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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.

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.

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.

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.

Background: In contrast to hypertrophic cardiomyopathy, there has been reported no specific pattern of cardiomyocyte array in dilated cardiomyopathy (DCM). This might be because it is assessed only on heart tissue sections two-dimensionally (2D). To address this, we sought to evaluate cardiomyocyte alignment in DCM three-dimensionally (3D) using intravital heart imaging techniques. Methods and Results: We observed cardiomyocytes of membrane fluorescent reporter mice up to 100μm depth by an intravital imaging system with two-photon microscopy. On each 2D images taken at 1μm interval, the angles of cardiomyocytes from a vertical line were measured and their distribution was plotted. Then the plots of all layers were merged so that layer-to-layer change of angle distribution can be visualized. In these merged plots, cardiomyocytes exhibited several peaks with a certain spread around each peak, suggesting that cardiomyocytes change their alignments by every layer in 3D and position twistedly even in a single layer (Figure). We next assessed cardiac mutant Troponin T knock-in mice as a DCM model. The angle distribution in these mice was less various within a single layer and between layers as well. These results indicate that cardiomyocytes of DCM model mice align homogeneously both in 2D and in 3D. To determine how homogeneous alignment contributes to cardiomyocyte contractility, we captured the motion of cultured cardiomyocytes and found that cardiomyocytes seeded on the top of linearly aligned fibers show greater motion than those seeded on randomly aligned fibers. Conclusion: Using intravital imaging, we have provided a first evidence of cardiomyocyte array in 3D and demonstrated that cardiomyocytes of DCM model mice align homogeneously both in 2D and in 3D. Homogeneous alignment of those mice might be the consequences of impaired cardiac function as a way to increase let ventricular contractility.

Bacterial biofilms formed through secretion of extracellular polymeric substances (EPS) have been implicated in many serious infections and can increase antibiotic resistance by a factor of more than 1000. Here, we examined the abilities of the antimicrobial peptide HPA3NT3-A2 to inhibit and reduce biofilm formation, eliminate EPS, and suppress inflammation in mice infected with clinical isolates of drug-resistant Pseudomonas aeruginosa strains. HPA3NT3-A2 was developed from a desirable analogue peptide, HPA3NT3, derived from residues 2–20 of the Helicobacter pylori ribosomal protein L1. HPA3NT3-A2 showed stronger activity against planktonic cells (MIC: 8 μM) compared to ciprofloxacin or tobramycin (>512 μM), and a favorable minimum biofilm inhibition and elimination concentration. This peptide also neutralized LPS; decreased levels of EPS; inhibited the production of pro-inflammatory cytokines in the lung, kidney, and spleen; decreased white blood cell counts; and increased survival among infected mice.

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.

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

// Jong-Kook Lee 1, 3 , Tudor Luchian 2 and Yoonkyung Park 1, 3 1 Research Center for Proteinaceous Materials, Chosun University, Gwangju, Korea 2 Department of Physics, Alexandru I. Cuza University, Iasi, Romania 3 Department of Biomedical Science, Chosun University, Gwangju, Korea Correspondence to: Yoonkyung Park, email: y_k_park@chosun.ac.kr Tudor Luchian, email: luchian@uaic.ro Keywords: Staphylococcus aureus; clavaspirin peptide (CSP); toll-like receptor-2 (TLR-2); nuclear factor-kappa B (NF-κB); pro-inflammatory cytokine Received: November 03, 2017 Accepted: February 20, 2018 Epub: February 26, 2018 Published: March 20, 2018 ABSTRACT Clavaspirin peptide (CSP) is derived from the pharyngeal tissues of the tunicate Styela clava . The 23-amino acid peptide is histidine-rich and amidated at the N-terminus. CSP possesses low antimicrobial and high hemolytic activity at pH 7.4. Therefore, we designed 4 CSP analogs with substituted hydrophobic amino acids to reduce hydrophobic amino acid interactions. These modifications reduced the aggregation and cytotoxicity of the analogs at pH 7.4. The analogs also showed potent antimicrobial activity by accumulating on bacterial cell surfaces and inducing the lytic mechanism against gram-negative and gram-positive cells at pH 5.5 and 7.4. Moreover, exposure to the CSP-4 analog for up to 29 passages did not induce drug resistance in Staphylococcus aureus . Application of CSP-4 to inflamed skin of hairless mice infected with drug-resistant S. aureus (DRSA) significantly reduced skin infections without damaging dermal collagen or elastin. Topically applied CSP-4 penetrated 25–40 μm in the dermis within 30 min, reducing the levels of Toll-like receptor-2, nuclear factor kappa B (NF-κB), and the pro-inflammatory cytokines tumor necrosis factor- α (TNF-α) and interleukin-1β (IL-1 β). These results suggest that CSP-4 could be a promising topical antimicrobial agent for skin diseases caused by DRSA such as S. aureus CCARM 0027.

Although engineered cardiac tissues (ECTs) derived from induced pluripotent stem cells (iPSCs) are promising for myocardial regenerative therapy, the appropriate ratio of cardiomyocytes to non-cardiomyocytes is not fully understood. Here, we determined whether ECT-cell content is a key determinant of its structure/function, thereby affecting ECT therapeutic potential for advanced heart failure. Scaffold-free ECTs containing different ratios (25%, 50%, 70%, or 90%) of iPSC-derived cardiomyocytes were generated by magnetic-activated cell sorting by using cardiac-specific markers. Notably, ECTs showed synchronized spontaneous beating when cardiomyocytes constituted ≥50% of total cells, with the electrical-conduction velocity increasing depending on cardiomyocyte ratio; however, ECTs containing 90% cardiomyocytes failed to form stable structures. ECTs containing 25% or 50% cardiomyocytes predominantly expressed collagen and fibronectin, whereas ECTs containing 70% cardiomyocytes predominantly expressed laminin and exhibited the highest contractile/relaxation properties. Furthermore, transplantation of ECTs containing 50% or 70% cardiomyocytes into a rat chronic myocardial infarction model led to a more profound functional recovery as compared with controls. Notably, transplanted ECTs showed electrical synchronization with the native heart under Langendorff perfusion. Collectively, these results indicate that the quantity of non-cardiomyocytes is critical in generating functional iPSC-derived ECTs as grafts for cardiac-regeneration therapy, with ECTs containing 50–70% cardiomyocytes exhibiting stable structures and increased cardiotherapeutic potential.

The antimicrobial peptide HPA3 shows anticancer activity in gastric cancer and leukaemia. However, how HPA3 exerts its anticancer activity, as well as whether it also exhibits activity in other cancers, remains unknown. Therefore, the aim of this study was to evaluate the anticancer activity of HPA3 and its analogues in colon cancer and to elucidate the mechanisms responsible for this activity. HPA3P decreased cell viability, whereas HPA3 and HPA3P2 did not decrease cell viability in colon cancer cells compared with control cells. This reduction in cell viability occurred through necrosis, a conclusion supported by our observation of the release of cellular contents, our intracellular PI staining results, and our observation of the release of HMGB1. Moreover, RIPK3 inhibition blocks the reduction of cell viability by HPA3P. Consistent with this finding, we found that knocking down RIPK3 and MLKL, key necroptosis proteins, attenuates the reductions in cell viability induced by HPA3P. Furthermore, HPA3P can improve the anticancer activity of chemotherapeutic agents and exhibits anticancer activity in other cancer cells. These results suggest that HPA3P may have potential as an anticancer agent in the treatment of colon cancer.

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.

Absorption curves for water are very low in the ultraviolet and visible range and the absorption curve rises in the near-infrared range and is the highest in the far-infrared. The absorption curve of water in far-infrared peaks at 2940 nm and then decreases, then gradually increases (Fig. 10.1) [2].

Melasma is a common hypermelanosis that occurs in areas exposed to sunlight, especially the face. It occurs rarely before puberty and is commonly observed in women, especially women of childbearing age. It is also observed in 10% of men. New melasma often arises and existing melasma may worsen during pregnancy. Symptoms appear as brown macules with irregular borders and occur symmetrically in the photodistribution of sunlight on the face. They are often fused in a reticular pattern. They are darkened by sunlight, which makes them worse during summer and improves during winter, i.e., there is a seasonal change. Melasma is common among Asian and Hispanic women who live in areas with strong sunlight. It occurs frequently in patients with Fitzpatrick skin types IV, V, and VI [1].

Although there are many side effects that can occur after laser treatment, the most common and problematic side effect in darker skin including Koreans is postinflammatory hyperpigmentation (PIH). Therefore, a full understanding and management of PIH are necessary. In this chapter, we go through PIH in detail.

Many papers and dermatology laser texts usually describe lasers about white skin. In separate articles or chapters laser treatment of “darker skin” or ethnic, nonwhite, pigmented skin is described [1–4]. Here, the term “darker skin” refers to all races except Caucasians, including Chinese, Japanese, Indian, Pakistani, Hispanic, Latino, African, African-Caribbean, and African American. Fitzpatrick skin types are III, IV, V, and VI [5].

Skin rejuvenation is a treatment that targets age-related pigmentation disorders and telangiectasia or improves wrinkles and skin texture [1]. It can be classified by treatment methods; ablative rejuvenation (AR) by CO2 laser and erbium-doped yttrium aluminium garnet (Er:YAG) laser or nonablative rejuvenation (NAR), which induces a selective thermal reaction to the dermis and subcutaneous fat while sparing the epidermis. It can also be classified by treatment targets; type I rejuvenation targets pigmentation and vascular disorders while type II rejuvenation focuses on improving wrinkles and skin texture. For example, AR and NAR are both treatment modalities for type II rejuvenation (Table 9.1). Generally, NAR refers to rejuvenation treatment that uses nonablative methods to improve wrinkles and skin texture (type II rejuvenation).

Hair is a cylindrical fiber consisting of tightly adhered, cornified epithelial cells that only mammals have [1]. It is present anywhere on the skin except on palms and soles, distal ends of fingers and toes, boundaries of mucous membranes, and the glans penis.

There are many types of scars. They vary according to the shape of the scar surface, shape of the scar itself, color, and chronology. Scars may be classified by scar surface (flat, elevated, depressed, or irregular) by morphology (atrophic, normorophic, hypertrophic, or keloid) by color (depigmented, hypopigmented, hyperpigmented, or discolored) or by chronology (acute, chronic, immature, or mature) [1].

Hemoglobin (Hb) includes oxygen-bound oxyhemoglobin (HbO2), oxygen-free deoxyhemoglobin (deoxyHb), and methemoglobin (metHb), which underwent protein denaturation by heat. The absorption curve of HbO2 reaches a peak at 418 nm, reaches a small peak at 542 and 577 nm, then decreases, and finally forms a smaller peak at 940 nm. DeoxyHb reaches peaks at 430 and 555 nm and reaches a peak once again between 800 and 1200 nm (Fig. 6.1) [1, 2].

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.

Ectopic foci from pulmonary veins (PVs) comprise the main trigger associated with the initiation of atrial fibrillation (AF). An abrupt anatomical narrow-to-wide transition, modeled as in vitro geometrical patterning with similar configuration in the present study, is located at the junction of PVs and the left atrium (LA). Complex cellular composition, i.e., constituent cell heterogeneity, is also observed in PVs and the PVs-LA junction. High frequency triggers accompanied with anatomical irregularity and constituent cell heterogeneity provoke impaired conduction, a prerequisite for AF genesis. However, few experiments investigating the effects of these factors on electrophysiological properties using human-based cardiomyocytes (CMs) with atrial properties have been reported. The aim of the current study was to estimate whether geometrical patterning and constituent cell heterogeneity under high frequency stimuli undergo conduction disturbance utilizing an in vitro two-dimensional (2D) monolayer preparation consisting of atrial-like CMs derived from human induced pluripotent stem cells (hiPSCs) and atrial fibroblasts (Fbs). We induced hiPSCs into atrial-like CMs using a directed cardiac differentiation protocol with the addition of all-trans retinoic acid (ATRA). The atrial-like hiPSC-derived CMs (hiPSC-CMs) and atrial Fbs were transferred in defined ratios (CMs/Fbs: 100%/0% or 70%/30%) on manually fabricated plates with or without geometrical patterning imitating the PVs-LA junction. High frequency field stimulation emulating repetitive ectopic foci originated in PVs were delivered, and the electrical propagation was assessed by optical mapping. We generated high purity CMs with or without the ATRA application. ATRA-treated hiPSC-CMs exhibited significantly higher atrial-specific properties by immunofluorescence staining, gene expression patterns, and optical action potential parameters than those of ATRA-untreated hiPSC-CMs. Electrical stimuli at a higher frequency preferentially induced impaired electrical conduction on atrial-like hiPSC-CMs monolayer preparations with an abrupt geometrical transition than on those with uniform geometry. Additionally, the application of human atrial Fbs to the geometrically patterned atrial-like hiPSC-CMs tended to further deteriorate the integrity of electrical conduction compared with those using the atrial-like hiPSC-CM alone preparations. Thus, geometrical narrow-to-wide patterning under high frequency stimuli preferentially jeopardized electrical conduction within in vitro atrial-like hiPSC-CM monolayers. Constituent cell heterogeneity represented by atrial Fbs also contributed to the further deterioration of conduction stability.

Antimicrobial peptides (AMPs) are a promising new class of antibacterial compounds. However, their applications in the treatment of intracellular pathogenic bacteria have been limited by their in vivo instability and low penetrating ability into mammalian cells. Here, we report that gold nanoparticles conjugated with DNA aptamer (AuNP-Apt) efficiently delivered AMPs into mammalian living systems with enhanced stability of the AMPs. C-terminally hexahistidine-tagged A3-APO (A3-APO His ) AMPs were loaded onto AuNPs conjugated with His-tag DNA aptamer (AuNP-Apt His ) by simple mixing and were delivered into Salmonella enterica serovar Typhimurium ( S. Typhimurium)-infected HeLa cells, resulting in the increased viability of host cells due to the elimination of intracellular S. Typhimurium cells. Furthermore, the intravenous injection of AuNP-Apt His loaded with A3-APO His into S. Typhimurium -infected mice resulted in a complete inhibition of S. Typhimurium colonization in the mice organs, leading to 100% survival of the mice. Therefore, AuNP-Apt His can serve as an innovative platform for AMP therapeutics to treat intracellular bacterial infections in mammals.

A thermochemical nonequilibrium analysis was performed under the low enthalpy shock-tunnel flows. A quasi-one-dimensional flow calculation was employed by dividing the flow calculations into two parts, for the shock-tube and the Mach 6 nozzle. To describe the thermochemical nonequilibrium of the low enthalpy shock-tunnel flows, a three-temperature model is proposed. The three-temperature model treats the vibrational nonequilibrium of O2 and NO separately from the single nonequilibrium energy mode of the previous two-temperature model. In the three-temperature model, electron-electronic energies and vibrational energy of N2 are grouped as one energy mode, and vibrational energies of O2, O2+, and NO are grouped as another energy mode. The results for the shock-tunnel flows calculated using the three-temperature model were then compared with existing experimental data and the results obtained from one- and two-temperature models, for various operating conditions of the K1 shock-tunnel facility. The results of the thermochemical nonequilibrium analysis of the low enthalpy shock-tunnel flows suggest that the nonequilibrium characteristics of N2 and O2 need to be treated separately. The vibrational relaxation of O2 is much faster than that of N2 in low enthalpy condition, and the dissociation rate of O2 is manly influenced by the species vibrational temperature of O2. The proposed three-temperature model is able to describe the thermochemical nonequilibrium characteristics of N2 and O2 behind the incident and reflected shock waves, and the rapid vibrational freezing of N2 in nozzle expanding flows.

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&ndash, 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.

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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.

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene which encodes dystrophin protein. Dystrophin defect affects cardiac muscle as well as skeletal muscle. Cardiac dysfunction is observed in all patients with DMD over 18 years of age, but there is no curative treatment for DMD cardiomyopathy. To establish novel experimental platforms which reproduce the cardiac phenotype of DMD patients, here we established iPS cell lines from T lymphocytes donated from two DMD patients, with a protocol using Sendai virus vectors. We successfully conducted the differentiation of the DMD patient-specific iPS cells into beating cardiomyocytes. DMD patient-specific iPS cells and iPS cell-derived cardiomyocytes would be a useful in vitro experimental system with which to investigate DMD cardiomyopathy.

Hydroxyapatite (HA) is widely used as a bioactive ceramics as it forms a chemical bond with bone. However, the drawback to using this material is its inferior mechanical properties. In this research, surface corrosion and disintegration of nanoscaled HA in a dog were studied, and the mechanism by which phase-pure HA dissolved In Vivo was investigated. Biological properties of HA In Vivo are affected by the grain-boundary dissolution followed by a surface corrosion and microstructural disintegration. This kind of dissolution process, apparently evidenced at the grain boundary, causes particle generation, which indicates that both long-term bone in-growth and mechanical properties can dramatically deteriorate. Implant dissolution by osteoclasts In Vivo is also observed on the surface of hydroxyapatite. Implant surface showed an aggressive corrosion by an osteoclast resorption. Severe and deeper dissolution underwent close to osteoclast resulting in formation of smaller and more round particle shape.

A series of experimental study has been conducted to investigate the flow associated with a moving throat plug in a shock tunnel testing. Bird et al [1] employed the moving throat plug to prevent upstream diaphragm fragments from entering the nozzle and blasting the model in the shock tunnel testing.

Background The sympathetic nervous system is critical in maintaining the normal physiological function of the heart. Its dysfunction in pathological states may exacerbate the substrate for arrhythmias. Obviously, knowledge of its three-dimensional (3D) structure is important, however, it has been revealed by conventional methods only to a limited extent. In this study, a new method of tissue clearance in combination with immunostaining unravels the 3D structure of the sympathetic cardiac network as well as its changes after myocardial infarction. Methods and results Hearts isolated from adult male mice were optically cleared using the CUBIC-perfusion protocol. After making the hearts transparent, sympathetic nerves and coronary vessels were immunofluorescently labeled, and then images were acquired. The spatial distribution of sympathetic nerves was visualized not only along the epicardial surface, but also transmurally. They were distributed over the epicardial surface and penetrated into the myocardium to twist around coronary vessels, but also independent from the coronary vasculature. At 2 weeks after myocardial infarction, we were able to quantify both denervation distal from the site of infarction and nerve sprouting (hyperinnervation) at the ischemic border zone of the hearts in a 3D manner. The nerve density at the ischemic border zone was more than doubled in hearts with myocardial infarction compared to intact mice hearts (3D analyses; n = 5, p

The DNA damage response (DDR) plays a pivotal role in maintaining genome integrity. DNA damage and DDR activation are observed in the failing heart, however, the type of DNA damage and its role in the pathogenesis of heart failure remain elusive. Here we show the critical role of DNA single-strand break (SSB) in the pathogenesis of pressure overload-induced heart failure. Accumulation of unrepaired SSB is observed in cardiomyocytes of the failing heart. Unrepaired SSB activates DDR and increases the expression of inflammatory cytokines through NF-κB signalling. Pressure overload-induced heart failure is more severe in the mice lacking XRCC1, an essential protein for SSB repair, which is rescued by blocking DDR activation through genetic deletion of ATM, suggesting the causative role of SSB accumulation and DDR activation in the pathogenesis of heart failure. Prevention of SSB accumulation or persistent DDR activation may become a new therapeutic strategy against heart failure., DNA damage response (DDR) is activated in cardiomyocytes of the failing heart, but the type of DNA damage leading to DDR is unclear. Higo et al. show that in mice heart failure is caused in part by unrepaired DNA single-strand breaks in cardiomyocytes, which activate persistent DDR and trigger an NF-κB-dependent cardiac inflammation.

Engineering of three-dimensional (3D) cardiac tissues using decellularized extracellular matrix could be a new technique to create an "organ-like" structure of the heart. To engineer artificial hearts functionally comparable to native hearts, however, much remain to be solved including stable excitation-propagation. To elucidate the points, we examined conduction properties of engineered tissues. We repopulated the decellularized hearts with neonatal rat cardiac cells and then, we observed excitation-propagation of spontaneous beatings using high resolution cameras. We also conducted immunofluorescence staining to examine morphological aspects. Live tissue imaging revealed that GFP-labeled-isolated cardiac cells were migrated into interstitial spaces through extravasation from coronary arteries. Engineered hearts repopulated with Ca(2+)-indicating protein (GCaMP2)-expressing cardiac cells were subjected to optical imaging experiments. Although the engineered hearts generally showed well-organized stable excitation-propagation, the hearts also demonstrated arrhythmogenic propensity such as disorganized propagation. Immunofluorescence study revealed randomly-mixed alignment of cardiomyocytes, endothelial cells and smooth muscle cells. The recellularized hearts also showed disarray of cardiomyocytes and markedly decreased expression of connexin43. In conclusion, we successfully demonstrated that the recellularized hearts showed dynamic excitation-propagation as a "whole organ". Our strategy could provide prerequisite information to construct a 3D-engineered heart, functionally comparable to the native heart.

Notch signaling is involved in an intercellular communication mechanism that is essential for coordinated cell fate determination and tissue morphogenesis. The biological effects of Notch signaling are context-dependent. We investigated the functional and hierarchical relationship between angiotensin (Ang) II receptor signaling and Notch signaling in vascular smooth muscle cells (VSMCs). A fluorogenic substrate assay revealed directly that the enzymatic activity of γ-secretase was enhanced after 10 min of Ang II stimulation in HEK293 cells expressing Ang II type 1 receptor. Notch cleavage by γ-secretase was consistently induced and peaked at 10 min after Ang II stimulation, and the Ang II-stimulated increase in Notch intracellular domain production was significantly suppressed by treatment with the γ-secretase inhibitor DAPT. Treatment with DAPT also significantly reduced the Ang II-stimulated proliferation and migration of human aortic VSMCs, as revealed by BrdU incorporation and the Boyden chamber assay, respectively. Systemic administration of the γ-secretase inhibitor dibenzazepine reduced Ang II-induced medial thickening and perivascular fibrosis in the aortas of wild-type mice. These findings suggest that the hierarchical Ang II receptor-Notch signaling pathway promotes the proliferation and migration of VSMCs, and thereby contributes to the progression of vascular remodeling.

Amphotericin B (AmB) has been widely used against fungal infections throughout almost the entire body, including the skin, nails, oral cavity, respiratory tract, and urinary tract. However, the development of AmB-loaded nanoparticles demands a novel technique that reduces its toxicity and other associated problems. Here, we developed a pH-responsive and redox-sensitive polymer-based AmB-delivery carrier system. In particular, this system was functionalized by conjugation with the antifungal peptide histatin 5, which acts both as a targeting ligand and a synergistic antifungal molecule against Candida albicans, a major systemic fungal pathogen of humans. Our results in vitro and in vivo suggest that this drug-delivery system may serve as a novel tool to facilitate the use of antimicrobial peptides as targeting ligands to pathogenic microbes, which would open new avenues of investigation in the field of drug delivery.

We present herein a first proof of concept demonstrating the potential of a protein nanopore-based technique for real-time detection of selected Gram-negative bacteria (Pseudomonas aeruginosa or Escherichia coli) at a concentration of 1.2 × 108 cfu/mL. The anionic charge on the bacterial outer membrane promotes the electrophoretically driven migration of bacteria towards a single α-hemolysin nanopore isolated in a lipid bilayer, clamped at a negative electric potential, and followed by capture at the nanopore’s mouth, which we found to be described according to the classical Kramers’ theory. By using a specific antimicrobial peptide as a putative molecular biorecognition element for the bacteria used herein, we suggest that the detection system can combine the natural sensitivity of the nanopore-based sensing techniques with selective biological recognition, in aqueous samples, and highlight the feasibility of the nanopore-based platform to provide portable, sensitive analysis and monitoring of bacterial pathogens. Electronic supplementary material The online version of this article (doi:10.1186/s11671-016-1715-z) contains supplementary material, which is available to authorized users.

Cardiac fibrosis is a pathological feature of myocardium of failing heart and plays causative roles in arrhythmia and cardiac dysfunction, but its regulatory mechanisms remain largely elusive. In this study, we investigated the effects of the novel EP4 receptor agonist ONO-0260164 on cardiac fibrosis in hypertrophied heart and explored the regulatory mechanisms in cardiac fibroblasts.In a mouse model of cardiac hypertrophy generated by transverse aortic constriction (TAC), ONO-0260164 treatment significantly prevented systolic dysfunction and progression of myocardial fibrosis at 5 weeks after TAC. In cultured neonatal rat cardiac fibroblasts, transforming growth factor-β1 (TGF-β1) induced upregulation of collagen type 1, alpha 1 (Col1a1) and type 3, alpha 1 (Col3a1), which was inhibited by ONO-0260164 treatment. ONO-0260164 activated protein kinase A (PKA) in the presence of TGF-β1 in the cardiac fibroblasts. PKA activation suppressed an increase in collagen expression induced by TGF-β1, indicating the important inhibitory roles of PKA activation in TGF-β1mediated collagen induction.We have demonstrated for the first time the antifibrotic effects of the novel EP4 agonist ONO-0260164 in vivo and in vitro, and the important role of PKA activation in the effects.

Activation of β-catenin-dependent canonical Wnt signaling in endothelial cells plays a key role in angiogenesis during development and ischemic diseases, however, other roles of Wnt/β-catenin signaling in endothelial cells remain poorly understood. Here, we report that sustained activation of β-catenin signaling in endothelial cells causes cardiac dysfunction through suppressing neuregulin-ErbB pathway in the heart. Conditional gain-of-function mutation of β-catenin, which activates Wnt/β-catenin signaling in Bmx-positive arterial endothelial cells (Bmx/CA mice) led to progressive cardiac dysfunction and 100% mortality at 40 weeks after tamoxifen treatment. Electron microscopic analysis revealed dilatation of T-tubules and degeneration of mitochondria in cardiomyocytes of Bmx/CA mice, which are similar to the changes observed in mice with decreased neuregulin-ErbB signaling. Endothelial expression of Nrg1 and cardiac ErbB signaling were suppressed in Bmx/CA mice. The cardiac dysfunction of Bmx/CA mice was ameliorated by administration of recombinant neuregulin protein. These results collectively suggest that sustained activation of Wnt/β-catenin signaling in endothelial cells might be a cause of heart failure through suppressing neuregulin-ErbB signaling and that the Wnt/β-catenin/NRG axis in cardiac endothelial cells might become a therapeutic target for heart failure.

Objective To evaluate the protective effect of Ginkgo biloba extract (GBE) on gentamicin (GM)-induced morphological damage of an artificial otoconia. Materials and methods An artificial otoconia powder was placed in a 12-well culture plate containing artificial endolymph. GM (500 μL; 40 mg/mL) was added to the first four wells. GM (500 μL; 40 mg/mL) with GBE (500 μL) was added to the next four wells. PBS (500 μL) was added to the remaining four wells as a control. The levels of nitric oxide (NO) synthesis were determined in the supernatants of each group. Alteration in surface morphology and calcium content were determined by scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX), respectively. Results NO concentration increased in the GM-treated group compared with that in the control group. When the calcite-gelatin composite was treated with GBE, GM-stimulated NO production significantly decreased. The surface of the calcite-gelatin composite exposed to GM showed erosive fissures and had a honeycomb-like appearance. GBE showed a protective effect against dissolution. EDX showed that the calcium content of the GM-treated group significantly decreased. However, the GBE-treated group demonstrated an insignificant change in the calcium concentration compared with the control. Conclusion From these results, we can conclude that GBE may have a protective effect against GM-induced NO production and superficial structural changes.

Natural hydroxyapatite powder was obtained from the calcination of bovine bones and its porous compacts were fabricated by pressureless sintering at 1100 and for 1h. To evaluate and compare their biocompatibility with porosity, we investigated the support of osteoblast cells growth and cytotoxicity using the MG-63 cell line model in vitro. Sintered hydroxyapatite ceramics have a porous microstructure with a relative density of 65 % at and 82 % at . Cells adherence to the surface of hydroxyapatite ceramics was observed in a day after the cell culture, and the spreading of cytoplasm around the nucleus was shown after 3 day cell culture. Most of cells were extended to the surface of hydroxyapatite through the wide area. Cell viability was nearly the same till 3 days culturing. But the rate of cell growth is higher in the specimen sintered at than that of . It indicates that the porosity is an important factor to enhance the cell viability in the porous hydroxyapatite ceramics derived from bovine bones.

A throat plug is a device to prevent fragments produced by the bursting of the primary diaphragm of a shock tunnel from entering the nozzle and damaging the model. An experimental study has been carried out to investigate the flow in the KAIST shock tunnel with two stationary throat plugs at primary shock velocities of 1.19 and 1.28 km/s. These shocks generate tailored conditions when helium and air are used as the driver and driven gases, respectively, at room temperature. Nozzle reservoir pressures and Pitot pressures at the nozzle exit are measured to examine the influence of the stationary throat plug on these properties. The throat plug that has an areal blockage of 6.2 % is found to have negligible effects on shock tunnel flow. However, the throat plug of 19.4 % blockage shows an appreciable influence by generating a pressure bump. Although it retards the establishment time of the nozzle flow, after the transient period, it does not cause deviation of the pressure from that for the clean nozzle configuration. It is found that the reduction of the cross-sectional area, which results from the presence of the throat plug, causes the pressure bump. By increasing the diameter of the driven tube in the region where the throat plug is located appropriately, the magnitude of the pressure bump is reduced significantly.

Congestive heart failure (CHF) predisposes to ventricular fibrillation (VF) in association with electrical remodeling of the ventricle. However, much remains unknown about the rate-dependent electrophysiological properties in a failing heart. Action potential properties in the left ventricular subepicardial muscles during dynamic pacing were examined with optical mapping in pacing-induced CHF ( n = 18) and control ( n = 17) rabbit hearts perfused in vitro. Action potential durations (APDs) in CHF were significantly longer than those observed for controls at basic cycle lengths (BCLs) >1,000 ms but significantly shorter at BCLs

Rat superior cervical ganglion (SCG) neurons and the ventricular myocytes (VMs) were cocultured in chambers made of polydimethylsiloxane. The chambers were placed on a microelectrode array (MEA) substrate and connected with a pathway. Twenty-four hours after dissemination of the VMs, neurites of the SCG neurons outgrew through the pathway and reached the VMs. Spontaneous electrical activities of the SCG neurons and the VMs were observed several days after the dissemination. Constant-voltage stimulation (1 V, 1 ms, biphasic square pulses) was applied to the SCG neurons at the frequency of 10 Hz using 32 electrodes. Contraction rate of the VMs increased by 153 ± 110% immediately after the stimulation to the SCG neurons was stopped. Then contraction rate gradually decreased and returned to almost the same rate as before the stimulation 5 min after the stimulation. Propranolol (β-adrenergic receptor antagonist) prevented contraction rate of the VMs from increasing after electrical stimulation to the SCG neurons. These results suggest that neuromuscular junctions were formed between the SCG neurons and the VMs and that this coculture system can be utilized in the research of cell–cell interactions between the sympathetic neurons and the cardiomyocytes. © 2011 Institute of Electrical Engineers of Japan. Published by John Wiley & Sons, Inc.