Low Forces Push the Maturation of Neural Precursors into Neurons.

Mechanical stimulation modulates neural development and neuronal activity. In a previous study, magnetic "nano-pulling" is proposed as a tool to generate active forces. By loading neural cells with magnetic nanoparticles (MNPs), a precise force vector is remotely generated through static magnetic fields. In the present study, human neural stem cells (NSCs) are subjected to a standard differentiation protocol, in the presence or absence of nano-pulling. Under mechanical stimulation, an increase in the length of the neural processes which showed an enrichment in microtubules, endoplasmic reticulum, and mitochondria is found. A stimulation lasting up to 82 days induces a strong remodeling at the level of synapse density and a re-organization of the neuronal network, halving the time required for the maturation of neural precursors into neurons. The MNP-loaded NSCs are then transplanted into mouse spinal cord organotypic slices, demonstrating that nano-pulling stimulates the elongation of the NSC processes and modulates their orientation even in an ex vivo model. Thus, it is shown that active mechanical stimuli can guide the outgrowth of NSCs transplanted into the spinal cord tissue. The findings suggest that mechanical forces play an important role in neuronal maturation which could be applied in regenerative medicine.

Axonal plasticity in response to active forces generated through magnetic nano-pulling.

Mechanical force is crucial in guiding axon outgrowth before and after synapse formation. This process is referred to as "stretch growth." However, how neurons transduce mechanical input into signaling pathways remains poorly understood. Another open question is how stretch growth is coupled in time with the intercalated addition of new mass along the entire axon. Here, we demonstrate that active mechanical force generated by magnetic nano-pulling induces remodeling of the axonal cytoskeleton. Specifically, the increase in the axonal density of microtubules induced by nano-pulling leads to an accumulation of organelles and signaling vesicles, which, in turn, promotes local translation by increasing the probability of assembly of the "translation factories." Modulation of axonal transport and local translation sustains enhanced axon outgrowth and synapse maturation.

Quantum computing algorithms: getting closer to critical problems in computational biology.

The recent biotechnological progress has allowed life scientists and physicians to access an unprecedented, massive amount of data at all levels (molecular, supramolecular, cellular and so on) of biological complexity. So far, mostly classical computational efforts have been dedicated to the simulation, prediction or de novo design of biomolecules, in order to improve the understanding of their function or to develop novel therapeutics. At a higher level of complexity, the progress of omics disciplines (genomics, transcriptomics, proteomics and metabolomics) has prompted researchers to develop informatics means to describe and annotate new biomolecules identified with a resolution down to the single cell, but also with a high-throughput speed. Machine learning approaches have been implemented to both the modelling studies and the handling of biomedical data. Quantum computing (QC) approaches hold the promise to resolve, speed up or refine the analysis of a wide range of these computational problems. Here, we review and comment on recently developed QC algorithms for biocomputing, with a particular focus on multi-scale modelling and genomic analyses. Indeed, differently from other computational approaches such as protein structure prediction, these problems have been shown to be adequately mapped onto quantum architectures, the main limit for their immediate use being the number of qubits and decoherence effects in the available quantum machines. Possible advantages over the classical counterparts are highlighted, along with a description of some hybrid classical/quantum approaches, which could be the closest to be realistically applied in biocomputation.

Human Microglia Extracellular Vesicles Derived from Different Microglia Cell Lines: Similarities and Differences.

Microglial cells are a component of the innate immune system in the brain that support cell-to-cell communication via secreted molecules and extracellular vesicles (EVs). EVs can be divided into two major populations: large (LEVs) and small (SEVs) EVs, carrying different mediators, such as proteins, lipids, and miRNAs. The microglia EVs cargo crucially reflects the status of parental cells and can lead to both beneficial and detrimental effects in many physiopathological states. Herein, a workflow for the extraction and characterization of SEVs and LEVs from human C20 and HMC3 microglia cell lines derived, respectively, from adult and embryonic microglia is reported. EVs were gathered from the culture media of the two cell lines by sequential ultracentrifugation steps and their biochemical and biophysical properties were analyzed by Western blot, transmission electron microscopy, and dynamic light scattering. Although the C20- and HMC3-derived EVs shared several common features, C20-derived EVs were slightly lower in number and more polydispersed. Interestingly, C20- but not HMC3-SEVs were able to interfere with the proliferation of U87 glioblastoma cells. This correlated with the different relative levels of eight miRNAs involved in neuroinflammation and tumor progression in the C20- and HMC3-derived EVs, which in turn reflected a different basal activation state of the two cell types. Our data fill a gap in the community of microglia EVs, in which the preparations from human cells have been poorly characterized so far. Furthermore, these results shed light on both the differences and similarities of EVs extracted from different human microglia cell models, underlining the need to better characterize the features and biological effects of EVs for therein useful and correct application.

Magnetically-actuated microposts stimulate axon growth.

New strategies to promote neuronal regeneration should aim to increase the speed of axonal elongation. Biochemical signaling is a key factor in axon growth, but recent discoveries have shown that mechanical force, through a process referred to as stretch growth, can significantly influence the elongation rate. Here, we develop a method to apply forces to primary hippocampal neurons from mice using magnetic microposts that actuate in response to an external magnetic field. Neurons are cultured onto these microposts and subjected to an average displacement of 0.2 µm at a frequency of 5 Hz. We find that the mechanical stimulation promotes an increase in the length of the axons compared to control conditions. In addition, there is an increase in the density of microtubules and in the amount of cisternae of the endoplasmic reticulum, providing evidence that stretch growth is accompanied by a mass addition to the neurite. Together, these results indicate that magnetically-actuated microposts can accelerate the rate of axon growth, paving the way for future applications in neuronal regeneration. VIDEO ABSTRACT.

Cerium oxide nanoparticles administration during machine perfusion of discarded human livers: A pilot study.

The combined approach of ex situ normothermic machine perfusion (NMP) and nanotechnology represents a strategy to mitigate ischemia/reperfusion injury in liver transplantation (LT). We evaluated the uptake, distribution, and efficacy of antioxidant cerium oxide nanoparticles (nanoceria) during normothermic perfusion of discarded human livers. A total of 9 discarded human liver grafts were randomized in 2 groups and underwent 4 h of NMP: 5 grafts were treated with nanoceria conjugated with albumin (Alb-NC; 50 µg/ml) and compared with 4 untreated grafts. The intracellular uptake of nanoceria was analyzed by electron microscopy (EM) and inductively coupled plasma-mass spectrometry (ICP-MS). The antioxidant activity of Alb-NC was assayed in liver biopsies by glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) assay, telomere length, and 4977-bp common mitochondrial DNA deletion (mtDNA4977 deletion). The cytokine profile was evaluated in perfusate samples. EM and ICP-MS confirmed Alb-NC internalization, rescue of mitochondrial phenotype, decrease of lipid droplet peroxidation, and lipofuscin granules in the treated grafts. Alb-NC exerted an antioxidant activity by increasing GSH levels (percentage change: +94% ± 25%; p = 0.01), SOD (+17% ± 4%; p = 0.02), and CAT activity (51% ± 23%; p = 0.03), reducing the occurrence of mtDNA4977 deletion (-67.2% ± 11%; p = 0.03), but did not affect cytokine release. Alb-NC during ex situ perfusion decreased oxidative stress, upregulating graft antioxidant defense. They could be a tool to improve quality grafts during NMP and represent an antioxidant strategy aimed at protecting the graft against reperfusion injury during LT.

Microscopes, tools, probes, and protocols: A guide in the route of correlative microscopy for biomedical investigation.

In the last decades, the advancements of microscopes technology, together with the development of new imaging approaches, are trying to address some biological questions that have been unresolved in the past: the need to combine in the same analysis temporal, functional and morphological information on the biological sample has become pressing. For this reason, the use of correlative microscopy, in which two or more imaging techniques are combined in the same analysis, is getting increasingly widespread. In fact, correlative microscopy can overcome limitations of a single imaging method, giving access to a larger amount of information from the same specimen. However, correlative microscopy can be challenging, and appropriate protocols for sample preparation and imaging methods must be selected. Here we review the state of the art of correlating electron microscopy with different imaging methods, focusing on sample preparation, tools, and labeling methods, with the aim to provide a comprehensive guide for those scientists who are approaching the field of correlative methods.

Extended criteria grafts and emerging therapeutics strategy in liver transplantation. The unstable balance between damage and repair.

Due to increasing demand for donor organs, "extended criteria" donors are increasingly considered for liver transplantation, including elderly donors and donors after cardiac death. The grafts of this subgroup of donors share a major risk to develop significant features of ischemia reperfusion injury, that may eventually lead to graft failure. Ex-situ machine perfusion technology has gained much interest in liver transplantation, because represents both a useful tool for improving graft quality before transplantation and a platform for the delivery of therapeutics directly to the organ. In this review, we survey ongoing clinical evidences supporting the use of elderly and DCD donors in liver transplantation, and the underlying mechanistic aspects of liver aging and ischemia reperfusion injury that influence graft quality and transplant outcome. Finally, we highlight evidences in the field of new therapeutics to test in MP in the context of recent findings of basic and translational research.

Induction of Axonal Outgrowth in Mouse Hippocampal Neurons via Bacterial Magnetosomes.

Magnetosomes are membrane-enclosed iron oxide crystals biosynthesized by magnetotactic bacteria. As the biomineralization of bacterial magnetosomes can be genetically controlled, they have become promising nanomaterials for bionanotechnological applications. In the present paper, we explore a novel application of magnetosomes as nanotool for manipulating axonal outgrowth via stretch-growth (SG). SG refers to the process of stimulation of axonal outgrowth through the application of mechanical forces. Thanks to their superior magnetic properties, magnetosomes have been used to magnetize mouse hippocampal neurons in order to stretch axons under the application of magnetic fields. We found that magnetosomes are avidly internalized by cells. They adhere to the cell membrane, are quickly internalized, and slowly degrade after a few days from the internalization process. Our data show that bacterial magnetosomes are more efficient than synthetic iron oxide nanoparticles in stimulating axonal outgrowth via SG.

Proteomics analysis of serum small extracellular vesicles for the longitudinal study of a glioblastoma multiforme mouse model.

Longitudinal analysis of disease models enables the molecular changes due to disease progression or therapeutic intervention to be better resolved. Approximately 75 µl of serum can be drawn from a mouse every 14 days. To date no methods have been reported that are able to analyze the proteome of small extracellular vesicles (sEV's) from such low serum volumes. Here we report a method for the proteomics analysis of sEV's from 50 µl of serum. Two sEV isolation procedures were first compared; precipitation based purification (PPT) and size exclusion chromatography (SEC). The methodological comparison confirmed that SEC led to purer sEV's both in terms of size and identified proteins. The procedure was then scaled down and the proteolytic digestion further optimized. The method was then applied to a longitudinal study of serum-sEV proteome changes in a glioblastoma multiforme (GBM) mouse model. Serum was collected at multiple time points, sEV's isolated and their proteins analyzed. The protocol enabled 274 protein groups to be identified and quantified. The longitudinal analysis revealed 25 deregulated proteins in GBM serum sEV's including proteins previously shown to be associated with GBM progression and metastasis (Myh9, Tln-1, Angpt1, Thbs1).

Electron Diffraction on Flash-Frozen Cowlesite Reveals the Structure of the First Two-Dimensional Natural Zeolite.

Cowlesite, ideally Ca6Al12Si18O60·36H2O, is to date the only natural zeolite whose structure could not be determined by X-ray methods. In this paper, we present the ab initio structure determination of this mineral obtained by three-dimensional (3D) electron diffraction data collected from single-crystal domains of a few hundreds of nanometers. The structure of cowlesite consists of an alternation of rigid zeolitic layers and low-density interlayers supported by water and cations. This makes cowlesite the only two-dimensional (2D) zeolite known in nature. When cowlesite gets in contact with a transmission electron microscope vacuum, a phase transition to a conventional 3D zeolite framework occurs in few seconds. The original cowlesite structure could be preserved only by adopting a cryo-plunging sample preparation protocol usually employed for macromolecular samples. Such a protocol allows the investigation by 3D electron diffraction of very hydrated and very beam-sensitive inorganic materials, which were previously considered intractable by transmission electron microscopy crystallographic methods.

Uranium-free X solution: a new generation contrast agent for biological samples ultrastructure.

Biological samples are mainly composed of elements with a low atomic number which show a relatively low electron scattering power. For Transmission Electron Microscopy analysis, biological samples are generally embedded in resins, which allow thin sectioning of the specimen. Embedding resins are also composed by light atoms, thus the contrast difference between the biological sample and the surrounding resin is minimal. Due to that reason in the last decades, several staining solutions and approaches, performed with heavy metal salts, have been developed with the purpose of enhancing both the intrinsic sample contrast and the differences between the sample and resin. The best staining was achieved with the uranyl acetate (UA) solution, which has been the election method for the study of morphology in biological samples. More recently several alternatives for UA have been proposed to get rid of its radiogenic issues, but to date none of these solutions has achieved efficiencies comparable to UA. In this work, we propose a different staining solution (X Solution or X SOL), characterized by lanthanide polyoxometalates (LnPOMs) as heavy atoms source, which could be used alternatively to UA in negative staining (NS), in en bloc staining, and post sectioning staining (PSS) of biological samples. Furthermore, we show an extensive chemical characterization of the LnPOM species present in the solution and the detailed work for its final formulation, which brought remarkable results, and even better performances than UA.

Extremely Low Forces Induce Extreme Axon Growth.

Stretch-growth has been defined as a process that extends axons via the application of mechanical forces. In the present article, we used a protocol based on magnetic nanoparticles (NPs) for labeling the entire axon tract of hippocampal neurons, and an external magnetic field gradient to generate a dragging force. We found that the application of forces below 10 pN induces growth at a rate of 0.66 ± 0.02 µm h-1 pN-1 Calcium imaging confirmed the strong increase in elongation rate, in comparison with the condition of tip-growth. Enhanced growth in stretched axons was also accompanied by endoplasmic reticulum (ER) accumulation and, accordingly, it was blocked by an inhibition of translation. Stretch-growth was also found to stimulate axonal branching, glutamatergic synaptic transmission, and neuronal excitability. Moreover, stretched axons showed increased microtubule (MT) density and MT assembly was key to sustaining stretch-growth, suggesting a possible role of tensile forces in MT translocation/assembly. Additionally, our data showed that stretched axons do not respond to BDNF signaling, suggesting interference between the two pathways. As these extremely low mechanical forces are physiologically relevant, stretch-growth could be an important endogenous mechanism of axon growth, with a potential for designing novel strategies for axonal regrowth.SIGNIFICANCE STATEMENT Axon growth involves motion, and motion is driven by forces. The growth cone (GC) itself can generate very low intracellular forces by inducing a drastic cytoskeleton remodeling, in response to signaling molecules. Here, we investigated the key role of intracellular force as an endogenous regulator of axon outgrowth, which it has been neglected for decades because of the lack of methodologies to investigate the topic. Our results indicate a critical role of force in promoting axon growth by facilitating microtubule (MT) polymerization.

Endogenously-Activated Ultrasmall-in-Nano Therapeutics: Assessment on 3D Head and Neck Squamous Cell Carcinomas.

Negative or positive HPV-associated Head and Neck Squamous Cell Carcinomas (HNSCCs) are high recurrence neoplasms usually resulting in a poor prognosis, mainly due to metastasis formation. Despite the low overall patient survival rate and the severe side effects, the treatment of choice is still cisplatin-based chemotherapy. Here, we report a straightforward protocol for the production of high throughput 3D models of negative or positive HPV-associated HNSCCs, together with their employment in the therapeutic evaluation of gold ultrasmall-in-nano architectures comprising an endogenously-activatable cisplatin prodrug. Beyond enhancing the biosafety of cisplatin, our approach paves the way for the establishment of synergistic co-therapies for HNSCCs based on excretable noble metals.

Effects of fixatives on myelin molecular order probed with RP-CARS microscopy.

When live imaging is not feasible, sample fixation allows preserving the ultrastructure of biological samples for subsequent microscopy analysis. This process could be performed with various methods, each one affecting differently the biological structure of the sample. While these alterations were well-characterized using traditional microscopy, little information is available about the effects of the fixatives on the spatial molecular orientation of the biological tissue. We tackled this issue by employing rotating-polarization coherent anti-Stokes Raman scattering (RP-CARS) microscopy to study the effects of different fixatives on the myelin sub-micrometric molecular order and micrometric morphology. RP-CARS is a novel technique derived from CARS microscopy that allows probing spatial orientation of molecular bonds while maintaining the intrinsic chemical selectivity of CARS microscopy. By characterizing the effects of the fixation procedures, the present work represents a useful guide for the choice of the best fixation technique(s), in particular for polarization-resolved CARS microscopy. Finally, we show that the combination of paraformaldehyde and glutaraldehyde can be effectively employed as a fixative for RP-CARS microscopy, as long as the effects on the molecular spatial distribution, here characterized, are taken into account.

Graphene Promotes Axon Elongation through Local Stall of Nerve Growth Factor Signaling Endosomes.

Several works reported increased differentiation of neuronal cells grown on graphene; however, the molecular mechanism driving axon elongation on this material has remained elusive. Here, we study the axonal transport of nerve growth factor (NGF), the neurotrophin supporting development of peripheral neurons, as a key player in the time course of axonal elongation of dorsal root ganglion neurons on graphene. We find that graphene drastically reduces the number of retrogradely transported NGF vesicles in favor of a stalled population in the first 2 days of culture, in which the boost of axon elongation is observed. This correlates with a mutual charge redistribution, observed via Raman spectroscopy and electrophysiological recordings. Furthermore, ultrastructural analysis indicates a reduced microtubule distance and an elongated axonal topology. Thus, both electrophysiological and structural effects can account for graphene action on neuron development. Unraveling the molecular players underneath this interplay may open new avenues for axon regeneration applications.

Production of 3D Tumor Models of Head and Neck Squamous Cell Carcinomas for Nanotheranostics Assessment.

As a first approach, standard 2D cell culture techniques are usually employed for the screening of drugs and nanomaterials. Despite the easy handling, findings achieved on 2D cultures are often not efficiently translatable to in vivo preclinical investigations. Furthermore, although animal models are pivotal in preclinical studies, more strict directives have been implemented to promote the use of alternative biological systems. In this context, the development and integration into preclinical research workflow of 3D neoplasm models is particularly appealing to promote the advancement and success of therapeutics in clinical trials while reducing the number of in vivo models. Indeed, 3D tumor models bridge several discrepancies between 2D cell culture and in vivo models, among which are morphology, polarity, drug penetration, osmolality, and gene expressions. Here, we comprehensively describe a robust and high-throughput hanging drop protocol for the production of 3D models of both Human Papillomavirus (HPV)-positive and HPV-negative head and neck squamous cell carcinomas (HNSCCs). We also report the standard cascade assays for their characterization and demonstrate their significance in investigations on these aggressive neoplasms. The employment of relevant 3D cancer models is pivotal to produce more reliable and robust findings in terms of biosafety, theranostic efficacy, and biokinetics as well as to promote further knowledge on HNSCC pathophysiology.

Ag@TiO2 nanogranular films by gas phase synthesis as hybrid SERS platforms.

The synthesis of hybrid metallic-dielectric substrates as reliable SERS platforms relies on core-shell nanoparticles, obtained by wet chemistry, with an outer dielectric shell composed of SiO2 or TiO2. Apart from the shell composition, the nanoparticle density and aggregation type strongly affect the surface-enhanced SERS. Going beyond a single layer by building random aggregates of hybrid NPs would result in a step forward in the production of reliable hybrid SERS platforms. Here we achieve the fabrication of a 3D nanogranular film of Ag metallic cores not fully enclosed in a TiO2 capping layer, defined as a Ag@TiO2 quasi-shell-isolated Raman substrate (Ag@TiO2 QuaSIRS) by an environmentally friendly gas phase synthesis technique (SCBD). The Ag core drives the electromagnetic enhancement with plasmonic hotspots while the TiO2 shell passivates it and leads to different possible surface functionalization. The SERS capabilities of the Ag@TiO2 QuaSIRS peak at a film thickness of 60 nm providing a detection limit of 10-9 M concentration for Methylene Blue at 632.81 nm. The importance of the nanogranular 3D morphology is evidenced by the very good detection of analytes dispersed in aqueous solutions, since the liquid can penetrate the pores hence exploiting most of the plasmonic hotspots present in the film. The versatility of SCBD to deposit such reliable hybrid SERS platforms by a single step at room temperature over different substrates provides an opportunity to design a new generation of hybrid SERS-active substrates based on hybrid nanoparticles.

Effects of cerium oxide nanoparticles on hemostasis: Coagulation, platelets, and vascular endothelial cells.

Cerium oxide nanoparticles (nanoceria [NC]) have attracted much attention in biomedicine due to their surface composition that confers interesting redox activities and regenerative properties. Studies have demonstrated that the application of NPs in biomedicine can influence components of hemostatic system, inducing blood clotting, alterations of blood cells, and endothelial cell functions. NC were tested in vitro to assess their hemocompatibility and anticoagulant, anti-inflammatory, and anti-senescence activity in human endothelial cells. Hemocompatibility has been evaluated in vitro looking at the impact of NC on coagulation times, fibrinogen, and platelet aggregation. The effect of NC on vascular endothelial cells were assayed by testing cell viability, antioxidant activity, anticoagulant (tissue factor [TF]-mRNA expression) and anti-inflammatory properties (VCAM-1 exposure, cytokine release), and senescence (telomere shortening). NC did not show significant effects on coagulation process, hemolysis, or platelet aggregation. In endothelial cells, NC did not affect cell viability, reduced oxidative stress, inhibited mRNA-TF expression, VCAM-1 expression, and cytokine release. Moreover, NC reduce telomere shortening, possibly counteracting premature senescence. The hemocompatibility combined with anticoagulant and anti-inflammatory phenotype and the ability of counteract the premature senescence in vascular cells make NC a promising therapeutic tool in oxidative stress-related conditions. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2019.

Insulin secretory granules labelled with phogrin-fluorescent proteins show alterations in size, mobility and responsiveness to glucose stimulation in living ß-cells.

The intracellular life of insulin secretory granules (ISGs) from biogenesis to secretion depends on their structural (e.g. size) and dynamic (e.g. diffusivity, mode of motion) properties. Thus, it would be useful to have rapid and robust measurements of such parameters in living ß-cells. To provide such measurements, we have developed a fast spatiotemporal fluctuation spectroscopy. We calculate an imaging-derived Mean Squared Displacement (iMSD), which simultaneously provides the size, average diffusivity, and anomalous coefficient of ISGs, without the need to extract individual trajectories. Clustering of structural and dynamic quantities in a multidimensional parametric space defines the ISGs' properties for different conditions. First, we create a reference using INS-1E cells expressing proinsulin fused to a fluorescent protein (FP) under basal culture conditions and validate our analysis by testing well-established stimuli, such as glucose intake, cytoskeleton disruption, or cholesterol overload. After, we investigate the effect of FP-tagged ISG protein markers on the structural and dynamic properties of the granule. While iMSD analysis produces similar results for most of the lumenal markers, the transmembrane marker phogrin-FP shows a clearly altered result. Phogrin overexpression induces a substantial granule enlargement and higher mobility, together with a partial de-polymerization of the actin cytoskeleton, and reduced cell responsiveness to glucose stimulation. Our data suggest a more careful interpretation of many previous ISG-based reports in living ß-cells. The presented data pave the way to high-throughput cell-based screening of ISG structure and dynamics under various physiological and pathological conditions.