Search

The question of how Zika virus (ZIKV) changed from a seemingly mild virus to a human pathogen capable of microcephaly and sexual transmission remains unanswered. The unexpected emergence of ZIKV's pathogenicity and capacity for sexual transmission may be due to genetic changes, and future changes in phenotype may continue to occur as the virus expands its geographic range. Alternatively, the sheer size of the 2015–16 epidemic may have brought attention to a pre-existing virulent ZIKV phenotype in a highly susceptible population. Thus, it is important to identify patterns of genetic change that may yield a better understanding of ZIKV emergence and evolution. However, because ZIKV has an RNA genome and a polymerase incapable of proofreading, it undergoes rapid mutation which makes it difficult to identify combinations of mutations associated with viral emergence. As next generation sequencing technology has allowed whole genome consensus and variant sequence data to be generated for numerous virus samples, the task of analyzing these genomes for patterns of mutation has become more complex. However, understanding which combinations of mutations spread widely and become established in new geographic regions versus those that disappear relatively quickly is essential for defining the trajectory of an ongoing epidemic. In this study, multiscale analysis of the wealth of genomic data generated over the course of the epidemic combined with in vivo laboratory data allowed trends in mutations and outbreak trajectory to be assessed. Mutations were detected throughout the genome via deep sequencing, and many variants appeared in multiple samples and in some cases become consensus. Similarly, amino acids that were previously consensus in pre-outbreak samples were detected as low frequency variants in epidemic strains. Protein structural models indicate that most of the mutations associated with the epidemic transmission occur on the exposed surface of viral proteins. At the macroscale level, consensus data was organized into large and interactive databases to allow the spread of individual mutations and combinations of mutations to be visualized and assessed for temporal and geographical patterns. Thus, the use of multiscale modeling for identifying mutations or combinations of mutations that impact epidemic transmission and phenotypic impact can aid the formation of hypotheses which can then be tested using reverse genetics. [ABSTRACT FROM AUTHOR]

Loss of Sostdc1, a growth factor paralogous to Sost, causes the formation of ectopic incisors, fused molars, abnormal hair follicles, and resistance to kidney disease. Sostdc1 is expressed in the periosteum, a source of osteoblasts, fibroblasts and mesenchymal progenitor cells, which are critically important for fracture repair. Here, we investigated the role of Sostdc1 in bone metabolism and fracture repair. Mice lacking Sostdc1 ( Sostdc1 − / − ) had a low bone mass phenotype associated with loss of trabecular bone in both lumbar vertebrae and in the appendicular skeleton. In contrast, Sostdc1 − / − cortical bone measurements revealed larger bones with higher BMD, suggesting that Sostdc1 exerts differential effects on cortical and trabecular bone. Mid-diaphyseal femoral fractures induced in Sostdc1 − / − mice showed that the periosteal population normally positive for Sostdc1 rapidly expands during periosteal thickening and these cells migrate into the fracture callus at 3 days post fracture. Quantitative analysis of mesenchymal stem cell (MSC) and osteoblast populations determined that MSCs express Sostdc1 , and that Sostdc1 − / − 5 day calluses harbor > 2-fold more MSCs than fractured wildtype controls. Histologically a fraction of Sostdc1 -positive cells also expressed nestin and α-smooth muscle actin, suggesting that Sostdc1 marks a population of osteochondral progenitor cells that actively participate in callus formation and bone repair. Elevated numbers of MSCs in D5 calluses resulted in a larger, more vascularized cartilage callus at day 7, and a more rapid turnover of cartilage with significantly more remodeled bone and a thicker cortical shell at 21 days post fracture. These data support accelerated or enhanced bone formation/remodeling of the callus in Sostdc1 − / − mice, suggesting that Sostdc1 may promote and maintain mesenchymal stem cell quiescence in the periosteum. [ABSTRACT FROM AUTHOR]

Abstract: WNT signaling is critical in most aspects of skeletal development and homeostasis, and antagonists of WNT signaling are emerging as key regulatory proteins with great promise as therapeutic agents for bone disorders. Here we show that Sost and its paralog Sostdc1 emerged through ancestral genome duplication and their expression patterns have diverged to delineate non-overlapping domains in most organ systems including musculoskeletal, cardiovascular, nervous, digestive, reproductive and respiratory. In the developing limb, Sost and Sostdc1 display dynamic expression patterns with Sost being restricted to the distal ectoderm and Sostdc1 to the proximal ectoderm and the mesenchyme. While Sostdc1 −/− mice lack any obvious limb or skeletal defects, Sost −/− mice recapitulate the hand defects described for Sclerosteosis patients. However, elevated WNT signaling in Sost −/− ; Sostdc1 −/− mice causes misregulation of SHH signaling, ectopic activation of Sox9 in the digit 1 field and preaxial polydactyly in a Gli1- and Gli3-dependent manner. In addition, we show that the syndactyly documented in Sclerosteosis is present in both Sost −/− and Sost −/− ; Sostdc1 −/− mice, and is driven by misregulation of Fgf8 in the AER, a region lacking Sost and Sostdc1 expression. This study highlights the complexity of WNT signaling in skeletal biology and disease and emphasizes how redundant mechanism and non-cell autonomous effects can synergize to unveil new intricate phenotypes caused by elevated WNT signaling. [Copyright &y& Elsevier]

The Wnt antagonist Sost has emerged as a key regulator of bone homeostasis through the modulation of Lrp4/5/6 Wnt coreceptors. In humans, lack of Sclerostin causes sclerosteosis and van Buchem (VB) disease, two generalized skeletal hyperostosis disorders that result from hyperactive Wnt signaling. Unlike sclerosteosis, VB patients lack SOST coding mutations but carry a homozygous 52 kb noncoding deletion that is essential for the transcriptional activation of SOST in bone. We recently identified a putative bone enhancer, ECR5, in the VB deletion region, and showed that the transcriptional activity of ECR5 is controlled by Mef2C transcription factor in vitro. Here we report that mice lacking ECR5 or Mef2C through Coll-Cre osteoblast/osteocyte-specific ablation result in high bone mass (HBM) due to elevated bone formation rates. We conclude that the absence of the Sost-specific long-range regulatory element ECRS causes VB disease in rodents, and that Mef2C is the main transcription factor responsible for ECR5-dependent Sost transcriptional activation in the adult skeleton. [ABSTRACT FROM AUTHOR]

Detailed information about stage-specific changes in gene expression is crucial for understanding the gene regulatory networks underlying development and the various signal transduction pathways contributing to morphogenesis. Here we describe the global gene expression dynamics during early murine limb development, when cartilage, tendons, muscle, joints, vasculature and nerves are specified and the musculoskeletal system of limbs is established. We used whole-genome microarrays to identify genes with differential expression at 5 stages of limb development (E9.5 to 13.5), during fore- and hind-limb patterning. We found that the onset of limb formation is characterized by an up-regulation of transcription factors, which is followed by a massive activation of genes during E10.5 and E11.5 which levels off at later time points. Among the 3520 genes identified as significantly up-regulated in the limb, we find ∼30% to be novel, dramatically expanding the repertoire of candidate genes likely to function in the limb. Hierarchical and stage-specific clustering identified expression profiles that are likely to correlate with functional programs during limb development and further characterization of these transcripts will provide new insights into specific tissue patterning processes. Here, we provide for the first time a comprehensive analysis of developmentally regulated genes during murine limb development, and provide some novel insights into the expression dynamics governing limb morphogenesis. [ABSTRACT FROM AUTHOR]

Abstract: SOST is a negative regulator of bone formation, and mutations in human SOST are responsible for sclerosteosis. In addition to high bone mass, sclerosteosis patients occasionally display hand defects, suggesting that SOST may function embryonically. Here we report that overexpression of SOST leads to loss of posterior structures of the zeugopod and autopod by perturbing anterior–posterior and proximal–distal signaling centers in the developing limb. Mutant mice that overexpress SOST in combination with Grem1 and Lrp6 mutations display more severe limb defects than single mutants alone, while Sost − / − significantly rescues the Lrp6 − / − skeletal phenotype, signifying that SOST gain-of-function impairs limb patterning by inhibiting the WNT signaling through LRP5/6. [Copyright &y& Elsevier]

The 2014–2016 Zika virus (ZIKV) epidemic in the Americas resulted in large deposits of next-generation sequencing data from clinical samples. This resource was mined to identify emerging mutations and trends in mutations as the outbreak progressed over time. Information on transmission dynamics, prevalence, and persistence of intra-host mutants, and the position of a mutation on a protein were then used to prioritize 544 reported mutations based on their ability to impact ZIKV phenotype. Using this criteria, six mutants (representing naturally occurring mutations) were generated as synthetic infectious clones using a 2015 Puerto Rican epidemic strain PRVABC59 as the parental backbone. The phenotypes of these naturally occurring variants were examined using both cell culture and murine model systems. Mutants had distinct phenotypes, including changes in replication rate, embryo death, and decreased head size. In particular, a NS2B mutant previously detected during in vivo studies in rhesus macaques was found to cause lethal infections in adult mice, abortions in pregnant females, and increased viral genome copies in both brain tissue and blood of female mice. Additionally, mutants with changes in the region of NS3 that interfaces with NS5 during replication displayed reduced replication in the blood of adult mice. This analytical pathway, integrating both bioinformatic and wet lab experiments, provides a foundation for understanding how naturally occurring single mutations affect disease outcome and can be used to predict the of severity of future ZIKV outbreaks. To determine if naturally occurring individual mutations in the Zika virus epidemic genotype affect viral virulence or replication rate in vitro or in vivo, we generated an infectious clone representing the epidemic genotype of stain Puerto Rico, 2015. Using this clone, six mutants were created by changing nucleotides in the genome to cause one to two amino acid substitutions in the encoded proteins. The six mutants we generated represent mutations that differentiated the early epidemic genotype from genotypes that were either ancestral or that occurred later in the epidemic. We assayed each mutant for changes in growth rate, and for virulence in adult mice and pregnant mice. Three of the mutants caused catastrophic embryo effects including increased embryonic death or significant decrease in head diameter. Three other mutants that had mutations in a genome region associated with replication resulted in changes in in vitro and in vivo replication rates. These results illustrate the potential impact of individual mutations in viral phenotype. [ABSTRACT FROM AUTHOR]

Highlights • Naphthalene DNA adducts were detected in primate and mouse airways. • Female mouse (4.7-fold) and primate (2.1-fold) airway adducts exceeded male levels. • Few naphthalene DNA adducts were detected in rat airway or nasal epithelium. • Naphthoquinone DNA adducts were 70 to 80-fold higher than naphthalene DNA adducts. Abstract Naphthalene (NA) is a ubiquitous environmental pollutant and possible human carcinogen that forms tumors in rodents with tissue/regional and species selectivity. This study seeks to determine whether NA is able to directly adduct DNA in an ex vivo culture system. Metabolically active lung tissue was isolated and incubated in explant culture with carbon-14 labeled NA (0, 25, 250 μM) or 1,2-naphthoquinone (NQ), followed by AMS analyses of metabolite binding to DNA. Despite relatively low metabolic bioactivation in the primate airway, dose-dependent NA-DNA adduct formation was detected. More airway adducts were detected in female mice (4.7-fold) and primates (2.1-fold) than in males of the same species. Few adducts were detected in rat airway or nasal epithelium. NQ, which is a metabolic product of NA, proved to be even more potent, with levels of adduct formation 70–80-fold higher than seen when tissues were incubated with the parent compound NA. This is the first study to demonstrate NA-DNA adduct formation at a site of carcinogenesis, the mouse lung. Adducts were also detected in non-human primate lung and with a NQ metabolite of NA. Taken together, this suggests that NA may contribute to in vivo carcinogenesis through a genotoxic mechanism. [ABSTRACT FROM AUTHOR]

Abstract Naphthalene (NA) is a respiratory toxicant and possible human carcinogen. NA is a ubiquitous combustion product and significant component of jet fuel. The National Toxicology Program found that NA forms tumors in two species, in rats (nose) and mice (lung). However, it has been argued that NA does not pose a cancer risk to humans because NA is bioactivated by cytochrome P450 monooxygenase enzymes that have very high efficiency in the lung tissue of rodents but low efficiency in the lung tissue of humans. It is thought that NA carcinogenesis in rodents is related to repeated cycles of lung epithelial injury and repair, an indirect mechanism. Repeated in vivo exposure to NA leads to development of tolerance, with the emergence of cells more resistant to NA insult. We tested the hypothesis that tolerance involves reduced susceptibility to the formation of NA-DNA adducts. NA-DNA adduct formation in tolerant mice was examined in individual, metabolically-active mouse airways exposed ex vivo to 250 μM 14C-NA. Ex vivo dosing was used since it had been done previously and the act of creating a radioactive aerosol of a potential carcinogen posed too many safety and regulatory obstacles. Following extensive rinsing to remove unbound 14C-NA, DNA was extracted and 14C-NA-DNA adducts were quantified by accelerator mass spectrometry (AMS). The tolerant mice appeared to have slightly lower NA-DNA adduct levels than non-tolerant controls, but intra-group variations were large and the difference was statistically insignificant. It appears the tolerance may be more related to other mechanisms, such as NA-protein interactions in the airway, than DNA-adduct formation. [ABSTRACT FROM AUTHOR]

ABSTRACT: Osteophytes are a typical radiographic finding during osteoarthritis (OA), but the mechanisms leading to their formation are not well known. Comparatively, fracture calluses have been studied extensively; therefore, drawing comparisons between osteophytes and fracture calluses may lead to a deeper understanding of osteophyte formation. In this study, we compared the time courses of osteophyte and fracture callus formation, and investigated mechanisms contributing to development of these structure. Additionally, we investigated the effect of mechanical unloading on the formation of both fracture calluses and osteophytes. Mice underwent either transverse femoral fracture or non‐invasive anterior cruciate ligament rupture. Fracture callus and osteophyte size and ossification were evaluated after 3, 5, 7, 14, 21, or 28 days. Additional mice were subjected to hindlimb unloading after injury for 3, 7, or 14 days. Protease activity and gene expression profiles after injury were evaluated after 3 or 7 days of normal ambulation or hindlimb unloading using in vivo fluorescence reflectance imaging (FRI) and quantitative PCR. We found that fracture callus and osteophyte growth achieved similar developmental milestones, but fracture calluses formed and ossified at earlier time points. Hindlimb unloading ultimately led to a threefold decrease in chondro/osteophyte area, and a twofold decrease in fracture callus area. Unloading was also associated with decreased inflammation and protease activity in injured limbs detected with FRI, particularly following ACL rupture. qPCR analysis revealed disparate cellular responses in fractured femurs and injured joints, suggesting that fracture calluses and osteophytes may form via different inflammatory, anabolic, and catabolic pathways. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:699–710, 2018. [ABSTRACT FROM AUTHOR]

Type 1 diabetes mellitus (T1DM) patients have osteopenia and impaired fracture healing due to decreased osteoblast activity. Further, no adequate treatments are currently available that can restore impaired healing in T1DM; hence a significant need exists to investigate new therapeutics for treatment of orthopedic complications. Sclerostin (SOST), a WNT antagonist, negatively regulates bone formation, and SostAb is a potent bone anabolic agent. To determine whether SOST antibody (SostAb) treatment improves fracture healing in streptozotocin (STZ) induced T1DM mice, we administered SostAb twice weekly for up to 21 days post-fracture, and examined bone quality and callus outcomes at 21 days and 42 days post-fracture (11 and 14 weeks of age, respectively). Here we show that SostAb treatment improves bone parameters; these improvements persist after cessation of antibody treatment. Markers of osteoblast differentiation such as Runx2, collagen I, osteocalcin, and DMP1 were reduced, while an abundant number of SP7/osterix-positive early osteoblasts were observed on the bone surface of STZ calluses. These results suggest that STZ calluses have poor osteogenesis resulting from failure of osteoblasts to fully differentiate and produce mineralized matrix, which produces a less mineralized callus. SostAb treatment enhanced fracture healing in both normal and STZ groups, and in STZ + SostAb mice, also reversed the lower mineralization seen in S TZ calluses. Micro-CT analysis of calluses revealed improved bone parameters with SostAb treatment, and the mineralized bone was comparable to Controls . Additionally, we found sclerostin levels to be elevated in STZ mice and β-catenin activity to be reduced. Consistent with its function as a WNT antagonist, SostAb treatment enhanced β-catenin activity, but also increased the levels of SOST in the callus and in circulation. Our results indicate that SostAb treatment rescues the impaired osteogenesis seen in the STZ induced T1DM fracture model by facilitating osteoblast differentiation and mineralization of bone. [ABSTRACT FROM AUTHOR]

Inhibitors of Wnt signaling have been shown to be involved in prostate cancer (PC) metastasis; however the role of Sclerostin (Sost) has not yet been explored. Here we show that elevated Wnt signaling derived from Sost deficient osteoblasts promotes PC invasion, while rhSOST has an inhibitory effect. In contrast, rhDKK1 promotes PC elongation and filopodia formation, morphological changes characteristic of an invasive phenotype. Furthermore, rhDKK1 was found to activate canonical Wnt signaling in PC3 cells, suggesting that SOST and DKK1 have opposing roles on Wnt signaling in this context. Gene expression analysis of PC3 cells co-cultured with OBs exhibiting varying amounts of Wnt signaling identified CRIM1 as one of the transcripts upregulated under highly invasive conditions. We found CRIM1 overexpression to also promote cell-invasion. These findings suggest that bone-derived Wnt signaling may enhance PC tropism by promoting CRIM1 expression and facilitating cancer cell invasion and adhesion to bone. We concluded that SOST and DKK1 have opposing effects on PC3 cell invasion and that bone-derived Wnt signaling positively contributes to the invasive phenotypes of PC3 cells by activating CRIM1 expression and facilitating PC-OB physical interaction. As such, we investigated the effects of high concentrations of SOST in vivo. We found that PC3-cells overexpressing SOST injected via the tail vein in NSG mice did not readily metastasize, and those injected intrafemorally had significantly reduced osteolysis, suggesting that targeting the molecular bone environment may influence bone metastatic prognosis in clinical settings. [ABSTRACT FROM AUTHOR]

Abstract: Wnt signaling is critical for skeletal development and homeostasis. Sclerostin (Sost) has emerged as a potent inhibitor of Wnt signaling and, thereby, bone formation. Thus, strategies to reduce sclerostin expression may be used to treat osteoporosis or non-union fractures. Transforming growth factor-beta (TGF-β) elicits various effects upon the skeleton both in vitro and in vivo depending on the duration and timing of administration. In vitro and in vivo studies demonstrate that TGF-β increases osteoprogenitor differentiation but decreases matrix mineralization of committed osteoblasts. Because sclerostin decreases matrix mineralization, this study aimed to examine whether TGF-β achieves such inhibitory effects via transcriptional modulation of Sost. Using the UMR106.01 mature osteoblast cell line, we demonstrated that TGF-βTGF-β1-β2-β3 and Activin A increase Sost transcript expression. Pharmacologic inhibition of Alk4/5/7 in vitro and in vivo decreased endogenous Sost expression, and siRNA against Alk4 and Alk5 demonstrated their requirement for endogenous Sost expression. TGF-β1 targeted the Sost bone enhancer ECR5 and did not affect the transcriptional activity of the endogenous Sost promoter. These results indicate that TGF-β1 controls Sost transcription in mature osteoblasts, suggesting that sclerostin may mediate the inhibitory effect of TGF-β upon osteoblast differentiation. [Copyright &y& Elsevier]

Mutations in distant regulatory elements can have a negative impact on human development and health, yet because of the difficulty of detecting these critical sequences, we predominantly focus on coding sequences for diagnostic purposes. We have undertaken a comparative sequence-based approach to characterize a large noncoding region deleted in patients affected by Van Buchem (VB) disease, a severe sclerosing bone dysplasia. Using BAC recombination and transgenesis, we characterized the expression of human sclerostin (SOST) from normal (SOSTwt) or Van Buchem (SOSTvbΔ) alleles. Only the SOSTwt allele faithfully expressed high levels of human SOST in the adult bone and had an impact on bone metabolism, consistent with the model that the VB noncoding deletion removes a SOST-specific regulatory element. By exploiting cross-species sequence comparisons with in vitro and in vivo enhancer assays, we were able to identify a candidate enhancer element that drives human SOST expression in osteoblast-like cell lines in vitro and in the skeletal anlage of the embryonic day 14.5 (E14.5) mouse embryo, and discovered a novel function for sclerostin during limb development. Our approach represents a framework for characterizing distant regulatory elements associated with abnormal human phenotypes. [ABSTRACT FROM AUTHOR]