Murine IRF8 Mutation Offers New Insight into Osteoclast and Root Resorption.

Interferon regulatory factor 8 (IRF8), a transcription factor expressed in immune cells, functions as a negative regulator of osteoclasts and helps maintain dental and skeletal homeostasis. Previously, we reported that a novel mutation in the IRF8 gene increases susceptibility to multiple idiopathic cervical root resorption (MICRR), a form of tooth root resorption mediated by increased osteoclast activity. The IRF8 G388S variant in the highly conserved C-terminal motif is predicted to alter the protein structure, likely impairing IRF8 function. To investigate the molecular basis of MICRR and IRF8 function in osteoclastogenesis, we generated Irf8 knock-in (KI) mice using CRISPR/Cas9 technique modeling the human IRF8G388S mutation. The heterozygous (Het) and homozygous (Homo) Irf8 KI mice showed no gross morphological defects, and the development of hematopoietic cells was unaffected and similar to wild-type (WT) mice. The Irf8 KI Het and Homo mice showed no difference in macrophage gene signatures important for antimicrobial defenses and inflammatory cytokine production. Consistent with the phenotype observed in MICRR patients, Irf8 KI Het and Homo mice demonstrated significantly increased osteoclast formation and resorption activity in vivo and in vitro when compared to WT mice. The oral ligature-inserted Het and Homo mice displayed significantly increased root resorption and osteoclast-mediated alveolar bone loss compared to WT mice. The increased osteoclastogenesis noted in KI mice is due to the inability of IRF8G388S mutation to inhibit NFATc1-dependent transcriptional activation and downstream osteoclast specific transcripts, as well as its impact on autophagy-related pathways of osteoclast differentiation. This translational study delineates the IRF8 domain important for osteoclast function and provides novel insights into the IRF8 mutation associated with MICRR. IRF8G388S mutation mainly affects osteoclastogenesis while sparing immune cell development and function. These insights extend beyond oral health and significantly advance our understanding of skeletal disorders mediated by increased osteoclast activity and IRF8's role in osteoclastogenesis.

Delivery of Alkaline Phosphatase Promotes Periodontal Regeneration in Mice.

Factors regulating the ratio of pyrophosphate (PPi) to phosphate (Pi) modulate biomineralization. Tissue-nonspecific alkaline phosphatase (TNAP) is a key promineralization enzyme that hydrolyzes the potent mineralization inhibitor PPi. The goal of this study was to determine whether TNAP could promote periodontal regeneration in bone sialoprotein knockout mice (Ibsp-/- mice), which are known to have a periodontal disease phenotype. Delivery of TNAP was accomplished either systemically (through a lentiviral construct expressing a mineral-targeted TNAP-D10 protein) or locally (through addition of recombinant human TNAP to a fenestration defect model). Systemic TNAP-D10 delivered by intramuscular injection at 5 d postnatal (dpn) increased circulating alkaline phosphatase (ALP) levels in Ibsp-/- mice by 5-fold at 30 dpn, with levels returning to normal by 60 dpn when tissues were evaluated by micro-computed tomography and histology. Local delivery of recombinant human TNAP to fenestration defects in 5-wk-old wild type (WT) and Ibsp-/- mice did not alter long-term circulating ALP levels, and tissues were evaluated by micro-computed tomography and histology at postoperative day 45. Systemic and local delivery of TNAP significantly increased alveolar bone volume (20% and 37%, respectively) and cementum thickness (3- and 42-fold) in Ibsp-/- mice, with evidence for periodontal ligament attachment and bone/cementum marker localization. Local delivery significantly increased regenerated cementum and bone in WT mice. Addition of 100-µg/mL bovine intestinal ALP to culture media to increase ALP in vitro increased media Pi concentration, mineralization, and Spp1 and Dmp1 marker gene expression in WT and Ibsp-/- OCCM.30 cementoblasts. Use of phosphonoformic acid, a nonspecific inhibitor of sodium Pi cotransport, indicated that effects of bovine intestinal ALP on mineralization and marker gene expression were in part through Pi transport. These findings show for the first time through multiple in vivo and in vitro approaches that pharmacologic modulation of Pi/PPi metabolism can overcome periodontal breakdown and accomplish regeneration.

Ablation of Pyrophosphate Regulators Promotes Periodontal Regeneration.

Biomineralization is regulated by inorganic pyrophosphate (PPi), a potent physiological inhibitor of hydroxyapatite crystal growth. Progressive ankylosis protein (ANK) and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) act to increase local extracellular levels of PPi, inhibiting mineralization. The periodontal complex includes 2 mineralized tissues, cementum and alveolar bone (AB), both essential for tooth attachment. Previous studies demonstrated that loss of function of ANK or ENPP1 (reducing PPi) resulted in increased cementum formation, suggesting PPi metabolism may be a target for periodontal regenerative therapies. To compare the effects of genetic ablation of Ank, Enpp1, and both factors concurrently on cementum and AB regeneration, mandibular fenestration defects were created in Ank knockout (Ank KO), Enpp1 mutant (Enpp1asj/asj), and double KO (dKO) mice. Genetic ablation of Ank, Enpp1, or both factors increased cementum regeneration compared to controls at postoperative days (PODs) 15 and 30 (Ank KO: 8-fold, 3-fold; Enpp1asj/asj: 7-fold, 3-fold; dKO: 11-fold, 4-fold, respectively) associated with increased fluorochrome labeling and expression of mineralized tissue markers, dentin matrix protein 1 (Dmp1/DMP1), osteopontin (Spp1/OPN), and bone sialoprotein (Ibsp/BSP). Furthermore, dKO mice featured increased cementum thickness compared to single KOs at POD15 and Ank KO at POD30. No differences were noted in AB volume between genotypes, but osteoblast/osteocyte markers were increased in all KOs, partially mineralized osteoid volume was increased in dKO versus controls at POD15 (3-fold), and mineral density was decreased in Enpp1asj/asj and dKOs at POD30 (6% and 9%, respectively). Increased numbers of osteoclasts were present in regenerated AB of all KOs versus controls. These preclinical studies suggest PPi modulation as a potential and novel approach for cementum regeneration, particularly targeting ENPP1 and/or ANK. Differences in cementum and AB regeneration in response to reduced PPi conditions highlight the need to consider tissue-specific responses in strategies targeting regeneration of the entire periodontal complex.

Phylogenetic relationships of living and recently extinct bandicoots based on nuclear and mitochondrial DNA sequences.

Bandicoots (Peramelemorphia) are a major order of australidelphian marsupials, which despite a fossil record spanning at least the past 25 million years and a pandemic Australasian range, remain poorly understood in terms of their evolutionary relationships. Many living peramelemorphians are critically endangered, making this group an important focus for biological and conservation research. To establish a phylogenetic framework for the group, we compiled a concatenated alignment of nuclear and mitochondrial DNA sequences, comprising representatives of most living and recently extinct species. Our analysis confirmed the currently recognised deep split between Macrotis (Thylacomyidae), Chaeropus (Chaeropodidae) and all other living bandicoots (Peramelidae). The mainly New Guinean rainforest peramelids were returned as the sister clade of Australian dry-country species. The wholly New Guinean Peroryctinae was sister to Echymiperinae. The poorly known and perhaps recently extinct Seram Bandicoot (Rhynchomeles) is sister to Echymipera. Estimates of divergence times from relaxed-clock Bayesian methods suggest that living bandicoots originated in the late Oligocene or early Miocene, much earlier than currently thought based on fossils. Subsequent radiations within Peramelemorphia probably took place on the Australian mainland during the Miocene, with diversification of rainforest taxa on the newly emergent New Guinean landmasses through the middle-late Miocene and complete establishment of modern lineages by the early Pliocene.

Aircraft gas turbine materials and processes.

Materials and processing innovations that have been incorporated into the manufacture of critical components for high-performance aircraft gas turbine engines are described. The materials of interest are the nickel- and cobalt-base superalloys for turbine and burner sections of the engine, and titanium alloys and composites for compressor and fan sections of the engine. Advanced processing methods considered include directional solidification, hot isostatic pressing, superplastic foring, directional recrystallization, and diffusion brazing. Future trends in gas turbine technology are discussed in terms of materials availability, substitution, and further advances in air-cooled hardware.