Skeletal Myogenic Progenitors Originating from Embryonic Dorsal Aorta Coexpress Endothelial and Myogenic Markers and Contribute to Postnatal Muscle Growth and Regeneration

Skeletal muscle in vertebrates is derived from somites, epithelial structures of the paraxial mesoderm, yet many unrelated reports describe the occasional appearance of myogenic cells from tissues of nonsomite origin, suggesting either transdifferentiation or the persistence of a multipotent progenitor. Here, we show that clonable skeletal myogenic cells are present in the embryonic dorsal aorta of mouse embryos. This finding is based on a detailed clonal analysis of different tissue anlagen at various developmental stages. In vitro, these myogenic cells show the same morphology as satellite cells derived from adult skeletal muscle, and express a number of myogenic and endothelial markers. Surprisingly, the latter are also expressed by adult satellite cells. Furthermore, it is possible to clone myogenic cells from limbs of mutant c-Met-/- embryos, which lack appendicular muscles, but have a normal vascular system. Upon transplantation, aorta-derived myogenic cells participate in postnatal muscle growth and regeneration, and fuse with resident satellite cells. The potential of the vascular system to generate skeletal muscle cells may explain observations of nonsomite skeletal myogenesis and raises the possibility that a subset of satellite cells may derive from the vascular system

Characterization of a synthetic bacterial self-destruction device for programmed cell death and for recombinant proteins release

<p>Abstract</p> <p>Background</p> <p>Bacterial cell lysis is a widely studied mechanism that can be achieved through the intracellular expression of phage native lytic proteins. This mechanism can be exploited for programmed cell death and for gentle cell disruption to release recombinant proteins when <it>in vivo </it>secretion is not feasible. Several genetic parts for cell lysis have been developed and their quantitative characterization is an essential step to enable the engineering of synthetic lytic systems with predictable behavior.</p> <p>Results</p> <p>Here, a BioBrick™ lysis device present in the Registry of Standard Biological Parts has been quantitatively characterized. Its activity has been measured in <it>E. coli </it>by assembling the device under the control of a well characterized N-3-oxohexanoyl-L-homoserine lactone (HSL) -inducible promoter and the transfer function, lysis dynamics, protein release capability and genotypic and phenotypic stability of the device have been evaluated. Finally, its modularity was tested by assembling the device to a different inducible promoter, which can be triggered by heat induction.</p> <p>Conclusions</p> <p>The studied device is suitable for recombinant protein release as 96% of the total amount of the intracellular proteins was successfully released into the medium. Furthermore, it has been shown that the device can be assembled to different input devices to trigger cell lysis in response to a user-defined signal. For this reason, this lysis device can be a useful tool for the rational design and construction of complex synthetic biological systems composed by biological parts with known and well characterized function. Conversely, the onset of mutants makes this device unsuitable for the programmed cell death of a bacterial population.</p

A standard vector for the chromosomal integration and characterization of BioBrick™ parts in Escherichia coli

BACKGROUND: The chromosomal integration of biological parts in the host genome enables the engineering of plasmid-free stable strains with single-copy insertions of the desired gene networks. Although different integrative vectors were proposed, no standard pre-assembled genetic tool is available to carry out this task. Synthetic biology concepts can contribute to the development of standardized and user friendly solutions to easily produce engineered strains and to rapidly characterize the desired genetic parts in single-copy context. RESULTS: In this work we report the design of a novel integrative vector that allows the genomic integration of biological parts compatible with the RFC10, RFC23 and RFC12 BioBrick™ standards in Escherichia coli. It can also be specialized by using BioBrick™ parts to target the desired integration site in the host genome. The usefulness of this vector has been demonstrated by integrating a set of BioBrick™ devices in two different loci of the E. coli chromosome and by characterizing their activity in single-copy. Construct stability has also been evaluated and compared with plasmid-borne solutions. CONCLUSIONS: Physical modularity of biological parts has been successfully applied to construct a ready-to-engineer BioBrick™ vector, suitable for a stable chromosomal insertion of standard parts via the desired recombination method, i.e. the bacteriophage integration mechanism or homologous recombination. In contrast with previously proposed solutions, it is a pre-assembled vector containing properly-placed restriction sites for the direct transfer of various formats of BioBrick™ parts. This vector can facilitate the characterization of parts avoiding copy number artefacts and the construction of antibiotic resistance-free engineered microbes, suitable for industrial use

Low-Power Ultrasounds as a Tool to Culture Human Osteoblasts inside Cancellous Hydroxyapatite

Bone graft substitutes and cancellous biomaterials have been widely used to heal critical-size long bone defects due to trauma, tumor resection, and tissue degeneration. In particular, porous hydroxyapatite is widely used in reconstructive bone surgery owing to its biocompatibility. In addition, the in vitro modification of cancellous hydroxyapatite with osteogenic signals enhances the tissue regeneration in vivo, suggesting that the biomaterial modification could play an important role in tissue engineering. In this study, we have followed a tissue-engineering strategy where ultrasonically stimulated SAOS-2 human osteoblasts proliferated and built their extracellular matrix inside a porous hydroxyapatite scaffold. The ultrasonic stimulus had the following parameters: average power equal to 149 mW and frequency of 1.5 MHz. In comparison with control conditions, the ultrasonic stimulus increased the cell proliferation and the surface coating with bone proteins (decorin, osteocalcin, osteopontin, type-I collagen, and type-III collagen). The mechanical stimulus aimed at obtaining a better modification of the biomaterial internal surface in terms of cell colonization and coating with bone matrix. The modified biomaterial could be used, in clinical applications, as an implant for bone repair

Evaluation of the effects of specific karate exercises during multilateral training in children of primary school

The early specialization in the development of sport skills is a point of discussion among researchers, even if the general trend is to encourage multilateral activities in children. The aim of this study was to evaluate the effect of specific karate exercises added during a program of multilateral exercises in a group of school age children. A sample of 82 primary school children (39 females, 6.4 ± 0.3 y and 43 males, 6.3 ± 0.3 y) were randomly assigned to two groups: Multilateral (MG) and Special (SG). MG was composed of 19 females (MGf, 6,4 ± 0,3 y) and 22 males (MGm, 6,3 ± 0,3 y), while SG was composed of 20 females (SGf, 6,3 ± 0,3 y) and 21 males (SGm, 6,4 ± 0,3 y). During the training period of eight weeks, the MG group has played only multilateral activities, while the SG group has also done specific exercises of Karate. At the end of the training period both groups were subjected to some physical evaluation test and the results was statistically analyzed (ANOVA). Although both groups (Mg and SG) have improved significantly (p &lt; 0.05) compared to the initial stage, the comparison between the two groups (MG vs SG) has not revealed significant differences in relation to the considered motor skills (speed, agility, strength, coordination), with the exception of the ability of static balance, in which the SG group showed a significant improvement compared to the MG group (p = 0.019). In particular, the improvement appears to be due mainly to the female component (SGf vs MGf: p = 0,012; SGm vs MGm p = 0,20). The fact that the improvement was mainly dependent on the female group deserves future investigations The results seem to confirm the fact that the multilateral activities would be sufficient to improve motor skills in primary school children, although some neuromotor abilities could be improved through more specific exercises without creating particular damag

Safe use of human anatomical preparations in frontal and interactive teaching

In the institute of Human Anatomy of Pavia, the use of cadaver dissection is not economically feasible. In order to improve students’ preparation related to topography of the central nervous system, we decided to use formalin-fixed brains and cranial sections belonging to the collection of cadaveric specimens. These specimens, preserved in formalin, however cannot be manipulated as such by the students because formalin can cause headaches, burning sensation in the throat, difficult breathing and can trigger or aggravate asthma symptoms [1, 2]. Furthermore, formalin is known to be a human carcinogen [3]. In order to minimize toxic effects, whole brains were extensively washed in running water and then sliced according to different reference planes using a “home-made” device enabling cuts according to parallel planes. Finally, the resulting sections were inserted into transparent plastic envelopes, immerged in a solution composed by 0.5% agar and 1% sodium azide as preservative. Medical students can now use these human brain sections to test their own ability to recognize nervous system structures. This strategy optimize specimen’s choice and focalize student’s attention on peculiar, selected human samples in full compliance with current security laws

Met-activating genetically improved chimeric factor-1 promotes angiogenesis and hypertrophy in adult myogenesis

BACKGROUND: Myogenic progenitor cells (activated satellite cells) are able to express both HGF and its receptor cMet. After muscle injury, HGF-Met stimulation promotes activation and primary division of satellite cells. MAGIC-F1 (Met-Activating Genetically Improved Chimeric Factor-1) is an engineered protein that contains two human Met-binding domains that promotes muscle hypertrophy. MAGIC-F1 protects myogenic precursors against apoptosis and increases their fusion ability enhancing muscle differentiation. Hemizygous and homozygous Magic-F1 transgenic mice displayed constitutive muscle hypertrophy. METHODS: Here we describe microarray analysis on Magic-F1 myogenic progenitor cells showing an altered gene signatures on muscular hypertrophy and angiogenesis compared to wild-type cells. In addition, we performed a functional analysis on Magic-F1+/+ transgenic mice versus controls using treadmill test. RESULTS: We demonstrated that Magic-F1+/+ mice display an increase in muscle mass and cross-sectional area leading to an improvement in running performance. Moreover, the presence of MAGIC-F1 affected positively the vascular network, increasing the vessel number in fast twitch fibers. Finally, the gene expression profile analysis of Magic-F1+/+ satellite cells evidenced transcriptomic changes in genes involved in the control of muscle growth, development and vascularisation. CONCLUSION: We showed that MAGIC -F1-induced muscle hypertrophy affects positively vascular network, increasing vessel number in fast twitch fibers. This was due to unique features of mammalian skeletal muscle and its remarkable ability to adapt promptly to different physiological demands by modulating the gene expression profile in myogenic progenitors

Human Bone RIgeneration in MAXillo-facial area using an innovative medical device for Tissue engineering (BRIMAX)

Bone regeneration today is one of the most important challenges for medicine and the need for this is particularly evident in the maxillo-facial area: our clinical trial will be based on a model of bone defect as in alveolar socket preservation and sinus lift augmentation, well described surgical techniques. The RIGENERA® system permits extraction of stem cells from a small sample of connective tissue obtained from the patient’s lingual mucosa or from a post-extraction surgical site (where an endosseous implant may be inserted), dental pulp or dental follicle. Our project is to demonstrate the efficacy in the maxillo-facial area of an innovative clinical protocol of bone tissue engineering based on a new medical device called Rigeneracons (CE certified Class I). Our clinical trial use already acquired technologies in comparation with new technologies (new selection methods, new Bio-compatible materials etc.) produced by us. Besides, we perform an in-vitro test to quantify the proliferative capacity of a cellular suspension obtained after disaggregation of connective tissue originating from the oral cavity using the RIGENERA® system, a biologic tissue disaggregator (Human Brain Wave–Torino, Italy) that recently came on the market. Evaluation of the histologic characteristics of neo-formed osseous tissue will be shown and discussed