The quest for an optimized protocol for whole-heart decellularization: a comparison of three popular and a novel decellularization technique and their diverse effects on crucial extracellular matrix qualities

Decellularized cardiac extracellular matrix (ECM) has been introduced as a template for cardiac tissue engineering, providing the advantages of a prevascularized scaffold that mimics native micro- and macroarchitecture to a degree difficult to achieve with synthetic materials. Nonetheless, the decellularization protocols used to create acellular myocardial scaffolds vary widely throughout the literature. In this study we performed a direct comparison of three previously described protocols while introducing and evaluating a novel, specifically developed fourth protocol, by decellularizing whole rat hearts through software-controlled automatic coronary perfusion. Although all protocols preserved the macroarchitecture of the hearts and all resulting scaffolds could successfully be reseeded with C2C12 myoblasts, assessing their biocompatibility for three-dimensional in vitro studies, we found striking differences concerning the microcomposition of the ECM scaffolds on a histological and biochemical level. While laminin could still be detected in all groups, other crucial ECM components, like elastin and collagen IV, were completely removed by at least one of the protocols. Further, only three protocols maintained a glycosaminoglycan content comparable to native tissue, whereas the remaining DNA content within the ECM varied highly throughout all four tested protocols. This study showed that the degree of acellularity and resulting ECM composition of decellularized myocardial scaffolds strongly differs depending on the decellularization protocol.     

A novel miniaturized multimodal bioreactor for continuous in situ assessment of bioartificial cardiac tissue during stimulation and maturation

Stem cell-based cardiac tissue engineering is a promising approach for regenerative therapy of the injured heart. At present, the small number of stem cell-derived cardiomyocytes that can be obtained using current culture and enrichment techniques represents one of the key limitations for the development of functional bioartificial cardiac tissue (BCT). We have addressed this problem by construction of a novel bioreactor with functional features of larger systems that enables the generation and in situ monitoring of miniaturized BCTs. BCTs were generated from rat cardiomyocytes to demonstrate advantages and usefulness of the bioreactor. Tissues showed spontaneous, synchronized contractions with cell orientation along the axis of strain. Cyclic stretch induced cardiomyocyte hypertrophy, demonstrated by a shift of myosin heavy chain expression from the alpha to beta isoform, together with elevated levels of atrial natriuretic factor. Stretch led to a moderate increase in systolic force (1.42?±?0.09?mN vs. 0.96?±?0.09?mN in controls), with significantly higher forces observed after β-adrenergic stimulation with noradrenalin (2.54?±?0.11?mN). Combined mechanical and β-adrenergic stimulation had no synergistic effect. This study demonstrates for the first time that mechanical stimulation and direct real-time contraction force measurement can be combined into a single multimodal bioreactor system, including electrical stimulation of excitable tissue, perfusion of the culture chamber, and the possibility of (fluorescence) microscopic assessment during continuous cultivation. Thus, this bioreactor represents a valuable tool for monitoring tissue development and, ultimately, the optimization of stem cell-based tissue replacement strategies in regenerative medicine.     

Biallelic loss of function variants in SYT2 cause a treatable congenital onset presynaptic myasthenic syndrome

Synaptotagmins are integral synaptic vesicle membrane proteins that function as calcium sensors and regulate neurotransmitter release at the presynaptic nerve terminal. Synaptotagmin‐2 (SYT2), is the major isoform expressed at the neuromuscular junction. Recently, dominant missense variants in SYT2 have been reported as a rare cause of distal motor neuropathy and myasthenic syndrome, manifesting with stable or slowly progressive distal weakness of variable severity along with presynaptic NMJ impairment. These variants are thought to have a dominant‐negative effect on synaptic vesicle exocytosis, although the precise pathomechanism remains to be elucidated. Here we report seven patients of five families, with biallelic loss of function variants in SYT2, clinically manifesting with a remarkably consistent phenotype of severe congenital onset hypotonia and weakness, with variable degrees of respiratory involvement. Electrodiagnostic findings were consistent with a presynaptic congenital myasthenic syndrome (CMS) in some. Treatment with an acetylcholinesterase inhibitor pursued in three patients showed clinical improvement with increased strength and function. This series further establishes SYT2 as a CMS‐disease gene and expands its clinical and genetic spectrum to include recessive loss‐of‐function variants, manifesting as a severe congenital onset presynaptic CMS with potential treatment implications.     

Robust H∞ output feedback design for networked control systems with partially known delay probabilities

This article develops a predictive robust H_∞ static output feedback control approach for networked control systems where random network-induced delays in both forward and feedback communication channels are modeled as two mutually uncorrelated Markov chains. By making use of the system augmentation method, the closed-loop system is formulated as a singular Markovian jump system with two modes, wherein the transition probability matrices of the underlying Markov chains are considered to be partially accessible. Necessary and sufficient conditions for the stochastic admissibility and robust H_∞ performance of the closed-loop system are given under the assumption of partially known transition probability matrices. A linear matrix inequality condition is proposed to determine the two-mode-dependent static output feedback controller gains to compensate for the random network-induced delays efficiently and provide the desired control performance. Finally, a numerical example is provided to demonstrate the effectiveness of the proposed approach.     

Epidemiology of gastroschisis: A population-based study in California from 1995 to 2012

Background

Although the incidence of gastroschisis is increasing, risk factors are not clearly identified.

Long‐lasting post‐mortem activity of spinal microglia in situ in mice

As CNS macrophages, microglia show a high spontaneous motility of their processes, continuously surveying their microenvironment. Upon CNS injury, microglia react by immediate cellular polarization and process extension toward the lesion site as well as by subsequent amoeboid lesion‐directed migration and phagocytosis. To determine the ability of microglia to fulfill their role within distinctively lesioned tissue in the absence of life support, we investigated microglial activity and responsiveness to laser‐induced axonal injuries in the spinal dorsal columns in situ after cardiac and respiratory arrest, i.e., post‐mortem, in the progressively degrading nervous tissue. For this purpose, we used time‐lapse two‐photon laser scanning microscopy in double transgenic mice expressing enhanced green fluorescent protein in microglia and enhanced yellow fluorescent protein in projection neurons. Depending on the premortal condition of the animal, microglial activity and responsiveness remain for up to5–10 hr post‐mortem. Thereby, the continuously decreasing glial reaction is independent of oxygen and glucose supply but requires residual ATP, suggesting a parasitic form of energy, such as a transmembrane uptake of ATP released from injured nervous tissue. Even though initially microglia are able to detect axonal injury after disruption of the blood supply, the later aspects of glial reaction, for example amoeboid conversion and migration, are absent post‐ mortem, corresponding to the failure of microglia to prevent secondary damage after injury of nervous tissue. © 2010 Wiley‐Liss, Inc.     

Intravascular T‐cell lymphoma: A rare,poorly characterized entity with cytotoxic phenotype

Intravascular T‐cell lymphomas are rare, poorly characterized lesions. We discuss the clinical, radiologic and especially the laboratory characteristics of a lesion which presented in a 62‐year‐old woman with a history of progressive CNS abnormalities. Throughout the course of the disease, radiologic findings consisted mainly of multifocal mixed areas of ischemia and vasogenic edema involving cortical and subcortical regions. A brain biopsy identified an abnormal T‐cell population confined to lumens of vessels. These T‐cells were abnormal cytotoxic cells, positive for CD3, CD8, and negative for CD2, CD4, CD5, CD7 and CD30. While flow cytometry and immunohistochemistry failed to identify a similar population in the blood or bone marrow, molecular studies showed a clonal T‐cell population in both the brain and the bone marrow. No other organs were involved. In spite of aggressive treatment, the patient's medical condition continued to progress and she passed away. In conclusion, this abnormal population of cytotoxic T‐cells with intravascular localization probably represents a specific type of T‐cell lymphoma with specific clinical, radiologic, molecular and immunophenotypic characteristics.