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.

Modeling Reduced Contractility and Stiffness Using iPSC-Derived Cardiomyocytes Generated From Female Becker Muscular Dystrophy Carrier.

Study investigators encountered a female Becker muscular dystrophy (BMD) carrier with advanced heart failure (HF) and identified a stop-gain variant in procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 (PLOD3) as a potential second-hit variant. Isogenic induced pluripotent stem cells (iPSCs) with dominant expression of WT-DMD, Δ45-48-DMD, or Δ45-48-DMD with corrected PLOD3 variant were established. Microforce testing using 3-dimensional self-organized tissue rings (SOTRs) generated from iPSC-derived cardiomyocytes (iPSC-CMs) demonstrated that correction of the heterozygous PLOD3 variant did not improve the reduced force, but it significantly recovered the reduced stiffness in Δ45-48-DMD SOTRs. Correction of the PLOD3 variant restored collagen synthesis in iPSC-CMs. Our findings revealed the pathogenesis underlying advanced HF in a female BMD carrier.

Anti-Biofilm Effects of Rationally Designed Peptides against Planktonic Cells and Pre-Formed Biofilm of Pseudomonas aeruginosa.

Biofilms are resistant to antibiotics and are a major source of persistent and recurring infections by clinically important pathogens. Drugs used for biofilm-associated infections are limited because biofilm-embedded or biofilm-matrix bacteria are difficult to kill or eradiate. Therefore, many researchers are developing new and effective antibiofilm agents. Among them, antimicrobial peptides have an attractive interest in the development of antibiofilm agents. The present study evaluated the effects of 10 synthetic peptides on growth inhibition, inhibition of biofilm formation, and biofilm elimination in drug-resistant Pseudomonas aeruginosa. The planktonic cell growth and biofilm formation were dose-dependently inhibited by most of the peptides. WIK-14 eliminated preformed biofilm masses by removing carbohydrates, extracellular nucleic acids, proteins, and lipids constituting extracellular polymeric substances. The results demonstrated that WIK-14 and WIKE-14 peptides might provide novel therapeutic drugs to overcome multidrug resistance in biofilm-associated infections.

Antifungal and Anti-Inflammatory Activities of PS1-2 Peptide against Fluconazole-Resistant Candida albicans.

Clinically, fungal pneumonia rarely occurs in adults, and invasive fungal infections can cause substantial morbidity, and mortality due to sepsis and septic shock. In the present study, we have designed peptides that exhibit potent antifungal activities against fluconazole-resistant Candida albicans in physiological monovalent, and divalent ionic buffers, with minimal fungicidal concentrations ranging from 16 to 32 µM. None of these tested peptides resulted in the development of drug resistance similar to fluconazole. Among them, the PS1-2 peptide did not induce stimulation of macrophages by C. albicans, and it exerted antifungal and anti-inflammatory effects against C. albicans-induced intratracheal infection, in an acute lung injury mouse model. PS1-2 is likely a novel therapeutic agent for the control, and prevention of drug-resistant C. albicans infection, and our findings may be useful for designing antimicrobial peptides to combat fungal infection.

Design of Antimicrobial Peptides with Cell-Selective Activity and Membrane-Acting Mechanism against Drug-Resistant Bacteria.

Antimicrobial peptides (AMPs) can combat drug-resistant bacteria with their unique membrane-disruptive mechanisms. This study aimed to investigate the antibacterial effects of several membrane-acting peptides with amphipathic structures and positional alterations of two tryptophan residues. The synthetic peptides exhibited potent antibacterial activities in a length-dependent manner against various pathogenic drug-resistant and susceptible bacteria. In particular, the location of tryptophan near the N-terminus of AMPs simultaneously increases their antibacterial activity and toxicity. Furthermore, the growth inhibition mechanisms of these newly designed peptides involve cell penetration and destabilization of the cell membrane. These findings provide new insights into the design of peptides as antimicrobial agents and suggest that these peptides can be used as substitutes for conventional antibiotics.

Potent Anti-Inflammatory Effects of a Helix-to-Helix Peptide against Pseudomonas aeruginosa Endotoxin-Mediated Sepsis.

Although considerable scientific research data is available for sepsis and cytokine storm syndrome, there is a need to develop new treatments or drugs for sepsis management. Antimicrobial peptides (AMPs) possess anti-bacterial and anti-inflammatory activity, neutralizing toxins such as lipopolysaccharides (LPS, endotoxin). Most AMPs have been designed as a substitute for conventional antibiotics, which kill drug-resistant pathogens. The present study aimed to determine the anti-inflammatory potential of 10 designed XIW (X: lysine, arginine, or glutamic acid) α-helical peptides in macrophages and a mouse model in the presence of LPS. Among them, WIKE-14, a peptide with a helix-to-helix structure, having the 12th amino acid substituted with glutamic acid, suppressed pro-inflammatory cytokines in RAW 264.7 macrophages. This reaction was mediated by the inhibition of the binding between LPS and macrophages. In addition, the WIKE-14 peptide exhibited a potent anti-inflammatory activity in mice ears and lungs inflamed using LPS. Thus, our results may provide useful insights for the development of anti-sepsis agents via the sequence and structure information of the WIKE-14 peptide.

Candidacidal and Antibiofilm Activity of PS1-3 Peptide against Drug-Resistant Candida albicans on Contact Lenses.

The recent emergence of antibiotic-resistant fungi has accelerated research on novel antifungal agents. In particular, Candida albicans infections are related to biofilm formation on medical devices, such as catheters, stents, and contact lenses, resulting in high morbidity and mortality. In this study, we aimed to elucidate the antifungal and antibiofilm effects of a peptide against drug-resistant C. albicans. α-Helical peptides in which the sequence of KWYK was repeated twice and four times, designated peptide series 1 (PS1)-1 and PS1-3, respectively, were generated, and the candidacidal activities of PS1-1, PS1-3, and fluconazole against drug-resistant C. albicans cells were assessed. The PS1-3 peptide showed higher killing activity than PS1-1 or fluconazole and acted via a membranolytic mechanism. In addition, the PS1-3 peptide exhibited more potent activity than PS1-1 and fluconazole in terms of fungal biofilm inhibition and reduction at the minimum fungicidal concentration on the contact lens surface. Overall, these findings established PS1-3 as a potential candidacidal agent for applications on contact lenses.

Human-Induced Pluripotent Stem Cell-Derived Cardiomyocyte Model for TNNT2 Δ160E-Induced Cardiomyopathy.

BACKGROUND: The Δ160E mutation in TNNT2, which encodes troponin T, is a rare pathogenic variant identified in patients with hypertrophic cardiomyopathy and is associated with poor prognosis. Thus, a convenient human model recapitulating the pathological phenotype caused by TNNT2 Δ160E is required for therapeutic development. METHODS: We identified a heterozygous in-frame deletion mutation (c.478_480del, p.Δ160E) in TNNT2 in a patient with familial hypertrophic cardiomyopathy showing progressive left ventricular systolic dysfunction, leading to advanced heart failure. To investigate the pathological phenotype caused by Δ160E, we generated a set of isogenic induced pluripotent stem cells carrying the heterozygous Δ160E, homozygously corrected or homozygously introduced Δ160E using genome editing and differentiated them into cardiomyocytes (Hetero-Δ160E-, wild type-, and Homo-Δ160E-induced pluripotent stem cells [iPSC]-derived cardiomyocytes [iPSC-CMs]). RESULTS: Hetero-Δ160E-iPSC-CMs exhibited prolonged calcium decay, relaxation impairment, and hypertrophy compared to wild type-iPSC-CMs. Notably, these phenotypes were further exacerbated in Homo-Δ160E-iPSC-CMs. Overexpression of R-GECO-fused Δ160E mutant troponin T prolonged decay time and time to peak of the myofilament-localized calcium transient in iPSC-CMs, indicating that sarcomeric calcium retention with Δ160E may affect intracellular calcium concentration. High-content imaging analysis detected remarkable nuclear translocation of NFATc1, especially in Homo-Δ160E-iPSC-CMs, indicating that the Δ160E mutation promotes hypertrophic signaling pathway in a dose-dependent manner. Increased phosphorylation of CaMKIIδ (calcium/calmodulin-dependent protein kinase IIδ) and phospholamban at Thr17 was observed in Homo- and Hetero-Δ160E-iPSC-CMs. Epigallocatechin-3-gallate, a calcium desensitizing compound, shortened prolonged calcium decay and relaxation duration in Δ160E-iPSC-CMs. CONCLUSIONS: Isogenic iPSC-CMs recapitulate the prolonged calcium decay, relaxation impairment, and subsequent calcium-regulated signaling pathways caused by the TNNT2 Δ160E mutation and can serve as a human model for therapeutic development to prevent hypertrophic cardiomyopathy pathology.

Antifungal Mechanism of Vip3Aa, a Vegetative Insecticidal Protein, against Pathogenic Fungal Strains.

Discovering new antifungal agents is difficult, since, unlike bacteria, mammalian and fungal cells are both eukaryotes. An efficient strategy is to consider new antimicrobial proteins that have variety of action mechanisms. In this study, a cDNA encoding Bacillus thuringiensis Vip3Aa protein, a vegetative insecticidal protein, was obtained at the vegetative growth stage; its antifungal activity and mechanism were evaluated using a bacterially expressed recombinant Vip3Aa protein. The Vip3Aa protein demonstrated various concentration- and time-dependent antifungal activities, with inhibitory concentrations against yeast and filamentous fungi ranging from 62.5 to 125 µg/mL and 250 to 500 µg/mL, respectively. The uptake of propidium iodide and cellular distributions of rhodamine-labeled Vip3Aa into fungal cells indicate that its growth inhibition mechanism involves its penetration within cells and subsequent intracellular damage. Furthermore, we discovered that the death of Candida albicans cells was caused by the induction of apoptosis via the generation of mitochondrial reactive oxygen species and binding to nucleic acids. The presence of significantly enlarged Vip3Aa-treated fungal cells indicates that this protein causes intracellular damage. Our findings suggest that Vip3Aa protein has potential applications in the development of natural antimicrobial agents.

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.

High-Throughput Drug Screening System Based on Human Induced Pluripotent Stem Cell-Derived Atrial Myocytes ∼ A Novel Platform to Detect Cardiac Toxicity for Atrial Arrhythmias.

Evaluation of proarrhythmic properties is critical for drug discovery. In particular, QT prolongation in electrocardiograms has been utilized as a surrogate marker in many evaluation systems to assess the risk of torsade de pointes and lethal ventricular arrhythmia. Recently, new evaluation systems based on human iPS cell-derived cardiomyocytes have been established. On the other hand, in clinical situations, it has been reported that the incidence of atrial arrhythmias such as atrial fibrillation has been increasing every year, with the prediction of a persistent increase in the near future. As to the increased incidence of atrial arrhythmias, in addition to the increased population of geriatric patients, a wide variety of drug treatments may be related, as an experimental method to detect drug-induced atrial arrhythmia has not been established so far. In the present study, we characterized the atrial-like cardiomyocytes derived from human induced pluripotent stem cells and examined their potential for the evaluation of drug-induced atrial arrhythmia. Atrial-like cardiomyocytes were induced by adding retinoic acid (RA) during the process of myocardial differentiation, and their characteristics were compared to those of RA-free cardiomyocytes. Using gene expression and membrane potential analysis, it was confirmed that the cells with or without RA treatment have atrial or ventricular like cardiomyocytes, respectively. Using the ultra-rapid activating delayed rectifier potassium current (IKur) channel inhibitor, which is specific to atrial cardiomyocytes, Pulse width duration (PWD) 30cF prolongation was confirmed only in atrial-like cardiomyocytes. In addition, ventricular like cardiomyocytes exhibited an early after depolarization by treatment with rapidly activating delayed rectifier potassium current (IKr) channel inhibitor, which induces ventricular arrhythmia in clinical situations. Here, we have established a high-throughput drug evaluation system using human iPS cell-derived atrial-like cardiomyocytes. Based on the obtained data, the system might be a valuable platform to detect potential risks for drug-induced atrial arrhythmias.

Protocol for Morphological and Functional Phenotype Analysis of hiPS-Derived Cardiomyocytes.

Induced pluripotent stem cells (iPSCs) have been utilized to study physiological development and also the pathogenesis of heart diseases. iPS-derived cardiomyocytes and engineered cardiac tissues provide a promising capacity for investigating cardiac development and disease modeling. In addition to protocols for cardiac differentiation and 3D cardiac tissue construction, the establishment of protocols for the comprehensive evaluation of the physiological and/or pathophysiological properties for the iPS-derived cells/tissues are indispensable.

Homogeneous 2D and 3D alignment of cardiomyocyte in dilated cardiomyopathy revealed by intravital heart imaging.

In contrast to hypertrophic cardiomyopathy, there has been reported no specific pattern of cardiomyocyte array in dilated cardiomyopathy (DCM), partially because lack of alignment assessment in a three-dimensional (3D) manner. Here we have established a novel method to evaluate cardiomyocyte alignment in 3D using intravital heart imaging and demonstrated homogeneous alignment in DCM mice. Whilst cardiomyocytes of control mice changed their alignment by every layer in 3D and position twistedly even in a single layer, termed myocyte twist, cardiomyocytes of DCM mice aligned homogeneously both in two-dimensional (2D) and in 3D and lost myocyte twist. Manipulation of cultured cardiomyocyte toward homogeneously aligned increased their contractility, suggesting that homogeneous alignment in DCM mice is due to a sort of alignment remodelling as a way to compensate cardiac dysfunction. Our findings provide the first intravital evidence of cardiomyocyte alignment and will bring new insights into understanding the mechanism of heart failure.

Decreased YAP activity reduces proliferative ability in human induced pluripotent stem cell of duchenne muscular dystrophy derived cardiomyocytes.

Duchenne muscular dystrophy (DMD) is characterized by progressive muscle degeneration accompanied by dilated cardiomyopathy. Recently, abnormality of yes-associated protein (YAP) has been reported as the pathogenesis of muscle degeneration of DMD; however YAP activity remains unclear in dystrophic heart of DMD. Herein, we investigated YAP activity using disease-specific induced pluripotent stem cell (iPSC) derived cardiomyocytes (CMs) in DMD. DMD-iPSCs were generated from DMD patient with exon 48-54 deletion in DMD, and genome-edited (Ed)-DMD-iPSCs with in-frame (Ed-DMD-iPSCs) were created using CRISPR/Cas9. Nuclear translocation of YAP [nuclear (N)/cytoplasmic (C) ratio] was significantly lower in DMD-iPSC-CMs than in Ed-DMD-iPSC-CMs. In addition, Ki67 expression, indicating proliferative ability, was significantly lower in DMD-iPSC-CMs than Ed-DMD-iPSC-CMs. Therefore, immunofluorescent staining showed that actin stress fibers associated with YAP activity by mechanotransduction were disorganized in DMD-iPSC-CMs. Lysophosphatidic acid (LPA), a known lipid mediator on induction of actin polymerization, significantly increased YAP activity and actin dynamics in DMD-iPSC-CMs using live cell imaging. These results suggested that altered YAP activity due to impaired actin dynamics reduced proliferative ability in DMD-iPSC-CMs. Hence, decreased YAP activity in dystrophic heart may contribute to DMD-cardiomyopathy pathogenesis.

Scaffold-Mediated Developmental Effects on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Are Preserved After External Support Removal.

Human induced pluripotent stem (hiPS) cells have been used as a cell source for regenerative therapy and disease modeling. The purity of hiPS-cardiomyocytes (hiPS-CMs) has markedly improved with advancements in cell culture and differentiation protocols. However, the morphological features and molecular properties of the relatively immature cells are still unclear, which has hampered their clinical application. The aim of the present study was to investigate the extent to which topographic substrates actively influence hiPS-CMs. hiPS-CMs were seeded on randomized oriented fiber substrate (random), anisotropic aligned fiber substrate (align), and flat non-scaffold substrate (flat). After culturing for one week, the hiPS-CMs on the aligned patterns showed more mature-like properties, including elongated rod shape, shorter duration of action potential, accelerated conduction velocity, and elevated cardiac gene expression. Subsequently, to determine whether this development was irreversible or was altered after withdrawal of the structural support, the hiPS-CMs were harvested from the three different patterns and reseeded on the non-scaffold (flat) pattern. After culturing for one more week, the improvements in morphological and functional properties diminished, although hiPS-CMs pre-cultured on the aligned pattern retained the molecular features of development, which were even more significant as compared to that observed during the pre-culture stage. Our results suggested that the anisotropic fiber substrate can induce the formation of geometrical mimic-oriented heart tissue in a short time. Although the morphological and electrophysiological properties of hiPS-CMs obtained via facilitated maturation somehow rely on the existence of an exterior scaffold, the molecular developmental features were preserved even in the absence of the external support, which might persist throughout hiPS-CM development.

Bioactivity Improvement of Zirconia Substrate by Hydroxyapatite Coating Using Room Temperature Spray Processing.

Zirconia ceramics has a bioinert property with low bioactivity. So, it is necessary to improve its low bioactivity by the surface modification using effective coating methods. In this study, we fabricated the hydroxyapatite-coated zirconia substrate by room temperature spray processing to improve the bioactivity of the zirconia implant and investigated its coating effect on the biological performance of zirconia substrate via an in vitro test in simulated body fluid (SBF) solution. Before the room temperature spray coating was completed, size-controlled hydroxyapatite powder that had an average size of 4.5 µm, was obtained by the calcination and milling of a commercial powder. By controlling the processing parameters, such as spraying distance, and deposition cycles, we fabricated homogeneous and dense hydroxyapatite coatings on zirconia substrate. Surface morphology, coating thickness, and microstructure were dependent on deposition cycles, and were related to surface roughness and bioactivity. Zirconia substrates with wave-patterned and roughened hydroxyapatite coatings demonstrated high bioactivity in their in vitro tests. Via the immersion test in an SBF solution, surface dissolution and new precipitates of hydroxyapatite were observed on coated zirconia substrate, indicating the degree of bioactivity.

Slurry Coating of Hydroxyapatite on Zirconia Substrate.

Zirconia dental implants require excellent biocompatibility and high bonding strength. In this study, we attempted to fabricate biocompatible zirconia ceramics through surface modification by hydroxyapatite (HA) slurry coating. A hydroxyapatite slurry for spin coating was prepared using two sizes of hydroxyapatite particles. The hydroxyapatite slurry was obtained by adjusting the solid loading, pH range, and dispersant content. The surface roughness of the HA-coated layers on the zirconia substrate depended on the change in microstructural evolution and coating thickness. With repeated coating, the coating thickness gradually increased for both small and large particles. The specimen with two coatings had the maximum surface roughness but displayed different values depending on the size of the HA particles. High surface roughness (Ra; 0.49 µm) could be obtained from the slurry of small particles compared with that of the large particles (Ra; 0.35 µm). During a 14 days in vitro experiment in SBF solution at pH 7.4, no changes were observed in the surface microstructure of the HA coating layer on the zirconia substrate.

Sol-Gel Coating of Hydroxyapatite on Zirconia Substrate.

The zirconia used in dental implants requires excellent mechanical and chemical properties such as high strength, high biological performance, corrosion resistance, and phase stability. In this study, after we prepared a highly fluidized solution of calcium phosphate, we fabricated a hydroxyapatite (HA) coating layer on a zirconia substrate using the sol-gel method to enhance its biocompatibility and bone-bonding ability. We dipped the zirconia substrate into the calcium phosphate sol to obtain the HA-coated film, which was dried at room temperature. The phase change and microstructural evolution were examined while the coating dried and during heat treatment. The biological activity of the coated and as-received substrates was evaluated using an in vitro experiment and the results were compared. The HA-coated film showed a highly dense and uniform layer structure, while its physical and biological properties depended on the starting substrate, coating times, and processing conditions.

Sintered Characteristics of 3 Mole% Yttria-Stabilized Zirconia Polycrystals (3Y-TZP) Implants Manufactured by Slip-Casting and Computer Aided Design/Computer Aided Manufacturing (CAD/CAM).

Two types of dense and toughened 3Y-TZP zirconia implants were fabricated by the sintering of green compacts. Then, the sintered properties of the two implants were compared. Slip-cast and post-sintered all-ceramic zirconia implants were fabricated by the heat treatment at 1450 °C for 2 h using optimal slurry conditions (60 wt% solid content, 1 wt% dispersant, and pH 12). Computer-aided design and manufacturing (CAD/CAM)-machined and post-sintered zirconia crowns, supplied by a dental hospital, were obtained by sintering at 1650 °C for 5 h. The X-ray diffraction results indicated that the phase composition of the slip-casted specimen was completely tetragonal, but the CAD/CAM machined sample was composed of mixed phases of main tetragonal and minor monoclinic crystals. The sintered density and Vickers hardness of the slip-casted specimen were 6.07 g/cm³ and 1367 Hv, respectively, and these were higher than those of the CAD/CAM machined specimen. From the comparative results of the surface microstructure, hardness, and roughness between the two sintered specimens, the slip-casted specimen was found to have higher surface roughness and mechanical hardness, smaller grain size, and less surface micro-cracks than the CAD/CAM machined specimen.