Molecular Study of the Fukutin-Related Protein (FKRP) Gene in Patients from Southern Italy with Duchenne/Becker-like Phenotype.

Pathogenic variants localized in the gene coding for the Fukutin-Related Protein (FKRP) are responsible for Limb-Girdle Muscular Dystrophy type 9 (LGMDR9), Congenital Muscular Dystrophies type 1C (MDC1C), Walker-Warburg Syndrome (WWS), and Muscle-Eye-Brain diseases (MEBs). LGMDR9 is the fourth most common hereditary Limb Girdle Muscular Dystrophy in Italy. LGMDR9 patients with severe disease show an overlapping Duchenne/Becker phenotype and may have secondary dystrophin reduction on muscle biopsy. We conducted a molecular analysis of the FKRP gene by direct sequencing in 153 patients from Southern Italy (Calabria) with Duchenne/Becker-like phenotypes without confirmed genetic diagnosis. Mutational screening of the patients (112 men and 41 women, aged between 5 and 84 years), revealed pathogenic variants in 16 subjects. The most frequent variants identified were c.427C > A, p.R143S, and c.826C > A, p.L276I (NM_024301.5). The results obtained show that the Duchenne/Becker-like phenotype is frequently determined by mutations in the FKRP gene in our cohort and highlight the importance of considering LGMDR9 in the differential diagnosis of dystrophinopathies in Calabria. Finally, this study, which, to our knowledge, is the first conducted on Calabrian subjects, will contribute to the rapid identification and management of LGMDR9 patients.

The Role of Mitochondrial Copy Number in Neurodegenerative Diseases: Present Insights and Future Directions.

Neurodegenerative diseases are progressive disorders that affect the central nervous system (CNS) and represent the major cause of premature death in the elderly. One of the possible determinants of neurodegeneration is the change in mitochondrial function and content. Altered levels of mitochondrial DNA copy number (mtDNA-CN) in biological fluids have been reported during both the early stages and progression of the diseases. In patients affected by neurodegenerative diseases, changes in mtDNA-CN levels appear to correlate with mitochondrial dysfunction, cognitive decline, disease progression, and ultimately therapeutic interventions. In this review, we report the main results published up to April 2024, regarding the evaluation of mtDNA-CN levels in blood samples from patients affected by Alzheimer's (AD), Parkinson's (PD), and Huntington's diseases (HD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS). The aim is to show a probable link between mtDNA-CN changes and neurodegenerative disorders. Understanding the causes underlying this association could provide useful information on the molecular mechanisms involved in neurodegeneration and offer the development of new diagnostic approaches and therapeutic interventions.

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.

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.

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.

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.

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.

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.

Normal immunofluorescence pattern of skin basement membranes in a family with porencephaly due to COL4A1 G749S mutation.

COL4A1 mutations have been associated with cerebral small-vessel disease, including perinatal intracerebral hemorrhage with consequent porencephaly, microbleeds, and lacunar strokes. Moreover, involvement of multiple organs and tissues like kidney, muscle, and large vessels have been reported. Three related patients with porencephaly bearing the G749S mutation in the COL4A1 gene and one healthy control belonging to the same family underwent skin biopsy. Tissue was examined by means of immunofluorescence microscopy and immunoreactivity for collagen type IV in skin basement membranes was tested. In subjects with COL4A1 mutation, we did not detect significant alterations of immunofluorescence patterns in basal membranes of different skin structures. Heterozygous COL4A1 G749S mutation is associated with a normal immunofluorescence pattern of skin basement membranes. Further studies are needed to clarify the role of possible functional abnormalities of the basement membranes in patients with this mutation.

Eco-Friendly Production of Polyvinyl Alcohol/Carboxymethyl Cellulose Wound Healing Dressing Containing Sericin.

Wound dressing production represents an important segment in the biomedical healthcare field, but finding a simple and eco-friendly method that combines a natural compound and a biocompatible dressing production for biomedical application is still a challenge. Therefore, the aim of this study is to develop wound healing dressings that are environmentally friendly, low cost, and easily produced, using natural agents and a physical crosslinking technique. Hydrogel wound healing dressings were prepared from polyvinyl alcohol/carboxymethyl cellulose and sericin using the freeze-thawing method as a crosslinking method. The morphological characterization was carried out by scanning electron microscopy (SEM), whereas the mechanical analysis was carried out by dynamic mechanical analysis (DMA) to test the tensile strength and compression properties. Then, the healing property of the wound dressing material was tested by in vitro and ex vivo tests. The results show a three-dimensional microporous structure with no cytotoxicity, excellent stretchability with compressive properties similar to those of human skin, and excellent healing properties. The proposed hydrogel dressing was tested in vitro with HaCaT keratinocytes and ex vivo with epidermal tissues, demonstrating an effective advantage on wound healing acceleration. Accordingly, this study was successful in developing wound healing dressings using natural agents and a simple and green crosslinking method.

Enhancing cardiovascular health monitoring: Simultaneous multi-artery cardiac markers recording with flexible and bio-compatible AlN piezoelectric sensors.

Continuous monitoring of cardiovascular parameters like pulse wave velocity (PWV), blood pressure wave (BPW), stiffness index (SI), reflection index (RI), mean arterial pressure (MAP), and cardio-ankle vascular index (CAVI) has significant clinical importance for the early diagnosis of cardiovascular diseases (CVDs). Standard approaches, including echocardiography, impedance cardiography, or hemodynamic monitoring, are hindered by expensive and bulky apparatus and accessibility only in specialized facilities. Moreover, noninvasive techniques like sphygmomanometry, electrocardiography, and arterial tonometry often lack accuracy due to external electrical interferences, artifacts produced by unreliable electrode contacts, misreading from placement errors, or failure in detecting transient issues and trends. Here, we report a bio-compatible, flexible, noninvasive, low-cost piezoelectric sensor for continuous and real-time cardiovascular monitoring. The sensor, utilizing a thin aluminum nitride film on a flexible Kapton substrate, is used to extract heart rate, blood pressure waves, pulse wave velocities, and cardio-ankle vascular index from four arterial pulse sites: carotid, brachial, radial, and posterior tibial arteries. This simultaneous recording, for the first time in the same experiment, allows to provide a comprehensive cardiovascular patient's health profile. In a test with a 28-year-old male subject, the sensor yielded the SI = 7.1 ± 0.2 m/s, RI = 54.4 ± 0.5 %, MAP = 86.2 ± 1.5 mmHg, CAVI = 7.8 ± 0.2, and seven PWVs from the combination of the four different arterial positions, in good agreement with the typical values reported in the literature. These findings make the proposed technology a powerful tool to facilitate personalized medical diagnosis in preventing CVDs.

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.

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.

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.

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.

Proteomic profiling of cerebrospinal fluid in Creutzfeldt-Jakob disease.

Creutzfeldt-Jakob disease (CJD) is a rare fatal neurodegenerative disease belonging to the group of transmissible spongiform encephalopathies or prion diseases. The agent responsible for the disease is the prion protein in an altered conformational form. Although there have been countless studies performed on the prion protein, the mechanisms that induce the structural change of the normal protein, and the harmful action the altered protein has on nervous cells, are still not fully understood. Furthermore, the final diagnosis for CJD can only occur with a postmortem histopathological analysis of the brain; the antemortem diagnosis is only possible for some specific CJD forms. Finally, there is no current treatment able to stop or delay the progression of the disease. Studies directed at resolving these issues are, therefore, extremely relevant. The proteomic approach is a very good strategy to be applied in such contexts because it allows easy identification of proteins and peptides possibly involved in the disease processes. In this article, the existing data regarding prion infection, biomarkers for CJD diagnosis and the use of several modern proteomic technologies for the identification of new cerebrospinal fluid polypeptides involved in CJD are reviewed.

High-Purcell-factor dipolelike modes at visible wavelengths in H1 photonic crystal cavity.

The optimization of H1 photonic crystal cavities for applications in the visible spectral range is reported, with the goal to obtain a versatile photonic platform to explore strongly and weakly coupled systems. The resonators have been realized in silicon nitride and weakly coupled to both organic (fluorophores) and inorganic (colloidal nanocrystals) nanoparticles emitting in the visible spectral range. The theoretical Purcell factor of the two dipolelike modes in the defect has been increased up to approximately 90, and the experimental quality factor was measured to be approximately 750.

Quantification of thymosin beta(4) in human cerebrospinal fluid using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been applied to the analysis of a wide range of biomolecules. To date, there are two specific areas of application where MALDI-TOF-MS is viewed as impractical: analysis of low-mass analytes and relative quantitative applications. However, these limitations can be overcome and quantification can be routine. Increased levels of thymosin beta(4) (TB4) have been recently found in cerebrospinal fluid (CSF) from Creutzfeldt-Jakob disease (CJD) patients. Our objective was to apply a label-free quantitative application of MALDI-TOF-MS to measure TB4 levels in human CSF by adding the oxidized form of TB4 as an internal standard. The relative peak area or peak height ratios of the native TB4 to the added oxidized form were evaluated. Considering the relative peak area ratios, healthy individuals showed a mean value of 40.8+/-21.27 ng/ml, whereas CJD patients showed high values with a mean of 154+/-59.07 ng/ml, in agreement with the previous observation found in CJD patients. Similar results were obtained considering peak height ratios. The proposed method may provide a simple and rapid screening method for quantification on CSF of TB4 levels suitable for diagnostic purposes.