Efficacia del trattamento riabilitativo sugli outcome funzionali nei bambini con distrofia muscolare di Duchenne: revisione della letteratura.

Introduzione: la Distrofia muscolare di Duchenne (DMD) è una patologia neuromuscolare rara ad esito infausto con incidenza prevalente nel sesso maschile, caratterizzata da atrofia e debolezza muscolare a progressione rapida e da degenerazione dei muscoli scheletrici, lisci e cardiaci. Dal punto di vista del trattamento farmacologico sono in corso numerose ricerche, mentre i vari aspetti del trattamento fisioterapico sono ancora poco studiati. Il trattamento standard prevede mobilizzazioni, rinforzo selettivo, uso di ortesi e ausili, chirurgia funzionale e farmacoterapia. Obiettivo:ricercare evidenze di studi scientifici che indaghino l’efficacia del trattamento fisioterapico di tipo aerobico e funzionale in pazienti affetti da DMD, in cui si analizzi l’aspetto prettamente funzionale della patologia. Materiali e metodi:la ricerca è stata condotta nel periodo di tempo che va da Marzo 2020 ad Agosto 2020, consultando i database Pubmed, PEDro e Cochrane. Le parole chiave utilizzate sono state “Duchenne Dystrophy”, “Aerobic Training” e “Functional Outcomes”. Sono stati selezionati RCT in lingua inglese che trattano l’efficacia del trattamento in pazienti in età evolutiva affetti da DMD, comparandoli a trattamento noto e che analizzano outcome relativi alle funzioni. Risultati:la ricerca in letteratura e l’analisi degli studi attraverso i criteri di eleggibilità hanno condotto all’individuazione di tre studi RCT. Le misure di outcome prese in esame sono: cammino, resistenza e abilità funzionali sia degli arti inferiori che superiori. Conclusioni:gli studi analizzati hanno dimostrato che il trattamento aerobico e funzionale si è rivelato efficace nel miglioramento di alcuni outcome presi in esame e nel rallentamento del declino motorio e funzionale della patologia. Tuttavia, i limiti riscontrati in questa revisione rendono impossibile effettuare un confronto oggettivabile tra gli esiti dei diversi gruppi di intervento. Sono quindi necessari ulteriori studi a riguardo

De novo design of antimicrobial and antibiofilm peptides starting from desert truffle Tirmania pinoyi peptides

With the aim of discovering new routes in the research of antimicrobials, we focused on polypeptide- enriched extracts derived from edible desert truffle mushroom Tirmania pinoyi. The extracts showed an interesting activity with MIC=50 μg/mL against Staphylococcus aureus ATCC 29213 and Pseudomonas aeruginosa ATCC 15442. Through mass spectrometry analysis (RP-HPLC/ nESI-MS/MS) the following eight novel peptides FDVVPKTAANFRAL, AVTVGASTLADERA, FLVGGASLKPEF, VARIFAVFNDTF, HLVDEPQNLLK, LGEYGFQNALLR, FAVNGGCAKET, SREDLHPKL were detected. To characterize them online websites were used: IAMPpred, DPBAAS, Cell-PPD, ToxinPred, HemoPI, PeptideCutter and HLP. The analysis indicated that some peptides showed negative or neutral charge, hydrophobic ratio between 42% and 67%, Boman Index < 2.25 kCal/mol. According to the “APD3: Antimicrobial Peptide Calculator and Predictor” tool of the Antimicrobial Peptide Database (APD) similarities (around 30-40%) with known antimicrobial peptides (AMPs) identified in amphibians were also detected. In contrast, the predicted antimicrobial, antifungal and antibiofilm activity was not significant. In order to improve biological and physico-chemical properties, the sequences of natural peptides were modified using APD3, by replacing some hydrophilic and negative charged amino acids with hydrophobic and positive ones. The derivative sequences (GWDVVPKTWWKFRAL, KWTWGASTLAKKRA, FLRGGWSLKPKF, KWRIFWVFNKTF, HLVKRWQNLLK, KGKYRFWNALLR, FARWGGCAKRT, SRKWLHPWL) showed net positive charge between +2 and +4, hydrophobic ratio between 42% and 48%, Boman Index < 2.25 kCal/mol and high stability. Moreover, the predicted antimicrobial, antifungal and antibiofilm activity was high, without toxic or hemolytic effects. In conclusion, bioinformatic analysis has demonstrated that novel peptides discovered in T. pinoyi may be considered new platforms for the design of novel antimicrobial and antibiofilm peptides to counteract multi-drug resistant pathogens

New Bioactive Peptides from the Mediterranean Seagrass Posidonia oceanica (L.) Delile and Their Impact on Antimicrobial Activity and Apoptosis of Human Cancer Cells

The demand for new molecules to counter bacterial resistance to antibiotics and tumor cell resistance is increasingly pressing. The Mediterranean seagrass Posidonia oceanica is considered a promising source of new bioactive molecules. Polypeptide-enriched fractions of rhizomes and green leaves of the seagrass were tested against Gram-positive (e.g., Staphylococcus aureus, Enterococcus faecalis) and Gram-negative bacteria (e.g., Pseudomonas aeruginosa, Escherichia coli), as well as towards the yeast Candida albicans. The aforementioned extracts showed indicative MIC values, ranging from 1.61 μg/mL to 7.5 μg/mL, against the selected pathogens. Peptide fractions were further analyzed through a high-resolution mass spectrometry and database search, which identified nine novel peptides. Some discovered peptides and their derivatives were chemically synthesized and tested in vitro. The assays identified two synthetic peptides, derived from green leaves and rhizomes of P. oceanica, which revealed interesting antibiofilm activity towards S. aureus, E. coli, and P. aeruginosa (BIC50 equal to 17.7 μg/mL and 70.7 μg/mL). In addition, the natural and derivative peptides were also tested for potential cytotoxic and apoptosis-promoting effects on HepG2 cells, derived from human hepatocellular carcinomas. One natural and two synthetic peptides were proven to be effective against the “ in vitro” liver cancer cell model. These novel peptides could be considered a good chemical platform for developing potential therapeutics

In Vitro Cytotoxic Effect of Aqueous Extracts from Leaves and Rhizomes of the Seagrass Posidonia oceanica (L.) Delile on HepG2 Liver Cancer Cells: Focus on Autophagy and Apoptosis

Aqueous extracts from Posidonia oceanica’s green and brown (beached) leaves and rhizomes were prepared, submitted to phenolic compound and proteomic analysis, and examined for their potential cytotoxic effect on HepG2 liver cancer cells in culture. The chosen endpoints related to survival and death were cell viability and locomotory behavior, cell-cycle analysis, apoptosis and autophagy, mitochondrial membrane polarization, and cell redox state. Here, we show that 24 h exposure to both green-leaf- and rhizome-derived extracts decreased tumor cell number in a dose– response manner, with a mean half maximal inhibitory concentration (IC50) estimated at 83 and 11.5 µg of dry extract/mL, respectively. Exposure to the IC50 of the extracts appeared to inhibit cell motility and long-term cell replicating capacity, with a more pronounced effect exerted by the rhizomederived preparation. The underlying death-promoting mechanisms identified involved the downregulation of autophagy, the onset of apoptosis, the decrease in the generation of reactive oxygen species, and the dissipation of mitochondrial transmembrane potential, although, at the molecular level, the two extracts appeared to elicit partially differentiating effects, conceivably due to their diverse composition. In conclusion, P. oceanica extracts merit further investigation to develop novel promising prevention and/or treatment agents, as well as beneficial supplements for the formulation of functional foods and food-packaging material with antioxidant and anticancer propertie

Escherichia coli TatA and TatB Proteins Have N-out, C-in Topology in Intact Cells

The twin arginine protein transport (Tat) system translocates folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of chloroplasts. In Escherichia coli, TatA, TatB, and TatC are essential components of the machinery. A complex of TatB and TatC acts as the substrate receptor, whereas TatA is proposed to form the Tat transport channel. TatA and TatB are related proteins that comprise an N-terminal transmembrane helix and an adjacent amphipathic helix. Previous studies addressing the topological organization of TatA have given conflicting results. In this study, we have addressed the topological arrangement of TatA and TatB in intact cells by labeling of engineered cysteine residues with the membrane-impermeable thiol reagent methoxypolyethylene glycol maleimide. Our results show that TatA and TatB share an N-out, C-in topology, with no evidence that the amphipathic helices of either protein are exposed at the periplasmic side of the membrane. We further show that the N-out, C-in topology of TatA is fixed and is not affected by the absence of other Tat components or by the overproduction of a Tat substrate. These data indicate that topological reorganization of TatA is unlikely to accompany Tat-dependent protein transport

A Novel Peptide with Antifungal Activity from Red Swamp Crayfish Procambarus clarkii

The defense system of freshwater crayfish Procambarus clarkii as a diversified source of bioactive molecules with antimicrobial properties was studied. Antimicrobial activity of two polypeptideenriched extracts obtained from hemocytes and hemolymph of P. clarkii were assessed against Gram positive (Staphylococcus aureus, Enterococcus faecalis) and Gram negative (Pseudomonas aeruginosa, Escherichia coli) bacteria and toward the yeast Candida albicans. The two peptide fractions showed interesting MIC values (ranging from 11 to 700 g/mL) against all tested pathogens. Polypeptideenriched extracts were further investigated using a high-resolution mass spectrometry and database search and 14 novel peptides were identified. Some peptides and their derivatives were chemically synthesized and tested in vitro against the bacterial and yeast pathogens. The analysis identified a synthetic derivative peptide, which showed an interesting antifungal (MIC and MFC equal to 31.2 g/mL and 62.5 g/mL, respectively) and antibiofilm (BIC50 equal to 23.2 g/mL) activities against Candida albicans and a low toxicity in human cells

Removal and Reconstitution of the Carotenoid Antenna of Xanthorhodopsin

Salinixanthin, a C40-carotenoid acyl glycoside, serves as a light-harvesting antenna in the retinal-based proton pump xanthorhodopsin of Salinibacter ruber. In the crystallographic structure of this protein, the conjugated chain of salinixanthin is located at the protein–lipid boundary and interacts with residues of helices E and F. Its ring, with a 4-keto group, is rotated relative to the plane of the π-system of the carotenoid polyene chain and immobilized in a binding site near the β-ionone retinal ring. We show here that the carotenoid can be removed by oxidation with ammonium persulfate, with little effect on the other chromophore, retinal. The characteristic CD bands attributed to bound salinixanthin are now absent. The kinetics of the photocycle is only slightly perturbed, showing a 1.5-fold decrease in the overall turnover rate. The carotenoid-free protein can be reconstituted with salinixanthin extracted from the cell membrane of S. ruber. Reconstitution is accompanied by restoration of the characteristic vibronic structure of the absorption spectrum of the antenna carotenoid, its chirality, and the excited-state energy transfer to the retinal. Minor modification of salinixanthin, by reducing the carbonyl C=O double bond in the ring to a C-OH, suppresses its binding to the protein and eliminates the antenna function. This indicates that the presence of the 4-keto group is critical for carotenoid binding and efficient energy transfer

The Twin-Arginine Translocation Pathway in α-Proteobacteria Is Functionally Preserved Irrespective of Genomic and Regulatory Divergence

The twin-arginine translocation (Tat) pathway exports fully folded proteins out of the cytoplasm of Gram-negative and Gram-positive bacteria. Although much progress has been made in unraveling the molecular mechanism and biochemical characterization of the Tat system, little is known concerning its functionality and biological role to confer adaptive skills, symbiosis or pathogenesis in the α-proteobacteria class. A comparative genomic analysis in the α-proteobacteria class confirmed the presence of tatA, tatB, and tatC genes in almost all genomes, but significant variations in gene synteny and rearrangements were found in the order Rickettsiales with respect to the typically described operon organization. Transcription of tat genes was confirmed for Anaplasma marginale str. St. Maries and Brucella abortus 2308, two α-proteobacteria with full and partial intracellular lifestyles, respectively. The tat genes of A. marginale are scattered throughout the genome, in contrast to the more generalized operon organization. Particularly, tatA showed an approximately 20-fold increase in mRNA levels relative to tatB and tatC. We showed Tat functionality in B. abortus 2308 for the first time, and confirmed conservation of functionality in A. marginale. We present the first experimental description of the Tat system in the Anaplasmataceae and Brucellaceae families. In particular, in A. marginale Tat functionality is conserved despite operon splitting as a consequence of genome rearrangements. Further studies will be required to understand how the proper stoichiometry of the Tat protein complex and its biological role are achieved. In addition, the predicted substrates might be the evidence of role of the Tat translocation system in the transition process from a free-living to a parasitic lifestyle in these α-proteobacteria