Altered threshold of the mitochondrial permeability transition pore in Ullrich congenital muscular dystrophy

AbstractWe have studied the effects of rotenone in myoblasts from healthy donors and from patients with Ullrich congenital muscular dystrophy (UCMD), a severe muscle disease due to mutations in the genes encoding the extracellular matrix protein collagen VI. Addition of rotenone to normal myoblasts caused a very limited mitochondrial depolarization because the membrane potential was maintained by the F1FO synthase, as indicated by full depolarization following the subsequent addition of oligomycin. In UCMD myoblasts rotenone instead caused complete mitochondrial depolarization, which was followed by faster ATP depletion than in healthy myoblasts. Mitochondrial depolarization could be prevented by treatment with cyclosporin A and intracellular Ca2+ chelators, while it was worsened by depleting Ca2+ stores with thapsigargin. Thus, in UCMD myoblasts rotenone-induced depolarization is due to opening of the permeability transition pore rather than to inhibition of electron flux as such. These findings indicate that in UCMD myoblasts the threshold for pore opening is very close to the resting membrane potential, so that even a small depolarization causes permeability transition pore opening and precipitates ATP depletion

Extracellular Collagen VI Has Prosurvival and Autophagy Instructive Properties in Mouse Fibroblasts

Collagen VI (ColVI) is an abundant and distinctive extracellular matrix protein secreted by fibroblasts in different tissues. Human diseases linked to mutations on ColVI genes are primarily affecting skeletal muscle due to non-cell autonomous myofiber defects. To date, it is not known whether and how fibroblast homeostasis is affected by ColVI deficiency, a critical missing information as this may strengthen the use of patients’ fibroblasts for preclinical purposes. Here, we established primary and immortalized fibroblast cultures from ColVI null (Col6a1-/-) mice, the animal model of ColVI-related diseases. We found that, under nutrient-stringent condition, lack of ColVI affects fibroblast survival, leading to increased apoptosis. Moreover, Col6a1-/- fibroblasts display defects in the autophagy/lysosome machinery, with impaired clearance of autophagosomes and failure of Parkin-dependent mitophagy. Col6a1-/- fibroblasts also show an increased activation of the Akt/mTOR pathway, compatible with the autophagy impairment, and adhesion onto purified ColVI elicits a major effect on the autophagic flux. Our findings reveal that ColVI ablation in fibroblasts impacts on autophagy regulation and cell survival, thus pointing at the new concept that this cell type may contribute to the pathological features of ColVI-related diseases

First steps to define murine amniotic fluid stem cell microenvironment

Stem cell niche refers to the microenvironment where stem cells reside in living organisms. Several elements define the niche and regulate stem cell characteristics, such as stromal support cells, gap junctions, soluble factors, extracellular matrix proteins, blood vessels and neural inputs. In the last years, different studies demonstrated the presence of cKit+ cells in human and murine amniotic fluid, which have been defined as amniotic fluid stem (AFS) cells. Firstly, we characterized the murine cKit+ cells present both in the amniotic fluid and in the amnion. Secondly, to analyze the AFS cell microenvironment, we injected murine YFP+ embryonic stem cells (ESC) into the amniotic fluid of E13.5 wild type embryos. Four days after transplantation we found that YFP+ sorted cells maintained the expression of pluripotency markers and that ESC adherent to the amnion were more similar to original ESC in respect to those isolated from the amniotic fluid. Moreover, cytokines evaluation and oxygen concentration analysis revealed in this microenvironment the presence of factors that are considered key regulators in stem cell niches. This is the first indication that AFS cells reside in a microenvironment that possess specific characteristics able to maintain stemness of resident and exogenous stem cells

Structure, Chromosomal Localization, and Promoter Analysis of the Human Elastin MicrofibrilInterfase Located proteIN (EMILIN) Gene

Abstract Elastinmicrofibril interfase-located protein (EMILIN) is an extracellular matrix glycoprotein abundantly expressed in elastin-rich tissues such as the blood vessels, skin, heart, and lung. It occurs with elastic fibers at the interface between amorphous elastin and microfibrils. In vitroexperiments suggested a role for EMILIN in the process of elastin deposition. This multimodular protein consists of 995 amino acids; the domain organization includes a C1q-like globular domain at the C terminus, a short collagenous stalk, a region containing two leucine zippers, and at least four heptad repeats with a high potential for forming coiled-coil α-helices and, at the N terminus, a cysteine-rich sequence characterized by a partial epidermal growth factor-like motif and homologous to a region of multimerin. Here we report the complete characterization of the human and murine EMILIN gene, their chromosomal assignment, and preliminary functional data of the human promoter. A cDNA probe corresponding to the C terminus of EMILIN was used to isolate two genomic clones from a human BAC library. Sequencing of several derived subclones allowed the characterization of the whole gene that was found to be about 8 kilobases in size and to contain 8 exons and 7 introns. The internal exons range in size from 17 base pairs to 1929 base pairs. All internal intron/exon junctions are defined by canonical splice donor and acceptor sites, and the different domains potentially involved in the formation of a coiled-coil structure are clustered in the largest exon. The 3′-end of the EMILIN gene overlaps with the 5′-end of the promoter region of the ketohexokinase gene, whose chromosomal position is between markers D2S305 and D2S165 on chromosome 2. A 1600-base pair-long sequence upstream of the translation starting point was evaluated for its promoter activity; five deletion constructs were assayed after transfection in primary chicken fibroblasts and in a human rhabdomyosarcoma cell line. This analysis indicates the existence of two contiguous regions able to modulate luciferase expression in both cell types used, one with a strong activatory function, ranging from positions −204 to −503, and the other, ranging from positions −504 to −683, with a strong inhibitory function

Exploring the larval transcriptome of the common sole (Solea solea L.)

open7noBackground The common sole (Solea solea) is a promising candidate for European aquaculture; however, the limited knowledge of the physiological mechanisms underlying larval development in this species has hampered the establishment of successful flatfish aquaculture. Although the fact that genomic tools and resources are available for some flatfish species, common sole genomics remains a mostly unexplored field. Here, we report, for the first time, the sequencing and characterisation of the transcriptome of S. solea and its application for the study of molecular mechanisms underlying physiological and morphological changes during larval-to-juvenile transition. Results The S. solea transcriptome was generated from whole larvae and adult tissues using the Roche 454 platform. The assembly process produced a set of 22,223 Isotigs with an average size of 726 nt, 29 contigs and a total of 203,692 singletons. Of the assembled sequences, 75.2% were annotated with at least one known transcript/protein; these transcripts were then used to develop a custom oligo-DNA microarray. A total of 14,674 oligonucleotide probes (60 nt), representing 12,836 transcripts, were in situ synthesised onto the array using Agilent non-contact ink-jet technology. The microarray platform was used to investigate the gene expression profiles of sole larvae from hatching to the juvenile form. Genes involved in the ontogenesis of the visual system are up-regulated during the early stages of larval development, while muscle development and anaerobic energy pathways increase in expression over time. The gene expression profiles of key transcripts of the thyroid hormones (TH) cascade and the temporal regulation of the GH/IGF1 (growth hormone/insulin-like growth factor I) system suggest a pivotal role of these pathways in fish growth and initiation of metamorphosis. Pre-metamorphic larvae display a distinctive transcriptomic landscape compared to previous and later stages. Our findings highlighted the up-regulation of gene pathways involved in the development of the gastrointestinal system as well as biological processes related to folic acid and retinol metabolism. Additional evidence led to the formation of the hypothesis that molecular mechanisms of cell motility and ECM adhesion may play a role in tissue rearrangement during common sole metamorphosis. Conclusions Next-generation sequencing provided a good representation of the sole transcriptome, and the combination of different approaches led to the annotation of a high number of transcripts. The construction of a microarray platform for the characterisation of the larval sole transcriptome permitted the definition of the main processes involved in organogenesis and larval growth. Keywords: Solea solea; Flatfish; Larval development; Metamorphosis; Transcriptome; Gene expressionopenSerena Ferraresso; Alessio Bonaldo; Luca Parma; Stefano Cinotti; Paola Massi; Luca Bargelloni; Pier Paolo GattaSerena Ferraresso; Alessio Bonaldo; Luca Parma; Stefano Cinotti; Paola Massi; Luca Bargelloni; Pier Paolo Gatt

Effects of Bioactive Peptides from Atlantic Salmon Processing By-Products on Oxyntopeptic and Enteroendocrine Cells of the Gastric Mucosa of European Seabass and Gilthead Seabream

The present study was designed to evaluate the effects of dietary levels of bioactive peptides (BPs) derived from salmon processing by-products on the presence and distribution of peptic cells (oxyntopeptic cells, OPs) and enteric endocrine cells (EECs) that contain GHR, NPY and SOM in the gastric mucosa of European seabass and gilthead seabream. In this study, 27 seabass and 27 seabreams were divided into three experimental groups: a control group (CTR) fed a control diet and two groups fed different levels of BP to replace fishmeal: 5% BP (BP5%) and 10% BP (BP10%). The stomach of each fish was sampled and processed for immunohistochemistry. Some SOM, NPY and GHR-IR cells exhibited alternating “open type” and “closed type” EECs morphologies. The BP10% group (16.8 ± 7.5) showed an increase in the number of NPY-IR cells compared to CTR (CTR 8.5 ± 4.8) and BP5% (BP10% vs. CTR p ≤ 0.01; BP10% vs. BP5% p ≤ 0.05) in the seabream gastric mucosa. In addition, in seabream gastric tissue, SOM-IR cells in the BP 10% diet (16.8 ± 3.5) were different from those in CTR (12.5 ± 5) (CTR vs. BP 10% p ≤ 0.05) and BP 5% (12.9 ± 2.5) (BP 5% vs. BP 10% p ≤ 0.01). EEC SOM-IR cells increased at 10% BP (5.3 ± 0.7) compared to 5% BP (4.4 ± 0.8) (5% BP vs. 10% BP p ≤ 0.05) in seabass. The results obtained may provide a good basis for a better understanding of the potential of salmon BPs as feed ingredients for seabass and seabream.publishedVersio

Analysis of Regulatory Regions of Emilin1 Gene and Their Combinatorial Contribution to Tissue-specific Transcription

The location of regions that regulate transcription of the murine Emilin1 gene was investigated in a DNA fragment of 16.8 kb, including the entire gene and about 8.7 and 0.6 kb of 5'- and 3'-flanking sequences, respectively. The 8.7-kb segment contains the 5'-end of the putative 2310015E02Rik gene and the sequence that separates it from Emilin1, whereas the 0.6-kb fragment covers the region between Emilin1 and Ketohexokinase genes. Sequence comparison between species identified several conserved regions in the 5'-flanking sequence. Most of them contained chromatin DNase I-hypersensitive sites, which were located at about -950 (HS1), -3100 (HS2), -4750 (HS3), and -5150 (HS4) in cells expressing Emilin1 mRNA. Emilin1 transcription initiates at multiple sites, the major of which correspond to two Initiator sequences. Promoter assays suggest that core promoter activity was mainly dependent on Initiator1 and on Sp1-binding sites close to the Initiators. Moreover, one important regulatory region was contained between -1 and -169 bp and a second one between -630 bp and -1.1 kb. The latter harbors a putative binding site for transcription factor AP1 matching the location of HS1. The function of different regions was studied by expressing lacZ constructs in transgenic mice. The results show that the 16.8-kb segment contains regulatory sequences driving high level transcription in all the tissues where Emilin1 is expressed. Moreover, the data suggest that transcription in different tissues is achieved through combinatorial cooperation between various regions, rather than being dependent on a single cis-activating region specific for each tissue

Integrated study on production performance and quality traits of European sea bass ( Dicentrarchus labrax ) fed high plant protein diets

In the issue of fishmeal replacement, besides maintaining optimal growth, a key area of investigation for continuing to improve modern aquafeeds includes the evaluation of the effects of plant ingredients on fish quality. It is generally accepted that farmed fish quality can be influenced by the formulation of composition of their feed. Hence, the aim of the present research was to evaluate plant protein inclusion up to 84% of the overall protein content in an integrated study on growth and quality traits of European sea bass. Three diets were formulated to contain increasing plant protein levels (50, 67 and 84%; 50PP, 67PP and 84PP, respectively), with fishmeal dietary levels at 30, 20 and 10%, respectively. No significant differences due to reducing fishmeal content were observed after 118 days in terms of growth (final body weight and specific growth rate) and feed intake, even though a trend towards lower growth performance at higher fishmeal replacement levels was observed. Fish fed diet 50PP showed lower feed conversion rate in comparison to those fed diet 84PP, while no differences were recorded between diet 50PP and 67PP. No significant differences among treatments were found in protein efficiency rate. On the contrary, fish fed diet 84PP showed lower gross protein efficiency in comparison to those fed diet 50PP and 67PP. No significant differences in biometric indices and fillet composition were observed. No significant differences were found in pH, liquid holding capacity and skin colour measurements between treatments, while regarding fillet colour, significant differences were found only for H\ub0ab. In conclusion, our findings demonstrated that dietary plant proteins up to 84% of the overall protein content had no effects on quality traits of European sea bass in comparison with 50% and 67%. All experimental groups showed similar growth even though 84% plant protein inclusion negatively influenced feed and protein utilisation

Feeding common sole (Solea solea) juveniles with increasing dietary lipid levels affects growth, feed utilization and gut health

Knowledge about the nutritional requirements of common sole (Solea solea) is limited and no information regarding the optimal dietary lipid level is available yet. Thus, this study was undertaken to assess the growth response and feed utilization of common sole juveniles fed diets with increasing lipid levels. Four isonitrogenous (59% protein) pelletized diets with different dietary lipid levels (8%, 12%, 16% and 20%; L8, L12, L16 and L20, respectively) were fed to triplicate fish groups of 80 individuals to apparent satiation over 150 days. A one-way ANOVA, Tukey's post hoc test and linear regression were used to analyse the data (P 64 0.05). At the end of the trial, final body weight was significantly higher in fish fed L8 (40.7 \ub1 1.7 g), followed by those fed L12 (35.1 \ub1 1.2 g), L16 (27.9 \ub1 2.5 g) and L20 (22.1 \ub1 0.3 g). The specific growth rate was higher in fish fed L8 and L12, compared to the other treatments, and it was lowest in L20. Voluntary feed intake decreased with an increase in the dietary lipid level. The feed conversion rate, the protein efficiency ratio and the gross protein efficiency were lower in fish fed L20, while no significant differences were observed among L8, L12 and L16. Gross lipid efficiency was significantly higher in fish fed low lipid diets. Histological observations showed that 19 of 36 observed subjects had lipid droplets in the cytoplasm of enterocytes at the apex of the mucosal folds and, in some cases, also along the entire fold (intestinal steatosis). The number of fish with intestinal steatosis in groups L20 and L16 was significantly higher than the number in group L8. Ultrastructure showed large electrondense lipid droplets within the cytoplasm of enterocytes and warping of the cytoplasmic membrane (steatosis); in some cases, lipid droplets were also present within the Golgi apparatus. In conclusion, the results of this trial suggest that the diet for S. solea juveniles should include no more than 12% lipids. Higher lipid inclusions not only led to a substantial decline in performance but also affected gut health. This should be taken into consideration in formulating specific practical diets for common sole

Cyclosporine A in Ullrich Congenital Muscular Dystrophy: Long-Term Results

Six individuals with Ullrich congenital muscular dystrophy (UCMD) and mutations in the genes-encoding collagen VI, aging 5–9, received 3–5 mg/kg of cyclosporine A (CsA) daily for 1 to 3.2 years. The primary outcome measure was the muscle strength evaluated with a myometer and expressed as megalimbs. The megalimbs score showed significant improvement (P = 0.01) in 5 of the 6 patients. Motor function did not change. Respiratory function deteriorated in all. CsA treatment corrected mitochondrial dysfunction, increased muscle regeneration, and decreased the number of apoptotic nuclei. Results from this study demonstrate that long-term treatment with CsA ameliorates performance in the limbs, but not in the respiratory muscles of UCMD patients, and that it is well tolerated. These results suggest considering a trial of CsA or nonimmunosuppressive cyclosporins, that retains the PTP-desensitizing properties of CsA, as early as possible in UCMD patients when diaphragm is less compromised