Background To understand the intrinsic cardiac developmental and functional abnormalities in pulmonary atresia with intact ventricular septum (PAIVS) free from effects secondary to anatomic defects, ...we performed and compared single-cell transcriptomic and phenotypic analyses of patient- and healthy subject-derived human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and engineered tissue models. Methods and Results We derived hiPSC lines from 3 patients with PAIVS and 3 healthy subjects and differentiated them into hiPSC-CMs, which were then bioengineered into the human cardiac anisotropic sheet and human cardiac tissue strip custom-designed for electrophysiological and contractile assessments, respectively. Single-cell RNA sequencing (scRNA-seq) of hiPSC-CMs, human cardiac anisotropic sheet, and human cardiac tissue strip was performed to examine the transcriptomic basis for any phenotypic abnormalities using pseudotime and differential expression analyses. Through pseudotime analysis, we demonstrated that bioengineered tissue constructs provide pro-maturational cues to hiPSC-CMs, although the maturation and development were attenuated in PAIVS hiPSC-CMs. Furthermore, reduced contractility and prolonged contractile kinetics were observed with PAIVS human cardiac tissue strips. Consistently, single-cell RNA sequencing of PAIVS human cardiac tissue strips and hiPSC-CMs exhibited diminished expression of cardiac contractile apparatus genes. By contrast, electrophysiological aberrancies were absent in PAIVS human cardiac anisotropic sheets. Conclusions Our findings were the first to reveal intrinsic abnormalities of cardiomyocyte development and function in PAIVS free from secondary effects. We conclude that hiPSC-derived engineered tissues offer a unique method for studying primary cardiac abnormalities and uncovering pathogenic mechanisms that underlie sporadic congenital heart diseases.
Bamboo was converted into carbon fibre using a novel approach combining microwave pyrolysis, solvent extraction, and chemical impregnation. The employment of solvent extraction and chemical ...impregnation transformed natural bamboo into bamboo fibre with yield of 64 wt% that comprised of 87 wt% cellulose, 4 wt% lignin and 9 wt% hemicellulose deemed desirable for conversion into carbon fibre. The subsequent use of microwave pyrolysis transformed the bamboo fibre to form carbon fibre; the effects of microwave irradiation time and microwave power (two key process parameters) on properties of carbon fibre were investigated. Microwave pyrolysis provides a rapid heating rate (30 °C/min) with relatively shorter process time (20 min) compared to that performed by conventional pyrolysis method, thus leading to a relatively lower electric consumption and providing a potentially cost-saving and energy-efficient approach to produce carbon fibre. The production and properties of carbon fibre produced were affected by irradiation time and microwave power. The carbon fibre obtained was detected to have high fixed carbon (80 wt%), carbon element (87%), high BET surface area (475 m2/g) and fibres in small diameter (≤1 μm). It also shows desirable features comprising low content of moisture (<6 wt%), inorganic elements of Na, K, S, Mg (≤5%) and ash (1 wt%), allowing the carbon fibre to have more active sites and making it a cleaner product rather than polluting the environment whereas a high ash content could lead to undesired catalytic reactions and emission of potentially hazardous metallic components present in the ash. Combined with the detection of positive capacitive behaviour, the carbon fibre shows great potential to be used as an electrode for energy storage application. The results disclose that microwave pyrolysis combining with solvent extraction and chemical impregnation can be a cleaner approach to transform bamboo into carbon fibre with favourable physicochemical and capacitive properties.
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•A new approach combining microwave pyrolysis, solvent extraction, impregnation.•The microwave pyrolysis provides fast heating rate and a short process time.•Carbon fibre with favourable physicochemical and capacitive properties is obtained.•Carbon fibre has high fixed carbon, surface area, and small fibres.•Carbon fibre shows positive capacitive behaviour.
Left ventricular (LV) dysfunction is 1 of the major determinants of late adverse clinical outcomes in patients after surgical repair of tetralogy of Fallot (TOF). The aim of this study was to test ...the hypothesis that LV myocardial deformation is impaired in patients after TOF repair and related to right ventricular (RV) dilation and exercise capacity. Longitudinal, radial, and circumferential LV myocardial deformation was determined using speckle-tracking echocardiography in 23 postoperative patients with TOF and compared to that of 23 age-matched controls. Relations between LV strain and strain rate (SR) and RV volumes and exercise parameters were determined in patients. Compared to controls, patients had reduced global LV longitudinal, radial, and circumferential strain (all p values <0.05). Patients with significantly increased RV end-systolic volume (>2 SDs higher than normal; n = 17) had reduced global LV circumferential strain (p = 0.048) and SR (p = 0.038), but similar longitudinal and radial speckle-tracking echocardiographic parameters, compared to those without (n = 6). RV end-systolic volume was correlated inversely with global LV circumferential strain and SR (r = −0.58, p = 0.004, and r = −0.58, p = 0.005, respectively), while RV end-diastolic volume was correlated only with global LV circumferential strain (r = −0.43, p = 0.047). In patients, the LV ejection fraction was correlated with global LV circumferential strain (r = 0.54, p = 0.01) and SR (r = 0.66, p = 0.001) but not with longitudinal or radial speckle-tracking echocardiographic parameters. Using multivariate analysis, global LV circumferential SR (β = 0.66, p = 0.001) and male gender (β = 0.46, p = 0.012) were identified as independent predictors of peak oxygen consumption. In conclusion, the negative impact of RV dilation on LV function relates to its influence on LV circumferential strain and SR in patients after TOF repair.
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•Co-pyrolysis of cellulose and LLDPE over MgO catalysts was performed.•MgO catalysts with different surface area and acid/base properties were used.•MgO/C has largest surface area and ...adequate acid-base property.•MgO/C produced the largest amounts of aromatics.
Various MgO impregnated catalysts were used for the catalytic co-pyrolysis (CCP) of cellulose and linear low-density polyethylene (LLDPE) at 600 °C in ambient pressure. Micro reactor-gas chromatography, a semi-batch reactor, was used as a reactor and gas chromatogram/mass spectrometry/flame ionization detector were used for product detection. Three kinds of MgO impregnated catalysts, MgO/Carbon (MgO/C), MgO/Al2O3, and MgO/ZrO2, were prepared by the impregnation of MgO to different supporting materials, C, Al2O3, and ZrO2, respectively. Activities of the three MgO-impregnated catalysts were compared with 1:5 of feedstock to catalyst ratio. MgO/C produced the highest quality oil, which consisted of a large amount of aromatic hydrocarbons during the catalytic pyrolysis (CP) of cellulose. When the MgO catalysts were applied to CCP, the selectivity to aromatic hydrocarbons over MgO/C were 13.42%, which were also much higher than those over the other catalysts, including bulk MgO, demonstrating the effectiveness of the MgO/C catalyst on aromatics formation. Importantly, the experimental BTEXs (benzene, toluene, ethylbenzene, xylenes) yields of CCP over MgO/C were also much higher than the theoretical yields, highlighting the effectiveness of hydrogen-rich LLDPE on the CP of cellulose over MgO/C. Further investigation of the cellulose/LLDPE mixing ratio showed that the amount of BTEXs during the CCP of cellulose and LLDPE could be maximized by adjusting the cellulose to LLDPE mixing ratio to 25:75.
The clinical relevance of evaluating right ventricular (RV) myocardial deformation in congenital heart disease is increasingly recognized. The aim of this study was to explore, using ...three-dimensional (3D) speckle-tracking echocardiography, RV mechanics in terms of 3D global area strain and mechanical dyssynchrony in adults with repaired tetralogy of Fallot.
Twenty patients (12 men) aged 24.7 ± 8.6 years and 22 age-matched controls (11 men) were studied. Global RV peak area strain and area strain-derived systolic dyssynchrony index (SDI) were determined using 3D speckle-tracking echocardiography. RV end-diastolic volume and end-systolic volume, ejection fraction (EF), and pulmonary regurgitation fraction were measured in patients using cardiac magnetic resonance.
Coefficients of variation for intraobserver and interobserver measurements of RV global area strain were 6.1% and 7.9%, respectively, and those for SDI were 7.6% and 10.1%, respectively. Compared with controls, patients had significantly lower global area strain (P = .005) and greater SDI (P = .008). The prevalence of RV mechanical dyssynchrony (SDI > control mean + 2 SDs) in patients was 30%. In patients, global area strain correlated inversely with SDI (r = -0.42, P = .04), RV end-diastolic volume (r = -0.48, P = .032), and RV end-systolic volume (r = -0.48, P = .031) and positively with EF (r = -0.51, P = .02), while RV SDI correlated positively with RV end-systolic volume (r = 0.55, P = .012), pulmonary regurgitation fraction (r = 0.54, P = .031), and QRS duration (r = 0.51, P = .022) and negatively with RV EF (r = -0.62, P = .004). Multivariate analysis showed that RV EF (β = 0.22, P = .048) was a significant correlate of global area strain in patients.
In adults after tetralogy of Fallot repair, 3D RV deformation is impaired in association with RV dyssynchrony, volume overloading, and reduced EF.
Cardiomyocytes can be readily derived from human induced pluripotent stem cell (hiPSC) lines, yet its efficacy varies across different batches of the same and different hiPSC lines. To unravel the ...inconsistencies of in vitro cardiac differentiation, we utilized single cell transcriptomics on hiPSCs undergoing cardiac differentiation and identified cardiac and extra-cardiac lineages throughout differentiation. We further identified APLNR as a surface marker for in vitro cardiac progenitors and immunomagnetically isolated them. Differentiation of isolated in vitro APLNR+ cardiac progenitors derived from multiple hiPSC lines resulted in predominantly cardiomyocytes accompanied with cardiac mesenchyme. Transcriptomic analysis of differentiating in vitro APLNR+ cardiac progenitors revealed transient expression of cardiac progenitor markers before further commitment into cardiomyocyte and cardiac mesenchyme. Analysis of in vivo human and mouse embryo single cell transcriptomic datasets have identified APLNR expression in early cardiac progenitors of multiple lineages. This platform enables generation of in vitro cardiac progenitors from multiple hiPSC lines without genetic manipulation, which has potential applications in studying cardiac development, disease modelling and cardiac regeneration.
The cyclic dinucleotide c-di-GMP is a master regulator of bacterial virulence and biofilm formation. The activations of c-di-GMP metabolism proteins, diguanylate cyclases (DGCs) and ...phosophodiesterases (PDEs), usually lead to diametrically opposite phenotypes in bacteria. Analogues of c-di-GMP, which can selectively modulate the activities of c-di-GMP processing proteins, will be useful chemical tools for studying and altering bacterial behavior. Herein we report that a conservative modification of one of the phosphate groups in c-di-GMP with a bridging sulfur in the phosphodiester linkage affords an analogue called endo-S-c-di-GMP. Computational, NMR (including DOSY), and CD experiments all reveal that, unlike c-di-GMP, endo-S-c-di-GMP does not readily form higher aggregates. The lower propensity of endo-S-c-di-GMP to form aggregates (as compared to that of c-di-GMP) is probably due to a higher activation barrier to convert from the “open” conformer (where the two guanines are on opposite faces) to the “closed” conformer (where the two guanines are on the same face). Consequently, endo-S-c-di-GMP has selectivity for proteins that bind monomeric but not dimeric c-di-GMP, which form from the “closed” conformer. For example, endo-S-c-di-GMP can inhibit the hydrolysis of c-di-GMP by RocR (a PDE enzyme that binds monomeric c-di-GMP) but did not bind to Alg44 (a PilZ protein) or regulate WspR (a DGC enzyme that has been shown to bind to dimeric c-di-GMP). This work demonstrates that selective binding to different classes of c-di-GMP binding proteins could be achieved by altering analogue conformer populations (conformational steering). We provide important design principles for the preparation of selective PDE inhibitors and reveal the role played by the c-di-GMP backbone in c-di-GMP polymorphism and binding to processing proteins.
•Myocardial fibrosis occurs after atrial switch operation for TGA.•Ventricular basal–apical heterogeneity of myocardial fibrosis is evident.•Myocardial fibrosis is worse in the subpulmonary left ...ventricle.•Systemic RV and subpulmonary LV myocardial fibrosis is related.
This study aimed to assess diffuse myocardial fibrosis of the systemic right ventricle and subpulmonary left ventricle in patients after Senning or Mustard operation for complete transposition of the great artery (TGA) using cardiac magnetic resonance (CMR) T1 mapping.
Thirty-one adult TGA patients after Senning (n = 24) or Mustard (n = 7) operation were studied at the age of 33.3 ± 4.0 years. Systemic right ventricular (RV) and subpulmonary left ventricular (LV) volumes, ejection fraction, and myocardial T1 values and extracellular volume fraction (ECV) were determined using CMR.
The RV and LV ejection fractions were 47.0 ± 10.9% and 61.3 ± 7.4%, respectively. Compared to published normative values, patients had significantly greater RV and LV native T1 and ECV values (all p < 0.001). For each of the basal, mid, and apical segments, the LV native T1 and ECV values were significantly greater in the left than the right ventricle (all p < 0.05). There is a significant trend on progressive increase in ECV value from the basal towards the apical segments in both the right (p = 0.002) and the left (p < 0.001) ventricle. Modestly strong correlations were found between RV and LV native T1 (r = 0.60, p < 0.001) and ECV (r = 0.49, p = 0.005) values but not with ejection fractions of the respective ventricles.
Differential myocardial fibrosis, with greater involvement of the subpulmonary left ventricle than the systemic right ventricle, is present in patients with TGA after atrial switch operation. Associations between the magnitude of RV and LV fibrosis suggests adverse ventricular-ventricular interaction at the cardiac extracellular matrix level.
Abstract
Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart disease. Ventricular dysfunction and cardiac arrhythmias are well-documented complications in patients with repaired ...TOF. Whether intrinsic abnormalities exist in TOF cardiomyocytes is unknown. We establish human induced pluripotent stem cells (hiPSCs) from TOF patients with and without DiGeorge (DG) syndrome, the latter being the most commonly associated syndromal association of TOF. TOF-DG hiPSC-derived cardiomyocytes (hiPSC-CMs) show impaired ventricular specification, downregulated cardiac gene expression and upregulated neural gene expression. Transcriptomic profiling of the in vitro cardiac progenitors reveals early bifurcation, as marked by ectopic
RGS13
expression, in the trajectory of TOF-DG-hiPSC cardiac differentiation. Functional assessments further reveal increased arrhythmogenicity in TOF-DG-hiPSC-CMs. These findings are found only in the TOF-DG but not TOF-with no DG (ND) patient-derived hiPSC-CMs and cardiac progenitors (CPs), which have implications on the worse clinical outcomes of TOF-DG patients.
Cyclic-di-GMP (c-di-GMP) is a central regulator of bacterial behavior. Various studies have implicated c-di-GMP in biofilm formation and virulence factor production in multitudes of bacteria. Hence ...it is expected that the disruption of c-di-GMP signaling could provide an effective means to disrupt biofilm and/or virulence factor formation in several bacteria of clinical relevance. C-di-GMP achieves the regulation of bacterial phenotype via binding to several effector molecules including transcription factors, enzymes and riboswitches. Crystal structure analyses of c-di-GMP effector molecules, in complex with the ligand, reveal that various classes of c-di-GMP receptors recognize this dinucleotide using different sets of recognition elements. Therefore, it is plausible that different analogues of c-di-GMP could be used to selectively modulate a specific class of c-di-GMP binding receptors, and hence modulate the bacterial phenotype. Thus far only a detailed study of the differential binding of c-di-GMP analogues to riboswitches, but not proteins, has been reported. In this report, we prepared various 2′-modified analogues of c-di-GMP and studied both polymorphisms of these analogues using DOSY NMR and the binding to several effector proteins, such as PilZ-containing proteins, diguanylate cyclases (DGC) containing I-sites, and phoshphodiesterases (PDE). 2′-Modification of c-di-GMP did not adversely affect the propensity to form higher aggregates, such as octameric forms, in the presence of potassium salts. Interestingly, we find that the selective binding to different classes of c-di-GMP binding proteins could be achieved with the 2′-modified analogues and that 2′-F analogue of c-di-GMP binds to the I-site of DGCs better (four times) than the native dinucleotide, c-di-GMP, whereas c-di-GMP binds to PDEs better (10 times) than 2′-F-c-di-GMP. 2′-F-c-di-GMP potently inhibits c-di-GMP synthesis by DGCs and hence raises the potential that cell permeable analogues of 2′-F-c-di-GMP could be used to disrupt c-di-GMP signaling in bacteria.
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