Zebrafish Tric-b is required for skeletal development and bone cells differentiation

IntroductionTrimeric intracellular potassium channels TRIC-A and -B are endoplasmic reticulum (ER) integral membrane proteins, involved in the regulation of calcium release mediated by ryanodine (RyRs) and inositol 1,4,5-trisphosphate (IP3Rs) receptors, respectively. While TRIC-A is mainly expressed in excitable cells, TRIC-B is ubiquitously distributed at moderate level. TRIC-B deficiency causes a dysregulation of calcium flux from the ER, which impacts on multiple collagen specific chaperones and modifying enzymatic activity, leading to a rare form of osteogenesis imperfecta (OI Type XIV). The relevance of TRIC-B on cell homeostasis and the molecular mechanism behind the disease are still unknown.ResultsIn this study, we exploited zebrafish to elucidate the role of TRIC-B in skeletal tissue. We demonstrated, for the first time, that tmem38a and tmem38b genes encoding Tric-a and -b, respectively are expressed at early developmental stages in zebrafish, but only the latter has a maternal expression. Two zebrafish mutants for tmem38b were generated by CRISPR/Cas9, one carrying an out of frame mutation introducing a premature stop codon (tmem38b-/-) and one with an in frame deletion that removes the highly conserved KEV domain (tmem38bΔ120-7/Δ120-7). In both models collagen type I is under-modified and partially intracellularly retained in the endoplasmic reticulum, as described in individuals affected by OI type XIV. Tmem38b-/- showed a mild skeletal phenotype at the late larval and juvenile stages of development whereas tmem38bΔ120-7/Δ120-7 bone outcome was limited to a reduced vertebral length at 21 dpf. A caudal fin regeneration study pointed towards impaired activity of osteoblasts and osteoclasts associated with mineralization impairment.DiscussionOur data support the requirement of Tric-b during early development and for bone cell differentiation

Genetic Approach for the Fast Discovery of Phenazine Producing Bacteria

A fast and efficient approach was established to identify bacteria possessing the potential to biosynthesize phenazines, which are of special interest regarding their antimicrobial activities. Sequences of phzE genes, which are part of the phenazine biosynthetic pathway, were used to design one universal primer system and to analyze the ability of bacteria to produce phenazine. Diverse bacteria from different marine habitats and belonging to six major phylogenetic lines were investigated. Bacteria exhibiting phzE gene fragments affiliated to Firmicutes, Alpha- and Gammaproteobacteria, and Actinobacteria. Thus, these are the first primers for amplifying gene fragments from Firmicutes and Alphaproteobacteria. The genetic potential for phenazine production was shown for four type strains belonging to the genera Streptomyces and Pseudomonas as well as for 13 environmental isolates from marine habitats. For the first time, the genetic ability of phenazine biosynthesis was verified by analyzing the metabolite pattern of all PCR-positive strains via HPLC-UV/MS. Phenazine production was demonstrated for the type strains known to produce endophenazines, 2-hydroxy-phenazine, phenazine-1-carboxylic acid, phenazine-1,6-dicarboxylic acid, and chlororaphin as well as for members of marine Actinobacteria. Interestingly, a number of unidentified phenazines possibly represent new phenazine structures

Effect of short-term formaldehyde fixation on Raman spectral parameters of bone quality

Medical knowledge of the skeleton including its structures has improved constantly over the past decades. Advanced imaging methods, mechanical testing and optical techniques have revealed insights into bone architecture and composition. Most of these advancements were possible due to the ex vivo investigation of biological tissues. Investigations of fresh tissue are generally preferred over preserved or fixed samples. However, chemical fixation is sometimes inevitable due to histological procedures or logistical reasons. The aim of this study was to investigate whether short-term chemical fixation with formaldehyde affects bone quality parameters obtained from Raman spectroscopy and if these effects last for intermediate sample storage of several hours. As formaldehyde induces cross-links to the organic components in bone tissue, we hypothesized that collagen-related parameters are particularly affected. Femurs of eight 17-week-old C57BL/6 mice were extracted and divided into two groups (N  =  8  /  group). Samples of the first group were fixed by immersion in 4% formaldehyde (PFA-solution) for 12 h at 4°C (fixed group) while samples of the second group were left untreated (unfixed group). Raman spectroscopy was performed, and repeated after 4 h, to assess whether intermediate storage time influenced the obtained results. Based on resultant spectra, mineral-to-matrix ratio, carbonate-to-phosphate ratio, carbonate-to-amide I ratio, mineral crystallinity and collagen maturity were determined. Carbonate-to-phosphate ratio was the only parameter showing a significant difference between the first and the subsequent measurements. For both groups, ratios showed a decrease in carbonate substitution compared to the first measurement (percentage decrease: 3.1% in fixed, 4.7% in unfixed). Collagen maturity of samples, which were short-term fixed with formaldehyde, was significantly lower than of fresh, unfixed samples (percentage difference: 3.8%). Our study shows that Raman spectroscopy is able to detect changes in collagen structure initiated by formaldehyde and that changes in short-term fixed samples are minimally influencing bone material properties measured with Raman spectroscopy.ISSN:1083-3668ISSN:1560-228

The different distribution of enzymatic collagen cross-links found in adult and children bone result in different mechanical behavior of collagen

International audienceEnzymatic collagen cross-linking has been shown to play an important role in the macroscopic elastic and plastic deformation of bone across ages. However, its direct contribution to collagen fibril deformation is unknown. The aim of this study is to determine how covalent intermolecular connections from enzymatic collagen cross-links contribute to collagen fibril elastic and plastic deformation of adults and children's bone matrix. We used ex vivo data previously obtained from biochemical analysis of children and adults bone samples (n = 14; n = 8, respectively) to create 22 sample-specific computational models of cross-linked collagen fibrils. By simulating a tensile test for each fibril, we computed the modulus of elasticity (E), ultimate tensile and yield stress (σ u and σ y), and elastic, plastic and total work (W e , W p and W tot) for each collagen fibril. We present a novel difference between children and adult bone in the deformation of the collagen phase and suggest a link between collagen fibril scale and macroscale for elastic behavior in children bone under the influence of immature enzymatic cross-links. We show a parametric linear correlation between W e and immature enzymatic collagen cross-links at the collagen fibril scale in the children population that is similar to the one we found at the macroscale in our previous study. Finally, we suggest the key role of covalent intermolecular connections to stiffness parameters (e.g. elastic modulus and W e) in children's collagen fibril and to toughness parameters in adult's collagen fi-bril, respectively

In Vitro Feasibility Analysis of a New Sutureless Wound-Closure System Based on a Temperature-Regulated Laser and a Transparent Collagen Membrane for Laser Tissue Soldering (LTS)

For the post-surgical treatment of oral wounds and mucosal defects beyond a certain size, the gold standard is still an autologous skin or mucosal graft in combination with complex suturing techniques. A variety of techniques and biomaterials has been developed for sutureless wound closure including different tissue glues or collagen patches. However, no wound covering that enables for sutureless fixation has yet been introduced. Thus, a new system was developed that allows for sutureless wound covering including a transparent collagen membrane, which can be attached to the mucosa using a specially modified 2λ laser beam with integrated temperature sensors and serum albumin as bio-adhesive. The sutureless wound closure system was tested for its applicability and its cytocompatibility by an established in vitro model in the present study. The feasibility of the laser system was tested ex vivo on a porcine palate. The in vitro cytocompatibility tests excluded the potential release of toxic substances from the laser-irradiated collagen membrane and the bio-adhesive. The results of the ex vivo feasibility study using a porcine palate revealed satisfactory mean tensile strength of 1.2–1.5 N for the bonding of the membrane to the tissue fixed with laser of 980 nm. The results suggest that our newly developed laser-assisted wound closure system is a feasible approach and could be a first step on the way towards a laser based sutureless clinical application in tissue repair and oral surgery

Collagen Fiber Orientation Is Coupled with Specific Nano-Compositional Patterns in Dark and Bright Osteons Modulating Their Biomechanical Properties

Bone continuously adapts to its mechanical environment by structural reorganization to maintain mechanical strength. As the adaptive capabilities of bone are portrayed in its nano- and microstructure, the existence of dark and bright osteons with contrasting preferential collagen fiber orientation (longitudinal and oblique-angled, respectively) points at a required tissue heterogeneity that contributes to the excellent fracture resistance mechanisms in bone. Dark and bright osteons provide an exceptional opportunity to deepen our understanding of how nanoscale tissue properties influence and guide fracture mechanisms at larger length scales. To this end, a comprehensive structural, compositional, and mechanical assessment is performed using circularly polarized light microscopy, synchrotron nanocomputed tomography, focused ion beam/scanning electron microscopy, quantitative backscattered electron imaging, Fourier transform infrared spectroscopy, and nanoindentation testing. To predict how the mechanical behavior of osteons is affected by shifts in collagen fiber orientation, finite element models are generated. Fundamental disparities between both osteon types are observed: dark osteons are characterized by a higher degree of mineralization along with a higher ratio of inorganic to organic matrix components that lead to higher stiffness and the ability to resist plastic deformation under compression. On the contrary, bright osteons contain a higher fraction of collagen and provide enhanced ductility and energy dissipation due to lower stiffness and hardness

The different distribution of enzymatic collagen cross-links found in adult and children bone result in different mechanical behavior of collagen

Enzymatic collagen cross-linking has been shown to play an important role in the macroscopic elastic and plastic deformation of bone across ages. However, its direct contribution to collagen fibril deformation is unknown. The aim of this study is to determine how covalent intermolecular connections from enzymatic collagen cross-links contribute to collagen fibril elastic and plastic deformation of adults and children's bone matrix. We used ex vivo data previously obtained from biochemical analysis of children and adults bone samples (n = 14; n = 8, respectively) to create 22 sample-specific computational models of cross-linked collagen fibrils. By simulating a tensile test for each fibril, we computed the modulus of elasticity (E), ultimate tensile and yield stress (σu and σy), and elastic, plastic and total work (We, Wp and Wtot) for each collagen fibril. We present a novel difference between children and adult bone in the deformation of the collagen phase and suggest a link between collagen fibril scale and macroscale for elastic behavior in children bone under the influence of immature enzymatic cross-links. We show a parametric linear correlation between We and immature enzymatic collagen cross-links at the collagen fibril scale in the children population that is similar to the one we found at the macroscale in our previous study. Finally, we suggest the key role of covalent intermolecular connections to stiffness parameters (e.g. elastic modulus and We) in children's collagen fibril and to toughness parameters in adult's collagen fibril, respectively.Wellcome Trust (Grant WT097347MA)NIH (Grants TUFTS-5U01EB014976 and WUSTL-5U01EB016422