A novel framework for functional annotation of variants of uncertain significance in ID/ASD risk gene CC2D1A.

Intellectual disability (ID) and autism spectrum disorder (ASD) are genetically heterogeneous with hundreds of identified risk genes, most affecting only a few patients. Novel missense variants in these genes are being discovered as clinical exome sequencing is now routinely integrated into diagnosis, yet most of them are annotated as variants of uncertain significance (VUS). VUSs are a major roadblock in using patient genetics to inform clinical action. We developed a framework to characterize VUSs in Coiled-coil and C2 domain containing 1A (CC2D1A), a gene causing autosomal recessive ID with comorbid ASD in 40% of cases. We analyzed seven VUSs (p.Pro319Leu, p.Ser327Leu, p.Gly441Val, p.Val449Met, p.Thr580Ile, p.Arg886His and p.Glu910Lys) from four cases of individuals with ID and ASD. Variants were cloned and overexpressed in HEK293 individually and in their respective heterozygous combination. CC2D1A is a signaling scaffold that positively regulates PKA-CREB signaling by repressing phosphodiesterase 4D (PDE4D) to prevent cAMP degradation. After testing multiple parameters including direct interaction between PDE4D and CC2D1A, cAMP levels and CREB activation, we found that the most sensitive readout was CREB transcriptional activity using a luciferase assay. Compared to WT CC2D1A, five VUSs (p.Pro319Leu, p.Gly441Val, p.Val449Met, p.Thr580Ile, and p.Arg886His) led to significantly blunted response to forskolin induced CREB activation. This luciferase assay approach can be scaled up to annotate ~150 CC2D1A VUSs that are currently listed in ClinVar. Since CREB activation is a common denominator for multiple ASD/ID genes, our paradigm can also be adapted for their VUSs.

Ultrasound Stimulation of Piezoelectric Nanocomposite Hydrogels Boosts Chondrogenic Differentiation in Vitro, in Both a Normal and Inflammatory Milieu.

The use of piezoelectric nanomaterials combined with ultrasound stimulation is emerging as a promising approach for wirelessly triggering the regeneration of different tissue types. However, it has never been explored for boosting chondrogenesis. Furthermore, the ultrasound stimulation parameters used are often not adequately controlled. In this study, we show that adipose-tissue-derived mesenchymal stromal cells embedded in a nanocomposite hydrogel containing piezoelectric barium titanate nanoparticles and graphene oxide nanoflakes and stimulated with ultrasound waves with precisely controlled parameters (1 MHz and 250 mW/cm2, for 5 min once every 2 days for 10 days) dramatically boost chondrogenic cell commitment in vitro. Moreover, fibrotic and catabolic factors are strongly down-modulated: proteomic analyses reveal that such stimulation influences biological processes involved in cytoskeleton and extracellular matrix organization, collagen fibril organization, and metabolic processes. The optimal stimulation regimen also has a considerable anti-inflammatory effect and keeps its ability to boost chondrogenesis in vitro, even in an inflammatory milieu. An analytical model to predict the voltage generated by piezoelectric nanoparticles invested by ultrasound waves is proposed, together with a computational tool that takes into consideration nanoparticle clustering within the cell vacuoles and predicts the electric field streamline distribution in the cell cytoplasm. The proposed nanocomposite hydrogel shows good injectability and adhesion to the cartilage tissue ex vivo, as well as excellent biocompatibility in vivo, according to ISO 10993. Future perspectives will involve preclinical testing of this paradigm for cartilage regeneration.

Functionalized aliphatic polyketones with germicide activity.

The COVID-19 pandemic has further confirmed to the community that direct contact with contaminated surfaces and objects represents an important source of pathogen spreading among humans. Therefore, it is of paramount importance to design effective germicidal paints to ensure a rapid and potent disinfectant action of surfaces. In this work, we design novel germicide polymeric coatings by inserting quaternary ammonium and sugar groups on the macromolecular backbone, thus respectively endowing the polymer with germicide features and hydrophilicity to interact with the surfaces. An aliphatic polyketone was selected as a starting polymer matrix that was functionalized with primary amine derivatives via the Paal-Knorr reaction. The resulting polymers were deposited on cellulose filter papers and checkboard charts with excellent coating yield and substrate coverage as determined by scanning electron microscopy for cellulose. Remarkably, the substrates coated by the functional polymers bearing quaternary ammonium compounds showed excellent bactericide properties with antibacterial rate of 99% and logarithmic reduction of >3. Notably, the polymers with higher hydrophobicity showed better retention on the substrate after being treated with water at neutral pH.

Piezoelectric nanocomposite bioink and ultrasound stimulation modulate early skeletal myogenesis.

Despite the significant progress in bioprinting for skeletal muscle tissue engineering, new stimuli-responsive bioinks to boost the myogenesis process are highly desirable. In this work, we developed a printable alginate/Pluronic-based bioink including piezoelectric barium titanate nanoparticles (nominal diameter: ∼60 nm) for the 3D bioprinting of muscle cell-laden hydrogels. The aim was to investigate the effects of the combination of piezoelectric nanoparticles with ultrasound stimulation on early myogenic differentiation of the printed structures. After the characterization of nanoparticles and bioinks, viability tests were carried out to investigate three nanoparticle concentrations (100, 250, and 500 µg mL-1) within the printed structures. An excellent cytocompatibility was confirmed for nanoparticle concentrations up to 250 µg mL-1. TEM imaging demonstrated the internalization of BTNPs in intracellular vesicles. The combination of piezoelectric nanoparticles and ultrasound stimulation upregulated the expression of MYOD1, MYOG, and MYH2 and enhanced cell aggregation, which is a crucial step for myoblast fusion, and the presence of MYOG in the nuclei. These results suggest that the direct piezoelectric effect induced by ultrasound on the internalized piezoelectric nanoparticles boosts myogenesis in its early phases.

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.

Analysis of the Damage Mechanism around the Crack Tip for Two Rubber-Toughened PLA-Based Blends.

The toughening mechanisms of poly(lactic acid; PLA) blended with two different elastomers, namely poly (butylene adipate-co-terephtalate; PBAT) and polyolefin elastomers with grafted glycidyl methacrylate (POE-g-GMA), at 10 and 20 wt.%, were investigated. Tensile and Charpy impact tests showed a general improvement in the performance of the PLA. The morphology of the dispersed phases showed that PBAT is in the form of spheres while POE-g-GMA has a dual sphere/fibre morphology. To correlate the micromechanical deformation mechanism with the macroscopical mechanical behaviour, the analysis of the subcritical crack tip damaged zone of double-notched specimens subjected to a four-point bending test (according to the single-edge double-notch four-point bend (SEDN-4PB) technique) was carried out using several microscopic techniques (SEM, polarized TOM and TEM). The damage was mainly generated by shear yielding deformation although voids associated with dilatational bands were observed.

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

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

Targeted Dendrimer-Coated Magnetic Nanoparticles for Selective Delivery of Therapeutics in Living Cells.

Nanoparticles are widely used as theranostic agents for the treatment of various pathologies, including cancer. Among all, dendrimers-based nanoparticles represent a valid approach for drugs delivery, thanks to their controllable size and surface properties. Indeed, dendrimers can be easily loaded with different payloads and functionalized with targeting agents. Moreover, they can be used in combination with other materials such as metal nanoparticles for combinatorial therapies. Here, we present the formulation of an innovative nanostructured hybrid system composed by a metallic core and a dendrimers-based coating that is able to deliver doxorubicin specifically to cancer cells through a targeting agent. Its dual nature allows us to transport nanoparticles to our site of interest through the magnetic field and specifically increase internalization by exploiting the T7 targeting peptide. Our system can release the drug in a controlled pH-dependent way, causing more than 50% of cell death in a pancreatic cancer cell line. Finally, we show how the system was internalized inside cancer cells, highlighting a peculiar disassembly of the nanostructure at the cell surface. Indeed, only the dendrimeric portion is internalized, while the metal core remains outside. Thanks to these features, our nanosystem can be exploited for a multistage magnetic vector.

An immune response to the avascular lens following wounding of the cornea involves ciliary zonule fibrils.

The lens and central cornea are avascular. It was assumed that the adult lens had no source of immune cells and that the basement membrane capsule surrounding the lens was a barrier to immune cell migration. Yet, microfibril-associated protein-1 (MAGP1)-rich ciliary zonules that originate from the vasculature-rich ciliary body and extend along the surface of the lens capsule, form a potential conduit for immune cells to the lens. In response to cornea debridement wounding, we find increased expression of MAGP1 throughout the central corneal stroma. The immune cells that populate this typically avascular region after wounding closely associate with this MAGP1-rich matrix. These results suggest that MAGP1-rich microfibrils support immune cell migration post-injury. Using this cornea wound model, we investigated whether there is an immune response to the lens following cornea injury involving the lens-associated MAGP1-rich ciliary zonules. Our results provide the first evidence that following corneal wounding immune cells are activated to travel along zonule fibers that extend anteriorly along the equatorial surface of the lens, from where they migrate across the anterior lens capsule. These results demonstrate that lens-associated ciliary zonules are directly involved in the lens immune response and suggest the ciliary body as a source of immune cells to the avascular lens.

Single-Cell Metabolic Profiling: Metabolite Formulas from Isotopic Fine Structures in Heterogeneous Plant Cell Populations.

Characterization of the metabolic heterogeneity in cell populations requires the analysis of single cells. Most current methods in single-cell analysis rely on cell manipulation, potentially altering the abundance of metabolites in individual cells. A small sample volume and the chemical diversity of metabolites are additional challenges in single-cell metabolomics. Here, we describe the combination of fiber-based laser ablation electrospray ionization (f-LAESI) with 21 T Fourier transform ion cyclotron resonance mass spectrometry (21TFTICR-MS) for in situ single-cell metabolic profiling in plant tissue. Single plant cells infected by bacteria were selected and sampled directly from the tissue without cell manipulation through mid-infrared ablation with a fine optical fiber tip for ionization by f-LAESI. Ultrahigh performance 21T-FTICR-MS enabled the simultaneous capture of isotopic fine structures (IFSs) for 47 known and 11 unknown compounds, thus elucidating their elemental compositions from single cells and providing information on metabolic heterogeneity in the cell population.

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.

Axonal debris accumulates in corneal epithelial cells after intraepithelial corneal nerves are damaged: A focused Ion Beam Scanning Electron Microscopy (FIB-SEM) study.

The intraepithelial corneal nerves (ICNs) that innervate the corneal epithelium are maintained through interactions with corneal epithelial cells and the extracellular matrix they produce. One to several axons bundle together within the basal cell layer and extend parallel to the ocular surface or branch and extend apically. Here we use 3-dimentional (3D) ultrastructural reconstructions of control and trephine injured mouse corneal epithelium and stroma produced using Focused Ion Beam Scanning Electron Microscope (FIB-SEM) to determine whether corneal epithelial or immune cells resident in the epithelium remove axonal debris and degrade it in their lysosomes after trephine injury to the cornea. We demonstrate that axonal fragments are internalized in the corneal epithelium and accumulate within electron dense structures consistent with lysosomes 3 h after trephine injury in both epithelial and immune cells located among the basal cells of the trephine injured cornea. Confocal imaging showed fewer CD45+ immune cells within the corneal epithelium after trephine injury compared to controls. The resolution obtained using FIB-SEM also allowed us to show that the presence of sensory axons at the basal aspect of the epithelial basal cells close to the anterior aspect of the epithelial basement membrane (EBM) is associated with a focal reduction in EBM thickness. In addition, we show using FIB-SEM and confocal imaging that superficial trephine injuries that do not penetrate the stroma, damage the integrity of anterior stromal nerves. These studies are the first to look at the mouse cornea following nerve injury using FIB-SEM.

Mass Spectrometry Imaging of Lipids in Human Skin Disease Model Hidradenitis Suppurativa by Laser Desorption Ionization from Silicon Nanopost Arrays.

Neutral lipids have been implicated in a host of potentially debilitating human diseases, such as heart disease, type-2 diabetes, and metabolic syndrome. Matrix-assisted laser desorption ionization (MALDI), the method-of-choice for mass spectrometry imaging (MSI), has led to remarkable success in imaging several lipid classes from biological tissue sections. However, due to ion suppression by phospholipids, MALDI has limited ability to efficiently ionize and image neutral lipids, such as triglycerides (TGs). To help overcome this obstacle, we have utilized silicon nanopost arrays (NAPA), a matrix-free laser desorption ionization (LDI) platform. Hidradenitis suppurativa (HS) is a chronic, recurrent inflammatory skin disease of the apocrine sweat glands. The ability of NAPA to efficiently ionize lipids is exploited in the analysis of human skin samples from sufferers of HS. Ionization by LDI from NAPA allows for the detection and imaging of a number of neutral lipid species, including TGs comprised of shorter, odd-chain fatty acids, which strongly suggests an increased bacterial load within the host tissue, as well as hexosylceramides (HexCers) and galabiosyl-/lactosylceramides that appear to be correlated with the presence of HS. Our results demonstrate that NAPA-LDI-MSI is capable of imaging and potentially differentiating healthy and diseased human skin tissues based on changes in detected neutral lipid composition.

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

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

Cerium oxide nanoparticles: the regenerative redox machine in bioenergetic imbalance.

AIM: Owing to their catalytic properties as reactive oxygen species scavengers, cerium oxide nanoparticles (nanoceria) have become an extremely promising candidate for medical applications, especially in the treatment of diseases where oxidative stress has been proposed as one of the main pathogenesis factors. MATERIALS & METHODS: In this work, nanoceria antioxidant power has been tested in primary cultured skin fibroblasts, derived from healthy individuals, by evaluating the mitochondrial function both in basal condition and after an oxidative insult. RESULTS & CONCLUSION: Combined with a clear lack of toxicity, antioxidant activity makes nanoceria promising in a wide range of clinical applications sharing the common signature of a global bioenergetic dysfunction.

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.

Ultrastructural Characterization of the Lower Motor System in a Mouse Model of Krabbe Disease.

Krabbe disease (KD) is a neurodegenerative disorder caused by the lack of ß- galactosylceramidase enzymatic activity and by widespread accumulation of the cytotoxic galactosyl-sphingosine in neuronal, myelinating and endothelial cells. Despite the wide use of Twitcher mice as experimental model for KD, the ultrastructure of this model is partial and mainly addressing peripheral nerves. More details are requested to elucidate the basis of the motor defects, which are the first to appear during KD onset. Here we use transmission electron microscopy (TEM) to focus on the alterations produced by KD in the lower motor system at postnatal day 15 (P15), a nearly asymptomatic stage, and in the juvenile P30 mouse. We find mild effects on motorneuron soma, severe ones on sciatic nerves and very severe effects on nerve terminals and neuromuscular junctions at P30, with peripheral damage being already detectable at P15. Finally, we find that the gastrocnemius muscle undergoes atrophy and structural changes that are independent of denervation at P15. Our data further characterize the ultrastructural analysis of the KD mouse model, and support recent theories of a dying-back mechanism for neuronal degeneration, which is independent of demyelination.