Sunitinib-mediated inhibition of STAT3 in skeletal muscle and spinal cord does not affect the disease in a mouse model of ALS.

Variability in disease onset and progression is a hallmark of amyotrophic lateral sclerosis (ALS), both in sporadic and genetic forms. Recently, we found that SOD1-G93A transgenic mice expressing the same amount of mutant SOD1 but with different genetic backgrounds, C57BL/6JOlaHsd and 129S2/SvHsd, show slow and rapid muscle wasting and disease progression, respectively. Here, we investigated the different molecular mechanisms underlying muscle atrophy. Although both strains showed similar denervation-induced degradation of muscle proteins, only the rapidly progressing mice exhibited early and sustained STAT3 activation that preceded atrophy in gastrocnemius muscle. We therefore investigated the therapeutic potential of sunitinib, a tyrosine kinase inhibitor known to inhibit STAT3 and prevent cancer-induced muscle wasting. Although sunitinib treatment reduced STAT3 activation in the gastrocnemius muscle and lumbar spinal cord, it did not preserve spinal motor neurons, improve neuromuscular impairment, muscle atrophy and disease progression in the rapidly progressing SOD1-G93A mice. Thus, the effect of sunitinib is not equally positive in different diseases associated with muscle wasting. Moreover, given the complex role of STAT3 in the peripheral and central compartments of the neuromuscular system, the present study suggests that its broad inhibition may lead to opposing effects, ultimately preventing a potential positive therapeutic action in ALS.

Integrated procedures for accelerating, deepening, and leading genetic inquiry: A first application on human muscle secretome.

PURPOSE: Beyond classical procedures, bioinformatic-assisted approaches and computational biology offer unprecedented opportunities for scholars. However, these amazing possibilities still need epistemological criticism, as well as standardized procedures. Especially those topics with a huge body of data may benefit from data science (DS)-assisted methods. Therefore, the current study dealt with the combined expert-assisted and DS-assisted approaches to address the broad field of muscle secretome. We aimed to apply DS tools to fix the literature research, suggest investigation targets with a data-driven approach, predict possible scenarios, and define a workflow. METHODS: Recognized scholars with expertise on myokines were invited to provide a list of the most important myokines. GeneRecommender, GeneMANIA, HumanNet, and STRING were selected as DS tools. Networks were built on STRING and GeneMANIA. The outcomes of DS tools included the top 5 recommendations. Each expert-led discussion has been then integrated with an DS-led approach to provide further perspectives. RESULTS: Among the results, 11 molecules had already been described as bona-fide myokines in literature, and 11 molecules were putative myokines. Most of the myokines and the putative myokines recommended by the DS tools were described as present in the cargo of extracellular vesicles. CONCLUSIONS: Including both supervised and unsupervised learning methods, as well as encompassing algorithms focused on both protein interaction and gene represent a comprehensive approach to tackle complex biomedical topics. DS-assisted methods for reviewing existent evidence, recommending targets of interest, and predicting original scenarios are worth exploring as in silico recommendations to be integrated with experts' ideas for optimizing molecular studies.

Will there be large or small gifts to PDM3 attendees and EJTM authors in March and June 2023?

The fall of 2022 approaches with the need to finalize our plans for next year. This is urgent for the 2023 Meeting of the Padua Days of Muscle and Mobility Medicine, (PDM3) to be held March 29 to April 1, 2023 at the Hotel Petrarca in the Thermae of Euganean Hills (Padua), Italy, but there are also news related to the inclusion of the European Journal of Translational Myology (EJTM) in the Web of Science: Emerging Sources Citation Index - Clarivate (ESCI) database. A preliminary PDM3 flyer is almost ready with session program, organzers and keynote speakers. Some are the traditional organizers of the PDM3 sessions dedicated to molecular and cellular myology, but there will also be interesting new entries, including those of Rehabilitation Sessions. No doubt that PDM3 2023 will be a great gift for all Participants, as is the tradition of PDM3. The other big news scheduled for June 2023 is the Impact Factor 2022 that Clarivate will release next year. It could be a big or small gift. As Authors who publish in other Magazines, but even more as Referees we could contribute in the next months of 2022 to make a small gift from Clarivate bigger. In any case, it will be a great gift that has been awaited for many years for one of us, who is approaching 80 years of age next February 2023.

Transforming Growth Factor-Beta Signaling in Cancer-Induced Cachexia: From Molecular Pathways to the Clinics.

Cachexia is a metabolic syndrome consisting of massive loss of muscle mass and function that has a severe impact on the quality of life and survival of cancer patients. Up to 20% of lung cancer patients and up to 80% of pancreatic cancer patients are diagnosed with cachexia, leading to death in 20% of them. The main drivers of cachexia are cytokines such as interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), macrophage inhibitory cytokine 1 (MIC-1/GDF15) and transforming growth factor-beta (TGF-ß). Besides its double-edged role as a tumor suppressor and activator, TGF-ß causes muscle loss through myostatin-based signaling, involved in the reduction in protein synthesis and enhanced protein degradation. Additionally, TGF-ß induces inhibin and activin, causing weight loss and muscle depletion, while MIC-1/GDF15, a member of the TGF-ß superfamily, leads to anorexia and so, indirectly, to muscle wasting, acting on the hypothalamus center. Against this background, the blockade of TGF-ß is tested as a potential mechanism to revert cachexia, and antibodies against TGF-ß reduced weight and muscle loss in murine models of pancreatic cancer. This article reviews the role of the TGF-ß pathway and to a minor extent of other molecules including microRNA in cancer onset and progression with a special focus on their involvement in cachexia, to enlighten whether TGF-ß and such other players could be potential targets for therapy.

The p97-Nploc4 ATPase complex plays a role in muscle atrophy during cancer and amyotrophic lateral sclerosis.

BACKGROUND: The p97 complex participates in the degradation of muscle proteins during atrophy upon fasting or denervation interacting with different protein adaptors. We investigated whether and how it might also be involved in muscle wasting in cancer, where loss of appetite occurs, or amyotrophic lateral sclerosis (ALS), where motoneuron death causes muscle denervation and fatal paralysis. METHODS: As cancer cachexia models, we used mice bearing colon adenocarcinoma C26, human renal carcinoma RXF393, or Lewis lung carcinoma, with breast cancer 4T1-injected mice as controls. As ALS models, we employed 129/SvHsd mice carrying the mutation G93A in human SOD1. The expression of p97 and its adaptors was analysed in their muscles by quantitative real-time polymerase chain reaction (qPCR) and western blot. We electroporated plasmids into muscles or treated mice with disulfiram (DSF) to test the effects of inhibiting p97 and nuclear protein localization protein 4 (Nploc4), one of its adaptors, on atrophy. RESULTS: The mRNA levels of p97 were induced by 1.5-fold to 2-fold in tibialis anterior (TA) of all the cachectic models but not in the non-cachectic 4T1 tumour-bearing mice (P ≤ 0.05). Similarly, p97 was high both in mRNA and protein in TA from 17-week-old SOD1G93A mice (P ≤ 0.01). Electroporation of a shRNA for murine p97 into mouse muscle reduced the fibre atrophy caused by C26 (P = 0.0003) or ALS (P ≤ 0.01). When we interrogated a microarray, we had previously generated for the expression of p97 adaptors, we found Derl1, Herpud1, Nploc4, Rnf31, and Hsp90ab1 induced in cachectic TA from C26-mice (Fold change > 1.2, adjusted P ≤ 0.05). By qPCR, we validated their inductions in TA of cachectic and ALS models and selected Nploc4 as the one also induced at the protein level by 1.5-fold (P ≤ 0.01). Electroporation of a CRISPR/Cas9 vector against Nploc4 into muscle reduced the fibre atrophy caused by C26 (P = 0.01) or ALS (P ≤ 0.0001). Because DSF uncouples p97 from Nploc4, we treated atrophying myotubes with DSF, and found accumulated mono and polyubiquitinated proteins and reduced degradation of long-lived proteins by 35% (P ≤ 0.0001), including actin (P ≤ 0.05). DSF halves Nploc4 in the soluble muscle fraction (P ≤ 0.001) and given to C26-bearing mice limited the body and muscle weight loss (P ≤ 0.05), with no effect on tumour growth. CONCLUSIONS: Overall, cancer cachexia and ALS seem to display similar mechanisms of muscle wasting at least at the catabolic level. The p97-Nploc4 complex appears to have a crucial role in muscle atrophy during these disorders and disrupting this complex might serve as a novel drug strategy.

Apelin Resistance Contributes to Muscle Loss during Cancer Cachexia in Mice.

Cancer cachexia consists of dramatic body weight loss with rapid muscle depletion due to imbalanced protein homeostasis. We found that the mRNA levels of apelin decrease in muscles from cachectic hepatoma-bearing rats and three mouse models of cachexia. Furthermore, apelin expression inversely correlates with MuRF1 in muscle biopsies from cancer patients. To shed light on the possible role of apelin in cachexia in vivo, we generated apelin 13 carrying all the last 13 amino acids of apelin in D isomers, ultimately extending plasma stability. Notably, apelin D-peptides alter cAMP-based signaling in vitro as the L-peptides, supporting receptor binding. In vitro apelin 13 protects myotube diameter from dexamethasone-induced atrophy, restrains rates of degradation of long-lived proteins and MuRF1 expression, but fails to protect mice from atrophy. D-apelin 13 given intraperitoneally for 13 days in colon adenocarcinoma C26-bearing mice does not reduce catabolic pathways in muscles, as it does in vitro. Puzzlingly, the levels of circulating apelin seemingly deriving from cachexia-inducing tumors, increase in murine plasma during cachexia. Muscle electroporation of a plasmid expressing its receptor APJ, unlike apelin, preserves myofiber area from C26-induced atrophy, supporting apelin resistance in vivo. Altogether, we believe that during cachexia apelin resistance occurs, contributing to muscle wasting and nullifying any possible peptide-based treatment.

TRIM32: A Multifunctional Protein Involved in Muscle Homeostasis, Glucose Metabolism, and Tumorigenesis.

Human tripartite motif family of proteins 32 (TRIM32) is a ubiquitous multifunctional protein that has demonstrated roles in differentiation, muscle physiology and regeneration, and tumor suppression. Mutations in TRIM32 result in two clinically diverse diseases. A mutation in the B-box domain gives rise to Bardet-Biedl syndrome (BBS), a disease whose clinical presentation shares no muscle pathology, while mutations in the NHL (NCL-1, HT2A, LIN-41) repeats of TRIM32 causes limb-girdle muscular dystrophy type 2H (LGMD2H). TRIM32 also functions as a tumor suppressor, but paradoxically is overexpressed in certain types of cancer. Recent evidence supports a role for TRIM32 in glycolytic-mediated cell growth, thus providing a possible mechanism for TRIM32 in the accumulation of cellular biomass during regeneration and tumorigenesis, including in vitro and in vivo approaches, to understand the broad spectrum of TRIM32 functions. A special emphasis is placed on the utility of the Drosophila model, a unique system to study glycolysis and anabolic pathways that contribute to the growth and homeostasis of both normal and tumor tissues.

Costameric integrin and sarcoglycan protein levels are altered in a Drosophila model for Limb-girdle muscular dystrophy type 2H.

Mutations in two different domains of the ubiquitously expressed TRIM32 protein give rise to two clinically separate diseases, one of which is Limb-girdle muscular dystrophy type 2H (LGMD2H). Uncovering the muscle-specific role of TRIM32 in LGMD2H pathogenesis has proven difficult, as neurogenic phenotypes, independent of LGMD2H pathology, are present in TRIM32 KO mice. We previously established a platform to study LGMD2H pathogenesis using Drosophila melanogaster as a model. Here we show that LGMD2H disease-causing mutations in the NHL domain are molecularly and structurally conserved between fly and human TRIM32. Furthermore, transgenic expression of a subset of myopathic alleles (R394H, D487N, and 520fs) induce myofibril abnormalities, altered nuclear morphology, and reduced TRIM32 protein levels, mimicking phenotypes in patients afflicted with LGMD2H. Intriguingly, we also report for the first time that the protein levels of ßPS integrin and sarcoglycan δ, both core components of costameres, are elevated in TRIM32 disease-causing alleles. Similarly, murine myoblasts overexpressing a catalytically inactive TRIM32 mutant aberrantly accumulate α- and ß-dystroglycan and α-sarcoglycan. We speculate that the stoichiometric loss of costamere components disrupts costamere complexes to promote muscle degeneration.

Nutraceuticals and Exercise against Muscle Wasting during Cancer Cachexia.

Cancer cachexia (CC) is a debilitating multifactorial syndrome, involving progressive deterioration and functional impairment of skeletal muscles. It affects about 80% of patients with advanced cancer and causes premature death. No causal therapy is available against CC. In the last few decades, our understanding of the mechanisms contributing to muscle wasting during cancer has markedly increased. Both inflammation and oxidative stress (OS) alter anabolic and catabolic signaling pathways mostly culminating with muscle depletion. Several preclinical studies have emphasized the beneficial roles of several classes of nutraceuticals and modes of physical exercise, but their efficacy in CC patients remains scant. The route of nutraceutical administration is critical to increase its bioavailability and achieve the desired anti-cachexia effects. Accumulating evidence suggests that a single therapy may not be enough, and a bimodal intervention (nutraceuticals plus exercise) may be a more effective treatment for CC. This review focuses on the current state of the field on the role of inflammation and OS in the pathogenesis of muscle atrophy during CC, and how nutraceuticals and physical activity may act synergistically to limit muscle wasting and dysfunction.

Trabectedin and Lurbinectedin Extend Survival of Mice Bearing C26 Colon Adenocarcinoma, without Affecting Tumor Growth or Cachexia.

Trabectedin (ET743) and lurbinectedin (PM01183) limit the production of inflammatory cytokines that are elevated during cancer cachexia. Mice carrying C26 colon adenocarcinoma display cachexia (i.e., premature death and body wasting with muscle, fat and cardiac tissue depletion), high levels of inflammatory cytokines and subsequent splenomegaly. We tested whether such drugs protected these mice from cachexia. Ten-week-old mice were inoculated with C26 cells and three days later randomized to receive intravenously vehicle or 0.05 mg/kg ET743 or 0.07 mg/kg PM01183, three times a week for three weeks. ET743 or PM01183 extended the lifespan of C26-mice by 30% or 85%, respectively, without affecting tumor growth or food intake. Within 13 days from C26 implant, both drugs did not protect fat, muscle and heart from cachexia. Since PM01183 extended the animal survival more than ET743, we analyzed PM01183 further. In tibialis anterior of C26-mice, but not in atrophying myotubes, PM01183 restrained the NF-κB/PAX7/myogenin axis, possibly reducing the pro-inflammatory milieu, and failed to limit the C/EBPß/atrogin-1 axis. Inflammation-mediated splenomegaly of C26-mice was inhibited by PM01183 for as long as the treatment lasted, without reducing IL-6, M-CSF or IL-1ß in plasma. ET743 and PM01183 extend the survival of C26-bearing mice unchanging tumor growth or cachexia but possibly restrain muscle-related inflammation and C26-induced splenomegaly.

Oxidation of HMGB1 Is a Dynamically Regulated Process in Physiological and Pathological Conditions.

Acute inflammation is a complex biological response of tissues to harmful stimuli, such as pathogens or cell damage, and is essential for immune defense and proper healing. However, unresolved inflammation can lead to chronic disorders, including cancer and fibrosis. The High Mobility Group Box 1 (HMGB1) protein is a Damage-Associated Molecular Pattern (DAMP) molecule that orchestrates key events in inflammation by switching among mutually exclusive redox states. Fully reduced HMGB1 (frHMGB1) supports immune cell recruitment and tissue regeneration, while the isoform containing a disulphide bond (dsHMGB1) promotes secretion of inflammatory mediators by immune cells. Although it has been suggested that the tissue itself determines the redox state of the extracellular space and of released HMGB1, the dynamics of HMGB1 oxidation in health and disease are unknown. In the present work, we analyzed the expression of HMGB1 redox isoforms in different inflammatory conditions in skeletal muscle, from acute injury to muscle wasting, in tumor microenvironment, in spleen, and in liver after drug intoxication. Our results reveal that the redox modulation of HMGB1 is tissue-specific, with high expression of dsHMGB1 in normal spleen and liver and very low in muscle, where it appears after acute damage. Similarly, dsHMGB1 is highly expressed in the tumor microenvironment while it is absent in cachectic muscles from the same tumor-bearing mice. These findings emphasize the accurate and dynamic regulation of HMGB1 redox state, with the presence of dsHMGB1 tightly associated with leukocyte infiltration. Accordingly, we identified circulating, infiltrating, and resident leukocytes as reservoirs and transporters of dsHMGB1 in tissue and tumor microenvironment, demonstrating that the redox state of HMGB1 is controlled at both tissue and cell levels. Overall, our data point out that HMGB1 oxidation is a timely and spatially regulated process in physiological and pathological conditions. This precise modulation might play key roles to finetune inflammatory and regenerative processes.

Musclin, A Myokine Induced by Aerobic Exercise, Retards Muscle Atrophy During Cancer Cachexia in Mice.

Physical activity improves the prognosis of cancer patients, partly by contrasting the associated muscle wasting (cachexia), through still unknown mechanisms. We asked whether aerobic exercise causes secretion by skeletal muscles of proteins (myokines) that may contrast cachexia. Media conditioned by peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α)-expressing myotubes, reproducing some metabolic adaptations of aerobic exercise, as increased mitochondrial biogenesis and oxidative phosphorylation, restrained constitutively active Forkhead box-containing subfamily O3 (caFoxO3)-induced proteolysis. Microarray analysis identified amphiregulin (AREG), natriuretic peptide precursor B (NppB), musclin and fibroblast growth factor 18 (FGF18) as myokines highly induced by PGC1α. Notably, only musclin tended to be low in muscle of mice with a rare human renal carcinoma; it was reduced in plasma and in muscles of C26-bearing mice and in atrophying myotubes, where PGC1α expression is impaired. Therefore, we electroporated the Tibialis Anterior (TA) of C26-bearing mice with musclin or (its receptor) natriuretic peptide receptor 3 (Npr3)-encoding plasmids and found a preserved fiber area, as a result of restrained proteolysis. Musclin knockout (KO) mice lose more muscle tissue during growth of two distinct cachexia-causing tumors. Running protected C26-bearing mice from cachexia, not changing tumor growth, and rescued the C26-induced downregulation of musclin in muscles and plasma. Musclin expression did not change in overloaded plantaris of mice, recapitulating partially muscle adaptations to anaerobic exercise. Musclin might, therefore, be beneficial to cancer patients who cannot exercise and are at risk of cachexia and may help to explain how aerobic exercise alleviates cancer-induced muscle wasting.

Exercise-Induced Myokines With Therapeutic Potential for Muscle Wasting.

Skeletal muscle is a highly vascularized tissue that can secrete proteins called myokines. These muscle-secreted factors exert biological functions in muscle itself (autocrine effect) or on short- or long-distant organs (paracrine/endocrine effects) and control processes such as metabolism, angiogenesis, or inflammation. Widely differing diseases ranging from genetic myopathies to cancers are emerging as causing dysregulated secretion of myokines from skeletal muscles. Myokines are also involved in the control of muscle size and may be important to be restored to normal levels to alleviate muscle wasting in various conditions, such as cancer, untreated diabetes, chronic obstructive pulmonary disease, aging, or heart failure. Interestingly, many myokines are induced by exercise (muscle-derived exerkines) and some even by specific types of physical activity, but more studies are needed on this issue. Most exercise-induced myokines travel throughout the body by means of extracellular vesicles. Restoring myokines by physical activity may be added to the list of mechanisms by which exercise exerts preventative or curative effects against a large number of diseases, including the deleterious muscle wasting they may cause. Extending our understanding about which myokines could be usefully restored in certain diseases might help in prescribing more tailored exercise or myokine-based drugs.

Micro-computed tomography for non-invasive evaluation of muscle atrophy in mouse models of disease.

Muscle wasting occurs during various chronic diseases and precedes death in humans as in mice. The evaluation of the degree of muscle atrophy in diseased mouse models is often overlooked since it requires the sacrifice of the animals for muscle examination or expensive instrumentation and highly qualified personnel, such as Magnetic Resonance Imaging (MRI). Very often behavioral tests for muscle strength evaluation are used as an outcome measurement in preclinical therapeutic trials. However, these tests are easy to perform serially, but not enough sensitive to detect early muscle changes during disease progression. Monitoring muscle loss in living animals could allow to perform more informative preclinical trials with a better evaluation of therapeutic benefit with respect to muscle wasting. We developed a non-invasive procedure based on micro-computed tomography (micro-CT) without contrast agents to monitor hind limb muscle wasting in mouse models of amyotrophic lateral sclerosis (ALS) and cancer cachexia: the transgenic SOD1G93A mouse and the colon adenocarcinoma C26-bearing mouse, respectively. We established the scanning procedure and the parameters to consider in the reconstructed images to calculate the Index of Muscle Mass (IMM). The coefficient of variance for the whole procedure was 2.2%. We performed longitudinally micro-CT scan of hind limbs in SOD1G93A mice at presymptomatic and symptomatic stages of the disease and calculated the IMM. We found that IMM in SOD1G93A mice was lower than age-matched controls even before symptom onset. We also detected a further decrease in IMM as disease progresses, most markedly just before disease onset. We performed the same analyses in the C26-based mouse model losing quickly body and muscle mass because of cancer cachexia. Overall, we found that the reduced muscle content detected by micro-CT mirrored the reduced muscle weight in both disease models. We developed a fast, precise and easy-to-conduct imaging procedure to monitor hind limb muscle mass, useful in therapeutic preclinical trials but also in proof-of-principle studies to identify the onset of muscle wasting. This method could be widely applied to other disease models characterized by muscle wasting, to assist drug development and search for early biomarkers of muscle atrophy. Moreover, reducing the number of mice needed for the experiments and being less distressing are in line with the 3R principle embodied in national and international directives for animal research.

Group I Paks support muscle regeneration and counteract cancer-associated muscle atrophy.

BACKGROUND: Skeletal muscle is characterized by an efficient regeneration potential that is often impaired during myopathies. Understanding the molecular players involved in muscle homeostasis and regeneration could help to find new therapies against muscle degenerative disorders. Previous studies revealed that the Ser/Thr kinase p21 protein-activated kinase 1 (Pak1) was specifically down-regulated in the atrophying gastrocnemius of Yoshida hepatoma-bearing rats. In this study, we evaluated the role of group I Paks during cancer-related atrophy and muscle regeneration. METHODS: We examined Pak1 expression levels in the mouse Tibialis Anterior muscles during cancer cachexia induced by grafting colon adenocarcinoma C26 cells and in vitro by dexamethasone treatment. We investigated whether the overexpression of Pak1 counteracts muscle wasting in C26-bearing mice and in vitro also during interleukin-6 (IL6)-induced or dexamethasone-induced C2C12 atrophy. Moreover, we analysed the involvement of group I Paks on myogenic differentiation in vivo and in vitro using the group I chemical inhibitor IPA-3. RESULTS: We found that Pak1 expression levels are reduced during cancer-induced cachexia in the Tibialis Anterior muscles of colon adenocarcinoma C26-bearing mice and in vitro during dexamethasone-induced myotube atrophy. Electroporation of muscles of C26-bearing mice with plasmids directing the synthesis of PAK1 preserves fiber size in cachectic muscles by restraining the expression of atrogin-1 and MuRF1 and possibly by inducing myogenin expression. Consistently, the overexpression of PAK1 reduces the dexamethasone-induced expression of MuRF1 in myotubes and increases the phospho-FOXO3/FOXO3 ratio. Interestingly, the ectopic expression of PAK1 counteracts atrophy in vitro by restraining the IL6-Stat3 signalling pathway measured in luciferase-based assays and by reducing rates of protein degradation in atrophying myotubes exposed to IL6. On the other hand, we observed that the inhibition of group I Paks has no effect on myotube atrophy in vitro and is associated with impaired muscle regeneration in vivo and in vitro. In fact, we found that mice treated with the group I inhibitor IPA-3 display a delayed recovery from cardiotoxin-induced muscle injury. This is consistent with in vitro experiments showing that IPA-3 impairs myogenin expression and myotube formation in vessel-associated myogenic progenitors, C2C12 myoblasts, and satellite cells. Finally, we observed that IPA-3 reduces p38α/ß phosphorylation that is required to proceed through various stages of satellite cells differentiation: activation, asymmetric division, and ultimately myotube formation. CONCLUSIONS: Our data provide novel evidence that is consistent with group I Paks playing a central role in the regulation of muscle homeostasis, atrophy and myogenesis.

Bioreducible Hydrophobin-Stabilized Supraparticles for Selective Intracellular Release.

One of the main hurdles in nanomedicine is the low stability of drug-nanocarrier complexes as well as the drug delivery efficiency in the region-of-interest. Here, we describe the use of the film-forming protein hydrophobin HFBII to organize dodecanethiol-protected gold nanoparticles (NPs) into well-defined supraparticles (SPs). The obtained SPs are exceptionally stable in vivo and efficiently encapsulate hydrophobic drug molecules. The HFBII film prevents massive release of the encapsulated drug, which, instead, is activated by selective SP disassembly triggered intracellularly by glutathione reduction of the protein film. As a consequence, the therapeutic efficiency of an encapsulated anticancer drug is highly enhanced (2 orders of magnitude decrease in IC50). Biodistribution and pharmacokinetics studies demonstrate the high stability of the loaded SPs in the bloodstream and the selective release of the payloads once taken up in the tissues. Overall, our results provide a rationale for the development of bioreducible and multifunctional nanomedicines.

The ubiquitin ligase tripartite-motif-protein 32 is induced in Duchenne muscular dystrophy.

Activation of the proteasome pathway is one of the secondary processes of cell damage, which ultimately lead to muscle degeneration and necrosis in Duchenne muscular dystrophy (DMD). In mdx mice, the proteasome inhibitor bortezomib up-regulates the membrane expression of members of the dystrophin complex and reduces the inflammatory reaction. However, chronic inhibition of the 26S proteasome may be toxic, as indicated by the systemic side-effects caused by this drug. Therefore, we sought to determine the components of the ubiquitin-proteasome pathway that are specifically activated in human dystrophin-deficient muscles. The analysis of a cohort of patients with genetically determined DMD or Becker muscular dystrophy (BMD) unveiled a selective up-regulation of the ubiquitin ligase tripartite motif-containing protein 32 (TRIM32). The induction of TRIM32 was due to a transcriptional effect and it correlated with disease severity in BMD patients. In contrast, atrogin1 and muscle RING-finger protein-1 (MuRF-1), which are strongly increased in distinct types of muscular atrophy, were not affected by the DMD dystrophic process. Knock-out models showed that TRIM32 is involved in ubiquitination of muscle cytoskeletal proteins as well as of protein inhibitor of activated STAT protein gamma (Piasγ) and N-myc downstream-regulated gene, two inhibitors of satellite cell proliferation and differentiation. Accordingly, we showed that in DMD/BMD muscle tissue, TRIM32 induction was more pronounced in regenerating myofibers rather than in necrotic muscle cells, thus pointing out a role of this protein in the regulation of human myoblast cell fate. This finding highlights TRIM32 as a possible therapeutic target to favor skeletal muscle regeneration in DMD patients.