Graphene displays properties that make it appealing for neuroregenerative medicine, yet its interaction with peripheral neurons has been scarcely investigated. Here, we culture on graphene two established models for peripheral neurons: PC12 cells and DRG primary neurons. We perform a nano-resolved analysis of polymeric coatings on graphene and combine optical microscopy and viability assays to assess the material cytocompatibility and influence on differentiation. We find that differentiated PC12 cells display a remarkably increased neurite length on graphene (up to 27%) with respect to controls. Notably, DRG primary neurons survive both on bare and coated graphene. They present dense axonal networks on coated graphene, while they form cell islets characterized by dense axonal bundles on uncoated graphene. These findings indicate that graphene holds potential for nerve tissue regeneration and might pave the road to novel concepts of active nerve conduits.

Green fluorescent protein (GFP) and its variants have been used as fluorescent reporters in a variety of applications for monitoring dynamic processes in cells and organisms, including gene expression, protein localization, and intracellular dynamics. GFP fluorescence is stable, species-independent, and can be monitored noninvasively in living cells by fluorescence microscopy, flow cytometry, or macroscopic imaging techniques. Owing to the presence of a phenol group on the chromophore, most GFP variants display pH-sensitive absorption and fluorescence bands. Such behavior has been exploited to genetically engineer encodable pH indicators for studies of pH regulation within specific intracellular compartments that cannot be probed using conventional pH-sensitive dyes. These pH indicators contributed to shedding light on a number of cell functions for which intracellular pH is an important modulator. In this review we discuss the photophysical properties that make GFPs so special as pH indicators for in vivo use and we describe the probes that are utilized most by the scientific community.

Intracellular chloride ([Cl−] i ) and pH (pH i ) are fundamental regulators of neuronal excitability. They exert wide-ranging effects on synaptic signaling and plasticity and on development and disorders of the brain. The ideal technique to elucidate the underlying ionic mechanisms is quantitative and combined two-photon imaging of [Cl − ] i and pH i , but this has never been performed at the cellular level in vivo. Here, by using a genetically encoded fluorescent sensor that includes a spectroscopic reference (an element insensitive to Cl − and pH), we show that ratiometric imaging is strongly affected by the optical properties of the brain. We have designed a method that fully corrects for this source of error. Parallel measurements of [Cl − ] i and pH i at the single-cell level in the mouse cortex showed the in vivo presence of the widely discussed developmental fall in [Cl − ] i and the role of the K-Cl cotransporter KCC2 in this process. Then, we introduce a dynamic twophoton excitation protocol to simultaneously determine the changes of pH i and [Cl − ] i in response to hypercapnia and seizure activity.I ntracellular ion concentrations are controlled by plasmalemmal transporters and channels, which generate and dissipate ionic electrochemical gradients, respectively (1). In recent years, regulation of the intracellular Cl − concentration ([Cl − ] i ) in neurons has attracted lots of attention, because it is the main ion that carries current across GABA A (and also, glycine) receptors. Changes in [Cl − ] i exert an immediate effect on the reversal potential of GABAergic currents (E GABA ) and, thereby, on the properties of GABA A receptor-mediated transmission (2-4). The "ionic plasticity" of GABAergic signaling involves not only the passive flux of Cl − ions through membrane channels but also, a number of ion transporters that regulate [Cl − ] i . Furthermore, this mechanism is under the control of intracellular signaling cascades that regulate the expression patterns as well as functional properties of ion transporters and channels (5, 6). With regard to long-term ionic modulation of GABAergic transmission, a case in point is the decrease in [Cl − ] i that is generally thought to take place during maturation of most central neurons. According to this widely accepted scenario, the Na-K-2Cl cotransporter NKCC1 accumulates Cl − in immature neurons, thereby promoting depolarizing GABA responses (3, 7-9), which is followed by developmental upregulation of the neuron-specific K-Cl cotransporter KCC2 that is required for the generation of classical hyperpolarizing inhibitory postsynaptic potentials (IPSPs) (10).A wealth of electrophysiological evidence dating back to the work in vivo by Eccles and coworkers (11) has provided evidence for active regulation of [Cl − ] i in mammalian central neurons and its crucial effect on the driving force of Cl − in inhibitory synapses (1). However, thus far, there are no direct data on neuronal [Cl − ] i measured in vivo at the single-cell level in the living brain, and for in...

Accumulation of inorganic nanostructures in the excretory system organs increases their likelihood of toxicity and interference with common medical diagnoses. Thus, one of the major concerns regarding their clinical translation is related to their persistence in organisms. Here the authors demonstrate that nano‐architectures composed by hollow silica nanocapsules embedding arrays of ultrasmall gold nanoparticles undergo biodegradation in cellular environment affording small, potentially clearable building blocks. Furthermore, the authors present their exploitation in glutathione‐triggered release of covalently loaded cisplatin prodrug. This endogenously triggered release leads to high cytotoxicity to human pancreatic carcinoma cells, setting the way for promising applications to synergistic dual chemo/radio‐therapy and radio‐imaging.

SummaryThe neurotrophin receptor TrkA (also known as NTRK1) is known to be crucially involved in several physio-pathological processes. However, a clear description of the early steps of ligand-induced TrkA responses at the cell plasma membrane is missing. We have exploited single particle tracking and TIRF microscopy to study TrkA membrane lateral mobility and changes of oligomerization state upon binding of diverse TrkA agonists (NGF, NGF R100E HSANV mutant, proNGF and NT-3). We show that, in the absence of ligands, most of the TrkA receptors are fast moving monomers characterized by an average diffusion coefficient of 0.47 mm 2 /second; about 20% of TrkA molecules move at least an order of magnitude slower and around 4% are almost immobile within regions of about 0.6 mm diameter. Ligand binding results in increased slow and/or immobile populations over the fast one, slowing down of non-immobile trajectories and reduction of confinement areas, observations that are consistent with the formation of receptor dimeric and oligomeric states. We demonstrate that the extent of TrkA lateral mobility modification is strictly ligand dependent and that each ligand promotes distinct trajectory patterns of TrkA receptors at the cell membrane (ligand 'fingerprinting' effect). This ligand signature of receptor dynamics results from a differential combination of receptor-binding affinity, intracellular effectors recruited in the signalling platforms and formation of signalling and/or recycling endosome precursors. Thus, our data uncover a close correlation between the initial receptor membrane dynamics triggered upon binding and the specific biological outcomes induced by different ligands for the same receptor.

SignificanceNeurotrophins (NTs) are homodimeric growth factors displaying fundamental roles in the nervous system. Their activity stems from binding and activation of 3 different receptor types in nervous cell membranes. The p75 NT receptor (p75NTR) was the first to be discovered in 1986; nevertheless, for the numerous structural and functional facets so far reported, its activation mechanisms have remained elusive. Here, we demonstrate that its pleiotropic functions are regulated by different redistributions of the receptors, which crucially depend on the available NT and on the involved subcellular compartment but are unrelated to its oligomerization state. Single-particle studies proved receptors to be monomers with a fast-diffusive behavior in the membrane with, at most, transient self-interactions on the millisecond time scale.

The transferrin receptor (TfR) is a promising target in cancer therapy owing to its overexpression in most solid tumors and on the blood-brain barrier. Nanostructures chemically derivatized with transferrin are employed in TfR targeting but often lose their functionality upon injection in the bloodstream. As an alternative strategy, we rationally designed a peptide coating able to bind transferrin on suitable pockets not involved in binding to TfR or iron by using an iterative multiscale-modeling approach coupled with quantitative structure-activity and relationship (QSAR) analysis and evolutionary algorithms. We tested that selected sequences have low aspecific protein adsorption and high binding energy toward transferrin, and one of them is efficiently internalized in cells with a transferrin-dependent pathway. Furthermore, it promotes transferrin-mediated endocytosis of gold nanoparticles by modifying their protein corona and promoting oriented adsorption of transferrin. This strategy leads to highly effective nanostructures, potentially useful in diagnostic and therapeutic applications, which exploit (and do not suffer) the protein solvation for achieving a better targeting.

Noble metal nanostructures have demonstrated a number of intriguing features for both medicine and catalysis. However, accumulation issues have prevented their clinical translation, while their use in catalysis has shown serious efficiency and stability hurdles. Here we introduce a simple and robust synthetic protocol for passion fruit-like nano-architectures composed by a silica shell embedding polymeric arrays of ultrasmall noble metal nanoparticles. These nano-architectures show interesting features for both oncology and catalysis. They avoid the issue of persistence in organism thanks to their fast biodegradation in renal clearable building blocks. Furthermore, their calcination results in yolk-shell structures composed by naked metal or alloy nanospheres shielded from aggregation by a silica shell.