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.

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.

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.

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.

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.

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.

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.

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.

Pectin-coated boron nitride nanotubes: In vitro cyto-/immune-compatibility on RAW 264.7 macrophages.

BACKGROUND: Boron nitride nanotubes (BNNTs) represent a new opportunity for drug delivery and clinical therapy. The present work has the objective to investigate pectin-coated BNNTs (P-BNNTs) for their biocompatibility on macrophage cultures, since these cells are among the first components of the immune system to interact with administered nanoparticles. METHODS: As first step, the potential toxicity of P-BNNTs is verified in terms of proliferation, oxidative stress induction and apoptosis/necrosis phenomena. Thereafter, the modulation of immune cell response following P-BNNT exposure is evaluated at gene and protein level, in particular focusing on cytokine release. Finally, P-BNNT internalization is assessed through transmission electron microscopy and confocal microscopy. RESULTS: The results proved that P-BNNTs are not toxic for macrophages up to 50 µg/ml after 24 h of incubation. The cytokine expression is not affected by P-BNNT administration both at gene and protein level. Moreover, P-BNNTs are internalized by macrophages without impairments of the cell structures. CONCLUSIONS: Collected data suggest that P-BNNTs cause neither adverse effects nor inflammation processes in macrophages. GENERAL SIGNIFICANCE: These findings represent the first and fundamental step in immune compatibility evaluation of BNNTs, mandatory before any further pre-clinical testing.

Gelatin/nanoceria nanocomposite fibers as antioxidant scaffolds for neuronal regeneration.

BACKGROUND: The design of efficient nerve conduits able to sustain the axonal outgrowth and its guidance towards appropriate targets is of paramount importance in nerve tissue engineering. METHODS: In this work, we propose the preparation of highly aligned nanocomposite fibers of gelatin/cerium oxide nanoparticles (nanoceria), prepared by electrospinning. Nanoceria are powerful self-regenerative antioxidant nanomaterials, that behave as strong reactive oxygen species scavengers, and among various beneficial effects, they have been proven to inhibit the cell senescence and to promote the neurite sprouting. RESULTS: After a detailed characterization of the developed substrates, they have been tested on neuron-like SH-SY5Y cells, demonstrating strong antioxidant properties and beneficial multi-cue effects in terms of neurite development and alignment. CONCLUSIONS: Obtained findings suggest efficiency of the proposed substrates in providing combined topographical stimuli and antioxidant effects to cultured cells. GENERAL SIGNIFICANCE: Proposed nanocomposite scaffolds represent a promising approach for nerve tissue engineering and regenerative medicine.

Design, synthesis and docking study of novel coumarin ligands as potential selective acetylcholinesterase inhibitors.

New coumaryl-thiazole derivatives with the acetamide moiety as a linker between the alkyl chains and/or the heterocycle nucleus were synthesized and in vitro tested as acetylcholinesterase (AChE) inhibitors. 2-(diethylamino)-N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)acetamide (6c, IC50 value of 43 nM) was the best AChE inhibitor with a selectivity index of 4151.16 over BuChE. Kinetic study of AChE inhibition revealed that 6c was a mixed-type inhibitor. Moreover, the result of H4IIE hepatoma cell toxicity assay for 6c showed negligible cell death. Molecular docking studies were also carried out to clarify the inhibition mode of the more active compounds. Best pose of compound 6c is positioned into the active site with the coumarin ring wedged between the residues of the CAS and catalytic triad of AChE. In addition, the coumarin ring is anchored into the gorge of the enzyme by H-bond with Tyr130.

Neuromuscular Junction Dismantling in Amyotrophic Lateral Sclerosis.

Neuromuscular junction assembly and plasticity during embryonic, postnatal, and adult life are tightly regulated by the continuous cross-talk among motor nerve endings, muscle fibers, and glial cells. Altered communications among these components is thought to be responsible for the physiological age-related changes at this synapse and possibly for its destruction in pathological states. Neuromuscular junction dismantling plays a crucial role in the onset of Amyotrophic Lateral Sclerosis (ALS). ALS is characterized by the degeneration and death of motor neurons leading to skeletal muscle denervation, atrophy and, most often, death of the patient within five years from diagnosis. ALS is a non-cell autonomous disease as, besides motor neuron degeneration, glial cells, and possibly muscle fibers, play a role in its onset and progression. Here, we will review the recent literature regarding the mechanisms leading to neuromuscular junction disassembly and muscle denervation focusing on the role of the three players of this peripheral tripartite synapse.

Effects of cerium oxide nanoparticles on PC12 neuronal-like cells: proliferation, differentiation, and dopamine secretion.

PURPOSE: Oxidative stress has been found to play a key role in several diseases, that range from cancer to neurodegenerative disorders. Besides traditional anti-oxidant agents, in recent years much attention has been focused on nanotechnological solutions, including cerium oxide nanoparticles (nanoceria). METHODS: Thanks to its extraordinary catalytic properties, nanoceria mimics the activity of superoxide dismutase and of catalase, therefore acting as a reactive oxygen species (ROS) scavenger in many biological contexts. In this paper, we report on nanoceria interactions with PC12 cell line, that represents a valuable model for many features of central dopaminergic neurons. RESULTS: Nanoceria confirmed a strong anti-ROS action but, most interestingly, also showed beneficial effects on both cell differentiation and dopamine production. CONCLUSIONS: Even if deeper examinations will be necessary in order to better clarify the mechanisms at the base of the documented effects, nanoceria demonstrated a significant potential as pharmacological agent in the treatment of neurological disorders.

Analysis of neuromuscular junctions and effects of anabolic steroid administration in the SOD1G93A mouse model of ALS.

Several lines of evidence indicate that neuromuscular junction (NMJ) destruction and disassembly is an early phenomenon in amyotrophic lateral sclerosis (ALS). Here we analyzed by confocal and electron microscopy the NMJ structure in the diaphragm of SOD1G93A mice at symptom onset. In these mice, which provide a model for familial ALS, diaphragm denervation (~50%) as well as gastrocnemius denervation (~40%) was found. In addition, the size of the synaptic vesicle pool was reduced and alterations of mitochondria were observed in approximately 40% of the remaining presynaptic terminals. Chronic treatment of SOD1G93A mice with the anabolic steroid nandrolone during the presymptomatic stage preserved the diaphragm muscle mass and features indicative of synaptic activity. These features were represented by the number of vesicles docked within 200 nm from the presynaptic membrane and area of acetylcholine receptor clusters. Structural preservation of mitochondria was documented in presynaptic terminals. However, innervation of diaphragm muscle fibers was only slightly increased in nandrolone-treated SOD1-mutant mice. Altogether the results point out and define fine structural alterations of diaphragm NMJs in the murine model of familial ALS at symptom onset, and indicate that nandrolone may prevent or delay structural alterations in NMJ mitochondria and stimulate presynaptic activity but does not prevent muscle denervation during the disease.

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.

Cholesterol reduction impairs exocytosis of synaptic vesicles.

Cholesterol and sphingolipids are abundant in neuronal membranes, where they help the organisation of the membrane microdomains involved in major roles such as axonal and dendritic growth, and synapse and spine stability. The aim of this study was to analyse their roles in presynaptic physiology. We first confirmed the presence of proteins of the exocytic machinery (SNARES and Ca(v)2.1 channels) in the lipid microdomains of cultured neurons, and then incubated the neurons with fumonisin B (an inhibitor of sphingolipid synthesis), or with mevastatin or zaragozic acid (two compounds that affect the synthesis of cholesterol by inhibiting HMG-CoA reductase or squalene synthase). The results demonstrate that fumonisin B and zaragozic acid efficiently decrease sphingolipid and cholesterol levels without greatly affecting the viability of neurons or the expression of synaptic proteins. Electron microscopy showed that the morphology and number of synaptic vesicles in the presynaptic boutons of cholesterol-depleted neurons were similar to those observed in control neurons. Zaragozic acid (but not fumonisin B) treatment impaired synaptic vesicle uptake of the lipophilic dye FM1-43 and an antibody directed against the luminal epitope of synaptotagmin-1, effects that depended on the reduction in cholesterol because they were reversed by cholesterol reloading. The time-lapse confocal imaging of neurons transfected with ecliptic SynaptopHluorin showed that cholesterol depletion affects the post-depolarisation increase in fluorescence intensity. Taken together, these findings show that reduced cholesterol levels impair synaptic vesicle exocytosis in cultured neurons.

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.

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.

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.