dynamic, supramolecular polymers, which are among the most challenging molecular systems to image.
da Silva, Ricardo M. P., van der Zwaag, Daan, Albertazzi, Lorenzo, Lee, Sungsoo S., Meijer, E. W., Stupp, Samuel I., (2016). Super-resolution microscopy reveals structural diversity in molecular exchange among peptide amphiphile nanofibres Nature Communications 7, 11561
The dynamic behaviour of supramolecular systems is an important dimension of their potential functions. Here, we report on the use of stochastic optical reconstruction microscopy to study the molecular exchange of peptide amphiphile nanofibres, supramolecular systems known to have important biomedical functions. Solutions of nanofibres labelled with different dyes (Cy3 and Cy5) were mixed, and the distribution of dyes inserting into initially single-colour nanofibres was quantified using correlative image analysis. Our observations are consistent with an exchange mechanism involving monomers or small clusters of molecules inserting randomly into a fibre. Different exchange rates are observed within the same fibre, suggesting that local cohesive structures exist on the basis of [beta]-sheet discontinuous domains. The results reported here show that peptide amphiphile supramolecular systems can be dynamic and that their intermolecular interactions affect exchange patterns. This information can be used to generate useful aggregate morphologies for improved biomedical function.
De Koker, Stefaan, Cui, Jiwei, Vanparijs, Nane, Albertazzi, Lorenzo, Grooten, Johan, Caruso, Frank, De Geest, Bruno G., (2016). Engineering polymer hydrogel nanoparticles for lymph node-targeted delivery Angewandte Chemie - International Edition , 55, (4), 1334-1339
The induction of antigen-specific adaptive immunity exclusively occurs in lymphoid organs. As a consequence, the efficacy by which vaccines reach these tissues strongly affects the efficacy of the vaccine. Here, we report the design of polymer hydrogel nanoparticles that efficiently target multiple immune cell subsets in the draining lymph nodes. Nanoparticles are fabricated by infiltrating mesoporous silica particles (ca. 200 nm) with poly(methacrylic acid) followed by disulfide-based crosslinking and template removal. PEGylation of these nanoparticles does not affect their cellular association in vitro, but dramatically improves their lymphatic drainage in vivo. The functional relevance of these observations is further illustrated by the increased priming of antigen-specific T cells. Our findings highlight the potential of engineered hydrogel nanoparticles for the lymphatic delivery of antigens and immune-modulating compounds.
Keywords: Dendritic cells, Disulfides, Hydrogels, Nanoparticles, Vaccines
Li, Hui, Fierens, Kaat, Zhang, Zhiyue, Vanparijs, Nane, Schuijs, Martijn J., Van Steendam, Katleen, Feiner Gracia, Natàlia, De Rycke, Riet, De Beer, Thomas, De Beuckelaer, Ans, De Koker, Stefaan, Deforce, Dieter, Albertazzi, Lorenzo, Grooten, Johan, Lambrecht, Bart N., De Geest, Bruno G., (2016). Spontaneous protein adsorption on graphene oxide nanosheets allowing efficient intracellular vaccine protein delivery ACS Applied Materials & Interfaces 8, (2), 1147-1155
Nanomaterials hold potential of altering the interaction between therapeutic molecules and target cells or tissues. High aspect ratio nanomaterials in particular have been reported to possess unprecedented properties and are intensively investigated for their interaction with biological systems. Graphene oxide (GOx) is a water-soluble graphene derivative that combines high aspect ratio dimension with functional groups that can be exploited for bioconjugation. Here, we demonstrate that GOx nanosheets can spontaneously adsorb proteins by a combination of interactions. This property is then explored for intracellular protein vaccine delivery, in view of the potential of GOx nanosheets to destabilize lipid membranes such as those of intracellular vesicles. Using a series of in vitro experiments, we show that GOx nanosheet adsorbed proteins are efficiently internalized by dendritic cells (DCs: the most potent class of antigen presenting cells of the immune system) and promote antigen cross-presentation to CD8 T cells. The latter is a hallmark in the induction of potent cellular antigen-specific immune responses against intracellular pathogens and cancer. Nanomaterials hold potential of altering the interaction between therapeutic molecules and target cells or tissues. High aspect ratio nanomaterials in particular have been reported to possess unprecedented properties and are intensively investigated for their interaction with biological systems. Graphene oxide (GOx) is a water-soluble graphene derivative that combines high aspect ratio dimension with functional groups that can be exploited for bioconjugation. Here, we demonstrate that GOx nanosheets can spontaneously adsorb proteins by a combination of interactions. This property is then explored for intracellular protein vaccine delivery, in view of the potential of GOx nanosheets to destabilize lipid membranes such as those of intracellular vesicles. Using a series of in vitro experiments, we show that GOx nanosheet adsorbed proteins are efficiently internalized by dendritic cells (DCs: the most potent class of antigen presenting cells of the immune system) and promote antigen cross-presentation to CD8 T cells. The latter is a hallmark in the induction of potent cellular antigen-specific immune responses against intracellular pathogens and cancer.
van der Zwaag, Daan, Vanparijs, Nane, Wijnands, Sjors, De Rycke, Riet, De Geest, Bruno G., Albertazzi, Lorenzo, (2016). Super resolution imaging of nanoparticles cellular uptake and trafficking ACS Applied Materials & Interfaces 8, (10), 6391-6399
Understanding the interaction between synthetic nanostructures and living cells is of crucial importance for the development of nanotechnology-based intracellular delivery systems. Fluorescence microscopy is one of the most widespread tools owing to its ability to image multiple colors in native conditions. However, due to the limited resolution, it is unsuitable to address individual diffraction-limited objects. Here we introduce a combination of super-resolution microscopy and single-molecule data analysis to unveil the behavior of nanoparticles during their entry into mammalian cells. Two-color Stochastic Optical Reconstruction Microscopy (STORM) addresses the size and positioning of nanoparticles inside cells and probes their interaction with the cellular machineries at nanoscale resolution. Moreover, we develop image analysis tools to extract quantitative information about internalized particles from STORM images. To demonstrate the potential of our methodology, we extract previously inaccessible information by the direct visualization of the nanoparticle uptake mechanism and the intracellular tracking of nanoparticulate model antigens by dendritic cells. Finally, a direct comparison between STORM, confocal microscopy, and electron microscopy is presented, showing that STORM can provide novel and complementary information on nanoparticle cellular uptake. Understanding the interaction between synthetic nanostructures and living cells is of crucial importance for the development of nanotechnology-based intracellular delivery systems. Fluorescence microscopy is one of the most widespread tools owing to its ability to image multiple colors in native conditions. However, due to the limited resolution, it is unsuitable to address individual diffraction-limited objects. Here we introduce a combination of super-resolution microscopy and single-molecule data analysis to unveil the behavior of nanoparticles during their entry into mammalian cells. Two-color Stochastic Optical Reconstruction Microscopy (STORM) addresses the size and positioning of nanoparticles inside cells and probes their interaction with the cellular machineries at nanoscale resolution. Moreover, we develop image analysis tools to extract quantitative information about internalized particles from STORM images. To demonstrate the potential of our methodology, we extract previously inaccessible information by the direct visualization of the nanoparticle uptake mechanism and the intracellular tracking of nanoparticulate model antigens by dendritic cells. Finally, a direct comparison between STORM, confocal microscopy, and electron microscopy is presented, showing that STORM can provide novel and complementary information on nanoparticle cellular uptake.
Beuwer, Michael A., Knopper, M. F., Albertazzi, Lorenzo, van der Zwaag, Daan, Ellenbroek, Wouter G., Meijer, E. W., Prins, Menno W. J., Zijlstra, Peter, (2016). Mechanical properties of single supramolecular polymers from correlative AFM and fluorescence microscopy Polymer Chemistry , 7, (47), 7260-7268
We characterize the structure and mechanical properties of 1,3,5-benzenetricarboxamide (BTA) supramolecular polymers using correlative AFM and fluorescence imaging. AFM allows for nanoscale structural investigation but we found that statistical analysis is difficult because these structures are easily disrupted by the AFM tip. We therefore correlate AFM and fluorescence microscopy to couple nanoscale morphological information to far-field optical images. A fraction of the immobilized polymers are in a clustered or entangled state, which we identify based on diffraction limited fluorescence images. We find that clustered and entangled polymers exhibit a significantly longer persistence length that is broader distributed than single unentangled polymers. By comparison with numerical simulations we find significant heterogeneity in the persistence length of single unentangled polymers, which we attribute to polymer-substrate interactions and the presence of structural diversity within the polymer.
Nikon NSTORM Super Resolution Microscope
Single particles tracking
TIRF fluorescence imaging
Roey Amir
Tel Aviv University, Israel
Mika Linden
Ulm University, Germany
Ilja Voets
Eindhoven University of Technology, The Netherlands
Giovanni Pavan
SUPSI, Switzerland
Bruno De Geest
University of Ghent, Belgium
Salvador Borros
IQS, Barcelona
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Why should I prepare International Affairs?
How to approach the Dynamic portion?
Because in UPSC General Studies Mains (Paper II), they ask questions worth more than 100 marks, from this topic.
In the Essay paper, they regulary ask essays directly or indirectly related to Globalization and International relations.
IGNOU Political science module MPSE-001 India and the World (Click me).
That pdf file set contains pretty much all the essential information necessary to cover the static/historic portion of India's foreign relations after independence.
In your head, visualize following. What exactly are you supposed to prepare?
Fall of USSR and LPG reforms in 1991.
Now superimpose both, and you've prepared India and the world.
^upto point number 5, I would classify under "static" question / theory portion.
Lately the UPSC has stopped asking questions from it, after all how much can they ask from NAM or Panchsheel! Whatever they could ask, has been asked in last 25 years GS papers, just look at the old papers and try to frame skeletal answer.
But since UPSC has a nasty habit of throwing unexpected surprizes, so one should go prepared.
So, how to approach the Static portion?
From the given IGNOU ZIP file, prepare a very short note, highlighting keywords of India's relations through each region and each phase. There are 193 countries in UN, you don't have to prepare each and every one of them but only the important ones, given in the "Based on Geography".
This pretty much sums up the "static" portion.
Now what about the "dynamic" (i.e. current based). Well past two years, majority of questions are coming from the dynamic portion only.
What types of questions can be asked from this portion? To understand it, just browse through the old question papers and prepare the current affairs on that line.
First make list of bilateral visits (you can find information on website of Ministry of External Affairs) Then dig up on google, What trade-pacts or treaties were signed. You don't have to do Ph.D on each and everything, for example of PM of Nicargua visited India, and signed a few trade pacts, it's hardly of any importance but when PM of Bangladesh visits India and discusses something on land tranfer, border or river dispute – that part is important. Again no need to mug up how many crores were given as loan or what was the exact date of meeting etc.
The list of all important international events happened last year, where India was not involved, can be found in your monthly issue of Chronicle, Wizard, Pratiyogita Darpan.
Kamla Prasad Bissesar became PM of Trinidad and Tobago last year, so prepare that region.
Crisis for Indian Workers in Tripoli, Middle east, Libya etc.
Schemes of Government for the protection and welfare of Indian workers abroad. And so on.
^this list is not exhaustive, just as an example.
You cannot prepare the international relations in a week, it's a continuous process, keep a habit of reading newspapers daily, particularly the Columns related to International relations. But at the same time, you don't need ball by ball commentary on what is going on, for example Political turmoil of Nepal, their Prime minister keeps changing every fortnight, so by the time it is October November, they'd be 25-30 Prime ministers already (!), you try to mug up the names and timelines, it won't do you any good.
Secondly, If you've to attempt an Essay on Globalization or international affairs, you'll need fodder material (facts and viewpoints), therefore reading newspapers and columns Is must.
But like I said earlier, there is no need to do Ph.D, else you won't be left with any time or energy for other subjects. Always keep the syllabus in mind, you've to prepare the topics, not the books or magazines.
This part will include questions to test candidate's awareness of India's relation- ship with the world in various spheres such as the following:- Foreign Affairs with special emphasis on India's relations with neighbouring countries and in the region. Security and defence related matters.
In this part, questions will be on economic and trade issues such as foreign trade, foreign investment; economic and diplo- macy issues relating to oil, gas and energy flows; the role and functions of I.M.F., World Bank, W.T.O., WIPO etc. which influence India's economic interaction with other countries and international institutions.
I am a second year medical student. And m so confused with what I should start as I have4 years to graduate and hence to appear in exam. I am just not able to plan. I am reading newspapers regularly but want to start with the books as well but there are some fears like what if i get bored after 3 years of reading same things, or what goes I read so much and accumulate more waste than productive ??? Please help .
thanks foe the guidance u gave..


text:
Dulce María Loynaz, née le  à La Havane à Cuba et morte le  dans la même ville, est une poétesse cubaine. Elle a reçu le prix Cervantes en 1992.

Biographie
Dulce María Loynaz Muñoz naît au sein d'une famille appartenant à la bourgeoisie libérale. Elle obtient en 1927 un doctorat en droit civil à l'université de La Havane et exerce ensuite la profession d'avocate. Elle fait la connaissance, en 1930, de Federico García Lorca, qui réside dans la demeure de sa famille lors de son séjour à Cuba. Elle se lie par la suite d'amitié avec le poète espagnol Juan Ramón Jiménez.

Elle est élue membre de l'académie des Arts et Lettres de Cuba en 1951, et de l'Académie cubaine de la langue, dont elle devient présidente, en 1959.

Œuvre
Dulce María Loynaz commence très jeune à écrire de la poésie. Ses premiers poèmes paraissent dans le journal La Nación de La Havane en 1919 ; elle est ensuite publiée dans plusieurs anthologies de la poésie cubaine. Un voyage autour de la Méditerranée, commencé en 1929, la conduit en Égypte, où elle découvre le tombeau de Toutânkhamon. Fascinée par le pharaon, elle lui écrit à cette occasion une lettre d'amour, sous la forme d'un poème, Carta de amor a Tut-Ank-Amen.

En 1938 paraît son premier recueil, Versos. 1920-1938.

Dulce María Loynaz est l'auteur d'un pamphlet, Les Corridas à Cuba, contre l'instauration de la tauromachie sur l'île, paru en 1950.

Liste partielle des œuvres

Poésie
 Canto a la mujer estéril (1937).
 Versos. 1920-1938 (1938).
 Carta de amor a Tut-Ank-Amen (1938).
 Juegos de agua (1947).
 Poemas sin nombre (1953).
 Dos poemas inéditos (1958).
 La Novia de Lázaro. Fragmentos (1991).

Prose
 Les Corridas à Cuba (1950)
 Jardin (roman, 1951)

Notes et références

Voir aussi

Bibliographie
 Dulce María Loynaz, La Fille prodigue, anthologie traduite par Claude Couffon, La Différence, collection « Orphée », 1994

Articles connexes
 Littérature cubaine

Liens externes
  Dulce María Loynaz, sur l'encyclopédie EcuRed (Cuba)
  Dulce María Loynaz, sur le site de l'université d'Alicante (Espagne)

Naissance en décembre 1902
Naissance à La Havane
Décès en avril 1997
Décès à La Havane
Décès à 94 ans
Poétesse cubaine
Personnalité opposée à la corrida
Étudiant de l'université de La Havane
Lauréat du prix Cervantes
Grand-croix de l'ordre d'Alphonse X le Sage
Chevalier de l'ordre d'Isabelle la Catholique

redpajama_set_name: RedPajamaWikipedia


This paper considers actuator-fault estimation for discrete-time descriptor systems with unknown but bounded system disturbance and measurement noise. A zonotopic fault estimation filter is designed based on the analysis of fault detectability indexes. To ensure estimation accuracy, the filter gain in the zonotopic fault estimation filter is optimized through the zonotope minimization. The designed zonotopic filter not only can estimate fault magnitudes, but it also provides fault estimation results in an interval, i.e. the upper and lower bounds of fault magnitudes. Moreover, the proposed fault estimation filter has a non-singular structure and hence is easy to implement. Finally, simulation results are provided to illustrate the effectiveness of the proposed method.
CitationWang, Y., Wang, Z., Puig, V., Cembrano, M. Zonotopic fault estimation filter design for discrete-time descriptor systems. "IFAC-PapersOnLine", 1 Juliol 2017, vol. 50, núm. 1, p. 5055-5060.


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Crunch Time for Calbright
California's new online-only community college grabbed headlines again with its CEO's departure. While some see the college as a doomed venture, others say it's too soon to pass judgment.
Madeline St. Amour
Courtesy California Community Colleges
Some observers are raising red flags after the unexpected departure of the president and CEO of California's new online-only community college. But others chalk it up to the normal growing pains associated with a start-up and say it's too soon to judge whether the college will be successful.
Calbright College, an initiative started by Jerry Brown, California's former governor, opened its programs to students in October. It's aimed at adult learners who don't have degrees and are underemployed. Calbright is completely online, statewide and competency-based. It doesn't offer degrees but instead features certificates based on skills that could lead to middle-income jobs.
Its first leader, Heather Hiles, announced this week that she will step down in March after a year on the job. A statement from Hiles said she plans to return to previous ventures now that Calbright is operational.
The news has raised some eyebrows and reignited the discussion of whether the college can be successful.
"Our legislative mandate remains that we have to serve these students, and that's what brings everyone to work every day," said Taylor Huckaby, a spokesman for Calbright. "We'll roll with the punches."
'I Don't Think We Can Wait'
So far, 464 students are enrolled in what Calbright calls its "beta cohort," which includes three programs. In each track, students enroll in college skills courses first before moving on to a "core curriculum" designed for the specific program. So far, 22 students have enrolled in the core curriculum portion, which the college's three deans are teaching as it works toward hiring faculty members.
Huckaby said they are "comfortable" with the number of enrolled students because Calbright is still hiring faculty and establishing partnerships with businesses.
The college was pushed to open before getting these pieces in place because of legislative deadlines, Huckaby said. The state required it to start programs by the fourth quarter of 2019.
Right now is "very much a research and development phase," Huckaby said. After the beta phase, he said, the college will work under its intended model. Programs would start with an employer agreement. The college would create a curriculum based on the workforce demands for that employer, which would then offer a set number of jobs and apprenticeships to the students who graduate.
The college has had more than 100 meetings with potential employers since last summer, Huckaby said, but it's not ready to announce any partnerships.
Before the programs even opened, critics have argued that the new college is unnecessary.
"We have existing colleges that do all these same programs," said Evan Hawkins, executive director of the Faculty Association of California Community Colleges.
Hawkins also finds the college's inherent model "flawed." Calbright plans to work with employers to train people in relevant skills. It would charge employers to train employees, reducing its reliance on taxpayer money, which Hawkins said is "incredibly problematic" for a public institution.
"From the very beginning, you have the idea of a start-up as opposed to a college," he said.
Hawkins and the faculty union believe the state's community colleges should be fully funded, as they are now the lowest funded of all institution types in the state. With more state support, the colleges could focus on similar programs he believes would be more effective locally than statewide.
"I think faculty are pretty frustrated," he said. "This is one thing that all faculty are in agreement on -- that this is really a waste of resources."
Sally Johnstone disagrees.
"What everyone's saying -- that's what they want to happen," said Johnstone, president of the National Center for Higher Education Management Systems and one of the consultants who helped design the Calbright model.
Despite losing Hiles, she said, the college has strong leaders on the ground who are "keeping things rolling." She believes Calbright is likely to succeed.
Johnstone said designers considered several models for the project, including asking existing colleges to use this model, but ultimately decided something new was needed.
"The colleges have not radically rethought what it is that the workplace needs and how could they work with employers in ways that are meaningful for adult learners," she said, adding that it doesn't make sense to wait "around to try and get the colleges that now exist to change a whole bunch of what they're doing within structures and systems that are not conducive to change."
She added that the "governor didn't want to wait, because I don't think we can wait."
Johnstone also said Calbright is a "threatening model" for traditional academic institutions.
"If it succeeds, it may well challenge the basic tenets of how you do things," she said, adding that those in higher education now may have their "hearts in the right places," but they're operating within a structure that might not be as relevant as it was in the past.
"And that's scary," she said.
Still, without substantive changes, Johnstone said, higher education won't be able to serve the population of people that Calbright is trying to help.
Start-Ups Take Time
As for how the college is doing so far, "it is a start-up," Johnstone said.
She compared it to Western Governors University, a nonprofit, private online university founded in 1997 that now enrolls nearly 120,000 students and has 170,000 alumni.
"People in higher education in the western states thought it was the worst idea in the world," Johnstone said of WGU. "So I'm not surprised to be hearing what I'm hearing about Calbright right now."
When looking at a history of WGU, some of the similarities are clear. It worked with employers, many in technology, to get funding; few students signed up in the beginning; it took a few years to find a leader who was more permanent; and much of the early news media coverage described it as a doomed venture.
As an outsider looking at Calbright, Scott Pulsipher, WGU's president, said it's too early to judge the college's success.
In WGU's case, it took five years to reach 1,000 students.
"That early phase of any new endeavor like this, that's not some quick turnaround," Pulsipher said.
With any start-up, Pulsipher said, there are two questions to answer: Is the product needed and valuable, and is the entity able to execute the product?
Calbright is offering something different in the form of nondegree credentials that focus on workforce needs and provide more flexibility to students.
Now, he said, the college must prove the demand and that it can scale the idea, which will take time.
And, while WGU serves thousands of Californians and has articulation agreements with the state's community colleges, Pulsipher doesn't see Calbright as competition.
"There are far more individuals that need to be served than there is capacity to serve them," he said. "It's bad to see this as a zero-sum game."
An 'Early Experiment'
Some experts have doubts about whether the idea itself can work.
"I'm not sure that only nondegree credentials on their own could sustain a business model for most institutions," said Sean Gallagher, executive director of the Center for the Future of Higher Education and Talent Strategy at Northeastern University. "You'd have to be operating at a very large scale."
Because the state of California is large, it does provide that opportunity. But Gallagher said Calbright may struggle to convince enough students to buy in to the idea.
Calbright's intended targets are less likely to enroll in online programs, Gallagher said. Adults without degrees tend not to try online higher education as often as do those who hold degrees.
"The comfort with online learning and the means and time to pursue it has been historically greater at higher levels of the job market," he said. "That means there's a special challenge, at times, in enrolling students at this level of a program and in certain fields in the online education market."
Calbright also must grapple with establishing a new brand, which will take time.
But there are advantages to starting anew.
"My sense is that there are aspects of the structure of higher education in California that have, at times, made it difficult to scale online," Gallagher said.
All of these issues make Calbright an "early experiment" in this field, Gallagher said, though he expects to see more ventures like it.
David Schejbal, vice president and chief of digital learning at Marquette University, also has doubts about the model, particularly its focus on nondegree credentials.
"Degrees are still the coin of the realm," Schejbal said. "The reality is that we don't have any kind of common medium the way we do with credit hours and degrees" that allows for easy credit transfer or understanding from employers.
While he thinks these new kinds of models are needed for the nation as a whole, getting the public and employers to latch on to the idea is difficult.
To change these perceptions, he said federal Title IV regulations that regulate financial aid funds need to change, too.
Because of the broader shifts needed to make real change, Schejbal said, new ventures should follow the current structure. The experiment won't change the broader culture, he said. And without that change, the ventures are likely to fail.
"We, as a nation, are not good at education strategy," he said. "It would be great if we got this one right. The future of the country depends on more educated citizens."
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redpajama_set_name: RedPajamaCommonCrawl


text:
        <meta http-equiv="Content-Type" content="text/html; charset=utf-8"/>

        <title>
      Design Fiction:
      A Method Toolbox for Design Research in a Complex World
    </title>

            Design Fiction:
      A Method Toolbox for Design Research in a Complex World

      Authors

      Simon Grand and Martin Wiedmer

      Notes

      Field of Study

      Concepts

      Quotations

      "Design and design research do not primarily focus on explaining the
      world as it is;

      they share with engineering a fundamental interest in focusing on the
      world as it could be. Design fiction is a new approach, which

      integrates these two perspectives, in order to develop a method toolbox
      for design research for a complex world"

      "Design and design research do not primarily focus

      on the world as it is, like most scientific disciplines, trying to
      develop descriptions,

      interpretations, and explanations of existing objects, processes, and
      activities; design and

      design research share with engineering a fundamental interest in focusing
      on the world as it

      could be, on the imagination and realization of possible futures, as well
      as on the disclosure

      of new worlds."

      References

      Methodology

redpajama_set_name: RedPajamaGithub


text:
Harper Shows Contempt for Workers by Passing Anti-Union law
In its essence, the bill is an act of contempt for unions, brought in at the behest of employer associations and well-funded right-wing think tanks. Bill C-377 imposes heavy reporting obligations on unions under the pretext that workers are entitled to a tax credit for their union dues.
Richard Perron, Contributor
President of Quebec Union of Public Employees (SPGQ)
07/08/2015 05:37am EDT | Updated July 8, 2016
COLE BURSTON via Getty Images
Canada's Prime Minister Stephen Harper speaks alongside India's Prime Minister Narendra Modi (not pictured) on Parliament Hill in Ottawa, Canada on April 15, 2015. Prime Minister Modi will continue his official visit to Canada in Toronto and Vancouver. AFP PHOTO/ COLE BURSTON (Photo credit should read Cole Burston/AFP/Getty Images)
It's hard not to reflect on the profound contempt Stephen Harper's Conservative government has consistently demonstrated towards all those who for purely ideological reasons it has designated as its enemies.
There's contempt for those who think Canada should be a force for peace on the world scene, and for those who think environmental protection should be a priority. Contempt for those who think controlling firearms might prevent bloodshed, and contempt for those who think fiscal measures that benefit the well-to-do to the detriment of everyone else are an abandonment of our long-held commitment to social justice. And there's the government's contempt for its own scientists. By muzzling them to keep the public in the dark about facts and science, the government seeks to focus political and economic discourse on ideology and religion. And now by railroading Bill C-377 into law, the Conservative government has laid another brick in this wall of contempt. Contempt for workers and their means of acting in solidarity: unions.
Contempt has marked the history of the bill since it was first introduced in 2012. Though it is claimed to make unions more transparent, Bill C-377 is actually a carbon copy of an anti-union salvo fired off by George W. Bush's Republicans in 2003. American workers continue to pay the price.
In its essence, the bill is an act of contempt for unions, brought in at the behest of employer associations and well-funded right-wing think tanks. Bill C-377 imposes heavy reporting obligations on unions under the pretext that workers are entitled to a tax credit for their union dues. Yet no one proposes such obligations for employer associations and right-wing think tanks -- which enjoy the tax advantages of charitable organization status!
By getting an obscure backbencher to propose the bill in order to duck the normal constitutional and charter validation process, the Conservatives showed their contempt for Canada's parliamentary institutions. The SPGQ in fact shed light before the Senate Committee on the key role of Mr. Harper's office in the affair. By scandalously underestimating the exorbitant cost of the monitoring system forced on the Canada Revenue Agency, as the parliamentary budget officer has revealed, the Conservatives showed their contempt for taxpayers, even as they continued to proclaim their bottom-line commitment to taxpayer interests.
By sweeping aside the near-unanimous opinions of constitutionalists, the Canadian Bar Association, and the Quebec Bar, all of whom have judged the bill unconstitutional and illegal, Mr. Harper's office clearly showed its contempt for the Canadian legal community. By ignoring the seven provinces representing over 80 per cent of Canada's population--including Ontario, Quebec, and Alberta--who oppose Bill C-377, it showed its contempt for the governments of those provinces and the people who elected them.
By proroguing parliament in summer 2013 and so making the amendments proposed by a conservative senator (Hugh Segal, former chief of staff under Brian Mulroney) disappear in order to reintroduce the bill in its original form and ram it through by packing the Senate with partisan Conservative nominees, Mr. Harper demonstrated his profound contempt for that upper chamber he claimed such respect for not long ago.
And as the final desecration of democracy, they changed the rules of the Senate, which involved quashing a decision made by the speaker of the Senate -- a Conservative! And by putting a gag order on debate and allowing Bill C-377 to pass at the last moment, just before Canada Day and the start of summer vacation when attention is focused elsewhere, Stephen Harper's Conservatives consummated their act of contempt for workers and those who defend their rights.
We must not stand idly by in the face of this sneaky, underhanded, malicious, illegal, and -- to call a spade a spade -- contemptible attack on our democratic values.
Richard Perron
SPGQ President
MORE ON HUFFPOST:
5 Recent Supreme Court Decisions That Went Against The Harper Government
bill C-377harperPoliticsrichard perronspgq


Productivity is a critical part of any business. A website report that is fast to build and simple to review improves productivity. A report that is complex and time consuming will deplete a critical chunk of time.
I recently found myself with a report that had evolved into a massive document with plenty of great information. Unfortunately, it had so much information that I started having trouble juggling all of the priorities it was telling me to pursue. The report, which I updated weekly, also started taking more and more time to put together.
When it became clear it was unwieldy, I deleted the trailing 13 weeks of entries that I was tracking for many dozens of data points on nearly 15 pages of material. Instead, I now one have column for all of 2017, one column for the most recent quarter, one for the previous full month and one for the last 30 days. I also combined some pages and deleted quite a few secondary rows of data.
The leaner report now focuses on the most important priorities. It saves me time because it is quicker to put together. It also allows me to look more closely at trends by comparing the last 30 days to the previous month, quarter and year instead of just 13 individual weeks that jump around in results.
If the comparison shows a downward trend, I know I have a problem to address. If it shows an upward trend, I can look for a cause and try to duplicate the success elsewhere. The new report is better in multiple ways.
My way of analytic reporting is not the only way or the best way. Rather, it's the best way for my business and how I track results.
Some businesses may use something a lot simpler or they may want to update the report once a month instead of once a week. Others with larger staffs involved in the website may use even more information and parse it out to multiple employees.
Regardless, the digital environment keeps changing quickly, and reporting should evolve with the changing environment, the needs of the business and a basic commitment to continuous improvement.
Never changing at all is a mistake. Any change is positive as long as a benefit is provable. Constantly thinking about ways to make it better is always a good thing to do.


Description: The Copystar CS-750c Color Multifunctional System encompasses the consistent high image quality, robust performance and unsurpassed reliability that fast-paced business environments demand. Designed to become an essential partner to your customers' operation where color documents play a critical role, the CS 750c features a sleek design on the outside and innovative imaging technology on the inside. At highly productive output speeds of up to 75 ppm black and 65 ppm color the powerful combination of user-friendly operation, flexible paper handling and advanced product configuration assures superior document output. That is the confidence of knowing that your customers' document imaging needs are successfully achieved, the first time, and every time. That's the Copystar CS-750c.


Saint Michael The Archangel, Patron of Police Officers and Soldiers.
The ONLY Police Council in the Knights of Columbus and open to ALL Practical/Practing Catholic Gentlemen.
This (the Original council) website is my way of showing how Proud I am of being a 4th Degree Member of the Knights of Columbus and Council 12173 St. Michael the Archangel Council, Patron of Police Officers and Soldiers. The ONLY Police council of its kind for Practical/Practicing Catholic Gentlemen who participate in CHARITY, UNITY, FRATERNITY and PATRIOTISM.
The doors are open to ALL Law Enforcement Communities.
Including: Active and Retired Officers, All Departments throughout the Chicagoland area and the U.S., Law Enforcement Agencies, Bureaus and Military members Active and Retired.
Meetings are the 4th Wednesday or the month at the FOP Hall, 1412 W. Washington Blvd. in Chicago at 1900 hrs.
Knights are Catholic men, 18 years of age and older, who are committed to making their community a better place while supporting their Church. Being a Knight is more than camaraderie; it is being involved with your community; it is supporting your local Catholic Church while enhancing your own faith; it is about protecting and enhancing your family life.
Our eight-page Overview Booklet (pdf, 415k) gives additional information about what it means to be a member of the Knights of Columbus.
© 2016- 2019 Former District Deputy Sir Knight Paul Piraino - All Rights Reserved.