COVID-19 infection in patients with history of pediatric heart transplant in Germany, Austria, and Switzerland.

COVID-19 is a heterogenous infection-asymptomatic to fatal. While the course of pediatric COVID-19 infections is usually mild or even asymptomatic, individuals after adult heart transplantation are at high risk of a severe infection. We conducted a retrospective, multicenter survey of 16 pediatric heart transplant centers in Germany, Austria and Switzerland to evaluate the risk of a severe COVID-19 infection after pediatric heart transplantation between 02/2020 and 06/2021. Twenty-six subjects (11 male) with a median age of 9.77 years at time of transplantation and a median of 4.65 years after transplantation suffered from COVID-19 infection. The median age at time of COVID-10 infection was 17.20 years. Fourteen subjects had an asymptomatic COVID-19 infection. The most frequent symptoms were myalgia/fatigue (n = 6), cough (n = 5), rhinitis (n = 5), and loss of taste (n = 5). Only one subject showed dyspnea. Eleven individuals needed therapy in an outpatient setting, four subjects were hospitalized. One person needed oxygen supply, none of the subjects needed non-invasive or invasive mechanical ventilation. No specific signs for graft dysfunction were found by non-invasive testing. In pediatric heart transplant subjects, COVID-19 infection was mostly asymptomatic or mild. There were no SARS-CoV-2 associated myocardial dysfunction in heart transplant individuals.

TANGO2: expanding the clinical phenotype and spectrum of pathogenic variants.

PURPOSE: TANGO2-related disorders were first described in 2016 and prior to this publication, only 15 individuals with TANGO2-related disorder were described in the literature. Primary features include metabolic crisis with rhabdomyolysis, encephalopathy, intellectual disability, seizures, and cardiac arrhythmias. We assess whether genotype and phenotype of TANGO2-related disorder has expanded since the initial discovery and determine the efficacy of exome sequencing (ES) as a diagnostic tool for detecting variants. METHODS: We present a series of 14 individuals from 11 unrelated families with complex medical and developmental histories, in whom ES or microarray identified compound heterozygous or homozygous variants in TANGO2. RESULTS: The initial presentation of patients with TANGO2-related disorders can be variable, including primarily neurological presentations. We expand the phenotype and genotype for TANGO2, highlighting the variability of the disorder. CONCLUSION: TANGO2-related disorders can have a more diverse clinical presentation than previously anticipated. We illustrate the utility of routine ES data reanalysis whereby discovery of novel disease genes can lead to a diagnosis in previously unsolved cases and the need for additional copy-number variation analysis when ES is performed.

Correction: TANGO2: expanding the clinical phenotype and spectrum of pathogenic variants.

The original version of this Article contained an error in the spelling of the author J. Lawrence Merritt, which was incorrectly given as Lawrence Merritt. This has now been corrected in both the PDF and HTML versions of the Article.

Reverse-engineering post-transcriptional regulation of gap genes in Drosophila melanogaster.

Systems biology proceeds through repeated cycles of experiment and modeling. One way to implement this is reverse engineering, where models are fit to data to infer and analyse regulatory mechanisms. This requires rigorous methods to determine whether model parameters can be properly identified. Applying such methods in a complex biological context remains challenging. We use reverse engineering to study post-transcriptional regulation in pattern formation. As a case study, we analyse expression of the gap genes Krüppel, knirps, and giant in Drosophila melanogaster. We use detailed, quantitative datasets of gap gene mRNA and protein expression to solve and fit a model of post-transcriptional regulation, and establish its structural and practical identifiability. Our results demonstrate that post-transcriptional regulation is not required for patterning in this system, but is necessary for proper control of protein levels. Our work demonstrates that the uniqueness and specificity of a fitted model can be rigorously determined in the context of spatio-temporal pattern formation. This greatly increases the potential of reverse engineering for the study of development and other, similarly complex, biological processes.

Transcriptome analysis of AAV-induced retinopathy models expressing human VEGF, TNF-α, and IL-6 in murine eyes.

Retinopathies are multifactorial diseases with complex pathologies that eventually lead to vision loss. Animal models facilitate the understanding of the pathophysiology and identification of novel treatment options. However, each animal model reflects only specific disease aspects and understanding of the specific molecular changes in most disease models is limited. Here, we conducted transcriptome analysis of murine ocular tissue transduced with recombinant Adeno-associated viruses (AAVs) expressing either human VEGF-A, TNF-α, or IL-6. VEGF expression led to a distinct regulation of extracellular matrix (ECM)-associated genes. In contrast, both TNF-α and IL-6 led to more comparable gene expression changes in interleukin signaling, and the complement cascade, with TNF-α-induced changes being more pronounced. Furthermore, integration of single cell RNA-Sequencing data suggested an increase of endothelial cell-specific marker genes by VEGF, while TNF-α expression increased the expression T-cell markers. Both TNF-α and IL-6 expression led to an increase in macrophage markers. Finally, transcriptomic changes in AAV-VEGF treated mice largely overlapped with gene expression changes observed in the oxygen-induced retinopathy model, especially regarding ECM components and endothelial cell-specific gene expression. Altogether, our study represents a valuable investigation of gene expression changes induced by VEGF, TNF-α, and IL-6 and will aid researchers in selecting appropriate animal models for retinopathies based on their agreement with the human pathophysiology.

Pulmonary vascular disease in Fontan circulation-is there a rationale for pulmonary vasodilator therapies?

The Fontan circulation is a palliative concept for patients with univentricular hearts. The central veins are connected directly to the pulmonary arteries (cavo-pulmonary connection) to separate the pulmonary and the systemic circulation. There is no sub-pulmonary ventricle that generates pressure to drive blood through the pulmonary arteries. Pulmonary blood flow is determined by central venous pressure (CVP) and pulmonary vascular resistance (PVR). The capability of the Fontan circulation to compensate for alterations in PVR is limited, as CVP can only be increased within narrow ranges without adverse clinical consequences. Consequently, systemic ventricular preload and cardiac output are dependent on a healthy lung with low PVR. Failure of the Fontan circulation is relatively common. In addition to ventricular dysfunction, maladaptive pulmonary vascular remodeling resulting in increased pulmonary resistance may play a key role. The pathophysiology of the maladaptive vascular processes remains largely unclear and diagnosis of an increased PVR is challenging in Fontan circulation as accurate measurement of pulmonary arterial blood flow is difficult. In the absence of a sub-pulmonary ventricle, pulmonary artery pressure will almost never reach the threshold conventionally used to define pulmonary arterial hypertension. There is a need for markers of pulmonary vascular disease complementary to invasive hemodynamic data in Fontan patients. In order to treat or prevent failure of the Fontan circulation, pathophysiological considerations support the use of pulmonary vasodilators to augment pulmonary blood flow and systemic ventricular preload and lower CVP. However, to date the available trial data have neither yielded enough evidence to support routine use of pulmonary vasodilators in every Fontan patient nor have they been helpful in defining subgroups of patients that might benefit from such therapies. This review discusses potential pathomechanisms of pulmonary vascular disease; it summarizes the current knowledge of the effects and efficacy of pulmonary vasodilator therapy in Fontan patients and tries to outline areas of potential future research on the diagnosis and treatment of pulmonary vascular disease and Fontan failure.

Pulmonary sling in a patient with common arterial trunk.

We report a rare association of common arterial trunk with left pulmonary artery sling and highlight the importance of cross-sectional imaging in complex congenital cardiac lesions. The patient was antenatally diagnosed with common arterial trunk and underwent surgical repair in the neonatal period. At the age of 20 months, the patient presented with respiratory symptoms and increased right ventricular pressure. Multislice computed tomography demonstrated a pulmonary sling with compression of the distal trachea. Surgical correction of the pulmonary sling and change of the right ventricular to pulmonary artery conduit to a bigger size was performed.

Evaluation of an Intravitreal Rho-Associated Kinase Inhibitor Depot Formulation in a Rat Model of Diabetic Retinopathy.

Rho-associated kinase (ROCK) activation was shown to contribute to microvascular closure, retinal hypoxia, and to retinal pigment epithelium (RPE) barrier disruption in a rat model of diabetic retinopathy. Fasudil, a clinically approved ROCK inhibitor, improved retinal perfusion and reduced edema in this model, indicating that ROCK inhibition could be a promising new therapeutic approach for the treatment of diabetic retinopathy. However, due to its short intravitreal half-life, fasudil is not suitable for long-term treatment. In this study, we evaluated a very potent ROCK1/2 inhibitor (BIRKI) in a depot formulation administered as a single intravitreal injection providing a slow release for at least four weeks. Following BIRKI intravitreal injection in old Goto-Kakizaki (GK) type 2 diabetic rats, we observed a significant reduction in ROCK1 activity in the retinal pigment epithelium/choroid complex after 8 days and relocation of ROCK1 to the cytoplasm and nucleus in retinal pigment epithelium cells after 28 days. The chronic ROCK inhibition by the BIRKI depot formulation restored retinal pigment epithelial cell morphology and distribution, favored retinal capillaries dilation, and reduced hypoxia and inner blood barrier leakage observed in the diabetic retina. No functional or morphological negative effects were observed, indicating suitable tolerability of BIRKI after intravitreous injection. In conclusion, our data suggest that sustained ROCK inhibition, provided by BIRKI slow-release formulation, could be a valuable treatment option for diabetic retinopathy, especially with regard to the improvement of retinal vascular infusion and protection of the outer retinal barrier.

In-depth transcriptomic analysis of human retina reveals molecular mechanisms underlying diabetic retinopathy.

Diabetic Retinopathy (DR) is among the major global causes for vision loss. With the rise in diabetes prevalence, an increase in DR incidence is expected. Current understanding of both the molecular etiology and pathways involved in the initiation and progression of DR is limited. Via RNA-Sequencing, we analyzed mRNA and miRNA expression profiles of 80 human post-mortem retinal samples from 43 patients diagnosed with various stages of DR. We found differentially expressed transcripts to be predominantly associated with late stage DR and pathways such as hippo and gap junction signaling. A multivariate regression model identified transcripts with progressive changes throughout disease stages, which in turn displayed significant overlap with sphingolipid and cGMP-PKG signaling. Combined analysis of miRNA and mRNA expression further uncovered disease-relevant miRNA/mRNA associations as potential mechanisms of post-transcriptional regulation. Finally, integrating human retinal single cell RNA-Sequencing data revealed a continuous loss of retinal ganglion cells, and Müller cell mediated changes in histidine and ß-alanine signaling. While previously considered primarily a vascular disease, attention in DR has shifted to additional mechanisms and cell-types. Our findings offer an unprecedented and unbiased insight into molecular pathways and cell-specific changes in the development of DR, and provide potential avenues for future therapeutic intervention.

PREMER: A Tool to Infer Biological Networks.

Inferring the structure of unknown cellular networks is a main challenge in computational biology. Data-driven approaches based on information theory can determine the existence of interactions among network nodes automatically. However, the elucidation of certain features-such as distinguishing between direct and indirect interactions or determining the direction of a causal link-requires estimating information-theoretic quantities in a multidimensional space. This can be a computationally demanding task, which acts as a bottleneck for the application of elaborate algorithms to large-scale network inference problems. The computational cost of such calculations can be alleviated by the use of compiled programs and parallelization. To this end, we have developed PREMER (Parallel Reverse Engineering with Mutual information & Entropy Reduction), a software toolbox that can run in parallel and sequential environments. It uses information theoretic criteria to recover network topology and determine the strength and causality of interactions, and allows incorporating prior knowledge, imputing missing data, and correcting outliers. PREMER is a free, open source software tool that does not require any commercial software. Its core algorithms are programmed in FORTRAN 90 and implement OpenMP directives. It has user interfaces in Python and MATLAB/Octave, and runs on Windows, Linux, and OSX (https://sites.google.com/site/premertoolbox/).

Quantifying post-transcriptional regulation in the development of Drosophila melanogaster.

Even though proteins are produced from mRNA, the correlation between mRNA levels and protein abundances is moderate in most studies, occasionally attributed to complex post-transcriptional regulation. To address this, we generate a paired transcriptome/proteome time course dataset with 14 time points during Drosophila embryogenesis. Despite a limited mRNA-protein correlation (ρ = 0.54), mathematical models describing protein translation and degradation explain 84% of protein time-courses based on the measured mRNA dynamics without assuming complex post transcriptional regulation, and allow for classification of most proteins into four distinct regulatory scenarios. By performing an in-depth characterization of the putatively post-transcriptionally regulated genes, we postulate that the RNA-binding protein Hrb98DE is involved in post-transcriptional control of sugar metabolism in early embryogenesis and partially validate this hypothesis using Hrb98DE knockdown. In summary, we present a systems biology framework for the identification of post-transcriptional gene regulation from large-scale, time-resolved transcriptome and proteome data.

Identifying Novel Transcriptional Regulators with Circadian Expression.

Organisms adapt their physiology and behavior to the 24-h day-night cycle to which they are exposed. On a cellular level, this is regulated by intrinsic transcriptional-translational feedback loops that are important for maintaining the circadian rhythm. These loops are organized by members of the core clock network, which further regulate transcription of downstream genes, resulting in their circadian expression. Despite progress in understanding circadian gene expression, only a few players involved in circadian transcriptional regulation, including transcription factors, epigenetic regulators, and long noncoding RNAs, are known. Aiming to discover such genes, we performed a high-coverage transcriptome analysis of a circadian time course in murine fibroblast cells. In combination with a newly developed algorithm, we identified many transcription factors, epigenetic regulators, and long intergenic noncoding RNAs that are cyclically expressed. In addition, a number of these genes also showed circadian expression in mouse tissues. Furthermore, the knockdown of one such factor, Zfp28, influenced the core clock network. Mathematical modeling was able to predict putative regulator-effector interactions between the identified circadian genes and may help for investigations into the gene regulatory networks underlying circadian rhythms.

Two-dimensional global longitudinal strain rate is a preload independent index of systemic right ventricular contractility in hypoplastic left heart syndrome patients after Fontan operation.

BACKGROUND: Assessment of systemic right ventricular (RV) function in patients with hypoplastic left heart syndrome is important during long-term follow-up after Fontan repair. Traditional echocardiographic parameters to evaluate systolic ventricular function are affected by loading conditions. The only generally accepted load-independent parameter of systolic function, end systolic elastance (Ees), requires invasive catheterization. Therefore, we sought to determine if parameters obtained by 2-dimensional speckle tracking (2DST) were affected by acute changes in preload and correlated with catheterization-derived indices of RV contractility in hypoplastic left heart syndrome patients after Fontan palliation. METHODS AND RESULTS: Fifty-two patients with hypoplastic left heart syndrome (median age, 6.6; range 2.9-22.2 years) were prospectively enrolled to have echocardiography and conductance catheter studies performed simultaneously. We compared traditional echo, 2-dimensional speckle tracking and catheterization-derived parameters during different states of preload at baseline and during dobutamine infusion. Global longitudinal strain (S) showed a tendency to decrease with preload reduction, whereas global longitudinal strain rate (SR) did not change (S: -17.7 ± 3.4% versus -16.9 ± 3.8%, P=0.08; SR: -1.30 ± 0.29 versus -1.34 ± 0.34 s(-1), P=0.3). S did not change with dobutamine infusion (-17.7 ± 3.4% versus -18.4 ± 3.9%, P=0.24), whereas SR increased significantly (-1.30 ± 0.29 versus -2.26 ± 0.49 s(-1), P<0.001). RV Ees correlated with SR (rs= -0.47, P<0.001), but not with S (rs=0.07, P=0.5) or other echocardiographic parameters. CONCLUSIONS: In contrast to S, SR was not affected by preload and correlated with Ees of the systemic RV. SR may be a useful noninvasive surrogate of RV contractility and suitable for follow-up of patients with hypoplastic left heart syndrome after Fontan palliation.

Interventional creation of an atrial septal defect and its impact on right ventricular function: an animal study with the pressure-volume conductance system.

BACKGROUND: The aim of our study was to assess the suitability of different interventional techniques to create an atrial septal defect (ASD) and to evaluate the short-term effects of right ventricular (RV) volume overload on RV contractility in the growing swine. METHODS: Thirteen ASD and six control animals were studied. An ASD was created by balloon dilatation (BD) of the fossa ovalis (n = 4) or by implantation of a multi-perforated Amplatzer Septal Occluder (n = 4) or a patch-less nitinol device (n = 5). After 4.8 (3.9-6.0) weeks, the amounts of left-to-right shunting (Qp/Qs) and RV contractility (end systolic elastance - Ees) were assessed. RESULTS: In the ASD group, a significant left-to-right shunt could be documented (Qp/Qs 1.5 ± ± 0.4). However, a shunt was absent in the BD subgroup (Qp/Qs 1.1 ± 0.1). In animals with devices implanted, a significant relationship between the post-mortem ASD area and Qp/Qs was found (r = 0.68, p < 0.05). Compared to controls, RV contractility was not significantly impaired at rest and during dobutamine in ASD animals (Ees: 0.40 ± 0.20 vs 0.54 ± 0.12 and 0.75 ± 0.29 vs 1.04 ± 0.24 mm Hg/mL, p = NS for both). CONCLUSIONS: Device implantation is necessary to create a patent ASD resulting in significant left-to-right shunting. In an experimental ASD model, a five week period of chronic RV volume overload does not alter RV contractility significantly.

Cardiolipin deficiency in X-linked cardioskeletal myopathy and neutropenia (Barth syndrome, MIM 302060): a study in cultured skin fibroblasts.

We determined cardiolipin concentrations in cultured skin fibroblasts of 5 patients with X-linked cardioskeletal myopathy and neutropenia (Barth syndrome, MIM 302060) and in two groups of control patients. High-performance liquid chromatography-electrospray mass spectrometry was used to quantify total cardiolipin and subclasses of cardiolipin molecular species in cultured skin fibroblasts. Total cardiolipin and cardiolipin subclasses were decreased in patients with Barth syndrome as compared with normal control patients and disease control patients. Patients with Barth syndrome have a specific decrease of various cardiolipin molecular species, foremost tetralineoyl-cardiolipin. Therefore the analysis of cardiolipin in fibroblasts offers a specific biochemical approach to detect this disorder.

Neutrophils in Barth syndrome (BTHS) avidly bind annexin-V in the absence of apoptosis.

Barth syndrome (BTHS) is a rare X-linked disease characterized by a triad of dilated cardiomyopathy, skeletal myopathy, and neutropenia. The disease is associated with mutations of the TAZ gene, resulting in defective cardiolipin (CL), an important inner mitochondrial membrane component. Untreated boys die in infancy or early childhood from septicemia or cardiac failure. To date, neutrophil function has never been studied. Directed motility and killing activity of neutrophils was investigated in 7 BTHS patients and found normal in those tested. The circulating neutrophils and eosinophils (but not monocytes or lymphocytes) showed annexin-V binding, suggesting phosphatidylserine (PS) exposure due to apoptosis. However, caspase activity was absent in fresh BTHS cells. Unexpectedly, the near absence of CL impacted neither the mitochondrial mass and shape in fresh BTHS neutrophils nor mitochondrial clustering and Bax translocation upon apoptosis. Annexin-V binding to BTHS neutrophils was not caused by phospholipid scrambling. Moreover, freshly purified BTHS neutrophils were not phagocytosed by macrophages. In sum, a massive number of circulating annexin-V-binding neutrophils in the absence of apoptosis can be demonstrated in BTHS. These neutrophils expose an alternative substrate for annexin-V different from PS and not recognized by macrophages, excluding early clearance as an explanation for the neutropenia.