Genomics landscape of mitochondrial DNA variations in patients from South Italy affected by mitochondriopathies.

Mitochondrial DNA (mtDNA) is a 16,569 base pairs, double-stranded, circular molecule that contains 37 genes coding for 13 subunits of the respiratory chain plus 2 rRNAs and 22 tRNAs. Mutations in these genes have been identified in patients with a variety of disorders affecting every system in the body. The advent of next generation sequencing technologies has provided the possibility to perform the whole mitochondrial DNA sequencing, allowing the identification of disease-causing pathogenic variants in a single platform. In this study, the whole mtDNA of 100 patients from South Italy affected by mitochondrial diseases was analyzed by using an amplicon-based approach and then the enriched libraries were deeply sequenced on the ION Torrent platform (Thermofisher Scientific Waltham, MA, USA). After bioinformatics analysis and filtering, we were able to find 26 nonsynonymous variants with a MAF <1% that were associated with different pathological phenotypes, expanding the mutational spectrum of these diseases. Moreover, among the new mutations found, we have also analyzed the 3D structure of the MT-ATP6 A200T gene variation in order to confirm suspected functional alterations. This work brings light on new variants possibly associated with several mitochondriopathies in patients from South Italy and confirms that deep sequencing approach, compared to the standard methods, is a reliable and time-cost reducing strategy to detect all the variants present in the mitogenome, making the possibility to create a genomics landscape of mitochondrial DNA variations in human diseases.

A Novel Homozygous Variant in DYSF Gene Is Associated with Autosomal Recessive Limb Girdle Muscular Dystrophy R2/2B.

Mutations in the DYSF gene, encoding dysferlin, are responsible for Limb Girdle Muscular Dystrophy type R2/2B (LGMDR2/2B), Miyoshi myopathy (MM), and Distal Myopathy with Anterior Tibialis onset (MDAT). The size of the gene and the reported inter and intra familial phenotypic variability make early diagnosis difficult. Genetic analysis was conducted using Next Gene Sequencing (NGS), with a panel of 40 Muscular Dystrophies associated genes we designed. In the present study, we report a new missense variant c.5033G>A, p.Cys1678Tyr (NM_003494) in the exon 45 of DYSF gene related to Limb Girdle Muscular Dystrophy type R2/2B in a 57-year-old patient affected with LGMD from a consanguineous family of south Italy. Both healthy parents carried this variant in heterozygosity. Genetic analysis extended to two moderately affected sisters of the proband, showed the presence of the variant c.5033G>A in both in homozygosity. These data indicate a probable pathological role of the variant c.5033G>A never reported before in the onset of LGMDR2/2B, pointing at the NGS as powerful tool for identifying LGMD subtypes. Moreover, the collection and the networking of genetic data will increase power of genetic-molecular investigation, the management of at-risk individuals, the development of new therapeutic targets and a personalized medicine.

Tapered fibertrodes for optoelectrical neural interfacing in small brain volumes with reduced artefacts.

Deciphering the neural patterns underlying brain functions is essential to understanding how neurons are organized into networks. This deciphering has been greatly facilitated by optogenetics and its combination with optoelectronic devices to control neural activity with millisecond temporal resolution and cell type specificity. However, targeting small brain volumes causes photoelectric artefacts, in particular when light emission and recording sites are close to each other. We take advantage of the photonic properties of tapered fibres to develop integrated 'fibertrodes' able to optically activate small brain volumes with abated photoelectric noise. Electrodes are positioned very close to light emitting points by non-planar microfabrication, with angled light emission allowing the simultaneous optogenetic manipulation and electrical read-out of one to three neurons, with no photoelectric artefacts, in vivo. The unconventional implementation of two-photon polymerization on the curved taper edge enables the fabrication of recoding sites all around the implant, making fibertrodes a promising complement to planar microimplants.

Hereditary Hyperekplexia: A New Family and a Systematic Review of GLRA1 Gene-Related Phenotypes.

Hereditary hyperekplexia (HPX) is a genetic neurodevelopmental disorder recently defined by the triad of (1) neonatal hypertonia, (2) excessive startle reflexes, and (3) generalized stiffness following the startle. Defects in GLRA1 are the most common cause of HPX, inherited both in an autosomal dominant and autosomal recessive manner. GLRA1 mutations can also cause milder phenotypes in the startle syndromes spectrum, but the prevalence is uncertain and no clear genotype-phenotype correlation has emerged yet. Moreover, the prevalence of neurodevelopmental outcomes has not been clearly defined. Here we report a new family of patients with a typical HPX phenotype, linked to a novel GLRA1 mutation, inherited with a recessive pattern. We then perform a systematic review of the literature of GLRA1-related HPX, describing the main epidemiological features of 210 patients. We found that GLRA1-related phenotypes do not necessarily fulfill the current criteria for HPX, including also milder and later-onset phenotypes. Among clinical features of the disease, neurodevelopmental issues were reported in a third of the sample; interestingly, we found that these problems, particularly when severe, were more common in homozygous than in heterozygous patients. Additional clinical and preclinical studies are needed to define predictors of adverse neurodevelopmental outcomes and underlying mechanisms.

Conformable AlN Piezoelectric Sensors as a Non-invasive Approach for Swallowing Disorder Assessment.

Deglutition disorders (dysphagia) are common symptoms of a large number of diseases and can lead to severe deterioration of the patient's quality of life. The clinical evaluation of this problem involves an invasive screening, whose results are subjective and do not provide a precise and quantitative assessment. To overcome these issues, alternative possibilities based on wearable technologies have been proposed. We explore the use of ultrathin, compliant, and flexible piezoelectric patches that are able to convert the laryngeal movement into a well-defined electrical signal, with extremely low anatomical obstruction and high strain resolution. The sensor is based on an aluminum nitride thin film, grown on a soft Kapton substrate, integrated with an electrical charge amplifier and low-power, wireless connection to a smartphone. An ad-hoc designed laryngeal motion simulator (LMS), which is able to mimic the motions of the laryngeal prominence, was used to evaluate its performances. The physiological deglutition waveforms were then extrapolated on a healthy volunteer and compared with the sEMG (surface electromyography) of the submental muscles. Finally, different tests were conducted to assess the ability of the sensor to provide clinically relevant information. The reliability of these features permits an unbiased evaluation of the swallowing ability, paving the way to the creation of a system that is able to provide a point-of-care automatic, unobtrusive, and real-time extrapolation of the patient's swallowing quality even during normal behavior.

Metal-Free Multilayer Hybrid PENG Based on Soft Electrospun/-Sprayed Membranes with Cardanol Additive for Harvesting Energy from Surgical Face Masks.

Disposable surgical face masks are usually used by medical/nurse staff but the current Covid-19 pandemic has caused their massive use by many people. Being worn closely attached to the people's face, they are continuously subjected to routine movements, i.e., facial expressions, breathing, and talking. These motional forces represent an unusual source of wasted mechanical energy that can be rather harvested by electromechanical transducers and exploited to power mask-integrated sensors. Typically, piezoelectric and triboelectric nanogenerators are exploited to this aim; however, most of the current devices are too thick or wide, not really conformable, and affected by humidity, which make them hardly embeddable in a mask, in contact with skin. Different from recent attempts to fabricate smart energy-harvesting cloth masks, in this work, a wearable energy harvester is rather enclosed in the mask and can be reused and not disposed. The device is a metal-free hybrid piezoelectric nanogenerator (hPENG) based on soft biocompatible materials. In particular, poly(vinylidene fluoride) (PVDF) membranes in the pure form and with a biobased plasticizer (cardanol oil, CA) are electrospun onto a laser-ablated polyimide flexible substrate attached on a skin-conformable elastomeric blend of poly(dimethylsiloxane) (PDMS) and Ecoflex. The multilayer structure of the device harnesses the piezoelectricity of the PVDF nanofibers and the friction triboelectric effects. The ultrasensitive mechanoelectrical transduction properties of the composite device are determined by the strong electrostatic behavior of the membranes and the plasticization effect of cardanol. In addition, encapsulation based on PVDF, PDMS, CA, and parylene C is used, allowing the hPENG to exhibit optimal reliability and resistance against the wet and warm atmosphere around the face mask. The proposed device reveals potential applications for the future development of smart masks with coupled energy-harvesting devices, allowing to use them not only for anti-infective protection but also to supply sensors or active antibacterial/viral devices.

LC-MALDI-TOF ISD MS analysis is an effective, simple and rapid method of investigation for histones characterization: Application to EBV lymphoblastoid cell lines.

This contribution is the result of our progressive engagement to develop and to apply a top-down liquid chromatography (LC) matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) (LC-MALDI-TOF) analysis for the histone post-translational modifications (PTMs) and variants characterization, mainly in order to provide comprehensive and fast results. The histone post-translational modifications and the differential expression of the histone variants play an essential role both in the DNA packaging mechanism in chromosomes and in the regulation of gene expression in different cellular processes, also in response to molecular agents of environmental origin. This epigenetic mechanism is widely studied in different field such as cellular differentiation, development and in the understanding of mechanisms underlying diseases. The characterization of histone PTMs has traditionally performed by antibodies-based assay, but immunological methods have significant limits, and today systems that use mass spectrometry are increasingly employed. We evaluated an in-source decay (ISD) analysis for the histone investigation on human lymphoblastoid cells, and by this approach, we were able to identify and quantify several PTMs such as the di-methylation in the lysine 20 and the acetylation in the lysine 16 in H4 and the mono-methylation, di-methylation and trimethylations at K9 of the histone H3.1. Moreover, we detected and quantified in the same H2B spectrum the prevalent H2B 1C/2E type but also the minor H2B 1D, 1M and 1B/1L/1N, 1O/2F, 1J/1K variants. In this work, we show that MALDI-ISD represents an excellent methodology to obtain global information on histone PTMs and variants from cells in culture, with rapidity and simplicity of execution. Finally, this is a useful approach to get label-free relative quantitative data of histone variants and PTMs.

The Mitochondrial Dysfunction Hypothesis in Autism Spectrum Disorders: Current Status and Future Perspectives.

Autism spectrum disorders (ASDs) constitute a set of heterogeneous neurodevelopmental conditions, characterized by a wide genetic variability that has led to hypothesize a polygenic origin. The metabolic profiles of patients with ASD suggest a possible implication of mitochondrial pathways. Although different physiological and biochemical studies reported deficits in mitochondrial oxidative phosphorylation in subjects with ASD, the role of mitochondrial DNA variations has remained relatively unexplored. In this review, we report and discuss very recent evidence to demonstrate the key role of mitochondrial disorders in the development of ASD.

High transmission from 2D periodic plasmonic finite arrays with sub-20 nm gaps realized with Ga focused ion beam milling.

Fabricating plasmonic nanostructures with good optical performances often requires lengthy and challenging patterning processes that can hardly be transferred to unconventional substrates, such as optical fiber tips or curved surfaces. Here we investigate the use of a single Ga focused ion beam process to fabricate 2D arrays of gold nanoplatelets for nanophotonic applications. While observing that focused ion beam milling of crossing tapered grooves inherently produces gaps below 20 nm, we provide experimental and theoretical evidence for the spectral features of grooves terminating with a sharp air gap. We show that transmission near 10% can be obtained via two-dimensional nano-focusing in a finite subset of 2D arrays of gold nanoplatelets. This enables the application of our nanostructure to detect variations in the refractive index of thin films using either reflected or transmitted light when a small number of elements are engaged.

Conformable Nanowire-in-Nanofiber Hybrids for Low-Threshold Optical Gain in the Ultraviolet.

The miniaturization of diagnostic devices that exploit optical detection schemes requires the design of light sources combining small size, high performance for effective excitation of chromophores, and mechanical flexibility for easy coupling to components with complex and nonplanar shapes. Here, ZnO nanowire-in-fiber hybrids with internal architectural order are introduced, exhibiting a combination of polarized stimulated emission, low propagation losses of light modes, and structural flexibility. Ultrafast transient absorption experiments on the electrospun material show optical gain which gives rise to amplified spontaneous emission with a threshold lower than the value found in films. These systems are highly flexible and can conveniently conform to curved surfaces, which makes them appealing active elements for various device platforms, such as bendable lasers, optical networks, and sensors, as well as for application in bioimaging, photo-cross-linking, and optogenetics.

Piezoelectricity and Biocompatibility of Flexible ScxAl(1-x)N Thin Films for Compliant MEMS Transducers.

There is huge research activity in the development of flexible and biocompatible piezoelectric materials for next-generation compliant micro electro-mechanical systems (MEMS) transducers to be exploited in wearable devices and implants. This work reports for the first time on the development of flexible ScxAl(1-x)N films deposited by sputtering technique onto polyimide substrates, assessing their piezoelectricity and biocompatibility. Flexible ScxAl(1-x)N films have been analyzed in terms of morphological, structural, and piezoelectric properties. ScxAl(1-x)N layer exhibits a good surface roughness of 4.40 nm and moderate piezoelectricity with an extracted effective piezoelectric coefficient (d33eff) value of 1.87 ± 0.06 pm/V, in good agreement with the diffraction pattern analysis results. Cell viability assay, performed to study the interaction of the ScxAl(1-x)N films with human cell lines, shows that this material does not have significant effects on tested cells. Furthermore, the ScxAl(1-x)N layer, integrated onto a flexible device and analyzed by bending/unbending measurements, shows a peak-to-peak open-circuit voltage (VOC) of 0.32 V and a short-circuit current (ISC) of 0.27 µA, with a generated power of 19.28 nW under optimal resistive load, thus demonstrating the potential of flexible ScxAl(1-x)N films as active layers for next-generation wearable/implantable piezoelectrics.

Tunable Near-Infrared Localized Surface Plasmon Resonance of F, In-Codoped CdO Nanocrystals.

Nanocrystals (NCs) of transparent conducting oxides with a localized surface plasmon resonance (LSPR) in the near-infrared (NIR) spectral region show promising electrochromic properties for the development of a new generation of dynamic "smart windows". In this regard, we exploit thin films of F, In-codoped CdO (FICO) NCs as active coatings for electrochromic devices. The control over the dopants concentration in FICO NCs results in fine tuning of their LSPR across the NIR region of the electromagnetic spectrum. Highly transparent mesoporous electrodes were prepared from colloidal FICO NCs by in situ ligand exchange of the pristine organic capping ligands. This approach preserves the optical and electrical properties of native NCs and delivers highly homogeneous, nonscattering films with a good electronic coupling between the NCs. We achieved a dynamic control over the LSPR frequency by reversible electrochemical doping, hence a spectrally selective modulation of the optical transmittance in the NIR region of the solar spectrum, which carries nearly 50% of the whole solar heat. Spectroelectrochemical characterization, coloration efficiency, and switching kinetics results indicate that thin film based on FICO NCs are potential candidates for plasmonic electrochromic applications. Moreover, the high electron mobility and wide optical bandgap of FICO makes NCs of this material suitable for large-area devices capable of dynamically controlling the heat load coming from the solar infrared radiation, without affecting the visible light transmittance.

Focused ion beam nanomachining of tapered optical fibers for patterned light delivery.

With the advent of optogenetic techniques, a major need for precise and versatile light-delivery techniques has arisen from the neuroscience community. Driven by this demand, research on innovative illuminating devices has opened previously inaccessible experimental paths. However, tailoring light delivery to functionally and anatomically diverse brain structures still remains a challenging task. We progressed in this endeavor by micro-structuring metal-coated tapered optical fibers and exploiting the resulting mode-division multiplexing/demultiplexing properties. To do this, a non-conventional Focused Ion Beam (FIB) milling method was developed in order to pattern the non-planar surface of the taper around the full 360°, by equipping the FIB chamber with a micromanipulation system. This led us to develop three novel typologies of micro-structured illuminating tools: (a) a tapered fiber that emits light from a narrow slot of adjustable length; (b) a tapered fiber that emits light from four independently addressable optical windows; (c) a tapered fiber that emits light from an annular aperture with 360° symmetry. The result is a versatile technology enabling reconfigurable light-delivery that can be tailored to specific experimental needs.

Design and Fabrication by Thermal Imprint Lithography and Mechanical Characterization of a Ring-Based PDMS Soft Probe for Sensing and Actuating Forces in Biological Systems.

In this paper, the design, fabrication and mechanical characterization of a novel polydimethylsiloxane (PDMS) soft probe for delivering and sensing forces in biological systems is proposed. On the basis of preliminary finite element (FEM) analysis, the design takes advantage of a suitable core geometry, characterized by a variable spring-like ring. The compliance of probes can be finely set in a wide range to measure forces in the micronewton to nanonewton range. In particular, this is accomplished by properly resizing the ring geometry and/or exploiting the mixing ratio-based elastic properties of PDMS. Fabrication by the thermal imprint lithography method allows fast and accurate tuning of ring sizes and tailoring of the contact section to their targets. By only varying geometrical parameters, the stiffness ranges from 1080 mNm-1 to 50 mNm-1, but by changing the base-curing agent proportion of the elastomer from 10:1 to 30:1, the stiffness drops to 37 mNm-1. With these compliances, the proposed device will provide a new experimental tool for investigating force-dependent biological functions in sensory systems.

Bidirectional biomimetic flow sensing with antiparallel and curved artificial hair sensors.

Background: Flow stimuli in the natural world are varied and contain a wide variety of directional information. Nature has developed morphological polarity and bidirectional arrangements for flow sensing to filter the incoming stimuli. Inspired by the neuromasts found in the lateral line of fish, we present a novel flow sensor design based on two curved cantilevers with bending orientation antiparallel to each other. Antiparallel cantilever pairs were designed, fabricated and compared to a single cantilever based hair sensor in terms of sensitivity to temperature changes and their response to changes in relative air flow direction. Results: In bidirectional air flow, antiparallel cantilever pairs exhibit an axially symmetrical sensitivity between 40 µV/(m s-1) for the lower air flow velocity range (between ±10-20 m s-1) and 80 µV/(m s-1) for a higher air flow velocity range (between ±20-32 m s-1). The antiparallel cantilever design improves directional sensitivity and provides a sinusoidal response to flow angle. In forward flow, the single sensor reaches its saturation limitation, flattening at 67% of the ideal sinusoidal curve which is earlier than the antiparallel cantilevers at 75%. The antiparallel artificial hair sensor better compensates for temperature changes than the single sensor. Conclusion: This work demonstrated the successive improvement of the bidirectional sensitivity, that is, improved temperature compensation, decreased noise generation and symmetrical response behaviour. In the antiparallel configuration, one of the two cantilevers always extends out into the free stream flow, remaining sensitive to directional flow and preserving a sensitivity to further flow stimuli.

Actively Targeted and Redox Responsive Delivery of Anticancer Drug by Chitosan Nanoparticles.

The clinical efficacy of methotrexate (MTX) is limited by its poor water solubility, its low bioavailability, and the development of resistance in cancer cells. Herein, we developed novel folate redox-responsive chitosan (FTC) nanoparticles for intracellular MTX delivery. l-Cysteine and folic acid molecules were selected to be covalently linked to chitosan in order to confer it redox responsiveness and active targeting of folate receptors (FRs). NPs based on these novel polymers could possess tumor specificity and a controlled drug release due to the overexpression of FRs and high concentration of reductive agents in the microenvironment of cancer cells. Nanoparticles (NPs) were prepared using an ionotropic gelation technique and characterized in terms of size, morphology, and loading capacity. In vitro drug release profiles exhibited a glutathione (GSH) dependence. In the normal physiological environment, NPs maintained good stability, whereas, in a reducing environment similar to tumor cells, the encapsulated MTX was promptly released. The anticancer activity of MTX-loaded FTC-NPs was also studied by incubating HeLa cells with formulations for various time and concentration intervals. A significant reduction in viability was observed in a dose- and time-dependent manner. In particular, FTC-NPs showed a better inhibition effect on HeLa cancer cell proliferation compared to non-target chitosan-based NPs used as control. The selective cellular uptake of FTC-NPs via FRs was evaluated and confirmed by fluorescence microscopy. Overall, the designed NPs provide an attractive strategy and potential platform for efficient intracellular anticancer drug delivery.

Notch3 protein expression in skin fibroblasts from CADASIL patients.

AIM: CADASIL is an inherited cerebrovascular disease caused by mutations in the NOTCH3 gene. Notch signaling is involved in a broad spectrum of function, from the cell proliferation to apoptosis. Thus far, because the molecular mechanism underlying the pathological alterations remains unclear and taking into account that fibroblasts contribute to the integrity of the vasculature, our aims was to establish whether fibroblasts, in subjects carrying different NOTCH3 mutations, show abnormalities in the protein expression. METHODS: We performed the investigation on skin fibroblasts in culture obtained from three CADASIL patients and normal subjects. The patients were genetically characterized, and carried a p.R61W, a p.C174T, and p.R103X, mutation respectively. Notch3 expression was first evaluated on fibroblasts by immunofluorescence analysis, then western blot on cellular extract was utilized to validate the immunofluorescence results. RESULTS: The Notch3 immunoreactivity was clearly detected along the cellular body and in the cellular nuclei of the control fibroblasts. We observed a marked, statistically significant, reduction of the fluorescence immunoreactivity in the fibroblasts from patient with the classical C174T cysteine mutation and a less pronounced reduction in the other two subject's samples with respect to the normal controls. These data were confirmed by the immunoblot analysis. CONCLUSIONS: Our results show that the investigated three NOTCH3 mutations are associated with a reduction of the levels of Notch3 expression in vitro. Because the smooth muscle cells appear to be predominantly involved in this cerebrovascular disease, our result, despite the limitation of the sample size examinated, clearly suggest that also fibroblasts, directly involved in making the vascular basal lamina and in maintaining the vascular integrity, may play an important role in the mechanism responsible for the disease.

Nitride-Based Materials for Flexible MEMS Tactile and Flow Sensors in Robotics.

The response to different force load ranges and actuation at low energies is of considerable interest for applications of compliant and flexible devices undergoing large deformations. We present a review of technological platforms based on nitride materials (aluminum nitride and silicon nitride) for the microfabrication of a class of flexible micro-electro-mechanical systems. The approach exploits the material stress differences among the constituent layers of nitride-based (AlN/Mo, Si x N y /Si and AlN/polyimide) mechanical elements in order to create microstructures, such as upwardly-bent cantilever beams and bowed circular membranes. Piezoresistive properties of nichrome strain gauges and direct piezoelectric properties of aluminum nitride can be exploited for mechanical strain/stress detection. Applications in flow and tactile sensing for robotics are described.

Near-infrared selective dynamic windows controlled by charge transfer impedance at the counter electrode.

Recent developments in the exploitation of transparent conductive oxide nanocrystals paved the way to the realization of a new class of electrochemical systems capable of selectively shielding the infrared heat loads carried by sunlight and prospected the blooming of a key enabling technology to be implemented in the next generation of "zero-energy" building envelopes. Here we report the fabrication of a set of electrochromic devices embodying an engineered nanostructured electrode made by high aspect-ratio tungsten oxide nanorods, which allow for selectively and dynamically controlling sunlight transmission over the near-infrared to visible range. Varying the intensity of applied voltage makes the spectral response of the device change across three different optical regimes, namely fully transparent, near-infrared only blocking and both visible and near-infrared blocking. It is demonstrated that the degree of reversible modulation of the thermal radiation entering the glazing element can approach a remarkable 85%, accompanied by only a modest reduction in the luminous transmittance.