Mutation-specific differences in arrhythmias and drug responses in CPVT patients : simultaneous patch clamp and video imaging of iPSC derived cardiomyocytes

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited cardiac disease characterized by arrhythmias under adrenergic stress. Mutations in the cardiac ryanodine receptor (RYR2) are the leading cause for CPVT. We characterized electrophysiological properties of CPVT patient-specific induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying different mutations in RYR2 and evaluated effects of carvedilol and flecainide on action potential (AP) and contractile properties of hiPSC-CMs. iPSC-CMs were generated from skin biopsies of CPVT patients carrying exon 3 deletion (E3D) and L4115F mutation in RYR2. APs and contractile movement were recorded simultaneously from the same hiPSC-CMs. Differences in AP properties of ventricular like CMs were seen in CPVT and control CMs: APD90 of both E3D (n = 20) and L4115F (n = 25) CPVT CMs was shorter than in control CMs (n = 15). E3D-CPVT CMs had shortest AP duration, lowest AP amplitude, upstroke velocity and more depolarized diastolic potential than controls. Adrenaline had positive and carvedilol and flecainide negative chronotropic effect in all hiPSC CMs. CPVT CMs had increased amount of delayed after depolarizations (DADs) and early after depolarizations (EADs) after adrenaline exposure. E3D CPVT CMs had the most DADs, EADs, and tachyarrhythmia. Discordant negatively coupled alternans was seen in L4115F CPVT CMs. Carvedilol cured almost all arrhythmias in L4115F CPVT CMs. Both drugs decreased contraction amplitude in all hiPSC CMs. E3D CPVT CMs have electrophysiological properties, which render them more prone to arrhythmias. iPSC-CMs provide a unique platform for disease modeling and drug screening for CPVT. Combining electrophysiological measurements, we can gain deeper insight into mechanisms of arrhythmias.Peer reviewe

Antiarrhythmic Effects of Carvedilol and Flecainide in Cardiomyocytes Derived from Catecholaminergic Polymorphic Ventricular Tachycardia Patients

Mutations in the cardiac ryanodine receptor (RYR2) are the leading cause for catecholaminergic polymorphic ventricular tachycardia (CPVT). In this study, we evaluated antiarrhythmic efficacy of carvedilol and flecainide in CPVT patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) carrying different mutations in RYR2. iPSC-CMs were generated from skin biopsies of CPVT patients carrying exon 3 deletion and IA115 or V4653F mutation in RYR2 and of a healthy individual. Ca2+ kinetics and drug effects were studied with Fluo-4 AM indicator. Carvedilol abolished Ca2+ abnormalities in 31% of L4115F, 36% of V4653F, and 46% of exon 3 deletion carrying CPVT cardiomyocytes and flecainide 33%, 30%, and 52%, respectively. Both drugs lowered the intracellular Ca2+ level and beating rate of the cardiomyocytes significantly. Moreover, flecainide caused abnormal Ca2+ transients in 61% of controls compared to 26% of those with carvedilol. Carvedilol and flecainide were equally effective in CPVT iPSC-CMs. However, flecainide induced arrhythmias in 61% of control cells. CPVT cardiomyocytes carrying the exon 3 deletion had the most severe Ca2+ abnormalities, but they had the best response to drug therapies. According to this study, the arrhythmia-abolishing effect of neither of the drugs is optimal. iPSC-CMs provide a unique platform for testing drugs for CPVT.Peer reviewe

Al-neal Degrades Al2O3 Passivation of Silicon Surface

Atomic layer deposited (ALD) aluminum oxide (Al2O3) has emerged as a useful material for silicon devices due to its capability for effective surface passivation and ability to generate p(+) region underneath the oxide as active or passive component in semiconductor devices. However, it is uncertain how Al2O3 films tolerate the so-called Al-neal treatment that is a necessary process step in devices that also contain silicon dioxide (SiO2) passivation layers. Herein, it is reported that the Al-neal process is harmful for the passivation performance of Al2O3 causing over eightfold increase in surface recombination velocity (SRV) (from 0.9 to 7.3 cm s(-1)). Interestingly, it is also observed that the stage at which the so-called activation of Al2O3 passivation is performed impacts the final degradation strength. The best result is obtained when the activation step is done at the end of the process together with the Al-neal thermal treatment, which results in SRV of 1.7 cm s(-1). The results correlate well with the measured interface defect density, indicating that the Al-neal affects defects at the Si/SiO x /Al2O3 interface. The root causes for the defect reactions are discussed and possible reasons for the observed phenomena are suggested.Peer reviewe

Defoliation and patchy nutrient return drive grazing effects on plant and soil properties in a dairy cow pasture

Large herbivores can influence plant and soil properties in grassland ecosystems, but especially for belowground biota and processes, the mechanisms that explain these effects are not fully understood. Here, we examine the capability of three grazing mechanisms-plant defoliation, dung and urine return, and physical presence of animals (causing trampling and excreta return in patches)-to explain grazing effects in Phleum pratense-Festuca pratensis dairy cow pasture in Finland. Comparison of control plots and plots grazed by cows showed that grazing maintained original plant-community structure, decreased shoot mass and root N and P concentrations, increased shoot N and P concentrations, and had an inconsistent effect on root mass. Among soil fauna, grazing increased the abundance of fungivorous nematodes and Aporrectodea earthworms and decreased the abundance of detritivorous enchytraeids and Lumbricus earthworms. Grazing also increased soil density and pH but did not affect average soil inorganic-N concentration. To reveal the mechanisms behind these effects, we analyzed results from mowed plots and plots that were both mowed and treated with a dung and urine mixture. This comparison revealed that grazing effects on plant attributes were almost entirely explained by defoliation, with only one partly explained by excreta return. Among belowground attributes, however, the mechanisms were more mixed, with effects explained by defoliation, patchy excreta return, and cow trampling. Average soil inorganic-N concentration was not affected by grazing because it was simultaneously decreased by defoliation and increased by cow presence. Presence of cows created great spatial heterogeneity in soil N availability and abundance of fungivorous nematodes. A greenhouse trial revealed a grazing-induced soil feedback on plant growth, which was explained by patchiness in N availability rather than changes in soil biota. Our results show that grazing effects on plant attributes can be satisfactorily predicted using the effects of defoliation, whereas those on soil fauna and soil N availability need understanding of other mechanisms as well. The results indicate that defoliation-induced changes in plant ecophysiology and the great spatial variation in N availability created by grazers are the two key mechanisms through which large herbivores can control grassland ecosystems.Large herbivores can influence plant and soil properties in grassland ecosystems, but especially for belowground biota and processes, the mechanisms that explain these effects are not fully understood. Here, we examine the capability of three grazing mechanisms-plant defoliation, dung and urine return, and physical presence of animals (causing trampling and excreta return in patches)-to explain grazing effects in Phleum pratense-Festuca pratensis dairy cow pasture in Finland. Comparison of control plots and plots grazed by cows showed that grazing maintained original plant-community structure, decreased shoot mass and root N and P concentrations, increased shoot N and P concentrations, and had an inconsistent effect on root mass. Among soil fauna, grazing increased the abundance of fungivorous nematodes and Aporrectodea earthworms and decreased the abundance of detritivorous enchytraeids and Lumbricus earthworms. Grazing also increased soil density and pH but did not affect average soil inorganic-N concentration. To reveal the mechanisms behind these effects, we analyzed results from mowed plots and plots that were both mowed and treated with a dung and urine mixture. This comparison revealed that grazing effects on plant attributes were almost entirely explained by defoliation, with only one partly explained by excreta return. Among belowground attributes, however, the mechanisms were more mixed, with effects explained by defoliation, patchy excreta return, and cow trampling. Average soil inorganic-N concentration was not affected by grazing because it was simultaneously decreased by defoliation and increased by cow presence. Presence of cows created great spatial heterogeneity in soil N availability and abundance of fungivorous nematodes. A greenhouse trial revealed a grazing-induced soil feedback on plant growth, which was explained by patchiness in N availability rather than changes in soil biota. Our results show that grazing effects on plant attributes can be satisfactorily predicted using the effects of defoliation, whereas those on soil fauna and soil N availability need understanding of other mechanisms as well. The results indicate that defoliation-induced changes in plant ecophysiology and the great spatial variation in N availability created by grazers are the two key mechanisms through which large herbivores can control grassland ecosystems.Large herbivores can influence plant and soil properties in grassland ecosystems, but especially for belowground biota and processes, the mechanisms that explain these effects are not fully understood. Here, we examine the capability of three grazing mechanisms-plant defoliation, dung and urine return, and physical presence of animals (causing trampling and excreta return in patches)-to explain grazing effects in Phleum pratense-Festuca pratensis dairy cow pasture in Finland. Comparison of control plots and plots grazed by cows showed that grazing maintained original plant-community structure, decreased shoot mass and root N and P concentrations, increased shoot N and P concentrations, and had an inconsistent effect on root mass. Among soil fauna, grazing increased the abundance of fungivorous nematodes and Aporrectodea earthworms and decreased the abundance of detritivorous enchytraeids and Lumbricus earthworms. Grazing also increased soil density and pH but did not affect average soil inorganic-N concentration. To reveal the mechanisms behind these effects, we analyzed results from mowed plots and plots that were both mowed and treated with a dung and urine mixture. This comparison revealed that grazing effects on plant attributes were almost entirely explained by defoliation, with only one partly explained by excreta return. Among belowground attributes, however, the mechanisms were more mixed, with effects explained by defoliation, patchy excreta return, and cow trampling. Average soil inorganic-N concentration was not affected by grazing because it was simultaneously decreased by defoliation and increased by cow presence. Presence of cows created great spatial heterogeneity in soil N availability and abundance of fungivorous nematodes. A greenhouse trial revealed a grazing-induced soil feedback on plant growth, which was explained by patchiness in N availability rather than changes in soil biota. Our results show that grazing effects on plant attributes can be satisfactorily predicted using the effects of defoliation, whereas those on soil fauna and soil N availability need understanding of other mechanisms as well. The results indicate that defoliation-induced changes in plant ecophysiology and the great spatial variation in N availability created by grazers are the two key mechanisms through which large herbivores can control grassland ecosystems.Peer reviewe

S53P4 bioactive glass scaffolds induce BMP expression and integrative bone formation in a critical-sized diaphysis defect treated with a single-staged induced membrane technique

Surgical management of critical-sized diaphyseal defects involves multiple challenges, and up to 10% result in delayed or non-union. The two-staged induced membrane technique is successfully used to treat these defects, but it is limited by the need of several procedures and bone graft. Repeated procedures increase costs and morbidity, while grafts are subject to donor-site complications and scarce availability. To transform this two-staged technique into one graft-independent procedure, we developed amorphous porous scaffolds sintered from the clinically used bioactive glass S53P4. This work constitutes the first evaluation of such scaffolds in vivo in a critical-sized diaphyseal defect in the weight-bearing rabbit femur. We provide important knowledge and prospects for future development of sintered S53P4 scaffolds as a bone substitute. Critical-sized diaphysis defects are complicated by inherent sub-optimal healing conditions. The two staged induced membrane technique has been used to treat these challenging defects since the 1980 & rsquo;s. It involves temporary implantation of a membrane-inducing spacer and subsequent bone graft defect filling. A single-staged, graft-independent technique would reduce both socio-economic costs and patient morbidity. Our aim was to enable such single-staged approach through development of a strong bioactive glass scaffold that could replace both the spacer and the graft filling. We constructed amorphous porous scaffolds of the clinically used bioactive glass S53P4 and evaluated them in vivo using a critical sized defect model in the weight-bearing femur diaphysis of New Zealand White rabbits. S53P4 scaffolds and standard polymethylmethacrylate spacers were implanted for 2, 4, and 8 weeks. Induced membranes were confirmed histologically, and their osteostimulative activity was evaluated through RT-qPCR of bone morphogenic protein 2, 4, and 7 (BMPs). Bone formation and osseointegration were examined using histology, scanning electron microscopy, energy-dispersive X-ray analysis, and micro-computed tomography imaging. Scaffold integration, defect union and osteosynthesis were assessed manually and with X-ray projections. We demonstrated that S53P4 scaffolds induce osteostimulative membranes and produce osseointegrative new bone formation throughout the scaffolds. We also demonstrated successful stable scaffold integration with early defect union at 8 weeks postoperative in critical-sized segmental diaphyseal defects with implanted sintered amorphous S53P4 scaffolds. This study presents important considerations for future research and the potential of the S53P4 bioactive glass as a bone substitute in large diaphyseal defects. Statement of significance Surgical management of critical-sized diaphyseal defects involves multiple challenges, and up to 10% result in delayed or non-union. The two-staged induced membrane technique is successfully used to treat these defects, but it is limited by the need of several procedures and bone graft. Repeated procedures increase costs and morbidity, while grafts are subject to donor-site complications and scarce availability. To transform this two-staged technique into one graft-independent procedure, we developed amorphous porous scaffolds sintered from the clinically used bioactive glass S53P4. This work constitutes the first evaluation of such scaffolds in vivo in a critical-sized diaphyseal defect in the weight-bearing rabbit femur. We provide important knowledge and prospects for future development of sintered S53P4 scaffolds as a bone substitute. (c) 2021 The Author(s). Published by Elsevier Ltd on behalf of Acta Materialia Inc. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ )Peer reviewe

S53P4 bioactive glass scaffolds induce BMP expression and integrative bone formation in a critical-sized diaphysis defect treated with a single-stage d induce d membrane technique

Surgical management of critical-sized diaphyseal defects involves multiple challenges, and up to 10% result in delayed or non-union. The two-staged induced membrane technique is successfully used to treat these defects, but it is limited by the need of several procedures and bone graft. Repeated procedures increase costs and morbidity, while grafts are subject to donor-site complications and scarce availability. To transform this two-staged technique into one graft-independent procedure, we developed amorphous porous scaffolds sintered from the clinically used bioactive glass S53P4. This work constitutes the first evaluation of such scaffolds in vivo in a critical-sized diaphyseal defect in the weight-bearing rabbit femur. We provide important knowledge and prospects for future development of sintered S53P4 scaffolds as a bone substitute. Critical-sized diaphysis defects are complicated by inherent sub-optimal healing conditions. The two staged induced membrane technique has been used to treat these challenging defects since the 1980 & rsquo;s. It involves temporary implantation of a membrane-inducing spacer and subsequent bone graft defect filling. A single-staged, graft-independent technique would reduce both socio-economic costs and patient morbidity. Our aim was to enable such single-staged approach through development of a strong bioactive glass scaffold that could replace both the spacer and the graft filling. We constructed amorphous porous scaffolds of the clinically used bioactive glass S53P4 and evaluated them in vivo using a critical sized defect model in the weight-bearing femur diaphysis of New Zealand White rabbits. S53P4 scaffolds and standard polymethylmethacrylate spacers were implanted for 2, 4, and 8 weeks. Induced membranes were confirmed histologically, and their osteostimulative activity was evaluated through RT-qPCR of bone morphogenic protein 2, 4, and 7 (BMPs). Bone formation and osseointegration were examined using histology, scanning electron microscopy, energy-dispersive X-ray analysis, and micro-computed tomography imaging. Scaffold integration, defect union and osteosynthesis were assessed manually and with X-ray projections. We demonstrated that S53P4 scaffolds induce osteostimulative membranes and produce osseointegrative new bone formation throughout the scaffolds. We also demonstrated successful stable scaffold integration with early defect union at 8 weeks postoperative in critical-sized segmental diaphyseal defects with implanted sintered amorphous S53P4 scaffolds. This study presents important considerations for future research and the potential of the S53P4 bioactive glass as a bone substitute in large diaphyseal defects. Statement of significance Surgical management of critical-sized diaphyseal defects involves multiple challenges, and up to 10% result in delayed or non-union. The two-staged induced membrane technique is successfully used to treat these defects, but it is limited by the need of several procedures and bone graft. Repeated procedures increase costs and morbidity, while grafts are subject to donor-site complications and scarce availability. To transform this two-staged technique into one graft-independent procedure, we developed amorphous porous scaffolds sintered from the clinically used bioactive glass S53P4. This work constitutes the first evaluation of such scaffolds in vivo in a critical-sized diaphyseal defect in the weight-bearing rabbit femur. We provide important knowledge and prospects for future development of sintered S53P4 scaffolds as a bone substitute. (c) 2021 The Author(s). Published by Elsevier Ltd on behalf of Acta Materialia Inc. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ )Peer reviewe