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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.

Human RAD52 is a DNA-binding protein involved in many DNA repair mechanisms and genomic stability maintenance. In the last few years, this protein was discovered to be a promising novel pharmacological target for anticancer synthetic lethality strategies since its inhibition or modulation, under specific genetic conditions, was proved to enhance therapies efficacy in various cancer cell types. Although the interest in RAD52 has exponentially grown in the last decade, most information about its structure and mechanism of action is still missing. This work provides novel insights into full-length RAD52 (RAD52 FL) protein, focusing on its structural and functional characterization. The Cryo-Electron Microscopy (Cryo-EM) structure of RAD52 FL, here presented at a resolution (2.16 Å) higher than the one currently available for RAD52 N-terminal X-ray structure, allows hypothesizing the role of individual amino acid residues. While the N-terminal region of RAD52 FL is structured in an undecameric ring, the C-terminal part is intrinsically disordered as fully characterized through SAXS and biophysical analyses. These detailed (atomic level) structural analyses will substantially impact future characterizations of RAD52 mechanisms of action and inhibitors development, particularly in the context of novel approaches to synthetic lethality.

Nanoscale echogenic bubbles (NBs), can be used as a theranostic platform for the localizeddelivery of encapsulated drugs. However, the generation of NBs is challenging, because they havelifetimes as short as milliseconds in solution. The aim of this work has been the optimization of apreparation method for the generation of stable NBs, characterized by measuring: a) acousticefficiency, b) nano-size, to ensure passive tumour targeting, c) stability during storage and afterinjection d) ability to entrap drugs. NBs are monodisperse and ultra-stable, their stability achievedby generation of an amphiphilic multilamellar shell able to efficiently retain the PFC gas. The NBsperform as good acoustic enhancers over a wide frequency range and out of resonant conditions,as tested in both in vitro and in vivo experiments, proving to be a potential platform for theproduction of versatile carriers to be used in ultrasound-assisted diagnostic, therapeutic andtheranostic applications.

Oxygenic phototrophs perform carbon fixation through the Calvin–Benson cycle. Different mechanisms adjust the cycle and the light-harvesting reactions to rapid environmental changes. Photosynthetic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a key enzyme of the cycle. In land plants, different photosynthetic GAPDHs exist: the most abundant formed by hetero-tetramers of A and B-subunits, and the homo-tetramer A4. Regardless of the subunit composition, GAPDH is the major consumer of photosynthetic NADPH and for this reason is strictly regulated. While A4-GAPDH is regulated by CP12, AB-GAPDH is autonomously regulated through the C-terminal extension (CTE) of B-subunits. Reversible inactivation of AB-GAPDH occurs via oxidation of a cysteine pair located in the CTE, and substitution of NADP(H) with NAD(H) in the cofactor binding domain. These combined conditions lead to a change in the oligomerization state and enzyme inactivation. SEC-SAXS and single-particle cryoEM analysis disclosed the structural basis of this regulatory mechanism. Both approaches revealed that (A2B2)n-GAPDH oligomers with n=1, 2, 4 and 5 co-exist in a dynamic system. B-subunits mediate the contacts between adjacent A2B2 tetramers in A4B4 and A8B8 oligomers. The CTE of each B-subunit penetrates into the active site of a B-subunit of the adjacent tetramer, while the CTE of this subunit moves in the opposite direction, effectively preventing the binding of the substrate 1,3-bisphosphoglycerate in the B-subunits. The whole mechanism is made possible, and eventually controlled, by pyridine nucleotides. In fact, NAD(H) by removing NADP(H) from A-subunits allows the entrance of the CTE in B-subunits active sites and hence inactive oligomer stabilization.Significance StatementIn land plants, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) unique sink of reducing power of the entire Calvin-Benson cycle, is finely regulated. Based on the redox state and substrates concentration, its heteromeric form AB-GAPDH oscillates between a fully active heterotetramer (A2B2) and inactive oligomers. Experimental evidence demonstrates that GAPDH inactivation depends on the formation of dimers, tetramers or pentamers of A2B2-modules, linked together by C-terminal extensions (CTE) of B-subunits that extrude from one modular tetramer and occupy two active sites of the adjacent one. This molecular mechanism along with the unexpected observed dynamism of the system, shed light on how the Calvin-Benson cycle is modulated in function of the light environmental changes.

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(e-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

Astrocytic involvement in dopamine dynamics related to motivational and sensorimotor gating abilities is unknown. We found that dysbindin-1 (Dys1) hypofunction increases the activity of astrocytes, which express only the isoform Dys1A that is reduced in the caudate of patients with schizophrenia. Astrocytic Dys1A disruption resulted in avolition and sensorimotor gating deficits, increased astrocytic dopamine D2 receptors and decreased dopaminergic tone within basal ganglia. Notably, astrocytic Dys1A hypofunction disrupted dopamine dynamics linked to reward expectancy and interconnected with astrocytes Ca2+responses mainly in the globus pallidus externus (GPe). Finally, we proved these phenotypes were mediated by D2 receptors in astrocytes as their selective deletion in astrocytes either in GPe or SNc/VTA enhanced motivation and sensorimotor gating abilities as well as dopaminergic release in the GPe. Therefore, astrocytes control motivational and sensorimotor gating processes through Dys1A/D2-dependent mechanisms within the basal ganglia.

Herein, we have developed pH-responsive shape-persistent polymersomes made of well-defined amphiphilic poly(ethylene oxide)-block-poly(diisopropylaminoethyl methacrylate-co-furfuryl methacrylate)s PEO-b-P(DPAco- FMA) by exploiting Diels-Alder chemistry as a robust and simple crosslinking method. Using Photo-induced Copper Mediated Reversible Deactivation Radical Polymerization, we synthesized PEO-b-P(DPA-co-FMA) with optimal block ratios that favor the formation of polymersomes in aqueous media having pH-responsive P(DPAco- FMA) membranes. Owing to the existence of furfuryl pendant groups within the polymersome membranes, the crosslinking of pH-responsive P(DPA-co-FMA) chains can be achieved via Diels-Alder chemistry. Interestingly, the resulting crosslinked polymersomes swell when the pH in the solution is decreased so that it lies in a biologically relevant range, as was demonstrated by cryogenic transmission electron microscopy. Here, the polymersomes' ability to swell can be controlled by adjusting the amount of crosslinker. A minimum threshold of crosslinking density is needed for the polymersomes to swell while an excess amount of crosslinker quenched their ability to swell. Furthermore, crosslinked polymersomes are capable of encapsulating hydrophilic model drug, such as Rhodamine B. At pH 7.20, due to the compact and hydrophobic membrane, the diffusion of the dye from the interior of the polymersomes to the media is minimized, while the pronation of PDPA at an acidic pH of 4.00, enables a permeable membrane, allowing the loaded cargo to leak much more quickly. The shape-persistent polymersomes that we developed herein, are a promising nano-platform for use in drug delivery applications.

RAD51, a key player in the homologous recombination (HR) mechanism, is a critical protein to preserve genomic stability. BRCA2, upon DNA damage, promotes RAD51 fibrils disassembly and its nuclear recruitment.Here, we use BRC4, a peptide derived from the fourth BRC repeat of BRCA2; BRC4 induces RAD51 defibrillation through a ‘domino’ effect, eroding fibrils from their termini, and yielding monomeric RAD51. The congruence among several techniques (static and dynamic light scattering, negative staining transmission electron microscopy (TEM), and microscale thermophoresis) allows an accurate estimation of the kinetic and thermodynamic parameters of this process. BRC4 lacks, however, a nuclear localization sequence; therefore, it cannot transport RAD51 into the nucleus, thus behaving as a RAD51 inhibitor. Cellular assays (BxPC-3, pancreatic cancer cells) indeed show that BRC4 efficiently inhibits HR and enhances the cytotoxic effect of cisplatin, a DNA-damaging drug.The present study sheds further light on the complexity of the HR pathway, paving the way for designing peptide and small organic molecule inhibitors of RAD51 as innovative anticancer and chemo/radiosensitizer compounds.

The formation of block copolymer nanoparticles is commonly obtained by switching solvent quality from good solvent for both blocks to a selective solvent for one block. The effect of the selective solvent on particle dimension, shape and inner structure has been analyzed in detail, instead less attention has been focused on the role of the common solvent. Poly(ethylene oxide)-block-poly(lactic acid) (PEO-b-PLA) is widely used for the preparation of nanometric drug delivery systems and the control of the particle morphology has relevant effects on bioactivity. In this work we investigate the effect of using different common solvents (acetone, dioxane, tetrahydrofuran, dimethylformamide) on the self-assembly behavior of narrowly dispersed PEO-b-PLA copolymers, synthesized by metal free ring-opening polymerization, with large variation of molecular weight of the hydrophobic block (from 65 to 1300 lactic units). Dynamic light scattering (DLS) and cryo-electron microscopy (cryo-EM) analyses of PEO-b-PLA nanoparticles reveal that their size and shape are strongly dependent on the hydrogen bonding ability of the common solvent used in the self-assembly process. As consequence of these variations, also the cytoxicity of the nanoparticles is affected.

Understanding and controlling the interaction between nanoparticles and biological entities is fundamental to the development of nanomedicine applications. In particular, the possibility to realize nanoparticles capable of directly targeting neutral lipid membranes would be advantageous to numerous applications aiming at delivering nanoparticles and their cargos into cells and biological vesicles. Here, we use experimental and computational methodologies to analyze the interaction between liposomes and gold nanoparticles (AuNPs) featuring cationic headgroups in their protecting monolayer. We find that in contrast to nanoparticles decorated with other positively charged headgroups, guanidinium-coated AuNPs can bind to neutral phosphatidylcholine liposomes, inducing nondisruptive membrane permeabilization. Atomistic molecular simulations reveal that this ability is due to the multivalent H-bonding interaction between the phosphate residues of the liposome’s phospholipids and the guanidinium groups. Our results demonstrate that the peculiar properties of arginine magic, an effect responsible for the membranotropic properties of some naturally occurring peptides, are also displayed by guanidinium-bearing functionalized AuNPs. Guanidinium-containing macromolecules, such as arginine-rich peptides, have peculiar properties in their interactions with membranes, including efficient translocation. Here the authors show that cationic gold nanoparticles functionalized with guanidinium groups can specifically and nondisruptively interact with phospholipid bilayer membranes.

In plant grana thylakoid membranes Photosystem II (PSII) associates with a variable number of antenna proteins (LHCII) to form different types of supercomplexes (PSII-LHCII), whose organization is dynamically adjusted in response to light cues, with the C2S2 more abundant in high-light and the C2S2M2 in low-light. Paired PSII-LHCII supercomplexes interacting at their stromal surface from adjacent thylakoid membranes were previously suggested to mediate grana stacking. Here, we present the cryo-electron microscopy maps of paired C2S2 and C2S2M2 supercomplexes isolated from pea plants grown in high-light and low-light, respectively. These maps show a different rotational offset between the two supercomplexes in the pair, responsible for modifying their reciprocal interaction and energetic connectivity. This evidence reveals a different way by which paired PSII-LHCII supercomplexes can mediate grana stacking at diverse irradiances. Electrostatic stromal interactions between LHCII trimers almost completely overlapping in the paired C2S2 can be the main determinant by which PSII-LHCII supercomplexes mediate grana stacking in plants grown in high-light, whereas the mutual interaction of stromal N-terminal loops of two facing Lhcb4 subunits in the paired C2S2M2 can fulfil this task in plants grown in low-light. The high-light induced accumulation of the Lhcb4.3 protein in PSII-LHCII supercomplexes has been previously reported. Our cryo-electron microscopy map at 3.8 &Aring, resolution of the C2S2 supercomplex isolated from plants grown in high-light suggests the presence of the Lhcb4.3 protein revealing peculiar structural features of this high-light-specific antenna important for photoprotection.

Chemokine-induced chemotaxis mediates physiological and pathological immune cell trafficking, as well as several processes involving cell migration. Among them, the role of CXCL12/CXCR4 signaling in cancer and metastasis is well known, and CXCR4 has been often targeted with small molecule-antagonists or short CXCL12-derived peptides to limit the pathological processes of cell migration and invasion. To reduce CXCR4-mediated chemotaxis, we adopted a different approach. We manufactured poly(lactic acid-co-glycolic acid) (PLGA)/Pluronic F127 nanoparticles through microfluidics-assisted nanoprecipitation and functionalized them with streptavidin to docking a biotinylated CXCL12 to be exposed on the nanoparticle surface. Our results show that CXCL12-decorated nanoparticles are non-toxic and do not induce inflammatory cytokine release in THP-1 monocytes cultured in fetal bovine and human serum-supplemented media. The cell internalization of our chemokine receptor-targeting particles increases in accordance with CXCR4 expression in FBS/medium. We demonstrated that CXCL12-decorated nanoparticles do not induce cell migration on their own, but their pre-incubation with THP-1 significantly decreases CXCR4+-cell migration, thereby antagonizing the chemotactic action of CXCL12. The use of biodegradable and immune-compatible chemokine-mimetic nanoparticles to reduce cell migration opens the way to novel antagonists with potential application in cancer treatments and inflammation.

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

The construction of energetically autonomous artificial protocells is one of the most urgent and challenging requirements in bottom-up synthetic biology. Here we show a hybrid multi-compartment approach to build Artificial Simplified-Autotroph Protocells (ASAPs) in an effective manner. Chromatophores obtained fromRhodobacter sphaeroidesaccomplish the photophosphorylation of ADP to ATP functioning as nanosized photosynthetic organellae when encapsulated inside artificial giant phospholipid vesicles. Under continuous illumination chromatophores produce ATP that in turn sustains the transcription of a DNA gene by T7 RNA polymerase inside ASAPs. Cryo-EM and time-resolved spectroscopy were used for characterizing the chromatophore morphology and the orientation of the photophosphorylation proteins, which allow high ATP production rates (up to ~100 ATP/s per ATP synthase). mRNA biosynthesis inside individual vesicles has been determined by confocal microscopy. The hybrid multi-compartment approach here proposed appears at the same time convenient and effective, and thus very promising for the construction of full-fledged artificial protocells.

A method for the aqueous synthesis of stable and biocompatible citrate-coated palladium nanoparticles (PdNPs) in the size range comparable to natural enzymes (4&ndash, 8 nm) has been developed. The toxicological profile of PdNPs was assessed by different assays on several cell lines demonstrating their safety in vitro also at high particle concentrations. To elucidate their cellular fate upon uptake, the localization of PdNPs was analyzed by Transmission Electron Microscopy (TEM). Moreover, crucial information about their intracellular stability and oxidation state was obtained by Sputtering-Enabled Intracellular X-ray Photoelectron Spectroscopy (SEI-XPS). TEM/XPS results showed significant stability of PdNPs in the cellular environment, an important feature for their biocompatibility and potential for biomedical applications. On the catalytic side, these PdNPs exhibited strong and broad antioxidant activities, being able to mimic the three main antioxidant cellular enzymes, i.e., peroxidase, catalase, and superoxide dismutase. Remarkably, using an experimental model of a human oxidative stress-related disease, we demonstrated the effectiveness of PdNPs as antioxidant nanozymes within the cellular environment, showing that they are able to completely re-establish the physiological Reactive Oxygen Species (ROS) levels in highly compromised intracellular redox conditions.

Despite its great potentiality, little attention has been paid to modelling gastrointestinal symptoms of Parkinson's disease (PD) in Drosophila melanogaster (Dm). Our previous studies on standardized Mucuna pruriens extract (Mpe) have shown usefulness in the Drosophila model of PD. In this communication, we provide new information on the effect of Mpe on basal and serotonin treated contractions in the crop (i.e., an important and essential part of the gut) in Drosophila PD mutant for PTEN-induced putative kinase 1 (PINK1B9) gene. The effect of Mpe on PINK1B9 supplied with standard diet to larvae and/or adults, were assayed on 10–15 days old flies. Conversely from what we observed in the wild type flies, recordings demonstrated that exogenous applications of serotonin on crop muscles of untreated PINK1B9 affect neither the frequency nor the amplitude of the crop contraction, while the same muscle parameters are enhanced following brain injections of serotonin, thus suggesting that PINK1B9 mutants may likely have an impairment in the serotonergic pathways. Also, the mitochondrial morphology in the crop muscles is strongly compromised, as demonstrated by the transmission electron microscopy analysis. The Mpe treatment rescued the crop muscle parameters and also the mitochondrial morphology when supplied to both larvae and adults. Overall, this study strengthens the relevance of using PINK1B9 Dm as a translational model to study the gastrointestinal symptoms in PD and also confirms the useful employment of M. pruriens for PD treatment.

Nanoparticles (NPs) are increasingly used in biomedical applications, but the factors that influence their interactions with living cells need to be elucidated. Here, wereveal the role of NP surface charge in determining theirneuronal interactions and electrical responses. We discoveredthat 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.

Corticosteroids, such as dexamethasone (DEX), are the mainstays for the treatment of moderate to severe inflammatory bowel disease (IBD). However, their relatively poor bioavailability and lack of specificity is often the origin of short and long-term adverse effects. Here, spherical polymeric nanoconstructs (SPNs) encapsulating dexamethasone are proposed for the systemic treatment of IBD. In a mouse model of colitis, the accumulation of SPNs within the inflamed intestine is firstly assessed using near infra-red fluorescent (NIRF) imaging at different stages of the disease - 5, 7 and 10 days of Dextran Sulfate Sodium (DSS) administration. Then, the efficacy of DEX-SPNs is tested in vitro over macrophages and in vivo by monitoring the animal weight, food and water intake; expression of inflammatory cytokines (TNF-α, IL-1β, IL-6); intestinal density of macrophages; rectal bleeding and histological scoring. 150 nm DEX-SPNs are shown to deposit within the hyper-permeable inflamed intestine in a disease severity-dependent fashion. DEX-SPNs exposed to LPS-stimulated RAW 264.7 cells reduce the expression of inflammatory cytokines as rapidly as free DEX. In DSS-administered mice, DEX-SPNs treatments improve weight loss, reduce the macrophage infiltration, expression of inflammatory cytokines, rectal bleeding and histological scoring, as compared to free DEX. Moreover, DEX-SPNs exert a strong systemic anti-inflammatory effect and facilitate animal recovery. This work confirms the benefits of using sufficiently small nanoconstructs for targeting inflamed, hyper-permeable tissues and efficiently delivering high doses of corticosteroids for the treatment of intestinal and systemic inflammation.

Hybrids composed of liposomes (L) and metallic nanoparticles (NP) hold great potential for imaging and drug delivery purposes. However, the efficient incorporation of metallic nanoparticles 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 nanoparticles of size 2-4 nm (Au) using a microfluidic assisted self-assembly process. The incorporation of increasing amounts of Au nanoparticles 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, smaller polydispersity index, and a 3-fold 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 confirmed qualitatively by transmission electron microscopy. The higher loading of gold nanoparticles 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, respectively. Structural characterization of the liposomal-nanoparticle hybrids with cryo-electron microscopy revealed the co-existence of membrane-embedded and interdigitated nanoparticle-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-nanoparticle hybrids with high loading capacity highlighting the utility of microfluidics to improve the payload of metallic nanoparticles within liposomes, thereby enhancing their application for imaging and drug delivery.

In this contribution we discuss the possible strategies to synthesize photo-autotrophic artificial protocells starting from scratch, following the semi-synthetic bottom up approach. The main aim is to build up artificial compartmentalized systems able to mimic living cell behavior in the transduction of light energy in chemical energy. Some preliminary results and future perspective are presented and discussed.

Sensitized triplet–triplet annihilation based photon up-conversion (TTA-UC) greatly improves the scope and applicability of fluorescence bioimaging by enabling anti-Stokes detection at low powers, thus eliminating the background autofluorescence and limiting the potential damage of the living tissues. Here the authors present a facile, one-step protocol to prepare dual dye-doped, TTA-UC active nanomicelles starting from the commercially available surfactant Kolliphor EL, a component of several FDA approved preparations. These nanosized micelles show an unprecedented up-conversion yield of 6.5% under 0.1 W cm−2 excitation intensity in an aqueous, nondeaerated dispersion. The supramolecular architecture obtained preserves the embedded dyes from oxygen quenching, allowing satisfactory anti-Stokes fluorescence imaging of 3T3 cells. This is the first example of efficient multicomponent up-converters prepared using highly biocompatible materials approved by the international authority, paving the way for the development of new complex and multifunctional materials for advanced theranostics.

Neuroblastoma (NB) tumor substantially contributes to childhood cancer mortality. The design of novel drugs targeted to specific molecular alterations becomes mandatory, especially for high-risk patients burdened by chemoresistant relapse. The dysregulated expression of MYCN, ALK, and LIN28B and the diminished levels of miR-34a and let-7b are oncogenic in NB. Due to the ability of miRNA-mimics to recover the tumor suppression functions of miRNAs underexpressed into cancer cells, safe and efficient nanocarriers selectively targeted to NB cells and tested in clinically relevant mouse models are developed. The technology exploits the nucleic acids negative charges to build coated-cationic liposomes, then functionalized with antibodies against GD2 receptor. The replenishment of miR-34a and let-7b by NB-targeted nanoparticles, individually and more powerfully in combination, significantly reduces cell division, proliferation, neoangiogenesis, tumor growth and burden, and induces apoptosis in orthotopic xenografts and improves mice survival in pseudometastatic models. These functional effects highlight a cooperative down-modulation of MYCN and its down-stream targets, ALK and LIN28B, exerted by miR-34a and let-7b that reactivate regulatory networks leading to a favorable therapeutic response. These findings demonstrate a promising therapeutic efficacy of miR-34a and let-7b combined replacement and support its clinical application as adjuvant therapy for high-risk NB patients.

Although conceptually obvious, the effective delivery of proteins in therapeutic applications is far from being a routine practice. The major limitation is the conservation of protein physicochemical identity during the transport to the target site. In this regard, nanoparticle-based systems offer new intriguing possibilities, provided that (i) the harsh and denaturating conditions typically used for nanoparticle synthesis are avoided or mitigated, and (ii) nanoparticle biocompatibility and degradation (for protein release) are optimized. Here, we tackle these issues by starting from a nanoparticle architecture already tested for small chemical compounds. In particular, silica-shielded liposomes are produced and loaded with a test protein (i.e., Green Fluorescent Protein) in an aqueous environment. We demonstrate promising results concerning protein encapsulation, protection during intracellular trafficking and final release triggered by nanoparticle degradations in acidic organelles. We believe this proof of principle may open new applications and developments for targeted and efficient protein delivery.

Monoaminergic modulation of cortical and thalamic inputs to the dorsal striatum (DS) is crucial for reward-based learning and action control. While dopamine has been extensively investigated in this context, the synaptic effects of serotonin (5-HT) have been largely unexplored. Here, we investigated how serotonergic signaling affects associative plasticity at glutamatergic synapses on the striatal projection neurons of the direct pathway (dSPNs). Combining chemogenetic and optogenetic approaches reveals that impeding serotonergic signaling preferentially gates spike-timing-dependent long-term depression (t-LTD) at thalamostriatal synapses. This t-LTD requires dampened activity of the 5-HT4 receptor subtype, which we demonstrate controls dendritic Ca2+ signals by regulating BK channel activity, and which preferentially localizes at the dendritic shaft. The synaptic effects of 5-HT signaling at thalamostriatal inputs provide insights into how changes in serotonergic levels associated with behavioral states or pathology affect striatal-dependent processes.

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.

Targeted delivery of anticancer drugs with nanocarriers can reduce side effects and ameliorate therapeutic efficacy. However, poorly perfused and dysfunctional tumor vessels limit the transport of the payload into solid tumors. The use of tumor-penetrating nanocarriers might enhance tumor uptake and antitumor effects. A peptide containing a tissue-penetrating (TP) consensus motif, capable of recognizing neuropilin-1, is here fused to a neuroblastoma-targeting peptide (pep) previously developed. Neuroblastoma cell lines and cells derived from both xenografts and high-risk neuroblastoma patients show overexpression of neuropilin-1. In vitro studies reveal that TP-pep binds cell lines and cells derived from neuroblastoma patients more efficiently than pep. TP-pep, after coupling to doxorubicin-containing stealth liposomes (TP-pep-SL[doxorubicin]), enhances their uptake by cells and cytotoxic effects in vitro, while increasing tumor-binding capability and homing in vivo. TP-pep-SL[doxorubicin] treatment enhances the Evans Blue dye accumulation in tumors but not in nontumor tissues, pointing to selective increase of vascular permeability in tumor tissues. Compared to pep-SL[doxorubicin], TP-pep-SL[doxorubicin] shows an increased antineuroblastoma activity in three neuroblastoma animal models mimicking the growth of neuroblastoma in humans. The enhancement of drug penetration in tumors by TP-pep-targeted nanoparticles may represent an innovative strategy for neuroblastoma.

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 ultrastructure of mature spermatozoa from three leech species, Haemadipsa zeylanica ( Moquin-Tandon, 1826 ) (Haemadipsidae), Theromyzon tessulatum ( Muller, 1774 ) and Placobdella costata (Fr. Muller, 1846 ) (Glossiphoniidae), was investigated by means of transmission electron microscopy. While the three species showed the general features of hirudinid spermatozoa, considerable variation was found in nearly all characters examined: the anterior acrosome varied in length and in shape; the nuclei also varied in size and show a very complicated shape which is maintained for the whole length in the two glossiphonids, while in the haemadipsid, it showed two different morphological regions. The single mitochondrion, which is surrounded by an electron-dense sheath, is straight in H and T. tessulatum, but twisted in P. costata. The flagellum in the three studied species is of the prominent central sheath type. The basal body is absent in the three species and the central apparatus of the flagellum was observed penetrating to the mitochondrion. By comparing the present data with those from “oligochaeta”, Branchiobdellida and Acanthobdellida, we found that only the presence of an anterior acrosome characterizes the true leeches and at present should be regarded as an autapomorphic character of Euhirudinea.

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. Stem Cells 2015;33:35–44