Ex vivo determination of bone tissue strains for an in vivo mouse tibial loading model

AbstractPrevious studies introduced the digital image correlation (DIC) as a viable technique for measuring bone strain during loading. In this study, we investigated the sensitivity of a DIC system in determining surface strains in a mouse tibia while loaded in compression through the knee joint. Specifically, we examined the effect of speckle distribution, facet size and overlap, initial vertical alignment of the bone into the loading cups, rotation with respect to cameras, and ex vivo loading configurations on the strain contour maps measured with a DIC system.We loaded tibiae of C57BL/6 mice (12 and 18 weeks old male) up to 12N at 8N/min. Images of speckles on the bone surface were recorded at 1N intervals and DIC was used to compute strains. Results showed that speckles must have the correct size and density with respect to the facet size of choice for the strain distribution to be computed and reproducible. Initial alignment of the bone within the loading cups does not influence the strain distribution measured during peak loading, but bones must be placed in front of the camera with the same orientation in order for strains to be comparable. Finally, the ex vivo loading configurations with the tibia attached to the entire mouse, or to the femur and foot, or only to the foot, showed different strain contour maps.This work provides a better understanding of parameters affecting full field strain measurements from DIC in ex vivo murine tibial loading tests

Modulation of 5' splice site selection using tailed oligonucleotides carrying splicing signals

BACKGROUND: We previously described the use of tailed oligonucleotides as a means of reprogramming alternative pre-mRNA splicing in vitro and in vivo. The tailed oligonucleotides that were used interfere with splicing because they contain a portion complementary to sequences immediately upstream of the target 5' splice site combined with a non-hybridizing 5' tail carrying binding sites for the hnRNP A1/A2 proteins. In the present study, we have tested the inhibitory activity of RNA oligonucleotides carrying different tail structures. RESULTS: We show that an oligonucleotide with a 5' tail containing the human β-globin branch site sequence inhibits the use of the 5' splice site of Bcl-xL, albeit less efficiently than a tail containing binding sites for the hnRNP A1/A2 proteins. A branch site-containing tail positioned at the 3' end of the oligonucleotide also elicited splicing inhibition but not as efficiently as a 5' tail. The interfering activity of a 3' tail was improved by adding a 5' splice site sequence next to the branch site sequence. A 3' tail carrying a Y-shaped branch structure promoted similar splicing interference. The inclusion of branch site or 5' splice site sequences in the Y-shaped 3' tail further improved splicing inhibition. CONCLUSION: Our in vitro results indicate that a variety of tail architectures can be used to elicit splicing interference at low nanomolar concentrations, thereby broadening the scope and the potential impact of this antisense technology

Age and Sex Differences in Load-Induced Tibial Cortical Bone Surface Strain Maps

Bone adapts its architecture to the applied load; however, it is still unclear how bone mechano-adaptation is coordinated and why potential for adaptation adjusts during the life course. Previous animal models have suggested strain as the mechanical stimulus for bone adaptation, but yet it is unknown how mouse cortical bone load-related strains vary with age and sex. In this study, full-field strain maps (at 1 N increments up to 12 N) on the bone surface were measured in young, adult, and old (aged 10, 22 weeks, and 20 months, respectively), male and female C57BL/6J mice with load applied using a noninvasive murine tibial model. Strain maps indicate a nonuniform strain field across the tibial surface, with axial compressive loads resulting in tension on the medial side of the tibia because of its curved shape. The load-induced surface strain patterns and magnitudes show sexually dimorphic changes with aging. A comparison of the average and peak tensile strains indicates that the magnitude of strain at a given load generally increases during maturation, with tibias in female mice having higher strains than in males. The data further reveal that postmaturation aging is linked to sexually dimorphic changes in average and maximum strains. The strain maps reported here allow for loading male and female C57BL/6J mouse legs in vivo at the observed ages to create similar increases in bone surface average or peak strain to more accurately explore bone mechano-adaptation differences with age and sex

Age and Sex Differences in Load-Induced Tibial Cortical Bone Surface Strain Maps

Bone adapts its architecture to the applied load; however, it is still unclear how bone mechano-adaptation is coordinated and why potential for adaptation adjusts during the life course. Previous animal models have suggested strain as the mechanical stimulus for bone adaptation, but yet it is unknown how mouse cortical bone load-related strains vary with age and sex. In this study, full-field strain maps (at 1 N increments up to 12 N) on the bone surface were measured in young, adult, and old (aged 10, 22 weeks, and 20 months, respectively), male and female C57BL/6J mice with load applied using a noninvasive murine tibial model. Strain maps indicate a nonuniform strain field across the tibial surface, with axial compressive loads resulting in tension on the medial side of the tibia because of its curved shape. The load-induced surface strain patterns and magnitudes show sexually dimorphic changes with aging. A comparison of the average and peak tensile strains indicates that the magnitude of strain at a given load generally increases during maturation, with tibias in female mice having higher strains than in males. The data further reveal that postmaturation aging is linked to sexually dimorphic changes in average and maximum strains. The strain maps reported here allow for loading male and female C57BL/6J mouse legs in vivo at the observed ages to create similar increases in bone surface average or peak strain to more accurately explore bone mechano-adaptation differences with age and sex

Reference point indentation is not indicative of whole mouse bone measures of stress intensity fracture toughness

AbstractBone fragility is a concern for aged and diseased bone. Measuring bone toughness and understanding fracture properties of the bone are critical for predicting fracture risk associated with age and disease and for preclinical testing of therapies. A reference point indentation technique (BioDent) has recently been developed to determine bone's resistance to fracture in a minimally invasive way by measuring the indentation distance increase (IDI) between the first and last indentations over cyclic indentations in the same position. In this study, we investigate the relationship between fracture toughness KC and reference point indentation parameters (i.e. IDI, total indentation distance (TID) and creep indentation distance (CID)) in bones from 38 mice from six types (C57Bl/6, Balb, oim/oim, oim/+, Phospho1−/− and Phospho1 wild type counterpart). These mice bone are models of healthy and diseased bone spanning a range of fracture toughness from very brittle (oim/oim) to ductile (Phospho1−/−). Left femora were dissected, notched and tested in 3-point bending until complete failure. Contralateral femora were dissected and indented in 10 sites of their anterior and posterior shaft surface over 10 indentation cycles. IDI, TID and CID were measured. Results from this study suggest that reference point indentation parameters are not indicative of stress intensity fracture toughness in mouse bone. In particular, the IDI values at the anterior mid-diaphysis across mouse types overlapped, making it difficult to discern differences between mouse types, despite having extreme differences in stress intensity based toughness measures. When more locations of indentation were considered, the normalised IDIs could distinguish between mouse types. Future studies should investigate the relationship of the reference point indentation parameters for mouse bone in other material properties of the bone tissue in order to determine their use for measuring bone quality

Overexpression of TIMP-3 in Chondrocytes Produces Transient Reduction in Growth Plate Length but Permanently Reduces Adult Bone Quality and Quantity

Bone development and length relies on the growth plate formation, which is dependent on degradative enzymes such as MMPs. Indeed, deletion of specific members of this enzyme family in mice results in important joint and bone abnormalities, suggesting a role in skeletal development. As such, the control of MMP activity is vital in the complex process of bone formation and growth. We generated a transgenic mouse line to overexpress TIMP3 in mouse chondrocytes using the Col2a1-chondrocyte promoter. This overexpression in cartilage resulted in a transient shortening of growth plate in homozygote mice but bone length was restored at eight weeks of age. However, tibial bone structure and mechanical properties remained compromised. Despite no transgene expression in adult osteoblasts from transgenic mice in vitro, their differentiation capacity was decreased. Neonates, however, did show transgene expression in a subset of bone cells. Our data demonstrate for the first time that transgene function persists in the chondro-osseous lineage continuum and exert influence upon bone quantity and quality

Power estimation for non-standardized multisite studies

AbstractA concern for researchers planning multisite studies is that scanner and T1-weighted sequence-related biases on regional volumes could overshadow true effects, especially for studies with a heterogeneous set of scanners and sequences. Current approaches attempt to harmonize data by standardizing hardware, pulse sequences, and protocols, or by calibrating across sites using phantom-based corrections to ensure the same raw image intensities. We propose to avoid harmonization and phantom-based correction entirely. We hypothesized that the bias of estimated regional volumes is scaled between sites due to the contrast and gradient distortion differences between scanners and sequences. Given this assumption, we provide a new statistical framework and derive a power equation to define inclusion criteria for a set of sites based on the variability of their scaling factors. We estimated the scaling factors of 20 scanners with heterogeneous hardware and sequence parameters by scanning a single set of 12 subjects at sites across the United States and Europe. Regional volumes and their scaling factors were estimated for each site using Freesurfer's segmentation algorithm and ordinary least squares, respectively. The scaling factors were validated by comparing the theoretical and simulated power curves, performing a leave-one-out calibration of regional volumes, and evaluating the absolute agreement of all regional volumes between sites before and after calibration. Using our derived power equation, we were able to define the conditions under which harmonization is not necessary to achieve 80% power. This approach can inform choice of processing pipelines and outcome metrics for multisite studies based on scaling factor variability across sites, enabling collaboration between clinical and research institutions

SARS-CoV-2 susceptibility and COVID-19 disease severity are associated with genetic variants affecting gene expression in a variety of tissues

Variability in SARS-CoV-2 susceptibility and COVID-19 disease severity between individuals is partly due to genetic factors. Here, we identify 4 genomic loci with suggestive associations for SARS-CoV-2 susceptibility and 19 for COVID-19 disease severity. Four of these 23 loci likely have an ethnicity-specific component. Genome-wide association study (GWAS) signals in 11 loci colocalize with expression quantitative trait loci (eQTLs) associated with the expression of 20 genes in 62 tissues/cell types (range: 1:43 tissues/gene), including lung, brain, heart, muscle, and skin as well as the digestive system and immune system. We perform genetic fine mapping to compute 99% credible SNP sets, which identify 10 GWAS loci that have eight or fewer SNPs in the credible set, including three loci with one single likely causal SNP. Our study suggests that the diverse symptoms and disease severity of COVID-19 observed between individuals is associated with variants across the genome, affecting gene expression levels in a wide variety of tissue types

A first update on mapping the human genetic architecture of COVID-19

peer reviewe

Chemical synthesis and biological evaluation of circular, branched and lariat oligonucleotides

This thesis highlights novel synthetic routes towards the facile synthesis of lariat DNA and RNA oligonucleotides, and the utilities of branched (bNAs) and circular (i.e. dumbbell-shaped) nucleic acids for targeting biologically relevant processes (i.e. HIV proliferation, alternative RNA splicing) with potential therapeutic applications.An innovative synthetic strategy for the synthesis and cyclization of a medium-sized (21-nucleotide) DNA lariat starting from a CPG-tethered, convergently synthesized branched DNA (bDNA) molecule was devised in Chapter 2. This synthetic route exploited the differential cleavage rates of two CPG-oligonucleotide tethers, namely the base-labile hydroquinone-O,O'-diacetate (Q-linker) and the more robust succinate (S-linker ) linkages, as well as phosphitylation of the 5'-oligonucleotide terminus and cyclization under standard phosphoramidite coupling conditions to effect new phosphodiester bond construction. The results clearly indicate a disadvantageous correlation between high branching efficiency on a densely loaded CPG and the production of dendrimeric (i.e. hyperbranched) oligonucleotide species rather than effective cyclization.Given the entropic disadvantage of synthesizing medium-sized DNA lariats on solid-support using the method described in Chapter 2, unique intermolecular (i.e. DNA dumbbells) and intermolecular template-mediated approaches for lariat cyclization commencing with convergently and divergently synthesized bDNAs and bRNAs were developed in Chapter 3. Both methods lead to the exclusive and high-yielding formation of medium sized (46--57 nucleotides) DNA and RNA lariats. Parameters for successful phosphodiester bond construction were also elucidated in both systems.A novel class of highly specific and potent oligonucleotide-based HIV-1 reverse transcriptase inhibitors, RNA dumbbells, comprising of a 10 base-pair stem and two flanking UUCG hairpin-loop motifs are described in Chapter 4. Explicitly, such constructs were capable of selectively hampering the RNase-H mediated activity of the retroviral enzyme without consequence to its DNA polymerase function with an IC50 in the 3 muM range. Its precise interaction with the RNase H domain of RT was authenticated via a UV-crosslinking assay. Furthermore, the RNA dumbbells did not inflict any effect on mammalian RNase H activity, suggesting that such compounds would not obstruct cellular RNase H function.Chapter 5 describes the utility of synthetic bRNA for the inhibition and modulation of pre-mRNA splicing in yeast and mammalian in vitro systems. Most notably, synthetic bNAs can be suitably exploited as agents for the study of branchpoint recognition during in vitro splicing of a pre-mRNA transcript. The results clearly indicate the requirement for a fully formed branchpoint (i.e. 5 '-, 2'- and 3'-extensions; Y-shaped molecules) off the conserved branchpoint adenosine for efficient splicing inhibition. Specific methods for stabilizing bNAs against ubiquitous cellular exo- and endonucleases as well as the 2'-scissle (2'-debranching) activity present in the HeLa extract milieu are also described