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Previous work in several laboratories has demonstrated that the epitranscriptomic addition of m6A to viral transcripts promotes the replication and pathogenicity of a wide range of DNA and RNA viruses, yet the underlying mechanisms responsible for this positive effect have remained unclear. It is known that m6A function is largely mediated by cellular m6A binding proteins or readers, yet how m6A readers regulate viral gene expression in general, and HIV-1 gene expression in particular, has been controversial. Here, we confirm that m6A addition indeed regulates HIV-1 RNA expression and demonstrate that this effect is in large part mediated by the the nuclear m6A reader YTHDC1 and the cytoplasmic m6A reader YTHDF2. Both YTHDC1 and YTHDF2 bind to multiple distinct and overlapping sites on the HIV-1 RNA genome, with YTHDC1 recruitment serving to regulate the alternative splicing of HIV-1 RNAs while YTHDF2 binding correlates with increased HIV-1 transcript stability.Author SummaryThis manuscript reports that the expression of mRNAs encoded by the pathogenic human retrovirus HIV-1 is regulated by the methylation of a small number of specific adenosine residues. These in turn recruit a nuclear RNA binding protein, called YTHDC1, which modulates the alternative splicing of HIV-1 transcripts, as well as a cytoplasmic RNA binding protein, called YTHDF2, which stabilizes viral mRNAs. The regulation of HIV-1 gene expression by adenosine methylation is therefore critical for the effective and ordered expression of HIV-1 mRNAs and could represent a novel target for antiviral development.

Human immunodeficiency virus 1 (HIV-1) is a retrovirus with a ten-kilobase single-stranded RNA genome. HIV-1 must express all of its gene products from a single primary transcript, which undergoes alternative splicing to produce diverse protein products that include structural proteins and regulatory factors1,2. Despite the critical role of alternative splicing, the mechanisms that drive the choice of splice site are poorly understood. Synonymous RNA mutations that lead to severe defects in splicing and viral replication indicate the presence of unknown cis-regulatory elements3. Here we use dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) to investigate the structure of HIV-1 RNA in cells, and develop an algorithm that we name 'detection of RNA folding ensembles using expectation-maximization' (DREEM), which reveals the alternative conformations that are assumed by the same RNA sequence. Contrary to previous models that have analysed population averages4, our results reveal heterogeneous regions of RNA structure across the entire HIV-1 genome. In addition to confirming that in vitro characterized5 alternative structures for the HIV-1 Rev responsive element also exist in cells, we discover alternative conformations at critical splice sites that influence the ratio of transcript isoforms. Our simultaneous measurement of splicing and intracellular RNA structure provides evidence for the long-standing hypothesis6-8 that heterogeneity in RNA conformation regulates splice-site use and viral gene expression.

Alternative splicing of the HIV transcriptome is controlled through cis regulatory elements functioning as enhancers or silencers depending on their context and the type of host RNA binding proteins they recruit. Splice site acceptor A3 (ssA3) is one of the least used acceptor sites in the HIV transcriptome and its activity determines the levels of tat mRNA. Splice acceptor 3 is regulated by a combination of cis regulatory sequences, auxiliary splicing factors, and presumably RNA structure. The mechanisms by which these multiple regulatory components coordinate to determine the frequency in which ssA3 is utilized is poorly understood. By NMR spectroscopy and phylogenetic analysis, we show that the ssA3 regulatory locus is conformationally heterogeneous and that the sequences that encompass the locus are conserved across most HIV isolates. Despite the conformational heterogeneity, the major stem loop (A3SL1) observed in vitro folds to base pair the Polypyrimdine Tract (PPyT) to the Exon Splicing Silencer 2p (ESS2p) element and to a conserved downstream linker. The 3D structure as determined by NMR spectroscopy further reveals that the A3 consensus cleavage site is embedded within a unique stereochemical environment within the apical loop, where it is surrounded by alternating base-base interactions. Despite being described as a receptor for hnRNP H, the ESS2p element is sequestered by base pairing to the 3’ end of the PPyT and within this context it cannot form a stable complex with hnRNP H. By comparison, hnRNP A1 directly binds to the A3 consensus cleavage site located within the apical loop, suggesting that it can directly modulate U2AF assembly. Sequence mutations designed to destabilize the PPyT:ESS2p helix results in an increase usage of ssA3 within HIV-infected cells, consistent with the PPyT becoming more accessible for U2AF recognition. Additional mutations introduced into the downstream ESS2 element synergize with ESS2p to cause further increases in ssA3 usage. When taken together, our work provides a unifying picture by which cis regulatory sequences, splicing auxiliary factors and RNA structure cooperate to provide stringent control over ssA3. We describe this as the pair-and-lock mechanism to restrict access of the PPyT, and posit that it operates to regulate a subset of the heterogenous structures encompassing the ssA3 regulatory locus.

Previous work has demonstrated that the epitranscriptomic addition of m(6)A to viral transcripts can promote the replication and pathogenicity of a wide range of DNA and RNA viruses, including HIV-1, yet the underlying mechanisms responsible for this effect have remained unclear. It is known that m(6)A function is largely mediated by cellular m(6)A binding proteins or readers, yet how these regulate viral gene expression in general, and HIV-1 gene expression in particular, has been controversial. Here, we confirm that m(6)A addition indeed regulates HIV-1 RNA expression and demonstrate that this effect is largely mediated by the nuclear m(6)A reader YTHDC1 and the cytoplasmic m(6)A reader YTHDF2. Both YTHDC1 and YTHDF2 bind to multiple distinct and overlapping sites on the HIV-1 RNA genome, with YTHDC1 recruitment serving to regulate the alternative splicing of HIV-1 RNAs. Unexpectedly, while YTHDF2 binding to m(6)A residues present on cellular mRNAs resulted in their destabilization as previously reported, YTHDF2 binding to m(6)A sites on HIV-1 transcripts resulted in a marked increase in the stability of these viral RNAs. Thus, YTHDF2 binding can exert diametrically opposite effects on RNA stability, depending on RNA sequence context.

HIV-1 transcribes only one kind of transcript – the full length genomic RNA. To make the mRNA transcripts for the accessory proteins Tat and Rev, the genomic RNA must completely splice. The mRNA transcripts for Vif, Vpr, and Env must splice but not completely. Genomic RNA (which also functions as mRNA for the Gag and Gag/Pro/Pol precursor polyproteins) must not splice at all. HIV-1 can tolerate a surprising range in the relative abundance of individual transcript types, and a surprising amount of aberrant and even odd splicing; however, it must not over-splice, which results in the loss of full length genomic RNA and has a dramatic fitness cost. Loss of full length genomic transcripts through over-splicing has a dramatic fitness cost. Cells typically do not tolerate unspliced/incompletely spliced transcripts, so HIV-1 has to circumvent this cell policing mechanism to allow some splicing while suppressing most. Splicing is controlled by RNA secondary structure, cis-acting regulatory sequences which bind splicing factors, and the viral protein Rev. There is still much work to be done to clarify the combinatorial effects of these splicing regulators. These control mechanisms represent attractive targets to induce over-splicing as an antiviral strategy. Finally, splicing has been implicated in latency, but to date there is little supporting evidence for such a mechanism. In this review we apply what is known of cellular splicing to understand splicing in HIV-1, and also present data from our newer and more sensitive deep sequencing assays quantifying the different HIV-1 transcript types.

Covalent modifications added to individual nucleotides on mRNAs, called epitranscriptomic modifications, have recently emerged as key regulators of both cellular and viral mRNA function1,2 and RNA methylation has now been shown to enhance the replication of human immunodeficiency virus 1 (HIV-1) and several other viruses3–11. Recently, acetylation of the N4 position of cytidine (ac4C) was reported to boost cellular mRNA function by increasing mRNA translation and stability12. We therefore hypothesized that ac4C and N-acetyltransferase 10 (NAT10), the cellular enzyme that adds ac4C to RNAs, might also have been subverted by HIV-1 to increase viral gene expression. We now confirm that HIV-1 transcripts are indeed modified by addition of ac4C at multiple discreet sites and demonstrate that silent mutagenesis of a subset of these ac4C addition sites inhibits HIV-1 gene expression in cis. Moreover, reduced expression of NAT10, and the concomitant decrease in the level of ac4C on viral RNAs, inhibits HIV-1 replication by reducing HIV-1 RNA stability. Interestingly Remodelin, a previously reported inhibitor of NAT10 function13,14, also inhibits HIV-1 replication without affecting cell viability, thus raising the possibility that the addition of ac4C to viral mRNAs might emerge as a novel cellular target for antiviral drug development.

Alternative splicing of HIV-1 mRNAs increases viral coding potential and controls the levels and timing of gene expression. HIV-1 splicing is regulated in part by heterogeneous nuclear ribonucleoproteins (hnRNPs) and their viral target sequences, which typically repress splicing when studied outside their native viral context. Here, we determined the location and extent of hnRNP binding to HIV-1 mRNAs and their impact on splicing in a native viral context. Notably, hnRNP A1, hnRNP A2, and hnRNP B1 bound to many dispersed sites across viral mRNAs. Conversely, hnRNP H1 bound to a few discrete purine-rich sequences, a finding that was mirrored in vitro. hnRNP H1 depletion and mutation of a prominent viral RNA hnRNP H1 binding site decreased the use of splice acceptor A1, causing a deficit in Vif expression and replicative fitness. This quantitative framework for determining the regulatory inputs governing alternative HIV-1 splicing revealed an unexpected splicing enhancer role for hnRNP H1 through binding to its target element. IMPORTANCE Alternative splicing of HIV-1 mRNAs is an essential yet quite poorly understood step of virus replication that enhances the coding potential of the viral genome and allows the temporal regulation of viral gene expression. Although HIV-1 constitutes an important model system for general studies of the regulation of alternative splicing, the inputs that determine the efficiency with which splice sites are utilized remain poorly defined. Our studies provide an experimental framework to study an essential step of HIV-1 replication more comprehensively and in much greater detail than was previously possible and reveal novel cis-acting elements regulating HIV-1 splicing.

How the covalent modification of mRNA ribonucleotides, termed epitranscriptomic modifications, alters mRNA function remains unclear. One issue has been the difficulty of quantifying these modifications. Using purified HIV-1 genomic RNA, we show that this RNA bears more epitranscriptomic modifications than the average cellular mRNA, with 5-methylcytosine (m(5)C) and 2’O-methyl modifications being particularly prevalent. The methyltransferase NSUN2 serves as the primary writer for m(5)C on HIV-1 RNAs. NSUN2 inactivation not only inhibits m(5)C addition to HIV-1 transcripts but also viral replication. This inhibition results from reduced HIV-1 protein, but not mRNA, expression, which in turn correlates with reduced ribosome binding to viral mRNAs. In addition, loss of m(5)C dysregulates the alternative splicing of viral RNAs. These data identify m(5)C as a post-transcriptional regulator of both splicing and function of HIV-1 mRNA, thereby affecting directly viral gene expression.

How the covalent modification of individual mRNA nucleotides, termed epitranscriptomic modification, alters mRNA function remains unclear. One road block has been the difficulty of quantifying epitranscriptomic modifications. Using purified HIV-1 genomic RNA, we show that this RNA bears more epitranscriptomic modifications than cellular mRNAs, with 2’O-methyl and 5-methylcytosine (m5C) modifications being particularly high. The primary writer for m5C on HIV-1 RNAs was identified as NSUN2 and inactivation of NSUN2 inhibits HIV-1 gene expression without affecting HIV-1 RNA levels. We identify MBD2 as an m5C reader and demonstrate that MBD2 not only has a higher affinity for m5C modified RNAs but also that MBD2 binding sites on the HIV-1 genome coincide with m5C addition sites. Loss of MBD2 function phenocopies loss of NSUN2 in that both reduce HIV-1 gene expression and perturb alternative splicing of HIV-1 RNAs. These data identify m5C as a post-transcriptional regulator of both mRNA splicing and function.

How the covalent modification of mRNA ribonucleotides, termed epitranscriptomic modifications, alters mRNA function remains unclear. One issue has been the difficulty of quantifying these modifications. Using purified HIV-1 genomic RNA, we show that this RNA bears many more epitranscriptomic modifications than the average cellular mRNA, with 5-methylcytosine (m5C) and 2’O-methyl modifications being particularly prevalent. The primary writer for m5C on HIV-1 RNAs was identified as NSUN2 and inactivation of NSUN2 inhibited not only m5C addition to HIV-1 transcripts but also viral replication. This inhibition resulted from reduced HIV-1 protein, but not mRNA, expression, which in turn correlated with reduced ribosome binding to viral mRNAs. In addition, loss of m5C dysregulated the alternative splicing of viral RNAs. These data identify m5C as a post-transcriptional regulator of both mRNA splicing and function.

The present study applied self-determination theory to examine the onset, maintenance, and cessation of non-suicidal self-injury (NSSI) in adolescents. Specifically, the study examined the relationship between the basic psychological needs of autonomy, competence, and relatedness, and NSSI status. Participants were classified into the NSSI Maintain (n = 30), NSSI Start (n = 44), NSSI Stop (n = 21), or Control (n = 98) groups based on NSSI status over 2 time points within a 12-month period. Repeated measures multiple analysis of variance was employed. Satisfaction of the need for competence decreased over time in all adolescents. Adolescents who maintained NSSI behavior reported significantly lower levels of need satisfaction compared to adolescents reporting no history of NSSI engagement, and adolescents who began NSSI over the course of the study reported significantly lower levels of need satisfaction compared to those reporting no history of NSSI engagement. The findings suggest that need satisfaction varies as a function of NSSI status.

Non-suicidal self-injury (NSSI) is a public health concern that affects young adults at alarming rates. The present study examines the role of satisfaction of self-determination theory's three basic needs for autonomy, competence, and relatedness in young adults' NSSI engagement. University students who reported ever having engaged in NSSI (n = 40, 85 % female; Mage = 20.10, SD = 1.66) reported significantly lower levels of the satisfaction of all three needs, as well as more difficulties with all aspects of emotion regulation (non-acceptance of emotional responses, difficulty engaging in goal directed behavior, impulse control, lack of emotional awareness, limited access to regulation strategies, lack of emotional clarity), compared to students with no history of NSSI (n = 46, 91 % female; Mage = 19.79, SD = 1.37). Results of a logistic regression analysis revealed that need satisfaction added to the prediction of NSSI group membership after controlling for the effects of emotion regulation. Satisfaction of the need for competence and limited access to emotion regulation strategies accounted for significant variance in NSSI in the final model. The findings suggest that self-determination theory may be a useful framework under which to conceptualize NSSI and that the need for competence may be particularly salient for University students.

The ~9.5 kilobase HIV-1 genome contains RNA sequences and structures that control many aspects of viral replication, including transcription, splicing, nuclear export, translation, packaging and reverse transcription. Nonetheless, chemical probing and other approaches suggest that the HIV-1 genome may contain many more RNA secondary structures of unknown importance and function. To determine whether there are additional, undiscovered cis-acting RNA elements in the HIV-1 genome that are important for viral replication, we undertook a global silent mutagenesis experiment. Sixteen mutant proviruses containing clusters of ~50 to ~200 synonymous mutations covering nearly the entire HIV-1 protein coding sequence were designed and synthesized. Analyses of these mutant viruses resulted in their division into three phenotypic groups. Group 1 mutants exhibited near wild-type replication, Group 2 mutants exhibited replication defects accompanied by perturbed RNA splicing, and Group 3 mutants had replication defects in the absence of obvious splicing perturbation. The three phenotypes were caused by mutations that exhibited a clear regional bias in their distribution along the viral genome, and those that caused replication defects all caused reductions in the level of unspliced RNA. We characterized in detail the underlying defects for Group 2 mutants. Second-site revertants that enabled viral replication could be derived for Group 2 mutants, and generally contained point mutations that reduced the utilization of proximal splice sites. Mapping of the changes responsible for splicing perturbations in Group 2 viruses revealed the presence of several RNA sequences that apparently suppressed the use of cryptic or canonical splice sites. Some sequences that affected splicing were diffusely distributed, while others could be mapped to discrete elements, proximal or distal to the affected splice site(s). Overall, our data indicate complex negative regulation of HIV-1 splicing by RNA elements in various regions of the HIV-1 genome that enable balanced splicing and viral replication., Author summary In addition to encoding viral proteins, the HIV-1 genome contains sequence elements that act at the level of RNA to enable replication. We undertook an experiment to discover new RNA elements that act in this way by altering nearly the entire coding sequence of the viral genome so as to change the RNA sequence without changing protein sequences. This experiment uncovered two classes of defective mutants. One class had profound defects in RNA splicing, the other had no obvious defects in splicing. Through an analysis of the splicing-defective mutants, we found several previously RNA sequences in the viral genome that affected splicing, enabling a nearly complete catalogue of signals that regulate HIV-1 alternative splicing in infected cells to be derived. Because these newly described sequences lack sequence motifs that are known to bind to canonical splicing-regulatory proteins, they may function through novel mechanisms.

The purpose of the present study was to test the applicability of self-determination theory (Deci and Ryan in J Res Pers 19:109–134. doi: 10.1016/0092-6566(85)90023-6 , 1985; Can Psychol 49:182–185. doi: 10.1037/a0012801 , 2008) across developmental periods by differentiating children and adolescents on the importance of individual needs (i.e., autonomy, competence, relatedness) and the role of balance across contexts (i.e., home, school, peers) in predicting depressive symptoms. Participants completed the Children’s Intrinsic Need Satisfaction Scale (Koestner and Veronneau in The Children’s Intrinsic Needs Satisfaction Scale. McGill University, Montreal, 2001) and the Children’s Depression Inventory (Kovacs in Children’s depression inventory manual. Multi-Health Systems, North Tonawanda, 1992). Results indicated that only the need for competence was significantly related to depressive symptoms in the child sample (n = 149) whereas, the satisfaction of autonomy and relatedness were significant predictors in the adolescent sample (n = 153). In both samples, need balance across contexts was a significant predictor over and above the level of satisfaction of each individual need. Implications for clinical practice and for theory will be presented.

Viral protein R (Vpr) is an HIV-1 accessory protein whose function remains poorly understood. In this report, we sought to determine the requirement of Vpr for facilitating HIV-1 infection of monocyte-derived dendritic cells (MDDCs), one of the first cell types to encounter virus in the peripheral mucosal tissues. In this report, we characterize a significant restriction of Vpr-deficient virus replication and spread in MDDCs alone and in cell-to-cell spread in MDDC-CD4 + T cell cocultures. This restriction of HIV-1 replication in MDDCs was observed in a single round of virus replication and was rescued by the expression of Vpr in trans in the incoming virion. Interestingly, infections of MDDCs with viruses that encode Vpr mutants unable to interact with either the DCAF1/DDB1 E3 ubiquitin ligase complex or a host factor hypothesized to be targeted for degradation by Vpr also displayed a significant replication defect. While the extent of proviral integration in HIV-1-infected MDDCs was unaffected by the absence of Vpr, the transcriptional activity of the viral long terminal repeat (LTR) from Vpr-deficient proviruses was significantly reduced. Together, these results characterize a novel postintegration restriction of HIV-1 replication in MDDCs and show that the interaction of Vpr with the DCAF1/DDB1 E3 ubiquitin ligase complex and the yet-to-be-identified host factor might alleviate this restriction by inducing transcription from the viral LTR. Taken together, these findings identify a robust in vitro cell culture system that is amenable to addressing mechanisms underlying Vpr-mediated enhancement of HIV-1 replication. IMPORTANCE Despite decades of work, the function of the HIV-1 protein Vpr remains poorly understood, primarily due to the lack of an in vitro cell culture system that demonstrates a deficit in replication upon infection with viruses in the absence of Vpr. In this report, we describe a novel cell infection system that utilizes primary human dendritic cells, which display a robust decrease in viral replication upon infection with Vpr-deficient HIV-1. We show that this replication difference occurs in a single round of infection and is due to decreased transcriptional output from the integrated viral genome. Viral transcription could be rescued by virion-associated Vpr. Using mutational analysis, we show that domains of Vpr involved in binding to the DCAF1/DDB1/E3 ubiquitin ligase complex and prevention of cell cycle progression into mitosis are required for LTR-mediated viral expression, suggesting that the evolutionarily conserved G 2 cell cycle arrest function of Vpr is essential for HIV-1 replication.

OBJECTIVE: We applied self-determination theory to examine a model whereby perceived parental autonomy support directly and indirectly affects nonsuicidal self-injury (NSSI) through difficulties in emotion regulation. METHOD: 639 participants (53% female) with a mean age of 13.38 years (SD = 0.51) completed the How I Deal with Stress Questionnaire as a screener for NSSI, the Perceptions of Parents Scale, and the Difficulties in emotion Regulation Scale. Participants who indicated having ever hurt themselves on purpose without the intent to die (n = 116, 66% female) were classified in the NSSI lifetime group. RESULTS: A mediation analysis with bootstrapping procedure revealed that adolescents who reported their parents as being less supportive of their need for autonomy were more likely to have engaged in NSSI. Further, this relationship was partially mediated by emotion regulation. CONCLUSION: Adolescents who do not perceive autonomy support from their parents, have more difficulties regulating their emotions, and may turn to NSSI as a means to cope. Clinical implications of the findings suggest involving the family, and specifically, targeting parental autonomy support may be beneficial when working with young adolescents who self-injure. (PsycINFO Database Record(c) 2017 APA, all rights reserved). Language: en

Full-length human immunodeficiency virus type 1 (HIV-1) RNA serves as the genome or as an mRNA, or this RNA undergoes splicing using four donors and 10 acceptors to create over 50 physiologically relevant transcripts in two size classes (1.8 kb and 4 kb). We developed an assay using Primer ID-tagged deep sequencing to quantify HIV-1 splicing. Using the lab strain NL4-3, we found that A5 ( env / nef ) is the most commonly used acceptor (about 50%) and A3 ( tat ) the least used (about 3%). Two small exons are made when a splice to acceptor A1 or A2 is followed by activation of donor D2 or D3, and the high-level use of D2 and D3 dramatically reduces the amount of vif and vpr transcripts. We observed distinct patterns of temperature sensitivity of splicing to acceptors A1 and A2. In addition, disruption of a conserved structure proximal to A1 caused a 10-fold reduction in all transcripts that utilized A1. Analysis of a panel of subtype B transmitted/founder viruses showed that splicing patterns are conserved, but with surprising variability of usage. A subtype C isolate was similar, while a simian immunodeficiency virus (SIV) isolate showed significant differences. We also observed transsplicing from a downstream donor on one transcript to an upstream acceptor on a different transcript, which we detected in 0.3% of 1.8-kb RNA reads. There were several examples of splicing suppression when the env intron was retained in the 4-kb size class. These results demonstrate the utility of this assay and identify new examples of HIV-1 splicing regulation. IMPORTANCE During HIV-1 replication, over 50 conserved spliced RNA variants are generated. The splicing assay described here uses new developments in deep-sequencing technology combined with Primer ID-tagged cDNA primers to efficiently quantify HIV-1 splicing at a depth that allows even low-frequency splice variants to be monitored. We have used this assay to examine several features of HIV-1 splicing and to identify new examples of different mechanisms of regulation of these splicing patterns. This splicing assay can be used to explore in detail how HIV-1 splicing is regulated and, with moderate throughput, could be used to screen for structural elements, small molecules, and host factors that alter these relatively conserved splicing patterns.

Summary How the covalent modification of mRNA ribonucleotides, termed epitranscriptomic modifications, alters mRNA function remains unclear. One issue has been the difficulty of quantifying these modifications. Using purified HIV-1 genomic RNA, we show that this RNA bears more epitranscriptomic modifications than the average cellular mRNA, with 5-methylcytosine (m5C) and 2′O-methyl modifications being particularly prevalent. The methyltransferase NSUN2 serves as the primary writer for m5C on HIV-1 RNAs. NSUN2 inactivation inhibits not only m5C addition to HIV-1 transcripts but also viral replication. This inhibition results from reduced HIV-1 protein, but not mRNA, expression, which in turn correlates with reduced ribosome binding to viral mRNAs. In addition, loss of m5C dysregulates the alternative splicing of viral RNAs. These data identify m5C as a post-transcriptional regulator of both splicing and function of HIV-1 mRNA, thereby affecting directly viral gene expression.

To investigate the role of bone morphogenetic protein (BMP) signaling in osteoclastogenesis in vivo, we eliminated BMPRII in osteoclasts by creating a BMPRII(fl/fl);lysM-Cre mouse strain. Conditional knock-out (cKO) mice are osteopetrotic when compared with WT controls due to a decrease in osteoclast activity. Bone marrow macrophages (BMMs) isolated from cKO mice are severely inhibited in their capacity to differentiate into mature osteoclasts in the presence of M-CSF and receptor activator of NF-κB (RANK) ligand. We also show that BMP noncanonical (MAPK) and canonical (SMAD) pathways are utilized at different stages of osteoclast differentiation. BMP2 induces p38 phosphorylation in pre-fusion osteoclasts and increases SMAD phosphorylation around osteoclast precursor fusion. Phosphorylation of MAPKs was decreased in differentiated BMMs from cKO animals. Treating BMMs with the SMAD inhibitor dorsomorphin confirms the requirement for the canonical pathway around the time of fusion. These results demonstrate the requirement for BMP signaling in osteoclasts for proper bone homeostasis and also explore the complex signaling mechanisms employed by BMP signaling during osteoclast differentiation.

The Rb and Pten tumor suppressor genes are important regulators of bone development and both are frequently mutated in the bone cancer osteosarcoma (OS). To determine if Rb1 and Pten synergize as tumor suppressor genes for osteosarcoma, we co-deleted them in osteoprogenitor cells. Surprisingly, we observed rapid development of adipogenic but not osteosarcoma tumors in the ΔRb1/Pten mice. ΔPten solo deleted mice also developed lipoma tumors but at a much reduced frequency and later onset than those co-deleted for Rb1. Pten deletion also led to a marked increase in adipocytes in the bone marrow. To better understand the function of Pten in bone development in vivo, we conditionally deleted Pten in OSX(+) osteoprogenitor cells using OSX-Cre mice. μCT analysis revealed a significant thickening of the calvaria and an increase in trabeculae volume and number in the femur, consistent with increased bone formation in these mice. To determine if Pten and Rb1 deletion actively promotes adipogenic differentiation, we isolated calvarial cells from Pten(fl/fl) and Pten(fl/fl); Rb1(fl/fl) mice, infected them with CRE or GFP expressing adenovirus, treated with differentiation media. We observed slightly increased adipogenic, and osteogenic differentiation in the ΔPten cells. Both phenotypes were greatly increased upon Rb1/Pten co-deletion. This was accompanied by an increase in expression of genes required for adipogenesis. These data indicate that Pten deletion in osteoblast precursors is sufficient to promote frequent adipogenic, but only rare osteogenic tumors. Rb1 hetero- or homo-zygous co-deletion greatly increases the incidence and the rapidity of onset of adipogenic tumors, again, with only rare osteosarcoma tumors.

There were two aims of the research with 190 Australian clinical psychologists: (1) to investigate the construct validity of the Therapist Belief Scale (TBS), and (2) to examine the relative contribution of demographics, workplace variables, and individual factors to burnout. Construct validity was examined using exploratory and confirmatory factor analyses and associations between the variables. Multivariate regressions were used to examine the relative contributions to burnout. The TBS showed three factors related to distress, inflexibility, and control, all of which were significantly associated with lower levels of personal accomplishment. Multivariate analyses showed emotional exhaustion to be associated being a woman, working for the government, having less personal resources, and endorsing more therapist beliefs related to control. Higher levels of personal accomplishment were significantly associated with a lower annual income, not having a mixed caseload, having more personal resources, and endorsing lower levels of therapist beliefs related to inflexibility and control.