Expression analysis of thg1l during Xenopus laevis development.

The tRNA-histidine guanylyltransferase 1-like (THG1L), also known as induced in high glucose-1 (IHG-1), encodes for an essential mitochondria-associated protein highly conserved throughout evolution, that catalyses the 3'-5' addition of a guanine to the 5'-end of tRNA-histidine (tRNAHis). Previous data indicated that THG1L plays a crucial role in the regulation of mitochondrial biogenesis and dynamics, in ATP production, and is critically involved in the modulation of apoptosis, cell-cycle progression and survival, as well as in cellular stress responses and redox homeostasis. Dysregulations of THG1L expression play a central role in various pathologies, including nephropathies, and neurodevelopmental disorders often characterized by developmental delay and cerebellar ataxia. Despite the essential role of THG1L, little is known about its expression during vertebrate development. Herein, we examined the detailed spatio-temporal expression of this gene in the developing Xenopus laevis. Our results show that thg1l is maternally inherited and its temporal expression suggests a role during the earliest stages of embryogenesis. Spatially, thg1l mRNA localizes in the ectoderm and marginal zone mesoderm during early stages of development. Then, at tadpole stages, thg1l transcripts mostly localise in neural crests and their derivatives, somites, developing kidney and central nervous system, therefore largely coinciding with territories displaying intense energy metabolism during organogenesis in Xenopus.

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.

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.

Soft Hydrogel Zwitterionic Coatings Minimize Fibroblast and Macrophage Adhesion on Polyimide Substrates.

Minimizing the foreign body reaction to polyimide-based implanted devices plays a pivotal role in several biomedical applications. In this work, we propose materials exhibiting nonbiofouling properties and a Young's modulus reflecting that of soft human tissues. We describe the synthesis, characterization, and in vitro validation of poly(carboxybetaine) hydrogel coatings covalently attached to polyimide substrates via a photolabile 4-azidophenyl group, incorporated in poly(carboxybetaine) chains at two concentrations of 1.6 and 3.1 mol %. The presence of coatings was confirmed by attenuated total reflectance Fourier transform infrared spectroscopy. White light interferometry was used to evaluate the coating continuity and thickness (between 3 and 6 µm under dry conditions). Confocal laser scanning microscopy allowed us to quantify the thickness of the swollen hydrogel coatings that ranged between 13 and 32 µm. The different hydrogel formulations resulted in stiffness values ranging from 2 to 19 kPa and led to different fibroblast and macrophage responses in vitro. Both cell types showed a minimum adhesion on the softest hydrogel type. In addition, both the overall macrophage activation and cytotoxicity were observed to be negligible for all of the tested material formulations. These results are a promising starting point toward future advanced implantable systems. In particular, such technology paves the way for novel neural interfaces able to minimize the fibrotic reaction, once implanted in vivo, and to maximize their long-term stability and functionality.

Peptide-based coatings for flexible implantable neural interfaces.

In the last decade, the use of flexible biosensors for neuroprosthetic and translational applications has widely increased. Among them, the polyimide (PI)-based thin-film electrodes got a large popularity. However, the usability of these devices is still hampered by a non-optimal tissue-device interface that usually compromises the long-term quality of neural signals. Advanced strategies able to improve the surface properties of these devices have been developed in the recent past. Unfortunately, most of them are not easy to be developed and combined with micro-fabrication processes, and require long-term efforts to be testable with human subjects. Here we show the results of the design and in vitro testing of an easy-to-implement and potentially interesting coating approach for thin-film electrodes. In particular, two biocompatible coatings were obtained via covalent conjugation of a laminin-derived peptide, CAS-IKVAV-S (IKV), with polyimide sheets that we previously functionalized with vinyl- and amino- groups (PI_v and PI_a respectively). Both the engineered coatings (PI_v+IKV and PI_a+IKV) showed morphological and chemical properties able to support neuronal adhesion, neurite sprouting, and peripheral glial cell viability while reducing the fibroblasts contamination of the substrate. In particular, PI_v+IKV showed promising results that encourage further in vivo investigation and pave the way for a new generation of peptide-coated thin-film electrodes.

Segregation of motor and sensory axons regenerating through bicompartmental tubes by combining extracellular matrix components with neurotrophic factors.

Segregation of regenerating motor and sensory axons may be a good strategy to improve selective functionality of regenerative interfaces to provide closed-loop commands. Provided that extracellular matrix components and neurotrophic factors exert guidance effects on different neuronal populations, we assessed in vivo the potential of separating sensory and motor axons regenerating in a bicompartmental Y-type tube, with each branch prefilled with an adequate combination of extracellular matrix and neurotrophic factors. The severed rat sciatic nerve was repaired using a bicompartmental tube filled with a collagen matrix enriched with fibronectin (FN) and brain-derived neurotrophic factor (BDNF) encapsulated in poly-lactic co-glycolic acid microspheres (FN + MP.BDNF) in one compartment to preferentially attract motor axons and collagen enriched with laminin (LM) and nerve growth factor (NGF) and neurotrophin-3 (NT-3) in microspheres (LM + MP.NGF/NT-3) in the other compartment for promoting sensory axons regeneration. Control animals were implanted with the same Y-tube with a collagen matrix with microspheres (MP) containing PBS (Col + MP.PBS). By using retrotracer labelling, we found that LM + MP.NGF/NT-3 did not attract higher number of regenerated sensory axons compared with controls, and no differences were observed in sensory functional recovery. However, FN + MP.BDNF guided a higher number of regenerating motor axons compared with controls, improving also motor recovery. A small proportion of sensory axons with large soma size, likely proprioceptive neurons, was also attracted to the FN + MP.BDNF compartment. These results demonstrate that muscular axonal guidance can be modulated in vivo by the addition of fibronectin and BDNF.

Preferential Enhancement of Sensory and Motor Axon Regeneration by Combining Extracellular Matrix Components with Neurotrophic Factors.

After peripheral nerve injury, motor and sensory axons are able to regenerate but inaccuracy of target reinnervation leads to poor functional recovery. Extracellular matrix (ECM) components and neurotrophic factors (NTFs) exert their effect on different neuronal populations creating a suitable environment to promote axonal growth. Here, we assessed in vitro and in vivo the selective effects of combining different ECM components with NTFs on motor and sensory axons regeneration and target reinnervation. Organotypic cultures with collagen, laminin and nerve growth factor (NGF)/neurotrophin-3 (NT3) or collagen, fibronectin and brain-derived neurotrophic factor (BDNF) selectively enhanced sensory neurite outgrowth of DRG neurons and motor neurite outgrowth from spinal cord slices respectively. For in vivo studies, the rat sciatic nerve was transected and repaired with a silicone tube filled with a collagen and laminin matrix with NGF/NT3 encapsulated in poly(lactic-co-glycolic acid) (PLGA) microspheres (MP) (LM + MP.NGF/NT3), or a collagen and fibronectin matrix with BDNF in PLGA MPs (FN + MP.BDNF). Retrograde labeling and functional tests showed that LM + MP.NGF/NT3 increased the number of regenerated sensory neurons and improved sensory functional recovery, whereas FN + MP.BDNF preferentially increased regenerated motoneurons and enhanced motor functional recovery. Therefore, combination of ECM molecules with NTFs may be a good approach to selectively enhance motor and sensory axons regeneration and promote appropriate target reinnervation.

Focal release of neurotrophic factors by biodegradable microspheres enhance motor and sensory axonal regeneration in vitro and in vivo.

Neurotrophic factors (NTFs) promote nerve regeneration and neuronal survival after peripheral nerve injury. However, drawbacks related with administration and bioactivity during long periods limit their therapeutic application. In this study, PLGA microspheres (MPs) were used to locally release different NTFs and evaluate whether they accelerate axonal regeneration in comparison with free NTFs or controls. ELISA, SEM, UV/visible light microscopy, organotypic cultures of DRG explants and spinal cord slices were used to characterize MP properties and the bioactivity of the released NTFs. Results of organotypic cultures showed that encapsulated NTFs maintain longer bioactivity and enhance neurite regeneration of both sensory and motor neurons compared with free NTFs. For in vivo assays, the rat sciatic nerve was transected and repaired with a silicone tube filled with collagen gel or collagen mixed with PBS encapsulated MPs (control groups) and with free or encapsulated NGF, BDNF, GDNF or FGF-2. After 20 days, a retrotracer was applied to the regenerated nerve to quantify motor and sensory axonal regeneration. NTF encapsulation in MPs improved regeneration of both motor and sensory axons, as evidenced by increased numbers of retrolabeled neurons. Hence, our results show that slow release of NTFs with PLGA MP enhance nerve regeneration.

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.

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.

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.

Mechanisms of toxicity induced by SiO2 nanoparticles of in vitro human alveolar barrier: effects on cytokine production, oxidative stress induction, surfactant proteins A mRNA expression and nanoparticles uptake.

An in vitro human alveolar barrier established by the coculture of epithelial human cell line NCI-H441 with endothelial human cell line ISO-HAS1 was used to evaluate the effects of amorphous silicon dioxide nanoparticles (SiNPs), in the presence or absence of THP-1 cells (monocytes). SiNPs exposure induced production of proinflammatory cytokine and oxidative stress. A high release of TNF-α and IL-8 by epithelial/endothelial cells, potentiated in the presence of THP-1 cells could contribute to the observed downregulation of surfactant proteins A mRNA expression resulting in the damage of the alveolar barrier. The obtained results suggested that in vitro approach can be used to study pulmonary toxicity as long as the applied in vitro model mimics closely the complexity of in vivo situation.

Comparative study of ZnO and TiO2 nanoparticles: physicochemical characterisation and toxicological effects on human colon carcinoma cells.

Despite human gastrointestinal exposure to nanoparticles (NPs), data on NPs toxicity in intestinal cells are quite scanty. In this study we evaluated the toxicity induced by zinc oxide (ZnO) and titanium dioxide (TiO2) NPs on Caco-2 cells. Only ZnO NPs produced significant cytotoxicity, evaluated by two different assays. The presence of foetal calf serum in culture medium significantly reduced ZnO NPs toxicity as well as ion leakage and NP-cell interaction. The two NPs increased the intracellular amount of reactive oxygen species (ROS) after 6 h treatment. However, only ZnO NPs increased ROS and induced IL-8 release both after 6 and 24 h. Experimental data indicate a main role of chemical composition and solubility in ZnO NPs toxicity. Moreover our results suggest a key role of oxidative stress in ZnO NPs cytotoxicity induction related both to ion leakage and to cell interaction with NPs in serum-free medium.

Amorphous silica nanoparticles do not induce cytotoxicity, cell transformation or genotoxicity in Balb/3T3 mouse fibroblasts.

Although amorphous silica nanoparticles (aSiO(2)NPs) are believed to be non-toxic and are currently used in several industrial and biomedical applications including cosmetics, food additives and drug delivery systems, there is still no conclusive information on their cytotoxic, genotoxic and carcinogenic potential. For this reason, this work has investigated the effects of aSiO(2)NPs on Balb/3T3 mouse fibroblasts, focusing on cytotoxicity, cell transformation and genotoxicity. Results obtained using aSiO(2)NPs, with diameters between 15 nm and 300 nm and exposure times up to 72 h, have not shown any cytotoxic effect on Balb/3T3 cells as measured by the MTT test and the Colony Forming Efficiency (CFE) assay. Furthermore, aSiO(2)NPs have induced no morphological transformation in Balb/3T3 cells and have not resulted in genotoxicity, as shown by Cell Transformation Assay (CTA) and Micronucleus (MN) assay, respectively. To understand whether the absence of any toxic effect could result from a lack of internalization of the aSiO(2)NPs by Balb/3T3 cells, we have investigated the uptake and the intracellular distribution following exposure to 85 nm fluorescently-labelled aSiO(2)NPs. Using fluorescence microscopy, it was observed that fluorescent aSiO(2)NPs are internalized and located exclusively in the cytoplasmic region. In conclusion, we have demonstrated that although aSiO(2)NPs are internalized in vitro by Balb/3T3 mouse fibroblasts, they do not trigger any cytotoxic or genotoxic effect and do not induce morphological transformation, suggesting that they might be a useful component in industrial applications.

Online monitoring of cell metabolism to assess the toxicity of nanoparticles: the case of cobalt ferrite.

Different in vitro assays are successfully used to determine the relative cytotoxicity of a broad range of compounds. Nevertheless, different research groups have pointed out the difficulty in using the same tests to assess the toxicity of nanoparticles (NPs). In this study, we evaluated the possible use of a microphysiometer, Bionas 2500 analyzing system Bionas GmbH®, to detect in real time changes in cell metabolisms linked to NPs exposure. We focused our work on response changes of fibroblast cultures linked to exposure by cobalt ferrite NPs and compared the results to conventional in vitro assays. The measurements with the microphysiometer showed a cobalt ferrite cytotoxic effect, confirmed by the Colony Forming Efficiency assay. In conclusion, this work demonstrated that the measurement of metabolic parameters with a microphysiometer is a promising method to assess the toxicity of NPs and offers the advantage to follow on-line the cell metabolic changes.

Colony Forming Efficiency and microscopy analysis of multi-wall carbon nanotubes cell interaction.

In this work, we present a complete physicochemical characterization of multi-wall carbon nanotubes (mwCNTs) in order to assess their potential toxicological effects in in vitro cell models using Colony Forming Efficiency (CFE) assay. We verified that Dimethyl Sulfoxide (DMSO) was a more suitable solvent to disperse mwCNTs compared to culture medium guaranteeing reproducibility in the preparation of testing dilutions. The CFE assay was carried out on five mammalian cell lines representing the potentially exposed and/or target organs for nanomaterials (lung, liver, kidney, intestine, skin), as well as on mouse fibroblasts cell line, which usually is considered a sensitive model to verify in vitro cytotoxicity of test compounds. A statistically significant toxic effect was found only in human alveolar basal epithelial cells and immortalized mouse fibroblasts, for which the interaction between mwCNTs and cells was additionally studied by Atomic Force and Scanning Electron Microscopy. In this study, we considered and suggested the CFE assay as a promising test for screening studies of cytotoxicity. In addition, combining in vitro tests with physicochemical analysis, this work underlines basic points to be considered when research on nanomaterials has to be carried out, to set up, in our opinion, well-defined and suitable experimental planning and procedures.