Complement C1q activates canonical Wnt signaling and promotes aging-related phenotypes.

Wnt signaling plays critical roles in development of various organs and pathogenesis of many diseases, and augmented Wnt signaling has recently been implicated in mammalian aging and aging-related phenotypes. We here report that complement C1q activates canonical Wnt signaling and promotes aging-associated decline in tissue regeneration. Serum C1q concentration is increased with aging, and Wnt signaling activity is augmented during aging in the serum and in multiple tissues of wild-type mice, but not in those of C1qa-deficient mice. C1q activates canonical Wnt signaling by binding to Frizzled receptors and subsequently inducing C1s-dependent cleavage of the ectodomain of Wnt coreceptor low-density lipoprotein receptor-related protein 6. Skeletal muscle regeneration in young mice is inhibited by exogenous C1q treatment, whereas aging-associated impairment of muscle regeneration is restored by C1s inhibition or C1qa gene disruption. Our findings therefore suggest the unexpected role of complement C1q in Wnt signal transduction and modulation of mammalian aging.

Effects of structural changes on antibacterial activity and cytotoxicity due to proline substitutions in chimeric peptide HnMc.

Owing to the rapidly increasing emergence of multidrug-resistant pathogens, antimicrobial peptides (AMPs) are being explored as next-generation antibiotics. However, AMPs present in nature are highly toxic and exhibit low antibacterial activity. Simple modifications, such as amino acid substitution, can enhance antimicrobial activity and cell selectivity. Herein, we show that HnMc-W, substituted by the Phe1Trp analog of HnMc, a chimeric peptide, resulted in membranolytic antibacterial action and enhanced salt tolerance, whereas HnMc-WP1 with one Ser9Pro substitution resulted in a proline-kink helical structure that increased salt-tolerant antibacterial effects and reduced cytotoxicity. In addition, the HnMc-WP2 peptide, designed with a PXXP motif, had a flexible central hinge in its α-helical structure due to the introduction of two Pro and two Gln (X positions, by deletion of two Gln at positions 16 and 17) residues instead of Ser at position. HnMc-WP2 exhibited excellent antibacterial effects without cytotoxicity in vitro. Moreover, its potent antibacterial activity was demonstrated in a drug-resistant Pseudomonas aeruginosa-infected mouse model in vivo. Our findings provide valuable information for the design of peptides with high antibacterial activity and cell selectivity.

Aberrant accumulation of TMEM43 accompanied by perturbed transmural gene expression in arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) caused by TMEM43 p.S358L is a fully penetrant heart disease that results in impaired cardiac function or fatal arrhythmia. However, the molecular mechanism of ACM caused by the TMEM43 variant has not yet been fully elucidated. In this study, we generated knock-in (KI) rats harboring a Tmem43 p.S358L mutation and established induced pluripotent stem cells (iPSCs) from patients based on the identification of TMEM43 p.S358L variant from a family with ACM. The Tmem43-S358L KI rats exhibited ventricular arrhythmia and fibrotic myocardial replacement in the subepicardium, which recapitulated the human ACM phenotype. The four-transmembrane protein TMEM43 with the p.S358L variant (TMEM43S358L ) was found to be modified by N-linked glycosylation in both KI rat cardiomyocytes and patient-specific iPSC-derived cardiomyocytes. TMEM43S358L glycosylation increased under the conditions of enhanced endoplasmic reticulum (ER) stress caused by pharmacological stimulation or age-dependent decline of the ER function. Intriguingly, the specific glycosylation of TMEM43S358L resulted from the altered membrane topology of TMEM43. Moreover, unlike TMEM43WT , which is mainly localized to the ER, TMEM43S358L accumulated at the nuclear envelope of cardiomyocytes with the increase in glycosylation. Finally, our comprehensive transcriptomic analysis demonstrated that the regional differences in gene expression patterns between the inner and outer layers observed in the wild type myocardium were partially diminished in the KI myocardium prior to exhibiting histological changes indicative of ACM. Altogether, these findings suggest that the aberrant accumulation of TMEM43S358L underlies the pathogenesis of ACM caused by TMEM43 p.S358L variant by affecting the transmural gene expression within the myocardium.

All d-Lysine Analogues of the Antimicrobial Peptide HPA3NT3-A2 Increased Serum Stability and without Drug Resistance.

Novel antibiotic drugs are urgently needed because of the increase in drug-resistant bacteria. The use of antimicrobial peptides has been suggested to replace antibiotics as they have strong antimicrobial activity and can be extracted from living organisms such as insects, marine organisms, and mammals. HPA3NT3-A2 ([Ala1,8] HPA3NT3) is an antimicrobial peptide that is an analogue of the HP (2-20) peptide derived from Helicobacter pylori ribosomal protein L1. Although this peptide was shown to have strong antimicrobial activity against drug-resistant bacteria, it also showed lower toxicity against sheep red blood cells (RBCs) and HaCaT cells compared to HPA3NT3. The l-Lys residues of HPA3NT3-A2 was substituted with d-Lys residues (HPA3NT3-A2D; [d-Lys2,5,6,9,10,15] HPA3NT3-A2) to prevent the cleavage of peptide bonds by proteolytic enzymes under physiological conditions. This peptide showed an increased half-life and maintained its antimicrobial activity in the serum against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) (pathogen). Furthermore, the antimicrobial activity of HPA3NT3-A2D was not significantly affected in the presence of mono- or divalent ions (Na+, Mg2+, Ca2+). Finally, l- or d-HPA3NT3-A2 peptides exhibited the strongest antimicrobial activity against antibiotic-resistant bacteria and failed to induce resistance in Staphylococcus aureus after 12 passages.

Effect of Slurry Viscosity and Dispersant Agent on the Sintering of 3Y-TZP Ceramics Fabricated by Slip Casting.

Dental zirconia implants are typically manufactured by mechanical machining of a zirconia block into a tooth shape. Many cracks are created by this machining process on the surface of the green body, which induces deterioration of mechanical strength and reliability of the sintered zirconia implant. In this study, we fabricated a dense zirconia specimen by slip casting and sintering. The zirconia slurry for slip casting was prepared by mixing yttria-stabilized zirconia powder with an average particle size of 20 nm, distilled water, and dispersant. Slurry viscosity was controlled by varying pH, dispersant concentration, and solid content; the lower viscosity being achieved at pH 11 controlled by ammonium hydroxide. With a dispersant content of 0.4-0.8 wt%, the viscosity was reduced from 190 cP to 40 cP at pH 11. After casting and sintering at 1550 °C for 2 h, the sintered body reached a density of 5.70-6.01 g/cm³ and a grain size of 300-700 nm, depending on the slurry preparation conditions.

Enhancement of the Surface Roughness by Powder Spray Coating on Zirconia Substrate.

Highly surface-roughened zirconia substrates were obtained from additive zirconia powder coating by room temperature spray processing. Homogeneous and dense zirconia coatings were deposited on sintered zirconia substrates with strong bonding by a powder spray coating method. The thickness and surface roughness of the coating layers on zirconia substrates increased with increasing coating cycles, which was confirmed from atomic force microscopy (AFM) and roughness analyses. The tetragonal phase and chemical composition of the zirconia coating layers were similar to those of the raw 3Y-TZP powder used as a raw material, indicating that no phase or composition changes occurred during the spray process.

Effect of Solid Loading on the Sintered Properties of 3 mol% Yttria-Stabilized Tetragonal Zirconia Polycrystals (3Y-TZP) Ceramics via Slip Casting.

Dental zirconia implants fabricated by the mechanical machining and sintering of zirconia blocks have many surface cracks that lead to the deterioration of mechanical strength and the failure of the implant in the body. In this study, we attempted to manufacture an extremely dense and crack-free zirconia specimen by slip casting and pressureless sintering. After the preparation of zirconia slurry by control of its viscosity and by solid loading, highly dense zirconia specimens could be obtained by pressureless sintering at 1450 °C for 2 h. Slurry viscosity was controlled by adjusting the mixing ratio of 3Y-TZP powder, a dispersant, and a pH adjustment agent. Highly dense 3Y-TZP specimens with a relative density of 99% and small grain size of 200-400 nm could be obtained at a solid loading of 50-65 wt%. An optimally dense specimen was fabricated from zirconia slurry with 60 wt% solid loading that had the highest apparent density of 6.07 g/cm³ (99.5%).

Separation of Halloysite Nano Tubes (HNTs) by Homogenization of Quartz Contaminated Kaolins.

Natural halloysite kaolin contains a lot of impurities such as quartz phases and varies in morphology and size during their formation in the earth. So to utilize as a new type of natural nano material, removing quartz impurities from kaolin clays without scathe the tube morphologies are necessary. So to remove quartz impurities from kaolin by forming a well deflocculated aqueous slip without fracturing the morphology of tubes, the slip of homogenized halloysite clay was recovered by adding polyvalent metallic cations and anionic polyelectrolyte flocculants simultaneously to selectively flocculate the mixture of quartz and halloysite, whereby the halloysite particles form floes and the tubular halloysite remains in suspensions. Then, the uniform size and tubular shape of halloysite was obtained which could be suitably used as a container or a carrier to encapsulate nanomaterials.

Activation of DNA Damage Response and Cellular Senescence in Cardiac Fibroblasts Limit Cardiac Fibrosis After Myocardial Infarction.

Cardiac fibrosis plays an important role in cardiac remodeling after myocardial infarction (MI). The molecular mechanisms that promote cardiac fibrosis after MI are well studied; however, the mechanisms by which the progression of cardiac fibrosis becomes attenuated after MI remain poorly understood. Recent reports show the role of cellular senescence in limiting tissue fibrosis. In the present study, we tested whether cellular senescence of cardiac fibroblasts (CFs) plays a role in attenuating the progression of cardiac fibrosis after MI. We found that the number of γH2AX-positive CFs increased up to day 7, whereas the number of proliferating CFs peaked at day 4 after MI. Senescent CFs were also observed at day 7, suggesting that attenuation of CF proliferation occurred simultaneously with the activation of the DNA damage response (DDR) system and the appearance of senescent CFs. We next cultured senescent CFs with non-senescent CFs and showed that senescent CFs suppressed proliferation of the surrounding non-senescent CFs in a juxtacrine manner. We also found that the blockade of DDR by Atm gene deletion sustained the proliferation of CFs and exacerbated the cardiac fibrosis at the early stage after MI. Our results indicate the role of DDR activation and cellular senescence in limiting cardiac fibrosis after MI. Regulation of cellular senescence in CFs may become one of the therapeutic strategies for preventing cardiac remodeling after MI.

Generation of Fabry cardiomyopathy model for drug screening using induced pluripotent stem cell-derived cardiomyocytes from a female Fabry patient.

BACKGROUND: Fabry disease is an X-linked disease caused by mutations in α-galactosidase A (GLA); these mutations result in the accumulation of its substrates, mainly globotriaosylceramide (Gb3). The accumulation of glycosphingolipids induces pathogenic changes in various organs, including the heart, and Fabry cardiomyopathy is the most frequent cause of death in patients with Fabry disease. Existing therapies to treat Fabry disease have limited efficacy, and new approaches to improve the prognosis of patients with Fabry cardiomyopathy are required. METHODS AND RESULTS: We generated induced pluripotent stem cell (iPSC) lines from a female patient and her son. Each iPSC clone from the female patient showed either deficient or normal GLA activity, which could be used as a Fabry disease model or its isogenic control, respectively. Erosion of the inactivated X chromosome developed heterogeneously among clones, and mono-allelic expression of the GLA gene was maintained for a substantial period in a subset of iPSC clones. Gb3 accumulation was observed in iPSC-derived cardiomyocytes (iPS-CMs) from GLA activity-deficient iPSCs by mass-spectrometry and immunofluorescent staining. The expression of ANP was increased, but the cell surface area was decreased in iPS-CMs from the Fabry model, suggesting that cardiomyopathic change is ongoing at the molecular level in Fabry iPS-CMs. We also established an algorithm for selecting proper Gb3 staining that could be used for high-content analysis-based drug screening. CONCLUSIONS: We generated a Fabry cardiomyopathy model and a drug screening system by using iPS-CMs from a female Fabry patient. Drug screening using our system may help discover new drugs that would improve the prognosis of patients with Fabry cardiomyopathy.

Anti-endotoxin mechanism of the KW4 peptide in inflammation in RAW 264.7 cells induced by LTA and drug-resistant Staphylococcus aureus 1630.

Drug-resistant microorganism infections cause serious disease and can lead to mortality and morbidity. In particular, Staphylococcus aureus induces pyrogenic and toxigenic infections, and drug-resistance occurs rapidly. Multidrug-resistant S. aureus, such as methicillin-resistant S. aureus and methicillin-sensitive S. aureus, can also cause immunodeficiency and immune deficiency syndrome from lipoteichoic acid. However, antimicrobial peptides, such as KW4, have strong antimicrobial activity, low cytotoxicity, and high neutralization activity against endotoxin substances from Gram-negative bacteria. The objective of this study was to use a synthetic KW4 antimicrobial peptide to evaluate the inhibition of drug-resistance development, antimicrobial activity, and neutralizing activity in S. aureus Gram-positive bacteria. The KW4 peptide showed strong antimicrobial activity against drug-resistant S. aureus strains and significantly increased the anti-neutralizing activity of lipoteichoic acid in S. aureus 1630 drug-resistant bacteria. In addition, S. aureus ATCC 29213 did not develop resistance to KW4 as with other antibiotic drugs. These results suggest that the KW4 peptide is an effective antibiotic and anti-neutralizing agent against multidrug-resistant S. aureus strains.

In vitro platform of allogeneic stem cell-derived cardiomyocyte transplantation for cardiac conduction defects.

Aims: The aim of the present study is to develop in vitro experimental analytical method for the electrophysiological properties of allogeneic induced pluripotent stem cell-derived cardiomyocytes (CMs) in cardiac conduction defect model. Methods and results: Cardiomyocytes were derived from rat induced pluripotent stem cells CMs (riPSC-CMs) using an embryoid body-based differentiation method with the serial application of growth factors including activin-A, bone morphogenetic protein 4 (BMP-4), and inhibitor of wnt production 2 (IWP-2). Flow cytometry analysis showed that 74.0 ± 2.7% of riPSC-CMs expressed cardiac troponin-T (n = 3). Immunostaining analysis revealed organized sarcomeric structure in riPSC-CMs and the expression of connexin 43 between riPSC-CMs and neonatal rat ventricular CMs (NRVMs). Ca2+ transient recordings revealed the simultaneous excitement of riPSC-CMs and NRVMs, and prolonged Ca2+ transient duration of riPSC-CMs as compared with NRVMs (731 ± 15.9 vs. 610 ± 7.72 ms, P < 0.01, n = 3). Isolated NRVMs were cultured in two discrete regions to mimic cardiac conduction defects on multi-electrode array dish, and riPSC-CMs were seeded in the channel between the two discrete regions. Membrane potential imaging with di-8-ANEPPS discerned the propagation of the electrical impulse from one NRVM region to the other through a riPSC-CM pathway. This pathway had significantly longer action potential duration as compared with NRVMs. Electrophysiological studies using a multi-electrode array platform demonstrated the longer conduction time and functional refractory period of the riPSC-CM pathway compared with the NRVM pathway. Conclusion: Using an in vitro experimental system to mimic cardiac conduction defect, transplanted allogeneic riPSC-CMs showed electrical coupling between two discrete regions of NRVMs. Electrophysiological testing using our platform will enable electrophysiological screening prior to transplantation of stem cell-derived CMs.

Effect of Initial ZrOCl2 Concentration on the Homogeneous Precipitation of Nanoscale 3Y-TZP Powder.

Nanoscale yttria-stabilized zirconia (Y-TZP) powder was synthesized by homogeneous precipitation via urea hydrolysis, and the influence of a dispersing agent and the initial ZrOCl2 concentration on the powder characteristics was investigated. A precipitated gel was obtained from the reaction of the precursor solution with zirconium oxychloride, yttrium chloride, and urea with heating at 110 °C for 5 h. The initial ZrOCl2 concentration was controlled from 0.25 to 1 M. To observe the effect of adding a dispersing agent on the agglomeration of primary particles, we used two starting compositions, one with and the other without a dispersing agent, ammonium polymethacrylate. Two crystalline powders were obtained after drying, calcination, and milling the gel, and we investigated the powder characteristics, such as particle agglomeration, the specific surface area, the microstructure, and phase composition. Two scales of agglomerates were observed in the particle size distribution, namely, 190 to 362 nm at the primary scale and 1.6-4.0 µm at the secondary scale. The amount of secondary agglomerate increased from 6 to 20 vol% with the increasing initial ZrOCl2 concentration. The size of both types of agglomerate and the amount of secondary agglomerates decreased due to the addition of the dispersing agent, especially the primary agglomerate size. The sintered density and microstructure of Y-TZP were affected by the agglomeration behavior, especially the amount of secondary agglomerates.

Synthesis and Thermal Stability of Nanocrystalline Tetragonal Zirconia by Hydrolysis with Ethylene Diamine.

Nanocrystalline zirconia with high surface area and pure tetragonal crystalline phase was prepared using ethylene diamine (EDA), which acts as both precipitating agent and dispersant of the zirconium precursor. The yttria stabilized zirconia (Y-TZP) is a very attractive material due to its excellent biocompatibility, high fracture toughness, high strength and low wear rates. So zirconyl chloride octahydrate (ZrOCl2 · 8H2O) and yttrium chloride hexahydrate (YCl3 · 6H2O) in different molar ratios were used as starting solution. The detailed effects of various process parameters such as reaction time, concentration of the precursor solution, amount of ethylene diamine, and calcination temperature on the structural properties of the zirconia powders were investigated. The preparation conditions significantly affected the structural stability, crystal size, and crystal phase of the final material. Increases in the reaction time and amount of ethylene diamine led to a substantial increase of the crystal growth rate, the specific surface area, and the tetragonal content of the zirconia.

Synthesis of Nanoscale Fully Stabilized Zirconia Powders by Urea Hydrolysis, Sintering and Electrical Characterization.

Active nanoscale powders of cubic phase zirconia stabilized with yttria, gadolinia, and scandia were successfully prepared by urea hydrolysis. Synthetic cubic zirconia powders had homogeneous, nanoscale, and less-agglomeration characteristics. Dense pellets of grain size about 0.4 µm exhibited grain boundary blocking resistance compared to the high frequency bulk resistance. Gadolinium doped system exhibited highest ionic resistivity. Yttria stabilized zirconia by urea hydrolysis in this work showed smaller ionic resistivity than the sample prepared from the commercial powder.

In-Vitro Mechanical Performance Study of Biodegradable Polylactic Acid/Hydroxyapatite Nanocomposites for Fixation Medical Devices.

Osteoconductive, biocompatible, and resorbable organic/inorganic composites are most commonly used in fixation medical devices, such as suture anchors and interference screws, because of their unique physical and chemical properties. Generally, studies on biodegradable composites have focused on their mechanical properties based on the composition and the individual roles of organic and inorganic biomaterials. In this study, we prepared biodegradable organic/inorganic nanocomposite materials using the solvent mixing process and conventional molding. We used polylactic acid (PLA) as the matrix and nano-sized hydroxyapatite (nano-HAp) as the osteoconductive filler. The content of nano-HAp was varied in 0-30 wt% and its influence on the In-Vitro mechanical performance of PLA/HAp nanocomposites was evaluated. The In-Vitro mechanical properties of nanocomposites were evaluated using standardized tensile and flexural tests after different immersion times in simulated body fluid.

Polylactic Acid/Nanostructured Si-Substituted ß-Tricalcium Phosphate Composites for Biodegradable Fixation Medical Devices.

Organic/inorganic biocomposite materials for biodegradable fixation medical devices require osteoconductivity, biocompatibility, and adequate mechanical properties with biodegradation behavior. The objective of this study was to investigate the effect of Si ions substituted in ß-tricalcium phosphate (ß-TCP) on the mechanical properties of organic/inorganic biocomposites. Biodegradable composite materials were prepared with polylactic acid (PLA) as the matrix and nano Si-substituted ß-TCP as the osteoconductive filler by solvent mixing and conventional molding. The nanostructured Si-substituted ß-TCP powders were synthesized by co-precipitation, controlling the quantity of Si ions. The amount of nanostructured Si-substituted ß-TCP powders in composites was varied in the 0-40 wt% range and the material properties were compared with those of pure ß-TCP/PLA composites. The influence of Si ions on the mechanical properties of the composites was evaluated by tensile and flexural tests.

Phenotypic Screening Using Patient-Derived Induced Pluripotent Stem Cells Identified Pyr3 as a Candidate Compound for the Treatment of Infantile Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a genetic disorder that is characterized by hypertrophy of the myocardium. Some of the patients are diagnosed for HCM during infancy, and the prognosis of infantile HCM is worse than general HCM. Nevertheless, pathophysiology of infantile HCM is less investigated and remains largely unknown. In the present study, we generated induced pluripotent stem cells (iPSCs) from two patients with infantile HCM: one with Noonan syndrome and the other with idiopathic HCM. We found that iPSC-derived cardiomyocytes (iPSC-CMs) from idiopathic HCM patient were significantly larger and showed higher diastolic intracellular calcium concentration compared with the iPSC-CMs from healthy subject. Unlike iPSC-CMs from the adult/adolescent HCM patient, arrhythmia was not observed as a disease-related phenotype in iPSC-CMs from idiopathic infantile HCM patient. Phenotypic screening revealed that Pyr3, a transient receptor potential channel 3 channel inhibitor, decreased both the cell size and diastolic intracellular calcium concentration in iPSC-CMs from both Noonan syndrome and idiopathic infantile HCM patients, suggesting that the target of Pyr3 may play a role in the pathogenesis of infantile HCM, regardless of the etiology. Further research may unveil the possibility of Pyr3 or its derivatives in the treatment of infantile HCM.

Interfacial Property of Dental Cobalt­Chromium Alloys and Their Bonding Strength with Porcelains.

Porcelain-fused-to-metal crown is one of the widely-used prostheses among the dental porcelain restorations. Nonprecious metals like Ni­Cr and Co­Cr have extensively been used for metal-ceramic restorations due to advantages such as inexpensive price, hardness, durability, resistance to deformation, thin thickness of metal of porcelain area, and other mechanical and physical properties. However, the immediate advantage of the Co­Cr alloy is comparable performance to other base metal alloys, but without an allergenic nickel component. To achieve clinical longevity of porcelain-fused-to-metal (PFM) crowns, it is essential to have adequate bond strength between the metal substrate and porcelain. Any type of metal-ceramic fracture failure can become a costly and timeconsuming problem, both in the clinic and laboratory. Therefore, the suitability of the Co­Cr alloy for dental applications is critically associated with its ceramic bonding capacity. In this study, Co­Cr metal alloys modified by acid-etching and sandblasting, oxide layer was formed for subsequent bonding to porcelain ceramics. By both acid-etching and sandblasting oxide layer was formed and showed higher bonding strength at a proper condition, but debonding was occurred at porcelain layer so that they showed highest bonding strength by combined these two kind of surface treatment. Because the oxide film was formed more densely in a vacuum at the portions where more sophisticated concavo-convex were formed on the surface of a metal.

The topogenic function of S4 promotes membrane insertion of the voltage-sensor domain in the KvAP channel.

Voltage-dependent K+ (KV) channels control K+ permeability in response to shifts in the membrane potential. Voltage sensing in KV channels is mediated by the positively charged transmembrane domain S4. The best-characterized KV channel, KvAP, lacks the distinct hydrophilic region corresponding to the S3-S4 extracellular loop that is found in other K+ channels. In the present study, we evaluated the topogenic properties of the transmembrane regions within the voltage-sensing domain in KvAP. S3 had low membrane insertion activity, whereas S4 possessed a unique type-I signal anchor (SA-I) function, which enabled it to insert into the membrane by itself. S4 was also found to function as a stop-transfer signal for retention in the membrane. The length and structural nature of the extracellular S3-S4 loop affected the membrane insertion of S3 and S4, suggesting that S3 membrane insertion was dependent on S4. Replacement of charged residues within the transmembrane regions with residues of opposite charge revealed that Asp72 in S2 and Glu93 in S3 contributed to membrane insertion of S3 and S4, and increased the stability of S4 in the membrane. These results indicate that the SA-I function of S4, unique among K+ channels studied to date, promotes the insertion of S3 into the membrane, and that the charged residues essential for voltage sensing contribute to the membrane-insertion of the voltage sensor domain in KvAP.