Despite substantial attention given to the development of osteoregenerative biomaterials, severe deficiencies remain in current products. These limitations include an inability to adequately, rapidly, and reproducibly regenerate new bone; high costs and limited manufacturing capacity; and lack of surgical ease of handling. To address these shortcomings, we generated a new, synthetic osteoregenerative biomaterial, hyperelastic “bone” (HB). HB, which is composed of 90 weight % (wt %) hydroxyapatite and 10 wt % polycaprolactone or poly(lactic-co-glycolic acid), could be rapidly three-dimensionally (3D) printed (up to 275 cm³/hour) from room temperature extruded liquid inks. The resulting 3D-printed HB exhibited elastic mechanical properties (~32 to 67% strain to failure, ~4 to 11 MPa elastic modulus), was highly absorbent (50% material porosity), supported cell viability and proliferation, and induced osteogenic differentiation of bone marrow–derived human mesenchymal stem cells cultured in vitro over 4 weeks without any osteo-inducing factors in the medium. We evaluated HB in vivo in a mouse subcutaneous implant model for material biocompatibility (7 and 35 days), in a rat posterolateral spinal fusion model for new bone formation (8 weeks), and in a large, non-human primate calvarial defect case study (4 weeks). HB did not elicit a negative immune response, became vascularized, quickly integrated with surrounding tissues, and rapidly ossified and supported new bone growth without the need for added biological factors.

Biological systems have evolved to utilize numerous proteins with capacity to bind polysaccharides for the purpose of optimizing their function. A well-known subset of these proteins with binding domains for the highly diverse sulfated polysaccharides are important growth factors involved in biological development and tissue repair. We report here on supramolecular sulfated glycopeptide nanostructures, which display a trisulfated monosaccharide on their surfaces and bind five critical proteins with different polysaccharide-binding domains. Binding does not disrupt the filamentous shape of the nanostructures or their internal β-sheet backbone, but must involve accessible adaptive configurations to interact with such different proteins. The glycopeptide nanostructures amplified signalling of bone morphogenetic protein 2 significantly more than the natural sulfated polysaccharide heparin, and promoted regeneration of bone in the spine with a protein dose that is 100-fold lower than that required in the animal model. These highly bioactive nanostructures may enable many therapies in the future involving proteins.

Background: The apoptotic speck-like protein containing a caspase recruitment domain (ASC) is the essential adaptor protein for caspase 1 mediated interleukin (IL)-1β and IL-18 processing in inflammasomes. It bridges activated Nod like receptors (NLRs), which are a family of cytosolic pattern recognition receptors of the innate immune system, with caspase 1, resulting in caspase 1 activation and subsequent processing of caspase 1 substrates. Hence, macrophages from ASC deficient mice are impaired in their ability to produce bioactive IL-1β. Furthermore, we recently showed that ASC translocates from the nucleus to the cytosol in response to inflammatory stimulation in order to promote an inflammasome response, which triggers IL-1β processing and secretion. However, the precise regulation of inflammasomes at the level of ASC is still not completely understood. In this study we identified and characterized three novel ASC isoforms for their ability to function as an inflammasome adaptor. Methods: To establish the ability of ASC and ASC isoforms as functional inflammasome adaptors, IL-1β processing and secretion was investigated by ELISA in inflammasome reconstitution assays, stable expression in THP-1 and J774A1 cells, and by restoring the lack of endogenous ASC in mouse RAW264.7 macrophages. In addition, the localization of ASC and ASC isoforms was determined by immunofluorescence staining. Results: The three novel ASC isoforms, ASC-b, ASC-c and ASC-d display unique and distinct capabilities to each other and to full length ASC in respect to their function as an inflammasome adaptor, with one of the isoforms even showing an inhibitory effect. Consistently, only the activating isoforms of ASC, ASC and ASC-b, co-localized with NLRP3 and caspase 1, while the inhibitory isoform ASC-c, co-localized only with caspase 1, but not with NLRP3. ASC-d did not co-localize with NLRP3 or with caspase 1 and consistently lacked the ability to function as an inflammasome adaptor and its precise function and relation to ASC will need further investigation. Conclusions: Alternative splicing and potentially other editing mechanisms generate ASC isoforms with distinct abilities to function as inflammasome adaptor, which is potentially utilized to regulate inflammasomes during the inflammatory host response.

Ubiquitin-like protein/sentrin-specific proteases (Ulp/SENPs) mediate both processing and deconjugation of small ubiquitin-like modifier proteins (SUMOs). Here, we show that Ulp/SENP family members SENP3 and SENP5 localize within the granular component of the nucleolus, a subnucleolar compartment that contains B23/nucleophosmin. B23/nucleophosmin is an abundant shuttling phosphoprotein, which plays important roles in ribosome biogenesis and which has been strongly implicated in hematopoietic malignancies. Moreover, we found that B23/nucleophosmin binds SENP3 and SENP5 in Xenopus laevis egg extracts and that it is essential for stable accumulation of SENP3 and SENP5 in mammalian tissue culture cells. After either codepletion of SENP3 and SENP5 or depletion of B23/nucleophosmin, we observed accumulation of SUMO proteins within nucleoli. Finally, depletion of these Ulp/SENPs causes defects in ribosome biogenesis reminiscent of phenotypes observed in the absence of B23/nucleophosmin. Together, these results suggest that regulation of SUMO deconjugation may be a major facet of B23/nucleophosmin function in vivo.

Multinucleated cells have been noted in pathophysiological states of the liver including infection with hepatitis B virus (HBV), the status of which is also closely associated with genomic instability in liver cancer. Here, we showed that hepatitis B virus X oncoprotein (HBx) expression in Chang cells results in a multinuclear phenotype and an abnormal number of centrosomes (n ≥ 3). Regulation of centrosome duplication in HBx-expressing ChangX-34 cells was defective and uncoupled from the cell cycle. HBx induced amplification of centrosomes, multipolar spindle formation, and chromosomal missegregation during mitosis and subsequently increased the generation of multinucleated cells and micronuclei formation. Treatment with PD98059, a mitogen-activated protein/extracellular signal-regulated kinase (MEK) 1/2 inhibitor, significantly reduced the number of cells with hyperamplified centrosomes and decreased the multinucleated cells and micronuclei formation. Consistently, the phospho-ERK level during cell progression was substantially higher in ChangX-34 cells than that of Chang cells. In contrast, neither wortmannin, an inhibitor of phosphoinositide-3 kinase, nor SB203589, an inhibitor of p38 mitogen-activated protein kinase (MAPK), showed any effects. Introduction of Ras dominant-negative (D/N) and MEK2 D/N genes into ChangX-34 cells significantly alleviated centrosome amplification, whereas introduction of the PKC D/N and PKB D/N genes did not. Thus, our results demonstrate that the HBx induced centrosome hyperamplification and mitotic aberration by activation of the Ras-MEK-MAPK. Intervention of this signaling pathway could suppress the centrosome amplification as well as mitotic aberration. These findings may provide a possible mechanism by which HBx promotes phenotypic progression by predisposing chromosomal alteration in HBV-infected liver.