DNA origami scaffold promoting nerve guidance and regeneration.

Self-assembly of biological elements into biomimetic cargo carriers for targeting and delivery is a promising approach. However, it still holds practical challenges. We developed a functionalization approach of DNA origami (DO) nanostructures with neuronal growth factor (NGF) for manipulating neuronal systems. NGF bioactivity and its interactions with the neuronal system were demonstrated in vitro and in vivo models. The DO elements fabricated by molecular self-assembly have manipulated the surrounding environment through static spatially and temporally controlled presentation of ligands to the cell surface receptors. Our data showed effective bioactivity in differentiating PC12 cells in vitro. Furthermore, the DNA origami NGF (DON) affected the growth directionality and spatial capabilities of dorsal root ganglion neurons in culture by introducing a chemotaxis effect along a gradient of functionalized DO structures. Finally, we showed that these elements provide enhanced axonal regeneration in a rat sciatic nerve injury model in vivo. This study is a proof of principle for the functionality of DO in neuronal manipulation and regeneration. The approach proposed here, of an engineered platform formed out of programmable nanoscale elements constructed of DO, could be extended beyond the nervous system and revolutionize the fields of regenerative medicine, tissue engineering, and cell biology.

The Beneficial Effect of the First COVID-19 Lockdown on Undergraduate Students of Education: Prospective Cohort Study.

BACKGROUND: The COVID-19 pandemic has been spreading consistently since the beginning of 2020. On February 27, 2020, the first patient with coronavirus was diagnosed in Israel. On March 14, 2020, the Israeli government declared a general lockdown that lasted about a month, which altered the lives of the entire population. OBJECTIVE: The objective of this paper is to evaluate the change in the well-being, physical activity, and sleep quality of undergraduate students of education at 2 time points: before (November 2019) and during (April 2020) the first COVID-19 lockdown. METHODS: In total, 533 undergraduate students of education submitted an online questionnaire before the lockdown and at its end. The questionnaire comprised 4 parts: a (1) sociodemographic and (2) weekly exercise questionnaire taken from the International Physical Activity Questionnaire-Short Form; (3) sleep quality, rated using the Mini Sleep Questionnaire; and (4) well-being, rated using the short version of the Mental Health Inventory. This was a pre-post prospective cohort questionnaire study. RESULTS: It was predicted that there would be a decrease in the aforementioned parameters. Contrary to all expectations, an increase was observed in all 3. Results showed that during the lockdown, there was an increase in the level of exercise students engaged in. Overall, 102 (61.4%) of 166 students engaged in a greater amount of physical activity during the COVID-19 lockdown compared to 150 (40.9%) of 367 students who engaged in a greater amount of physical activity before COVID-19. Levels of sleep quality (mean 5.34 [SD 0.92] vs mean 5.12 [SD 0.46], P=.02) and well-being (mean 3.79 [SD 0.62] vs mean 3.67 [SD 0.59], P=.02) were also higher during the COVID-19 lockdown. CONCLUSIONS: These findings indicate that undergraduate students seem to have taken advantage of the change in lifestyle due to the lockdown, directing the free time toward improving health by engaging in more physical activity, thus improving sleep quality and well-being.

Large-scale acoustic-driven neuronal patterning and directed outgrowth.

Acoustic manipulation is an emerging non-invasive method enabling precise spatial control of cells in their native environment. Applying this method for organizing neurons is invaluable for neural tissue engineering applications. Here, we used surface and bulk standing acoustic waves for large-scale patterning of Dorsal Root Ganglia neurons and PC12 cells forming neuronal cluster networks, organized biomimetically. We showed that by changing parameters such as voltage intensity or cell concentration we were able to affect cluster properties. We examined the effects of acoustic arrangement on cells atop 3D hydrogels for up to 6 days and showed that assembled cells spontaneously grew branches in a directed manner towards adjacent clusters, infiltrating the matrix. These findings have great relevance for tissue engineering applications as well as for mimicking architectures and properties of native tissues.

Magnetic Targeting of mTHPC To Improve the Selectivity and Efficiency of Photodynamic Therapy.

Photodynamic therapy (PDT) is a promising recognized treatment for cancer. To date, PDT drugs are injected systemically, and the tumor area is irradiated to induce cell death. Current clinical protocols have several drawbacks, including limited accessibility to solid tumors and insufficient selectivity of drugs. Herein, we propose an alternative approach to improve PDT effectiveness by magnetic targeting of responsive carriers conjugated to the PDT drug. We coordinatively attached a meso-tetrahydroxyphenylchlorin (mTHPC) photosensitizer to Ce-doped-γ-Fe2O3 maghemite nanoparticles (MNPs). These MNPs are superparamagnetic and biocompatible, and the resulting mTHPC-MNPs nanocomposites are stable in aqueous suspensions. MDA-MB231 (human breast cancer) cells incubated with the mTHPC-MNPs showed high uptake and high death rates in cell population after PDT. The exposure to external magnetic forces during the incubation period directed the nanocomposites to selected sites enhancing drug accumulation that was double that of cells with no magnetic exposure. Next, breast cancer tumors were induced subcutaneously in mice and treated magnetically. In vivo results showed accelerated drug accumulation in tumors of mice injected with mTHPC-MNP nanocomposites, compared to the free drug. PDT irradiation led to a decrease in tumor size of both groups, whereas treatment with the focused magnetic nanocomposites led to significant tumor regression. Our results demonstrate a method to improve the current PDT treatments by applying magnetic forces to effectively direct the drug to cancerous tissue. This approach leads to a highly localized and effective PDT process, opening new directions for clinical PDT protocols.

Neuroprotective Effect of Nerve Growth Factor Loaded in Porous Silicon Nanostructures in an Alzheimer's Disease Model and Potential Delivery to the Brain.

Nerve growth factor (NGF) plays a vital role in reducing the loss of cholinergic neurons in Alzheimer's disease (AD). However, its delivery to the brain remains a challenge. Herein, NGF is loaded into degradable oxidized porous silicon (PSiO2 ) carriers, which are designed to carry and continuously release the protein over a 1 month period. The released NGF exhibits a substantial neuroprotective effect in differentiated rat pheochromocytoma PC12 cells against amyloid-beta (Aß)-induced cytotoxicity, which is associated with Alzheimer's disease. Next, two potential localized administration routes of the porous carriers into murine brain are investigated: implantation of PSiO2 chips above the dura mater, and biolistic bombardment of PSiO2 microparticles through an opening in the skull using a pneumatic gene gun. The PSiO2 -implanted mice are monitored for a period of 8 weeks and no inflammation or adverse effects are observed. Subsequently, a successful biolistic delivery of these highly porous microparticles into a live-mouse brain is demonstrated for the first time. The bombarded microparticles are observed to penetrate the brain and reach a depth of 150 µm. These results pave the way for using degradable PSiO2 carriers as potential localized delivery systems for NGF to the brain.

Magnetic Targeting of Growth Factors Using Iron Oxide Nanoparticles.

Growth factors play an important role in nerve regeneration and repair. An attractive drug delivery strategy, termed "magnetic targeting", aims to enhance therapeutic efficiency by directing magnetic drug carriers specifically to selected cell populations that are suitable for the nervous tissues. Here, we covalently conjugated nerve growth factor to iron oxide nanoparticles (NGF-MNPs) and used controlled magnetic fields to deliver the NGF⁻MNP complexes to target sites. In order to actuate the magnetic fields a modular magnetic device was designed and fabricated. PC12 cells that were plated homogenously in culture were differentiated selectively only in targeted sites out of the entire dish, restricted to areas above the magnetic "hot spots". To examine the ability to guide the NGF-MNPs towards specific targets in vivo, we examined two model systems. First, we injected and directed magnetic carriers within the sciatic nerve. Second, we injected the MNPs intravenously and showed a significant accumulation of MNPs in mouse retina while using an external magnet that was placed next to one of the eyes. We propose a novel approach to deliver drugs selectively to injured sites, thus, to promote an effective repair with minimal systemic side effects, overcoming current challenges in regenerative therapeutics.

meso-Tetrahydroxyphenylchlorin-Conjugated Gold Nanoparticles as a Tool To Improve Photodynamic Therapy.

Photodynamic therapy (PDT) is a promising therapeutic modality for cancer. However, current protocols using bare drugs suffer from several limitations that impede its beneficial clinical effects. Here, we introduce a new approach for an efficient PDT treatment. It involves conjugating a PDT agent, meso-tetrahydroxyphenylchlorin (mTHPC) photosensitizer, to gold nanoparticles (AuNPs) that serve as carriers for the drug. AuNPs have a number of characteristics that make them highly suitable to function as drug carriers: they are biocompatible, serve as biomarkers, and function as contrast agents in vitro and in vivo. We synthesized AuNPs and covalently conjugated the mTHPC drug molecules through a linker. The resultant functional complex, AuNP-mTHPC, is a stable, soluble compound. SH-SY5Y human neuroblastoma cells were incubated with the complex, showing possible administration of higher doses of drug when conjugated to the AuNPs. Then cells were irradiated with a laser beam at 650 nm to mimic the PDT procedure. Our study shows higher rates of cell death in cells incubated with the AuNP-mTHPC complex compared to the incubation with the free drug. Using the new complex may form the basis for a better PDT strategy for a wide range of cancers.

Mechanically Oriented 3D Collagen Hydrogel for Directing Neurite Growth.

Recent studies in the field of neuro-tissue engineering have demonstrated the promising effects of aligned contact guidance cue to scaffolds of enhancement and direction of neuronal growth. In vivo, neurons grow and develop neurites in a complex three-dimensional (3D) extracellular matrix (ECM) surrounding. Studies have utilized hydrogel scaffolds derived from ECM molecules to better simulate natural growth. While many efforts have been made to control neuronal growth on 2D surfaces, the development of 3D scaffolds with an elaborate oriented topography to direct neuronal growth still remains a challenge. In this study, we designed a method for growing neurons in an aligned and oriented 3D collagen hydrogel. We aligned collagen fibers by inducing controlled uniaxial strain on gels. To examine the collagen hydrogel as a suitable scaffold for neuronal growth, we evaluated the physical properties of the hydrogel and measured collagen fiber properties. By combining the neuronal culture in 3D collagen hydrogels with strain-induced alignment, we were able to direct neuronal growth in the direction of the aligned collagen matrix. Quantitative evaluation of neurite extension and directionality within aligned gels was performed. The analysis showed neurite growth aligned with collagen matrix orientation, while maintaining the advantageous 3D growth.

Prolonged controlled delivery of nerve growth factor using porous silicon nanostructures.

Although nerve growth factor (NGF) is beneficial for the treatment of numerous neurological and non-neurological diseases, its therapeutic administration represents a significant challenge, due to the difficulty to locally deliver relevant doses in a safe and non-invasive manner. In this work, we employ degradable nanostructured porous silicon (PSi) films as carriers for NGF, allowing its continuous and prolonged release, while retaining its bioactivity. The PSi carriers exhibit high loading efficacy (up to 90%) of NGF and a continuous release, with no burst, over a period of>26days. The released NGF bioactivity is compared to that of free NGF in both PC12 cells and dissociated dorsal root ganglion (DRG) neurons. We show that the NGF has retained its bioactivity and induces neurite outgrowth and profound differentiation (of >50% for PC12 cells) throughout the period of release within a single administration. Thus, this proof-of-concept study demonstrates the immense therapeutic potential of these tunable carriers as long-term implants of NGF reservoirs and paves the way for new localized treatment strategies of neurodegenerative diseases.

Topographical impact of silver nanolines on the morphology of neuronal SH-SY5Y Cells.

An extracellular environment is critical in neuronal development and growth. Changes in neuronal morphology, neuron adhesion, and even the rate of neurite formation, can be modified by both the chemical and physical properties of interfacing substrates. Topography has a major impact on neuronal growth. Neuronal behavior and morphology are affected by the size, shape and pattern of the topographic elements. Combining topography with active materials may lead to enhanced influence. This paper demonstrates the effects of silver nanolines (AgNLs) on the growth pattern of SH-SY5Y cells. The morphology of the cells atop the nanotopographical substrates is measured, revealing a significant promoting effect. The number of neurites initiating from the soma is larger in SH-SY5Y cells plated on AgNLs than in control samples. The cells also exhibit an increase in neurite branching points towards more complex structures. These results indicate that substrates decorated with nanotopography affect cellular growth in a way that may be useful for enhanced regeneration, opening new possibilities for electrode and implant design.

De novo transcriptome assembly databases for the central nervous system of the medicinal leech.

The study of non-model organisms stands to benefit greatly from genetic and genomic data. For a better understanding of the molecular mechanisms driving neuronal development, and to characterize the entire leech Hirudo medicinalis central nervous system (CNS) transcriptome we combined Trinity for de-novo assembly and Illumina HiSeq2000 for RNA-Seq. We present a set of 73,493 de-novo assembled transcripts for the leech, reconstructed from RNA collected, at a single ganglion resolution, from the CNS. This set of transcripts greatly enriches the available data for the leech. Here, we share two databases, such that each dataset allows a different type of search for candidate homologues. The first is the raw set of assembled transcripts. This set allows a sequence-based search. A comprehensive analysis of which revealed 22,604 contigs with high e-values, aligned versus the Swiss-Prot database. This analysis enabled the production of the second database, which includes correlated sequences to annotated transcript names, with the confidence of BLAST best hit.

Promotion of neural sprouting using low-level green light-emitting diode phototherapy.

We irradiated neuroblastoma SH-SY5Y cell line with low-level light-emitting diode (LED) illumination at a visible wavelength of 520 nm (green) and intensity of 100 mW∕cm2. We captured and analyzed the cell morphology before LED treatment, immediately after, and 12 and 24 h after treatment. Our study demonstrated that LED illumination increases the amount of sprouting dendrites in comparison to the control untreated cells. This treatment also resulted in more elongated cells after treatment in comparison to the control cells and higher levels of expression of a differentiation related gene. This result is a good indication that the proposed method could serve in phototherapy treatment for increasing sprouting and enhancing neural network formation.

Spatial regulation dominates gene function in the ganglia chain.

MOTIVATION: To understand the molecular mechanisms of neurons, it is imperative to identify genomic dissimilarities within the heterogeneity of the neural system. This is especially true for neuronal disorders in which spatial considerations are of critical nature. For this purpose, Hirudo medicinalis provides here an ideal system in which we are able to follow gene expression along the central nervous system, to affiliate location with gene behavior. RESULTS: In all, 221.1 million high-quality short reads were sequenced on the Illumina Hiseq2000 platform at the single ganglion level. Thereafter, a de novo assembly was performed using two state-of-the-art assemblers, Trinity and Trans-ABySS, to reconstruct a comprehensive de novo transcriptome. Classification of Trinity and Trans-ABySS transcripts produced a non-redundant set of 76 845 and 268 355 transcripts (>200 bp), respectively. Remarkably, using Trinity, 82% of the published medicinal leech messenger RNAs was identified. For the innexin family, all of the 21 recently reported genes were identified. Spatial regulation analysis across three ganglia throughout the entire central nervous system revealed distinct patterns of gene expression. These transcriptome data were combined with expression distribution to produce a spatio-transcripto map along the ganglia chain. This study provides a resource for gene discovery and gene regulation in future studies.

Genetic manipulation of CD74 in mouse strains of different backgrounds can result in opposite responses to central nervous system injury.

The ability to recover from CNS injuries is strain dependent. Transgenic mice that weakly express the p41 CD74 isoform (an integral membrane protein functioning as a MHC class II chaperone) on an I-A(b) genetic background have normal CD4(+) T cell populations and normal surface expression of MHC class II, but their B cell development is arrested while the cells are still immature. After a CNS injury, these mice recover better than their matched wild-type controls. We generated p41-transgenic mice on an I-A(d) background (p41-I-A(d) mice), and found that their recovery from CNS injuries was worse than that of controls. A correlative inverse effect was seen with respect to the kinetics of T cell and B cell recruitment to the injured CNS and the expression of insulin-like growth factor at the lesion site. These results, besides verifying previous findings that B cells function in the damaged CNS, demonstrate that the outcome of a particular genetic manipulation may be strain dependent.

Post-intoxication vaccination for protection of neurons against the toxicity of nerve agents.

Nerve agents are highly toxic organophosphates (OPs) that can cause severe damage to the central and peripheral nervous systems. The central nervous system insult results in seizures and neuronal death. The glutamatergic system apparently contributes to the neuropathology. Using a model of OP intoxication causing death of retinal ganglion cells in the mouse eye, we show here that intoxication is exacerbated if the mice are devoid of mature T cells. The retinal neurons could be protected from these effects by vaccination, 7 days before or immediately after intoxication, with the copolymer glatiramer acetate (Cop-1), recently found to limit the usual consequences of an acute glutamate insult to the eye. These findings underlie a new therapeutic approach to protection against OP intoxication, based on the rationale that boosting of the adaptive immunity recruited at the site of intoxication helps the local cellular machinery such as resident microglia to withstand the neurotoxic effects.

A disaccharide derived from chondroitin sulphate proteoglycan promotes central nervous system repair in rats and mice.

Chondroitin sulphate proteoglycan (CSPG) inhibits axonal regeneration in the central nervous system (CNS) and its local degradation promotes repair. We postulated that the enzymatic degradation of CSPG generates reparative products. Here we show that an enzymatic degradation product of CSPG, a specific disaccharide (CSPG-DS), promoted CNS recovery by modulating both neuronal and microglial behaviour. In neurons, acting via a mechanism that involves the PKCalpha and PYK2 intracellular signalling pathways, CSPG-DS induced neurite outgrowth and protected against neuronal toxicity and axonal collapse in vitro. In microglia, via a mechanism that involves ERK1/2 and PYK2, CSPG-DS evoked a response that allowed these cells to manifest a neuroprotective phenotype ex vivo. In vivo, systemically or locally injected CSPG-DS protected neurons in mice subjected to glutamate or aggregated beta-amyloid intoxication. Our results suggest that treatment with CSPG-DS might provide a way to promote post-traumatic recovery, via multiple cellular targets.

Protective autoimmunity against the enemy within: fighting glutamate toxicity.

Glutamate, a key neurotransmitter, is pivotal to CNS function. Alterations in its concentration can be dangerous, as seen for example in acute injuries of the CNS, chronic neurodegenerative disorders and mental disorders. Its homeostasis is attributed to the efficient removal of glutamate from the extracellular milieu by reuptake via local transport mechanisms. Our recent studies suggest that glutamate, either directly or indirectly, elicits a purposeful systemic T-cell-mediated immune response directed against immunodominant self-antigens that reside at the site of glutamate-induced damage. We suggest that the harnessed autoimmunity (which we have termed 'protective autoimmunity') helps the resident microglia in their dual function as antigen-presenting cells (serving the immune system) and as cells that clear the damaged site of potentially harmful material (serving the nervous system). The interplay between glutamate and an adaptive immune response illustrates the bidirectional dialog between the immune and nervous systems, under both physiological and pathological conditions. These results point to the possible development of a therapeutic vaccination with self-antigens, or with antigens cross-reactive with self-antigens, as a way to augment autoimmunity without inducing an autoimmune disease, thus providing a safe method of limiting degeneration. This approach, which boosts a physiological mechanism for the regulation of glutamate, and possibly also that of other self-compounds, might prove to be a feasible strategy for therapeutic protection against glutamate-associated neurodegenerative or mental disorders.

Severe immunodeficiency has opposite effects on neuronal survival in glutamate-susceptible and -resistant mice: adverse effect of B cells.

The resistance of rats or mice to glutamate-induced toxicity depends on their ability to spontaneously manifest a T cell-dependent response to the insult. Survival of retinal ganglion cells (RGCs) exposed to glutamate in BALB/c SCID mice (a strain relatively resistant to glutamate toxicity) was significantly worse than in the wild type. In the susceptible C57BL/6J mouse strain, however, significantly more RGCs survived among SCID mutants than in the matched wild type. RGC survival in the SCID mutants of the two strains was similar. These results suggest 1) that immunodeficiency might be an advantage in strains incapable of spontaneously manifesting protective T cell-dependent immunity and 2) that B cells might be destructive in such cases. After exposure of RGCs to toxic glutamate concentrations in three variants of B cell-deficient C57BL/6J mice, namely muMT(-/-) (B cell knockout mice) and Ii(-/-) mice reconstituted with transgenically expressed low levels of Ii p31 isoforms (p31 mice) or Ii p41 isoforms (p41 mice), significantly more RGCs survived in these mice than in the wild type. The improved survival was diminished by replenishment of the B cell-deficient mice with B cells derived from the wild type. It thus seems that B cells have an adverse effect on neuronal recovery after injury, at least in a strain that is unable to spontaneously manifest a T cell-dependent protective mechanism. These findings have clear implications for the design of immune-based therapies for CNS injury.

Immune-related mechanisms participating in resistance and susceptibility to glutamate toxicity.

Glutamate is an essential neurotransmitter in the CNS. However, at abnormally high concentrations it becomes cytotoxic. Recent studies in our laboratory showed that glutamate evokes T cell-mediated protective mechanisms. The aim of the present study was to examine the nature of the glutamate receptors and signalling pathways that participate in immune protection against glutamate toxicity. We show, using the mouse visual system, that glutamate-induced toxicity is strain dependent, not only with respect to the amount of neuronal loss it causes, but also in the pathways it activates. In strains that are genetically endowed with the ability to manifest a T cell-dependent neuroprotective response to glutamate insult, neuronal losses due to glutamate toxicity were relatively small, and treatment with NMDA-receptor antagonist worsened the outcome of exposure to glutamate. In contrast, in mice devoid of T cell-dependent endogenous protection, NMDA receptor antagonist reduced the glutamate-induced neuronal loss. In all strains, blockage of the AMPA/KA receptor was beneficial. Pharmacological (with alpha2-adrenoceptor agonist) or molecular intervention (using either mice overexpressing Bcl-2, or DAP-kinase knockout mice) protected retinal ganglion cells from glutamate toxicity but not from the toxicity of NMDA. The results suggest that glutamate-induced neuronal toxicity involves multiple glutamate receptors, the types and relative contributions of which, vary among strains. We suggest that a multifactorial protection, based on an immune mechanism independent of the specific pathway through which glutamate exerts its toxicity, is likely to be a safer, more comprehensive, and hence more effective strategy for neuroprotection. It might suggest that, because of individual differences, the pharmacological use of NMDA-antagonist for neuroprotective purposes might have an adverse effect, even if the affinity is low.

The mesenchyme expresses T cell receptor mRNAs: relevance to cell growth control.

The mesenchyme plays a crucial regulatory role in organ formation and maintenance. However, comprehensive molecular characterization of these cells is lacking. We found unexpectedly that primary mesenchyme, as well as mesenchymal cell clones, express T cell receptor (TCR)alphabeta mRNAs, lacking the variable region. Immunological and genetic evidence support the expression of a corresponding TCRbeta protein. Additionally, mRNAs encoding TCR complex components including CD3 and zeta chain are present. A relatively higher expression of the mesenchymal TCRbeta mRNA by cultured mesenchymal cell clones correlates with fast growth, whereas poorly expressing cells are slow growers and are contact inhibited. The clones that express relatively higher amount of the TCR mRNA exhibit an increased capacity to form tumors in nude mice. However, the expression of this mRNA in the mesenchyme is not per se leading to tumorigenesis, as demonstrated by primary mesenchyme that does not form tumors in mice while expressing moderate amounts of the TCR transcripts. The expression of mesencymal TCRbeta was confined to the G2/M phases of the cell cycle in the MBA-13 mesenchymal cell line. This cell cycle dependent expression, considered together with the correlation between growth properties and the level of TCR expression by cell clones, implies association of mesenchymal TCR with cell growth control.