Kdm7a expression is spatiotemporally regulated in developing Xenopus laevis embryos, and its overexpression influences late retinal development.

BACKGROUND: Post-translational histone modifications are among the most common epigenetic modifications that orchestrate gene expression, playing a pivotal role during embryonic development and in various pathological conditions. Among histone lysine demethylases, KDM7A, also known as KIAA1718 or JHDM1D, catalyzes the demethylation of H3K9me1/2 and H3K27me1/2, leading to transcriptional regulation. Previous data suggest that KDM7A plays a central role in several biological processes, including cell proliferation, commitment, differentiation, apoptosis, and maintenance. However, information on the expression pattern of KDM7A in whole organisms is limited, and its functional role is still unclear. RESULTS: In Xenopus development, kdm7a is expressed early, undergoing spatiotemporal regulation in various organs and tissues, including the central nervous system and the eye. Focusing on retinal development, we found that kdm7a overexpression does not affect the expression of genes critically involved in early neural development and eye-field specification, whereas unbalances the distribution of neural cell subtypes in the mature retina by disfavoring the development of ganglion cells while promoting that of horizontal cells. CONCLUSIONS: Kdm7a is dynamically expressed during embryonic development, and its overexpression influences late retinal development, suggesting a potential involvement in the molecular machinery regulating the spatiotemporally ordered generation of retinal neuronal subtypes.

Neuroprotective Nanoparticles Targeting the Retina: A Polymeric Platform for Ocular Drug Delivery Applications.

Neuroprotective drug delivery to the posterior segment of the eye represents a major challenge to counteract vision loss. This work focuses on the development of a polymer-based nanocarrier, specifically designed for targeting the posterior eye. Polyacrylamide nanoparticles (ANPs) were synthesised and characterised, and their high binding efficiency was exploited to gain both ocular targeting and neuroprotective capabilities, through conjugation with peanut agglutinin (ANP:PNA) and neurotrophin nerve growth factor (ANP:PNA:NGF). The neuroprotective activity of ANP:PNA:NGF was assessed in an oxidative stress-induced retinal degeneration model using the teleost zebrafish. Upon nanoformulation, NGF improved the visual function of zebrafish larvae after the intravitreal injection of hydrogen peroxide, accompanied by a reduction in the number of apoptotic cells in the retina. Additionally, ANP:PNA:NGF counteracted the impairment of visual behaviour in zebrafish larvae exposed to cigarette smoke extract (CSE). Collectively, these data suggest that our polymeric drug delivery system represents a promising strategy for implementing targeted treatment against retinal degeneration.

Zebrafish Avatar to Develop Precision Breast Cancer Therapies.

BACKGROUND: Zebrafish (Danio rerio) is a vertebrate that has become a popular alternative model for the cellular and molecular study of human tumors and for drug testing and validating approaches. Notably, zebrafish embryos, thanks to their accessibility, allow rapid collection of in vivo results prodromal to validation in the murine models in respect to the 3R principles. The generation of tumor xenograft in zebrafish embryos and larvae, or zebrafish avatar, represents a unique opportunity to study tumor growth, angiogenesis, cell invasion and metastatic dissemination, interaction between tumor and host in vivo avoiding immunogenic rejection, representing a promising platform for the translational research and personalized therapies. OBJECTIVE: In this mini-review, we report recent advances in breast cancer research and drug testing that took advantage of the zebrafish xenograft model using both breast cancer cell lines and patient's biopsy. CONCLUSION: Patient derived xenograft, together with the gene editing, the omics biotechnology, the in vivo time lapse imaging and the high-throughput screening that are already set up and largely used in zebrafish, could represent a step forward towards precision and personalized medicine in the breast cancer research field.

Association of the Somatostatin Analog Octreotide With Magnetic Nanoparticles for Intraocular Delivery: A Possible Approach for the Treatment of Diabetic Retinopathy.

The somatostatin analog octreotide (OCT) displays important neuroprotective and anti-angiogenic properties that could make it an interesting candidate to treat diabetic retinopathy (DR). Unfortunately, systemic drug administration is hindered by severe side effects, therefore topical administration routes are preferable. However, drug delivery through eye drops may be difficult due to ocular barriers and, in the long term, could induce ocular damage. On the other hand, intraocular injections must be repeated to maintain drug concentration, and this may cause severe damage to the eye. To decrease injection frequency, long-term release and reduced biodegradation could be obtained by binding the drug to biodegradable polymeric nanoparticles. In the present study, we made a preparation of OCT bound to magnetic nanoparticles (MNP-OCT) and tested its possible use as an OCT delivery system to treat retinal pathologies such as DR. In particular, in vitro, ex vivo, and in vivo experimental models of the mammalian retina were used to investigate the possible toxicity of MNPs, possible effects of the binding to MNPs on OCT bioactivity, and the localization of MNP-OCT in the retina after intraocular injection. The results showed that, both in human retinal endothelial cells (HRECs) and in mouse retinal explants, MNPs were not toxic and the binding with MNPs did not influence OCT antiangiogenic or antiapoptotic activity. Rather, effects of MNP-OCT were observed at concentrations up to 100-fold (in HRECs) or 10-fold (in mouse retinal explants) lower compared to OCT, indicating that OCT bioactivity was enhanced in MNP-OCT. MNP-OCT in mouse retinas in vivo after intraocular delivery were initially localized mainly to the outer retina, at the level of the retinal pigment epithelium, while after 5 days they were observed throughout the retinal thickness. These observations demonstrate that MNP-OCT may be used as an OCT intraocular delivery system that may ensure OCT localization to the retina and enhanced OCT bioactivity. Further studies will be necessary to determine the OCT release rate in the retina and the persistence of drug effects in the long period.

Comparative analysis of p4ha1 and p4ha2 expression during Xenopus laevis development.

Collagen prolyl 4-hydroxylases (c-P4Hs) are evolutionary conserved enzymes whose activity is essential for the correct folding of stable triple helical molecules of collagen and collagen-like proteins. They play crucial roles in embryo development, connective tissue functional organization, tumor growth and metastasis. Despite the important function of these enzymes, little is known about their expression during vertebrate development. In this study, we determine and compare the previously undescribed spatio-temporal expression patterns of the p4ha1 and p4ha2 genes, which encode the main subunits containing the enzyme active site, during Xenopus development. The two genes are maternally inherited and share expression in dorsal mesoderm, branchial arches and their derivatives, as well as in the central nervous system, although with distinct spatio-temporal patterns. A major co-expression domain for p4ha1 and p4ha2 is represented by the developing notochord, where these genes are transcribed from early neurula stage to stage 42 tadpole, thus paralleling the profile of collagen II production and suggesting a coordination between collagen synthesis and its post-translational modifications.

Nano-topography: Quicksand for cell cycle progression?

The 3-D spatial and mechanical features of nano-topography can create alternative environments, which influence cellular response. In this paper, murine fibroblast cells were grown on surfaces characterized by protruding nanotubes. Cells cultured on such nano-structured surface exhibit stronger cellular adhesion compared to control groups, but despite the fact that stronger adhesion is generally believed to promote cell cycle progression, the time cells spend in G1 phase is doubled. This apparent contradiction is solved by confocal microscopy analysis, which shows that the nano-topography inhibits actin stress fiber formation. In turn, this impairs RhoA activation, which is required to suppress the inhibition of cell cycle progression imposed by p21/p27. This finding suggests that the generation of stress fibers, required to impose the homeostatic intracellular tension, rather than cell adhesion/spreading is the limiting factor for cell cycle progression. Indeed, nano-topography could represent a unique tool to inhibit proliferation in adherent well-spread cells.

Neurotrophin-conjugated nanoparticles prevent retina damage induced by oxidative stress.

Glaucoma and other optic neuropathies are characterized by a loss of retinal ganglion cells (RGCs), a cell layer located in the posterior eye segment. Several preclinical studies demonstrate that neurotrophins (NTs) prevent RGC loss. However, NTs are rarely investigated in the clinic due to various issues, such as difficulties in reaching the retina, the very short half-life of NTs, and the need for multiple injections. We demonstrate that NTs can be conjugated to magnetic nanoparticles (MNPs), which act as smart drug carriers. This combines the advantages of the self-localization of the drug in the retina and drug protection from fast degradation. We tested the nerve growth factor and brain-derived neurotrophic factor by comparing the neuroprotection of free versus conjugated proteins in a model of RGC loss induced by oxidative stress. Histological data demonstrated that the conjugated proteins totally prevented RGC loss, in sharp contrast to the equivalent dose of free proteins, which had no effect. The overall data suggest that the nanoscale MNP-protein hybrid is an excellent tool in implementing ocular drug delivery strategies for neuroprotection and therapy.

Magnetic nanoparticles: a strategy to target the choroidal layer in the posterior segment of the eye.

Despite the higher rate of blindness due to population aging, minimally invasive and selective drug delivery to the eye still remains an open challenge, especially in the posterior segment. The retina, the retinal pigment epithelium (RPE) and the choroid are posterior segment cell layers, which may be affected by several diseases. In particular, damages to the choroid are associated with poor prognosis in the most severe pathologies. A drug delivery approach, able to target the choroid, is still missing. Recently, we demonstrated that intravitreally injected magnetic nanoparticles (MNP) are able to rapidly and persistently localise within the RPE in an autonomous manner. In this work we functionalised the MNP surface with the vascular endothelial growth factor, a bioactive molecule capable of transcytosis from the RPE towards more posterior layers. Such functionalisation successfully addressed the MNPs to the choroid, while MNP functionalised with a control polypeptide (poly-L-lysine) showed the same localisation pattern of the naked MNP particles. These data suggest that the combination of MNP with different bioactive molecules could represent a powerful strategy for cell-specific targeting of the eye posterior segment.

Magnetic Nanoparticles for Efficient Delivery of Growth Factors: Stimulation of Peripheral Nerve Regeneration.

The only clinically approved alternative to autografts for treating large peripheral nerve injuries is the use of synthetic nerve guidance conduits (NGCs), which provide physical guidance to the regenerating stump and limit scar tissue infiltration at the injury site. Several lines of evidence suggest that a potential future strategy is to combine NGCs with cellular or molecular therapies to deliver growth factors that sustain the regeneration process. However, growth factors are expensive and have a very short half-life; thus, the combination approach has not been successful. In the present paper, we proposed the immobilization of growth factors (GFs) on magnetic nanoparticles (MNPs) for the time- and space-controlled release of GFs inside the NGC. We tested the particles in a rat model of a peripheral nerve lesion. Our results revealed that the injection of a cocktail of MNPs functionalized with nerve growth factor (NGF) and with vascular endothelial growth factor (VEGF) strongly accelerate the regeneration process and the recovery of motor function compared to that obtained using the free factors. Additionally, we found that injecting MNPs in the NGC is safe and does not impair the regeneration process, and the MNPs remain in the conduit for weeks.

A catechin nanoformulation inhibits WM266 melanoma cell proliferation, migration and associated neo-angiogenesis.

We validated the anticancer potential of a nanoformulation made by (+)-catechin, gelatin and carbon nanotubes in terms of inhibition of cancer cell proliferation, migration and associated neo-angiogenesis. Gelatin was selected to stabilize the catechin without compromising its anti-oxidant potential and the carbon nanotubes were used to increase its intracellular bioavailability. The anticancer potential of the resulting nanohybrid was validated on an aggressive melanoma cell line, in vitro and in zebrafish xenotransplants. The nanohybrid strongly enhances the cytotoxic effect of (+)-catechin. At a concentration of (+)-catechin 50µg/ml, the nanohybrid inhibited the ability of melanoma cells to proliferate (100% increase of cell doubling time and severe impairment in zebrafish xenotransplants), to migrate (totally inhibition in vitro and 50% reduction of cell motility in zebrafish xenotransplants) and to induce neo-angiogenesis (100% inhibition in zebrafish xenotransplants). Conversely, the free (+)-catechin and carrier (CNT:gel) had no statistically significant effects over the control, at any concentration tested. Our results suggest that the use of the nanohybrid, able to improve the therapeutic efficacy of the catechins, could represent a successful strategy for a future clinical translation.

Comparative expression analysis of pfdn6a and tcp1α during Xenopus development.

We recently identified pfdn6a and tcp1α (also known as cct-α) as genes coregulated by the transcription factor Rx1. The proteins encoded by these genes belong to two interacting complexes (Prefoldin and "chaperonin containing t-complex polypeptide 1"), which promote the folding of actin and tubulin and have more recently been reported to be involved in a variety of additional functions including cell cycle control and transcription regulation. However, little is known about the expression and function of these two genes during vertebrate development. To assess whether pfdn6a and tcp1α display a general coordinated expression during Xenopus development, we determined, by RT-PCR and in situ hybridization, the spatio-temporal expression pattern of pfnd6a, which was not previously described, and compared it to that of tcp1α, extending the analysis to stages not previously investigated for this gene. We detected maternal transcripts of pfnd6a in the animal hemisphere at early blastula stage. During gastrulation, pfdn6a was expressed in the involuting mesoderm and subsequently in the anterior and dorsal neural plate. At tailbud and tadpole stages, pfdn6a RNA was mainly detected in the forebrain, midbrain, eye vesicle, otic vesicle, branchial arches, and developing pronephros. The pfnd6a expression pattern largely overlaps with that of tcp1α indicating a spatio-temporal transcriptional coregulation of these genes in the majority of their expression sites, which is suggestive of a possible involvement in the same developmental events.

Sheets of vertically aligned BaTiO3 nanotubes reduce cell proliferation but not viability of NIH-3T3 cells.

All biomaterials initiate a tissue response when implanted in living tissues. Ultimately this reaction causes fibrous encapsulation and hence isolation of the material, leading to failure of the intended therapeutic effect of the implant. There has been extensive bioengineering research aimed at overcoming or delaying the onset of encapsulation. Nanotechnology has the potential to address this problem by virtue of the ability of some nanomaterials to modulate interactions with cells, thereby inducing specific biological responses to implanted foreign materials. To this effect in the present study, we have characterised the growth of fibroblasts on nano-structured sheets constituted by BaTiO3, a material extensively used in biomedical applications. We found that sheets of vertically aligned BaTiO3 nanotubes inhibit cell cycle progression - without impairing cell viability - of NIH-3T3 fibroblast cells. We postulate that the 3D organization of the material surface acts by increasing the availability of adhesion sites, promoting cell attachment and inhibition of cell proliferation. This finding could be of relevance for biomedical applications designed to prevent or minimize fibrous encasement by uncontrolled proliferation of fibroblastic cells with loss of material-tissue interface underpinning long-term function of implants.

Characterization of the Rx1-dependent transcriptome during early retinal development.

BACKGROUND: The transcription factor Rx1, also known as Rax, controls key properties of retinal precursors including migration behavior, proliferation, and maintenance of multipotency. However, Rx1 effector genes are largely unknown. RESULTS: To identify genes controlled by Rx1 in early retinal precursors, we compared the transcriptome of Xenopus embryos overexpressing Rx1 to that of embryos in which Rx1 was knocked-down. In particular, we selected 52 genes coherently regulated, i.e., actived in Rx1 gain of function and repressed in Rx1 loss of function experiments, or vice versa. RT-qPCR and in situ hybridization confirmed the trend of regulation predicted by microarray data for the selected genes. Most of the genes upregulated by Rx1 are coexpressed with this transcription factor, while downregulated genes are either not expressed or expressed at very low levels in the early developing retina. Putative direct Rx1 target genes, activated by GR-Rx1 in the absence of protein synthesis, include Ephrin B1 and Sh2d3c, an interactor of ephrinB1 receptor, which represent candidate novel effectors for the migration promoting activity of Rx1. CONCLUSIONS: This study identifies previously undescribed Rx1 regulated genes mainly involved in transcription regulation, cell migration/adhesion, and cell proliferation that contribute to delineate the molecular mechanisms underlying Rx1 activities.

The orientation of the neuronal growth process can be directed via magnetic nanoparticles under an applied magnetic field.

There is a growing body of evidence indicating the importance of physical stimuli for neuronal growth and development. Specifically, results from published experimental studies indicate that forces, when carefully controlled, can modulate neuronal regeneration. Here, we validate a non-invasive approach for physical guidance of nerve regeneration based on the synergic use of magnetic nanoparticles (MNPs) and magnetic fields (Ms). The concept is that the application of a tensile force to a neuronal cell can stimulate neurite initiation or axon elongation in the desired direction, the MNPs being used to generate this tensile force under the effect of a static external magnetic field providing the required directional orientation. In a neuron-like cell line, we have confirmed that MNPs direct the neurite outgrowth preferentially along the direction imposed by an external magnetic field, by inducing a net angle displacement (about 30°) of neurite direction. From the clinical editor: This study validates that non-invasive approaches for physical guidance of nerve regeneration based on the synergic use of magnetic nanoparticles and magnetic fields are possible. The hypothesis was confirmed by observing preferential neurite outgrowth in a cell culture system along the direction imposed by an external magnetic field.

Magnetic nanoparticles as intraocular drug delivery system to target retinal pigmented epithelium (RPE).

One of the most challenging efforts in drug delivery is the targeting of the eye. The eye structure and barriers render this organ poorly permeable to drugs. Quite recently the entrance of nanoscience in ocular drug delivery has improved the penetration and half-life of drugs, especially in the anterior eye chamber, while targeting the posterior chamber is still an open issue. The retina and the retinal pigment epithelium/choroid tissues, located in the posterior eye chamber, are responsible for the majority of blindness both in childhood and adulthood. In the present study, we used magnetic nanoparticles (MNPs) as a nanotool for ocular drug delivery that is capable of specific localization in the retinal pigmented epithelium (RPE) layer. We demonstrate that, following intraocular injection in Xenopus embryos, MNPs localize specifically in RPE where they are retained for several days. The specificity of the localization did not depend on particle size and surface properties of the MNPs used in this work. Moreover, through similar experiments in zebrafish, we demonstrated that the targeting of RPE by the nanoparticles is not specific for the Xenopus species.

Non-mammalian vertebrate embryos as models in nanomedicine.

Various in vivo biological models have been proposed for studying the interactions of nano-materials in biological systems. Unfortunately, the widely used small mammalian animal models (rodents) are costly and labor intensive and generate ethical issues and antagonism from the anti-vivisectionist movement. Recently, there has been increasing interest in the scientific community in the interactions between nano-materials and non-mammalian developmental organisms, which are now being recognized as valid models for the study of human disease. This review examines and discusses the biomedical applications and the interaction of nano-materials with embryonic systems, focusing on non-mammalian vertebrate models, such as chicken, zebrafish and Xenopus. FROM THE CLINICAL EDITOR: Animal models are critical components of preclinical biomedical research. This review discusses the feasibility and potential applications of non-mammalian vertebral animals, such as zebrafish, xenopus, and chicken as animal models in nanomedicine research.

Brief report: Rx1 defines retinal precursor identity by repressing alternative fates through the activation of TLE2 and Hes4.

The molecular mechanisms underlying the acquisition of retinal precursor identity are scarcely defined. Although the homeobox gene Rx1 (also known as Rax) plays a major role in specifying retinal precursors and maintaining their multipotent state, the involved mechanisms remain to be largely deciphered. Here, following a highthroughput screen for genes regulated by Rx1, we found that this transcription factor specifies the fate of retinal progenitors by repressing genes normally activated in adjacent ectodermal territories. Unexpectedly, we also observed that Rx1, mainly through the activation of the transcriptional repressors TLE2 and Hes4, is necessary and sufficient to inhibit endomesodermal gene expression in retinal precursors of the eye field. In particular, Rx1 knockdown leads retinogenic blastomeres to adopt an endomesodermal fate, indicating a previously undescribed function for Rx1 in preventing the expression of endomesoderm determinants known to inhibit retinal fate. Altogether these data suggest that an essential requirement to establish a retinal precursor identity is the active inhibition of pathways leading to alternative fates.