In the course of attempting to define the bone "secretome" using a signal-trap screening approach, we identified a gene encoding a small membrane protein novel to osteoblasts. Although previously identified in silico as ifitm5, no localization or functional studies had been undertaken on this gene. We characterized the expression patterns and localization of this gene in vitro and in vivo and assessed its role in matrix mineralization in vitro. The bone specificity and shown role in mineralization led us to rename the gene bone restricted ifitm-like protein (Bril). Bril encodes a 14.8-kDa 134 amino acid protein with two transmembrane domains. Northern blot analysis showed bone-specific expression with no expression in other embryonic or adult tissues. In situ hybridization and immunohistochemistry in mouse embryos showed expression localized on the developing bone. Screening of cell lines showed Bril expression to be highest in osteoblasts, associated with the onset of matrix maturation/mineralization, suggesting a role in bone formation. Functional evidence of a role in mineralization was shown by adenovirus-mediated Bril overexpression and lentivirus-mediated Bril shRNA knockdown in vitro. Elevated Bril resulted in dose-dependent increases in mineralization in UMR106 and rat primary osteoblasts. Conversely, knockdown of Bril in MC3T3 osteoblasts resulted in reduced mineralization. Thus, we identified Bril as a novel osteoblast protein and showed a role in mineralization, possibly identifying a new regulatory pathway in bone formation.

The chloroplasts of most dinoflagellates are unusual in that they are surrounded by three membranes and contain the carotenoid peridinin. The ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO) in dinoflagellate chloroplasts was found here to also be unusual. Unlike other eukaryotes, dinoflagellates containing peridinin use a form of RuBisCO (form II) previously found only in some species of proteobacteria. Furthermore, this RuBisCO is not encoded in the chloroplast DNA, as is the case in other organisms, but is encoded by the nuclear DNA. The unusual nature of this enzyme and location of its gene support the idea that dinoflagellate chloroplasts may have had a distinctive evolutionary origin.

Although a number of secreted factors have been demonstrated to be bone regulators, none of these are unique to bone. Using a viral-based signal-trap strategy we have identified a novel gene we have termed "osteocrin." A 1280-bp mRNA encodes osteocrin producing a mature protein of 103 amino acids with a molecular mass of 11.4 kDa. Osteocrin shows no homology with any known gene except for two conserved sequence motifs reminiscent of dibasic cleavage sites found in peptide hormone precursors. Immunofluorescence and Western blot analysis confirmed the secretory nature of osteocrin. Two protein species were identified in the medium of cells overexpressing osteocrin, a full-length 11.4 kDa species and a processed ϳ5 kDa species. Mutation of the 76 KKKR 79 dibasic cleavage site abolished the appearance of this smaller osteocrin fragment. By in situ hybridization in mouse embryos, osteocrin was expressed specifically in Cbfa-1-positive, osteocalcin-negative osteoblasts. Immunohistochemistry on adult mouse bone showed osteocrin localization in osteoblasts and young osteocytes. By Northern blot analysis, osteocrin expression was only detected in bone, expression peaking just after birth and decreasing markedly with age. In primary osteoblastic cell cultures osteocrin expression coincided with matrix formation then decreased in very mature cultures. Treatment of cultures with 1,25-dihydroxyvitamin D 3 resulted in a rapid dose-dependent down-regulation of osteocrin expression, suggesting direct regulation. Chronic treatment of primary cultures with osteocrin-conditioned media inhibited mineralization and reduced osteocalcin and alkaline phosphatase expression. These results suggest that osteocrin represents a novel, unique vitamin D-regulated bone-specific protein that appears to act as a soluble osteoblast regulator.Bone is a dynamic tissue that is continually being modeled and remodeled through the coordinated actions of the bone forming osteoblasts and bone resorbing osteoclasts. Such remodeling is necessary to respond to the continually changing mechanical and regulatory demands placed upon the skeleton (1-4). The balance of the activity of the osteoblasts and osteoclasts is tightly regulated at both the systemic and local levels through the actions of a number of secreted molecules. Systemically, calciotropic hormones such as 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) 1 and parathyroid hormone (PTH) can regulate both bone formation and resorption, acting on both osteoclastic and osteoblastic cell lineages (5-8), whereas 1,25(OH) 2 D 3 also acts via the intestine and kidney to regulate systemic calcium and phosphate availability (9, 10). Other hormones, such as growth hormone (11), estrogens (12), and thyroid hormone (13) also influence bone mass. More recently, it has been reported that the peptide hormone leptin exerts a strong systemic antiosteogenic effect when activating the sympathetic nervous system through a hypothalamic relay (14, 15).Remodeling occurs in discrete units, termed basic multicellular units, con...

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tested (L100I, K103N/Y181C, V106A, or Y188L). Notably, viruses encoding K65R were hypersusceptible to inhibition by compound A. Compound A also retained full activity against viruses encoding M184V. In vitro selection for resistant virus to compound A led to the selection of a single substitution within RT: W153L. A recombinant virus encoding the RT W153L was highly resistant to compound A (fold change, 160). W153 is a highly conserved residue in HIV RT and has not been previously associated with drug resistance. In summary, a novel NcRTI series with optimized antiviral activity, minimal cross-resistance to existing RT inhibitor classes, and a distinct resistance profile has been discovered. These results further establish NcRTIs as an emerging class of antiretroviral agents.

BackgroundCoexpression of CD160 and PD-1 on HIV-specific CD8+ T-cells defines a highly exhausted T-cell subset. CD160 binds to Herpes Virus Entry Mediator (HVEM) and blocking this interaction with HVEM antibodies reverses T-cell exhaustion. As HVEM binds both inhibitory and activatory receptors, our aim in the current study was to assess the impact of CD160-specific antibodies on the enhancement of T-cell activation.MethodsExpression of the two CD160 isoforms; glycosylphosphatidylinositol-anchored (CD160-GPI) and the transmembrane isoforms (CD160-TM) was assessed in CD4 and CD8 primary T-cells by quantitative RT-PCR and Flow-cytometry. Binding of these isoforms to HVEM ligand and the differential capacities of CD160 and HVEM specific antibodies to inhibit this binding were further evaluated using a Time-Resolved Fluorescence assay (TRF). The impact of both CD160 and HVEM specific antibodies on enhancing T-cell functionality upon antigenic stimulation was performed in comparative ex vivo studies using primary cells from HIV-infected subjects stimulated with HIV antigens in the presence or absence of blocking antibodies to the key inhibitory receptor PD-1.ResultsWe first show that both CD160 isoforms, CD160-GPI and CD160-TM, were expressed in human primary CD4+ and CD8+ T-cells. The two isoforms were also recognized by the HVEM ligand, although this binding was less pronounced with the CD160-TM isoform. Mechanistic studies revealed that although HVEM specific antibodies blocked its binding to CD160-GPI, surprisingly, these antibodies enhanced HVEM binding to CD160-TM, suggesting that potential antibody-mediated HVEM multimerization and/or induced conformational changes may be required for optimal CD160-TM binding. Triggering of CD160-GPI over-expressed on Jurkat cells with either bead-bound HVEM-Fc or anti-CD160 monoclonal antibodies enhanced cell activation, consistent with a positive co-stimulatory role for CD160-GPI. However, CD160-TM did not respond to this stimulation, likely due to the lack of optimal HVEM binding. Finally, ex vivo assays using PBMCs from HIV viremic subjects showed that the use of CD160-GPI-specific antibodies combined with blockade of PD-1 synergistically enhanced the proliferation of HIV-1 specific CD8+ T-cells upon antigenic stimulation.ConclusionsAntibodies targeting CD160-GPI complement the blockade of PD-1 to enhance HIV-specific T-cell responses and warrant further investigation in the development of novel immunotherapeutic approaches.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-014-0217-y) contains supplementary material, which is available to authorized users.

We have developed a functional genomics tool to identify the subset of cDNAs encoding secreted and membrane-bound proteins within a library (the 'secretome'). A Sindbis virus replicon was engineered such that the envelope protein precursor no longer enters the secretory pathway. cDNA fragments were fused to the mutant precursor and expression screened for their ability to restore membrane localization of envelope proteins. In this way, recombinant replicons were released within infectious viral particles only if the cDNA fragment they contain encodes a secretory signal. By using engineered viral replicons to selectively export cDNAs of interest in the culture medium, the methodology reported here efficiently filters genetic information in mammalian cells without the need to select individual clones. This adaptation of the 'signal trap' strategy is highly sensitive (1/200 000) and efficient. Indeed, of the 2546 inserts that were retrieved after screening various libraries, more than 97% contained a putative signal peptide. These 2473 clones encoded 419 unique cDNAs, of which 77% were previously annotated. Of the 94 cDNAs encoding proteins of unknown function, 24% either had no match in databases or contained a secretory signal that could not be predicted from electronic data.