Contractility assessment using aligned human iPSC-derived cardiomyocytes.

INTRODUCTION: Cardiac safety assessment, such as lethal arrhythmias and contractility dysfunction, is critical during drug development. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been shown to be useful in predicting drug-induced proarrhythmic risk through international validation studies. Although cardiac contractility is another key function, fit-for-purpose hiPSC-CMs in evaluating drug-induced contractile dysfunction remain poorly understood. In this study, we investigated whether alignment of hiPSC-CMs on nanopatterned culture plates can assess drug-induced contractile changes more efficiently than non-aligned monolayer culture. METHODS: Aligned hiPSC-CMs were obtained by culturing on 96-well culture plates with a ridge-groove-ridge nanopattern on the bottom surface, while non-aligned hiPSC-CMs were cultured on regular 96-well plates. Next-generation sequencing and qPCR experiments were performed for gene expression analysis. Contractility of the hiPSC-CMs was assessed using an imaging-based motion analysis system. RESULTS: When cultured on nanopatterned plates, hiPSC-CMs exhibited an aligned morphology and enhanced expression of genes encoding proteins that regulate contractility, including myosin heavy chain, calcium channel, and ryanodine receptor. Compared to cultures on regular plates, the aligned hiPSC-CMs also showed both enhanced contraction and relaxation velocity. In addition, the aligned hiPSC-CMs showed a more physiological response to positive and negative inotropic agents, such as isoproterenol and verapamil. DISCUSSION: Taken together, the aligned hiPSC-CMs exhibited enhanced structural and functional properties, leading to an improved capacity for contractility assessment compared to the non-aligned cells. These findings suggest that the aligned hiPSC-CMs can be used to evaluate drug-induced cardiac contractile changes.

Effects of cigarette smoke extract derived from heated tobacco products on the proliferation of lung cancer stem cells.

Epidemiological studies have suggested that cigarette smoking can increase a person's risk of developing several types of cancer, including lung cancer. Lung cancer originates from cancer stem cells (CSCs), which constitute a minor cell population in tumors, and contribute to drug resistance and recurrence. Heated tobacco products (HTPs) produce aerosols that contain nicotine and toxic chemicals. Current evidence, however, is insufficient to accurately determine if HTPs are less harmful than burned cigarettes. This study has investigated the effects of cigarette smoke extract (CSE) from HTPs on lung CSCs in lung cancer cell lines. We found that CSEs induced the proliferation of lung CSCs and increased the expression levels of stem cell markers. In addition, CSE induced epithelial-mesenchymal transition (EMT) expression and cytokine production. These results suggest that HTPs can induce lung CSCs in vitro.

Oligoarginine-Conjugated Peptide Foldamers Inhibiting Vitamin D Receptor-Mediated Transcription.

The vitamin D receptor (VDR) is a nuclear receptor, which is involved in several physiological processes, including differentiation and bone homeostasis. The VDR is a promising target for the development of drugs against cancer and bone-related diseases. To date, several VDR antagonists, which bind to the ligand binding domain of the VDR and compete with the endogenous agonist 1α,25(OH)D3, have been reported. However, these ligands contain a secosteroidal skeleton, which is chemically unstable and complicated to synthesize. A few VDR antagonists with a nonsecosteroidal skeleton have been reported. Alternative inhibitors against VDR transactivation that act via different mechanisms are desirable. Here, we developed peptide-based VDR inhibitors capable of disrupting the VDR-coactivator interaction. It was reported that helical SRC2-3 peptides strongly bound to the VDR and competed with the coactivator in vitro. Therefore, we designed and synthesized a series of SRC2-3 derivatives by the introduction of nonproteinogenic amino acids, such as ß-amino acids, and by side-chain stapling to stabilize helical structures and provide resistance against digestive enzymes. In addition, conjugation with a cell-penetrating peptide increased the cell membrane permeability and was a promising strategy for intracellular VDR inhibition. The nona-arginine-conjugated peptides 24 with side-chain stapling and 25 with cyclic ß-amino acids showed strong intracellular VDR inhibitory activity, resulting in suppression of the target gene expression and inhibition of the cell differentiation of HL-60 cells. Herein, the peptide design, structure-activity relationship (SAR) study, and biological evaluation of the peptides are described.

Can a Short-term Daily Oral Administration of Propolis Improve Muscle Fatigue and Recovery?

This study investigated the effect of 1-week oral administration of propolis on muscle fatigue and recovery after performing a fatigue task (total 100 maximal voluntary concentric knee extension repetitions). In this placebo-controlled, double-blind study, 18 young men consumed a formulation with high Brazilian green propolis dose (H-BGP), a formulation with low Brazilian green propolis dose, or a placebo, for 1 week before performing the fatigue task (an interval between each intervention: 1-2 weeks). Maximal voluntary contraction torque, central fatigue (voluntary activation and root mean square values of the surface electromyography amplitude), and peripheral fatigue (potentiated triplet torque) were assessed before, immediately after, and 2 minutes after the fatigue task. Maximal voluntary contraction torque decreased immediately after the fatigue task in all conditions (P<0.001); however, it recovered from immediately after to 2 minutes after the fatigue task only in the H-BGP condition (P<0.001). Furthermore, there was a significant decrease in voluntary activation (P<0.001) and root mean square values of the surface electromyography amplitude (P≤0.035) only in the placebo condition. No significant difference was observed in the time-course change in potentiated triplet torque between the conditions. These results suggest that oral administration of propolis promotes muscle fatigue recovery by reducing central fatigue.

Transforming Growth Factor Beta Promotes the Expansion of Cancer Stem Cells via S1PR3 by Ligand-Independent Notch Activation.

Growing evidence suggests that cancer originates from cancer stem cells (CSCs), which can be identified by aldehyde dehydrogenase (ALDH) activity-based flow cytometry. However, the regulation of CSC growth is not fully understood. In the present study, we investigated the effects of Transforming Growth Factor-ß (TGFß) in breast CSC expansion. Stimulation with TGFß increased the ALDH-positive breast CSC population via the phosphorylation of sphingosine kinase 1 (SphK1), a sphingosine-1-phosphate (S1P)-producing enzyme, and subsequent S1P-mediated S1P receptor 3 (S1PR3) activation. These data suggest that TGFß promotes breast CSC expansion via the ALK5/SphK1/S1P/S1PR3 signaling pathway. Our findings provide new insights into the role of TGFß in the regulation of CSCs.

Lysophosphatidic Acid Promotes the Expansion of Cancer Stem Cells via TRPC3 Channels in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a highly aggressive cancer for which targeted therapeutic agents are limited. Growing evidence suggests that TNBC originates from breast cancer stem cells (BCSCs), and elucidation of the molecular mechanisms controlling BCSC proliferation will be crucial for new drug development. We have previously reported that the lysosphingolipid sphingosine-1-phosphate mediates the CSC phenotype, which can be identified as the ALDH-positive cell population in several types of human cancer cell lines. In this study, we have investigated additional lipid receptors upregulated in BCSCs. We found that lysophosphatidic acid (LPA) receptor 3 was highly expressed in ALDH-positive TNBC cells. The LPAR3 antagonist inhibited the increase in ALDH-positive cells after LPA treatment. Mechanistically, the LPA-induced increase in ALDH-positive cells was dependent on intracellular calcium ion (Ca2+), and the increase in Ca2+ was suppressed by a selective inhibitor of transient receptor potential cation channel subfamily C member 3 (TRPC3). Moreover, IL-8 production was involved in the LPA response via the activation of the Ca2+-dependent transcriptional factor nuclear factor of activated T cells. Taken together, our findings provide new insights into the lipid-mediated regulation of BCSCs via the LPA-TRPC3 signaling axis and suggest several potential therapeutic targets for TNBC.

Lithocholic Acid Amides as Potent Vitamin D Receptor Agonists.

1α,25-Dihydroxyvitamin D3 [1α,25(OH)2D3, 1] is an active form of vitamin D3 and regulates various biological phenomena, including calcium and phosphate homeostasis, bone metabolism, and immune response via binding to and activation of vitamin D receptor (VDR). Lithocholic acid (LCA, 2) was identified as a second endogenous agonist of VDR, though its potency is very low. However, the lithocholic acid derivative 3 (Dcha-20) is a more potent agonist than 1α,25(OH)2D3, (1), and its carboxyl group has similar interactions to the 1,3-dihydroxyl groups of 1 with amino acid residues in the VDR ligand-binding pocket. Here, we designed and synthesized amide derivatives of 3 in order to clarify the role of the carboxyl group. The synthesized amide derivatives showed HL-60 cell differentiation-inducing activity with potency that depended upon the substituent on the amide nitrogen atom. Among them, the N-cyanoamide 6 is more active than either 1 or 3.

FTY720 Inhibits Expansion of Breast Cancer Stem Cells via PP2A Activation.

Growing evidence suggests that breast cancer originates from a minor population of cancer cells termed cancer stem cells (CSCs), which can be identified by aldehyde dehydrogenase (ALDH) activity-based flow cytometry analysis. However, novel therapeutic drugs for the eradication of CSCs have not been discovered yet. Recently, drug repositioning, which finds new medical uses from existing drugs, has been expected to facilitate drug discovery. We have previously reported that sphingosine kinase 1 (SphK1) induced proliferation of breast CSCs. In the present study, we focused on the immunosuppressive agent FTY720 (also known as fingolimod or Gilenya), since FTY720 is known to be an inhibitor of SphK1. We found that FTY720 blocked both proliferation of ALDH-positive cells and formation of mammospheres. In addition, we showed that FTY720 reduced the expression of stem cell markers such as Oct3/4, Sox2 and Nanog via upregulation of protein phosphatase 2A (PP2A). These results suggest that FTY720 is an effective drug for breast CSCs in vitro.

Relationship between resting medial gastrocnemius stiffness and drop jump performance.

Although the influence of the series elastic element of the muscle-tendon unit on jump performance has been investigated, the corresponding effect of the parallel elastic element remains unclear. This study examined the relationship between the resting calf muscle stiffness and drop jump performance. Twenty-four healthy men participated in this study. The shear moduli of the medial gastrocnemius and the soleus were measured at rest as an index of muscle stiffness using ultrasound shear wave elastography. The participants performed drop jumps from a 15 cm high box. The Spearman rank correlation coefficient was used to examine the relationships between shear moduli of the muscles and drop jump performance. The medial gastrocnemius shear modulus showed a significant correlation with the drop jump index (jump height/contact time) (r = 0.414, P = 0.044) and jump height (r = 0.411, P = 0.046), but not with contact time (P > 0.05). The soleus shear modulus did not correlate with these jump parameters (P > 0.05). These results suggest that the resting medial gastrocnemius stiffness can be considered as one of the factors that influence drop jump performance. Therefore, increase in resting muscle stiffness should enhance explosive athletic performance in training regimens.

Lithocholic Acid Derivatives as Potent Vitamin D Receptor Agonists.

Lithocholic acid (2) was identified as a second endogenous ligand of vitamin D receptor (VDR), though its activity is very weak. In this study, we designed novel lithocholic acid derivatives based on the crystal structure of VDR-ligand-binding domain (LBD) bound to 2. Among the synthesized compounds, 6 bearing a 2-hydroxy-2-methylprop-1-yl group instead of the 3-hydroxy group at the 3α-position of 2 showed dramatically increased activity in HL-60 cell differentiation assay, being at least 10 000 times more potent than lithocholic acid (2) and 3 times more potent than 1α,25-dihydroxyvitamin D3 (1). Although the binding affinities of 6 and its epimer 7 were less than that of 1, their transactivation activities were greater than that of 1. X-ray structure analyses of VDR LBD bound to 6 or 7 showed that the binding positions of these compounds in the ligand-binding pocket are similar to that of 1.

Relationship Between Drop Jump Training-Induced Changes in Passive Plantar Flexor Stiffness and Explosive Performance.

Passive muscle stiffness is positively associated with explosive performance. Drop jump training may be a strategy to increase passive muscle stiffness in the lower limb muscles. Therefore, the purpose of this study was to examine the effect of 8-week drop jump training on the passive stiffness in the plantar flexor muscles and the association between training-induced changes in passive muscle stiffness and explosive performance. This study was a randomized controlled trial. Twenty-four healthy young men were divided into two groups, control and training. The participants in the training group performed drop jumps (five sets of 20 repetitions each) 3days per week for 8weeks. As an index of passive muscle stiffness, the shear moduli of the medial gastrocnemius and soleus were measured by shear wave elastography before and after the intervention. The participants performed maximal voluntary isometric plantar flexion at an ankle joint angle of 0° and maximal drop jumps from a 15cm high box. The rate of torque development during isometric contraction was calculated. The shear modulus of the medial gastrocnemius decreased for the training group (before: 13.5±2.1kPa, after: 10.6±2.1kPa); however, such a reduction was not observed in the control group. There was no significant group (control and training groups)×time (before and after the intervention) interaction for the shear modulus of the soleus. The drop jump performance for the training group improved, while the rate of torque development did not change. Relative changes in these measurements were not correlated with each other in the training group. These results suggest that drop jump training decreases the passive stiffness in the medial gastrocnemius, and training-induced improvement in explosive performance cannot be attributed to change in passive muscle stiffness.

Eight-Week Low-Intensity Squat Training at Slow Speed Simultaneously Improves Knee and Hip Flexion and Extension Strength.

Considering that the squat exercise requires flexion and extension of the knee and hip joints, a resistance training program based on squat exercises should efficiently increase the flexion and extension strength of both the knee and hip. To our knowledge, however, no study has simultaneously investigated the effects of squat training on both flexion and extension strength in both the knee and hip. Low-intensity squat exercises at slow speeds can be expected to effectively and safely improve knee and hip flexion and extension strength in a wide range of individuals. This study aimed to clarify whether knee and hip flexion and extension strength improved after an 8-week low-intensity squat training program at slow speed. Twenty-four untrained young men were randomly assigned to a training or control group. Participants in the training group performed 40% one-repetition maximum parallel squats at slow speed (4 s for concentric/eccentric actions), 3 days per week for 8 weeks. Before and after the intervention, isometric peak torque of the knee and hip flexors and extensors during maximal voluntary contraction (MVC) was determined. For the knee flexors and extensors, muscle volume was also measured. There were significant training-induced increases in peak torque (P < 0.05). The training effects on knee and hip extension torque (effect size = 0.36-0.38) were higher than those on knee and hip flexion torque (effect size = 0.09-0.13). The squat training used here increased both knee and hip flexion and extension strength, but the training effects on the flexion strength were less than those on the extension strength. Regarding the knee extensors, a significant training-related increase in muscle volume was found (P < 0.05) without neuromuscular adaptations. In addition, there were significant correlations between the training-induced increases in muscle volume and peak torque of KE. These results suggest that muscle hypertrophy may be responsible for increased muscle strength of the knee extensors after an 8-week low-intensity squat training program at slow speed.

Carbon dioxide hydrate as a recovery tool after fatigue of the plantar flexors.

This study investigated the effects of cooling the triceps surae with carbon dioxide hydrate (CDH), which is a gas hydrate, a crystalline structure belonging to the clathrates, on the recovery from muscle fatigue. Thirty-six healthy young men were equally and randomly assigned to an ICE group, a CDH group, or a non-cooling (NON) group. All participants performed 80 maximal voluntary isometric contractions (MVCs) of the plantar flexors as a fatiguing task. MVC torque and voluntary activation were determined before, immediately after, and 20 min after the fatiguing task. Evoked torque was similarly assessed except for immediately after the task. In the ICE and CDH groups, the triceps surae was cooled for 5 min using ice and CDH, starting 5 min after the fatiguing task, respectively. The MVC torque and voluntary activation were higher in order of before >20 min after >immediately after the fatiguing task regardless of group, and those time-course changes did not differ between the groups. A decrease in the evoked torque from before to 20 min after the fatiguing task was observed in the ICE and NON groups but not in the CDH group. These results suggest that cooling muscle with CDH can facilitate recovery from peripheral muscle fatigue. This may be due to an increase in blood flow caused by carbon dioxide contained within the CDH, and indicates the potential of CDH as a recovery tool after fatiguing exercise.

Synthetic biology based construction of biological activity-related library of fungal decalin-containing diterpenoid pyrones.

A synthetic biology method based on heterologous biosynthesis coupled with genome mining is a promising approach for increasing the opportunities to rationally access natural product with novel structures and biological activities through total biosynthesis and combinatorial biosynthesis. Here, we demonstrate the advantage of the synthetic biology method to explore biological activity-related chemical space through the comprehensive heterologous biosynthesis of fungal decalin-containing diterpenoid pyrones (DDPs). Genome mining reveals putative DDP biosynthetic gene clusters distributed in five fungal genera. In addition, we design extended DDP pathways by combinatorial biosynthesis. In total, ten DDP pathways, including five native pathways, four extended pathways and one shunt pathway, are heterologously reconstituted in a genetically tractable heterologous host, Aspergillus oryzae, resulting in the production of 22 DDPs, including 15 new analogues. We also demonstrate the advantage of expanding the diversity of DDPs to probe various bioactive molecules through a wide range of biological evaluations.

Active recovery has a positive and acute effect on recovery from fatigue induced by repeated maximal voluntary contractions of the plantar flexors.

This study investigated the acute effect of active recovery (AR) following fatigue induced by 80 three-second maximal voluntary isometric plantar flexion contractions (MVICs) in 12 young men. AR consisted of a total of 180 voluntary isometric ramp contractions of the plantar flexors (0.75-s contraction/relaxation) targeting 10% of MVIC torque. MVIC torque, voluntary activation and root mean square values of electromyographic signals for the triceps surae normalized by each peak-to-peak amplitude of compound motor action potential were determined before, and immediately, 10, 20 and 30 min after the fatiguing task. Evoked torques were similarly assessed except for immediately after it. The AR and passive recovery were randomly performed on two days by each participant between 5 min and 10 min after the fatiguing task. For all the parameters other than MVIC torque, there was no significant difference between the conditions at any time point. MVIC torque decreased significantly immediately after the fatiguing task regardless of condition (P < 0.05), and the corresponding decrease in MVIC torque recovered 30 min after the fatiguing task only in AR (P < 0.05). These results suggest an acute positive effect of AR on recovery of neuromuscular function and/or contractile properties after fatigue.

Structural Development of Cell-Penetrating Peptides Containing Cationic Proline Derivatives.

We designed and synthesized a series of cell-penetrating peptides containing cationic proline derivatives (ProGu) that exhibited responsive changes in their secondary structures to the cellular environment. Effects of the peptide length and steric arrangement of the side chain in cationic proline derivatives [Pro4SGu and Pro4RGu] on their secondary structures and cell membrane permeability were investigated. Moreover, peptides 3 and 8 exhibited efficient intracellular delivery of plasmid DNA.

Tobacco nitrosamine NNK increases ALDH-positive cells via ROS-Wnt signaling pathway in A549 human lung cancer cells.

Epidemiological studies suggest that lung cancer, which is a major cause of cancer death, has a critical association with cigarette smoking. Tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in cigarette smoke is a major risk factor for carcinogenesis. However, the mechanisms by which NNK promotes cancer development have not been fully elucidated. Growing evidence suggests that lung cancer originates from cancer stem cells (CSCs), which are a minor population of lung cancer cells. In the present study, we investigated the effects of NNK on the CSCs in A549 human lung cancer cells using flow cytometry with aldehyde dehydrogenase (ALDH), a functional marker of CSCs. We found that NNK increased the proportion of ALDH-positive cells in a dose-dependent manner. A Wnt inhibitor PNU74654 reduced NNK-induced expression levels of Wnt target gene Dkk1 and increase in ALDH-positive cells. We next examined the signaling pathway that mediates the NNK-induced increase in ALDH-positive cells via Wnt signaling. DCF assay revealed that NNK induced reactive oxygen species (ROS) production. The ROS scavenger N-acetylcysteine (NAC) inhibited the NNK-induced Wnt activation and increase in ALDH-positive cells. These data suggest that NNK-induced ROS activate the Wnt signaling pathway in A549 cells. These findings would provide new insights into the role of NNK in the lung CSCs.

Tributyltin induces mitochondrial fission through Mfn1 degradation in human induced pluripotent stem cells.

Organotin compounds, such as tributyltin (TBT), are well-known endocrine disruptors. TBT is also known to cause various forms of cytotoxicity, including neurotoxicity and immunotoxicity. However, TBT toxicity has not been identified in normal stem cells. In the present study, we examined the effects of TBT on cell growth in human induced pluripotent stem cells (iPSCs). We found that exposure to nanomolar concentrations of TBT decreased intracellular ATP levels and inhibited cell viability in iPSCs. Because TBT suppressed energy production, which is a critical function of the mitochondria, we further assessed the effects of TBT on mitochondrial dynamics. Staining with MitoTracker revealed that nanomolar concentrations of TBT induced mitochondrial fragmentation. TBT also reduced the expression of mitochondrial fusion protein mitofusin 1 (Mfn1), and this effect was abolished by knockdown of the E3 ubiquitin ligase membrane-associated RING-CH 5 (MARCH5), suggesting that nanomolar concentrations of TBT could induce mitochondrial dysfunction via MARCH5-mediated Mfn1 degradation in iPSCs. Thus, mitochondrial function in normal stem cells could be used to assess cytotoxicity associated with metal exposure.

Tributyltin induces G2/M cell cycle arrest via NAD(+)-dependent isocitrate dehydrogenase in human embryonic carcinoma cells.

Organotin compounds, such as tributyltin (TBT), are well-known endocrine-disrupting chemicals (EDCs). We have recently reported that TBT induces growth arrest in the human embryonic carcinoma cell line NT2/D1 at nanomolar levels by inhibiting NAD(+)-dependent isocitrate dehydrogenase (NAD-IDH), which catalyzes the irreversible conversion of isocitrate to α-ketoglutarate. However, the molecular mechanisms by which NAD-IDH mediates TBT toxicity remain unclear. In the present study, we examined whether TBT at nanomolar levels affects cell cycle progression in NT2/D1 cells. Propidium iodide staining revealed that TBT reduced the ratio of cells in the G1 phase and increased the ratio of cells in the G2/M phase. TBT also reduced cell division cycle 25C (cdc25C) and cyclin B1, which are key regulators of G2/M progression. Furthermore, apigenin, an inhibitor of NAD-IDH, mimicked the effects of TBT. The G2/M arrest induced by TBT was abolished by NAD-IDHα knockdown. Treatment with a cell-permeable α-ketoglutarate analogue recovered the effect of TBT, suggesting the involvement of NAD-IDH. Taken together, our data suggest that TBT at nanomolar levels induced G2/M cell cycle arrest via NAD-IDH in NT2/D1 cells. Thus, cell cycle analysis in embryonic cells could be used to assess cytotoxicity associated with nanomolar level exposure of EDCs.

Nicotine induces mitochondrial fission through mitofusin degradation in human multipotent embryonic carcinoma cells.

Nicotine is considered to contribute to the health risks associated with cigarette smoking. Nicotine exerts its cellular functions by acting on nicotinic acetylcholine receptors (nAChRs), and adversely affects normal embryonic development. However, nicotine toxicity has not been elucidated in human embryonic stage. In the present study, we examined the cytotoxic effects of nicotine in human multipotent embryonal carcinoma cell line NT2/D1. We found that exposure to 10 µM nicotine decreased intracellular ATP levels and inhibited proliferation of NT2/D1 cells. Because nicotine suppressed energy production, which is a critical mitochondrial function, we further assessed the effects of nicotine on mitochondrial dynamics. Staining with MitoTracker revealed that 10 µM nicotine induced mitochondrial fragmentation. The levels of the mitochondrial fusion proteins, mitofusins 1 and 2, were also reduced in cells exposed to nicotine. These nicotine effects were blocked by treatment with mecamylamine, a nonselective nAChR antagonist. These data suggest that nicotine degrades mitofusin in NT2/D1 cells and thus induces mitochondrial dysfunction and cell growth inhibition in a nAChR-dependent manner. Thus, mitochondrial function in embryonic cells could be used to assess the developmental toxicity of chemicals.