Cryo-EM structures elucidate the multiligand receptor nature of megalin.

Megalin (low-density lipoprotein receptor-related protein 2) is a giant glycoprotein of about 600 kDa, mediating the endocytosis of more than 60 ligands, including those of proteins, peptides, and drug compounds [S. Goto, M. Hosojima, H. Kabasawa, A. Saito, Int. J. Biochem. Cell Biol. 157, 106393 (2023)]. It is expressed predominantly in renal proximal tubule epithelial cells, as well as in the brain, lungs, eyes, inner ear, thyroid gland, and placenta. Megalin is also known to mediate the endocytosis of toxic compounds, particularly those that cause renal and hearing disorders [Y. Hori et al., J. Am. Soc. Nephrol. 28, 1783-1791 (2017)]. Genetic megalin deficiency causes Donnai-Barrow syndrome/facio-oculo-acoustico-renal syndrome in humans. However, it is not known how megalin interacts with such a wide variety of ligands and plays pathological roles in various organs. In this study, we elucidated the dimeric architecture of megalin, purified from rat kidneys, using cryoelectron microscopy. The maps revealed the densities of endogenous ligands bound to various regions throughout the dimer, elucidating the multiligand receptor nature of megalin. We also determined the structure of megalin in complex with receptor-associated protein, a molecular chaperone for megalin. The results will facilitate further studies on the pathophysiology of megalin-dependent multiligand endocytic pathways in multiple organs and will also be useful for the development of megalin-targeted drugs for renal and hearing disorders, Alzheimer's disease [B. V. Zlokovic et al., Proc. Natl. Acad. Sci. U.S.A. 93, 4229-4234 (1996)], and other illnesses.

Active Release of eCIRP via Gasdermin D Channels to Induce Inflammation in Sepsis.

Extracellular cold-inducible RNA binding protein (eCIRP) is an inflammatory mediator that causes inflammation and tissue injury in sepsis. Gasdermin D (GSDMD) is a protein that, when cleaved, forms pores in the cell membrane, releasing intracellular contents into the extracellular milieu to exacerbate inflammation. We hypothesize that eCIRP is released actively from viable macrophages via GSDMD pores. We found that LPS induced eCIRP secretion from macrophages into the extracellular space. LPS significantly increased the expression of caspase-11 and cleavage of the GSDMD, as evidenced by increased N-terminal GSDMD expression in RAW 264.7 cells and mouse primary peritoneal macrophages. GSDMD inhibitor disulfiram decreased eCIRP release in vitro. Treatment with glycine to prevent pyroptosis-induced cell lysis did not significantly decrease eCIRP release from LPS-treated macrophages, indicating that eCIRP was actively released and was independent of pyroptosis. Downregulation of GSDMD gene expression by siRNA transfection suppressed eCIRP release in vitro after LPS stimulation. Moreover, GSDMD-/- peritoneal macrophages and mice had decreased levels of eCIRP in the culture supernatants and in blood treated with LPS in vitro and in vivo, respectively. GSDMD inhibitor disulfiram inhibited serum levels of eCIRP in endotoxemia and cecal ligation and puncture-induced sepsis. We conclude that eCIRP release from living macrophages is mediated through GSDMD pores, suggesting that targeting GSDMD could be a novel and potential therapeutic approach to inhibit eCIRP-mediated inflammation in sepsis.

Crack Detecting Method Based on Grid-Type Sensing Networks Using Electrical Signals.

Cracks have a primary effect on the failure of a structure. Therefore, the development of crack sensors with high accuracy and resolution and cracks detection method are important. In this study, the crack sensors were fabricated, and the crack locations were detected with the electrical signal of the crack sensor. First, a metal grid-type micro-crack sensor based on silver was fabricated. The sensor is made with electrohydrodynamics (EHD) inkjet printing technology, which is well known as the next generation of printed electronics technology. Optimal printing conditions were established through experiments, and a grid sensor was obtained. After that, single cracks and multiple cracks were simulated on the sensor, and electrical signals generated from the sensor were measured. The measured electrical signal tracked the location of the cracks in three steps: simple cross-calculation, interpolation, and modified P-SPICE. It was confirmed that cracks could be effectively found and displayed using the method presented in this paper.

Neutrophil trogocytosis during their trans-endothelial migration: role of extracellular CIRP.

BACKGROUND: Neutrophils are the most abundant innate immune cells in the circulating blood, and they act as the first responder against bacterial and fungal infection. However, accumulation of activated neutrophils can cause severe inflammation and tissue damage. Recently, neutrophil trogocytosis or membrane transfer with neighboring cells was reported to modulate immune responses. Extracellular cold-inducible RNA binding protein (eCIRP) is a newly identified damage-associated molecular pattern (DAMP). eCIRP can activate neutrophils to be more pro-inflammatory. This study aimed to identify the role of eCIRP in neutrophil trogocytosis during their trans-endothelial migration. METHODS: A trans-endothelial migration (TEM) assay using bone marrow neutrophils and mouse primary lung vascular endothelial cells was conducted using transwell chambers and neutrophil trogocytosis was assessed in vitro. In an in vivo mouse model of acute lung injury, neutrophil trogocytosis was assessed from bronchoalveolar lavage fluid. RESULTS: In TEM assay, the trogocytosis of neutrophils occurred during trans-endothelial migration and eCIRP significantly increased the percentage of these neutrophils. The trogocytosed neutrophils acquired the endothelial membrane containing junctional adhesion molecule-C (JAM-C) and VE-cadherin, and these membrane patches were polarized by Mac-1 binding. Furthermore, eCIRP-induced JAM-C positive trogocytosed neutrophils are more pro-inflammatory than the JAM-C negative counterpart. JAM-C positive trogocytosed neutrophils were also observed in the bronchoalveolar lavage fluid of a mouse model of acute lung injury. CONCLUSION: These data suggest that during the paracellular trans-endothelial migration of neutrophils in response to inflammation, eCIRP induces trogocytosis of neutrophils, and the trogocytosed neutrophils exhibit an exaggerated pro-inflammatory phenotype promoting acute lung injury.

Virtually-Planned Orthognathic Surgery Achieves an Accurate Condylar Position.

PURPOSE: Accuracy in orthognathic surgery with virtual planning has been reported, but detailed analysis of accuracy according to anatomic location, including the mandibular condyle, is insufficient. The purpose of this study was to compare the virtual plan and surgical outcomes and analyze the degree and distribution of errors according to each anatomic location. PATIENTS AND METHODS: This retrospective cohort study evaluated skeletal class III patients, treated with bimaxillary surgery. The primary predictor was anatomic locations that consisted of right and left condyles, maxilla, and the distal segment of the mandible. Other variables were age and gender. The primary outcome was surgical accuracy, defined as mean 3-dimensional distance error, mean absolute error, and mean error along the horizontal, vertical, and anteroposterior axes between the virtual plan and surgical outcomes. Landmarks were compared using a computational method based on affine transformation with a 1-time landmark setting. The mean errors were visualized with multidimensional scattergrams. Bivariate and regression statistics were computed. RESULTS: This study included 52 patients, 26 men and 26 women, with a mean age of 21 years and 3 months. The mean 3D distance errors for condylar landmarks, maxillary landmarks, and landmarks on the distal segment of the mandible were 1.03, 1.25, and 2.24 mm, respectively. Condylar landmarks, maxillary landmarks, and the landmarks on the distal segment of the mandible were positioned at 0.49 mm inferior, 0.28 mm anterior, and 1.25 mm inferior, respectively. The landmark errors for the distal segment of the mandible exhibited a wider distribution than those for condylar and maxillary landmarks. CONCLUSIONS: Agreement between the planned and actual outcome aided by virtual surgical planning was highest for the condyles, followed by the maxilla, and the distal segment of the mandible. It is important to consider the tendency for surgical errors in each anatomic location during operations.

After-Death Functions of Cell Death.

Cell death can occur through numerous regulated mechanisms, from apoptosis to necrosis, entosis, and others. Each has a distinct mode of regulation and effect on tissue homeostasis. While the elimination of individual cells is typically considered the relevant physiologic endpoint of cell death, in some cases the remnants left behind by death can also function to support tissue homeostasis. Here we discuss specific functions of the end products of cell death, and how "after-death" functions may contribute to the roles of programmed cell death in physiology.

Cutting Edge: Drebrin-Regulated Actin Dynamics Regulate IgE-Dependent Mast Cell Activation and Allergic Responses.

Mast cells play critical roles in allergic responses. Calcium signaling controls the function of these cells, and a role for actin in regulating calcium influx into cells has been suggested. We have previously identified the actin reorganizing protein Drebrin as a target of the immunosuppressant 3,5-bistrifluoromethyl pyrazole, which inhibits calcium influx into cells. In this study, we show that Drebrin(-/-) mice exhibit reduced IgE-mediated histamine release and passive systemic anaphylaxis, and Drebrin(-/-) mast cells also exhibit defects in FcεRI-mediated degranulation. Drebrin(-/-) mast cells exhibit defects in actin cytoskeleton organization and calcium responses downstream of the FcεRI, and agents that relieve actin reorganization rescue mast cell FcεRI-induced degranulation. Our results indicate that Drebrin regulates the actin cytoskeleton and calcium responses in mast cells, thus regulating mast cell function in vivo.

Postoperative Three-Dimensional Evaluation of Mandibular Contouring Surgery Using Computer-Assisted Simulation Planning and a Three-Dimensional-Printed Surgical Guide.

Mandibular contouring surgery was performed using computer-assisted simulation planning (CASP) and 3-dimensional printed surgical guide. The outcome of the surgery was evaluated by overlapping preoperative image. The patient underwent mandibular contouring surgery according to CASP for his residual facial asymmetry of the mandibular angle and mental area. The overall facial aesthetic of the patient was improved. In the overlapping image, the left mandibular border area was slightly overcorrected. However, the other portion was operated as planned. The overcorrection was due to the improper adaptation of the surgical guide adjacent to the mental foramen. In conclusion, usage of CASP and a surgical guide could reduce operation time and increase the accuracy of the operation. However, the design of the stent should be improved around the mental foramen to avoid nerve damage and improper adaptation.

Allele-sensitive mutant, Itkas, reveals that Itk kinase activity is required for Th1, Th2, Th17, and iNKT-cell cytokine production.

Itk(-/-) mice exhibit defects in the activation, development, and function of CD4(+) and CD8(+) T cells and iNKT cells. These and other defects in these mice make it difficult to uncouple the developmental versus functional requirement of Itk signaling. Here, we report an allele-sensitive mutant of Itk (Itkas) whose catalytic activity can be selectively inhibited by analogs of the PP1 kinase inhibitor. We show that Itkas behaves like WT Itk in the absence of the inhibitor and can rescue the development of Itk(-/-) T cells in mice. Using mice carrying Itkas, we show using its inhibitor that Itk activity is required not only for Th2, Th17, and iNKT-cell cytokine production, but also surprisingly, for Th1 cytokine production. This work has important implications for understanding the role of Itk signaling in the development versus function of iNKT cells, Th1, Th2, and Th17 cells.

Hydroxyapatite and silk combination-coated dental implants result in superior bone formation in the peri-implant area compared with hydroxyapatite and collagen combination-coated implants.

PURPOSE: The objective of this study was to compare bone formation after installation of uncoated (UC), hydroxyapatite-coated (HA), collagen plus HA-coated (CH), and silk plus HA-coated (SH) implants. MATERIALS AND METHODS: Implants in the UC group had acid-etched surfaces. Surface coating was applied using the aerosol deposition method. Cellular responses on the coated surfaces were examined with scanning electron microscopy. Cellular responses to the surfaces were studied with the corresponding coated discs and MG63 cells. Subsequently, 3-(4, 5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and alkaline phosphatase (ALP) assays were performed. Peri-implant bone formation was evaluated with the rabbit tibia model. Twenty-four implants from each group were installed. The animals were sacrificed 6 weeks after implant installation. Peri-implant bone formation and implant-to-bone contact were measured in histologic sections. Significance of differences across groups was evaluated using analysis of variance. RESULTS: Scanning electron microscopic images showed that the CH and SH groups exhibited cells that appeared more spread out than those in the other groups. The SH group exhibited the highest value in the MTT assay. The CH group exhibited the highest level of ALP activity. Comparisons of these modifications with the acid-etched surfaces showed that the CH and SH groups displayed significantly greater peri-implant bone formation (P < .001). CONCLUSION: The SH group displayed significantly greater new bone formation and bone-to-implant contact than did the other groups.

Extracellular CIRP promotes Kupffer cell inflammatory polarization in sepsis.

Introduction: Sepsis is a life-threatening inflammatory condition caused by dysregulated host responses to infection. Extracellular cold-inducible RNA-binding protein (eCIRP) is a recently discovered damage-associated molecular pattern that causes inflammation and organ injury in sepsis. Kupffer cells can be activated and polarized to the inflammatory M1 phenotype, contributing to tissue damage by producing proinflammatory mediators. We hypothesized that eCIRP promotes Kupffer cell M1 polarization in sepsis. Methods: We stimulated Kupffer cells isolated from wild-type (WT) and TLR4-/- mice with recombinant mouse (rm) CIRP (i.e., eCIRP) and assessed supernatant IL-6 and TNFα levels by ELISA. The mRNA expression of iNOS and CD206 for M1 and M2 markers, respectively, was assessed by qPCR. We induced sepsis in WT and CIRP-/- mice by cecal ligation and puncture (CLP) and assessed iNOS and CD206 expression in Kupffer cells by flow cytometry. Results: eCIRP dose- and time-dependently increased IL-6 and TNFα release from WT Kupffer cells. In TLR4-/- Kupffer cells, their increase after eCIRP stimulation was prevented. eCIRP significantly increased iNOS gene expression, while it did not alter CD206 expression in WT Kupffer cells. In TLR4-/- Kupffer cells, however, iNOS expression was significantly decreased compared with WT Kupffer cells after eCIRP stimulation. iNOS expression in Kupffer cells was significantly increased at 20 h after CLP in WT mice. In contrast, Kupffer cell iNOS expression in CIRP-/- mice was significantly decreased compared with WT mice after CLP. CD206 expression in Kupffer cells was not different across all groups. Kupffer cell M1/M2 ratio was significantly increased in WT septic mice, while it was significantly decreased in CIRP-/- mice compared to WT mice after CLP. Conclusion: Our data have clearly shown that eCIRP induces Kupffer cell M1 polarization via TLR4 pathway in sepsis, resulting in overproduction of inflammatory cytokines. eCIRP could be a promising therapeutic target to attenuate inflammation by preventing Kupffer cell M1 polarization in sepsis.

The unique allosteric property of crocodilian haemoglobin elucidated by cryo-EM.

The principal effect controlling the oxygen affinity of vertebrate haemoglobins (Hbs) is the allosteric switch between R and T forms with relatively high and low oxygen affinity respectively. Uniquely among jawed vertebrates, crocodilians possess Hb that shows a profound drop in oxygen affinity in the presence of bicarbonate ions. This allows them to stay underwater for extended periods by consuming almost all the oxygen present in the blood-stream, as metabolism releases carbon dioxide, whose conversion to bicarbonate and hydrogen ions is catalysed by carbonic anhydrase. Despite the apparent universal utility of bicarbonate as an allosteric regulator of Hb, this property evolved only in crocodilians. We report here the molecular structures of both human and a crocodilian Hb in the deoxy and liganded states, solved by cryo-electron microscopy. We reveal the precise interactions between two bicarbonate ions and the crocodilian protein at symmetry-related sites found only in the T state. No other known effector of vertebrate Hbs binds anywhere near these sites.

New bone formation between bare titanium surface and hydroxyapatite coating by the aerosol deposition technique in the nasal mucosal penetration model.

OBJECTIVE: The aim of this study was to compare new bone formation with titanium (Ti) surface and hydroxyapatite (HA)-coated titanium surface in mucosal perforation model. MATERIALS AND METHODS: HA coating to the Ti disc and implant were done by aerosol deposition technique. Alkaline phosphatase assay and cell migration assay were done in Ti and HA surface disc with MG63 cells. For the in vivo test, 5 New Zealand white rabbits were used. Two penetration defects were prepared in the nasal bone. Subsequently, 2 types of implants were installed into the defect (diameter: 3.0 mm, length: 6.0 mm). Approximately 5.0 mm of the fixture's surface penetrated into the nasal cavity. In the experimental group, HA-coated implants were used. The same design of implants without coating was used in the control group. The animals were sacrificed 8 weeks postoperatively. Subsequently, a histomorphometric analysis was done. RESULTS: Alkaline phosphatase activity was significantly higher in HA-coated surface than in titanium surface (P < 0.05). In addition, more cells were migrated into the HA-coated surface when compared to Ti surface. In the animal experiments, mean new bone formation was 30.68 ± 14.16% in the experimental group and 6.92 ± 5.12% in the control group (P = 0.001). Mean bone-to-implant contact was 31.71 ± 8.41% in the experimental group and 7.98 ± 5.58% in the control group (P < 0.001). Mean height of the bone regeneration was 3.70 ± 0.76 mm in the experimental group and 1.04 ± 0.67 mm in the control group. The difference between the 2 groups was statistically significant (P < 0.001). CONCLUSIONS: HA-coated implants exhibited more bone regeneration in the mucosal penetration model than the uncoated implants.

Redefining precision and efficiency in orthognathic surgery through virtual surgical planning and 3D printing: a narrative review.

Orthognathic surgery, essential for addressing jaw and facial skeletal irregularities, has historically relied on traditional surgical planning (TSP) involving a series of time-consuming steps including two-dimensional radiographs. The advent of virtual surgical planning (VSP) and 3D printing technologies has revolutionized this field, bringing unprecedented precision and customization to surgical processes. VSP facilitates 3D visualization of the surgical site, allowing for real-time adjustments and improving preoperative stress for patients by reducing planning time. 3D printing dovetails with VSP, offering the creation of anatomical models and surgical guides, enhancing the predictability of surgical outcomes despite higher initial setup and material costs. The integration of VSP and 3D printing promises innovative and effective solutions in orthognathic surgery, surpassing the limitations of traditional methods. Patient-reported outcomes show a positive post-surgery impact on the quality of life, underlining the significant role of these technologies in enhancing self-esteem and reducing anxiety. Economic analyses depict a promising long-term fiscal advantage with these modern technologies, notwithstanding the higher initial costs. The review emphasizes the need for large-scale randomized controlled trials to address existing research gaps and calls for a deeper exploration into the long-term impacts and ethical considerations of these technologies. In conclusion, while standing on the cusp of a technological renaissance in orthognathic surgery, it is incumbent upon the medical fraternity to foster a collaborative approach, balancing innovation with scrutiny to enhance patient care. The narrative review encourages the leveraging of VSP and 3D printing technologies for more efficient and patient-centric orthognathic surgery, urging the community to navigate uncharted territories in pursuit of precision and efficiency in the surgical landscape.

Extracellular CIRP Induces Calpain Activation in Neurons via PLC-IP3-Dependent Calcium Pathway.

Abnormal calcium homeostasis, activation of protease calpain, generation of p25 and hyperactivation of cyclin-dependent kinase 5 (Cdk5) have all been implicated in the pathogenesis of neurogenerative diseases including Alzheimer's disease. We have recently shown that extracellular cold-inducible RNA-binding protein (eCIRP) induces Cdk5 activation via p25. However, the precise molecular mechanism by which eCIRP regulates calcium signaling and calpain remains to be addressed. We hypothesized that eCIRP regulates p25 via Ca2+-dependent calpain activation. eCIRP increased calpain activity and decreased the endogenous calpain inhibitor calpastatin in Neuro 2a (N2a) cells. Calpain inhibition with calpeptin attenuated eCIRP-induced calpain activity and p25. eCIRP specifically upregulated cytosolic calpain 1, and calpain 1 silencing attenuated the eCIRP-induced increase in p25. eCIRP stimulation increased cytosolic free Ca2+, especially in hippocampal neuronal HT22 cells, which was attenuated by the eCIRP inhibitor Compound 23 (C23). Endoplasmic reticulum (ER) inositol 1,4,5-trisphosphate receptor (IP3R) inhibition using 2-aminoethoxy-diphenyl-borate or xestospongin-C (X-C), interleukin-6 receptor alpha (IL-6Rα)-neutralization, and phospholipase C (PLC) inhibition with U73122 attenuated eCIRP-induced Ca2+ increase, while Ca2+ influx across the plasma membrane remained unaffected by eCIRP. Finally, C23, IL-6Rα antibody, U73122 and X-C attenuated eCIRP-induced p25 in HT-22 cells. In conclusion, the current study uncovers eCIRP-triggered Ca2+ release from ER stores in an IL-6Rα/PLC/IP3-dependent manner as a novel molecular mechanism underlying eCIRP's induction of Cdk5 activity and potential involvement in neurodegeneration.

Evaluation of postoperative changes in condylar positions after orthognathic surgery using balanced orthognathic surgery system.

BACKGROUND: Many studies on maintaining the condyle in a normal or anatomical position during orthognathic surgery have been conducted to stabilize surgical outcomes and prevent iatrogenic temporomandibular joint complications. The aim of this study is to evaluate the changes in condylar positions after orthognathic surgery using virtual surgical planning via the balanced orthognathic surgery (BOS) system. METHODS: Postoperative changes in condylar position were retrospectively evaluated in 22 condyles of 11 patients with skeletal class III malocclusion who underwent orthognathic surgery using virtual surgical planning via the BOS system. The center point coordinates of the condylar head before and after orthognathic surgery were analyzed using voxel-based registration. RESULTS: Changes in the condylar position mainly occurred downward in the y-axis (-1.09 ± 0.62 mm) (P < 0.05). The change in the x-axis (0.02 ± 0.68 mm) and z-axis (0.01 ± 0.48 mm) showed no significant difference between before and after orthognathic surgery. CONCLUSION: These results indicate that the changes in the condylar positions after orthognathic surgery using virtual surgical planning via the BOS system mainly occurred downward in the y-axis, with slight changes in the x- and z-axes. The change in the condylar position after orthognathic surgery using the BOS system is clinically acceptable.

Ion transfer mechanisms in Mrp-type antiporters from high resolution cryoEM and molecular dynamics simulations.

Multiple resistance and pH adaptation (Mrp) cation/proton antiporters are essential for growth of a variety of halophilic and alkaliphilic bacteria under stress conditions. Mrp-type antiporters are closely related to the membrane domain of respiratory complex I. We determined the structure of the Mrp antiporter from Bacillus pseudofirmus by electron cryo-microscopy at 2.2 Å resolution. The structure resolves more than 99% of the sidechains of the seven membrane subunits MrpA to MrpG plus 360 water molecules, including ~70 in putative ion translocation pathways. Molecular dynamics simulations based on the high-resolution structure revealed details of the antiport mechanism. We find that switching the position of a histidine residue between three hydrated pathways in the MrpA subunit is critical for proton transfer that drives gated trans-membrane sodium translocation. Several lines of evidence indicate that the same histidine-switch mechanism operates in respiratory complex I.

Ca2+-mediated higher-order assembly of heterodimers in amino acid transport system b0,+ biogenesis and cystinuria.

Cystinuria is a genetic disorder characterized by overexcretion of dibasic amino acids and cystine, causing recurrent kidney stones and kidney failure. Mutations of the regulatory glycoprotein rBAT and the amino acid transporter b0,+AT, which constitute system b0,+, are linked to type I and non-type I cystinuria respectively and they exhibit distinct phenotypes due to protein trafficking defects or catalytic inactivation. Here, using electron cryo-microscopy and biochemistry, we discover that Ca2+ mediates higher-order assembly of system b0,+. Ca2+ stabilizes the interface between two rBAT molecules, leading to super-dimerization of b0,+AT-rBAT, which in turn facilitates N-glycan maturation and protein trafficking. A cystinuria mutant T216M and mutations of the Ca2+ site of rBAT cause the loss of higher-order assemblies, resulting in protein trapping at the ER and the loss of function. These results provide the molecular basis of system b0,+ biogenesis and type I cystinuria and serve as a guide to develop new therapeutic strategies against it. More broadly, our findings reveal an unprecedented link between transporter oligomeric assembly and protein-trafficking diseases.

Enhanced formation of disulfide-bridged dimer (Fab-PE38)2 utilizing repeats of the Fab binding domain of protein G.

Fab-PE38 used in this study is B3(Fab)-ext-PE38, and it is an antibody toxin that is made by fusing the Pseudomonas exotoxin to the Fab domain of B3 antibody. This antibody toxin selectively binds to cancer cells and kills the target cancer cells. B3(Fab)-ext-PE38 has a cysteine residue on the ext sequence, and (B3(Fab)-ext-PE38)(2) is the disulfide-bridged dimer of the B3(Fab)-ext-PE38 monomer. (B3(Fab)-ext-PE38)(2) has been found to have 11-fold higher cytotoxicity on the CRL-1739 cell line than monomeric B3(scFv)-PE38. We made a recombinant tandem repeat of the domain III of Streptococcal protein G that has Fab binding property up to seven repeats. Multiple monomers were found to form non-covalent complexes with this tandem repeat. Complexes were purified by size-exclusion chromatography, and we could enhance the production of the disulfide-bridged dimer by reduction and oxidation of the complexes. The tandem repeat makes close intermolecular interactions between monomers possible, and the use of it greatly enhances the yield of the disulfide-bridged dimer.

Aerosol deposition of hydroxyapatite and 4-hexylresorcinol coatings on titanium alloys for dental implants.

PURPOSE: Aerosol deposition is a newly developed technique, and it can deliver the drug from a hydroxyapatite (HA)-coated surface. 4-Hexylresorcinol (4-HR) is a well-known antiseptic. The influence of the 4-HR component of HA coatings on titanium surfaces was studied in vitro and in vivo. MATERIALS AND METHODS: X-ray diffraction and Fourier transform infrared techniques were used for the evaluation of the coating. The cellular response of the coating was evaluated by scanning electron microscopic study, MTT assay, alkaline phosphatase assay, and osteocalcin assay. In addition, the dental implant was coated with HA or HA + 4-HR. The implant was installed into the tibia of a rabbit after contamination by Aggregatibacter actinomycetemcomitans. The torque test and histologic analysis were then performed at 8 weeks after the operation. RESULTS: By use of an aerosol deposition technique, the combination of HA and 4-HR was successfully coated onto a titanium surface, which was confirmed by x-ray diffraction and Fourier transform infrared techniques. MG63 cells attached more rapidly to the HA + 4-HR coating than to the HA-only coating. The HA + 4-HR coating had significantly increased osteocalcin expression and alkaline phosphatase activity compared with the HA-only coating (P < .05). The dental implant coated with HA + 4-HR had a significantly higher removal torque value than that coated with HA alone at 8 weeks after surgery (P < .05). On histologic analysis, both the bone formation value and the bone-to-implant contact value were significantly higher in the HA + 4-HR group than in the HA-only group at 8 weeks after surgery (P < .05). CONCLUSIONS: Collectively, the HA + 4-HR-coated dental implant had clear advantages over the HA-coated dental implant. Therefore HA + 4-HR coatings can be considered for patients who need immediate implant installation after tooth extraction or who have poor-quality bone.