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This essay investigates the pictorial practices used by Edmund Sharpe (1809–77) to visualize the evolution of English Gothic architecture. Examining his 1849 Treatise on the Rise and Progress of th...

Osteoclastogenesis is essential for bone remodeling and normal skeletal maintenance. Receptor activator of NF-κB ligand (RANKL) promotes osteoclast differentiation and function but requires costimulation of immunoreceptor tyrosine-based activation motif (ITAM)-coupled immunoreceptors. Triggering receptor expressed on myeloid cells-2 (TREM2) coupled to ITAM-adaptor protein DNAX activation protein 12kDA (DAP12) provides costimulation of intracellular calcium signaling during osteoclastogenesis. Previously, we found that downstream of kinase-3 (DOK3) physically associates with DAP12 to inhibit toll-like receptor (TLR)-induced inflammatory signaling in macrophages. However, whether and how DOK3 modulates DAP12-dependent osteoclastogenesis is unknown and the focus of this study. Bone microarchitecture and histology of sex- and age-matched wild-type (WT) and DOK3-deficient (DOK3-/- ) mice were evaluated. Male and female DOK3-/- mice have significantly reduced trabecular bone mass compared with WT mice with increased TRAP+ osteoclasts in vivo. In vitro, DOK3-/- bone marrow-derived macrophages (BMMs) have increased macrophage colony-stimulating factor (M-CSF)-induced proliferation and increased sensitivity to RANKL-induced osteoclastogenesis. Compared with WT, DOK3-/- osteoclasts are significantly larger with more nuclei and have increased resorptive capacity. Mechanistically, DOK3 limits osteoclastogenesis by inhibiting activation of Syk and ERK in response to RANKL and M-CSF. DOK3 is phosphorylated in a DAP12-dependent manner and associates with Grb2 and Cbl. Compared with DAP12-/- mice with high bone mass, DOK3- and DAP12- doubly deficient mice (DKO) have normalized bone mass, indicating that DOK3 also limits DAP12-independent osteoclastogenesis in vivo. In vitro osteoclasts derived from DKO mice are mononuclear with poor resorptive capacity similar to DAP12-/- osteoclasts. Histomorphometry reveals that DOK3-/- mice also have reduced osteoblast parameters. DOK3-/- osteoblasts have reduced in vitro osteoblastogenesis and increased osteoprotegerin (OPG) to RANKL expression ratio compared with WT osteoblasts. Co-culture of WT and DOK3-/- osteoblasts with pre-osteoclasts reveals a reduced capacity of DOK3-/- osteoblasts to support osteoclastogenesis. These data indicate that DOK3 regulates bone remodeling by negatively regulating M-CSF- and RANKL-mediated osteoclastogenesis and positively regulating osteoblastogenesis. © 2017 American Society for Bone and Mineral Research.

This research suggests a strategy to address a difficult challenge: self-sustainability and the promotion of increasing the availability and accessibility of local food. This strategy has the opportunity to bring nutritious meals into our homes, place money into our local economies and promote self-sustaining communities. This study examines literature and case studies involving both primary and secondary sources. Cities and their local food programs were examined by travel within Havana, Cuba, and Kansas City, Missouri, and a field research study in Des Moines, Iowa. The study presents models that address urban agriculture, food accessibility, and design implementations to improve local food access. The ultimate goal of this research is to provide alternative, healthy, and sustainable means to make local food available to residents and create a model for future communities to initiate sustainable measures and promote the design of holistic food systems.

Rotaviruses (RVs) can evolve through the process of reassortment, whereby the 11 double-stranded RNA genome segments are exchanged among strains during co-infection. However, reassortment is limited in cases where the genes or encoded proteins of co-infecting strains are functionally incompatible. In this study, we employed a helper virus-based reverse genetics system to identify NSP2 gene regions that correlate with restricted reassortment into simian RV strain SA11. We show that SA11 reassortants with NSP2 genes from human RV strains Wa or DS-1 were efficiently rescued and exhibit no detectable replication defects. However, we could not rescue an SA11 reassortant with a human RV strain AU-1 NSP2 gene, which differs from that of SA11 by 186 nucleotides ( 36 amino acids). To map restriction determinants, we engineered viruses to contain chimeric NSP2 genes in which specific regions of AU-1 sequence were substituted with SA11 sequence. We show that a region spanning AU-1 NSP2 gene nucleotides 784-820 is critical for the observed restriction; yet additional determinants reside in other gene regions. In silico and in vitro analyses were used to predict how the 784-820 region may impact NSP2 gene/protein function, thereby informing an understanding of the reassortment restriction mechanism. Virginia Tech Carilion Research Institute; National Institutes of Health [R01-AI116815, R21-AI119588]; Translational Biology, Medicine, and Health Graduate Program at Virginia Tech The authors would like to thank members of the McDonald laboratory for intellectual and technical support. We also thank Dr. John Patton (University of Maryland, College Park) for the generous donation of reagents. This work was supported through start-up funding from the Virginia Tech Carilion Research Institute and through grants from the National Institutes of Health (R01-AI116815 and R21-AI119588). C.P.L. was also supported by the Translational Biology, Medicine, and Health Graduate Program at Virginia Tech.

Ca2+ signaling is essential for bone homeostasis and skeletal development. Here, we show that the transient receptor potential canonical 1 (TRPC1) channel and the inhibitor of MyoD family, I-mfa, function antagonistically in the regulation of osteoclastogenesis. I-mfa null mice have an osteopenic phenotype characterized by increased osteoclast numbers and surface, which are normalized in mice lacking both Trpc1 and I-mfa. In vitro differentiation of pre-osteoclasts derived from I-mfa-deficient mice leads to an increased number of mature osteoclasts and higher bone resorption per osteoclast. These parameters return to normal levels in osteoclasts derived from double mutant mice. Consistently, whole cell currents activated in response to the depletion of intracellular Ca2+ stores are larger in pre-osteoclasts derived from I-mfa knock-out mice compared with currents in wild type mice and normalized in cells derived from double mutant mice, suggesting a cell-autonomous effect of I-mfa on TRPC1 in these cells. A new splice variant of TRPC1 (TRPC1ϵ) was identified in early pre-osteoclasts. Heterologous expression of TRPC1ϵ in HEK293 cells revealed that it is unique among all known TRPC1 isoforms in its ability to amplify the activity of the Ca2+ release-activated Ca2+ (CRAC) channel, mediating store-operated currents. TRPC1ϵ physically interacts with Orai1, the pore-forming subunit of the CRAC channel, and I-mfa is recruited to the TRPC1ϵ-Orai1 complex through TRPC1ϵ suppressing CRAC channel activity. We propose that the positive and negative modulation of the CRAC channel by TRPC1ϵ and I-mfa, respectively, fine-tunes the dynamic range of the CRAC channel regulating osteoclastogenesis. Background: Ca2+ signaling is essential for osteoclastogenesis. Results: I-mfa negatively regulates TRPC1-mediated Ca2+ signaling and osteoclastogenesis. Conclusion: TRPC1 and I-mfa fine-tune the dynamic range of store-operated Ca2+ entry channels during osteoclastogenesis. Significance: The TRPC1/I-mfa interaction is biologically relevant in osteoclastogenesis.

Osteoclasts are bone-specific polykaryons derived from myeloid precursors under the stimulation of macrophage colony stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). E proteins are basic helix-loop-helix (bHLH) transcription factors that modulate lymphoid versus myeloid cell fate decisions. To study the role of E proteins in osteoclasts, myeloid-specific E protein gain-of-function transgenic mice were generated. These mice have high bone mass due to decreased osteoclast numbers and increased osteoclast apoptosis leading to overall reductions in resorptive capacity. The molecular mechanism of decreased osteoclast numbers and resorption is in part a result of elevated expression of CD38, a regulator of intracellular calcium pools with known antiosteoclastogenic properties, which increases sensitivity to apoptosis. In vivo, exogenous RANKL stimulation can overcome this inhibition to drive osteoclastogenesis and bone loss. In vitro-derived ET2 osteoclasts are more spread and more numerous with increases in RANK, triggering receptor expressed on myeloid cells 2 (TREM2), and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) compared to wild type. However, their resorptive capacity does not increase accordingly. Thus, E proteins participate in osteoclast maturation and survival in homeostatic bone remodeling.

CD731 is a GPI-anchored cell surface protein with ecto-5'-nucleotidase enzyme activity that plays a crucial role in adenosine production. While the roles of adenosine receptors (AR) on osteoblasts and osteoclasts have been unveiled to some extent, the roles of CD73 and CD73-generated adenosine in bone tissue are largely unknown. To address this issue, we first analyzed the bone phenotype of CD73-deficient (cd73(-/-)) mice. The mutant male mice showed osteopenia, with significant decreases of osteoblastic markers. Levels of osteoclastic markers were, however, comparable to those of wild-type mice. A series of in vitro studies revealed that CD73 deficiency resulted in impairment in osteoblast differentiation but not in the number of osteoblast progenitors. In addition, over expression of CD73 on MC3T3-E1 cells resulted in enhanced osteoblastic differentiation. Moreover, MC3T3-E1 cells expressed adenosine A(2A) receptors (A(2A)AR) and A(2B) receptors (A(2B)AR) and expression of these receptors increased with osteoblastic differentiation. Enhanced expression of osteocalcin (OC) and bone sialoprotein (BSP) observed in MC3T3-E1 cells over expressing CD73 were suppressed by treatment with an A(2B)AR antagonist but not with an A(2A) AR antagonist. Collectively, our results indicate that CD73 generated adenosine positively regulates osteoblast differentiation via A(2B)AR signaling.

Based on previous research indicating that character portrayals in video games and other media can influence users’ perceptions of social reality, systematic content analyses have examined demographic trends in the way video game characters are portrayed. Although these studies have extensively documented character portrayals in traditional console and computer video games, there is a lack of content analyses examining character portrayals in the very popular massively multiplayer online game (MMO) genre. Such studies are needed because many characters in MMOs are customized avatars created by users, which may lead to different trends in character demographics. This content analysis examined representations of gender and race among 417 unique characters appearing 1,356 times in 20 hours of recorded content from four popular commercial MMOs, which was generated by five recruited users. Characters tended to be disproportionately male and white, with females and racial minorities appearing much less often. Implications for potential effects on users’ perceptions of social reality are discussed.

DNAX-activating protein of 12 kD (DAP12) is an immunoreceptor tyrosine-based activation motif (ITAM)–containing adaptor protein found in myeloid cells and natural killer cells, and it couples to various receptors that mediate either cellular activation or inhibition. DAP12 inhibits Toll-like receptor (TLR) signaling, such as that of TLR4 in response to its ligand lipopolysaccharide (LPS), as well as cytokine responses by coupling to TREM2 (triggering receptor expressed on myeloid cells 2) at the plasma membrane. Understanding the mechanisms that inhibit inflammatory responses in macrophages is important for the development of therapies to treat inflammatory diseases. We show that inhibition of LPS responses by DAP12 is mediated by the adaptor protein DOK3 (downstream of kinase 3). DOK3 physically associated with the ITAM of DAP12 through its phosphotyrosine-binding domain. In response to LPS, DOK3 was phosphorylated in a DAP12- and Src-dependent manner, which led to translocation of phosphorylated DOK3 to the plasma membrane. DOK3-deficient cells exhibited increased production of proinflammatory cytokines and activation of extracellular signal–regulated kinase (ERK). Compared to wild-type mice, DOK3-deficient mice had increased susceptibility to challenge with a sublethal dose of LPS and produced increased serum concentrations of the inflammatory cytokine tumor necrosis factor–α (TNF-α). Together, these data suggest the mechanism by which DAP12 and TREM2 inhibit LPS signaling in macrophages to prevent inflammation.

Commercially pure titanium plates/coupons and pure titanium powders were soaked for 24 h in 5 M NaOH and 5 M KOH solutions, under identical conditions, over the temperature range of 37° to 90 °C. Wettability of the surfaces of alkali-treated cpTi coupons was studied by using contact angle goniometry. cpTi coupons soaked in 5 M NaOH or 5 M KOH solutions were found to have hydrophilic surfaces. Hydrous alkali titanate nanofibers and nanotubes were identified with SEM/EDXS and grazing incidence XRD. Surface areas of Ti powders increased > 50–220 times, depending on the treatment, when soaked in the above solutions. A solution was developed to coat amorphous calcium phosphate, instead of hydroxyapatite, on Ti coupon surfaces. In vitro cell culture tests were performed with osteoblast-like cells on the alkali-treated samples.

The study of bone and immunology (termed osteoimmunology) has led to the discovery of many important similarities between the two systems including shared niches, mechanisms, cytokines and receptors. The bone marrow provides a niche for hematopoietic cells including those of the lymphoid and myeloid lineage. Osteoclasts, specialized polykarons arising from myeloid precursors, bind to bone and resorb the organic and inorganic components through secretion of acid and proteases. Osteoclasts are differentiated and activated by cytokines that can be produced by immune cells and osteoclast activity can be dysregulated in states of autoimmunity or high inflammation. Similar to B and T cells, osteoclasts require coordinated co-stimulation of signaling pathways provided in the form of receptor-associated immunoreceptor tyrosine-based activation motif adaptor proteins, DAP12 and FcRγ, to drive differentiation and activation. In this review, we will cover the differentiation process of osteoclasts from the earliest precursors shown to have differentiation potential and the signals needed to drive these cells into osteoclast commitment and activation.

Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists.