Structural and functional glycosphingolipidomics by glycoblotting with aminooxy-functionalized gold nanoparticle

Glycosphingolipids (GSLs) synthesized in Golgi apparatus by sequential transfer of sugar residues to a ceramide lipid anchor are ubiquitously distributing on vertebrate plasma membranes. Standardized method allowing for high throughput structural profiling and functional characterization of living cell surface GSLs is of growing importance because they function as crucial signal transduction molecules in various processes of dynamic cellular recognitions. However, methods are not available for amplification of GSLs, while the genomic scale PCR amplification permits large-scale mammalian proteomic analysis.　Here we communicate such an approach to a novel "omics", namely glycosphingolipidomics based on the glycoblotting method. The method, which involves selective ozonolysis of the C-C double bond in ceramide moiety and subsequent enrichment of generated GSL-aldehydes by chemical ligation using aminooxy-functionalized gold nanoparticle (aoGNP) should be of widespread utility for identifying and characterizing whole GSLs present in the living cell surfaces. The present protocol using glycoblotting permitted MALDI-TOFMS-based high throughput structural profiling of mouse brain gangliosides such as GM1, GD1a/GD1b, and GT1b for adult or GD3 in case for embryonic mouse. When mouse melanoma B16 cells were subjected to this protocol, it was demonstrated that gangliosides enriched from the plasma membranes are only GM3 bearing microheteogeneity in the structure of N-acyl chain. Surface plasmon resonance analysis revealed that aoGNP displaying whole GSLs blotted from mouse B16 melanoma cell surfaces can be used directly for monitoring specific interaction with self-assembled monolayer (SAM) of Gg3Cer (gangliotriaosylceramide). Our results indicate that GSL-selective enrichment onto aoGNP from living cell surfaces allows for rapid reconstruction of plasma membrane models mimicking intact GSL-microdomain feasible for further structural and functional characterization

A new landscape of host–protozoa interactions involving the extracellular vesicles world

This version is free to view and download for private research and study only. Not for re-distribution, re-sale or use in derivative works. © Cambridge University Press 2018Extracellular vesicles (EVs) are released by a wide number of cells including blood cells, immune system cells, tumour cells, adult and embryonic stem cells. EVs are a heterogeneous group of vesicles (~30–1000 nm) including microvesicles and exosomes. The physiological release of EVs represents a normal state of the cell, raising a metabolic equilibrium between catabolic and anabolic processes. Moreover, when the cells are submitted to stress with different inducers or in pathological situations (malignancies, chronic diseases, infectious diseases.), they respond with an intense and dynamic release of EVs. The EVs released from stimulated cells vs those that are released constitutively may themselves differ, both physically and in their cargo. EVs contain protein, lipids, nucleic acids and biomolecules that can alter cell phenotypes or modulate neighbouring cells. In this review, we have summarized findings involving EVs in certain protozoan diseases. We have commented on strategies to study the communicative roles of EVs during host–pathogen interaction and hypothesized on the use of EVs for diagnostic, preventative and therapeutic purposes in infectious diseases. This kind of communication could modulate the innate immune system and reformulate concepts in parasitism. Moreover, the information provided within EVs could produce alternatives in translational medicine.Peer reviewedFinal Accepted Versio

Aqueous Amino Acids and Proteins Near the Surface of Gold in Hydrophilic and Hydrophobic Force Fields

We calculate potentials of the mean force for twenty amino acids in the vicinity of the (111) surface of gold, for several dipeptides, and for some analogs of the side chains, using molecular dynamics simulations and the umbrella sampling method. We compare results obtained within three different force fields: one hydrophobic (for a contaminated surface) and two hydrophilic. All of these fields lead to good binding with very different specificities and different patterns in the density and polarization of water. The covalent bond with the sulfur atom on cysteine is modeled by the Morse potential. We demonstrate that binding energies of dipeptides are different than the combined binding energies of their amino-acidic components. For the hydrophobic gold, adsorption events of a small protein are driven by attraction to the strongest binding amino acids. This is not so in the hydrophilic cases - a result of smaller specificities combined with the difficulty for proteins, but not for single amino acids, to penetrate the first layer of water. The properties of water near the surface sensitively depend on the force field

EPMA position paper in cancer:current overview and future perspectives

At present, a radical shift in cancer treatment is occurring in terms of predictive, preventive, and personalized medicine (PPPM). Individual patients will participate in more aspects of their healthcare. During the development of PPPM, many rapid, specific, and sensitive new methods for earlier detection of cancer will result in more efficient management of the patient and hence a better quality of life. Coordination of the various activities among different healthcare professionals in primary, secondary, and tertiary care requires well-defined competencies, implementation of training and educational programs, sharing of data, and harmonized guidelines. In this position paper, the current knowledge to understand cancer predisposition and risk factors, the cellular biology of cancer, predictive markers and treatment outcome, the improvement in technologies in screening and diagnosis, and provision of better drug development solutions are discussed in the context of a better implementation of personalized medicine. Recognition of the major risk factors for cancer initiation is the key for preventive strategies (EPMA J. 4(1):6, 2013). Of interest, cancer predisposing syndromes in particular the monogenic subtypes that lead to cancer progression are well defined and one should focus on implementation strategies to identify individuals at risk to allow preventive measures and early screening/diagnosis. Implementation of such measures is disturbed by improper use of the data, with breach of data protection as one of the risks to be heavily controlled. Population screening requires in depth cost-benefit analysis to justify healthcare costs, and the parameters screened should provide information that allow an actionable and deliverable solution, for better healthcare provision

Contribution of galectin-1, a glycan-binding protein, to gastrointestinal tumor progression

Gastrointestinal cancer is a group of tumors that affect multiple sites of the digestive system, including the stomach, liver, colon and pancreas. These cancers are very aggressive and rapidly metastasize, thus identifying effective targets is crucial for treatment. Galectin-1 (Gal-1) belongs to a family of glycan-binding proteins, or lectins, with the ability to cross-link specific glycoconjugates. A variety of biological activities have been attributed to Gal-1 at different steps of tumor progression. Herein, we summarize the current literature regarding the roles of Gal-1 in gastrointestinal malignancies. Accumulating evidence shows that Gal-1 is drastically up-regulated in human gastric cancer, hepatocellular carcinoma, colorectal cancer and pancreatic ductal adenocarcinoma tissues, both in tumor epithelial and tumor-associated stromal cells. Moreover, Gal-1 makes a crucial contribution to the pathogenesis of gastrointestinal malignancies, favoring tumor development, aggressiveness, metastasis, immunosuppression and angiogenesis. We also highlight that alterations in Gal-1-specific glycoepitopes may be relevant for gastrointestinal cancer progression. Despite the findings obtained so far, further functional studies are still required. Elucidating the precise molecular mechanisms modulated by Gal-1 underlying gastrointestinal tumor progression, might lead to the development of novel Gal-1-based diagnostic methods and/or therapies.Fil: Bacigalupo, Maria Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas ; ArgentinaFil: Carabias, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas ; ArgentinaFil: Troncoso, María Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas ; Argentin

Targeting Somatostatin Receptors with Peptide-functionalized Silica Nanoparticles

ABSTRACT Nano-sized synthetic drug carriers comprise a valuable addition to oncology armaments, opening new avenues for improved delivery of anticancer therapies. Nanocarriers designed to bind surface receptors of tumor cells by virtue of bearing cognate high affinity ligands, also called as actively-targeted nanocarriers, have drawn a lot of attention during the last decade, promising to enable selective tumor accumulation via ligand-receptor interactions. The present work encompasses development and early in vitro evaluation efforts with a particular type of such a targeted nanocarrier [nanoparticles of mesoporous silica (SiO2), MSN], functionalized with short peptide ligands of somatostatin receptors (SSTR), frequently abundant in tumors. The synthesized targeted MSN, as well as control inactive peptide–functionalized counterparts, were characterized by physicochemical means and evaluated for their ability to bind to SSTR and enter living cells in vitro. We sequentially studied MSN in protein-depleted and serumenriched media – the latter condition involved adsorption of proteins to MSN surface (formation of the so-called protein corona), which invariably happens in vivo and affects cellular interactions of nanomaterials. We demonstrate that the targeted MSN can bind SSTR not only under the rectified conditions, but also in the presence of protein adsorption. Ultimately, we show that SSTR targeting leads to a decreased cellular accumulation of MSN with protein corona, which highlights the complexity of nanobiointeractions and urges to re-consider the applicability of the conventional model of tumor receptor targeting with nanocarriers to somatostatin receptors. The work covers a number of chemical (MSN design, synthesis and surface functionalization) and molecular biology (in vitro targetability evaluation and generation of relevant tools, receptor signaling, endocytosis) aspects of high relevance for development and characterization of targeted nanocarriers and as such should be of interest to the broad community working with nanomedicines and targeted delivery. The discussion of the original experimental evidence is preceded by a critical literature review, introducing the current concepts of nanocarriermediated delivery in oncology. KEYWORDS: nanocarrier, tumor receptor targeting, ligand-receptor interaction, nanoparticle corona, mesoporous silica nanoparticle, somatostatin receptor, cAMP.TIIVISTELMÄ Nanokokoiset lääkkeiden kantajat avaavat uusia keinoja syöpähoitojen parantamiseksi. Nanokantajat, joiden pinnalla olevat korkean affiniteetin ligandit ovat suunniteltu sitomaan tuumorisolujen pintareseptoreita (ns. aktiivisesti kohdennetut nanokantajat), mahdollistavat nanohiukkasten selektiivisen kertymisen syöpäkasvaimeen ligandi-reseptori-vuorovaikutusten kautta. Tämä työ kattaa kehityksen ja varhaiset in vitro-kokeet kohdennetulla nanokantajahiukkasella [mesohuokoisen piidioksidin (SiO2) nanohiukkaset, MSN], jotka on funktionalisoitu somatostatiinireseptorien lyhyillä peptidiligandeilla (SSTR), joita esiintyy usein runsaasti kasvaimissa. Syntetisoidut kohdennetut MSN-hiukkaset sekä kontrollina käytetyt inaktiiviset peptidifunktionaaliset vastineet karakterisoitiin fysikaaliskemiallisilla menetelmillä sekä arvioitiin niiden kykyä sitoutua SSTR:ään ja päästä eläviin soluihin in vitro. Tutkimus suoritettiin MSN-hiukkasilla sekä proteiinipitoisessa että seerumilla rikastetussa ravintoliuoksessa – jälkimmäisessä tapauksessa proteiinit kiinnittyivät MSN:n pinnalle (ns. proteiinikoronan muodostuminen), joka tapahtuu in vivo ja vaikuttaa nanomateriaalin ja solun väliseen vuorovaikutukseen. Osoitamme, että kohdennettu MSN voi sitoa SSTR:ää paitsi puhdistetuissa olosuhteissa, myös proteiinikoronan läsnäollessa. Lopuksi osoitamme, että SSTR-kohdennuksessa proteiinikorona vähentää MSN:n kertymistä soluihin, mikä korostaa nanobiovuorovaikutusten monimutkaisuutta, ja kyseenalaistaa nanokantajien kanssa käytetyn tavanomaisen kasvainreseptorikohdennusmallin soveltuvuutta somatostatiinireseptoreihin. Työssä käytettiin kemiallisia (MSN-suunnittelu, synteesi ja pinnan funktionalisointi) ja molekyylibiologisia (kohdennettavuuden arviointi in vitro, työkalujen luominen, reseptorisignalointi ja endosytoosi) menetelmiä, jotka ovat olennaisia kohdennettujen nanokantajien kehitykselle ja karakterisoinnille. Tulosten oletetaan kiinnostavan laajaa yhteisöä, joka työskentelee nanolääkkeiden ja niiden kohdentamisen parissa. Kokeellista osuutta edeltää kirjallisuuskatsaus, jossa esitellään nanokantaja-välitteisen kuljetuksen nykyiset käsitteet syöpäbiologiassa. AVAINSANAT: nanokantaja, kasvainreseptorikohdistus, ligandi-reseptorivuorovaikutus, nanohiukkasen korona, mesohuokoinen piidioksidi-nanopartikkeli, somatostatiinireseptori, cAMP

Ultrasensitive Label-Free Nanosensing and High-Speed Tracking of Single Proteins

: Label-free detection, analysis, and rapid tracking of nanoparticles is crucial for future ultrasensitive sensing applications, ranging from understanding of biological interactions to the study of size-dependent classical-quantum transitions. Yet optical techniques to distinguish nanoparticles directly among their background remain challenging. Here we present amplified interferometric scattering microscopy (aiSCAT) as a new all-optical method capable of detecting individual nanoparticles as small as 15 kDa proteins that is equivalent to half a GFP. By balancing scattering and reflection amplitudes the interference contrast of the nanoparticle signal is amplified 1 to 2 orders of magnitude. Beyond high sensitivity, a-iSCAT allows high-speed image acquisition exceeding several hundreds of frames-per-second. We showcase the performance of our approach by detecting single Streptavidin binding events and by tracking single Ferritin proteins at 400 frames-per-second with 12 nm localization precision over seconds. Moreover, due to its extremely simple experimental realization, this advancement finally enables a cheap and routine implementation of label-free all-optical single nanoparticle detection platforms with sensitivity operating at the single protein level.Peer ReviewedPostprint (author's final draft

Nanopipettes as Monitoring Probes for the Single Living Cell: State of the Art and Future Directions in Molecular Biology.

Examining the behavior of a single cell within its natural environment is valuable for understanding both the biological processes that control the function of cells and how injury or disease lead to pathological change of their function. Single-cell analysis can reveal information regarding the causes of genetic changes, and it can contribute to studies on the molecular basis of cell transformation and proliferation. By contrast, whole tissue biopsies can only yield information on a statistical average of several processes occurring in a population of different cells. Electrowetting within a nanopipette provides a nanobiopsy platform for the extraction of cellular material from single living cells. Additionally, functionalized nanopipette sensing probes can differentiate analytes based on their size, shape or charge density, making the technology uniquely suited to sensing changes in single-cell dynamics. In this review, we highlight the potential of nanopipette technology as a non-destructive analytical tool to monitor single living cells, with particular attention to integration into applications in molecular biology

Nitric Oxide-Releasing Nanoparticles Prevent Propionibacterium acnes-Induced Inflammation by Both Clearing the Organism and Inhibiting Microbial Stimulation of the Innate Immune Response.

Propionibacterium acnes induction of IL-1 cytokines through the NLRP3 (NLR, nucleotide oligomerization domain-like receptor) inflammasome was recently highlighted as a dominant etiological factor for acne vulgaris. Therefore, therapeutics targeting both the stimulus and the cascade would be ideal. Nitric oxide (NO), a potent biological messenger, has documented broad-spectrum antimicrobial and immunomodulatory properties. To harness these characteristics to target acne, we used an established nanotechnology capable of generating/releasing NO over time (NO-np). P. acnes was found to be highly sensitive to all concentrations of NO-np tested, although human keratinocyte, monocyte, and embryonic zebra fish assays revealed no cytotoxicity. NO-np significantly suppressed IL-1β, tumor necrosis factor-α (TNF-α), IL-8, and IL-6 from human monocytes, and IL-8 and IL-6 from human keratinocytes, respectively. Importantly, silencing of NLRP3 expression by small interfering RNA did not limit NO-np inhibition of IL-1 β secretion from monocytes, and neither TNF-α nor IL-6 secretion, nor inhibition by NO-np was found to be dependent on this pathway. The observed mechanism by which NO-np impacts IL-1β secretion was through inhibition of caspase-1 and IL-1β gene expression. Together, these data suggest that NO-np can effectively prevent P. acnes-induced inflammation by both clearing the organism and inhibiting microbial stimulation of the innate immune response