Generation and Characterization of Mouse Models for Skeletal Disease

Our laboratories have used genetically engineered mouse models (GEMMs) to assess genetic contributions to skeletal diseases such as osteoporosis and osteoarthritis. Studies on the genetic contributions to OA are often done by assessing how GEMMs respond to surgical methods that induce symptoms modeling OA. Here, we will describe protocols outlining the induction of experimental OA in mice as well as detailed descriptions of methods for analyzing skeletal phenotypes using micro-computerized tomography and skeletal histomorphometry

Functional Insight into the C-Terminal Extension of Halolysin SptA from Haloarchaeon Natrinema sp. J7

Halolysin SptA from haloarchaeon Natrinema sp. J7 consists of a subtilisin-like catalytic domain and a C-terminal extension (CTE) containing two cysteine residues. In this report, we have investigated the function of the CTE using recombinant enzymes expressed in Haloferax volcanii WFD11. Deletion of the CTE greatly reduced but did not abolish protease activity, which suggests that the CTE is not essential for enzyme folding. Mutational analysis suggests that residues Cys303 and Cys338 within the CTE form a disulfide bond that make this domain resistant to autocleavage and proteolysis under hypotonic conditions. Characterization of full-length and CTE-truncation enzymes indicates the CTE not only confers extra stability to the enzyme but also assists enzyme activity on protein substrates by facilitating binding at high salinities. Interestingly, homology modeling of the CTE yields a β-jelly roll-like structure similar to those seen in Claudin-binding domain of Clostridium perfringens enterotoxin (clostridial C-CPE) and collagen binding domain (CBD), and the CTE also possesses collagen-binding activity, making it a potential candidate as an anchoring unit in drug delivery systems

Biology of moderately halophilic aerobic bacteria

The moderately halophilic heterotrophic aerobic bacteria form a diverse group of microorganisms. The property of halophilism is widespread within the bacterial domain. Bacterial halophiles are abundant in environments such as salt lakes, saline soils, and salted food products. Most species keep their intracellular ionic concentrations at low levels while synthesizing or accumulating organic solutes to provide osmotic equilibrium of the cytoplasm with the surrounding medium. Complex mechanisms of adjustment of the intracellular environments and the properties of the cytoplasmic membrane enable rapid adaptation to changes in the salt concentration of the environment. Approaches to the study of genetic processes have recently been developed for several moderate halophiles, opening the way toward an understanding of haloadaptation at the molecular level. The new information obtained is also expected to contribute to the development of novel biotechnological uses for these organisms

C/EBPβ Promotes Transition from Proliferation to Hypertrophic Differentiation of Chondrocytes through Transactivation of p57Kip2

BACKGROUND: Although transition from proliferation to hypertrophic differentiation of chondrocytes is a crucial step for endochondral ossification in physiological skeletal growth and pathological disorders like osteoarthritis, the underlying mechanism remains an enigma. This study investigated the role of the transcription factor CCAAT/enhancer-binding protein beta (C/EBPbeta) in chondrocytes during endochondral ossification. METHODOLOGY/PRINCIPAL FINDINGS: Mouse embryos with homozygous deficiency in C/EBPbeta (C/EBPbeta-/-) exhibited dwarfism with elongated proliferative zone and delayed chondrocyte hypertrophy in the growth plate cartilage. In the cultures of primary C/EBPbeta-/- chondrocytes, cell proliferation was enhanced while hypertrophic differentiation was suppressed. Contrarily, retroviral overexpression of C/EBPbeta in chondrocytes suppressed the proliferation and enhanced the hypertrophy, suggesting the cell cycle arrest by C/EBPbeta. In fact, a DNA cell cycle histogram revealed that the C/EBPbeta overexpression caused accumulation of cells in the G0/G1 fraction. Among cell cycle factors, microarray and real-time RT-PCR analyses have identified the cyclin-dependent kinase inhibitor p57(Kip2) as the transcriptional target of C/EBPbeta. p57(Kip2) was co-localized with C/EBPbeta in late proliferative and pre-hypertrophic chondrocytes of the mouse growth plate, which was decreased by the C/EBPbeta deficiency. Luciferase-reporter and electrophoretic mobility shift assays identified the core responsive element of C/EBPbeta in the p57(Kip2) promoter between -150 and -130 bp region containing a putative C/EBP motif. The knockdown of p57(Kip2) by the siRNA inhibited the C/EBPbeta-induced chondrocyte hypertrophy. Finally, when we created the experimental osteoarthritis model by inducing instability in the knee joints of adult mice of wild-type and C/EBPbeta+/- littermates, the C/EBPbeta insufficiency caused resistance to joint cartilage destruction. CONCLUSIONS/SIGNIFICANCE: C/EBPbeta transactivates p57(Kip2) to promote transition from proliferation to hypertrophic differentiation of chondrocytes during endochondral ossification, suggesting that the C/EBPbeta-p57(Kip2) signal would be a therapeutic target of skeletal disorders like growth retardation and osteoarthritis

Molecular changes in articular cartilage and subchondral bone in the rat anterior cruciate ligament transection and meniscectomized models of osteoarthritis

<p>Abstract</p> <p>Background</p> <p>Osteoarthritis (OA) is a debilitating, progressive joint disease.</p> <p>Methods</p> <p>Similar to the disease progression in humans, sequential events of early cartilage degradation, subchondral osteopenia followed by sclerosis, and late osteophyte formation were demonstrated in the anterior cruciate ligament transection (ACLT) or ACLT with partial medial meniscectomy (ACLT + MMx) rat OA models. We describe a reliable and consistent method to examine the time dependent changes in the gene expression profiles in articular cartilage and subchondral bone.</p> <p>Results</p> <p>Local regulation of matrix degradation markers was demonstrated by a significant increase in mRNA levels of aggrecanase-1 and MMP-13 as early as the first week post-surgery, and expression remained elevated throughout the 10 week study. Immunohistochemistry confirmed MMP-13 expression in differentiated chondrocytes and synovial fibroblasts at week-2 and cells within osteophytes at week-10 in the surgically-modified-joints. Concomitant increases in chondrocyte differentiation markers, Col IIA and Sox 9, and vascular invasion markers, VEGF and CD31, peaked around week-2 to -4, and returned to Sham levels at later time points in both models. Indeed, VEGF-positive cells were found in the deep articular chondrocytes adjacent to subchondral bone. Osteoclastic bone resorption markers, cathepsin K and TRAP, were also elevated at week-2. Confirming bone resorption is an early local event in OA progression, cathepsin K positive osteoclasts were found invading the articular cartilage from the subchondral region at week 2. This was followed by late disease events, including subchondral sclerosis and osteophyte formation, as demonstrated by the upregulation of the osteoanabolic markers runx2 and osterix, toward week-4 to 6 post-surgery.</p> <p>Conclusions</p> <p>In summary, this study demonstrated the temporal and cohesive gene expression changes in articular cartilage and subchondral bone using known markers of OA progression. The findings here support genome-wide profiling efforts to elucidate the sequential and complex regulation of the disease.</p

Identification of polyhydroxyalkanoates in Halococcus and other haloarchaeal species

Polyhydroxyalkanoates (PHAs) are accumulated in many prokaryotes. Several members of the Halobacteriaceae produce poly-3-hydroxybutyrate (PHB), but it is not known if this is a general property of the family. We evaluated identification methods for PHAs with 20 haloarchaeal species, three of them isolates from Permian salt. Staining with Sudan Black B, Nile Blue A, or Nile Red was applied to screen for the presence of PHAs. Transmission electron microscopy and 1H-nuclear magnetic resonance spectroscopy were used for visualization of PHB granules and chemical confirmation of PHAs in cell extracts, respectively. We report for the first time the production of PHAs by Halococcus sp. (Halococcus morrhuae DSM 1307T, Halococcus saccharolyticus DSM 5350T, Halococcus salifodinae DSM 8989T, Halococcus dombrowskii DSM 14522T, Halococcus hamelinensis JCM 12892T, Halococcus qingdaonensis JCM 13587T), Halorubrum sp. (Hrr. coriense DSM 10284T, Halorubrum chaoviator DSM 19316T, Hrr. chaoviator strains NaxosII and AUS-1), haloalkaliphiles (Natronobacterium gregoryi NCMB 2189T, Natronococcus occultus DSM 3396T) and Halobacterium noricense DSM 9758T. No PHB was detected in Halobacterium salinarum NRC-1 ATCC 700922, Hbt. salinarum R1 and Haloferax volcanii DSM 3757T. Most species synthesized PHAs when growing in synthetic as well as in complex medium. The polyesters were generally composed of PHB and poly-ß-hydroxybutyrate-co-3-hydroxyvalerate (PHBV). Available genomic data suggest the absence of PHA synthesis in some haloarchaea and in all other Euryarchaeota and Crenarchaeota. Homologies between haloarchaeal and bacterial PHA synthesizing enzymes had indicated to some authors probable horizontal gene transfer, which, considering the data obtained in this study, may have occurred already before Permian times

Thymic stromal lymphopoietin (TSLP) is associated with allergic rhinitis in children with asthma

<p>Abstract</p> <p>Background</p> <p>Allergic rhinitis (AR) affects up to 80% of children with asthma and increases asthma severity. Thymic stromal lymphopoietin (TSLP) is a key mediator of allergic inflammation. The role of the TSLP gene (<it>TSLP</it>) in the pathogenesis of AR has not been studied.</p> <p>Objective</p> <p>To test for associations between variants in <it>TSLP</it>, <it>TSLP</it>-related genes, and AR in children with asthma.</p> <p>Methods</p> <p>We genotyped 15 single nucleotide polymorphisms (SNPs) in <it>TSLP, OX40L, IL7R</it>, and <it>RXRα </it>in three independent cohorts: 592 asthmatic Costa Rican children and their parents, 422 nuclear families of North American children with asthma, and 239 Swedish children with asthma. We tested for associations between these SNPs and AR. As we previously reported sex-specific effects for <it>TSLP</it>, we performed overall and sex-stratified analyses. We additionally performed secondary analyses for gene-by-gene interactions.</p> <p>Results</p> <p>Across the three cohorts, the T allele of <it>TSLP </it>SNP rs1837253 was undertransmitted in boys with AR and asthma as compared to boys with asthma alone. The SNP was associated with reduced odds for AR (odds ratios ranging from 0.56 to 0.63, with corresponding Fisher's combined P value of 1.2 × 10^-4). Our findings were significant after accounting for multiple comparisons. SNPs in <it>OX40L, IL7R</it>, and <it>RXRα </it>were not consistently associated with AR in children with asthma. There were nominally significant interactions between gene pairs.</p> <p>Conclusions</p> <p><it>TSLP </it>SNP rs1837253 is associated with reduced odds for AR in boys with asthma. Our findings support a role for <it>TSLP </it>in the pathogenesis of AR in children with asthma.</p

Haloarchaea swim slowly for optimal chemotactic efficiency in low nutrient environments

Archaea have evolved to survive in some of the most extreme environments on earth. Life in extreme, nutrient-poor conditions gives the opportunity to probe fundamental energy limitations on movement and response to stimuli, two essential markers of living systems. Here we use three-dimensional holographic microscopy and computer simulations to reveal that halophilic archaea achieve chemotaxis with power requirements one hundred-fold lower than common eubacterial model systems. Their swimming direction is stabilised by their flagella (archaella), enhancing directional persistence in a manner similar to that displayed by eubacteria, albeit with a different motility apparatus. Our experiments and simulations reveal that the cells are capable of slow but deterministic chemotaxis up a chemical gradient, in a biased random walk at the thermodynamic limit

Increased Serum and Musculotendinous Fibrogenic Proteins following Persistent Low-Grade Inflammation in a Rat Model of Long-Term Upper Extremity Overuse.

We examined the relationship between grip strength declines and muscle-tendon responses induced by long-term performance of a high-repetition, low-force (HRLF) reaching task in rats. We hypothesized that grip strength declines would correlate with inflammation, fibrosis and degradation in flexor digitorum muscles and tendons. Grip strength declined after training, and further in weeks 18 and 24, in reach limbs of HRLF rats. Flexor digitorum tissues of reach limbs showed low-grade increases in inflammatory cytokines: IL-1β after training and in week 18, IL-1α in week 18, TNF-α and IL-6 after training and in week 24, and IL-10 in week 24, with greater increases in tendons than muscles. Similar cytokine increases were detected in serum with HRLF: IL-1α and IL-10 in week 18, and TNF-α and IL-6 in week 24. Grip strength correlated inversely with IL-6 in muscles, tendons and serum, and TNF-α in muscles and serum. Four fibrogenic proteins, TGFB1, CTGF, PDGFab and PDGFbb, and hydroxyproline, a marker of collagen synthesis, increased in serum in HRLF weeks 18 or 24, concomitant with epitendon thickening, increased muscle and tendon TGFB1 and CTGF. A collagenolytic gelatinase, MMP2, increased by week 18 in serum, tendons and muscles of HRLF rats. Grip strength correlated inversely with TGFB1 in muscles, tendons and serum; with CTGF-immunoreactive fibroblasts in tendons; and with MMP2 in tendons and serum. Thus, motor declines correlated with low-grade systemic and musculotendinous inflammation throughout task performance, and increased fibrogenic and degradative proteins with prolonged task performance. Serum TNF-α, IL-6, TGFB1, CTGF and MMP2 may serve as serum biomarkers of work-related musculoskeletal disorders, although further studies in humans are needed

Analysis of osteoarthritis in a mouse model of the progeroid human DNA repair syndrome trichothiodystrophy

The increasing average age in developed societies is paralleled by an increase in the prevalence of many age-related diseases such as osteoarthritis (OA), which is characterized by deformation of the joint due to cartilage damage and increased turnover of subchondral bone. Consequently, deficiency in DNA repair, often associated with premature aging, may lead to increased pathology of these two tissues. To examine this possibility, we analyzed the bone and cartilage phenotype of male and female knee joints derived from 52- to 104-week-old WT C57Bl/6 and trichothiodystrophy (TTD) mice, who carry a defect in the nucleotide excision repair pathway and display many features of premature aging. Using micro-CT, we found bone loss in all groups of 104-week-old compared to 52-week-old mice. Cartilage damage was mild to moderate in all mice. Surprisingly, female TTD mice had less cartilage damage, proteoglycan depletion, and osteophytosis compared to WT controls. OA severity in males did not significantly differ between genotypes, although TTD males had less osteophytosis. These results indicate that in premature aging TTD mice age-related changes in cartilage were not more severe compared to WT mice, in striking contrast with bone and many other tissues. This segmental aging character may be explained by a difference in vasculature and thereby oxygen load in cartilage and bone. Alternatively, a difference in impact of an anti-aging response, previously found to be triggered by accumulation of DNA damage, might help explain why female mice were protected from cartilage damage. These findings underline the exceptional segmental nature of progeroid conditions and provide an explanation for pro- and anti-aging features occurring in the same individual