The use of primary dermal fibroblast cultures to evaluate type I collagen expression in the oim model mouse

Abstract only availableOsteogenesis imperfecta type III is a heritable disorder leading to impaired connective tissue function in type I collagen containing tissues including bone fragility, blue-grey sclera, short stature, and hearing loss. Normal type I collagen is a heterotrimeric molecule containing two pro1(I) collagen chains and a similar but genetically distinct pro2(I) collagen chain. The osteogenesis imperfecta murine (oim) model mouse produces only homotrimeric type I collagen due to a single nucleotide deletion in the COL1A2 gene resulting in a non-functional pro2(I) collagen chain. The result is expression of an abnormal type I collagen molecule which leads to the above phenotype. This study is aimed at developing a methodology whereby dermal fibroblast cultures can be utilized for a variety of assays, including type I collagen RNA and protein quantification. For genotype identification, primers flanking the site of the single nucleotide deletion allow for differentiation of wild type, heterozygous and homozygous animals at the genomic level. Upon confirmation, skin was removed from both oim and wildtype mice and the dermal layer harvested. Fibroblasts originating from the dermal layer of each genotype were then cultured and grown to confluence as separate cultures and the RNA and/or protein harvested from both cell types. Total RNA was harvested using the Qiagen RNeasy kit and used to make cDNA, which was then used in conjunction with specific PCR primers to differentiate between wildtype and oim transcripts. For protein studies, the cells were treated with ascorbic acid to maximize the production of collagen prior to harvesting. Type I collagen expression was then confirmed via a western blot using a type I collagen-specific antibody. Initial results indicate the presence of both pro1(I) and pro2(I) collagen chains from harvested wildtype dermal fibroblasts, while media harvested from oim dermal fibroblasts indicated the presence of only the pro1(I) collagen chains. These results confirm the in vivo protein expression profile seen in skin of both oim and wildtype mice is exhibited in vitro in the respective dermal fibroblast cultures. These results will allow us to quantitate pro1(I) and pro2(I) collagen mRNA and protein expression levels. Future experiments will include the quantitation of type I collagen mRNA levels using RT-PCR, as well as the use of densitometry to quantitate type I collagen protein levels by western blot analysis. This in turn promises to provide insight into the mechanism of formation of abnormal type I collagen in the oim model mouse.Life Sciences Undergraduate Research Opportunity Progra

Multi-scale Investigation of Weight-bearing Exercise on Bone Biomechanical Integrity in the Osteogenesis Imperfecta Model (oim) Mouse

Comparative Medicine - OneHealth and Comparative Medicine Poster SessionOsteogenesis imperfecta (OI), a heritable connective tissue disorder generally due to type I collagen defects, is characterized by small stature, reduced bone mineral density, and frequent fractures. Bone is inherently mechanosensitive, responding and adapting to its mechanical environment. Bone formation occurs in response to high mechanical loads; often changing its geometry to strengthen the skeleton. In humans, during the normal 2 year prepubertal/pubertal growth period normal children attain 26% of their peak bone mass, and children which are physically active accrue 10-40% more bone (region specific) than inactive children. This suggests that sedentary lifestyle choices of children with OI are particularly detrimental to their bone health. We postulate that even though the OI bone material is biomechanically weaker, the OI bone will respond to exercise (muscle loading and/or gravitational ground force), especially during pubertal growth by altering bone geometry, architecture, and/or mineral:matrix physiochemistry to generate an inherently stronger bone. The potential benefits of therapeutic exercise to OI patients are significant, but the risks are real. It is critical that we first demonstrate the feasibility and potential success of an exercise therapy in a mouse model of OI for it to be considered a viable therapy for patients. To address this need we combined the unique strengths of two University of Missouri Campuses (Columbia and Kansas City) to create a collaborative research team from the Departments of Biochemistry (UMC) and Veterinary Pathobiology (UMC) and Oral Biology (UMKC School of Dentistry) to determine if weight bearing exercise will improve bone biomechanical integrity in a mouse model of osteogenesis imperfecta (oim), and to investigate the molecular, biochemical, physiochemical, structural and biomechanical impact of exercise on bone at the macro-, ultra- and nano-structural levels. The relationship of whole bone biomechanical integrity and geometry to the mineral:matrix composition, architecture, crystal geometry, and the matrix:mineral interactions of bone is poorly understood. Therefore, we examined femurs of wildtype and oim mice by multi-scale analyses characterizing geometry (muCT) and biomechanics (torsional loading to failure) in relation to the bone mineral and matrix, physicochemical and mechanical properties (FTIR, Raman and scanning acoustic microscopy). By muCT and torsional loading to failure we defined the geometric structural properties and the whole bone biomechanical properties (torsional ultimate strength, torsional stiffness, and strain energy until failure), which are a function of both the geometry and bone biomechanical material properties (tensile strength and shear modulus of elasticity). We used FTIR and Raman microscopy in conjunction with scanning acoustic microscopy to correlate the chemical structure and composition with mechanical integrity. We then performed the same analyses on femoral bones from wildtype and oim mice that underwent moderate weight bearing exercise (running on a treadmill) to determine if weight bearing exercise could alter the molecular structure of bone mineral:matrix and improve bone physicochemical and biomechanical properties. Our preliminary findings support the hypothesis that weight bearing exercise induces an adaptive response in oim mouse bone to alter its matrix/mineral composition, physiochemical structure/ property, and geometry to increase bone quality and biomechanical strength

Live Imaging of Type I Collagen Assembly Dynamics in Osteoblasts Stably Expressing GFP and mCherry-Tagged Collagen Constructs

Type I collagen is the most abundant extracellular matrix protein in bone and other connective tissues and plays key roles in normal and pathological bone formation as well as in connective tissue disorders and fibrosis. Although much is known about the collagen biosynthetic pathway and its regulatory steps, the mechanisms by which it is assembled extracellularly are less clear. We have generated GFPtpz and mCherry-tagged collagen fusion constructs for live imaging of type I collagen assembly by replacing the α2(I)-procollagen N-terminal propeptide with GFPtpz or mCherry. These novel imaging probes were stably transfected into MLO-A5 osteoblast-like cells and fibronectin-null mouse embryonic fibroblasts (FN-null-MEFs) and used for imaging type I collagen assembly dynamics and its dependence on fibronectin. Both fusion proteins co-precipitated with α1(I)-collagen and remained intracellular without ascorbate but were assembled into α1(I) collagen-containing extracellular fibrils in the presence of ascorbate. Immunogold-EM confirmed their ultrastuctural localization in banded collagen fibrils. Live cell imaging in stably transfected MLO-A5 cells revealed the highly dynamic nature of collagen assembly and showed that during assembly the fibril networks are continually stretched and contracted due to the underlying cell motion. We also observed that cell-generated forces can physically reshape the collagen fibrils. Using co-cultures of mCherry- and GFPtpz-collagen expressing cells, we show that multiple cells contribute collagen to form collagen fiber bundles. Immuno-EM further showed that individual collagen fibrils can receive contributions of collagen from more than one cell. Live cell imaging in FN-null-MEFs expressing GFPtpz-collagen showed that collagen assembly was both dependent upon and dynamically integrated with fibronectin assembly. These GFP-collagen fusion constructs provide a powerful tool for imaging collagen in living cells and have revealed novel and fundamental insights into the dynamic mechanisms for the extracellular assembly of collagen

Accurate PCR detection of influenza A/B and respiratory syncytial viruses by use of Cepheid Xpert Flu+RSV Xpress Assay in point-of-care settings: Comparison to Prodesse ProFlu+

ABSTRACT The Xpert Flu+RSV Xpress Assay is a fast, automated in vitro diagnostic test for qualitative detection and differentiation of influenza A and B viruses and respiratory syncytial virus (RSV) performed on the Cepheid GeneXpert Xpress System. The objective of this study was to establish performance characteristics of the Xpert Flu+RSV Xpress Assay compared to those of the Prodesse ProFlu+ real-time reverse transcription-PCR (RT-PCR) assay (ProFlu+) for the detection of influenza A and B viruses as well as RSV in a Clinical Laboratory Improvement Amendments (CLIA)-waived (CW) setting. Overall, the assay, using fresh and frozen nasopharyngeal (NP) swabs, demonstrated high concordance with results of the ProFlu+ assay in the combined CW and non-CW settings with positive percent agreements (PPA) (100%, 100%, and 97.1%) and negative percent agreements (NPA) (95.2%, 99.5%, and 99.6%) for influenza A and B viruses and RSV, respectively. In conclusion, this multicenter study using the Cepheid Xpert Flu+RSV Xpress Assay demonstrated high sensitivities and specificities for influenza A and B viruses and RSV in ∼60 min for use at the point-of-care in the CW setting. </jats:p

Characterization of muscle in OI Model mice

Abstract only availableOsteogenesis imperfecta (OI) is a congenital connective tissue disorder characterized by decreased bone mineral density and increased bone fragility and susceptibility to fracture. In addition to skeletal fragility, patients with OI reportedly have muscle weakness although currently no systematic evaluation of muscle function or morphology in humans or animal models of the disease has been performed. Normal type I collagen is coded for two genes located on different chromosomes: COL1A1 and COL1A2. The oim/oim mouse is homozygous for a null mutation in the COL1A2 gene and is a phenocopy of human type III OI (severe disease phenotype). Heterozygous mice (oim/+) harbor the null mutation in only one allele of the COL1A2 gene and model human patients with type I OI (mild disease phenotype). We wanted to determine whether the reported muscle weakness in OI patients is due to a muscle pathology. We analyzed the muscle mass, fiber morphology, and cross-sectional area of muscles fibers of the hind limb muscles (quadriceps, gastrocnemius, plantaris, tibialis anterior and soleus), as well as the fiber type composition of the soleus muscle of wildtype (wt), heterozygous (oim/+), and homozygous (oim/oim) mice. Our results demonstrate that the muscle mass/body mass, fiber morphology, cross-sectional area of hindlimb muscles, as well as fiber type composition of the soleus muscle of oim, oim/+ relative to wt (+/+) mouse muscles were not significantly different between the genotypes. We correlated our morphologic findings with a functional contractile assay and determined that muscle tension-force generation and nerve conduction are not impaired in oim/oim or oim/+ mice. These findings suggest that oim and oim/+ mice do not have inherent muscle pathology. This knowledge is important in our ultimate understanding of skeletal muscle in OI model mice and ultimately, humans with this disease.Life Sciences Undergraduate Research Opportunity Progra

Development of a protocol for the maintenance and mineralization of osteoblasts from oim mice [abstract]

Abstract only availableOsteogenesis imperfecta (OI) is a disease of type I collagen whose hallmark is extreme bone fragility manifested by numerous fractures throughout life. OI is usually caused by decreased amounts of type I collagen and/or the production of abnormal collagen, leading to biomechanically impaired extracellular matrix. Osteoblasts are cells that build the matrix constituents of bone by secreting collagen, which, along with hydroxyapatite crystals, forms the composite structure of bone. It is unclear how osteoblasts respond to a structurally compromised matrix at a cellular level. This project is a pilot study to explore how osteoblasts from the osteogenesis imperfecta mouse (oim) model differ from wildtype osteoblasts, and how these differences contribute to the structurally inferior bone matrix of oim mice. Because there is no established protocol for growing and mineralizing oim osteoblasts in tissue culture, the main focus of my summer research has been developing such a protocol. Osteoblasts were harvested from calvaria taken from oim and wildtype mice at three to four days of age using a trypsin-collagenase solution. The cells were then grown in -MEM in 12-well plates and encouraged to mineralize using -glycerol phosphate and ascorbic acid. Because the outcome measures are protein quantification, fibroblast cultures have been used to help develop the protocols for measuring hydroxyproline, an amino acid unique to collagen, as well as western blotting of type I collagen. While the protocol for growing and mineralizing osteoblasts is still in development, we have finalized our harvesting and plating procedures, allowing to us effectively grow both wildtype and oim osteoblasts in culture

Theoretical investigation of a genetic switch for metabolic adaptation

Membrane transporters carry key metabolites across the cell membrane and, from a resource standpoint, are hypothesized to be produced when necessary. The expression of membrane transporters in metabolic pathways is often upregulated by the transporter substrate. In E. coli, such systems include for example the lacY, araFGH, and xylFGH genes, which encode for lactose, arabinose, and xylose transporters, respectively. As a case study of a minimal system, we build a generalizable physical model of the xapABR genetic circuit, which features a regulatory feedback loop via membrane transport (positive feedback) and enzymatic degradation (negative feedback) of an inducer. Dynamical systems analysis and stochastic simulations show that the membrane transport makes the model system bistable in certain parameter regimes. Thus, it serves as a genetic “on-off” switch, enabling the cell to only produce a set of metabolic enzymes when the corresponding metabolite is present in large amounts. We find that the negative feedback from the degradation enzyme does not significantly disturb the positive feedback from the membrane transporter. We investigate hysteresis in the switching and discuss the role of cooperativity and multiple binding sites in the model circuit. Fundamentally, this work explores how a stable genetic switch for a set of enzymes is obtained from transcriptional auto-activation of a membrane transporter through its substrate

Do type I collagen defects that cause Osteogenesis Imperfecta result in an inherent muscle pathology? [abstract]

Abstract only availableOsteogenesis imperfecta (OI) is a congenital connective tissue disorder characterized by decreased bone mineral density and increased bone fragility and susceptibility to fracture. In addition to skeletal fragility, patients with OI reportedly have muscle weakness, although currently no systematic evaluation of muscle function or morphology in humans or animal models of the disease has been performed. Normal type I collagen is coded for by two genes located on different chromosomes: COL1A1 and COL1A2. The oim/oim mouse is homozygous for a null mutation in the COL1A2 gene and is a phenocopy of a human type III OI (severe disease phenotype). Heterozygous mice (oim/+) harbor the null mutation in only one allele of the COL1A2 gene and model human patients with type I OI (mild disease phenotype). One of our aims is to characterize and determine muscle mass and cross-sectional area of hind limb muscle fibers in wild type (+/+), heterozygous (oim/+), and homozygous (oim/oim) mice. We analyzed muscle mass, fiber morphology, cross-sectional area of hindlimb muscles, as well as fiber type composition of the soleus muscle of oim, oim/+ relative to +/+ mouse muscles and determined that significant differences do not exist between genotypes. We also determined that there is no evidence of necrosis, degeneration, regeneration, hypertrophy or atrophy in hindlimb muscles of oim/oim and oim/+ mice. We correlated our morphologic findings with a functional contractile assay and determined that muscle tension-force generation and nerve conduction are not impaired in oim mice. These findings suggest that oim and oim/+ mice do not have inherent muscle pathology. This knowledge is important in our ultimate understanding of skeletal muscle in OI model mice and ultimately, humans with this disease.Biochemistry Departmen