A novel, patient-derived RyR1 mutation impairs muscle function and calcium homeostasis in mice.

RYR1 is the most commonly mutated gene associated with congenital myopathies, a group of early-onset neuromuscular conditions of variable severity. The functional effects of a number of dominant RYR1 mutations have been established; however, for recessive mutations, these effects may depend on multiple factors, such as the formation of a hypomorphic allele, or on whether they are homozygous or compound heterozygous. Here, we functionally characterize a new transgenic mouse model knocked-in for mutations identified in a severely affected child born preterm and presenting limited limb movement. The child carried the homozygous c.14928C>G RYR1 mutation, resulting in the p.F4976L substitution. In vivo and ex vivo assays revealed that homozygous mice fatigued sooner and their muscles generated significantly less force compared with their WT or heterozygous littermates. Electron microscopy, biochemical, and physiological analyses showed that muscles from RyR1 p.F4976L homozygous mice have the following properties: (1) contain fewer calcium release units and show areas of myofibrillar degeneration, (2) contain less RyR1 protein, (3) fibers show smaller electrically evoked calcium transients, and (4) their SR has smaller calcium stores. In addition, single-channel recordings indicate that RyR1 p.F4976L exhibits higher Po in the presence of 100 µM [Ca2+]. Our mouse model partly recapitulates the clinical picture of the homozygous human patient and provides significant insight into the functional impact of this mutation. These results will help understand the pathology of patients with similar RYR1 mutations.

Self-isolation of an Italian long-term care facility during COVID-19 pandemic: A comparison study on care-related infectious episodes.

The aim of this retrospective cohort study is to understand if and how much the preventive self-isolation approach might have been a valid model to avoid care-related infection, not only from COVID-19 but also from other non-viral infectious diseases. From March to May 2020, the healthcare and management staff of the Villa Santa Maria long-term care facilities, located in the village of Montenero di Bisaccia (Campobasso, Molise, Italy), decided to carry out a preventive self-isolation plan to safeguard the residents from SARS-CoV-2. The impact on other infectious diseases was evaluated by analyzing the antibiotic therapies prescription trend among the inpatients. Our data showed that although self-isolation protected residents and caregivers from SARS-CoV-2, it can also be associated with mobility reduction, leading to an increase in bedridden pathologies, namely, pressure ulcers and pressure sores. The simultaneous isolation of residents and caregivers in the same location significantly reduced any outside influence as a cause of possible infections.

Voluntary wheel running mitigates disease in an Orai1 gain-of-function mouse model of tubular aggregate myopathy.

Tubular aggregate myopathy (TAM) is an inherited skeletal muscle disease associated with progressive muscle weakness, cramps, and myalgia. Tubular aggregates (TAs) are regular arrays of highly ordered and densely packed SR straight-tubes in muscle biopsies; the extensive presence of TAs represent a key histopathological hallmark of this disease in TAM patients. TAM is caused by gain-of-function mutations in proteins that coordinate store-operated Ca2+ entry (SOCE): STIM1 Ca2+ sensor proteins in the sarcoplasmic reticulum (SR) and Ca2+-permeable ORAI1 channels in the surface membrane. We have previously shown that voluntary wheel running (VWR) prevents formation of TAs in aging mice. Here, we assessed the therapeutic potential of endurance exercise (in the form of VWR) in mitigating the functional and structural alterations in a knock-in mouse model of TAM (Orai1G100S/+ or GS mice) based on a gain-of-function mutation in the ORAI1 pore. WT and GS mice were singly-housed for six months (from two to eight months of age) with either free-spinning or locked low profile wheels. Six months of VWR exercise significantly increased soleus peak tetanic specific force production, normalized FDB fiber Ca2+ store content, and markedly reduced TAs in EDL muscle from GS mice. Six months of VWR exercise normalized the expression of mitochondrial proteins found to be altered in soleus muscle of sedentary GS mice in conjunction with a signature of increased protein translation and biosynthetic processes. Parallel proteomic analyses of EDL muscles from sedentary WT and GS mice revealed changes in a tight network of pathways involved in formation of supramolecular complexes, which were also normalized following six months of VWR. In summary, sustained voluntary endurance exercise improved slow twitch muscle function, reduced the presence of TAs in fast twitch muscle, and normalized the muscle proteome of GS mice consistent with protective adaptions in proteostasis, mitochondrial structure/function, and formation of supramolecular complexes.

Structural Adaptation of the Excitation-Contraction Coupling Apparatus in Calsequestrin1-Null Mice during Postnatal Development.

The precise arrangement and peculiar interaction of transverse tubule (T-tubule) and sarcoplasmic reticulum (SR) membranes efficiently guarantee adequate contractile properties of skeletal muscle fibers. Fast muscle fibers from mice lacking calsequestrin 1 (CASQ1) are characterized by the profound ultrastructural remodeling of T-tubule/SR junctions. This study investigates the role of CASQ1, an essential component of calcium release units (CRUs), in the postnatal development of muscle fibers. By using CASQ1-knockout mice, we examined the maturation of CRUs and the involvement of different junctional proteins in the juxtaposition of the membrane system. Our morphological investigation of both wild-type (WT) and CASQ1-null extensor digitorum longus (EDL) fibers, from 1 week to 4 months of age, yielded noteworthy findings. Firstly, we observed that the absence of CASQ1 hindered the full maturation of CRUs, despite the correct localization of key junctional components (ryanodine receptor, dihydropyridine receptor, and triadin) to the junctional SR in adult animals. Furthermore, analysis of protein expression profiles related to T-tubule biogenesis and organization (junctophilin 1, amphiphysin 2, caveolin 3, and mitsugumin 29) demonstrated delayed progression in their expression during postnatal development in the absence of CASQ1, suggesting the impaired maturation of CRUs. The absence of CASQ1 directly impacts the proper assembly of CRUs during development and influences the expression and coordination of other proteins involved in T-tubule biogenesis and organization.

Efficacy and Microbiota Modulation Induced by LimpiAL 2.5%, a New Medical Device for the Inverse Psoriasis Treatment.

(1) Inverse psoriasis (IP), also known as intertriginous, typically affects the groin, armpits, navel, intergluteal fissure, and external genitalia. Skin lesions are erythematous plaques of inflammatory nature, smooth, well-delimited, non-scaly, and non-infiltrated. Lesions may be accompanied by itching, pain, or burning sensation. The aim of this study is both to investigate the modulation of the skin microbiota induced by IP and, on the other hand, to test the effectiveness of the new biotechnological product LimpiAL 2.5%. (2) Patients affected by IP were recruited in a private practice and treated for 4 weeks with LimpiAL 2.5% exclusively. The clinical effects on the lesion skin were evaluated, and the skin microbiotas before and after treatment were compared. (3) The clinical outcomes reveled a significant beneficial effect of the tested product. At the same time, LimpiAL increased the biological diversity of the skin microbiota and exerted a significant decrease of some Corynebacterium species, and the increase of some Staphylococcus species. (4) Together, the clinical outcomes and the microbiota analysis suggest that LimpiAL treatment improves the skin condition of affected patients, basically restoring the eubiosis conditions of the affected sites and modulating the bacterial composition of the resident microbiota.

A Wall Fragment of Cutibacterium acnes Preserves Junctional Integrity Altered by Staphylococcus aureus in an Ex Vivo Porcine Skin Model.

(1) Background alteration of the skin microbiota, dysbiosis, causes skin barrier impairment resulting in disease development. Staphylococcus aureus, the main pathogen associated with dysbiosis, secretes several virulence factors, including α-toxin that damages tight junctions and compromises the integrity of the skin barrier. The use of members of the resident microbiota to restore the skin barrier, bacteriotherapy, represents a safe treatment for skin conditions among innovative options. The aim of this study is the evaluation of a wall fragment derived from a patented strain of Cutibacterium acnes DSM28251 (c40) alone and conjugated to a mucopolysaccharide carrier (HAc40) in counteracting S. aureus pathogenic action on two tight junction proteins (Claudin-1 and ZO-1) in an ex vivo porcine skin infection model. Methods: skin biopsies were infected with live S. aureus strains ATCC29213 and DSM20491. Tissue was pre-incubated or co-incubated with c40 and HAc40. (3) Results: c40 and HAc40 prevent and counteract Claudin-1 and Zo-1 damage (4) Conclusions: c40 and the functional ingredient HAc40 represent a potential non-pharmacological treatment of skin diseases associated with cutaneous dysbiosis of S. aureus. These findings offer numerous avenues for new research.

Searching for Mechanisms Underlying the Assembly of Calcium Entry Units: The Role of Temperature and pH.

Store-operated Ca2+ entry (SOCE) is a mechanism that allows muscle fibers to recover external Ca2+, which first enters the cytoplasm and then, via SERCA pump, also refills the depleted intracellular stores (i.e., the sarcoplasmic reticulum, SR). We recently discovered that SOCE is mediated by Calcium Entry Units (CEUs), intracellular junctions formed by: (i) SR stacks containing STIM1; and (ii) I-band extensions of the transverse tubule (TT) containing Orai1. The number and size of CEUs increase during prolonged muscle activity, though the mechanisms underlying exercise-dependent formation of new CEUs remain to be elucidated. Here, we first subjected isolated extensor digitorum longus (EDL) muscles from wild type mice to an ex vivo exercise protocol and verified that functional CEUs can assemble also in the absence of blood supply and innervation. Then, we evaluated whether parameters that are influenced by exercise, such as temperature and pH, may influence the assembly of CEUs. Results collected indicate that higher temperature (36 °C vs. 25 °C) and lower pH (7.2 vs. 7.4) increase the percentage of fibers containing SR stacks, the n. of SR stacks/area, and the elongation of TTs at the I band. Functionally, assembly of CEUs at higher temperature (36 °C) or at lower pH (7.2) correlates with increased fatigue resistance of EDL muscles in the presence of extracellular Ca2+. Taken together, these results indicate that CEUs can assemble in isolated EDL muscles and that temperature and pH are two of the possible regulators of CEU formation.

Emerging Evidence on Tenebrio molitor Immunity: A Focus on Gene Expression Involved in Microbial Infection for Host-Pathogen Interaction Studies.

In recent years, the scientific community's interest in T. molitor as an insect model to investigate immunity and host-pathogen interactions has considerably increased. The reasons for this growing interest could be explained by the peculiar features of this beetle, which offers various advantages compared to other invertebrates models commonly used in laboratory studies. Thus, this review aimed at providing a broad view of the T. molitor immune system in light of the new scientific evidence on the developmental/tissue-specific gene expression studies related to microbial infection. In addition to the well-known cellular component and humoral response process, several studies investigating the factors associated with T. molitor immune response or deepening of those already known have been reported. However, various aspects remain still less understood, namely the possible crosstalk between the immune deficiency protein and Toll pathways and the role exerted by T. molitor apolipoprotein III in the expression of the antimicrobial peptides. Therefore, further research is required for T. molitor to be recommended as an alternative insect model for pathogen-host interaction and immunity studies.

Ablation of Calsequestrin-1, Ca2+ unbalance, and susceptibility to heat stroke.

Introduction: Ca2+ levels in adult skeletal muscle fibers are mainly controlled by excitation-contraction (EC) coupling, a mechanism that translates action potentials in release of Ca2+ from the sarcoplasmic reticulum (SR) release channels, i.e. the ryanodine receptors type-1 (RyR1). Calsequestrin (Casq) is a protein that binds large amounts of Ca2+ in the lumen of the SR terminal cisternae, near sites of Ca2+ release. There is general agreement that Casq is not only important for the SR ability to store Ca2+, but also for modulating the opening probability of the RyR Ca2+ release channels. The initial studies: About 20 years ago we generated a mouse model lacking Casq1 (Casq1-null mice), the isoform predominantly expressed in adult fast twitch skeletal muscle. While the knockout was not lethal as expected, lack of Casq1 caused a striking remodeling of membranes of SR and of transverse tubules (TTs), and mitochondrial damage. Functionally, CASQ1-knockout resulted in reduced SR Ca2+ content, smaller Ca2+ transients, and severe SR depletion during repetitive stimulation. The myopathic phenotype of Casq1-null mice: After the initial studies, we discovered that Casq1-null mice were prone to sudden death when exposed to halogenated anaesthetics, heat and even strenuous exercise. These syndromes are similar to human malignant hyperthermia susceptibility (MHS) and environmental-exertional heat stroke (HS). We learned that mechanisms underlying these syndromes involved excessive SR Ca2+ leak and excessive production of oxidative species: indeed, mortality and mitochondrial damage were significantly prevented by administration of antioxidants and reduction of oxidative stress. Though, how Casq1-null mice could survive without the most important SR Ca2+ binding protein was a puzzling issue that was not solved. Unravelling the mystery: The mystery was finally solved in 2020, when we discovered that in Casq1-null mice the SR undergoes adaptations that result in constitutively active store-operated Ca2+ entry (SOCE). SOCE is a mechanism that allows skeletal fibers to use external Ca2+ when SR stores are depleted. The post-natal compensatory mechanism that allows Casq1-null mice to survive involves the assembly of new SR-TT junctions (named Ca2+ entry units) containing Stim1 and Orai1, the two proteins that mediate SOCE.

Constitutive assembly of Ca2+ entry units in soleus muscle from calsequestrin knockout mice.

Calcium (Ca2+) entry units (CEUs) are junctions within the I band of the sarcomere between stacks of sarcoplasmic reticulum (SR) cisternae and extensions of the transverse (T)-tubule. CEUs contain STIM1 and Orai1 proteins, the molecular machinery of store-operated Ca2+ entry (SOCE). In extensor digitorum longus (EDL) fibers of wild-type (WT) mice, CEUs transiently assemble during acute exercise and disassemble several hours thereafter. By contrast, calsequestrin-1 (CASQ1) ablation induces a compensatory constitutive assembly of CEUs in EDL fibers, resulting in enhanced constitutive and maximum SOCE that counteracts SR Ca2+ depletion during repetitive activity. However, whether CEUs form in slow-twitch fibers, which express both the skeletal CASQ1 and the cardiac CASQ2 isoforms, is unknown. Herein, we compared the structure and function of soleus muscles from WT and knockout mice that lack either CASQ1 (CASQ1-null) or both CASQs (dCASQ-null). Ultrastructural analyses showed that SR/T-tubule junctions at the I band, virtually identical to CEUs in EDL muscle, were present and more frequent in CASQ1-null than WT mice, with dCASQ-null exhibiting the highest incidence. The greater incidence of CEUs in soleus from dCASQ-null mice correlated with increased specific force production during repetitive, high-frequency stimulation, which depended on Ca2+ entry. Consistent with this, Orai1 expression was significantly increased in soleus of CASQ1-null mice, but even more in dCASQ-null mice, compared with WT. Together, these results strengthen the concept that CEU assembly strongly depends on CASQ expression and provides an alternative source of Ca2+ needed to refill SR Ca2+ stores to maintain specific force production during sustained muscle activity.

The new invasive mosquito species Aedes koreicus as vector-borne diseases in the European area, a focus on Italian region: What we know from the scientific literature.

The increased mobility of goods, people, and animals worldwide has caused the spread of several arthropod vectors, leading to an increased risk of animal and human infections. Aedes koreicus is a common species in South Korea, China, Japan, and Russia. Due to its cold-resistant dormant eggs, the adults last from the late summer until the autumn seasons. For these reasons, it seems to be better adapted to colder temperatures, favoring its colonization of hilly and pre-alpine areas. Its first appearance in Europe was in 2008 in Belgium, where it is currently established. The species was subsequently detected in Italy in 2011, European Russia, Germany, the Swiss-Italian border region, Hungary, Slovenia, Crimea, Austria, the Republic of Kazakhstan, and the Netherlands. The role of A. koreicus in the transmission of vector-borne pathogens remains unclear. The available scientific evidence is very old, often not available in English or not indexed in international databases, and therefore difficult to find. According to the literature reviewed, A. koreicus can be considered a new invasive mosquito species in Europe, establishing populations on the European continent. In addition, experimental evidence demonstrated its vector competence for both Dirofilaria immitis and Chikungunya and is relatively low for ZIKA but not for Western Nile Virus. On the other hand, even if the field evidence does not confirm the experimental findings, it is currently not possible to exclude with absolute certainty the potential involvement of this species in the spread, emergence, or re-emergence of these vector-borne disease agents.

Oxygen Consumption and Basal Metabolic Rate as Markers of Susceptibility to Malignant Hyperthermia and Heat Stroke.

Calsequestrin 1 (CASQ1) and Ryanodine receptor 1 (RYR1) are two of the main players in excitation-contraction (EC) coupling. CASQ1-knockout mice and mice carrying a mutation in RYR1 (Y522S) linked to human malignant hyperthermia susceptibility (MHS) both suffer lethal hypermetabolic episodes when exposed to halothane (MHS crises) and to environmental heat (heat stroke, HS). The phenotype of Y522S is more severe than that of CASQ1-null mice. As MHS and HS are hypermetabolic responses, we studied the metabolism of adult CASQ1-null and Y522S mice using wild-type (WT) mice as controls. We found that CASQ1-null and Y522S mice have increased food consumption and higher core temperature at rest. By indirect calorimetry, we then verified that CASQ1-null and Y522S mice show an increased oxygen consumption and a lower respiratory quotient (RQ). The accelerated metabolism of CASQ1-null and Y522S mice was also accompanied with a reduction in body fat. Moreover, both mouse models displayed increased oxygen consumption and a higher core temperature during heat stress. The results collected suggest that metabolic rate, oxygen consumption, and body temperature at rest, all more elevated in Y522S than in CASQ1-null mice, could possibly be used as predictors of the level of susceptibility to hyperthermic crises of mice (and possibly humans).

Bioengineered Human Stromal Lenticule for Recombinant Human Nerve Growth Factor Release: A Potential Biocompatible Ocular Drug Delivery System.

Small incision lenticule extraction (SMILE), is a surgical procedure for the myopia correction, during which a corneal stromal lenticule is extracted. Given that we have previously demonstrated how this discarded tissue could be repurposed as a bio-scaffold for stromal engineering, this study aimed to explore its use as an ocular drug delivery system of active molecules, using neurotrophic factor Nerve Growth Factor (NGF). We employed human stromal lenticules directly collected from healthy donors undergoing SMILE. Following a sodium dodecylsulfate (SDS) treatment, decellularized lenticules were incubated with a suspension of polylactic-co-glycolic-acid (PLGA) microparticles (MPs) loaded with recombinant human NGF (rhNGF-MPs). Fluorescent MPs (Fluo-MPs) were used as control. Data demonstrated the feasibility to engineer decellularized lenticules with PLGA-MPs which remain incorporated both on the lenticules surface and in its stromal. Following their production, the in vitro release kinetic showed a sustained release for up to 1 month of rhNGF from MPs loaded to the lenticule. Interestingly, rhNGF was rapidly released in the first 24 h, but it was sustained up to the end of the experiment (1 month), with preservation of rhNGF activity (around 80%). Our results indicated that decellularized human stromal lenticules could represent a biocompatible, non-immunogenic natural scaffold potential useful for ocular drug delivery. Therefore, combining the advantages of tissue engineering and pharmaceutical approaches, this in vitro proof-of-concept study suggests the feasibility to use this scaffold to allow target release of rhNGF in vivo or other pharmaceutically active molecules that have potential to treat ocular diseases.

Identifying plant genes shaping microbiota composition in the barley rhizosphere.

A prerequisite to exploiting soil microbes for sustainable crop production is the identification of the plant genes shaping microbiota composition in the rhizosphere, the interface between roots and soil. Here, we use metagenomics information as an external quantitative phenotype to map the host genetic determinants of the rhizosphere microbiota in wild and domesticated genotypes of barley, the fourth most cultivated cereal globally. We identify a small number of loci with a major effect on the composition of rhizosphere communities. One of those, designated the QRMC-3HS, emerges as a major determinant of microbiota composition. We subject soil-grown sibling lines harbouring contrasting alleles at QRMC-3HS and hosting contrasting microbiotas to comparative root RNA-seq profiling. This allows us to identify three primary candidate genes, including a Nucleotide-Binding-Leucine-Rich-Repeat (NLR) gene in a region of structural variation of the barley genome. Our results provide insights into the footprint of crop improvement on the plant's capacity of shaping rhizosphere microbes.

High-Fat Diet Impairs Muscle Function and Increases the Risk of Environmental Heatstroke in Mice.

Environmental heat-stroke (HS) is a life-threatening response often triggered by hot and humid weather. Several lines of evidence indicate that HS is caused by excessive heat production in skeletal muscle, which in turn is the result of abnormal Ca2+ leak from the sarcoplasmic reticulum (SR) and excessive production of oxidative species of oxygen and nitrogen. As a high fat diet is known to increase oxidative stress, the objective of the present study was to investigate the effects of 3 months of high-fat diet (HFD) on the HS susceptibility of wild type (WT) mice. HS susceptibility was tested in an environmental chamber where 4 months old WT mice were exposed to heat stress (41 °C for 1 h). In comparison with mice fed with a regular diet, mice fed with HFD showed: (a) increased body weight and accumulation of adipose tissue; (b) elevated oxidative stress in skeletal muscles; (c) increased heat generation and oxygen consumption during exposure to heat stress; and finally, (d) enhanced sensitivity to both temperature and caffeine of isolated muscles during in-vitro contracture test. These data (a) suggest that HFD predisposes WT mice to heat stress and (b) could have implications for guidelines regarding food intake during periods of intense environmental heat.

Store-Operated Ca2+ Entry in Skeletal Muscle Contributes to the Increase in Body Temperature during Exertional Stress.

Exertional heat stroke (HS) is a hyperthermic crisis triggered by an excessive accumulation of Ca2+ in skeletal muscle fibers. We demonstrated that exercise leads to the formation of calcium entry units (CEUs), which are intracellular junctions that reduce muscle fatigue by promoting the recovery of extracellular Ca2+ via store-operated Ca2+ entry (SOCE). Here, we tested the hypothesis that exercise-induced assembly of CEUs may increase the risk of HS when physical activity is performed in adverse environmental conditions (high temperature and humidity). Adult mice were: (a) first, divided into three experimental groups: control, trained-1 month (voluntary running in wheel cages), and acutely exercised-1 h (incremental treadmill run); and (b) then subjected to an exertional stress (ES) protocol, a treadmill run in an environmental chamber at 34 °C and 40% humidity. The internal temperature of the mice at the end of the ES was higher in both pre-exercised groups. During an ES ex-vivo protocol, extensor digitorum longus(EDL) muscles from the trained-1 month and exercised-1 h mice generated greater basal tension than in the control and were those that contained a greater number of CEUs, assessed by electron microscopy. The data collected suggest that the entry of Ca2+ from extracellular space via CEUs could contribute to exertional HS when exercise is performed in adverse environmental conditions.

Prospective Evaluation of Ghrelin and Des-Acyl Ghrelin Plasma Levels in Children with Newly Diagnosed Epilepsy: Evidence for Reduced Ghrelin-to-Des-Acyl Ghrelin Ratio in Generalized Epilepsies.

Children with epilepsy and identified as responders to antiseizure medications (ASMs) were found to present markedly higher ghrelin plasma levels when compared to drug-resistant patients. However, it was undetermined if this phenotype could be influenced by the ASMs. Here, we prospectively investigated total ghrelin and des-acyl ghrelin (DAG) plasma levels by enzyme-linked immunosorbent assay before and after ASM administration. Inclusion criteria were: (i) subject with a suspicion of epilepsy; (ii) age ranging from 0 to 16 years; and (iii) informed consent signed by parents or caregivers. Exclusion criteria were acute or chronic metabolic disorders with occasional convulsions but without epilepsy. Fifty patients were followed over a period of one year in Italian neuropediatric centers. Apart from a few exceptions, the majority of children were responsive to ASMs. No differences were found in total ghrelin and DAG levels before and after the treatment, but total ghrelin levels were significantly lower in children with generalized epilepsy compared to those with combined focal and generalized epilepsy. Moreover, the ghrelin-to-DAG ratio was also markedly lower in generalized epilepsies compared to all the other types of epilepsy. Finally, ghrelin was unchanged by ASMs, including the first (e.g., carbamazepine), second (levetiracetam), and third (lacosamide) generation of anticonvulsants.

Improvement of muscle strength in a mouse model for congenital myopathy treated with HDAC and DNA methyltransferase inhibitors.

To date there are no therapies for patients with congenital myopathies, muscle disorders causing poor quality of life of affected individuals. In approximately 30% of the cases, patients with congenital myopathies carry either dominant or recessive mutations in the ryanodine receptor 1 (RYR1) gene; recessive RYR1 mutations are accompanied by reduction of RyR1 expression and content in skeletal muscles and are associated with fiber hypotrophy and muscle weakness. Importantly, muscles of patients with recessive RYR1 mutations exhibit increased content of class II histone deacetylases and of DNA genomic methylation. We recently created a mouse model knocked-in for the p.Q1970fsX16+ p.A4329D RyR1 mutations, which are isogenic to those carried by a severely affected child suffering from a recessive form of RyR1-related multi-mini core disease. The phenotype of the RyR1 mutant mice recapitulates many aspects of the clinical picture of patients carrying recessive RYR1 mutations. We treated the compound heterozygous mice with a combination of two drugs targeting DNA methylases and class II histone deacetylases. Here, we show that treatment of the mutant mice with drugs targeting epigenetic enzymes improves muscle strength, RyR1 protein content, and muscle ultrastructure. This study provides proof of concept for the pharmacological treatment of patients with congenital myopathies linked to recessive RYR1 mutations.

Proteomic Analysis of Marinesco-Sjogren Syndrome Fibroblasts Indicates Pro-Survival Metabolic Adaptation to SIL1 Loss.

Marinesco-Sjogren syndrome (MSS) is a rare multisystem pediatric disorder, caused by loss-of-function mutations in the gene encoding the endoplasmic reticulum cochaperone SIL1. SIL1 acts as a nucleotide exchange factor for BiP, which plays a central role in secretory protein folding. SIL1 mutant cells have reduced BiP-assisted protein folding, cannot fulfil their protein needs, and experience chronic activation of the unfolded protein response (UPR). Maladaptive UPR may explain the cerebellar and skeletal muscle degeneration responsible for the ataxia and muscle weakness typical of MSS. However, the cause of other more variable, clinical manifestations, such as mild to severe mental retardation, hypogonadism, short stature, and skeletal deformities, is less clear. To gain insights into the pathogenic mechanisms and/or adaptive responses to SIL1 loss, we carried out cell biological and proteomic investigations in skin fibroblasts derived from a young patient carrying the SIL1 R111X mutation. Despite fibroblasts not being overtly affected in MSS, we found morphological and biochemical changes indicative of UPR activation and altered cell metabolism. All the cell machineries involved in RNA splicing and translation were strongly downregulated, while protein degradation via lysosome-based structures was boosted, consistent with an attempt of the cell to reduce the workload of the endoplasmic reticulum and dispose of misfolded proteins. Cell metabolism was extensively affected as we observed a reduction in lipid synthesis, an increase in beta oxidation, and an enhancement of the tricarboxylic acid cycle, with upregulation of eight of its enzymes. Finally, the catabolic pathways of various amino acids, including valine, leucine, isoleucine, tryptophan, lysine, aspartate, and phenylalanine, were enhanced, while the biosynthetic pathways of arginine, serine, glycine, and cysteine were reduced. These results indicate that, in addition to UPR activation and increased protein degradation, MSS fibroblasts have profound metabolic alterations, which may help them cope with the absence of SIL1.

LimpiAD foam and the potential control of the pressure ulcers onset.

Pressure ulcers development is an undesirable event that often worsens the clinical condition of patients already affected by severe pathologies. Since the aetiology of this clinical complication is unclear yet, at current the primary approach to treat the problem is the adoption of suitable patients' assistance procedures. At the same time, the research focuses on finding medicaments or treatment strategies that could prevent the lesions and/or accelerate their healing. The international market's wide range of cosmetic/pharmaceuticals products is mainly topical preparations based on emollient agents to preserve or restore skin homeostasis. On the other hand, the skin microbiome's implication in the pressure ulcers occurrence is mainly unknown. Based on these assumptions, here we tested an innovative preparation, the LimpiAD foam, as a potential preventive strategy of pressure ulcers onset. The active component of this product is composed of hyaluronic acid conjugated with a bacterial cell wall fragment of C. acnes DSM 28251. For LimpiAD foam, we hypothesised a combined action of the two components on the skin tissue, an emollient effect due to the hyaluronic acid properties together with a modulatory effect on the skin microbiota carried out by the component of bacterial derivation. Our results supported the hypothesis and suggested a potential role of LimpiAD foam in pressure ulcers prevention.