Protein Interaction Mapping Identifies RBBP6 as a Negative Regulator of Ebola Virus Replication.

Ebola virus (EBOV) infection often results in fatal illness in humans, yet little is known about how EBOV usurps host pathways during infection. To address this, we used affinity tag-purification mass spectrometry (AP-MS) to generate an EBOV-host protein-protein interaction (PPI) map. We uncovered 194 high-confidence EBOV-human PPIs, including one between the viral transcription regulator VP30 and the host ubiquitin ligase RBBP6. Domain mapping identified a 23 amino acid region within RBBP6 that binds to VP30. A crystal structure of the VP30-RBBP6 peptide complex revealed that RBBP6 mimics the viral nucleoprotein (NP) binding to the same interface of VP30. Knockdown of endogenous RBBP6 stimulated viral transcription and increased EBOV replication, whereas overexpression of either RBBP6 or the peptide strongly inhibited both. These results demonstrate the therapeutic potential of biologics that target this interface and identify additional PPIs that may be leveraged for novel therapeutic strategies.

miRNA-mediated targeting of human cytomegalovirus reveals biological host and viral targets of IE2.

Human cytomegalovirus (HCMV) impacts more than one-half of the human population owing to its capacity to manipulate the cell and create latent reservoirs in the host. Despite an extensive understanding of HCMV biology during acute infection in fibroblasts, the molecular basis for latency in myeloid cells remains incomplete. This knowledge gap is due largely to the fact that the existing genetic systems require virus rescue in fibroblasts, precluding the study of genes that are essential during acute infection, yet likely play unique roles in myeloid cells or the establishment of latency. Here we present a solution to address this restriction. Through the exploitation of a hematopoietic-specific microRNA, we demonstrate a one-step recombineering approach that enables gene silencing only in cells associated with latency. As a proof of concept, here we describe a TB40/E variant that undergoes hematopoietic targeting of the Immediate Early-2 (IE2) gene to explore its function during infection of myeloid cells. While virus replication of the hematopoietic-targeted IE2 variant was unimpaired in fibroblasts, we observed a >100-fold increase in virus titers in myeloid cells. Virus replication in myeloid cells demonstrated that IE2 has a significant transcriptional footprint on both viral and host genes. These data implicate IE2 as an essential mediator of virus biology in myeloid cells and illustrate the utility of cell-specific microRNA-based targeting.

VP24-Karyopherin Alpha Binding Affinities Differ between Ebolavirus Species, Influencing Interferon Inhibition and VP24 Stability.

Zaire ebolavirus (EBOV), Bundibugyo ebolavirus (BDBV), and Reston ebolavirus (RESTV) belong to the same genus but exhibit different virulence properties. VP24 protein, a structural protein present in all family members, blocks interferon (IFN) signaling and likely contributes to virulence. Inhibition of IFN signaling by EBOV VP24 (eVP24) involves its interaction with the NPI-1 subfamily of karyopherin alpha (KPNA) nuclear transporters. Here, we evaluated eVP24, BDBV VP24 (bVP24), and RESTV VP24 (rVP24) interactions with three NPI-1 subfamily KPNAs (KPNA1, KPNA5, and KPNA6). Using purified proteins, we demonstrated that each VP24 binds to each of the three NPI-1 KPNAs. bVP24, however, exhibited approximately 10-fold-lower KPNA binding affinity than either eVP24 or rVP24. Cell-based assays also indicate that bVP24 exhibits decreased KPNA interaction, decreased suppression of IFN induced gene expression, and a decreased half-life in transfected cells compared to eVP24 or rVP24. Amino acid sequence alignments between bVP24 and eVP24 also identified residues within and surrounding the previously defined eVP24-KPNA5 binding interface that decrease eVP24-KPNA affinity or bVP24-KPNA affinity. VP24 mutations that lead to reduced KPNA binding affinity also decrease IFN inhibition and shorten VP24 half-lives. These data identify novel functional differences in VP24-KPNA interaction and reveal a novel impact of the VP24-KPNA interaction on VP24 stability. IMPORTANCE: The interaction of Ebola virus (EBOV) VP24 protein with host karyopherin alpha (KPNA) proteins blocks type I interferon (IFN) signaling, which is a central component of the host innate immune response to viral infection. Here, we quantitatively compared the interactions of VP24 proteins from EBOV and two members of the Ebolavirus genus, Bundibugyo virus (BDBV) and Reston virus (RESTV). The data reveal lower binding affinity of the BDBV VP24 (bVP24) for KPNAs and demonstrate that the interaction with KPNA modulates inhibition of IFN signaling and VP24 stability. The effect of KPNA interaction on VP24 stability is a novel functional consequence of this virus-host interaction, and the differences identified between viral species may contribute to differences in pathogenesis.

Innate Immune Responses of Bat and Human Cells to Filoviruses: Commonalities and Distinctions.

Marburg (MARV) and Ebola (EBOV) viruses are zoonotic pathogens that cause severe hemorrhagic fever in humans. The natural reservoir of MARV is the Egyptian rousette bat (Rousettus aegyptiacus); that of EBOV is unknown but believed to be another bat species. The Egyptian rousette develops subclinical productive infection with MARV but is refractory to EBOV. Interaction of filoviruses with hosts is greatly affected by the viral interferon (IFN)-inhibiting domains (IID). Our study was aimed at characterization of innate immune responses to filoviruses and the role of filovirus IID in bat and human cells. The study demonstrated that EBOV and MARV replicate to similar levels in all tested cell lines, indicating that permissiveness for EBOV at cell and organism levels do not necessarily correlate. Filoviruses, particularly MARV, induced a potent innate immune response in rousette cells, which was generally stronger than that in human cells. Both EBOV VP35 and VP24 IID were found to suppress the innate immune response in rousette cells, but only VP35 IID appeared to promote virus replication. Along with IFN-α and IFN-ß, IFN-γ was demonstrated to control filovirus infection in bat cells but not in human cells, suggesting host species specificity of the antiviral effect. The antiviral effects of bat IFNs appeared not to correlate with induction of IFN-stimulated genes 54 and 56, which were detected in human cells ectopically expressing bat IFN-α and IFN-ß. As bat IFN-γ induced the type I IFN pathway, its antiviral effect is likely to be partially induced via cross talk.IMPORTANCE Bats serve as reservoirs for multiple emerging viruses, including filoviruses, henipaviruses, lyssaviruses, and zoonotic coronaviruses. Although there is no evidence for symptomatic disease caused by either Marburg or Ebola viruses in bats, spillover of these viruses into human populations causes deadly outbreaks. The reason for the lack of symptomatic disease in bats infected with filoviruses remains unknown. The outcome of a virus-host interaction depends on the ability of the host immune system to suppress viral replication and the ability of a virus to counteract the host defenses. Our study is a comparative analysis of the host innate immune response to either MARV or EBOV infection in bat and human cells and the role of viral interferon-inhibiting domains in the host innate immune responses. The data are useful for understanding the interactions of filoviruses with natural and accidental hosts and for identification of factors that influence filovirus evolution.

Stability determinants of murine cytomegalovirus long noncoding RNA7.2.

Cytomegalovirus is a ubiquitous herpesvirus that persistently replicates in glandular epithelial tissue. Murine cytomegalovirus expresses a 7.2-kb-long noncoding RNA (RNA7.2) that is a determinant of viral persistence in the salivary gland. RNA7.2 is an extremely long-lived intron, yet the basis of its stability is unknown. We present data that localize key sequence determinants of RNA stability to the 3' end of RNA7.2 and suggest that stability is a result of sustained lariat conformation.

Molecular investigation of the 7.2 kb RNA of murine cytomegalovirus.

BACKGROUND: HCMV encodes a stable 5 kb RNA of unknown function that is conserved across cytomegalovirus species. In vivo studies of the MCMV orthologue, a 7.2 kb RNA, demonstrated that viruses that do not express the RNA fail to establish efficient persistent replication in the salivary glands of mice. To gain further insight into the function and properties of this conserved locus, we characterized the MCMV intron in finer detail. METHODS: We performed multiple analyses to evaluate transcript expression kinetics, identify transcript termini and promoter elements. The half-lives of intron locus RNAs were quantified by measuring RNA levels following actinomycin D treatment in a qRT-PCR-based assay. We also constructed a series of recombinant viruses to evaluate protein coding potential in the locus and test the role of putative promoter elements. These recombinant viruses were tested in both in vitro and in vivo assays. RESULTS: We show that the 7.2 kb RNA is expressed with late kinetics during productive infection of mouse fibroblasts. The termini of the precursor RNA that is processed to produce the intron were identified and we demonstrate that the m106 open reading frame, which resides on the spliced mRNA derived from precursor processing, can be translated during infection. Mapping the 5' end of the primary transcript revealed minimal promoter elements located upstream that contribute to transcript expression. Analysis of recombinant viruses with deletions in the putative promoter elements, however, revealed these elements exert only minor effects on intron expression and viral persistence in vivo. Low transcriptional output by the putative promoter element(s) is compensated by the long half-life of the 7.2 kb RNA of approximately 28.8 hours. Detailed analysis of viral spread prior to the establishment of persistence also showed that the intron is not likely required for efficient spread to the salivary gland, but rather enhances persistent replication in this tissue site. CONCLUSIONS: This data provides a comprehensive transcriptional analysis of the MCMV 7.2 kb intron locus. Our studies indicate that the 7.2 kb RNA is an extremely long-lived RNA, a feature which is likely to be important in its role promoting viral persistence in the salivary gland.

Marburg and Ebola Virus Infections Elicit a Complex, Muted Inflammatory State in Bats.

The Marburg and Ebola filoviruses cause a severe, often fatal, disease in humans and nonhuman primates but have only subclinical effects in bats, including Egyptian rousettes, which are a natural reservoir of Marburg virus. A fundamental question is why these viruses are highly pathogenic in humans but fail to cause disease in bats. To address this question, we infected one cohort of Egyptian rousette bats with Marburg virus and another cohort with Ebola virus and harvested multiple tissues for mRNA expression analysis. While virus transcripts were found primarily in the liver, principal component analysis (PCA) revealed coordinated changes across multiple tissues. Gene signatures in kidney and liver pointed at induction of vasodilation, reduction in coagulation, and changes in the regulation of iron metabolism. Signatures of immune response detected in spleen and liver indicated a robust anti-inflammatory state signified by macrophages in the M2 state and an active T cell response. The evolutionary divergence between bats and humans of many responsive genes might provide a framework for understanding the differing outcomes upon infection by filoviruses. In this study, we outline multiple interconnected pathways that respond to infection by MARV and EBOV, providing insights into the complexity of the mechanisms that enable bats to resist the disease caused by filoviral infections. The results have the potential to aid in the development of new strategies to effectively mitigate and treat the disease caused by these viruses in humans.

HDAC3 and HDAC7 have opposite effects on osteoclast differentiation.

Histone deacetylases (HDACs) are negative regulators of transcription. Endochondral bone formation including chondrocyte and osteoblast maturation is regulated by HDACs. Very little is known about the role HDACs play in osteoclast differentiation. It has been previously reported that HDAC inhibitors, trichostatin A and sodium butyrate, suppress osteoclast differentiation through multiple mechanisms. In this study, we report that suppression of HDAC3 expression similar to HDAC inhibitors inhibits osteoclast differentiation, whereas osteoclasts suppressed for HDAC7 expression had accelerated differentiation when compared with control cells. Mitf, a transcription factor, is necessary for osteoclast differentiation. We demonstrate that Mitf and HDAC7 interact in RAW 264 cells and osteoclasts. The transcriptional activity of Mitf is repressed by HDAC7. Lastly, we show that either the amino or the carboxyl terminus of HDAC7 is sufficient for transcriptional repression and that the repression of HDAC7 is insensitive to trichostatin A, indicating that HDAC7 represses Mitf at least in part by deacetylation-independent mechanism.

Digital Technologies for Children and Parents Sharing Self-Management in Childhood Chronic or Long-Term Conditions: A Scoping Review.

Worldwide, the prevalence of chronic (or long-term) conditions in children and young people from birth to 18 years (children) is increasing. Promoting competent and effective self-management skills early in the trajectory is important to improve adherence to treatment and optimise quality of life. Successful self-management, therefore, requires parents and children who are developmentally able to develop a range of complex skills, including the use of digital technologies. This scoping review aimed to identify primary research investigating digital technologies for children and parents sharing self-management in childhood chronic illnesses. A comprehensive search of electronic databases was conducted. Nineteen papers were included, assessed for quality and methodological rigour using the Hawker tool and thematically analysed. Three themes were identified: (i) the feasibility and acceptability of using technology, (ii) the usability of technologies and (iii) the effect of technologies on adherence and self-management skills. The results indicate that technologies such as mobile apps and websites can assist the management of long-term conditions, are an acceptable method of delivering information and can promote the development of effective self-management skills by parents and children. However, future technology design must include children and parents in all stages of development.

Valspodar limits human cytomegalovirus infection and dissemination.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that establishes a life-long infection affecting up to 80% of the US population. HCMV periodically reactivates leading to enhanced morbidity and mortality in immunosuppressed patients causing a range of complications including organ transplant failure and cognitive disorders in neonates. Therapeutic options for HCMV are limited to a handful of antivirals that target late stages of the virus life cycle and efficacy is often challenged by the emergence of mutations that confer resistance. In addition, these antiviral therapies may have adverse reactions including neutropenia in newborns and an increase in adverse cardiac events in HSCT patients. These findings highlight the need to develop novel therapeutics that target different steps of the viral life cycle. To this end, we screened a small molecule library against ion transporters to identify new antivirals against the early steps of virus infection. We identified valspodar, a 2nd-generation ABC transporter inhibitor, that limits HCMV infection as demonstrated by the decrease in IE2 expression of virus infected cells. Cells treated with increasing concentrations of valspodar over a 9-day period show minimal cytotoxicity. Importantly, valspodar limits HCMV plaque numbers in comparison to DMSO controls demonstrating its ability to inhibit viral dissemination. Collectively, valspodar represents a potential new anti-HCMV therapeutic that limits virus infection by likely targeting a host factor. Further, the data suggest that specific ABC transporters may participate in the HCMV life-cycle.

Added Diagnostic Utility of Clinical Metagenomics for the Diagnosis of Pneumonia in Immunocompromised Adults.

BACKGROUND: In the evaluation of community-acquired pneumonia, 30% to 60% of cases remain undiagnosed, despite extensive conventional microbiologic testing (CMT). Clinical metagenomics (CM) is an unbiased pathogen detection method that can increase diagnostic yield. RESEARCH QUESTION: Does adding clinical metagenomics to conventional microbiologic testing improve the diagnostic yield for pneumonia in immunocompromised adults? STUDY DESIGN AND METHODS: We performed a noninterventional prospective study of immunocompromised adults with pneumonia who underwent bronchoscopy and BAL over 2 years. CMT was performed per standard of care. A commercial CM test was performed on residual BAL fluid. Final microbiologic diagnoses were based on CMT vs CMT + CM. Final clinical diagnoses for CMT and CMT + CM were made based on laboratory results in conjunction with clinical and radiologic findings. Hypothetical impact of CMT + CM on management and antimicrobial stewardship was also assessed. RESULTS: A total of 30 immunocompromised adult patients (31 episodes of pneumonia) were included. Final microbiologic diagnoses were made in 11 cases (35%) with the use of CMT and in 18 cases (58%) with the use of CMT + CM. Bacterial pneumonia was diagnosed in five cases (16%) by CMT and in 13 cases (42%) by CMT + CM; fungal pneumonia was diagnosed in six cases (19%) by CMT and in seven cases (23%) by CMT + CM, and viral pneumonia was diagnosed in two cases (6%) by CMT and in five cases (16%) by CMT + CM. The hypothetical impact of CMT + CM on management was deemed probable in one case, possible in eight cases, and unlikely in two cases, whereas the impact on antimicrobial stewardship was possible in 13 cases and unlikely in seven cases. Final clinical diagnoses were made in 20 of 31 cases (65%) based on CMT and in 23 of 31 cases (74%) based on CMT + CM. INTERPRETATION: CMT + CM increased diagnostic yield in immunocompromised adults with pneumonia from 35% to 58%, mostly by the detection of additional bacterial causes but was less useful for fungal pneumonia.

Metagenomic Next-Generation Sequencing for Pathogen Detection and Transcriptomic Analysis in Pediatric Central Nervous System Infections.

BACKGROUND: Pediatric central nervous system (CNS) infections are potentially life-threatening and may incur significant morbidity. Identifying a pathogen is important, both in terms of guiding therapeutic management and in characterizing prognosis. Usual care testing by culture and polymerase chain reaction is often unable to identify a pathogen. We examined the systematic application of metagenomic next-generation sequencing (mNGS) for detecting organisms and transcriptomic analysis of cerebrospinal fluid (CSF) in children with central nervous system (CNS) infections. METHODS: We conducted a prospective multisite study that aimed to enroll all children with a CSF pleocytosis and suspected CNS infection admitted to 1 of 3 tertiary pediatric hospitals during the study timeframe. After usual care testing had been performed, the remaining CSF was sent for mNGS and transcriptomic analysis. RESULTS: We screened 221 and enrolled 70 subjects over a 12-month recruitment period. A putative organism was isolated from CSF in 25 (35.7%) subjects by any diagnostic modality. Metagenomic next-generation sequencing of the CSF samples identified a pathogen in 20 (28.6%) subjects, which were also all identified by usual care testing. The median time to result was 38 hours. CONCLUSIONS: Metagenomic sequencing of CSF has the potential to rapidly identify pathogens in children with CNS infections.

The 19S proteasomal lid subunit POH1 enhances the transcriptional activation by Mitf in osteoclasts.

The microphthalmia-associated transcription factor (Mitf) regulates gene expression required for osteoclast differentiation. Genes regulated by Mitf have been previously identified. However, proteins that interact and regulate Mitf's activity in osteoclasts are not well known. Here, we report that POH1, a subunit of the 19S proteasome lid is a regulator of Mitf. We show that POH1 and Mitf interact in osteoclasts and that this interaction is dependent on RANKL signaling. Overexpression of POH1 increased Mitf's activation of 5XGal4-TK and Acp5 promoters. The amino terminus of POH1 mediates the binding to Mitf and is sufficient to increase Mitf's transcriptional activity. Finally, we show that mutations in the JAMM motif of POH1 reduced Mitf activation of promoters. In summary, our results identify a novel mechanism of Mitf regulation in osteoclasts by POH1.

C-TAK1 interacts with microphthalmia-associated transcription factor, Mitf, but not the related family member Tfe3.

Microphthalmia-associated transcription factor, Mitf, has been shown to be necessary for regulating genes involved in osteoclast differentiation. Previously it was shown by others that Mitf translocates from the cytoplasm to the nucleus upon M-CSF/RANKL signaling in osteoclasts. Mitf's movement is regulated by its interaction with 14-3-3 and the kinase C-TAK1. Here we demonstrate that the related family member, Tfe3, does not shuttle from the cytoplasm to the nucleus and does not interact with C-TAK1. We also demonstrate that overexpression of C-TAK1 inhibits the expression of Acp5 while a kinase dead C-TAK1 or a Mitf mutant that cannot interact with C-TAK1 increased expression of Acp5. Finally, we show that the catalytic subunit of protein phosphatase 2A is up-regulated in osteoclasts with M-CSF/RANKL signaling, indicating a possible mechanism for dephosphorylating Mitf on its 14-3-3 binding site and allowing Mitf to be translocated to the nucleus of osteoclasts.

The Microtubule Inhibitor Podofilox Inhibits an Early Entry Step of Human Cytomegalovirus.

Human cytomegalovirus is a ubiquitous ß-herpesvirus that infects many different cell types through an initial binding to cell surface receptors followed by a fusion event at the cell membrane or endocytic vesicle. A recent high-throughput screen to identify compounds that block a step prior to viral gene expression identified podofilox as a potent and nontoxic inhibitor. Time-of-addition studies in combination with quantitative-PCR analysis demonstrated that podofilox limits an early step of virus entry at the cell surface. Podofilox was also able to drastically reduce infection by herpes simplex 1, an α-herpesvirus with a very similar entry process to CMV. Podofilox caused a reduced maximal plateau inhibition of infection by viruses with single step binding processes prior to fusion-like Newcastle disease virus, Sendai virus, and influenza A virus or viruses that enter via endocytosis like vesicular stomatitis virus and a clinical-like strain of CMV. These results indicate that microtubules appear to be participating in the post-binding step of virus entry including the pre- and post-penetration events. Modulation of the plasma membrane is required to promote virus entry for herpesviruses, and that podofilox, unlike colchicine or nocodazole, is able to preferentially target microtubule networks at the plasma membrane.

Functional screening for anti-CMV biologics identifies a broadly neutralizing epitope of an essential envelope protein.

The prototypic ß-herpesvirus human cytomegalovirus (CMV) establishes life-long persistence within its human host. The CMV envelope consists of various protein complexes that enable wide viral tropism. More specifically, the glycoprotein complex gH/gL/gO (gH-trimer) is required for infection of all cell types, while the gH/gL/UL128/130/131a (gH-pentamer) complex imparts specificity in infecting epithelial, endothelial and myeloid cells. Here we utilize state-of-the-art robotics and a high-throughput neutralization assay to screen and identify monoclonal antibodies (mAbs) targeting the gH glycoproteins that display broad-spectrum properties to inhibit virus infection and dissemination. Subsequent biochemical characterization reveals that the mAbs bind to gH-trimer and gH-pentamer complexes and identify the antibodies' epitope as an 'antigenic hot spot' critical for virus entry. The mAbs inhibit CMV infection at a post-attachment step by interacting with a highly conserved central alpha helix-rich domain. The platform described here provides the framework for development of effective CMV biologics and vaccine design strategies.

Dimerization Controls Marburg Virus VP24-dependent Modulation of Host Antioxidative Stress Responses.

Marburg virus (MARV), a member of the Filoviridae family that also includes Ebola virus (EBOV), causes lethal hemorrhagic fever with case fatality rates that have exceeded 50% in some outbreaks. Within an infected cell, there are numerous host-viral interactions that contribute to the outcome of infection. Recent studies identified MARV protein 24 (mVP24) as a modulator of the host antioxidative responses, but the molecular mechanism remains unclear. Using a combination of biochemical and mass spectrometry studies, we show that mVP24 is a dimer in solution that directly binds to the Kelch domain of Kelch-like ECH-associated protein 1 (Keap1) to regulate nuclear factor (erythroid-derived 2)-like 2 (Nrf2). This interaction between Keap1 and mVP24 occurs through the Kelch interaction loop (K-Loop) of mVP24 leading to upregulation of antioxidant response element transcription, which is distinct from other Kelch binders that regulate Nrf2 activity. N-terminal truncations disrupt mVP24 dimerization, allowing monomeric mVP24 to bind Kelch with higher affinity and stimulate higher antioxidative stress response element (ARE) reporter activity. Mass spectrometry-based mapping of the interface revealed overlapping binding sites on Kelch for mVP24 and the Nrf2 proteins. Substitution of conserved cysteines, C209 and C210, to alanine in the mVP24 K-Loop abrogates Kelch binding and ARE activation. Our studies identify a shift in the monomer-dimer equilibrium of MARV VP24, driven by its interaction with Keap1 Kelch domain, as a critical determinant that modulates host responses to pathogenic Marburg viral infections.

Enhanced osteoclastogenesis causes osteopenia in twisted gastrulation-deficient mice through increased BMP signaling.

The uncoupling of osteoblastic and osteoclastic activity is central to disorders such as osteoporosis, osteolytic malignancies, and periodontitis. Numerous studies have shown explicit functions for bone morphogenetic proteins (BMPs) in skeletogenesis. Their signaling activity has been shown in various contexts to be regulated by extracellular proteins, including Twisted gastrulation (TWSG1). However, experimental paradigms determining the effects of BMP regulators on bone remodeling are limited. In this study, we assessed the role of TWSG1 in postnatal bone homeostasis. Twsg1-deficient (Twsg1(-/-)) mice developed osteopenia that could not be explained by defective osteoblast function, because mineral apposition rate and differentiation markers were not significantly different compared with wildtype (WT) mice. Instead, we discovered a striking enhancement of osteoclastogenesis in Twsg1(-/-) mice, leading to increased bone resorption with resultant osteopenia. Enhanced osteoclastogenesis in Twsg1(-/-) mice was caused by increased cell fusion, differentiation, and function of osteoclasts. Furthermore, RANKL-mediated osteoclastogenesis and phosphorylated Smad1/5/8 levels were enhanced when WT osteoclasts were treated with recombinant BMP2, suggesting direct regulation of osteoclast differentiation by BMPs. Increase in detectable levels of phosphorylated Smad 1/5/8 was noted in osteoclasts from Twsg1(-/-) mice compared with WT mice. Furthermore, the enhanced osteoclastogenesis in Twsg1(-/-) mice was reversed in vitro in a dose-dependent manner with exposure to Noggin, a BMP antagonist, strongly suggesting that the enhanced osteoclastogenesis in Twsg1 mutants is attributable to increased BMP signaling. Thus, we present a novel and previously uncharacterized role for TWSG1 in inhibiting osteoclastogenesis through regulation of BMP activity.