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Background: Calcitonin gene-related peptide (CGRP), implicated in migraine pain, also possesses bone anabolic properties, which leads to the possibility that monoclonal antibodies targeting CGRP (anti-CGRPs) might increase the risk of bone density abnormalities., Objective: The objective of this study was to explore bone mineral density abnormalities in a cohort of migraine patients treated with anti-CGRPs., Methods: This was a single-center, cross-sectional, cohort study including migraine patients who underwent a densitometry assessment during anti-CGRP treatment. We assessed the frequency of osteopenia or osteoporosis (OSTEO+ status), defined as a bone mineral density T-score of -1 to -2.5, and <-2.5 standard deviations from the young female adult mean, respectively. Additionally, the association of OSTEO+ status with anti-CGRP treatment duration and primary osteoporosis' risk factors was investigated using logistic regression models., Results: Data from 51 patients (43 female, mean age 46 ± 13.9 years) were evaluated. The mean duration of anti-CGRP treatment was 15.7 (±11.8) months. Twenty-seven patients (53%) were OSTEO+ (n = 22 osteopenia; n = 5 osteoporosis). In the final model, menopause [odds ratio 11.641 (95% confidence interval 1.486-91.197), p = 0.019] and anti-seizure drug use [odds ratio 12.825 (95% confidence interval 1.162-141.569), p = 0.037] were associated with OSTEO+ status., Conclusions: In our cohort of migraine patients, no evidence of an association between anti-CGRP treatment duration and an increasing risk of bone mineral density abnormalities was found. However, these findings are preliminary and necessitate further longitudinal research with larger cohorts and extended follow-up to be validated., (© 2024. The Author(s).)

Broadly neutralizing antibodies (bNAbs) are promising candidates for the treatment and prevention of HIV-1 infections. Despite their critical importance, automatic detection of HIV-1 bNAbs from immune repertoires is still lacking. Here, we develop a straightforward computational method for the Rapid Automatic Identification of bNAbs (RAIN) based on machine learning methods. In contrast to other approaches, which use one-hot encoding amino acid sequences or structural alignment for prediction, RAIN uses a combination of selected sequence-based features for the accurate prediction of HIV-1 bNAbs. We demonstrate the performance of our approach on non-biased, experimentally obtained and sequenced BCR repertoires from HIV-1 immune donors. RAIN processing leads to the successful identification of distinct HIV-1 bNAbs targeting the CD4-binding site of the envelope glycoprotein. In addition, we validate the identified bNAbs using an in vitro neutralization assay and we solve the structure of one of them in complex with the soluble native-like heterotrimeric envelope glycoprotein by single-particle cryo-electron microscopy (cryo-EM). Overall, we propose a method to facilitate and accelerate HIV-1 bNAbs discovery from non-selected immune repertoires., (© 2024. The Author(s).)

Here we present a method to extract thermodynamic quantities for nanoparticle dispersions in solvents. The method is based on the study of tomograms obtained from cryogenic electron tomography (cryoET). The approach is demonstrated for gold nanoparticles (diameter < 5 nm). Tomograms are reconstructed from tilt-series 2D images. Once the three-dimensional (3D) coordinates for the centres of mass of all of the particles in the sample are determined, we calculate the pair distribution function g ( r ) and the potential of mean force U ( r ) without any assumption. Importantly, we show that further quantitative information from 3D tomograms is readily available as the spatial fluctuation in the particles' position can be efficiently determined. This in turn allows for the prompt derivation of the Kirkwood-Buff integrals with all their associated quantities such as the second virial coefficient. Finally, the structure factor and the agglomeration states of the particles are evaluated directly. These thermodynamic quantities provide key insights into the dispersion properties of the particles. The method works well both for dispersed systems containing isolated particles and for systems with varying degrees of agglomerations.

Control of the ongoing SARS-CoV-2 pandemic is endangered by the emergence of viral variants with increased transmission efficiency, resistance to marketed therapeutic antibodies, and reduced sensitivity to vaccine-induced immunity. Here, we screen B cells from COVID-19 donors and identify P5C3, a highly potent and broadly neutralizing monoclonal antibody with picomolar neutralizing activity against all SARS-CoV-2 variants of concern (VOCs) identified to date. Structural characterization of P5C3 Fab in complex with the spike demonstrates a neutralizing activity defined by a large buried surface area, highly overlapping with the receptor-binding domain (RBD) surface necessary for ACE2 interaction. We further demonstrate that P5C3 shows complete prophylactic protection in the SARS-CoV-2-infected hamster challenge model. These results indicate that P5C3 opens exciting perspectives either as a prophylactic agent in immunocompromised individuals with poor response to vaccination or as combination therapy in SARS-CoV-2-infected individuals., Competing Interests: Declaration of interests C.F., P.T., G.P., and D.T declare that they are co-inventors on a patent application titled “Neutralizing antibodies and use thereof in the treatment of SARS-CoV-2 infection” with filing number PCT/IB2021/050621 that covers newly identified antibodies described in this manuscript. The remaining authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Human cytomegalovirus (HCMV) is the primary viral cause of congenital birth defects and causes significant morbidity and mortality in immune-suppressed transplant recipients. Despite considerable efforts in vaccine development, HCMV infection still represents an unmet clinical need. In recent phase II trials, a MF59-adjuvanted gB vaccine showed only modest efficacy in preventing infection. These findings might be attributed to low level of antibodies (Abs) with a neutralizing activity induced by this vaccine. Here, we analyzed the immunogenicity of each gB antigenic domain (AD) and demonstrated that domain I of gB (AD5) is the main target of HCMV neutralizing antibodies. Furthermore, we designed, characterized and evaluated immunogenic responses to two different nanoparticles displaying a trimeric AD5 antigen. We showed that mice immunization with nanoparticles induces sera neutralization titers up to 100-fold higher compared to those obtained with the gB extracellular domain (gBECD). Collectively, these results illustrate with a medically relevant example the advantages of using a general approach combining antigen discovery, protein engineering and scaffold presentation for modern development of subunit vaccines against complex pathogens., Competing Interests: The authors have declared that no competing interests exist.

Centrioles are polarized microtubule-based organelles that seed the formation of cilia, and which assemble from a cartwheel containing stacked ring oligomers of SAS-6 proteins. A cryo-tomography map of centrioles from the termite flagellate Trichonympha spp. was obtained previously, but higher resolution analysis is likely to reveal novel features. Using sub-tomogram averaging (STA) in T. spp. and Trichonympha agilis, we delineate the architecture of centriolar microtubules, pinhead, and A-C linker. Moreover, we report ~25 Å resolution maps of the central cartwheel, revealing notably polarized cartwheel inner densities (CID). Furthermore, STA of centrioles from the distant flagellate Teranympha mirabilis uncovers similar cartwheel architecture and a distinct filamentous CID. Fitting the CrSAS-6 crystal structure into the flagellate maps and analyzing cartwheels generated in vitro indicate that SAS-6 rings can directly stack onto one another in two alternating configurations: with a slight rotational offset and in register. Overall, improved STA maps in three flagellates enabled us to unravel novel architectural features, including of centriole polarity and cartwheel stacking, thus setting the stage for an accelerated elucidation of underlying assembly mechanisms., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

KAP1 (KRAB domain-associated protein 1) plays a fundamental role in regulating gene expression in mammalian cells by recruiting different transcription factors and altering the chromatin state. In doing so, KAP1 acts both as a platform for macromolecular interactions and as an E3 small ubiquitin modifier ligase. This work sheds light on the overall organization of the full-length protein combining solution scattering data, integrative modeling, and single-molecule experiments. We show that KAP1 is an elongated antiparallel dimer with an asymmetry at the C-terminal domains. This conformation is consistent with the finding that the Really Interesting New Gene (RING) domain contributes to KAP1 auto-SUMOylation. Importantly, this intrinsic asymmetry has key functional implications for the KAP1 network of interactions, as the heterochromatin protein 1 (HP1) occupies only one of the two putative HP1 binding sites on the KAP1 dimer, resulting in an unexpected stoichiometry, even in the context of chromatin fibers., (© 2019 Fonti et al.)

Liquid biopsies allow monitoring of cancer progression and detection of relapse, but reliable biomarkers in melanoma are lacking. Because secreted factors preferentially drain to lymphatic vessels before dilution in the blood, we hypothesized that lymph should be vastly enriched in cancer biomarkers. We characterized postoperative lymphatic exudate and plasma of metastatic melanoma patients after lymphadenectomy and found a dramatic enrichment in lymphatic exudate of tumor-derived factors and especially extracellular vesicles containing melanoma-associated proteins and miRNAs, with unique protein signatures reflecting early versus advanced metastatic spread. Furthermore, lymphatic exudate was enriched in memory T cells, including tumor-reactive CD137 + and stem cell-like types. In mice, lymph vessels were the major route of extracellular vesicle transport from tumors to the systemic circulation. We suggest that lymphatic exudate provides a rich source of tumor-derived factors for enabling the discovery of novel biomarkers that may reflect disease stage and therapeutic response., (© 2019 Broggi et al.)

The development of synthetic nanomaterials that could embed within, penetrate, or induce fusion between membranes without permanent disruption would have great significance for biomedical applications. Here we describe structure-function relationships of highly water-soluble gold nanoparticles comprised of an ∼1.5-5 nm diameter metal core coated by an amphiphilic organic ligand shell, which exhibit membrane embedding and fusion activity mediated by the surface ligands. Using an environment-sensitive dye anchored within the ligand shell as a sensor of membrane embedding, we demonstrate that particles with core sizes of ∼2-3 nm are capable of embedding within and penetrating fluid bilayers. At the nanoscale, these particles also promote spontaneous fusion of liposomes or spontaneously embed within intact liposomal vesicles. These studies provide nanoparticle design and selection principles that could be used in drug delivery applications, as membrane stains, or for the creation of novel organic/inorganic nanomaterial self-assemblies.

Gellan nanohydrogel and phospholipid vesicles were combined to incorporate baicalin in new self-assembling core-shell gellan-transfersomes obtained by an easy, scalable method. The vesicles were small in size (~107 nm) and monodispersed (P.I. ≤ 0.24), forming a viscous system (~24 mPa/s) as compared to transfersomes (~1.6 mPa/s), as confirmed by rheological studies. Gellan was anchored to the bilayer domains through cholesterol, and the polymer chains were distributed onto the outer surface of the bilayer, thus forming a core-shell structure, as suggested by SAXS analyses. The optimal carrier ability of core-shell gellan-transfersomes was established by the high deposition of baicalin in the skin (~11% in the whole skin), especially in the deeper tissue (~8% in the dermis). Moreover, their ability to improve baicalin efficacy in anti-inflammatory and skin repair tests was confirmed in vivo in mice, providing the complete skin restoration and inhibiting all the studied inflammatory markers., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The target of rapamycin (TOR) is a eukaryotic serine/threonine protein kinase that functions in two distinct complexes, TORC1 and TORC2, to regulate growth and metabolism. GTPases, responding to signals generated by abiotic stressors, nutrients, and, in metazoans, growth factors, play an important but poorly understood role in TORC1 regulation. Here we report that, in budding yeast, glucose withdrawal (which leads to an acute loss of TORC1 kinase activity) triggers a similarly rapid Rag GTPase-dependent redistribution of TORC1 from being semi-uniform around the vacuolar membrane to a single, vacuole-associated cylindrical structure visible by super-resolution optical microscopy. Three-dimensional reconstructions of cryo-electron micrograph images of these purified cylinders demonstrate that TORC1 oligomerizes into a higher-level hollow helical assembly, which we name a TOROID (TORC1 organized in inhibited domain). Fitting of the recently described mammalian TORC1 structure into our helical map reveals that oligomerization leads to steric occlusion of the active site. Guided by the implications from our reconstruction, we present a TOR1 allele that prevents both TOROID formation and TORC1 inactivation in response to glucose withdrawal, demonstrating that oligomerization is necessary for TORC1 inactivation. Our results reveal a novel mechanism by which Rag GTPases regulate TORC1 activity and suggest that the reversible assembly and/or disassembly of higher-level structures may be an underappreciated mechanism for the regulation of protein kinases.

Reduced taste sensitivity to 6-n-propylthiouracil (PROP), a genetic trait regarded as a general index for oral chemosensory perception, has been associated with a calorie-rich food preference and lower circulating endocannabinoid levels in participants with normal weight (NW), which suggests an adaptive mechanism to maintain a lean phenotype. In this study, we assessed whether participants with obesity (OB) show different patterns of plasma endocannabinoids and lipid metabolism biomarkers from those of NW, with further categorization based on their PROP sensitivity. NW and OB were classified by their PROP taster status as non-tasters (NT), medium-tasters (MT) and supertasters (ST). The blood samples were analysed for plasma endocannabinoids, nonesterified fatty acids (NEFA) and retinol, which have been associated to metabolic syndrome. In OB, we found a higher BMI and lower circulating endocannabinoids in ST vs. OB NT. However, OB ST showed lower circulating NEFA and retinol levels, which suggested a more favourable lipid metabolism and body fat distribution than those of OB NT. We confirmed lower plasma endocannabinoid levels in NW NT than in NW ST. These data suggest that PROP taste sensitivity determines metabolic changes and ultimately body mass composition differently in OB and NW.

In this study we report on the interactions between the water-soluble fraction (WSF) of two municipal solid waste composts (C1- and C2-WSF) with Pb(II) and Cu(II) ions at pH 4.5. The Me(II) addition to the compost-WSFs led to the formation of soluble Me(II)-organic complexes (as highlighted by FT-IR spectroscopy), and to a decrease of the trace metals' solubility, which was greater for Pb(II) than Cu(II). This was due to the formation of insoluble Me(II) complexes involving the water-soluble organic carbon (WSOC) and the inorganic anions within both WSFs [1.10 and 0.62 mmol L -1 and 2.06 and 0.42 mmol L -1 of Pb(II) and Cu(II) precipitated from C1- and C2-WSF respectively, when 6.4 mmol L -1 Me(II) was added]. A loss of WSOC from both WSFs, i.e. ∼13% and <5%, was detected in the systems containing 6.4 mmol L -1 Pb(II) and Cu(II) respectively. A significant contribution in the formation of Pb(II) precipitates was also due to phosphate, chloride and sulphate anions, since their concentrations in the WSF decreased of 80, 25 and 90%, respectively, after the addition of 6.4 mmol L -1 Pb(II). A decrease of phosphate anions in both WSFs (∼30%) was found in the systems containing Cu(II)., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The target autoantigens in several organ-specific autoimmune diseases, including type 1 diabetes (T1D), are intracellular membrane proteins, whose initial encounter with the immune system is poorly understood. Here we propose a new model for how these proteins can initiate autoimmunity. We found that rat and human pancreatic islets release the intracellular β-cell autoantigens in human T1D, GAD65, IA-2, and proinsulin in exosomes, which are taken up by and activate dendritic cells. Accordingly, the anchoring of GAD65 to exosome-mimetic liposomes strongly boosted antigen presentation and T-cell activation in the context of the human T1D susceptibility haplotype HLA-DR4. Cytokine-induced endoplasmic reticulum stress enhanced exosome secretion by β-cells; induced exosomal release of the immunostimulatory chaperones calreticulin, Gp96, and ORP150; and increased exosomal stimulation of antigen-presenting cells. We propose that stress-induced exosomal release of intracellular autoantigens and immunostimulatory chaperones may play a role in the initiation of autoimmune responses in T1D., (© 2017 by the American Diabetes Association.)

The ability of two municipal solid waste composts (MSW-C) to sorb As(V) in the presence of Pb(II) and in acidic conditions was investigated. Sorption isotherms and kinetics showed that both MSW-C were able to sorb As(V) in a similar way (∼0.24mmolg -1 MSW-C), but only when Pb(II) was present (0.45mmolL -1 ). The concomitant sorption of Pb(II) by both MSW-C (∼0.40mmolg -1 ) suggested that the metal cation was likely acting as bridging element between the negatively charged functional groups of composts and As(V). SEM-EDX analysis of the MSW-C+Pb(II)+As(V) systems supported the association between Pb(II) and As(V), while sequential extraction procedures and organic acids treatment showed that As(V) was strongly retained by MSW-C+Pb(II) and suggested the presence of different interaction types between As(V) and Pb(II). Plant growth experiments highlighted the key role of Pb(II) in the reduction of As(V)-phytotoxicity for triticale plants (×Triticosecale Wittm.) in the presence of MSW-C., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Amyloid-beta (Aβ) peptide oligomerization plays a central role in the pathogenesis of Alzheimer's disease (AD), and Aβ oligomers are collectively considered an appealing therapeutic target for the treatment of AD. However, the molecular mechanisms leading to the pathologic accumulation of oligomers are unclear, and the exact structural composition of oligomers is being debated. Using targeted and quantitative mass spectrometry, we reveal site-specific Aβ autocleavage during the early phase of aggregation, producing a typical Aβ fragment signature and that truncated Aβ peptides can form stable oligomeric complexes with full-length Aβ peptide. We show that the use of novel anti-Aβ antibodies raised against these truncated Aβ isoforms allows for monitoring and targeting the accumulation of truncated Aβ fragments. Antibody-enabled screening of transgenic models of AD as well as human postmortem brain tissue and cerebrospinal fluid revealed that aggregation-associated Aβ cleavage is a highly relevant clinical feature of AD., (Copyright © 2016 The Alzheimer's Association. Published by Elsevier Inc. All rights reserved.)

New biocarriers, named santosomes, were formulated using Santolina insularis essential oil and hydrogenated phosphatidylcholine. They were modified by adding propylene glycol, a hydrophylic penetration enhancer, and loaded with phycocyanin, a protein found in cyanobacteria, which possesses antioxidant and antiinflammatory properties. The essential oil was expected to modify the bilayer structure and improve the delivery and efficacy of the protein due to a synergistic effect of the phospholipid and S. insularis terpenes. Santosomes were small in size (∼118nm), unilamellar and with polyhedral shape. SAXS patterns showed that phycocyanin strongly interacted with the polar heads of the vesicle bilayer. Phycocyanin-loaded vesicles did not show any toxic effect in vitro: cell viability was ∼100% in endothelial cells and ∼120% in keratinocytes, at all the concentrations tested. In addition, phycocyanin-loaded vesicles protected the cells against free radical damage. In vivo studies were performed to evaluate the ability of santosomes to inhibit chemically-induced oedema and inflammation in mice. Results demonstrated that the application of phycocyanin-loaded santosomes produced an evident amelioration of the skin lesion, confirming their great potential for wound healing., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Nanoscale carrier platforms promote immune responses to vaccination by facilitating delivery of vaccine components to immunologically relevant sites. The technique is particularly valuable for subunit vaccination, in which coadministration of immunostimulatory adjuvant is known to enhance immune responses to protein antigen. The fabrication of polymer-based nanoparticle vaccines commonly requires covalent attachment of vaccine components to the carrier surface. In contrast, we here describe a cationic micelle vaccination platform in which antigen and adjuvant loading is mediated by noncovalent molecular encapsulation and electrostatic complexation. Cationic micelles were generated through self-assembly of a polyarginine-conjugated poly(ethylene glycol)- b -poly(propylene sulfide) (PEG-PPS) diblock copolymer amphiphile, with or without encapsulation of monophosphoryl lipid A (MPLA), an amphiphilic experimental vaccine adjuvant. Micelle complexes were subsequently formed by complexation of ovalbumin (OVA) and CpG-B oligodeoxynucleotide (a second experimental adjuvant) to the cationic micelles. In a 35-day study in mouse, micelle-mediated codelivery of OVA antigen and CpG-B enhanced cellular and humoral responses to vaccination. These outcomes were highlighted in spleen and lymph node CD8 + T cells, with significantly increased populations of IFNγ + , TNFα + , and polyfunctional IFNγ + TNFα + cells. Elevated cytokine production is a hallmark of robust cytotoxic T lymphocyte (CTL) responses sought in next-generation vaccine technologies. Increased production of OVA-specific IgG1, IgG2c, and IgG3 also confirmed micelle enhancement of humoral responses. In a subsequent 35-day study, we explored micelle-mediated vaccination against OVA antigen coadministered with MPLA and CpG-B adjuvants. A synergistic effect of adjuvant coadministration was observed in micelle-free vaccination but not in groups immunized with micelle complexes. This outcome underlines the advantage of the micelle carrier: we achieved optimal cellular and humoral responses to vaccination by use of this nanoparticle platform with a single adjuvant. In particular, enhanced CTL responses support future development of the cationic micelle platform in experimental cancer vaccines and for vaccination against reticent viral pathogens.

Bulk and dispersed cubic liquid crystalline phases (cubosomes), present in the body and in living cell membranes, are believed to play an essential role in biological phenomena. Moreover, their biocompatibility is attractive for nutrient or drug delivery system applications. Here the three-dimensional organization of dispersed cubic lipid self-assembled phases is fully revealed by cryo-electron tomography and compared with simulated structures. It is demonstrated that the interior is constituted of a perfect bicontinuous cubic phase, while the outside shows interlamellar attachments, which represent a transition state between the liquid crystalline interior phase and the outside vesicular structure. Therefore, compositional gradients within cubosomes are inferred, with a lipid bilayer separating at least one water channel set from the external aqueous phase. This is crucial to understand and enhance controlled release of target molecules and calls for a revision of postulated transport mechanisms from cubosomes to the aqueous phase.

The aim of this work was to formulate Santolina insularis essential oil-loaded nanocarriers, namely Penetration Enhancer containing Vesicles (PEVs), evaluate the physico-chemical features and stability, and gain insights into their ability to deliver the oil to the skin. S. insularis essential oil was obtained by steam distillation, and was predominantly composed of terpenes, the most abundant being β-phellandrene (22.6%), myrcene (11.4%) and curcumenes (12.1%). Vesicles were prepared using phosphatidylcholine, and ethylene or propylene glycol were added to the water phase (10% (v/v)) to improve vesicle performances as delivery systems. Vesicles were deeply characterized by light scattering, cryogenic transmission electron microscopy and small/wide-angle X-ray scattering, the results showing polyhedral, faceted, unilamellar vesicles of ∼115 nm in size. The presence of the glycols improved vesicle stability under accelerated ageing conditions, without changes in size or migration phenomena (e.g. sedimentation and creaming). Confocal laser scanning microscopy images of pig skin treated with S. insularis formulations displayed a penetration ability of PEVs greater than that of control liposomes. Moreover, all formulations showed a marked in vitro biocompatibility in human keratinocytes. These findings suggest that the nanoformulation may be of value in enhancing the delivery of S. insularis essential oil to the skin, where it can exert its biological activities., (Copyright © 2015 Elsevier B.V. All rights reserved.)

With proper control over copolymer design and solvation conditions, self-assembled materials display impressive morphological variety that encompasses nanoscale colloids as well as bulk three-dimensional architectures. Here we take advantage of both hydrophobicity and crystallinity to mediate supramolecular self-assembly of spherical micellar, linear fibrillar, or hydrogel structures by a family of highly asymmetric poly(ethylene glycol)-b-oligo(ethylene sulfide) (PEG-OES) copolymers. Assembly structural polymorphism was achieved with modification of PEG-OES topology (linear versus multiarm) and with precise, monomer-by-monomer control of OES length. Notably, all three morphologies were accessed utilizing OES oligomers with degrees of polymerization as short as three. These exceptionally small assembly forming blocks represent the first application of ethylene sulfide oligomers in supramolecular materials. While the assemblies demonstrated robust aqueous stability over time, oxidation by hydrogen peroxide progressively converted ethylene sulfide residues to increasingly hydrophilic and amorphous sulfoxides and sulfones, causing morphological changes and permanent disassembly. We utilized complementary microscopic and spectroscopic techniques to confirm this chemical stimulus-responsive behavior in self-assembled PEG-OES colloidal dispersions and physical gels. In addition to inherent stimulus-responsive behavior, fibrillar assemblies demonstrated biologically relevant molecular delivery, as confirmed by the dose-dependent activation of murine bone marrow-derived dendritic cells following fibril-mediated delivery of the immunological adjuvant monophosphoryl lipid A. In physical gels composed of either linear or multiarm PEG-OES precursors, rheologic analysis also identified mechanical stimulus-responsive shear thinning behavior. Thanks to the facile preparation, user-defined morphology, aqueous stability, carrier functionality, and stimuli-responsive behaviors of PEG-OES supramolecular assemblies, our findings support a future role for these materials as injectable or implantable biomaterials.

We report on a generic method to detect and identify the molecular profile of exosomes either derived from cultured cell lines or isolated from biofluids. Exosomes are nanovesicles shed by cells into their microenvironment and carry the molecular identity of their mother cells. These vesicles are actively involved in intercellular communication under physiological conditions and ultimately in the spread of various diseases such as cancer. As they are accessible in most biofluids (e.g., blood, urine, or saliva), these biological entities are promising tools for cancer diagnostics, offering a non-invasive and remote access to the molecular state of the disease. The composition of exosomes derived from cancer cells depends on the sort and state of the tumor, requiring a screening of multiple antigens to fully characterize the disease. Here, we exploited the capacity of surface plasmon resonance biosensing to detect simultaneously multiple exosomal and cancer biomarkers on exosomes derived from breast cancer cells. We developed an immunosensor surface which provides efficient and specific capture of exosomes, together with their identification through their distinct molecular profiles. The successful analysis of blood samples demonstrated the suitability of our bioanalytical procedure for clinical use.

In this study, we investigated the ability of Fe- and Al-based water treatment residues (Fe- and Al-WTR) to accumulate Pb(II) and Cu(II) at pH 4.5. The role of the inorganic and organic fractions of WTRs in metals sorption was also assessed. Sorption isotherms showed a higher sorption of Pb(II) by both WTRs with respect to Cu(II) (e.g. 0.105 and 0.089 mmol g(-1) of Pb(II) and Cu(II) respectively sorbed by Fe-WTR). Fe-WTR revealed a stronger sorbent for both metals than Al-WTR. The amount of Pb(II) and Cu(II) sorbed by Fe-WTR was about the 69% and 63% higher than that sorbed by the Al-WTR. The organic matter of Fe- and Al-WTR contributed to about 26% and 8.5% respectively in the sorption of both metals. The sequential extraction procedure showed that the greatest amount of metals sorbed by both WTRs were tightly bound and not extractable, and this was particularly apparent for Cu(II). The FT-IR spectra indicated the formation of inner-sphere complexes between the Fe(Al)-O nucleus and Pb(II) and Cu(II). Moreover, the FT-IR spectra also suggested that the humic fraction of WTRs interacted, through the carboxylate groups, with Cu(II) and Pb(II) by forming mainly monodentate and bidentate complexes, respectively., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Cells secrete extracellular vesicles (EVs) into their microenvironment that act as mediators of intercellular communication under physiological conditions and in this context also actively participate in spreading various diseases. Large efforts are currently made to produce reliable EV samples and to develop, improve, and standardize techniques allowing their biophysical characterization. Here, we used ultrafiltration and size-exclusion chromatography for the isolation and a model-free fluorescence fluctuation analysis for the investigation of the physical and biological properties of EVs secreted by mammalian cells. Our purification strategy produced enriched samples of morphologically intact EVs free of extravesicular proteins and allowed labeling of marker molecules on the vesicle surface for single-vesicle analysis with single-molecule sensitivity. This novel approach provides information on the distribution profile of both EV size and relative expression level of a specific exosomal marker, deciphering the overall heterogeneity of EV preparations.

A major contemporary concern in developing effective liposome-nanoparticle hybrids is the present inclusion size limitation of nanoparticles between vesicle bilayers, which is considered to be around 6.5 nm in diameter. In this article, we present experimental observations backed by theoretical considerations which show that greater structures can be incorporated within vesicle membranes by promoting the clustering of nanoparticles before liposome formation. Cryo-transmission electron microscopy and cryo-electron tomography confirm these observations at unprecedented detail and underpin that the liposome membranes can accommodate flexible structures of up to 60 nm in size. These results imply that this material is more versatile in terms of inclusion capabilities and consequently widens the opportunities in developing multivalent vesicles for nanobiotechnology applications.

Background: Centrioles are cylindrical microtubule-based structures whose assembly is critical for the formation of cilia, flagella, and centrosomes. The centriole proximal region harbors a cartwheel that dictates the 9-fold symmetry of centrioles. Although the cartwheel architecture has been recently analyzed, how it connects to the peripheral microtubules is not understood. More generally, a high-resolution view of the proximal region of the centriole is lacking, thus limiting understanding of the underlying assembly mechanisms., Results: We report the complete architecture of the Trichonympha centriole proximal region using cryotomography. The resulting 3D map reveals several features, including additional densities in the cartwheel that exhibit a 9-fold symmetrical arrangement, as well as the structure of the Pinhead and the A-C linker that connect to microtubules. Moreover, we uncover striking chiral features that might impart directionality to the entire centriole. Furthermore, we identify Trichonympha SAS-6 and demonstrate that it localizes to the cartwheel in vivo., Conclusions: Our work provides unprecedented insight into the architecture of the centriole proximal region, which is key for a thorough understanding of the mechanisms governing centriole assembly., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The development of low-temperature carbonization procedures promises to provide novel nanostructured carbon materials that are of high current interest in materials science and technology. Here, we report a "wet-chemical" carbonization method that utilizes hexayne amphiphiles as metastable carbon precursors. Nearly perfect control of the nanoscopic morphology was achieved by self-assembly of the precursors into colloidal aggregates with tailored diameter in water. Subsequent carbonization furnished carbon nanocapsules with a carbon microstructure resembling graphite-like amorphous carbon materials.

In cells, DNA is routinely subjected to significant levels of bending and twisting. In some cases, such as under physiological levels of supercoiling, DNA can be so highly strained, that it transitions into non-canonical structural conformations that are capable of relieving mechanical stress within the template. DNA minicircles offer a robust model system to study stress-induced DNA structures. Using DNA minicircles on the order of 100 bp in size, we have been able to control the bending and torsional stresses within a looped DNA construct. Through a combination of cryo-EM image reconstructions, Bal31 sensitivity assays and Brownian dynamics simulations, we have been able to analyze the effects of biologically relevant underwinding-induced kinks in DNA on the overall shape of DNA minicircles. Our results indicate that strongly underwound DNA minicircles, which mimic the physical behavior of small regulatory DNA loops, minimize their free energy by undergoing sequential, cooperative kinking at two sites that are located about 180° apart along the periphery of the minicircle. This novel form of structural cooperativity in DNA demonstrates that bending strain can localize hyperflexible kinks within the DNA template, which in turn reduces the energetic cost to tightly loop DNA., (© The Author(s) 2011. Published by Oxford University Press.)

The 1-hexadecyl-3-vinylimidazolium bromide (hvimBr), a water-soluble long-chain imidazolium ionic liquid (IL) with surfactant properties, showed the ability to produce stable homogeneous aqueous dispersions of pristine Single-Walled Carbon Nanotubes (SWNTs). The purpose of this study is the improvement of SWNT dispersing ability by assessing the effect of different groups in position 3 of the imidazole ring. In this regard structural analogues were synthesized and, after characterization, their capability to dissolve SWNTs in water was investigated. Molecular Dynamics (MD) simulations have been performed to provide a semi-quantitative indication of the affinity of each dispersing agent toward SWNT and to attempt an explanation of the experimental results.

We designed block copolymer pro-amphiphiles and amphiphiles for providing very long-term release of nitric oxide (NO). A block copolymer of N-acryloylmorpholine (AM, as a hydrophile) and N-acryloyl-2,5-dimethylpiperazine (AZd, as a hydrophilic precursor) was synthesized. The poly(N-acryloyl-2,5-dimethylpiperazine) (PAZd) is water-soluble, but chemical reaction of the secondary amines with NO to form a N-diazeniumdiolate (NONOate) converts the hydrophilic PAZd into a hydrophobic poly(sodium-1-(N-acryloyl-2,5-dimethylpiperazin-1-yl)diazen-1-ium-1,2-diolate) (PAZd.NONOate), driving aggregation. The PAM block guides this process toward micellization, rather than precipitation, yielding ca. 50 nm spherical micelles. The hydrophobic core of the micelle shielded the NONOate from the presence of water, and thus protons, which are required for NO liberation, delaying release to a remarkable 7 d half-life. Release of the NO returned the original soluble polymer. The very small NO-loaded micelles were able to penetrate complex tissue structures, such as the arterial media, opening up a number of tissue targets to NO-based therapy.

One of the major engineering challenges for the implementation of block copolymer vesicles, or polymersomes, as therapeutic drug carriers is obtaining high encapsulation efficiencies for biomolecules. Here we present a novel method for encapsulation of proteins with high encapsulation efficiency within polymersomes formed from block copolymers of poly(ethylene glycol)-bl-poly(propylene sulfide). By formulation of the neat block copolymer with a low molecular weight poly(ethylene glycol), direct hydration of the formulated mixture yielded polymersomes. We were able to achieve encapsulation efficiencies for ovalbumin at 37%, bovine serum albumin at 19%, and bovine gamma-globulin at 15% when the proteins were included in the hydration solution. The formulation process and the dispersion of polymersomes from the preparation in phosphate-buffered saline were characterized using confocal microscopy, cryogenic transmission electron microscopy, and fluorimetry. We were also successful in the encapsulation of proteinase K, a proteolytic enzyme, and demonstrated by SDS-PAGE that the enzyme was contained inside polymersomes when dispersed in a solution of ovalbumin.

We present the formation of collagen-binding mixed micelles and their potential suitability to deliver therapeutic drugs to the vessel wall. We modified poly(ethylene oxide)-bl-poly(propylene oxide)-bl-poly(ethylene oxide) (Pluronic F-127) to display sulfate groups on the terminus of the PEO block to act as a heparin mimics and bind to collagen in the extracellular matrix. This functionalized macroamphiphile was incorporated into a mixed micelle with poly(propylene sulfide)-bl-poly(ethylene oxide), a macroamphiphile that demonstrates improved micellar stability relative to Pluronic F-127 micelles. The mixed micelles were examined using analytical ultracentrifugation, dynamic light scattering, transmission electron microscopy, and measures of the critical micellar concentration using surface tensiometry. Encapsulation and in vitro release of Sirolimus, an immunosuppressant drug of interest in coronary artery treatment, was considered as an example. Mixed micelles with the sulfate functionality demonstrated enhanced binding to collagen I coated surfaces, suggestive of the potential for binding to the extracellular milieu.

We use cryo-electron microscopy (cryo-EM) to study the 3D shapes of 94-bp-long DNA minicircles and address the question of whether cyclization of such short DNA molecules necessitates the formation of sharp, localized kinks in DNA or whether the necessary bending can be redistributed and accomplished within the limits of the elastic, standard model of DNA flexibility. By comparing the shapes of covalently closed, nicked and gapped DNA minicircles, we conclude that 94-bp-long covalently closed and nicked DNA minicircles do not show sharp kinks while gapped DNA molecules, containing very flexible single-stranded regions, do show sharp kinks. We corroborate the results of cryo-EM studies by using Bal31 nuclease to probe for the existence of kinks in 94-bp-long minicircles.

Micelles formed from amphiphilic block copolymers have been explored in recent years as carriers for hydrophobic drugs. In an aqueous environment, the hydrophobic blocks form the core of the micelle, which can host lipophilic drugs, while the hydrophilic blocks form the corona or outer shell and stabilize the interface between the hydrophobic core and the external medium. In the present work, mesophase behavior and drug encapsulation were explored in the AB block copolymeric amphiphile composed of poly(ethylene glycol) (PEG) as a hydrophile and poly(propylene sulfide) PPS as a hydrophobe, using the immunosuppressive drug cyclosporin A (CsA) as an example of a highly hydrophobic drug. Block copolymers with a degree of polymerization of 44 on the PEG and of 10, 20 and 40 on the PPS respectively (abbreviated as PEG44-b-PPS10, PEG44-b-PPS20, PEG44-b-PPS40) were synthesized and characterized. Drug-loaded polymeric micelles were obtained by the cosolvent displacement method as well as the remarkably simple method of dispersing the warm polymer melt, with drug dissolved therein, in warm water. Effective drug solubility up to 2 mg/mL in aqueous media was facilitated by the PEG- b-PPS micelles, with loading levels up to 19% w/w being achieved. Release was burst-free and sustained over periods of 9-12 days. These micelles demonstrate interesting solubilization characteristics, due to the low glass transition temperature, highly hydrophobic nature, and good solvent properties of the PPS block.