Customized bioreactor enables the production of 3D diaphragmatic constructs influencing matrix remodeling and fibroblast overgrowth

The production of skeletal muscle constructs useful for replacing large defects in vivo, such as in congenital diaphragmatic hernia (CDH), is still considered a challenge. The standard application of prosthetic material presents major limitations, such as hernia recurrences in a remarkable number of CDH patients. With this work, we developed a tissue engineering approach based on decellularized diaphragmatic muscle and human cells for the in vitro generation of diaphragmatic-like tissues as a proof-of-concept of a new option for the surgical treatment of large diaphragm defects. A customized bioreactor for diaphragmatic muscle was designed to control mechanical stimulation and promote radial stretching during the construct engineering. In vitro tests demonstrated that both ECM remodeling and fibroblast overgrowth were positively influenced by the bioreactor culture. Mechanically stimulated constructs also increased tissue maturation, with the formation of new oriented and aligned muscle fibers. Moreover, after in vivo orthotopic implantation in a surgical CDH mouse model, mechanically stimulated muscles maintained the presence of human cells within myofibers and hernia recurrence did not occur, suggesting the value of this approach for treating diaphragm defects

Nuovi approcci farmacologici per la cura di malattie da protein misfolding

Le malattie genetiche da protein misfolding sono caratterizzate dalla presenza di mutazioni che destabilizzano la struttura della proteina di interesse. Tali mutazioni si possono dividere in due grandi categorie, mutazioni loss of function e mutazioni gain of function. Nel primo caso il difetto pu\uf2 determinare la perdita della funzione legata alla proteina, e ci\uf2 avviene perch\ue9 l\u2019alterazione del folding porta o alla prematura eliminazione della proteina mutata da parte del sistema di controllo qualit\ue0 cellulare, o alla non corretta localizzazione della proteina; basti pensare alle mutazioni del canale del cloro (CFTR) responsabili della fibrosi cistica. Alla seconda categoria appartengono invece le mutazioni che, causando alterazioni del folding, provocano l\u2019accumulo di aggregati che possono divenire tossici per la cellula, con gli esempi pi\uf9 noti delle malattie neurodegenerative come Alzheimer o Parkinson. Per disegnare nuovi approcci terapeutici per queste malattie, al momento ancora incurabili, grande interesse \ue8 stato rivolto negli ultimi anni allo studio: 1) dei processi di folding, sostenuti da un gran numero di proteine ed enzimi; 2) del sistema di controllo qualit\ue0 cellulare che verifica la \u201cbont\ue0\u201d delle proteine di neosintesi; e 3) dei meccanismi molecolari responsabili della eliminazione delle proteine difettose. In questo ambito si colloca il nostro lavoro che ha consentito di definire il meccanismo patogenetico delle sarcoglicanopatie e della malattia di Brody, in effetti classificabili come patologie da protein misfolding. Inoltre, e ben pi\uf9 importante, per queste malattie rare abbiamo potuto proporre nuove strategie terapeutiche basate sull\u2019uso di piccole molecole e che abbiamo gi\ue0 validato con successo in vitro ed ora stiamo testando in vivo. Verranno illustrati gli esperimenti che hanno stabilito il coinvolgimento del sistema ubiquitina-proteasoma nella prematura eliminazione di mutanti missenso di \u3b1-sarcoglicano e SERCA1 e sar\ue0 mostrato come l\u2019inibizione di tale sistema consenta, in vitro, il recupero quantitativo e funzionale dei mutanti. Infine vedremo come la decifrazione del pathway degradativo di un mutante di \u3b1-sarcoglicano abbia permesso di ideare nuovi approcci terapeutici basati sull\u2019uso di piccole molecole in grado o di ridurre il tasso di degradazione del mutante (recupero del mutante), bloccando specifici target farmacologici, o di facilitare il folding del mutante (riparare il mutante), consentendo la sua corretta localizzazione. Quest\u2019ultimo approccio \ue8 stato gi\ue0 utilizzato in vitro con successo anche con mutanti di SERCA1, dimostrando la grande potenzialit\ue0 di azione per queste molecole, in grado di agire su proteine strutturalmente e funzionalmente molto diverse, ma che condividono simili problemi di folding

INHIBITION OF THE SYNTHESIS OF A CYTOCHROME-C-OXIDASE SUBUNIT ISOFORM BY ANTISENSE RNA

To investigate the role of subunit VIIe, an oxygen-regulated subunit isoform of Dictyostelium discoideum cytochrome-c oxidase, the full-length cDNA was inserted into an expression vector under the control of an actin promoter in the sense and antisense orientation. The DNA constructs were used for stable transformation of the slime mold amoebae. In most of the 28 antisense clones tested, the concentration of cytochrome-c oxidase was lowered compared to the wild type, while no significant changes were found in the sense mutants. Antisense RNA was abundantly expressed, leading to a drastic reduction of the steady-state level of the endogenous subunit VIIe mRNA, which was decreased up to 20-30% the level observed in parent cells. In these transformants, the amount of the target polypeptide and cytochrome c oxidase was 40-50% and 60-70% of control, respectively. A similar decrease was found in the level of the remaining nuclear and mitochondrial subunits. Unexpectedly, these changes affected neither basal nor uncoupled cell respiration suggesting an increase of the enzyme specific activity. Hypoxia completely relieved the cytochrome-c-oxidase deficit. These results indicate that subunit VII is needed for an efficient assembly of the protein complex and provide evidence for its involvement in the modulation of the enzyme activity

Emerging therapeutic strategies for sarcoglycanopathy

Introduction: Sarcoglycanopathy is the name shared by four rare autosomal recessive muscular dystrophies (LGMD2 C-F) that are usually characterized by early onset and rapid progression and an accompanying loss of independent walking since adolescence. Respiratory problems are frequent, and dilated cardiomyopathy may occur, although milder forms have also been described. However, sarcoglycanopathy is currently incurable, and we herein aim to describe the state of the art in the field of treatments for this disease. Areas covered: We summarize the pathogenesis of sarcoglycanopathy, with particular emphasis on the molecular mechanism(s) underlying the disease. We describe the very few published cases of symptomatic treatment with steroids and the gene therapy approaches that have entered phase I/II clinical trials. We then present emerging novel therapeutic strategies explored at the preclinical stage that are expected to replace the defective gene (cell therapy), address general effects of the disease, or address the primary events of the pathogenic mechanism (small molecule-based therapy). Expert opinion: Anti-inflammatory strategies, which are at present empirically applied, warrant further exploration. Although promising and currently being evaluated in clinical trials, gene therapy remains associated with concerns and requires additional confirmation. Thus, novel strategies targeting different aspects of the disease pathogenic mechanism are highly anticipated

Functional roles of dystrophin and of associated proteins. New insights for the sarcoglycans

The discovery of the dystrophin gene, whose mutations lead to Duchenne's and Becker's muscular dystrophy (DMD and BMD), represents the first important landmark by which, in the last ten years, molecular biology and genetic studies have revealed many of the molecular defects of the major muscular dystrophies. Very rapidly, several studies revealed the presence at skeletal and cardiac muscle sarcolemma of a group of proteins associated to dystrophin. This includes a set of five transmembrane glycoproteins, the sarcoglycans, whose physiological role, however, is still poorly understood. Dystrophin and the associated proteins are believed to play an important role in membrane stability and maintenance during muscle contraction and relaxation. However, the absence of sarcoglycans from sarcolemma does not appear to affect membrane integrity suggesting that these components of the dystrophin complex are recipients of other important functions. This review deals with recent advances in the knowledge of sarcoglycan function and organization that may give important insights into the pathogenetic mechanisms of muscular dystrophies

Novel aspects of chlorophyll a/b-binding proteins

The light-harvesting proteins (LHC) constitute a multigene family including, in higher plants, at least 12 members whose location, within the photosynthetic membrane, relative abundance and putative function appear to be very different. The major light-harvesting complex of photosystem II (LHCII) is the most abundant membrane protein in the biosphere and fulfil a constitutive light-harvesting function for photosystem II while the early light-induced proteins (ELIPs) are expressed in low amounts under stress conditions. Primary sequence analysis suggests that all these proteins share a common structure which was resolved at 3.7 \uc5 resolution by electron crystallography in the case of the major LHCII complex: Three transmembrane helices connected by hydrophilic loops coordinate seven chlorophyll a and five chlorophyll b molecules by histidine, glutamine, asparagine lateral chains as well as by charge compensated ionic pairs of glutamic acid and arginine residues; moreover, at least two xantophyll molecules are located at the centre of the structure in close contact with seven porphyrins, tentatively identified as chlorophyll a. The antenna system is also involved in the regulation of excitation energy transfer to reaction centre II. This function has been attributed to three members of the protein family, namely CP29, CP26 and CP24 (also called minor chlorophyll proteins) which have been recently characterised and shown to bind most of the xantophyll cycle carotenoids, thus suggesting that the non-photochemical quenching mechanism is acting in these proteins. Further support to this assignment comes from the recent identification of protonation sites in CP29 and CP26 by covalent dicyclohexhylcarbodiimide binding suggesting that these respond to low lumenal pH. In addition, CP29 is reversibly phosphorylated under light and cold stress conditions, undergoing conformational change, supporting the hypothesis that these subunits, present in low amounts in photosystem II, have a major regulatory role in the light-harvesting function and are thus important in environmental stress resistance

Evidence for the presence of two Homer 1 transcripts in skeletal and cardiac muscles

A family of proteins, Homers 1, 2 and 3, involved in activity-dependent control of signal transduction has been recently described in neurons [Xiao, B., Tu, C. J., Petralia, R. S., Yuan, J. P., Doan, A., Breder, C. D., Ruggiero, A., Lanahan, A. A., Wenthold, R. J., and Worley, P. F. (1998) Homer regulates the association of group 1 metabotropic glutamate receptors with multivalent complexes of Homer-related, synaptic proteins. Neuron 21, 707-716]. By RT-PCR and RNasePA, mRNAs transcripts for Homer 1a and Homer 1c, but not Homer 1b, are detected in both skeletal and cardiac muscles of the rat. Full-length cloning of Homer 1a and Homer 1c cDNAs has been accomplished: There is no tissue specificity, by comparing skeletal muscle, cardiac muscle and cerebellum, and there are a few species-specific base substitutions, by comparing rat and mouse sequences. The regulatory mechanism exerted via transition of Homer 1 isoform composition may be operative in striated muscles