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The mechanisms underlying the dichotomic cortical/basal ganglia dopaminergic abnormalities in schizophrenia are unclear. Astrocytes are important non-neuronal modulators of brain circuits, but their role in dopaminergic system remains poorly explored. Microarray analyses, immunohistochemistry, and two-photon laser scanning microscopy revealed that Dys1 hypofunction increases the reactivity of astrocytes, which express only the Dys1A isoform. Notably, behavioral and electrochemical assessments in mice selectively lacking the Dys1A isoform unraveled a more prominent impact of Dys1A in behavioral and dopaminergic/D2 alterations related to basal ganglia, but not cortical functioning. Ex vivo electron microscopy and protein expression analyses indicated that selective Dys1A disruption might alter intracellular trafficking in astrocytes, but not in neurons. In agreement, Dys1A disruption only in astrocytes resulted in decreased motivation and sensorimotor gating deficits, increased astrocytic dopamine D2 receptors and decreased dopaminergic tone within basal ganglia. These processes might have clinical relevance because the caudate, but not the cortex, of patients with schizophrenia shows a reduction of the Dys1A isoform. Therefore, we started to show a hitherto unknown role for the Dys1A isoform in astrocytic-related modulation of basal ganglia behavioral and dopaminergic phenotypes, with relevance to schizophrenia.

The poor transport of molecular and nanoscale agents through the blood–brain barrier together with tumour heterogeneity contribute to the dismal prognosis in patients with glioblastoma multiforme. Here, a biodegradable implant (μMESH) is engineered in the form of a micrometre-sized poly(lactic-co-glycolic acid) mesh laid over a water-soluble poly(vinyl alcohol) layer. Upon poly(vinyl alcohol) dissolution, the flexible poly(lactic-co-glycolic acid) mesh conforms to the resected tumour cavity as docetaxel-loaded nanomedicines and diclofenac molecules are continuously and directly released into the adjacent tumour bed. In orthotopic brain cancer models, generated with a conventional, reference cell line and patient-derived cells, a single μMESH application, carrying 0.75 mg kg−1of docetaxel and diclofenac, abrogates disease recurrence up to eight months after tumour resection, with no appreciable adverse effects. Without tumour resection, the μMESH increases the median overall survival (∼30 d) as compared with the one-time intracranial deposition of docetaxel-loaded nanomedicines (15 d) or 10 cycles of systemically administered temozolomide (12 d). The μMESH modular structure, for the independent coloading of different molecules and nanomedicines, together with its mechanical flexibility, can be exploited to treat a variety of cancers, realizing patient-specific dosing and interventions.

We show the first example of a synergic approach of oxidant (ROS) scavenging carrier and ROS-responsive drug release in the context of a potential therapy against osteoporosis, aiming to inhibit the differentiation of inflammatory cells into osteoclasts. In our “tandem” approach, a branched amphiphilic, PEGylated polysulfide (PPSES–PEG) was preferred over a linear analogue, because of improved homogeneity in the aggregates (spherical micelles vs mixture of wormlike and spherical), increased stability, and higher drug loading (up to ∼22 wt % of antiosteoclastic rapamycin). These effects are ascribed to the branching inhibiting crystallization in the polysulfide blocks. The ROS-scavenging micelles alone were already able to reduce osteoclastogenesis in a RAW 264.7 model, but the “drug” combination (the polymer itself + rapamycin released only under oxidation) completely abrogated the process. An important take-home message is that the synergic performance depended very strongly on the oxidant:oxidizable group molar ratio, a parameter to carefully tune in the perspective of targeting specific diseases.

The genus Ruminococcusis dominant in the human gut, but higher levels of some species, such as R. gnavus, R. torquesand R. bromii, have been linked to health or disease. In this study, we analyzed >9,000 RuminococcusMetagenome-Assembled Genomes (MAGs) reconstructed from >5,000 subjects and revealed significant links between the prevalence of some species/subspecies and geographic origin, age, lifestyle and disease, with subspecies prevalent in specific subpopulations showing divergent metabolic potential. Furthermore, Ruminococcusspecies from Lachnospiraceaeencoded for Carbohydrate Active Enzymes (CAZymes) potentially involved in the metabolism of human N- and O-glycans, whereas those from Oscillospiraceaeappear more adapted towards fiber metabolism. These new findings contribute to elucidate the potential functional role of Ruminococcusin specific lifestyles and diseases, and to decipher the diversity and the adaptation of members of this genus to the human gut.

Hybrids composed of liposomes (L) and metallic nanoparticles (NPs) hold great potential for imaging and drug delivery purposes. However, the efficient incorporation of metallic NPs into liposomes using conventional methodologies has so far proved to be challenging. In this study, we report the fabrication of hybrids of liposomes and hydrophobic gold NPs of size 2–4 nm (Au) using a microfluidic-assisted self-assembly process. The incorporation of increasing amounts of AuNPs into liposomes was examined using microfluidics and compared to L–AuNP hybrids prepared by the reverse-phase evaporation method. Our microfluidics strategy produced L–AuNP hybrids with a homogeneous size distribution, a smaller polydispersity index, and a threefold increase in loading efficiency when compared to those hybrids prepared using the reverse-phase method of production. Quantification of the loading efficiency was determined by ultraviolet spectroscopy, inductively coupled plasma mass spectroscopy, and centrifugal field flow fractionation, and qualitative validation was confirmed by transmission electron microscopy. The higher loading of gold NPs into the liposomes achieved using microfluidics produced a slightly thicker and more rigid bilayer as determined with small-angle neutron scattering. These observations were confirmed using fluorescent anisotropy and atomic force microscopy. Structural characterization of the liposomal–NP hybrids with cryo-electron microscopy revealed the coexistence of membrane-embedded and interdigitated NP-rich domains, suggesting AuNP incorporation through hydrophobic interactions. The microfluidic technique that we describe in this study allows for the automated production of monodisperse liposomal–NP hybrids with high loading capacity, highlighting the utility of microfluidics to improve the payload of metallic NPs within liposomes, thereby enhancing their application for imaging and drug delivery.

Here, we report a nanoplatform based on iron oxide nanocubes (IONCs) coated with a bioresorbable polymer that, upon exposure to lytic enzymes, can be disassembled increasing the heat performances in comparison with the initial clusters. We have developed two-dimensional (2D) clusters by exploiting benchmark IONCs as heat mediators for magnetic hyperthermia and a polyhydroxyalkanoate (PHA) copolymer, a biodegradable polymer produced by bacteria that can be digested by intracellular esterase enzymes. The comparison of magnetic heat performance of the 2D assemblies with 3D centrosymmetrical assemblies or single IONCs emphasizes the benefit of the 2D assembly. Moreover, the heat losses of 2D assemblies dispersed in water are better than the 3D assemblies but worse than for single nanocubes. On the other hand, when the 2D magnetic beads (2D-MNBs) are incubated with the esterase enzyme at a physiological temperature, their magnetic heat performances began to progressively increase. After 2 h of incubation, specific absorption rate values of the 2D assembly double the ones of individually coated nanocubes. Such an increase can be mainly correlated to the splitting of the 2D-MNBs into smaller size clusters with a chain-like configuration containing few nanocubes. Moreover, 2D-MNBs exhibited nonvariable heat performances even after intentionally inducing their aggregation. Magnetophoresis measurements indicate a comparable response of 3D and 2D clusters to external magnets (0.3 T) that is by far faster than that of single nanocubes. This feature is crucial for a physical accumulation of magnetic materials in the presence of magnetic field gradients. This system is the first example of a nanoplatform that, upon exposure to lytic enzymes, such as those present in a tumor environment, can be disassembled from the initial 2D-MNB organization to chain-like assemblies with clear improvement of the heat magnetic losses resulting in better heat dissipation performances. The potential application of 2D nanoassemblies based on the cleavable PHAs for preserving their magnetic losses inside cells will benefit hyperthermia therapies mediated by magnetic nanoparticles under alternating magnetic fields.

This study is about (1) nanomanufacturing (focusing on microfluidic-assisted nanoprecipitation), (2) advanced colloid characterization (focusing on field flow fractionation), and (3) the possible restructuring of surface disulfides. Disulfides are dynamic and exchangeable groups, and here we specifically focus, first, on their use to introduce biofunctional groups and, second, on their re-organization, which may lead to variable surface chemistries and uncontrolled cell interactions. The particles were obtained via microfluidic-assisted (flow-focused) nanoprecipitation of poly(ethylene glycol)-b-poly(ε-caprolactone) bearing or not a 2-pyridyl disulfide (PDS) terminal group, which quantitatively exchanges with thiols in solution. In this study, we have paid specific attention to size characterization, thereby also demonstrating the limitations of dynamic light scattering (DLS) as a stand-alone technique. By using asymmetric flow field flow fractionation coupled with DLS, static light scattering (SLS), and refractive index detectors, we show that relatively small amounts of >100 nm aggregates (cryogenic transmission electron microscopy and SLS/DLS comparison suggesting them to be wormlike micelles) dominated the stand-alone DLS results, whereas the “real” size distributions picked <50 nm. Our key result is that the kinetics of the conjugation based on PDS–thiol exchange was controlled by the thiol pKa, and this also determined the rate of the exchange between the resulting disulfides and glutathione (GSH). In particular, more acidic thiols (e.g., peptides, where a cysteine is flanked by cationic residues) react faster with PDS, but their disulfides hardly exchange with GSH; the reverse applies to thiols with a higher pKa. Disulfides that resist against restructuring via thiol–disulfide exchange allow for a stable bioconjugation, although they may be bad news for payload release under reducing conditions. However, experiments of both thiol release and nanoparticles uptake in cells (HCT116) show that also the disulfides formed from less-acidic and, therefore, less-reactive, and more exchangeable thiols were stable for at least a few hours even in a GSH-rich (10 mM) environment; this suggests a sufficiently long stability of surface groups to achieve, for example, a cell-targeting effect.

The investigation of the toxicological profile and biomedical potential of nanoparticles (NPs) requires a deep understanding of their intracellular fate. Various techniques are usually employed to characterize NPs upon cellular internalization, including high-resolution optical and electron microscopies. Here, we show a versatile method, named sputtering-enabled intracellular X-ray photoelectron spectroscopy, proving that it is able to provide valuable information about the behavior of metallic NPs in culture media as well as within cells, directly measuring their internalization, stability/degradation, and oxidation state, without any preparative steps. The technique can also provide nanoscale vertical resolution along with semiquantitative information about the cellular internalization of the metallic species. The proposed approach is easy-to-use and can become a standard technique in nanotoxicology/nanomedicine and in the rational design of metallic NPs. Two model cases were investigated: silver nanoparticles (AgNPs) and platinum nanoparticles (PtNPs) with the same size and coating. We observed that, after 48 h incubation, intracellular AgNPs were almost completely dissolved, forming nanoclusters as well as AgO, AgS, and AgCl complexes. On the other hand, PtNPs were resistant to the harsh endolysosomal environment, and only some surface oxidation was detected after 48 h.

One area where nanomedicine may offer superior performances and efficacy compared to current strategies is in the diagnosis and treatment of central nervous system (CNS) diseases. However, the application of nanomaterials in such complex arenas is still in its infancy and an optimal vector for the therapy of CNS diseases has not been identified. Graphitic carbon nano-onions (CNOs) represent a class of carbon nanomaterials that shows promising potential for biomedical purposes. To probe the possible applications of graphitic CNOs as a platform for therapeutic and diagnostic interventions on CNS diseases, fluorescently labeled CNOs were stereotaxically injected in vivo in mice hippocampus. Their diffusion within brain tissues and their cellular localization were analyzed ex vivo by confocal microscopy, electron microscopy, and correlative light-electron microscopy techniques. The subsequent fluorescent staining of hippocampal cells populations indicates they efficiently internalize the nanomaterial. Furthermore, the inflammatory potential of the CNOs injection was found comparable to sterile vehicle infusion, and it did not result in manifest neurophysiological and behavioral alterations of hippocampal-mediated functions. These results clearly demonstrate that CNOs can interface effectively with several cell types, which encourages further their development as possible brain disease-targeted diagnostics or therapeutics nanocarriers.

Nanoparticles (NPs) are increasingly used in biomedical applications, but the factors that influence their interactions with living cells need to be elucidated. Here, we reveal the role of NP surface charge in determining their neuronal interactions and electrical responses. We discovered that negatively charged NPs administered at low concentration (10 nM) interact with the neuronal membrane and at the synaptic cleft, whereas positively and neutrally charged NPs never localize on neurons. This effect is shape and material independent. The presence of negatively charged NPs on neuronal cell membranes influences the excitability of neurons by causing an increase in the amplitude and frequency of spontaneous postsynaptic currents at the single cell level and an increase of both the spiking activity and synchronous firing at neural network level. The negatively charged NPs exclusively bind to excitable neuronal cells, and never to nonexcitable glial cells. This specific interaction was also confirmed by manipulating the electrophysiological activity of neuronal cells. Indeed, the interaction of negatively charged NPs with neurons is either promoted or hindered by pharmacological suppression or enhancement of the neuronal activity with tetrodotoxin or bicuculline, respectively. We further support our main experimental conclusions by using numerical simulations. This study demonstrates that negatively charged NPs modulate the excitability of neurons, revealing the potential use of NPs for controlling neuron activity.

Microglia reactivity entails a large-scale remodeling of cellular geometry, but the behavior of the microtubule cytoskeleton during these changes remains unexplored. Here we show that activated microglia provide an example of microtubule reorganization from a non-centrosomal array of parallel and stable microtubules to a radial array of more dynamic microtubules. While in the homeostatic state microglia nucleate microtubules at Golgi outposts, activating signaling induces recruitment of nucleating material nearby the centrosome, a process inhibited by microtubule stabilization. Our results demonstrate that a hallmark of microglia reactivity is a striking remodeling of the microtubule cytoskeleton and suggest that while pericentrosomal microtubule nucleation may serve as a distinct marker of microglia activation, inhibition of microtubule dynamics may provide a different strategy to reduce microglia reactivity in inflammatory disease.

Lamin B1 depletion has detrimental effects on brain development. Lamin B1 loss of function affects neuronal development in primary mouse cortical neurons, strongly impairing dendrite length and complexity. This defective dendrite development stems from impaired nuclear ERK signaling and, possibly, mislocalization of nuclear pores.

Herein, we prepare nanohybrids by incorporating iron oxide nanocubes (cubic-IONPs) within a thermoresponsive polymer shell that can act as drug carriers for doxorubicin(doxo). The cubic-shaped nanoparticles employed are at the interface between superparamagnetic and ferromagnetic behavior and have an exceptionally high specific absorption rate (SAR), but their functionalization is extremely challenging compared to bare superparamagnetic iron oxide nanoparticles as they strongly interact with each other. By conducting the polymer grafting reaction using reversible addition–fragmentation chain transfer (RAFT) polymerization in a viscous solvent medium, we have here developed a facile approach to decorate the nanocubes with stimuli-responsive polymers. When the thermoresponsive shell is composed of poly(N-isopropylacrylamide-co-polyethylene glycolmethyl ether acrylate), nanohybrids have a phase transition temperature, the lower critical solution temperature (LCST), above 37 °C in physiological conditions. Doxo loaded nanohybrids exhibited a negligible drug release below 37 °C but showed a consistent release of their cargo on demand by exploiting the capability of the nanocubes to generate heat under an alternating magnetic field (AMF). Moreover, the drug free nanocarrier does not exhibit cytotoxicity even when administered at high concentration of nanocubes (1g/L of iron) and internalized at high extent (260 pg of iron per cell). We have also implemented the synthesis protocol to decorate the surface of nanocubes with poly(vinylpyridine) polymer and thus prepare pH-responsive shell coated nanocubes.

The cancer stem cell (CSC) model is describing tumors as a hierarchical organized system and CSCs are suggested to be responsible for cancer recurrence after therapy. The identification of specific markers of CSCs is therefore of paramount importance. Here, we show that high levels of lipid droplets (LDs) are a distinctive mark of CSCs in colorectal (CR) cancer. This increased lipid content was clearly revealed by label‐free Raman spectroscopy and it directly correlates with well‐accepted CR‐CSC markers as CD133 and Wnt pathway activity. By xenotransplantation experiments, we have finally demonstrated that CR‐CSCs overexpressing LDs retain most tumorigenic potential. A relevant conceptual advance in this work is the demonstration that a cellular organelle, the LD, is a signature of CSCs, in addition to molecular markers. A further functional characterization of LDs could lead soon to design new target therapies against CR‐CSCs. StemCells2015;33:35–44

Trifunctional polymer nanobeads are prepared by destabilization of a mixture of magnetic nanoparticles, quantum dots, and an amphiphilic polymer, followed by functionalization of the bead surface with folic acid molecules. The distribution of the nanoparticles within the nanobeads can be tuned using either acetonitrile or water as destabilizing solvent. The luminescence of the resulting beads can be tuned by varying the ratio of quantum dots per magnetic nanoparticles. The application of an external magnetic field (such as a small static magnet of 0.3 T) to the magnetic−fluorescent nanobeads allows the quantitative accumulation of the beads within a few hours depending on the total size of the beads. Furthermore, specific targeting of cancer cells overexpressing folate receptors is achieved thanks to the folic acid decorating the surface of the as-synthesized nanobeads. Folate receptor mediated cellular uptake of the folic acid-functionalized nanobeads is proven viaboth confocal imaging and transmission electron microscopy characterization. Cell sorting experiments performed with trifunctional nanobeads show quantitative recovering of targeted cells even when they are present at low percentage (up to 1%).

The spermatozoa of two species supposed to be basal to Gastrotricha Chaetonotida, Neodasys ciritus and Musellifer delamarei, were studied in order to supply further elements to the understanding of sperm evolution in Chaetonotida, a group in which a fully parthenogenetic reproduction is dominant. Two considerably different sperm patterns were found: the spermatozoon of N. ciritus has a simple, conical acrosome, a short, condensed nucleus, few conventional mitochondria randomly arranged along the sperm head, and a 9×2+2 flagellum perpendicular to the sperm major axis. The spermatozoon of M. delamarei is a filiform cell with a simple acrosome, a partially condensed nucleus, four mitochondria at the nuclear base, and a flagellum with a 9×2+2 axoneme and large accessory fibers. Some sperm features of M. delamarei are comparable to those of Xenotrichulidae, the only other Chaetonotida producing conventional spermatozoa, whereas the sperm of N. ciritus appears different from all the other patterns known among Gastrotricha, thus knowledge of it does not help in solving the problem of the discussed phylogenetic position of the genus.

Spermatogenesis, spermatozoa, seminal receptacles and the transformations of sperm after mating were examined for the first time in the polychaete annelid Aeolosoma singulare from Lake Baikal. The structure of the spermatozoon in the coelomic cavity is very similar to that already described for A. litorale, in particular in the presence of a cytoplasmic droplet containing lysosome-like vesicles around the nuclear basis. However, comparison of coelomic spermatozoa with those stored in the sperm receptacles revealed a progressive reduction of the cytoplasmic droplet. The cytoplasmic droplet can thus be considered as a sort of remnant of the spermiogenetic process in the mature spermatozoon. The seminal receptacles of A. singulare, like those of the other aeolo-somatids, are epidermal formations; but in this species they have a multicellular organisation and penetrate the muscular layers to end deep inside the coelomic cavity. The spermatozoon of A. singulare was compared with those of polychaetes considered close to aeolosomatids, and was found to have characters in common only with potamodrilids.

Glacier ice worms, Mesenchytraeus solifugus and related species, are the largest glacially obligate metazoans. As one component of cold temperature adaptation, ice worms maintain atypically high energy levels in an apparent mechanism to offset cold temperature-induced lethargy and death. To explore this observation at a mechanistic level, we considered the putative contribution of 5′ adenosine monophosphate deaminase (AMPD), a key regulator of energy metabolism in eukaryotes. We cloned cDNAs encoding ice worm AMPD, generating a fragment encoding 543 amino acids that included a short N-terminal region and complete C-terminal catalytic domain. The predicted ice worm AMPD amino acid sequence displayed conservation with homologues from other mesophilic eukaryotes with notable exceptions. In particular, an ice worm-specific K188E substitution proximal to the AMP binding site likely alters the architecture of the active site and negatively affects the enzyme's activity. Paradoxically, this would contribute to elevated intracellular ATP levels, which appears to be a signature of cold adapted taxa.