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Find video protocols related to scientific articles indexed in Pubmed.
A transgenic mouse model of OI type V supports a neomorphic mechanism of the IFITM5 mutation.
J. Bone Miner. Res.
PUBLISHED: 09-09-2014
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Osteogenesis Imperfecta (OI) type V is characterized by increased bone fragility, long bone deformities, hyperplastic callus formation and calcification of interosseous membranes. It is caused by a recurrent mutation in the 5' UTR of the IFITM5 gene (c.-14C>T). This mutation introduces an alternative start codon, adding 5 amino acid residues to the N-terminus of the protein. The mechanism whereby this novel IFITM5 protein causes OI type V is yet to be defined. To address this, we created transgenic mice expressing either the wild type or the OI type V mutant IFITM5 under the control of an osteoblast-specific Col1a1 2.3kb promoter. These mutant IFITM5 transgenic mice exhibited perinatal lethality, whereas wild-type IFITM5 transgenic mice showed normal growth and development. Skeletal preparations and radiographs performed on E15.5 and E18.5 OI type V transgenic embryos revealed delayed/abnormal mineralization and skeletal defects including abnormal rib cage formation, long bone deformities and fractures. Primary osteoblast cultures, derived from mutant mice calvaria at E18.5, showed decreased mineralization by Alizarin-Red staining and RNA isolated from calvaria showed reduced expression of osteoblast differentiation markers such as Osteocalcin, as compared to non-transgenic littermates and wild-type mice calvaria, consistent with the in vivo phenotype. Importantly, overexpression of wild-type Ifitm5 did not manifest a significant bone phenotype. Collectively, our results suggest that expression of mutant IFITM5 causes abnormal skeletal development, low bone mass, and abnormal osteoblast differentiation. Given that neither overexpression of the wild type Ifitm5, as shown in our model, nor knock-out of Ifitm5, as previously published, showed significant bone abnormalities, we conclude that the IFITM5 mutation in OI type V acts in a neomorphic fashion. © 2014 American Society for Bone and Mineral Research.
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Abnormal type I collagen post-translational modification and crosslinking in a cyclophilin B KO mouse model of recessive osteogenesis imperfecta.
PLoS Genet.
PUBLISHED: 06-01-2014
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Cyclophilin B (CyPB), encoded by PPIB, is an ER-resident peptidyl-prolyl cis-trans isomerase (PPIase) that functions independently and as a component of the collagen prolyl 3-hydroxylation complex. CyPB is proposed to be the major PPIase catalyzing the rate-limiting step in collagen folding. Mutations in PPIB cause recessively inherited osteogenesis imperfecta type IX, a moderately severe to lethal bone dysplasia. To investigate the role of CyPB in collagen folding and post-translational modifications, we generated Ppib-/- mice that recapitulate the OI phenotype. Knock-out (KO) mice are small, with reduced femoral areal bone mineral density (aBMD), bone volume per total volume (BV/TV) and mechanical properties, as well as increased femoral brittleness. Ppib transcripts are absent in skin, fibroblasts, femora and calvarial osteoblasts, and CyPB is absent from KO osteoblasts and fibroblasts on western blots. Only residual (2-11%) collagen prolyl 3-hydroxylation is detectable in KO cells and tissues. Collagen folds more slowly in the absence of CyPB, supporting its rate-limiting role in folding. However, treatment of KO cells with cyclosporine A causes further delay in folding, indicating the potential existence of another collagen PPIase. We confirmed and extended the reported role of CyPB in supporting collagen lysyl hydroxylase (LH1) activity. Ppib-/- fibroblast and osteoblast collagen has normal total lysyl hydroxylation, while increased collagen diglycosylation is observed. Liquid chromatography/mass spectrometry (LC/MS) analysis of bone and osteoblast type I collagen revealed site-specific alterations of helical lysine hydroxylation, in particular, significantly reduced hydroxylation of helical crosslinking residue K87. Consequently, underhydroxylated forms of di- and trivalent crosslinks are strikingly increased in KO bone, leading to increased total crosslinks and decreased helical hydroxylysine- to lysine-derived crosslink ratios. The altered crosslink pattern was associated with decreased collagen deposition into matrix in culture, altered fibril structure in tissue, and reduced bone strength. These studies demonstrate novel consequences of the indirect regulatory effect of CyPB on collagen hydroxylation, impacting collagen glycosylation, crosslinking and fibrillogenesis, which contribute to maintaining bone mechanical properties.
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Increased C-telopeptide cross-linking of tendon type I collagen in fibromodulin-deficient mice.
J. Biol. Chem.
PUBLISHED: 05-21-2014
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The controlled assembly of collagen monomers into fibrils, with accompanying intermolecular cross-linking by lysyl oxidase-mediated bonds, is vital to the structural and mechanical integrity of connective tissues. This process is influenced by collagen-associated proteins, including small leucine-rich proteins (SLRPs), but the regulatory mechanisms are not well understood. Deficiency in fibromodulin, an SLRP, causes abnormal collagen fibril ultrastructure and decreased mechanical strength in mouse tendons. In this study, fibromodulin deficiency rendered tendon collagen more resistant to nonproteolytic extraction. The collagen had an increased and altered cross-linking pattern at an early stage of fibril formation. Collagen extracts contained a higher proportion of stably cross-linked ?1(I) chains as a result of their C-telopeptide lysines being more completely oxidized to aldehydes. The findings suggest that fibromodulin selectively affects the extent and pattern of lysyl oxidase-mediated collagen cross-linking by sterically hindering access of the enzyme to telopeptides, presumably through binding to the collagen. Such activity implies a broader role for SLRP family members in regulating collagen cross-linking placement and quantity.
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Connective tissue alterations in Fkbp10-/- mice.
Hum. Mol. Genet.
PUBLISHED: 04-28-2014
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Osteogenesis imperfecta (OI) is an inherited brittle bone disorder characterized by bone fragility and low bone mass. Loss of function mutations in FK506-binding protein 10 (FKBP10), encoding the FKBP65 protein, result in recessive OI and Bruck syndrome, of which the latter is additionally characterized by joint contractures. FKBP65 is thought to act as a collagen chaperone, but it is unknown how loss of FKBP65 affects collagen synthesis and extracellular matrix formation. We evaluated the developmental and postnatal expression of Fkbp10 and analyzed the consequences of its generalized loss of function. Fkbp10 is expressed at low levels in E13.5 mouse embryos, particularly in skeletal tissues, and steadily increases through E17.5 with expression in not only skeletal tissues, but also in visceral tissues. Postnatally, expression is limited to developing bone and ligaments. In contrast to humans, with complete loss of function mutations, Fkbp10(-/-) mice do not survive birth, and embryos present with growth delay and tissue fragility. Type I calvarial collagen isolated from these mice showed reduced stable crosslink formation at telopeptide lysines. Furthermore, Fkbp10(-/-) mouse embryonic fibroblasts show retention of procollagen in the cell layer and associated dilated endoplasmic reticulum. These data suggest a requirement for FKBP65 function during embryonic connective tissue development in mice, but the restricted expression postnatally in bone, ligaments and tendons correlates with the bone fragility and contracture phenotype in humans.
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Excessive transforming growth factor-? signaling is a common mechanism in osteogenesis imperfecta.
Nat. Med.
PUBLISHED: 03-24-2014
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Osteogenesis imperfecta (OI) is a heritable disorder, in both a dominant and recessive manner, of connective tissue characterized by brittle bones, fractures and extraskeletal manifestations. How structural mutations of type I collagen (dominant OI) or of its post-translational modification machinery (recessive OI) can cause abnormal quality and quantity of bone is poorly understood. Notably, the clinical overlap between dominant and recessive forms of OI suggests common molecular pathomechanisms. Here, we show that excessive transforming growth factor-? (TGF-?) signaling is a mechanism of OI in both recessive (Crtap(-/-)) and dominant (Col1a2(tm1.1Mcbr)) OI mouse models. In the skeleton, we find higher expression of TGF-? target genes, higher ratio of phosphorylated Smad2 to total Smad2 protein and higher in vivo Smad2 reporter activity. Moreover, the type I collagen of Crtap(-/-) mice shows reduced binding to the small leucine-rich proteoglycan decorin, a known regulator of TGF-? activity. Anti-TGF-? treatment using the neutralizing antibody 1D11 corrects the bone phenotype in both forms of OI and improves the lung abnormalities in Crtap(-/-) mice. Hence, altered TGF-? matrix-cell signaling is a primary mechanism in the pathogenesis of OI and could be a promising target for the treatment of OI.
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Modulation of the secretory pathway rescues zebrafish polycystic kidney disease pathology.
J. Am. Soc. Nephrol.
PUBLISHED: 03-13-2014
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Mutations in polycystin 1 and polycystin 2 are responsible for autosomal dominant polycystic kidney disease, the most common heritable human disease. Polycystins function as calcium ion channels, but their impact on cell physiology is not fully known. Recent findings suggest that polycystins could function in the maintenance of extracellular matrix integrity. In zebrafish, polycystin 2 knockdown induces kidney cysts, hydrocephalus, left/right asymmetry defects, and strong dorsal axis curvature. Here, we show that increased notochord sheath collagen deposition in polycystin 2-deficient embryos is directly linked to axis defects. Increased collagen II protein accumulation did not associate with increased col2a1 mRNA or a decrease in matrix metalloproteinase activity but, instead, it associated with increased expression of the endoplasmic reticulum/Golgi transport coat protein complex II Sec proteins. sec24D knockdown prevented dorsal axis curvature and kidney cystogenesis in polycystin 2 morphants. Nontoxic doses of brefeldin A also prevented the dorsal axis curvature formation in polycystin 2 morphants and curly up polycystin 2 mutants. Brefeldin A treatment after the onset of polycystin deficiency phenotypes reversed the curved axis phenotype but not kidney cyst progression. Our results suggest that polycystin 2 deficiency causes increased collagen II synthesis with upregulation of secretory pathway coat protein complex II components. Restoration of normal rates of secretory protein synthesis and secretion may be a new target in the treatment of autosomal dominant polycystic kidney disease.
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Targeting the LRP5 pathway improves bone properties in a mouse model of osteogenesis imperfecta.
J. Bone Miner. Res.
PUBLISHED: 01-09-2014
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The cell surface receptor low-density lipoprotein receptor-related protein 5 (LRP5) is a key regulator of bone mass and bone strength. Heterozygous missense mutations in LRP5 cause autosomal dominant high bone mass (HBM) in humans by reducing binding to LRP5 by endogenous inhibitors, such as sclerostin (SOST). Mice heterozygous for a knockin allele (Lrp5(p.A214V) ) that is orthologous to a human HBM-causing mutation have increased bone mass and strength. Osteogenesis imperfecta (OI) is a skeletal fragility disorder predominantly caused by mutations that affect type I collagen. We tested whether the LRP5 pathway can be used to improve bone properties in animal models of OI. First, we mated Lrp5(+/p.A214V) mice to Col1a2(+/p.G610C) mice, which model human type IV OI. We found that Col1a2(+/p.G610C) ;Lrp5(+/p.A214V) offspring had significantly increased bone mass and strength compared to Col1a2(+/p.G610C) ;Lrp5(+/+) littermates. The improved bone properties were not a result of altered mRNA expression of type I collagen or its chaperones, nor were they due to changes in mutant type I collagen secretion. Second, we treated Col1a2(+/p.G610C) mice with a monoclonal antibody that inhibits sclerostin activity (Scl-Ab). We found that antibody-treated mice had significantly increased bone mass and strength compared to vehicle-treated littermates. These findings indicate increasing bone formation, even without altering bone collagen composition, may benefit patients with OI.
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3-D ultrastructure and collagen composition of healthy and overloaded human tendon: evidence of tenocyte and matrix buckling.
J. Anat.
PUBLISHED: 01-06-2014
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Achilles tendinopathies display focal tissue thickening with pain and ultrasonography changes. Whilst complete rupture might be expected to induce changes in tissue organization and protein composition, little is known about the consequences of non-rupture-associated tendinopathies, especially with regards to changes in the content of collagen type I and III (the major collagens in tendon), and changes in tendon fibroblast (tenocyte) shape and organization of the extracellular matrix (ECM). To gain new insights, we took biopsies from the tendinopathic region and flanking healthy region of Achilles tendons of six individuals with clinically diagnosed tendinopathy who had no evidence of cholesterol, uric acid and amyloid accumulation. Biochemical analyses of collagen III/I ratio were performed on all six individuals, and electron microscope analysis using transmission electron microscopy and serial block face-scanning electron microscopy were made on two individuals. In the tendinopathic regions, compared with the flanking healthy tissue, we observed: (i) an increase in the ratio of collagen III : I proteins; (ii) buckling of the collagen fascicles in the ECM; (iii) buckling of tenocytes and their nuclei; and (iv) an increase in the ratio of small-diameter : large-diameter collagen fibrils. In summary, load-induced non-rupture tendinopathy in humans is associated with localized biochemical changes, a shift from large- to small-diameter fibrils, buckling of the tendon ECM, and buckling of the cells and their nuclei.
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Evolutionary origins of C-terminal (GPP)n 3-hydroxyproline formation in vertebrate tendon collagen.
PLoS ONE
PUBLISHED: 01-01-2014
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Approximately half the proline residues in fibrillar collagen are hydroxylated. The predominant form is 4-hydroxyproline, which helps fold and stabilize the triple helix. A minor form, 3-hydroxyproline, still has no clear function. Using peptide mass spectrometry, we recently revealed several previously unknown molecular sites of 3-hydroxyproline in fibrillar collagen chains. In fibril-forming A-clade collagen chains, four new partially occupied 3-hydroxyproline sites were found (A2, A3, A4 and (GPP)n) in addition to the fully occupied A1 site at Pro986. The C-terminal (GPP)n motif has five consecutive GPP triplets in ?1(I), four in ?2(I) and three in ?1(II), all subject to 3-hydroxylation. The evolutionary origins of this substrate sequence were investigated by surveying the pattern of its 3-hydroxyproline occupancy from early chordates through amphibians, birds and mammals. Different tissue sources of type I collagen (tendon, bone and skin) and type II collagen (cartilage and notochord) were examined by mass spectrometry. The (GPP)n domain was found to be a major substrate for 3-hydroxylation only in vertebrate fibrillar collagens. In higher vertebrates (mouse, bovine and human), up to five 3-hydroxyproline residues per (GPP)n motif were found in ?1(I) and four in ?2(I), with an average of two residues per chain. In vertebrate type I collagen the modification exhibited clear tissue specificity, with 3-hydroxyproline prominent only in tendon. The occupancy also showed developmental changes in Achilles tendon, with increasing 3-hydroxyproline levels with age. The biological significance is unclear but the level of 3-hydroxylation at the (GPP)n site appears to have increased as tendons evolved and shows both tendon type and developmental variations within a species.
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Differential effects of collagen prolyl 3-hydroxylation on skeletal tissues.
PLoS Genet.
PUBLISHED: 01-01-2014
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Mutations in the genes encoding cartilage associated protein (CRTAP) and prolyl 3-hydroxylase 1 (P3H1 encoded by LEPRE1) were the first identified causes of recessive Osteogenesis Imperfecta (OI). These proteins, together with cyclophilin B (encoded by PPIB), form a complex that 3-hydroxylates a single proline residue on the ?1(I) chain (Pro986) and has cis/trans isomerase (PPIase) activity essential for proper collagen folding. Recent data suggest that prolyl 3-hydroxylation of Pro986 is not required for the structural stability of collagen; however, the absence of this post-translational modification may disrupt protein-protein interactions integral for proper collagen folding and lead to collagen over-modification. P3H1 and CRTAP stabilize each other and absence of one results in degradation of the other. Hence, hypomorphic or loss of function mutations of either gene cause loss of the whole complex and its associated functions. The relative contribution of losing this complex's 3-hydroxylation versus PPIase and collagen chaperone activities to the phenotype of recessive OI is unknown. To distinguish between these functions, we generated knock-in mice carrying a single amino acid substitution in the catalytic site of P3h1 (Lepre1(H662A) ). This substitution abolished P3h1 activity but retained ability to form a complex with Crtap and thus the collagen chaperone function. Knock-in mice showed absence of prolyl 3-hydroxylation at Pro986 of the ?1(I) and ?1(II) collagen chains but no significant over-modification at other collagen residues. They were normal in appearance, had no growth defects and normal cartilage growth plate histology but showed decreased trabecular bone mass. This new mouse model recapitulates elements of the bone phenotype of OI but not the cartilage and growth phenotypes caused by loss of the prolyl 3-hydroxylation complex. Our observations suggest differential tissue consequences due to selective inactivation of P3H1 hydroxylase activity versus complete ablation of the prolyl 3-hydroxylation complex.
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Deletion of Mecom in mouse results in early-onset spinal deformity and osteopenia.
Bone
PUBLISHED: 11-13-2013
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Recent studies have indicated a role for a MECOM allele in susceptibility to osteoporotic fractures in humans. We have generated a mutation in Mecom in mouse (termed ME(m1)) via lacZ knock-in into the upstream transcription start site for the gene, resulting in disruption of Mds1 and Mds1-Evi1 transcripts, but not of Evi1 transcripts. We demonstrate that ME(m1/m1) mice have severe kyphoscoliosis that is reminiscent of human congenital or primary kyphoscoliosis. ME(m1/m1) mice appear normal at birth, but by 2weeks, they exhibit a slight lumbar lordosis and narrowed intervertebral space. This progresses to severe lordosis with disc collapse and synostosis, together with kyphoscoliosis. Bone formation and strength testing show that ME(m1/m1) mice have normal bone formation and composition but are osteopenic. While endochondral bone development is normal, it is markedly dysplastic in its organization. Electron micrographs of the 1week postnatal intervertebral discs reveals marked disarray of collagen fibers, consistent with an inherent weakness in the non-osseous connective tissue associated with the spine. These findings indicate that lack of ME leads to a complex defect in both osseous and non-osseous musculoskeletal tissues, including a marked vertebral osteopenia, degeneration of the IVD, and disarray of connective tissues, which is likely due to an inherent inability to establish and/or maintain components of these tissues.
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Whole-Genome Sequencing Demonstrates That Fidaxomicin Is Superior to Vancomycin for Preventing Reinfection and Relapse of Infection With Clostridium difficile.
J. Infect. Dis.
PUBLISHED: 11-11-2013
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Whole-genome sequencing was used to determine whether the reductions in recurrence of Clostridium difficile infection observed with fidaxomicin in pivotal phase 3 trials occurred by preventing relapse of the same infection, by preventing reinfection with a new strain, or by preventing both outcomes. Paired isolates of C. difficile were available from 93 of 199 participants with recurrences (28 were treated with fidaxomicin, and 65 were treated with vancomycin). Given C. difficile evolutionary rates, paired samples ?2 single-nucleotide variants (SNVs) apart were considered relapses, paired samples >10 SNVs apart were considered reinfection, and those 3-10 SNVs apart (or without whole-genome sequences) were considered indeterminate in a competing risks survival analysis. Fidaxomicin reduced the risk of both relapse (competing risks hazard ratio [HR], 0.40 [95% confidence interval {CI}, .25-.66]; P = .0003) and reinfection (competing risks HR, 0.33 [95% CI, 0.11-1.01]; P = .05).
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Clostridium difficile surveillance: harnessing new technologies to control transmission.
Expert Rev Anti Infect Ther
PUBLISHED: 10-25-2013
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Clostridium difficile surveillance allows outbreaks of cases clustered in time and space to be identified and further transmission prevented. Traditionally, manual detection of groups of cases diagnosed in the same ward or hospital, often followed by retrospective reference laboratory genotyping, has been used to identify outbreaks. However, integrated healthcare databases offer the prospect of automated real-time outbreak detection based on statistically robust methods, and accounting for contacts between cases, including those distant to the ward of diagnosis. Complementary to this, rapid benchtop whole genome sequencing, and other highly discriminatory genotyping, has the potential to distinguish which cases are part of an outbreak with high precision and in clinically relevant timescales. These new technologies are likely to shape future surveillance.
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Diverse sources of C. difficile infection identified on whole-genome sequencing.
N. Engl. J. Med.
PUBLISHED: 09-27-2013
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It has been thought that Clostridium difficile infection is transmitted predominantly within health care settings. However, endemic spread has hampered identification of precise sources of infection and the assessment of the efficacy of interventions.
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Molecular properties and fibril ultrastructure of types II and XI collagens in cartilage of mice expressing exclusively the ?1(IIA) collagen isoform.
Matrix Biol.
PUBLISHED: 07-09-2013
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Until now, no biological tools have been available to determine if a cross-linked collagen fibrillar network derived entirely from type IIA procollagen isoforms, can form in the extracellular matrix (ECM) of cartilage. Recently, homozygous knock-in transgenic mice (Col2a1(+ex2), ki/ki) were generated that exclusively express the IIA procollagen isoform during post-natal development while type IIB procollagen, normally present in the ECM of wild type mice, is absent. The difference between these Col2a1 isoforms is the inclusion (IIA) or exclusion (IIB) of exon 2 that is alternatively spliced in a developmentally regulated manner. Specifically, chondroprogenitor cells synthesize predominantly IIA mRNA isoforms while differentiated chondrocytes produce mainly IIB mRNA isoforms. Recent characterization of the Col2a1(+ex2) mice has surprisingly shown that disruption of alternative splicing does not affect overt cartilage formation. In the present study, biochemical analyses showed that type IIA collagen extracted from ki/ki mouse rib cartilage can form homopolymers that are stabilized predominantly by hydroxylysyl pyridinoline (HP) cross-links at levels that differed from wild type rib cartilage. The findings indicate that mature type II collagen derived exclusively from type IIA procollagen molecules can form hetero-fibrils with type XI collagen and contribute to cartilage structure and function. Heteropolymers with type XI collagen also formed. Electron microscopy revealed mainly thin type IIA collagen fibrils in ki/ki mouse rib cartilage. Immunoprecipitation and mass spectrometry of purified type XI collagen revealed a heterotrimeric molecular composition of ?1(XI)?2(XI)?1(IIA) chains where the ?1(IIA) chain is the IIA form of the ?3(XI) chain. Since the N-propeptide of type XI collagen regulates type II collagen fibril diameter in cartilage, the retention of the exon 2-encoded IIA globular domain would structurally alter the N-propeptide of type XI collagen. This structural change may subsequently affect the regulatory function of type XI collagen resulting in the collagen fibril and cross-linking differences observed in this study.
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Collagen prolyl 3-hydroxylation: a major role for a minor post-translational modification?
Connect. Tissue Res.
PUBLISHED: 06-21-2013
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Prolyl 3-hydroxylation is a rare but conserved post-translational modification in many collagen types and, when defective, may be linked to a number of human diseases with musculoskeletal and potentially ocular and renal pathologies. Prolyl 3-hydroxylase-1 (P3H1), the enzyme responsible for converting proline to 3-hydroxyproline (3Hyp) in type I collagen, requires the coenzyme CRTAP for activity. Mass spectrometric analysis showed that the Crtap-/- mouse was missing 3-hydroxyproline in type I collagen ?-chains. This finding led to the discovery of mutations in genes encoding the P3H1 complex as a cause of recessively inherited osteogenesis imperfecta (brittle bone disease). Since then, many additional 3Hyp sites have been identified in various collagen types and classified based on observed substrate and tissue specificity. P3H1 is part of a family of gene products that also includes isoenzymes P3H2 and P3H3 as well as CRTAP and Sc65. It is believed these isoenzymes and coenzyme proteins have evolved different collagen substrate site and tissue specificities in their activities. The post-translational fingerprinting of collagens will be essential in understanding the basic role and extent of regulated variations of prolyl 3-hydroxylation in collagen. We believe that prolyl 3-hydroxylation is a functionally significant collagen post-translational modification and can be a cause of disease when absent.
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Detection of mixed infection from bacterial whole genome sequence data allows assessment of its role in Clostridium difficile transmission.
PLoS Comput. Biol.
PUBLISHED: 05-01-2013
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Bacterial whole genome sequencing offers the prospect of rapid and high precision investigation of infectious disease outbreaks. Close genetic relationships between microorganisms isolated from different infected cases suggest transmission is a strong possibility, whereas transmission between cases with genetically distinct bacterial isolates can be excluded. However, undetected mixed infections-infection with ?2 unrelated strains of the same species where only one is sequenced-potentially impairs exclusion of transmission with certainty, and may therefore limit the utility of this technique. We investigated the problem by developing a computationally efficient method for detecting mixed infection without the need for resource-intensive independent sequencing of multiple bacterial colonies. Given the relatively low density of single nucleotide polymorphisms within bacterial sequence data, direct reconstruction of mixed infection haplotypes from current short-read sequence data is not consistently possible. We therefore use a two-step maximum likelihood-based approach, assuming each sample contains up to two infecting strains. We jointly estimate the proportion of the infection arising from the dominant and minor strains, and the sequence divergence between these strains. In cases where mixed infection is confirmed, the dominant and minor haplotypes are then matched to a database of previously sequenced local isolates. We demonstrate the performance of our algorithm with in silico and in vitro mixed infection experiments, and apply it to transmission of an important healthcare-associated pathogen, Clostridium difficile. Using hospital ward movement data in a previously described stochastic transmission model, 15 pairs of cases enriched for likely transmission events associated with mixed infection were selected. Our method identified four previously undetected mixed infections, and a previously undetected transmission event, but no direct transmission between the pairs of cases under investigation. These results demonstrate that mixed infections can be detected without additional sequencing effort, and this will be important in assessing the extent of cryptic transmission in our hospitals.
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Kuskokwim syndrome, a recessive congenital contracture disorder, extends the phenotype of FKBP10 mutations.
Hum. Mutat.
PUBLISHED: 03-18-2013
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Recessive mutations in FKBP10 at 17q21.2, encoding FKBP65, cause both osteogenesis imperfecta (OI) and Bruck syndrome (OI plus congenital contractures). Contractures are a variable manifestation of null/missense FKBP10 mutations. Kuskokwim syndrome (KS) is an autosomal recessive congenital contracture disorder found among Yupik Eskimos. Linkage mapping of KS to chromosome 17q21, together with contractures as a feature of FKBP10 mutations, made FKBP10 a candidate gene. We identified a homozygous three-nucleotide deletion in FKBP10 (c.877_879delTAC) in multiple Kuskokwim pedigrees; 3% of regional controls are carriers. The mutation deletes the highly conserved p.Tyr293 residue in FKBP65s third peptidyl-prolyl cis-trans isomerase domain. FKBP10 transcripts are normal, but mutant FKBP65 is destabilized to a residual 5%. Collagen synthesized by KS fibroblasts has substantially decreased hydroxylation of the telopeptide lysine crucial for collagen cross-linking, with 2%-10% hydroxylation in probands versus 60% in controls. Matrix deposited by KS fibroblasts has marked reduction in maturely cross-linked collagen. KS collagen is disorganized in matrix, and fibrils formed in vitro had subtle loosening of monomer packing. Our results imply that FKBP10 mutations affect collagen indirectly, by ablating FKBP65 support for collagen telopeptide hydroxylation by lysyl hydroxylase 2, thus decreasing collagen cross-links in tendon and bone matrix. FKBP10 mutations may also underlie other arthrogryposis syndromes.
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Relationship between bacterial strain type, host biomarkers, and mortality in Clostridium difficile infection.
Clin. Infect. Dis.
PUBLISHED: 03-05-2013
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Despite substantial interest in biomarkers, their impact on clinical outcomes and variation with bacterial strain has rarely been explored using integrated databases.
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Bone collagen: new clues to its mineralization mechanism from recessive osteogenesis imperfecta.
Calcif. Tissue Int.
PUBLISHED: 03-01-2013
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Until 2006 the only mutations known to cause osteogenesis imperfecta (OI) were in the two genes coding for type I collagen chains. These dominant mutations affecting the expression or primary sequence of collagen ?1(I) and ?2(I) chains account for over 90% of OI cases. Since then a growing list of mutant genes causing the 5-10% of recessive cases has rapidly emerged. They include CRTAP, LEPRE1, and PPIB, which encode three proteins forming the prolyl 3-hydroxylase complex; PLOD2 and FKBP10, which encode, respectively, lysyl hydroxylase 2 and a foldase required for its activity in forming mature cross-links in bone collagen; SERPINH1, which encodes the collagen chaperone HSP47; SERPINF1, which encodes pigment epithelium-derived factor required for osteoid mineralization; and BMP1, which encodes the type I procollagen C-propeptidase. All cause fragile bone in infancy, which can include overmineralization or undermineralization defects as well as abnormal collagen posttranslational modifications. Consistently both dominant and recessive variants lead to abnormal cross-linking chemistry in bone collagen. These recent discoveries strengthen the potential for a common pathogenic mechanism of misassembled collagen fibrils. Of the new genes identified, eight encode proteins required for collagen posttranslational modification, chaperoning of newly synthesized collagen chains into native molecules, or transport through the endoplasmic reticulum and Golgi for polymerization, cross-linking, and mineralization. In reviewing these findings, we conclude that a common theme is emerging in the pathogenesis of brittle bone disease of mishandled collagen assembly with important insights on posttranslational features of bone collagen that have evolved to optimize it as a biomineral template.
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Asymptomatic Clostridium difficile Colonisation and Onward Transmission.
PLoS ONE
PUBLISHED: 01-01-2013
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Combined genotyping/whole genome sequencing and epidemiological data suggest that in endemic settings only a minority of Clostridium difficile infection, CDI, is acquired from other cases. Asymptomatic patients are a potential source for many unexplained cases.
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Short-term genome stability of serial Clostridium difficile ribotype 027 isolates in an experimental gut model and recurrent human disease.
PLoS ONE
PUBLISHED: 01-01-2013
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Clostridium difficile whole genome sequencing has the potential to identify related isolates, even among otherwise indistinguishable strains, but interpretation depends on understanding genomic variation within isolates and individuals.
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Clostridium difficile mixed infection and reinfection.
J. Clin. Microbiol.
PUBLISHED: 11-09-2011
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Isolates from consecutive Clostridium difficile infection (CDI) fecal samples underwent multilocus sequence typing. Potential reinfections with different genotypes were identified in 88/560 (16%) sample pairs taken 1 to 1,414 days (median, 24; interquartile range [IQR], 1 to 52 days) apart; odds of reinfection increased by 58% for every doubling of time between samples. Of 109 sample pairs taken on the same day, 3 (3%) had different genotypes. Considering samples 0 to 7 days apart as the same CDI, 7% of cases had mixed infections with >1 genotype.
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Identification of a mutation causing deficient BMP1/mTLD proteolytic activity in autosomal recessive osteogenesis imperfecta.
Hum. Mutat.
PUBLISHED: 09-12-2011
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Herein, we have studied a consanguineous Egyptian family with two children diagnosed with severe autosomal recessive osteogenesis imperfecta (AR-OI) and a large umbilical hernia. Homozygosity mapping in this family showed lack of linkage to any of the previously known AR-OI genes, but revealed a 10.27 MB homozygous region on chromosome 8p in the two affected sibs, which comprised the procollagen I C-terminal propeptide (PICP) endopeptidase gene BMP1. Mutation analysis identified both patients with a Phe249Leu homozygous missense change within the BMP1 protease domain involving a residue, which is conserved in all members of the astacin group of metalloproteases. Type I procollagen analysis in supernatants from cultured fibroblasts demonstrated abnormal PICP processing in patient-derived cells consistent with the mutation causing decreased BMP1 function. This was further confirmed by overexpressing wild type and mutant BMP1 longer isoform (mammalian Tolloid protein [mTLD]) in NIH3T3 fibroblasts and human primary fibroblasts. While overproduction of normal mTLD resulted in a large proportion of pro?1(I) in the culture media being C-terminally processed, pro?1(I) cleavage was not enhanced by an excess of the mutant protein, proving that the Phe249Leu mutation leads to a BMP1/mTLD protein with deficient PICP proteolytic activity. We conclude that BMP1 is an additional gene mutated in AR-OI.
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A role for prolyl 3-hydroxylase 2 in post-translational modification of fibril-forming collagens.
J. Biol. Chem.
PUBLISHED: 07-11-2011
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The fibrillar collagen types I, II, and V/XI have recently been shown to have partially 3-hydroxylated proline (3Hyp) residues at sites other than the established primary Pro-986 site in the collagen triple helical domain. These sites showed tissue specificity in degree of hydroxylation and a pattern of D-periodic spacing. This suggested a contributory role in fibril supramolecular assembly. The sites in clade A fibrillar ?1(II), ?2(V), and ?1(I) collagen chains share common features with known prolyl 3-hydroxylase 2 (P3H2) substrate sites in ?1(IV) chains implying a role for this enzyme. We pursued this possibility using the Swarm rat chondrosarcoma cell line (RCS-LTC) found to express high levels of P3H2 mRNA. Mass spectrometry determined that all the additional candidate 3Hyp substrate sites in the pN type II collagen made by these cells were highly hydroxylated. In RNA interference experiments, P3H2 protein synthesis was suppressed coordinately with prolyl 3-hydroxylation at Pro-944, Pro-707, and the C-terminal GPP repeat of the pN?1(II) chain, but Pro-986 remained fully hydroxylated. Furthermore, when P3H2 expression was turned off, as seen naturally in cultured SAOS-2 osteosarcoma cells, full 3Hyp occupancy at Pro-986 in ?1(I) chains was unaffected, whereas 3-hydroxylation of residue Pro-944 in the ?2(V) chain was largely lost, and 3-hydroxylation of Pro-707 in ?2(V) and ?2(I) chains were sharply reduced. The results imply that P3H2 has preferred substrate sequences among the classes of 3Hyp sites in clade A collagen chains.
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Murine tendon function is adversely affected by aggrecan accumulation due to the knockout of ADAMTS5.
J. Orthop. Res.
PUBLISHED: 05-24-2011
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The present study examined the effect of ADAMTS5 (TS5) knockout on the properties of murine flexor digitorum longus (FDL) and Achilles tendons. FDL and Achilles tendons were analyzed using biomechanical testing, histology, and immunohistochemistry; further characterization of FDL tendons was conducted using transmission electron microscopy (collagen fibril ultrastructure), SDS-PAGE (collagen content and type), fluorescence-assisted carbohydrate electrophoresis for chondroitin sulfate and hyaluronan, and Western blotting for aggrecan, versican, and decorin abundance and distribution. FDL tendons of TS5(-/-) mice showed a 33% larger cross-sectional area, increased collagen fibril area fraction, and decreased material properties relative to those of wild type mice. In TS5(-/-) mice, aggrecan accumulated in the pericellular matrix of tendon fibroblasts. In Achilles tendons, cross-sectional area, stress relaxation, and structural properties were similar in TS5(-/-) and wild type mice; however, the TS5(-/-) tendons exhibited a higher tensile modulus and a weakened enthesis. These results demonstrate that TS5 deficiency disturbs normal tendon collagen organization and alters biomechanical properties. Hence, the role of ADAMTS5 in tendon is to remove pericellular and interfibrillar aggrecan to maintain the molecular architecture responsible for normal tissue function.
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Insights on the evolution of prolyl 3-hydroxylation sites from comparative analysis of chicken and Xenopus fibrillar collagens.
PLoS ONE
PUBLISHED: 03-27-2011
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Recessive mutations that prevent 3-hydroxyproline formation in type I collagen have been shown to cause forms of osteogenesis imperfecta. In mammals, all A-clade collagen chains with a GPP sequence at the A1 site (P986), except ?1(III), have 3Hyp at residue P986. Available avian, amphibian and reptilian type III collagen sequences from the genomic database (Ensembl) all differ in sequence motif from mammals at the A1 site. This suggests a potential evolutionary distinction in prolyl 3-hydroxylation between mammals and earlier vertebrates. Using peptide mass spectrometry, we confirmed that this 3Hyp site is fully occupied in ?1(III) from an amphibian, Xenopus laevis, as it is in chicken. A thorough characterization of all predicted 3Hyp sites in collagen types I, II, III and V from chicken and xenopus revealed further differences in the pattern of occupancy of the A3 site (P707). In mammals only ?2(I) and ?2(V) chains had any 3Hyp at the A3 site, whereas in chicken all ?-chains except ?1(III) had A3 at least partially 3-hydroxylated. The A3 site was also partially 3-hydroxylated in xenopus ?1(I). Minor differences in covalent cross-linking between chicken, xenopus and mammal type I and III collagens were also found as a potential index of evolving functional differences. The function of 3Hyp is still unknown but observed differences in site occupancy during vertebrate evolution are likely to give important clues.
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Type IX collagen neo-deposition in degenerative discs of surgical patients whether genotyped plus or minus for COL9 risk alleles.
Spine
PUBLISHED: 02-12-2011
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Immunohistochemical analysis of type IX collagen in disc tissue from spinal fusion patients.
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Mutations in PPIB (cyclophilin B) delay type I procollagen chain association and result in perinatal lethal to moderate osteogenesis imperfecta phenotypes.
Hum. Mol. Genet.
PUBLISHED: 01-31-2011
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Recessive mutations in the cartilage-associated protein (CRTAP), leucine proline-enriched proteoglycan 1 (LEPRE1) and peptidyl prolyl cis-trans isomerase B (PPIB) genes result in phenotypes that range from lethal in the perinatal period to severe deforming osteogenesis imperfecta (OI). These genes encode CRTAP (encoded by CRTAP), prolyl 3-hydroxylase 1 (P3H1; encoded by LEPRE1) and cyclophilin B (CYPB; encoded by PPIB), which reside in the rough endoplasmic reticulum (RER) and can form a complex involved in prolyl 3-hydroxylation in type I procollagen. CYPB, a prolyl cis-trans isomerase, has been thought to drive the prolyl-containing peptide bonds to the trans configuration needed for triple helix formation. Here, we describe mutations in PPIB identified in cells from three individuals with OI. Cultured dermal fibroblasts from the most severely affected infant make some overmodified type I procollagen molecules. Pro?1(I) chains are slow to assemble into trimers, and abnormal procollagen molecules concentrate in the RER, and bind to protein disulfide isomerase (PDI) and prolyl 4-hydroxylase 1 (P4H1). These findings suggest that although CYPB plays a role in helix formation another effect is on folding of the C-terminal propeptide and trimer formation. The extent of procollagen accumulation and PDI/P4H1 binding differs among cells with mutations in PPIB, CRTAP and LEPRE1 with the greatest amount in PPIB-deficient cells and the least in LEPRE1-deficient cells. These findings suggest that prolyl cis-trans isomerase may be required to effectively fold the proline-rich regions of the C-terminal propeptide to allow pro? chain association and suggest an order of action for CRTAP, P3H1 and CYPB in procollagen biosynthesis and pathogenesis of OI.
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A novel 3-hydroxyproline (3Hyp)-rich motif marks the triple-helical C terminus of tendon type I collagen.
J. Biol. Chem.
PUBLISHED: 01-14-2011
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Because of its unique physical and chemical properties, rat tail tendon collagen has long been favored for crystallographic and biochemical studies of fibril structure. In studies of the distribution of 3-hydroxyproline in type I collagen of rat bone, skin, and tail tendon by mass spectrometry, the repeating sequences of Gly-Pro-Pro (GPP) triplets at the C terminus of ?1(I) and ?2(I) chains were shown to be heavily 3-hydroxylated in tendon but not in skin and bone. By isolating the tryptic peptides and subjecting them to Edman sequence analysis, the presence of repeating 3-hydroxyprolines in consecutive GPP triplets adjacent to 4-hydroxyproline was confirmed as a unique feature of the tendon collagen. A 1960s study by Piez et al. (Piez, K. A., Eigner, E. A., and Lewis, M. S. (1963) Biochemistry 2, 58-66) in which they compared the amino acid compositions of rat skin and tail tendon type I collagen chains indeed showed 3-4 residues of 3Hyp in tendon ?1(I) and ?2(I) chains but only one 3Hyp residue in skin ?1(I) and none in ?2(I). The present work therefore confirms this difference and localizes the additional 3Hyp to the GPP repeat at the C terminus of the triple-helix. We speculate on the significance in terms of a potential function in contributing to the unique assembly mechanism and molecular packing in tendon collagen fibrils and on mechanisms that could regulate 3-hydroxylation at this novel substrate site in a tissue-specific manner.
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Fibrochondrogenesis results from mutations in the COL11A1 type XI collagen gene.
Am. J. Hum. Genet.
PUBLISHED: 08-29-2010
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Fibrochondrogenesis is a severe, autosomal-recessive, short-limbed skeletal dysplasia. In a single case of fibrochondrogenesis, whole-genome SNP genotyping identified unknown ancestral consanguinity by detecting three autozygous regions. Because of the predominantly skeletal nature of the phenotype, the 389 genes localized to the autozygous intervals were prioritized for mutation analysis by correlation of their expression with known cartilage-selective genes via the UCLA Gene Expression Tool, UGET. The gene encoding the ?1 chain of type XI collagen (COL11A1) was the only cartilage-selective gene among the three candidate intervals. Sequence analysis of COL11A1 in two genetically independent fibrochondrogenesis cases demonstrated that each was a compound heterozygote for a loss-of-function mutation on one allele and a mutation predicting substitution for a conserved triple-helical glycine residue on the other. The parents who were carriers of missense mutations had myopia. Early-onset hearing loss was noted in both parents who carried a loss-of-function allele, suggesting COL11A1 as a locus for mild, dominantly inherited hearing loss. These findings identify COL11A1 as a locus for fibrochondrogenesis and indicate that there might be phenotypic manifestations among carriers.
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Mathematically modeling PCR: an asymptotic approximation with potential for optimization.
Math Biosci Eng
PUBLISHED: 05-14-2010
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A mathematical model for PCR (Polymerase Chain Reaction) is developed using the law of mass action and simplifying assumptions regarding the structure of the reactions. Differential equations are written from the chemical equations, preserving the detail of the complementary DNA single strand being extended one base pair at a time. The equations for the annealing stage are solved analytically. The method of multiple scales is used to approximate solutions for the extension stage, and a map is developed from the solutions to simulate PCR. The map recreates observed PCR well, and gives us the ability to optimize the PCR process. Our results suggest that dynamically optimizing the extension and annealing stages of individual samples may significantly reduce the total time for a PCR run. Moreover, we present a nearly optimal design that functions almost as well and does not depend on the specifics of a single reaction, and so would work for multi sample and multiplex applications.
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Type III collagen, a fibril network modifier in articular cartilage.
J. Biol. Chem.
PUBLISHED: 04-19-2010
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The collagen framework of hyaline cartilages, including articular cartilage, consists largely of type II collagen that matures from a cross-linked heteropolymeric fibril template of types II, IX, and XI collagens. In the articular cartilages of adult joints, type III collagen makes an appearance in varying amounts superimposed on the original collagen fibril network. In a study to understand better the structural role of type III collagen in cartilage, we find that type III collagen molecules with unprocessed N-propeptides are present in the extracellular matrix of adult human and bovine articular cartilages as covalently cross-linked polymers extensively cross-linked to type II collagen. Cross-link analyses revealed that telopeptides from both N and C termini of type III collagen were linked in the tissue to helical cross-linking sites in type II collagen. Reciprocally, telopeptides from type II collagen were recovered cross-linked to helical sites in type III collagen. Cross-linked peptides were also identified in which a trifunctional pyridinoline linked both an alpha1(II) and an alpha1(III) telopeptide to the alpha1(III) helix. This can only have arisen from a cross-link between three different collagen molecules, types II and III in register staggered by 4D from another type III molecule. Type III collagen is known to be prominent at sites of healing and repair in skin and other tissues. The present findings emphasize the role of type III collagen, which is synthesized in mature articular cartilage, as a covalent modifier that may add cohesion to a weakened, existing collagen type II fibril network as part of a chondrocyte healing response to matrix damage.
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Generalized connective tissue disease in Crtap-/- mouse.
PLoS ONE
PUBLISHED: 04-15-2010
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Mutations in CRTAP (coding for cartilage-associated protein), LEPRE1 (coding for prolyl 3-hydroxylase 1 [P3H1]) or PPIB (coding for Cyclophilin B [CYPB]) cause recessive forms of osteogenesis imperfecta and loss or decrease of type I collagen prolyl 3-hydroxylation. A comprehensive analysis of the phenotype of the Crtap-/- mice revealed multiple abnormalities of connective tissue, including in the lungs, kidneys, and skin, consistent with systemic dysregulation of collagen homeostasis within the extracellular matrix. Both Crtap-/- lung and kidney glomeruli showed increased cellular proliferation. Histologically, the lungs showed increased alveolar spacing, while the kidneys showed evidence of segmental glomerulosclerosis, with abnormal collagen deposition. The Crtap-/- skin had decreased mechanical integrity. In addition to the expected loss of proline 986 3-hydroxylation in alpha1(I) and alpha1(II) chains, there was also loss of 3Hyp at proline 986 in alpha2(V) chains. In contrast, at two of the known 3Hyp sites in alpha1(IV) chains from Crtap-/- kidneys there were normal levels of 3-hydroxylation. On a cellular level, loss of CRTAP in human OI fibroblasts led to a secondary loss of P3H1, and vice versa. These data suggest that both CRTAP and P3H1 are required to maintain a stable complex that 3-hydroxylates canonical proline sites within clade A (types I, II, and V) collagen chains. Loss of this activity leads to a multi-systemic connective tissue disease that affects bone, cartilage, lung, kidney, and skin.
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Maturation of collagen Ketoimine cross-links by an alternative mechanism to pyridinoline formation in cartilage.
J. Biol. Chem.
PUBLISHED: 04-02-2010
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The tensile strength of fibrillar collagens depends on stable intermolecular cross-links formed through the lysyl oxidase mechanism. Such cross-links based on hydroxylysine aldehydes are particularly important in cartilage, bone, and other skeletal tissues. In adult cartilages, the mature cross-linking structures are trivalent pyridinolines, which form spontaneously from the initial divalent ketoimines. We examined whether this was the complete story or whether other ketoimine maturation products also form, as the latter are known to disappear almost completely from mature tissues. Denatured, insoluble, bovine articular cartilage collagen was digested with trypsin, and cross-linked peptides were isolated by copper chelation chromatography, which selects for their histidine-containing sequence motifs. The results showed that in addition to the naturally fluorescent pyridinoline peptides, a second set of cross-linked peptides was recoverable at a high yield from mature articular cartilage. Sequencing and mass spectral analysis identified their origin from the same molecular sites as the initial ketoimine cross-links, but the latter peptides did not fluoresce and were nonreducible with NaBH(4). On the basis of their mass spectra, they were identical to their precursor ketoimine cross-linked peptides, but the cross-linking residue had an M+188 adduct. Considering the properties of an analogous adduct of identical added mass on a glycated lysine-containing peptide from type II collagen, we predicted that similar dihydroxyimidazolidine structures would form from their ketoimine groups by spontaneous oxidation and free arginine addition. We proposed the trivial name arginoline for the ketoimine cross-link derivative. Mature bovine articular cartilage contains about equimolar amounts of arginoline and hydroxylysyl pyridinoline based on peptide yields.
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Mutations in the gene encoding the RER protein FKBP65 cause autosomal-recessive osteogenesis imperfecta.
Am. J. Hum. Genet.
PUBLISHED: 02-01-2010
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Osteogenesis imperfecta is a clinically and genetically heterogeneous brittle bone disorder that results from defects in the synthesis, structure, or posttranslational modification of type I procollagen. Dominant forms of OI result from mutations in COL1A1 or COL1A2, which encode the chains of the type I procollagen heterotrimer. The mildest form of OI typically results from diminished synthesis of structurally normal type I procollagen, whereas moderately severe to lethal forms of OI usually result from structural defects in one of the type I procollagen chains. Recessively inherited OI, usually phenotypically severe, has recently been shown to result from defects in the prolyl-3-hydroxylase complex that lead to the absence of a single 3-hydroxyproline at residue 986 of the alpha1(I) triple helical domain. We studied a cohort of five consanguineous Turkish families, originating from the Black Sea region of Turkey, with moderately severe recessively inherited OI and identified a novel locus for OI on chromosome 17. In these families, and in a Mexican-American family, homozygosity for mutations in FKBP10, which encodes FKBP65, a chaperone that participates in type I procollagen folding, was identified. Further, we determined that FKBP10 mutations affect type I procollagen secretion. These findings identify a previously unrecognized mechanism in the pathogenesis of OI.
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Homozygosity for a missense mutation in SERPINH1, which encodes the collagen chaperone protein HSP47, results in severe recessive osteogenesis imperfecta.
Am. J. Hum. Genet.
PUBLISHED: 01-23-2010
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Osteogenesis imperfecta (OI) is characterized by bone fragility and fractures that may be accompanied by bone deformity, dentinogenesis imperfecta, short stature, and shortened life span. About 90% of individuals with OI have dominant mutations in the type I collagen genes COL1A1 and COL1A2. Recessive forms of OI resulting from mutations in collagen-modifying enzymes and chaperones CRTAP, LEPRE1, PPIB, and FKBP10 have recently been identified. We have identified an autosomal-recessive missense mutation (c.233T>C, p.Leu78Pro) in SERPINH1, which encodes the collagen chaperone-like protein HSP47, that leads to a severe OI phenotype. The mutation results in degradation of the endoplasmic reticulum resident HSP47 via the proteasome. Type I procollagen accumulates in the Golgi of fibroblasts from the affected individual and a population of the secreted type I procollagen is protease sensitive. These findings suggest that HSP47 monitors the integrity of the triple helix of type I procollagen at the ER/cis-Golgi boundary and, when absent, the rate of transit from the ER to the Golgi is increased and helical structure is compromised. The normal 3-hydroxylation of the prolyl residue at position 986 of the triple helical domain of proalpha1(I) chains places the role of HSP47 downstream from the CRTAP/P3H1/CyPB complex that is involved in prolyl 3-hydroxylation. Identification of this mutation in SERPINH1 gives further insight into critical steps of the collagen biosynthetic pathway and the molecular pathogenesis of OI.
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Lack of cyclophilin B in osteogenesis imperfecta with normal collagen folding.
N. Engl. J. Med.
PUBLISHED: 01-20-2010
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Osteogenesis imperfecta is a heritable disorder that causes bone fragility. Mutations in type I collagen result in autosomal dominant osteogenesis imperfecta, whereas mutations in either of two components of the collagen prolyl 3-hydroxylation complex (cartilage-associated protein [CRTAP] and prolyl 3-hydroxylase 1 [P3H1]) cause autosomal recessive osteogenesis imperfecta with rhizomelia (shortening of proximal segments of upper and lower limbs) and delayed collagen folding. We identified two siblings who had recessive osteogenesis imperfecta without rhizomelia. They had a homozygous start-codon mutation in the peptidyl-prolyl isomerase B gene (PPIB), which results in a lack of cyclophilin B (CyPB), the third component of the complex. The probands collagen had normal collagen folding and normal prolyl 3-hydroxylation, suggesting that CyPB is not the exclusive peptidyl-prolyl cis-trans isomerase that catalyzes the rate-limiting step in collagen folding, as is currently thought.
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Location of 3-hydroxyproline residues in collagen types I, II, III, and V/XI implies a role in fibril supramolecular assembly.
J. Biol. Chem.
PUBLISHED: 11-23-2009
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Collagen triple helices are stabilized by 4-hydroxyproline residues. No function is known for the much less common 3-hydroxyproline (3Hyp), although genetic defects inhibiting its formation cause recessive osteogenesis imperfecta. To help understand the pathogenesis, we used mass spectrometry to identify the sites and local sequence motifs of 3Hyp residues in fibril-forming collagens from normal human and bovine tissues. The results confirm a single, essentially fully occupied 3Hyp site (A1) at Pro(986) in A-clade chains alpha1(I), alpha1(II), and alpha2(V). Two partially modified sites (A2 and A3) were found at Pro(944) in alpha1(II) and alpha2(V) and Pro(707) in alpha2(I) and alpha2(V), which differed from A1 in sequence motif. Significantly, the distance between sites 2 and 3, 237 residues, is close to the collagen D-period (234 residues). A search for additional D-periodic 3Hyp sites revealed a fourth site (A4) at Pro(470) in alpha2(V), 237 residues N-terminal to site 3. In contrast, human and bovine type III collagen contained no 3Hyp at any site, despite a candidate proline residue and recognizable A1 sequence motif. A conserved histidine in mammalian alpha1(III) at A1 may have prevented 3-hydroxylation because this site in chicken type III was fully hydroxylated, and tyrosine replaced histidine. All three B-clade type V/XI collagen chains revealed the same three sites of 3Hyp but at different loci and sequence contexts from those in A-clade collagen chains. Two of these B-clade sites were spaced apart by 231 residues. From these and other observations we propose a fundamental role for 3Hyp residues in the ordered self-assembly of collagen supramolecular structures.
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In Saccharomyces cerevisiae, yKu and subtelomeric core X sequences repress homologous recombination near telomeres as part of the same pathway.
Genetics
PUBLISHED: 08-03-2009
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Unlike in meiosis where recombination near telomeres is repressed, subtelomeric regions appear to recombine with each other frequently in vegetative cells with no detrimental consequences. To test whether or not such recombination is prevented in the core of chromosomes for maintenance of genome stability, we measured allelic homologous recombination (HR) along chromosome arms and between different ectopic locations. We found that there is an increase of recombination at telomeres in wild-type cells compared with sequences at proximal subtelomeric and interstitial regions of the genome. We also screened for mutations that result in an increase in HR between a telomeric sequence and a more internal sequence, which normally exhibit very low rates of HR. YKU80 was hit most frequently in our screen, and we show that the yKu heterodimer specifically represses HR in the vicinity of telomeres. This repression of HR is not explained solely by the role of yKu in maintaining telomere length, silencing, or tethering to the nuclear periphery. Analysis of mutant strains harboring deleted core X sequences revealed a role for this subtelomeric element in preventing telomeric recombination. Furthermore, core X bestowed this protection as part of the same pathway as yKu. Our findings implicate a role for both yKu and core X in stabilizing the genome against recombination events involving telomeric sequences.
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Differences in chain usage and cross-linking specificities of cartilage type V/XI collagen isoforms with age and tissue.
J. Biol. Chem.
PUBLISHED: 05-12-2009
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Collagen type V/XI is a minor but essential component of collagen fibrils in vertebrates. We here report on age- and tissue-related variations in isoform usage in cartilages. With maturation of articular cartilage, the alpha1(V) chain progressively replaced the alpha2(XI) chain. A mix of the molecular isoforms, alpha1(XI)alpha1(V)alpha3(XI) and alpha1(XI)alpha2(XI)alpha3(XI), best explained this finding. A prominence of alpha1(V) chains is therefore characteristic and a potential biomarker of mature mammalian articular cartilage. Analysis of cross-linked peptides showed that the alpha1(V) chains were primarily cross-linked to alpha1(XI) chains in the tissue and hence an integral component of the V/XI polymer. From nucleus pulposus of the intervertebral disc (in which the bulk collagen monomer is type II as in articular cartilage), type V/XI collagen consisted of a mix of five genetically distinct chains, alpha1(XI), alpha2(XI), alpha3(XI), alpha1(V), and alpha2(V). These presumably were derived from several different molecular isoforms, including alpha1(XI)alpha2(XI)alpha3(XI), (alpha1(XI))(2)alpha2(V), and others. Meniscal fibrocartilage shows yet another V/XI phenotype. The findings support and extend the concept that the clade B subfamily of COL5 and COL11 gene products should be considered members of the same collagen subfamily, from which, in combination with clade A gene products (COL2A1 or COL5A2), a range of molecular isoforms has evolved into tissue-dependent usage. We propose an evolving role for collagen V/XI isoforms as an adaptable polymeric template of fibril macro-architecture.
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Microevolutionary analysis of Clostridium difficile genomes to investigate transmission.
Genome Biol.
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BACKGROUND: The control of Clostridium difficile infection is a major international healthcare priority, hindered by a limited understanding of transmission epidemiology for these bacteria. However, transmission studies of bacterial pathogens are rapidly being transformed by the advent of next generation sequencing. RESULTS: Here we sequence whole C. difficile genomes from 486 cases arising over four years in Oxfordshire. We show that we can estimate the times back to common ancestors of bacterial lineages with sufficient resolution to distinguish whether direct transmission is plausible or not. Time depths were inferred using a within-host evolutionary rate that we estimated at 1.4 mutations per genome per year based on serially isolated genomes. The subset of plausible transmissions was found to be highly associated with pairs of patients sharing time and space in hospital. Conversely, the large majority of pairs of genomes matched by conventional typing and isolated from patients within a month of each other were too distantly related to be direct transmissions. CONCLUSIONS: Our results confirm that nosocomial transmission between symptomatic C. difficile cases contributes far less to current rates of infection than has been widely assumed, which clarifies the importance of future research into other transmission routes, such as from asymptomatic carriers. With the costs of DNA sequencing rapidly falling and its use becoming more and more widespread, genomics will revolutionize our understanding of the transmission of bacterial pathogens.
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Recombinational switching of the Clostridium difficile S-layer and a novel glycosylation gene cluster revealed by large-scale whole-genome sequencing.
J. Infect. Dis.
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Clostridium difficile is a major cause of nosocomial diarrhea, with 30-day mortality reaching 30%. The cell surface comprises a paracrystalline proteinaceous S-layer encoded by the slpA gene within the cell wall protein (cwp) gene cluster. Our purpose was to understand the diversity and evolution of slpA and nearby genes also encoding immunodominant cell surface antigens.
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Whole-genome sequencing to delineate Mycobacterium tuberculosis outbreaks: a retrospective observational study.
Lancet Infect Dis
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Tuberculosis incidence in the UK has risen in the past decade. Disease control depends on epidemiological data, which can be difficult to obtain. Whole-genome sequencing can detect microevolution within Mycobacterium tuberculosis strains. We aimed to estimate the genetic diversity of related M tuberculosis strains in the UK Midlands and to investigate how this measurement might be used to investigate community outbreaks.
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Mutations in FKBP10, which result in Bruck syndrome and recessive forms of osteogenesis imperfecta, inhibit the hydroxylation of telopeptide lysines in bone collagen.
Hum. Mol. Genet.
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Although biallelic mutations in non-collagen genes account for <10% of individuals with osteogenesis imperfecta, the characterization of these genes has identified new pathways and potential interventions that could benefit even those with mutations in type I collagen genes. We identified mutations in FKBP10, which encodes the 65 kDa prolyl cis-trans isomerase, FKBP65, in 38 members of 21 families with OI. These include 10 families from the Samoan Islands who share a founder mutation. Of the mutations, three are missense; the remainder either introduce premature termination codons or create frameshifts both of which result in mRNA instability. In four families missense mutations result in loss of most of the protein. The clinical effects of these mutations are short stature, a high incidence of joint contractures at birth and progressive scoliosis and fractures, but there is remarkable variability in phenotype even within families. The loss of the activity of FKBP65 has several effects: type I procollagen secretion is slightly delayed, the stabilization of the intact trimer is incomplete and there is diminished hydroxylation of the telopeptide lysyl residues involved in intermolecular cross-link formation in bone. The phenotype overlaps with that seen with mutations in PLOD2 (Bruck syndrome II), which encodes LH2, the enzyme that hydroxylates the telopeptide lysyl residues. These findings define a set of genes, FKBP10, PLOD2 and SERPINH1, that act during procollagen maturation to contribute to molecular stability and post-translational modification of type I procollagen, without which bone mass and quality are abnormal and fractures and contractures result.
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Predictors of first recurrence of Clostridium difficile infection: implications for initial management.
Clin. Infect. Dis.
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Symptomatic recurrence of Clostridium difficile infection (CDI) occurs in approximately 20% of patients and is challenging to treat. Identifying those at high risk could allow targeted initial management and improve outcomes. Adult toxin enzyme immunoassay-positive CDI cases in a population of approximately 600,000 persons from September 2006 through December 2010 were combined with epidemiological/clinical data. The cumulative incidence of recurrence ? 14 days after the diagnosis and/or onset of first-ever CDI was estimated, treating death without recurrence as a competing risk, and predictors were identified from cause-specific proportional hazards regression models. A total of 1678 adults alive 14 days after their first CDI were included; median age was 77 years, and 1191 (78%) were inpatients. Of these, 363 (22%) experienced a recurrence ? 14 days after their first CDI, and 594 (35%) died without recurrence through March 2011. Recurrence risk was independently and significantly higher among patients admitted as emergencies, with previous gastrointestinal ward admission(s), last discharged 4-12 weeks before first diagnosis, and with CDI diagnosed at admission. Recurrence risk also increased with increasing age, previous total hours admitted, and C-reactive protein level at first CDI (all P < .05). The 4-month recurrence risk increased by approximately 5% (absolute) for every 1-point increase in a risk score based on these factors. Risk factors, including increasing age, initial disease severity, and hospital exposure, predict CDI recurrence and identify patients likely to benefit from enhanced initial CDI treatment.
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Absence of FKBP10 in recessive type XI osteogenesis imperfecta leads to diminished collagen cross-linking and reduced collagen deposition in extracellular matrix.
Hum. Mutat.
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Recessive osteogenesis imperfecta (OI) is caused by defects in genes whose products interact with type I collagen for modification and/or folding. We identified a Palestinian pedigree with moderate and lethal forms of recessive OI caused by mutations in FKBP10 or PPIB, which encode endoplasmic reticulum resident chaperone/isomerases FKBP65 and CyPB, respectively. In one pedigree branch, both parents carry a deletion in PPIB (c.563_566delACAG), causing lethal type IX OI in their two children. In another branch, a child with moderate type XI OI has a homozygous FKBP10 mutation (c.1271_1272delCCinsA). Proband FKBP10 transcripts are 4% of control and FKBP65 protein is absent from proband cells. Proband collagen electrophoresis reveals slight band broadening, compatible with ?10% over-modification. Normal chain incorporation, helix folding, and collagen T(m) support a minimal general collagen chaperone role for FKBP65. However, there is a dramatic decrease in collagen deposited in culture despite normal collagen secretion. Mass spectrometry reveals absence of hydroxylation of the collagen telopeptide lysine involved in cross-linking, suggesting that FKBP65 is required for lysyl hydroxylase activity or access to type I collagen telopeptide lysines, perhaps through its function as a peptidylprolyl isomerase. Proband collagen to organics ratio in matrix is approximately 30% of normal in Raman spectra. Immunofluorescence shows sparse, disorganized collagen fibrils in proband matrix.
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A pilot study of rapid benchtop sequencing of Staphylococcus aureus and Clostridium difficile for outbreak detection and surveillance.
BMJ Open
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To investigate the prospects of newly available benchtop sequencers to provide rapid whole-genome data in routine clinical practice. Next-generation sequencing has the potential to resolve uncertainties surrounding the route and timing of person-to-person transmission of healthcare-associated infection, which has been a major impediment to optimal management.
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A novel mutation in LEPRE1 that eliminates only the KDEL ER- retrieval sequence causes non-lethal osteogenesis imperfecta.
PLoS ONE
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Prolyl 3-hydroxylase 1 (P3H1), encoded by the LEPRE1 gene, forms a molecular complex with cartilage-associated protein (CRTAP) and cyclophilin B (encoded by PPIB) in the endoplasmic reticulum (ER). This complex is responsible for one step in collagen post-translational modification, the prolyl 3-hydroxylation of specific proline residues, specifically ?1(I) Pro986. P3H1 provides the enzymatic activity of the complex and has a Lys-Asp-Glu-Leu (KDEL) ER-retrieval sequence at the carboxyl terminus. Loss of function mutations in LEPRE1 lead to the Pro986 residue remaining unmodified and lead to slow folding and excessive helical post-translational modification of type I collagen, which is seen in both dominant and recessive osteogenesis imperfecta (OI). Here, we present the case of siblings with non-lethal OI due to novel compound heterozygous mutations in LEPRE1 (c.484delG and c.2155dupC). The results of RNA analysis and real-time PCR suggest that mRNA with c.2155dupC escapes from nonsense-mediated RNA decay. Without the KDEL ER- retrieval sequence, the product of the c.2155dupC variant cannot be retained in the ER. This is the first report of a mutation in LEPRE1 that eliminates only the KDEL ER-retrieval sequence, whereas other functional domains remain intact. Our study shows, for the first time, that the KDEL ER- retrieval sequence is essential for P3H1 functionality and that a defect in KDEL is sufficient for disease onset.
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Peptidyl 3-hydroxyproline binding properties of type I collagen suggest a function in fibril supramolecular assembly.
Biochemistry
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Proline residues in collagens are extensively hydroxylated post-translationally. A rare form of this modification, (3S,2S)-l-hydroxyproline (3Hyp), remains without a clear function. Disruption of the enzyme complex responsible for prolyl 3-hydroxylation results in severe forms of recessive osteogenesis imperfecta (OI). These OI types exhibit a loss of or reduction in the level of 3-hydroxylation at two proline residues, ?1(I) Pro986 and ?2(I) Pro707. Whether the resulting brittle bone phenotype is caused by the lack of the 3-hydroxyl addition or by another function of the enzyme complex is unknown. We have speculated that the most efficient mechanism for explaining the chemistry of collagen intermolecular cross-linking is for pairs of collagen molecules in register to be the subunit that assembles into fibrils. In this concept, the exposed hydroxyls from 3Hyp are positioned within mutually interactive binding motifs on adjacent collagen molecules that contribute through hydrogen bonding to the process of fibril supramolecular assembly. Here we report observations on the physical binding properties of 3Hyp in collagen chains from experiments designed to explore the potential for interaction using synthetic collagen-like peptides containing 3Hyp. Evidence of self-association was observed between a synthetic peptide containing 3Hyp and the CB6 domain of the ?1(I) chain, which contains the single fully 3-hydroxylated proline. Using collagen from a case of severe recessive OI with a CRTAP defect, in which Pro986 was minimally 3-hydroxylated, such binding was not observed. Further study of the role of 3Hyp in supramolecular assembly is warranted for understanding the evolution of tissue-specific variations in collagen fibril organization.
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Characterisation of Clostridium difficile hospital ward-based transmission using extensive epidemiological data and molecular typing.
PLoS Med.
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Clostridium difficile infection (CDI) is a leading cause of antibiotic-associated diarrhoea and is endemic in hospitals, hindering the identification of sources and routes of transmission based on shared time and space alone. This may compromise rational control despite costly prevention strategies. This study aimed to investigate ward-based transmission of C. difficile, by subdividing outbreaks into distinct lineages defined by multi-locus sequence typing (MLST).
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Evolutionary History of the Clostridium difficile Pathogenicity Locus.
Genome Biol Evol
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The symptoms of Clostridium difficile infection are caused by toxins expressed from its 19kb pathogenicity locus (PaLoc). Stable integration of the PaLoc is suggested by its single chromosomal location and the clade-specificity of its different genetic variants. However, the PaLoc is variably present, even among closely related strains, and thus resembles a mobile genetic element. Our aim was to explain these apparently conflicting observations by reconstructing the evolutionary history of the PaLoc. Phylogenetic analyses and annotation of the regions spanning the PaLoc were performed using C. difficile population-representative genomes chosen from a collection of 1,693 toxigenic (PaLoc present) and non-toxigenic (PaLoc absent) isolates. Comparison of the core genome and PaLoc phylogenies demonstrated an eventful evolutionary history, with distinct PaLoc variants acquired clade-specifically after divergence. In particular, our data suggest a relatively recent PaLoc acquisition in clade 4. Exchanges and losses of the PaLoc DNA have also occurred, via long homologous recombination events involving flanking chromosomal sequences. The most recent loss event occurred ~30 years ago within a clade 1 genotype. The genetic organisation of the clade 3 PaLoc was unique in containing a stably integrated novel transposon (designated Tn6218), variants of which were found at multiple chromosomal locations. Tn6218 elements were Tn916-related, but non-conjugative, and occasionally contained genes conferring resistance to clinically relevant antibiotics. The evolutionary histories of two contrasting, but clinically important genetic elements were thus characterised: the PaLoc, mobilised rarely via homologous recombination, and Tn6218, mobilised frequently through transposition.
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Sc65 is a novel endoplasmic reticulum protein that regulates bone mass homeostasis.
J. Bone Miner. Res.
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Members of the Leprecan family of proteins include enzymes, prolyl 3-hydroxylase 1 (P3h1), P3h2 and P3h3, and non-enzymatic proteins, Crtap and Sc65. Mutations in CRTAP and LEPRE1 (encoding P3H1) have been associated with human disease such as recessive osteogenesis imperfecta, however, the function of Sc65 which is closely related and highly homologous to Crtap is unknown. Sc65 has been described as a synaptonemal complex protein, a nucleolar protein, and a cytoplasmic adapter protein. In light of its high sequence similarity with Crtap, an endoplasmic reticulum (ER)-associated protein, and the importance of post-translational modifications such as collagen prolyl 3-hydroxylation in bone metabolism, we hypothesized that Sc65 was an ER-resident protein that would have an important role in bone homeostasis. In this study, we demonstrate that Sc65 is a previously unrecognized ER protein, and that it does not localize in the nucleus of somatic cells. Moreover, Sc65 is expressed and functional during skeletal development since loss of Sc65 results in a progressive osteopenia that affects both trabecular and cortical bone. Bone loss is due to increased bone resorption mediated by a non-cell autonomous effect on osteoclasts. Therefore, Sc65, like its related family member Crtap, is an important modulator of bone homeostasis, acting as a negative regulator of osteoclastogenesis.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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