Double-plating of ovine critical sized defects of the tibia: a low morbidity model enabling continuous in vivo monitoring of bone healing

<p>Abstract</p> <p>Background</p> <p>Recent studies using sheep critical sized defect models to test tissue engineered products report high morbidity and complications rates. This study evaluates a large bone defect model in the sheep tibia, stabilized with two, a novel Carbon fibre Poly-ether-ether-ketone (CF-PEEK) and a locking compression plate (LCP) which could sustain duration for up to 6 month with an acceptable low complication rate.</p> <p>Methods</p> <p>A large bone defect of 3 cm was performed in the mid diaphysis of the right tibia in 33 sheep. The defect was stabilised with the CF - PEEK plate and an LCP. All sheep were supported with slings for 8 weeks after surgery. The study was carried out for 3 months in 6 and for 6 months in 27 animals.</p> <p>Results</p> <p>The surgical procedure could easily be performed in all sheep and continuous in vivo radiographic evaluation of the defect was possible. This long bone critical sized defect model shows with 6.1% a low rate of complications compared with numbers mentioned in the literature.</p> <p>Conclusions</p> <p>This experimental animal model could serve as a standard model in comparative research. A well defined standard model would reduce the number of experimental animals needed in future studies and would therefore add to ethical considerations.</p

microPIR: An Integrated Database of MicroRNA Target Sites within Human Promoter Sequences

Background: microRNAs are generally understood to regulate gene expression through binding to target sequences within 39-UTRs of mRNAs. Therefore, computational prediction of target sites is usually restricted to these gene regions. Recent experimental studies though have suggested that microRNAs may alternatively modulate gene expression by interacting with promoters. A database of potential microRNA target sites in promoters would stimulate research in this field leading to more understanding of complex microRNA regulatory mechanism. Methodology: We developed a database hosting predicted microRNA target sites located within human promoter sequences and their associated genomic features, called microPIR (microRNA-Promoter Interaction Resource). microRNA seed sequences were used to identify perfect complementary matching sequences in the human promoters and the potential target sites were predicted using the RNAhybrid program..15 million target sites were identified which are located within 5000 bp upstream of all human genes, on both sense and antisense strands. The experimentally confirmed argonaute (AGO) binding sites and EST expression data including the sequence conservation across vertebrate species of each predicted target are presented for researchers to appraise the quality of predicted target sites. The microPIR database integrates various annotated genomic sequence databases, e.g. repetitive elements, transcription factor binding sites, CpG islands, and SNPs, offering users the facility to extensively explore relationships among target sites and other genomi

Assessing mechanical integrity of spinal fusion by in situ endochondral osteoinduction in the murine model

<p>Abstract</p> <p>Background</p> <p>Historically, radiographs, micro-computed tomography (micro-CT) exams, palpation and histology have been used to assess fusions in a mouse spine. The objective of this study was to develop a faster, cheaper, reproducible test to directly quantify the mechanical integrity of spinal fusions in mice.</p> <p>Methods</p> <p>Fusions were induced in ten mice spine using a previously described technique of in situ endochondral ossification, harvested with soft tissue, and cast in radiolucent alginate material for handling. Using a validated software package and a customized mechanical apparatus that flexed and extended the spinal column, the amount of intervertebral motion between adjacent vertebral discs was determined with static flexed and extended lateral spine radiographs. Micro-CT images of the same were also blindly reviewed for fusion.</p> <p>Results</p> <p>Mean intervertebral motion between control, non-fused, spinal vertebral discs was 6.1 ± 0.2° during spine flexion/extension. In fusion samples, adjacent vertebrae with less than 3.5° intervertebral motion had fusions documented by micro-CT inspection.</p> <p>Conclusions</p> <p>Measuring the amount of intervertebral rotation between vertebrae during spine flexion/extension is a relatively simple, cheap (<$100), clinically relevant, and fast test for assessing the mechanical success of spinal fusion in mice that compared favorably to the standard, micro-CT.</p

A Chaperone Trap Contributes to the Onset of Cystic Fibrosis

Protein folding is the primary role of proteostasis network (PN) where chaperone interactions with client proteins determine the success or failure of the folding reaction in the cell. We now address how the Phe508 deletion in the NBD1 domain of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein responsible for cystic fibrosis (CF) impacts the binding of CFTR with cellular chaperones. We applied single ion reaction monitoring mass spectrometry (SRM-MS) to quantitatively characterize the stoichiometry of the heat shock proteins (Hsps) in CFTR folding intermediates in vivo and mapped the sites of interaction of the NBD1 domain of CFTR with Hsp90 in vitro. Unlike folding of WT-CFTR, we now demonstrate the presence of ΔF508-CFTR in a stalled folding intermediate in stoichiometric association with the core Hsps 40, 70 and 90, referred to as a ‘chaperone trap’. Culturing cells at 30 C resulted in correction of ΔF508-CFTR trafficking and function, restoring the sub-stoichiometric association of core Hsps observed for WT-CFTR. These results support the interpretation that ΔF508-CFTR is restricted to a chaperone-bound folding intermediate, a state that may contribute to its loss of trafficking and increased targeting for degradation. We propose that stalled folding intermediates could define a critical proteostasis pathway branch-point(s) responsible for the loss of function in misfolding diseases as observed in CF