Ascorbic acid induces MLC2v protein expression and promotes ventricular-like cardiomyocyte subtype in human induced pluripotent stem cells derived cardiomyocytes.

Introduction: The potentially unlimited number of cardiomyocyte (CMs) derived from human induced pluripotent stem cells (hiPSCs) in vitro facilitates high throughput applications like cell transplantation for myocardial repair, disease modelling, and cardiotoxicity testing during drug development. Despite promising progress in these areas, a major disadvantage that limits the use of hiPSC derived CMs (hiPSC-CMs) is their immaturity. Methods: Three hiPSC lines (PCBC-hiPSC, DP3-hiPSCs, and MLC2v-mEGFP hiPSC) were differentiated into CMs (PCBC-CMs, DP3-CMs, and MLC2v-CMs, respectively) with or without retinoic acid (RA). hiPSC-CMs were either maintained up to day 30 of contraction (D30C), or D60C, or purified using lactate acid and used for experiments. Purified hiPSC-CMs were cultured in basal maturation medium (BMM) or BMM supplemented with ascorbic acid (AA) for 14 days. The AA treated and non-treated hiPSC-CMs were characterized for sarcomeric proteins (MLC2v, TNNI3, and MYH7), ion channel proteins (Kir2.1, Nav1.5, Cav1.2, SERCA2a, and RyR), mitochondrial membrane potential, metabolomics, and action potential. Bobcat339, a selective and potent inhibitor of DNA demethylation, was used to determine whether AA promoted hiPSC-CM maturation through modulating DNA demethylation. Results: AA significantly increased MLC2v expression in PCBC-CMs, DP3-CMs, MLC2v-CMs, and RA induced atrial-like PCBC-CMs. AA treatment significantly increased mitochondrial mass, membrane potential, and amino acid and fatty acid metabolism in PCBC-CMs. Patch clamp studies showed that AA treatment induced PCBC-CMs and DP3-CMs adaptation to a ventricular-like phenotype. Bobcat339 inhibited MLC2v protein expression in AA treated PCBC-CMs and DP3-CMs. DNA demethylation inhibition was also associated with reduced TET1 and TET2 protein expressions and reduced accumulation of the oxidative product, 5 hmC, in both PCBC-CMs and DP3-CMs, in the presence of AA. Conclusions: Ascorbic acid induced MLC2v protein expression and promoted ventricular-like CM subtype in hiPSC-CMs. The effect of AA on hiPSC-CM was attenuated with inhibition of TET1/TET2 mediated DNA demethylation.

The effect of side substitution and quantum interference on the performance of molecular thermoelectric devices: a brief review.

In recent years, researchers have shown great interest in organic thermoelectric materials that are economical, efficient, lightweight, and environmentally friendly. With advancements in experimental measurement techniques and theoretical calculations, investigations of the thermoelectric properties of molecular devices have become feasible. To regulate the thermoelectric properties of molecular devices, many strategies have been proposed. In this work, we review the theoretical analytical and experimental research methods used to study these properties. We then focus on two tuning strategies, side substitution, and quantum interface effects, which have demonstrated significant improvements in the thermoelectric performance of molecular devices. Finally, we discuss the challenges faced in experimental and theoretical studies and the future prospects of molecular thermoelectric devices.

Half-Heusler-like compounds with wide continuous compositions and tunable p- to n-type semiconducting thermoelectrics.

Half-Heusler and full-Heusler compounds were considered as independent phases with a natural composition gap. Here we report the discovery of TiRu1+xSb (x = 0.15 ~ 1.0) solid solution with wide homogeneity range and tunable p- to n-type semiconducting thermoelectrics, which bridges the composition gap between half- and full-Heusler phases. At the high-Ru end, strange glass-like thermal transport behavior with unusually low lattice thermal conductivity (~1.65 Wm-1K-1 at 340 K) is observed for TiRu1.8Sb, being the lowest among reported half-Heusler phases. In the composition range of 0.15 < x < 0.50, TiRu1+xSb shows abnormal semiconducting behaviors because tunning Ru composition results in band structure change and carrier-type variation simultaneously, which seemingly correlates with the localized d electrons. This work reveals the possibility of designing fascinating half-Heusler-like materials by manipulating the tetrahedral site occupancy, and also demonstrates the potential of tuning crystal and electronic structures simultaneously to realize intriguing physical properties.

SYBR® Green RT-qPCR for the Universal Detection of Citrus Viroids.

Quantitative polymerase chain reaction (qPCR) and reverse transcription (RT)-qPCR have now become the gold standard for molecular diagnostics because of its sensitivity, specificity, and reproducibility. In addition, qPCR diagnostics are flexible because they can be scaled for high- or low-throughput applications. Here we describe an optimized assay and workflow for the universal detection of eight citrus viroid species and their variants by RT-qPCR. The assay allows for quick and efficient molecular detection of viroids without the need to run RT-qPCR for each individual viroid species.

Angiopoietin-1 enhanced myocyte mitosis, engraftment, and the reparability of hiPSC-CMs for treatment of myocardial infarction.

AIMS: To examine whether transient over-expression of angiopoietin-1 (Ang-1) increases the potency of hiPSC-CMs for treatment of heart failure. METHODS AND RESULTS: Atrial hiPSC-CMs (hiPSC-aCMs) were differentiated from hiPSCs and purified by lactic acid and were transfected with Ang-1 (Ang-1-hiPSC-aCMs) plasmid using lipoSTEM. Ang-1 gene transfection efficiency was characterized in vitro. Gene transfected CMs (1×106) were seeded into a fibrin/thrombin patch and implanted on the rat-infarcted left ventricular (LV) anterior wall after myocardial infarction (MI). Echo function was determined at 1- and 6 weeks post-MI. Immunohistochemistry study was performed at 6 weeks post-MI. Ang-1 (20 and 40 ng/mL) protected hiPSC-aCMs from hypoxia through up-regulating pERK1/2 and inhibiting Bax protein expressions. Ang-1-hiPSC-aCMs transiently secreted Ang-1 protein up to 14 days, with peak level on day-2 post-transfection (24.39 ± 13.02 ng/mL) in vitro. Animal study showed that transplantation of Ang-1-hiPSC-aCM seeded patch more effectively limited rat heart apoptosis at 1 day post-MI as compared with LipoSTEM-Ang-1 or hiPSC-aCMs transplantation. Ang-1-hiPSC-aCMs transplantation induced host (rat) and donor (human) CM mitosis and arteriole formation, improved cell engraftment rate, more effectively limited LV dilation (EDV = 460.7 ± 96.1 µL and ESV = 219.8 ± 72.9 µL) and improved LV global pump function (EF = 53.1 ± 9%) as compared with the MI (EDV = 570.9 ± 91.8 µL, P = 0.033; ESV = 331.6 ± 71.2 µL, P = 0.011; EF = 42.3 ± 4.1%, P = 0.02) or the LipoSTEM-Ang-1 injected (EDV = 491.4 ± 100.4 µL, P = 0.854; ESV = 280.9 ± 71.5 µL, P = 0.287; EF = 43.2 ± 4.6, P = 0.039) or hiPSC-CM transplanted (EDV = 547.9 ± 55.5 µL, P = 0.095; ESV = 300.2 ± 88.4 µL, P = 0.075; EF = 46 ± 10.9%, P = 0.166) animal groups at 6 weeks post-MI and treatment. CONCLUSION: Transient over-expression of Ang-1 enhanced hiPSC-aCM mitosis and engraftment and increased the reparability potency of hiPSC-aCMs for treatment of MI.

Non-viral vector based gene transfection with human induced pluripotent stem cells derived cardiomyocytes.

Non-viral transfection of mammalian cardiomyocytes (CMs) is challenging. The current study aims to characterize and determine the non-viral vector based gene transfection efficiency with human induced pluripotent stem cells (hiPSCs) derived cardiomyocytes (hiPSC-CMs). hiPSC-CMs differentiated from PCBC hiPSCs were used as a cell model to be transfected with plasmids carrying green fluorescence protein (pGFP) using polyethylenimine (PEI), including Transporter 5 Transfection Reagent (TR5) and PEI25, and liposome, including lipofectamine-2000 (Lipo2K), lipofectamine-3000 (Lipo3K), and Lipofectamine STEM (LipoSTEM). The gene transfection efficiency and cell viability were quantified by flow cytometry. We found that the highest gene transfection efficiency in hiPSC-CMs on day 14 of contraction can be achieved by LipoSTEM which was about 32.5 ± 6.7%. However, it also cuased poor cell viability (60.1 ± 4.5%). Furthermore, a prolonged culture of (transfection on day 23 of contraction) hiPSC-CMs not only improved gene transfection (54.5 ± 8.9%), but also enhanced cell viability (74 ± 4.9%) by LipoSTEM. Based on this optimized gene transfection condition, the highest gene transfection efficiency was 55.6 ± 7.8% or 34.1 ± 4%, respectively, for P1C1 or DP3 hiPSC line that was derived from healthy donor (P1C1) or patient with diabetes (DP3). The cell viability was 80.8 ± 5.2% or 92.9 ± 2.24%, respectively, for P1C1 or DP3. LipoSTEM is a better non-viral vector for gene transfection of hiPSC-CMs. The highest pGFP gene transfection efficiency can reach >50% for normal hiPSC-CMs or >30% for diabetic hiPSC-CMs.

The prostaglandin H2 analog U-46619 improves the differentiation efficiency of human induced pluripotent stem cells into endothelial cells by activating both p38MAPK and ERK1/2 signaling pathways.

BACKGROUND: We have shown that the differentiation of human-induced pluripotent stem cells (hiPSCs) into endothelial cells (ECs) is more efficient when performed with a 3-dimensional (3D) scaffold of biomaterial than in monolayers. The current study aims to further increase hiPSC-EC differentiation efficiency by deciphering the signaling pathways in 3D scaffolds. METHODS AND RESULTS: We modified our 3D protocol by using U-46619 to upregulate both p38 mitogen-activated protein kinase (p38MAPK) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling, which increased the differentiation efficiency (as measured by CD31 expression) to as high as 89% in two established hiPSC lines. The differentiated cells expressed arteriovenous, but not lymphatic, markers; formed tubular structures and EC lumen in vitro; had significantly shorter population-doubling times than monolayer-differentiated hiPSC-ECs; and restored perfusion and vascularity in a murine hind limb ischemia model. The differentiation efficiency was also > 85% in three hiPSC lines that had been derived from patients with diseases or disease symptoms that have been linked to endothelial dysfunction. CONCLUSIONS: These observations demonstrate that activating both p38MAPK and ERK1/2 signaling pathways with U-46619 improves the efficiency of arteriovenous hiPSC-EC differentiation and produces cells with greater proliferative capacity.

Stem Cell Homing: a Potential Therapeutic Strategy Unproven for Treatment of Myocardial Injury.

Despite advances in the prevention and therapeutic modalities of ischemic heart disease, morbidity and mortality post-infarction heart failure remain big challenges in modern society. Stem cell therapy is emerging as a promising therapeutic strategy. Stem cell homing, the ability of stem cells to find their destination, is receiving more attention. Identification of specific cues and understanding the signaling pathways that direct stem cells to targeted destination will improve stem cell homing efficiency. This review discusses the cellular and molecular mechanism of stem cell homing at length in the light of literature and analyzes the problem and considerations of this approach as a treatment strategy for the treatment of ischemic heart disease clinically.

Truncations of the titin Z-disc predispose to a heart failure with preserved ejection phenotype in the context of pressure overload.

Titin (TTN) Truncating variants (TTNtv) in the A-band of TTN predispose the mouse heart to systolic dysfunction when subjected to pressure-loading. However, the effects of TTNtv of the Z-disc are largely unexplored. A rat model of pressure-loaded heart is developed by trans-aortic constriction (TAC). Rats with TTNtv of the Z-disc were randomly assigned to TAC (Z-TAC) or sham-surgery (Z-Sham) and wildtype (WT) littermates served as controls (WT-TAC or WT-Sham). Left ventricular (LV) function was assessed by echocardiography. Pressure volume (PV) loops, histology and molecular profiling were performed eight months after surgery. Pressure-load by TAC increased LV mass in all cases when compared with Sham animals. Notably, systolic function was preserved in TAC animals throughout the study period, which was confirmed by terminal PV loops. Diastolic function was impaired in Z-disc TTNtv rats at baseline as compared to WT and became impaired further after TAC (dp/dtmin, mmHg/s): Z-TAC = -3435±763, WT-TAC = -6497±1299 (p<0.01). Z-TAC animals had greater cardiac fibrosis, with elevated collagen content and decreased vascular density as compared to WT-TAC animals associated with enhanced apoptosis of myocyte and non-myocyte populations. In the context of pressure overload, Z-disc TTNtv is associated with cardiac fibrosis, diastolic dysfunction, and capillary rarefaction in the absence of overt systolic dysfunction.

Controllable unzipping for intramolecular junctions of graphene nanoribbons and single-walled carbon nanotubes.

Graphene is often regarded as one of the most promising candidates for future nanoelectronics. As an indispensable component in graphene-based electronics, the formation of junctions with other materials not only provides utility functions and reliable connexions, but can also improve or alter the properties of pristine graphene, opening up possibilities for new applications. Here we demonstrate an intramolecular junction produced by the controllable unzipping of single-walled carbon nanotubes, which combines a graphene nanoribbon and single-walled carbon nanotube in a one-dimensional nanostructure. This junction shows a strong gate-dependent rectifying behaviour. As applications, we demonstrate the use of the junction in prototype directionally dependent field-effect transistors, logic gates and high-performance photodetectors, indicating its potential in future graphene-based electronics and optoelectronics.