Defective cell migration causes delayed wound healing (WH) and chronic skin lesions. Autologous micrograft (AMG) therapies have recently emerged as a new effective and affordable treatment able to ...improve wound healing capacity. However, the precise molecular mechanism through which AMG exhibits its beneficial effects remains unrevealed. Herein we show that AMG improves skin re-epithelialization by accelerating the migration of fibroblasts and keratinocytes. More specifically, AMG-treated wounds showed improvement of indispensable events associated with successful wound healing such as granulation tissue formation, organized collagen content, and newly formed blood vessels. We demonstrate that AMG is enriched with a pool of WH-associated growth factors that may provide the starting signal for a faster endogenous wound healing response. This work links the increased cell migration rate to the activation of the extracellular signal-regulated kinase (ERK) signaling pathway, which is followed by an increase in matrix metalloproteinase expression and their extracellular enzymatic activity. Overall we reveal the AMG-mediated wound healing transcriptional signature and shed light on the AMG molecular mechanism supporting its potential to trigger a highly improved wound healing process. In this way, we present a framework for future improvements in AMG therapy for skin tissue regeneration applications.
Muscular regeneration is a complex biological process that occurs during acute injury and chronic degeneration, implicating several cell types. One of the earliest events of muscle regeneration is ...the inflammatory response, followed by the activation and differentiation of muscle progenitor cells. However, the process of novel neuromuscular junction formation during muscle regeneration is still largely unexplored. Here, we identify by single-cell RNA sequencing and isolate a subset of vessel-associated cells able to improve myogenic differentiation. We termed them 'guide' cells because of their remarkable ability to improve myogenesis without fusing with the newly formed fibers. In vitro, these cells showed a marked mobility and ability to contact the forming myotubes. We found that these cells are characterized by CD44 and CD34 surface markers and the expression of Ng2 and Ncam2. In addition, in a murine model of acute muscle injury and regeneration, injection of guide cells correlated with increased numbers of newly formed neuromuscular junctions. Thus, we propose that guide cells modulate de novo generation of neuromuscular junctions in regenerating myofibers. Further studies are necessary to investigate the origin of those cells and the extent to which they are required for terminal specification of regenerating myofibers.
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 in vivo 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.
Here, a BioBrick™ lysis device present in the Registry of Standard Biological Parts has been quantitatively characterized. Its activity has been measured in E. coli 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.
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.
Inducible promoters are widely spread genetic tools for triggering, tuning and optimizing the expression of recombinant genes in engineered biological systems. Most of them are controlled by the ...addition of a specific exogenous chemical inducer that indirectly regulates the promoter transcription rate in a concentration-dependent fashion. In order to have a robust and predictable degree of control on promoter activity, the degradation rate of such chemicals should be considered in many applications like recombinant protein production.
In this work, we use whole-cell biosensors to assess the half-life of three commonly used chemical inducers for recombinant Escherichia coli: Isopropyl β-D-1-thiogalactopyranoside (IPTG), anhydrotetracycline (ATc) and N-(3-oxohexanoyl)-L-homoserine lactone (HSL). A factorial study was conducted to investigate the conditions that significantly contribute to the decay rate of these inducers. Temperature has been found to be the major factor affecting ATc, while medium and pH have been found to highly affect HSL. Finally, no significant degradation was observed for IPTG among the tested conditions.
We have quantified the decay rate of IPTG, ATc and HSL in many conditions, some of which were not previously tested in the literature, and the main effects affecting their degradation were identified via a statistics-based framework. Whole-cell biosensors were successfully used to conduct this study, yielding reproducible measurements via simple multiwell-compatible assays. The knowledge of inducer degradation rate in several contexts has to be considered in the rational design of synthetic biological systems for improving the predictability of induction effects, especially for prolonged experiments.
Tm7sf2
gene encodes 3β‐hydroxysterol Δ
14
‐reductase (C14SR, DHCR14), an endoplasmic reticulum enzyme acting on Δ
14
‐unsaturated sterol intermediates during the conversion of lanosterol to ...cholesterol. The C‐terminal domain of lamin B receptor, a protein of the inner nuclear membrane mainly involved in heterochromatin organization, also possesses sterol Δ
14
‐reductase activity. The subcellular localization suggests a primary role of C14SR in cholesterol biosynthesis. To investigate the role of C14SR and lamin B receptor as 3β‐hydroxysterol Δ
14
‐reductases,
Tm7sf2
knockout mice were generated and their biochemical characterization was performed. No
Tm7sf2
mRNA was detected in the liver of knockout mice. Neither C14SR protein nor 3β‐hydroxysterol Δ
14
‐reductase activity were detectable in liver microsomes of
Tm7sf2
(−/−)
mice, confirming the effectiveness of gene inactivation. C14SR protein and its enzymatic activity were about half of control levels in the liver of heterozygous mice. Normal cholesterol levels in liver membranes and in plasma indicated that, despite the lack of C14SR,
Tm7sf2
(−/−)
mice are able to perform cholesterol biosynthesis. Lamin B receptor 3β‐hydroxysterol Δ
14
‐reductase activity determined in liver nuclei showed comparable values in wild‐type and knockout mice. These results suggest that lamin B receptor, although residing in nuclear membranes, may contribute to cholesterol biosynthesis in
Tm7sf2
(−/−)
mice. Affymetrix microarray analysis of gene expression revealed that several genes involved in cell‐cycle progression are downregulated in the liver of
Tm7sf2
(−/−)
mice, whereas genes involved in xenobiotic metabolism are upregulated.
Tm7sf2 gene encodes 3β-hydroxysterol Δ¹⁴-reductase (C14SR, DHCR14), an endoplasmic reticulum enzyme acting on Δ¹⁴-unsaturated sterol intermediates during the conversion of lanosterol to cholesterol. ...The C-terminal domain of lamin B receptor, a protein of the inner nuclear membrane mainly involved in heterochromatin organization, also possesses sterol Δ¹⁴-reductase activity. The subcellular localization suggests a primary role of C14SR in cholesterol biosynthesis. To investigate the role of C14SR and lamin B receptor as 3β-hydroxysterol Δ¹⁴-reductases, Tm7sf2 knockout mice were generated and their biochemical characterization was performed. No Tm7sf2 mRNA was detected in the liver of knockout mice. Neither C14SR protein nor 3β-hydroxysterol Δ¹⁴-reductase activity were detectable in liver microsomes of Tm7sf2⁽⁻/⁻⁾ mice, confirming the effectiveness of gene inactivation. C14SR protein and its enzymatic activity were about half of control levels in the liver of heterozygous mice. Normal cholesterol levels in liver membranes and in plasma indicated that, despite the lack of C14SR, Tm7sf2⁽⁻/⁻⁾ mice are able to perform cholesterol biosynthesis. Lamin B receptor 3β-hydroxysterol Δ¹⁴-reductase activity determined in liver nuclei showed comparable values in wild-type and knockout mice. These results suggest that lamin B receptor, although residing in nuclear membranes, may contribute to cholesterol biosynthesis in Tm7sf2⁽⁻/⁻⁾ mice. Affymetrix microarray analysis of gene expression revealed that several genes involved in cell-cycle progression are downregulated in the liver of Tm7sf2⁽⁻/⁻⁾ mice, whereas genes involved in xenobiotic metabolism are upregulated.
Duchenne muscular dystrophy remains an untreatable genetic disease that severely limits motility and life expectancy in affected children. The only animal model specifically reproducing the ...alterations in the dystrophin gene and the full spectrum of human pathology is the golden retriever dog model. Affected animals present a single mutation in intron 6, resulting in complete absence of the dystrophin protein, and early and severe muscle degeneration with nearly complete loss of motility and walking ability. Death usually occurs at about 1 year of age as a result of failure of respiratory muscles. Here we report that intra-arterial delivery of wild-type canine mesoangioblasts (vessel-associated stem cells) results in an extensive recovery of dystrophin expression, normal muscle morphology and function (confirmed by measurement of contraction force on single fibres). The outcome is a remarkable clinical amelioration and preservation of active motility. These data qualify mesoangioblasts as candidates for future stem cell therapy for Duchenne patients.
Tm7sf2 gene encodes 3beta-hydroxysterol Delta14-reductase (C14SR, DHCR14), an endoplasmic reticulum enzyme acting on Delta14-unsaturated sterol intermediates during the conversion of lanosterol to ...cholesterol. The C-terminal domain of lamin B receptor, a protein of the inner nuclear membrane mainly involved in heterochromatin organization, also possesses sterol Delta14-reductase activity. The subcellular localization suggests a primary role of C14SR in cholesterol biosynthesis. To investigate the role of C14SR and lamin B receptor as 3beta-hydroxysterol Delta14-reductases, Tm7sf2 knockout mice were generated and their biochemical characterization was performed. No Tm7sf2 mRNA was detected in the liver of knockout mice. Neither C14SR protein nor 3beta-hydroxysterol Delta14-reductase activity were detectable in liver microsomes of Tm7sf2(-/-) mice, confirming the effectiveness of gene inactivation. C14SR protein and its enzymatic activity were about half of control levels in the liver of heterozygous mice. Normal cholesterol levels in liver membranes and in plasma indicated that, despite the lack of C14SR, Tm7sf2(-/-) mice are able to perform cholesterol biosynthesis. Lamin B receptor 3beta-hydroxysterol Delta14-reductase activity determined in liver nuclei showed comparable values in wild-type and knockout mice. These results suggest that lamin B receptor, although residing in nuclear membranes, may contribute to cholesterol biosynthesis in Tm7sf2(-/-) mice. Affymetrix microarray analysis of gene expression revealed that several genes involved in cell-cycle progression are downregulated in the liver of Tm7sf2(-/-) mice, whereas genes involved in xenobiotic metabolism are upregulated. PUBLICATION ABSTRACT
In 2002 we published an article describing a population of vessel-associated progenitors that we termed mesoangioblasts (MABs). During the past decade evidence had accumulated that during muscle ...development and regeneration things may be more complex than a simple sequence of binary choices (e.g., dorsal vs. ventral somite). LacZ expressing fibroblasts could fuse with unlabelled myoblasts but not among themselves or with other cell types. Bone marrow derived, circulating progenitors were able to participate in muscle regeneration, though in very small percentage. Searching for the embryonic origin of these progenitors, we identified them as originating at least in part from the embryonic aorta and, at later stages, from the microvasculature of skeletal muscle. While continuing to investigate origin and fate of MABs, the fact that they could be expanded
(also from human muscle) and cross the vessel wall, suggested a protocol for the cell therapy of muscular dystrophies. We tested this protocol in mice and dogs before proceeding to the first clinical trial on Duchenne Muscular Dystrophy patients that showed safety but minimal efficacy. In the last years, we have worked to overcome the problem of low engraftment and tried to understand their role as auxiliary myogenic progenitors during development and regeneration.
The titanium surfaces with micro-roughness have been studied to substitute machined titanium, with the focus on enhancing the bone apposition onto the implant. In this study we have followed a ...biomimetic strategy where human SAOS-2 osteoblasts proliferated and built their extracellular matrix on a sandblasted titanium surface modified with plastic deformation. In comparison with sandblasted titanium surface, the plastic deformation increased the cell proliferation and the surface coating with bone matrix. The superficially modified biomaterial could be used, in clinical applications, as an implant for bone repair.
PDF
