New strontium-based coatings show activity against pathogenic bacteria in spine infection.

Infections of implants and prostheses represent relevant complications associated with the implantation of biomedical devices in spine surgery. Indeed, due to the length of the surgical procedures and the need to implant invasive devices, infections have high incidence, interfere with osseointegration, and are becoming increasingly difficult to threat with common therapies due to the acquisition of antibiotic resistance genes by pathogenic bacteria. The application of metal-substituted tricalcium phosphate coatings onto the biomedical devices is a promising strategy to simultaneously prevent bacterial infections and promote osseointegration/osseoinduction. Strontium-substituted tricalcium phosphate (Sr-TCP) is known to be an encouraging formulation with osseoinductive properties, but its antimicrobial potential is still unexplored. To this end, novel Sr-TCP coatings were manufactured by Ionized Jet Deposition technology and characterized for their physiochemical and morphological properties, cytotoxicity, and bioactivity against Escherichia coli ATCC 8739 and Staphylococcus aureus ATCC 6538P human pathogenic strains. The coatings are nanostructured, as they are composed by aggregates with diameters from 90 nm up to 1 µm, and their morphology depends significantly on the deposition time. The Sr-TCP coatings did not exhibit any cytotoxic effects on human cell lines and provided an inhibitory effect on the planktonic growth of E. coli and S. aureus strains after 8 h of incubation. Furthermore, bacterial adhesion (after 4 h of exposure) and biofilm formation (after 24 h of cell growth) were significantly reduced when the strains were cultured on Sr-TCP compared to tricalcium phosphate only coatings. On Sr-TCP coatings, E. coli and S. aureus cells lost their organization in a biofilm-like structure and showed morphological alterations due to the toxic effect of the metal. These results demonstrate the stability and anti-adhesion/antibiofilm properties of IJD-manufactured Sr-TCP coatings, which represent potential candidates for future applications to prevent prostheses infections and to promote osteointegration/osteoinduction.

The microbiota characterizing huge carbonatic moonmilk structures and its correlation with preserved organic matter.

BACKGROUND: Moonmilk represents complex secondary structures and model systems to investigate the interaction between microorganisms and carbonatic rocks. Grotta Nera is characterized by numerous moonmilk speleothems of exceptional size hanging from the ceiling, reaching over two meters in length. In this work we combined microbiological analyses with analytical pyrolysis and carbon stable isotope data to determine the molecular composition of these complex moonmilk structures as well as the composition of the associated microbiota. RESULTS: Three moonmilk structures were dissected into the apical, lateral, and core parts, which shared similar values of microbial abundance, richness, and carbon isotopes but different water content, microbiota composition, and organic matter. Moonmilk parts/niches showed higher values of microbial biomass and biodiversity compared to the bedrock (not showing moonmilk development signs) and the waters (collected below dripping moonmilk), indicating the presence of more complex microbial communities linked to carbonate rock interactions and biomineralization processes. Although each moonmilk niche was characterized by a specific microbiota as well as a distinct organic carbon profile, statistical analyses clustered the samples in two main groups, one including the moonmilk lateral part and the bedrock and the other including the core and apical parts of the speleothem. The organic matter profile of both these groups showed two well-differentiated organic carbon groups, one from cave microbial activity and the other from the leaching of vascular plant litter above the cave. Correlation between organic matter composition and microbial taxa in the different moonmilk niches were found, linking the presence of condensed organic compounds in the apical part with the orders Nitrospirales and Nitrosopumilales, while different taxa were correlated with aromatic, lignin, and polysaccharides in the moonmilk core. These findings are in line with the metabolic potential of these microbial taxa suggesting how the molecular composition of the preserved organic matter drives the microbiota colonizing the different moonmilk niches. Furthermore, distinct bacterial and archaeal taxa known to be involved in the metabolism of inorganic nitrogen and C1 gases (CO2 and CH4) (Nitrospira, Nitrosopumilaceae, Nitrosomonadaceae, Nitrosococcaceae, and novel taxa of Methylomirabilota and Methanomassiliicoccales) were enriched in the core and apical parts of the moonmilk, probably in association with their contribution to biogeochemical cycles in Grotta Nera ecosystem and moonmilk development. CONCLUSIONS: The moonmilk deposits can be divided into diverse niches following oxygen and water gradients, which are characterized by specific microbial taxa and organic matter composition originating from microbial activities or deriving from soil and vegetation above the cave. The metabolic capacities allowing the biodegradation of complex polymers from the vegetation above the cave and the use of inorganic nitrogen and atmospheric gases might have fueled the development of complex microbial communities that, by interacting with the carbonatic rock, led to the formation of these massive moonmilk speleothems in Grotta Nera.

Digenic Leigh syndrome on the background of the m.11778G>A Leber hereditary optic neuropathy variant.

Leigh syndrome spectrum (LSS) is a primary mitochondrial disorder defined neuropathologically by a subacute necrotizing encephalomyelopathy and characterised by bilateral basal ganglia and/or brainstem lesions. LSS is associated with variants in several mitochondrial DNA (mtDNA) genes and more than 100 nuclear genes, most often related to mitochondrial complex I (CI) dysfunction. Rarely, LSS has been reported in association with primary Leber hereditary optic neuropathy (LHON) variants of the mtDNA, coding for CI subunits (m.3460G>A in MT-ND1, m.11778G>A in MT-ND4, and m.14484T>C in MT-ND6). The underlying mechanism by which these variants manifest as LSS, a severe neurodegenerative disease, as opposed to the LHON phenotype of isolated optic neuropathy, remains an open question. Here, we analyse the exome sequencing of six probands with LSS carrying primary LHON variants, and report digenic co-occurrence of the m.11778G>A variant with damaging heterozygous variants in nuclear disease genes encoding CI subunits as a plausible explanation. Our findings suggest a digenic mechanism of disease for m.11778G>A-associated LSS, consistent with recent reports of digenic disease in individuals manifesting with LSS due to biallelic variants in the recessive LHON-associated disease gene DNAJC30 in combination with heterozygous variants in CI subunits.

Biallelic USP14 variants cause a syndromic neurodevelopmental disorder.

PURPOSE: Imbalances in protein homeostasis affect human brain development, with the ubiquitin-proteasome system (UPS) and autophagy playing crucial roles in neurodevelopmental disorders (NDD). This study explores the impact of biallelic USP14 variants on neurodevelopment, focusing on its role as a key hub connecting UPS and autophagy. METHODS: Here, we identified biallelic USP14 variants in 4 individuals from 3 unrelated families: 1 fetus, a newborn with a syndromic NDD and 2 siblings affected by a progressive neurological disease. Specifically, the 2 siblings from the latter family carried 2 compound heterozygous variants c.8T>C p.(Leu3Pro) and c.988C>T p.(Arg330∗), whereas the fetus had a homozygous frameshift c.899_902del p.(Lys300Serfs∗24) variant, and the newborn patient harbored a homozygous frameshift c.233_236del p.(Leu78Glnfs∗11) variant. Functional studies were conducted using sodium dodecyl-sulfate polyacrylamide gel electrophoresis, western blotting, and mass spectrometry analyses in both patient-derived and CRISPR-Cas9-generated cells. RESULTS: Our investigations indicated that the USP14 variants correlated with reduced N-terminal methionine excision, along with profound alterations in proteasome, autophagy, and mitophagy activities. CONCLUSION: Biallelic USP14 variants in NDD patients perturbed protein degradation pathways, potentially contributing to disorder etiology. Altered UPS, autophagy, and mitophagy activities underscore the intricate interplay, elucidating their significance in maintaining proper protein homeostasis during brain development.

Ionized jet deposition of silver nanostructured coatings: Assessment of chemico-physical and biological behavior for application in orthopedics.

Infection is one of the main issues connected to implantation of biomedical devices and represents a very difficult issue to tackle, for clinicians and for patients. This study aimed at tackling infection through antibacterial nanostructured silver coatings manufactured by Ionized Jet Deposition (IJD) for application as new and advanced coating systems for medical devices. Films composition and morphology depending on deposition parameters were investigated and their performances evaluated by correlating these properties with the antibacterial and antibiofilm efficacy of the coatings, against Escherichia coli and Staphylococcus aureus strains and with their cytotoxicity towards human cell line fibroblasts. The biocompatibility of the coatings, the nanotoxicity, and the safety of the proposed approach were evaluated, for the first time, in vitro and in vivo by rat subcutaneous implant models. Different deposition times, corresponding to different thicknesses, were selected and compared. All silver coatings exhibited a highly homogeneous surface composed of nanosized spherical aggregates. All coatings having a thickness of 50 nm and above showed high antibacterial efficacy, while none of the tested options caused cytotoxicity when tested in vitro. Indeed, silver films impacted on bacterial strains viability and capability to adhere to the substrate, in a thickness-dependent manner. The nanostructure obtained by IJD permitted to mitigate the toxicity of silver, conferring strong antibacterial and anti-adhesive features, without affecting the coatings biocompatibility. At the explant, the coatings were still present although they showed signs of progressive dissolution, compatible with the release of silver, but no cracking, delamination or in vivo toxicity was observed.

A natural biogenic fluorapatite as a new biomaterial for orthopedics and dentistry: antibacterial activity of lingula seashell and its use for nanostructured biomimetic coatings.

Calcium phosphates are widely studied in orthopedics and dentistry, to obtain biomimetic and antibacterial implants. However, the multi-substituted composition of mineralized tissues is not fully reproducible from synthetic procedures. Here, for the first time, we investigate the possible use of a natural, fluorapatite-based material, i.e., Lingula anatina seashell, resembling the composition of bone and enamel, as a biomaterial source for orthopedics and dentistry. Indeed, thanks to its unique mineralization process and conditions, L. anatina seashell is among the few natural apatite-based shells, and naturally contains ions having possible antibacterial efficacy, i.e., fluorine and zinc. After characterization, we explore its deposition by ionized jet deposition (IJD), to obtain nanostructured coatings for implantable devices. For the first time, we demonstrate that L. anatina seashells have strong antibacterial properties. Indeed, they significantly inhibit planktonic growth and cell adhesion of both Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. The two strains show different susceptibility to the mineral and organic parts of the seashells, the first being more susceptible to zinc and fluorine in the mineral part, and the second to the organic (chitin-based) component. Upon deposition by IJD, all films exhibit a nanostructured morphology and sub-micrometric thickness. The multi-doped, complex composition of the target is maintained in the coating, demonstrating the feasibility of deposition of coatings starting from biogenic precursors (seashells). In conclusion, Lingula seashell-based coatings are non-cytotoxic with strong antimicrobial capability, especially against Gram-positive strains, consistently with their higher susceptibility to fluorine and zinc. Importantly, these properties are improved compared to synthetic fluorapatite, showing that the films are promising for antimicrobial applications.

A Novel Pathogenic Variant in the SCA25-Related Gene Expanding the Etiology of Early-Onset and Progressive Cerebellar Ataxia in Childhood.

Spinocerebellar ataxias (SCAs) are heterogeneous autosomal dominant progressive ataxic disorders. SCA25 has been linked to PNPT1 pathogenic variants. Although pediatric onset is not unusual, to date only one patient with onset in the first years of life has been reported. This study presents an additional case, wherein symptoms emerged during the toddler phase, accompanied by the identification of a novel PNPT1 variant. The child was seen at 3 years because of frequent falls. Neurological examination revealed cerebellar signs and psychomotor delay. Brain MRI showed cerebellar atrophy (CA), cerebellar cortex, and dentate nuclei hyperintensities. Metabolic and genetic testing was inconclusive. At follow-up (age 6), the child had clinically and radiologically worsened; electroneurography (ENG) revealed axonal sensory neuropathy. Screening of genes associated with ataxias and mitochondrial disease identified a novel, heterozygous variant in PNPT1, which was probably pathogenic. This variant was also detected in the proband's mother and maternal grandmother, both asymptomatic, which aligns with the previously documented incomplete penetrance of heterozygous PNPT1 variants. Our study confirms that SCA25 can have onset in early childhood and characterizes natural history in pediatric cases: progressive cerebellar ataxia with sensory neuropathy, which manifests during the course of the disease. We report for the first time cerebellar gray matter hyperintensities, suggesting that SCA25 should be included in the differential diagnosis of cerebellar ataxias associated with such brain imaging features. In summary, SCA25 should be considered in the diagnostic workup of early onset pediatric progressive ataxias. Additionally, we confirm an incomplete penetrance and highly variable expressivity of PNPT1-associated SCA25.

PPAR-gamma agonist pioglitazone recovers mitochondrial quality control in fibroblasts from PITRM1-deficient patients.

Introduction: Biallelic variants in PITRM1 are associated with a slowly progressive syndrome characterized by intellectual disability, spinocerebellar ataxia, cognitive decline and psychosis. The pitrilysin metallopeptidase 1 (PITRM1) is a mitochondrial matrix enzyme, which digests diverse oligopeptides, including the mitochondrial targeting sequences (MTS) that are cleaved from proteins imported across the inner mitochondrial membrane by the mitochondrial processing peptidase (MPP). Mitochondrial peptidases also play a role in the maturation of Frataxin, the protein affected in Friedreich's ataxia. Recent studies in yeast indicated that the mitochondrial matrix protease Ste23, which is a homologue of the human insulin-degrading enzyme (IDE), cooperates with Cym1 (homologue of PITRM1) to ensure the proper functioning of the preprotein processing machinery. In humans, IDE could be upregulated by Peroxisome Proliferator-Activated Receptor Gamma (PPARG) agonists. Methods: We investigated preprotein processing, mitochondrial membrane potential and MTS degradation in control and patients' fibroblasts, and we evaluated the pharmacological effect of the PPARG agonist Pioglitazone on mitochondrial proteostasis. Results: We discovered that PITRM1 dysfunction results in the accumulation of MTS, leading to the disruption and dissipation of the mitochondrial membrane potential. This triggers a feedback inhibition of MPP activity, consequently impairing the processing and maturation of Frataxin. Furthermore, we found that the pharmacological stimulation of PPARG by Pioglitazone upregulates IDE and also PITRM1 protein levels restoring the presequence processing machinery and improving Frataxin maturation and mitochondrial function. Discussion: Our findings provide mechanistic insights and suggest a potential pharmacological strategy for this rare neurodegenerative mitochondrial disease.

Evaluation of Mitochondrial Dysfunction and Idebenone Responsiveness in Fibroblasts from Leber's Hereditary Optic Neuropathy (LHON) Subjects.

Leber's hereditary optic neuropathy (LHON) is a disease that affects the optical nerve, causing visual loss. The diagnosis of LHON is mostly defined by the identification of three pathogenic variants in the mitochondrial DNA. Idebenone is widely used to treat LHON patients, but only some of them are responders to treatment. In our study, we assessed the maximal respiration rate (MRR) and other respiratory parameters in eight fibroblast lines from subjects carrying LHON pathogenic variants. We measured also the effects of idebenone treatment on cell growth and mtDNA amounts. Results showed that LHON fibroblasts had significantly reduced respiratory parameters in untreated conditions, but no significant gain in MRR after idebenone supplementation. No major toxicity toward mitochondrial function and no relevant compensatory effect in terms of mtDNA quantity were found for the treatment at the tested conditions. Our findings confirmed that fibroblasts from subjects harboring LHON pathogenic variants displayed impaired respiration, regardless of the disease penetrance and severity. Testing responsiveness to idebenone treatment in cultured cells did not fully recapitulate in vivo data. The in-depth evaluation of cellular respiration in fibroblasts is a good approach to evaluating novel mtDNA variants associated with LHON but needs further evaluation as a potential biomarker for disease prognosis and treatment responsiveness.

Expanding the spectrum of neonatal-onset AIFM1-associated disorders.

OBJECTIVES: Pathogenic variants in AIFM1 have been associated with a wide spectrum of disorders, spanning from CMT4X to mitochondrial encephalopathy. Here we present a novel phenotype and review the existing literature on AIFM1-related disorders. METHODS: We performed EEG recordings, brain MRI and MR Spectroscopy, metabolic screening, echocardiogram, clinical exome sequencing (CES) and family study. Effects of the variant were established on cultured fibroblasts from skin punch biopsy. RESULTS: The patient presented with drug-resistant, electro-clinical, multifocal seizures 6 h after birth. Brain MRI revealed prominent brain swelling of both hemispheres and widespread signal alteration in large part of the cortex and of the thalami, with sparing of the basal nuclei. CES analysis revealed the likely pathogenic variant c.5T>C; p.(Phe2Ser) in the AIFM1 gene. The affected amino acid residue is located in the mitochondrial targeting sequence. Functional studies on cultured fibroblast showed a clear reduction in AIFM1 protein amount and defective activities of respiratory chain complexes I, III and IV. No evidence of protein mislocalization or accumulation of precursor protein was observed. Riboflavin, Coenzyme Q10 and thiamine supplementation was therefore given. At 6 months of age, the patient exhibited microcephaly but did not experience any further deterioration. He is still fed orally and there is no evidence of muscle weakness or atrophy. INTERPRETATION: This is the first AIFM1 case associated with neonatal seizures and diffuse white matter involvement with relative sparing of basal ganglia, in the absence of clinical signs suggestive of myopathy or motor neuron disease.

Nanopore long-read next-generation sequencing for detection of mitochondrial DNA large-scale deletions.

Primary mitochondrial diseases are progressive genetic disorders affecting multiple organs and characterized by mitochondrial dysfunction. These disorders can be caused by mutations in nuclear genes coding proteins with mitochondrial localization or by genetic defects in the mitochondrial genome (mtDNA). The latter include point pathogenic variants and large-scale deletions/rearrangements. MtDNA molecules with the wild type or a variant sequence can exist together in a single cell, a condition known as mtDNA heteroplasmy. MtDNA single point mutations are typically detected by means of Next-Generation Sequencing (NGS) based on short reads which, however, are limited for the identification of structural mtDNA alterations. Recently, new NGS technologies based on long reads have been released, allowing to obtain sequences of several kilobases in length; this approach is suitable for detection of structural alterations affecting the mitochondrial genome. In the present work we illustrate the optimization of two sequencing protocols based on long-read Oxford Nanopore Technology to detect mtDNA structural alterations. This approach presents strong advantages in the analysis of mtDNA compared to both short-read NGS and traditional techniques, potentially becoming the method of choice for genetic studies on mtDNA.

NGS-Based Genetic Analysis in a Cohort of Italian Patients with Suspected Inherited Myopathies and/or HyperCKemia.

Introduction/Aims HyperCKemia is considered a hallmark of neuromuscular diseases. It can be either isolated or associated with cramps, myalgia, weakness, myoglobinuria, or rhabdomyolysis, suggesting a metabolic myopathy. The aim of this work was to investigate possible genetic causes in order to help diagnose patients with recurrent hyperCKemia or clinical suspicion of inherited metabolic myopathy. Methods A cohort of 139 patients (90 adults and 49 children) was analyzed using a custom panel containing 54 genes associated with hyperCKemia. Results A definite genetic diagnosis was obtained in 15.1% of cases, while candidate variants or variants of uncertain significance were found in a further 39.5%. Similar percentages were obtained in patients with infantile or adult onset, with some different causative genes. RYR1 was the gene most frequently identified, either with single or compound heterozygous variants, while ETFDH variants were the most common cause for recessive cases. In one patient, mRNA analysis allowed identifying a large LPIN1 deletion missed by DNA sequencing, leading to a certain diagnosis. Conclusion These data confirm the high genetic heterogeneity of hyperCKemia and metabolic myopathies. The reduced diagnostic yield suggests the existence of additional genes associated with this condition but also allows speculation that a significant number of cases presenting with hyperCKemia or muscle symptoms are due to extrinsic, not genetic, factors.

Phenotyping mitochondrial DNA-related diseases in childhood: A cohort study of 150 patients.

BACKGROUND AND PURPOSE: Mitochondrial diseases (MDs) are heterogeneous disorders caused by mutations in nuclear DNA (nDNA) or mitochondrial DNA (mtDNA) associated with specific syndromes. However, especially in childhood, patients often display heterogeneity. Several reports on the biochemical and molecular profiles in children have been published, but studies tend not to differentiate between mtDNA- and nDNA-associated diseases, and focus is often on a specific phenotype. Thus, large cohort studies specifically focusing on mtDNA defects in the pediatric population are lacking. METHODS: We reviewed the clinical, metabolic, biochemical, and neuroimaging data of 150 patients with MDs due to mtDNA alterations collected at our neurological institute over the past 20 years. RESULTS: mtDNA impairment is less frequent than nDNA impairment in pediatric MDs. Ocular involvement is extremely frequent in our cohort, as is classical Leber hereditary optic neuropathy, especially with onset before 12 years of age. Extraneurological manifestations and isolated myopathy appear to be rare, unlike adult phenotypes. Deep gray matter involvement, early disease onset, and specific phenotypes, such as Pearson syndrome and Leigh syndrome, represent unfavorable prognostic factors. Phenotypes related to single large scale mtDNA deletions appear to be very frequent in the pediatric population. Furthermore, we report for the first time an mtDNA pathogenic variant associated with cavitating leukodystrophy. CONCLUSIONS: We report on a large cohort of pediatric patients with mtDNA defects, adding new data on the phenotypical characterization of mtDNA defects and suggestions for diagnostic workup and therapeutic approach.

Variants in ATP5F1B are associated with dominantly inherited dystonia.

ATP5F1B is a subunit of the mitochondrial ATP synthase or complex V of the mitochondrial respiratory chain. Pathogenic variants in nuclear genes encoding assembly factors or structural subunits are associated with complex V deficiency, typically characterized by autosomal recessive inheritance and multisystem phenotypes. Movement disorders have been described in a subset of cases carrying autosomal dominant variants in structural subunits genes ATP5F1A and ATP5MC3. Here, we report the identification of two different ATP5F1B missense variants (c.1000A>C; p.Thr334Pro and c.1445T>C; p.Val482Ala) segregating with early-onset isolated dystonia in two families, both with autosomal dominant mode of inheritance and incomplete penetrance. Functional studies in mutant fibroblasts revealed no decrease of ATP5F1B protein amount but severe reduction of complex V activity and impaired mitochondrial membrane potential, suggesting a dominant-negative effect. In conclusion, our study describes a new candidate gene associated with isolated dystonia and confirms that heterozygous variants in genes encoding subunits of the mitochondrial ATP synthase may cause autosomal dominant isolated dystonia with incomplete penetrance, likely through a dominant-negative mechanism.

Expanding the Spectrum of NUBPL-Related Leukodystrophy.

Mitochondrial leukodystrophies constitute a group of different conditions presenting with a wide range of clinical presentation but with some shared neuroradiological features. Genetic defects in NUBPL have been recognized as cause of a pediatric onset mitochondrial leukodystrophy characterized by onset at the end of the first year of life with motor delay or regression and cerebellar signs, followed by progressive spasticity. Early magnetic resonance imagings (MRIs) show white matter abnormalities with predominant involvement of frontoparietal regions and corpus callosum. A striking cerebellar involvement is usually observed. Later MRIs show spontaneous improvement of white matter abnormalities but worsening of the cerebellar involvement evolving to global atrophy and progressive involvement of brainstem. After the 7 cases initially described, 11 more subjects were reported. Some of them were similar to patients from the original series while few others broadened the phenotypic spectrum. We performed a literature review and report on a new patient who further expand the spectrum of NUBPL-related leukodystrophy. With our study we confirm that the association of cerebral white matter and cerebellar cortex abnormalities is a feature commonly observed in early stages of the disease but beside the original and so far prevalent presentation, there are also uncommon phenotypes: clinical onset can be earlier and more severe than previously thought and signs of extraneurological involvement can be observed. Brain white matter can be diffusely abnormal without anteroposterior gradient, can progressively worsen, and cystic degeneration can be present. Thalami can be involved. Basal ganglia can also become involved during disease evolution.

Customized biofilm device for antibiofilm and antibacterial screening of newly developed nanostructured silver and zinc coatings.

BACKGROUND: Bacterial colonisation on implantable device surfaces is estimated to cause more than half of healthcare-associated infections. The application of inorganic coatings onto implantable devices limits/prevents microbial contaminations. However, reliable and high-throughput deposition technologies and experimental trials of metal coatings for biomedical applications are missing. Here, we propose the combination of the Ionized Jet Deposition (IJD) technology for metal-coating application, with the Calgary Biofilm Device (CBD) for high-throughput antibacterial and antibiofilm screening, to develop and screen novel metal-based coatings. RESULTS: The films are composed of nanosized spherical aggregates of metallic silver or zinc oxide with a homogeneous and highly rough surface topography. The antibacterial and antibiofilm activity of the coatings is related with the Gram staining, being Ag and Zn coatings more effective against gram-negative and gram-positive bacteria, respectively. The antibacterial/antibiofilm effect is proportional to the amount of metal deposited that influences the amount of metal ions released. The roughness also impacts the activity, mostly for Zn coatings. Antibiofilm properties are stronger on biofilms developing on the coating than on biofilms formed on uncoated substrates. This suggests a higher antibiofilm effect arising from the direct contact bacteria-coating than that associated with the metal ions release. Proof-of-concept of application to titanium alloys, representative of orthopaedic prostheses, confirmed the antibiofilm results, validating the approach. In addition, MTT tests show that the coatings are non-cytotoxic and ICP demonstrates that they have suitable release duration (> 7 days), suggesting the applicability of these new generation metal-based coatings for the functionalization of biomedical devices. CONCLUSIONS: The combination of the Calgary Biofilm Device with the Ionized Jet Deposition technology proved to be an innovative and powerful tool that allows to monitor both the metal ions release and the surface topography of the films, which makes it suitable for the study of the antibacterial and antibiofilm activity of nanostructured materials. The results obtained with the CBD were validated with coatings on titanium alloys and extended by also considering the anti-adhesion properties and biocompatibility. In view of upcoming application in orthopaedics, these evaluations would be useful for the development of materials with pleiotropic antimicrobial mechanisms.

Mitochondrial DNA Sequencing and Heteroplasmy Quantification by Next Generation Sequencing.

Over the last 10 years, next generation sequencing (NGS) became the gold standard for both diagnosis and discovery of new disease genes responsible for heterogeneous disorders, such as mitochondrial encephalomyopathies. The application of this technology to mtDNA mutations poses extra challenges compared to other genetic conditions because of the peculiarities of mitochondrial genetics and the requirement for proper NGS data management and analysis. Here, we describe a detailed, clinically relevant protocol to sequence the whole mtDNA and quantify heteroplasmy levels of mtDNA variants, starting from total DNA through the generation of a single PCR amplicon.

Insights into the microbial life in silica-rich subterranean environments: microbial communities and ecological interactions in an orthoquartzite cave (Imawarì Yeuta, Auyan Tepui, Venezuela).

Microbial communities inhabiting caves in quartz-rich rocks are still underexplored, despite their possible role in the silica cycle. The world's longest orthoquartzite cave, Imawarì Yeuta, represents a perfect arena for the investigation of the interactions between microorganisms and silica in non-thermal environments due to the presence of extraordinary amounts of amorphous silica speleothems of different kinds. In this work, the microbial diversity of Imawarì Yeuta was dissected by analyzing nineteen samples collected from different locations representative of different silica amorphization phases and types of samples. Specifically, we investigated the major ecological patterns in cave biodiversity, specific taxa enrichment, and the main ecological clusters through co-occurrence network analysis. Water content greatly contributed to the microbial communities' composition and structures in the cave leading to the sample clustering into three groups DRY, WET, and WATER. Each of these groups was enriched in members of Actinobacteriota, Acidobacteriota, and Gammaproteobacteria, respectively. Alpha diversity analysis showed the highest value of diversity and richness for the WET samples, while the DRY group had the lowest. This was accompanied by the presence of correlation patterns including either orders belonging to various phyla from WET samples or orders belonging to the Actinobacteriota and Firmicutes phyla from DRY group samples. The phylogenetic analysis of the dominant species in WET and DRY samples showed that Acidobacteriota and Actinobacteriota strains were affiliated with uncultured bacteria retrieved from various oligotrophic and silica/quartz-rich environments, not only associated with subterranean sites. Our results suggest that the water content greatly contributes to shaping the microbial diversity within a subterranean quartzite environment. Further, the phylogenetic affiliation between Imawarì Yeuta dominant microbes and reference strains retrieved from both surface and subsurface silica- and/or CO2/CO-rich environments, underlines the selective pressure applied by quartz as rock substrate. Oligotrophy probably in association with the geochemistry of silica/quartz low pH buffering activity and alternative energy sources led to the colonization of specific silica-associated microorganisms. This study provides clues for a better comprehension of the poorly known microbial life in subsurface and surface quartz-dominated environments.

Bi-allelic LETM1 variants perturb mitochondrial ion homeostasis leading to a clinical spectrum with predominant nervous system involvement.

Leucine zipper-EF-hand containing transmembrane protein 1 (LETM1) encodes an inner mitochondrial membrane protein with an osmoregulatory function controlling mitochondrial volume and ion homeostasis. The putative association of LETM1 with a human disease was initially suggested in Wolf-Hirschhorn syndrome, a disorder that results from de novo monoallelic deletion of chromosome 4p16.3, a region encompassing LETM1. Utilizing exome sequencing and international gene-matching efforts, we have identified 18 affected individuals from 11 unrelated families harboring ultra-rare bi-allelic missense and loss-of-function LETM1 variants and clinical presentations highly suggestive of mitochondrial disease. These manifested as a spectrum of predominantly infantile-onset (14/18, 78%) and variably progressive neurological, metabolic, and dysmorphic symptoms, plus multiple organ dysfunction associated with neurodegeneration. The common features included respiratory chain complex deficiencies (100%), global developmental delay (94%), optic atrophy (83%), sensorineural hearing loss (78%), and cerebellar ataxia (78%) followed by epilepsy (67%), spasticity (53%), and myopathy (50%). Other features included bilateral cataracts (42%), cardiomyopathy (36%), and diabetes (27%). To better understand the pathogenic mechanism of the identified LETM1 variants, we performed biochemical and morphological studies on mitochondrial K+/H+ exchange activity, proteins, and shape in proband-derived fibroblasts and muscles and in Saccharomyces cerevisiae, which is an important model organism for mitochondrial osmotic regulation. Our results demonstrate that bi-allelic LETM1 variants are associated with defective mitochondrial K+ efflux, swollen mitochondrial matrix structures, and loss of important mitochondrial oxidative phosphorylation protein components, thus highlighting the implication of perturbed mitochondrial osmoregulation caused by LETM1 variants in neurological and mitochondrial pathologies.