Problems or prospects? Being a parent in the early phase of the COVID-19 pandemic in Germany.

Background: In the early phase of the COVID-19 pandemic, many restrictions hit people in ways never seen before. Mental wellbeing was affected and burden was high, especially for high-risk groups such as parents. However, to our knowledge no research has yet examined whether being a parent was not only a risk for psychological burden but also a way to cope with the COVID-19 pandemic. Methods: An online survey was used to collect data from 1,121 participants from April to June 2020. In addition to demographic variables, risk factors (financial burden, problems complying with COVID-19 restrictions, and pre-treatment due to mental health problems) and protective factors (emotion regulation, humor, and crisis self-efficacy) were collected. The dataset was divided into three groups: parents whose children lived at home (n = 395), parents whose children did not (no longer) live at home (n = 165), and people who were not parents (n = 561). Results: A linear mixed effect model showed that parents had no higher burden than non-parents, and even less when children did not live at home. Expected risk factors were generally less important, and there were no differences between parents and non-parents. In contrast, parents had advantages in protective factors. Conclusion: In the early phase of the COVID-19 pandemic, it was shown that parents (with and without their children at home) were not necessarily at risk due to additional burden, but also had prospects of coping better with the situation than people without children.

Non-Human Primate iPSC Generation, Cultivation, and Cardiac Differentiation under Chemically Defined Conditions.

Non-human primates (NHP) are important surrogate models for late preclinical development of advanced therapy medicinal products (ATMPs), including induced pluripotent stem cell (iPSC)-based therapies, which are also under development for heart failure repair. For effective heart repair by remuscularization, large numbers of cardiomyocytes are required, which can be obtained by efficient differentiation of iPSCs. However, NHP-iPSC generation and long-term culture in an undifferentiated state under feeder cell-free conditions turned out to be problematic. Here we describe the reproducible development of rhesus macaque (Macaca mulatta) iPSC lines. Postnatal rhesus skin fibroblasts were reprogrammed under chemically defined conditions using non-integrating vectors. The robustness of the protocol was confirmed using another NHP species, the olive baboon (Papio anubis). Feeder-free maintenance of NHP-iPSCs was essentially dependent on concurrent Wnt-activation by GSK-inhibition (Gi) and Wnt-inhibition (Wi). Generated NHP-iPSCs were successfully differentiated into cardiomyocytes using a combined growth factor/GiWi protocol. The capacity of the iPSC-derived cardiomyocytes to self-organize into contractile engineered heart muscle (EHM) was demonstrated. Collectively, this study establishes a reproducible protocol for the robust generation and culture of NHP-iPSCs, which are useful for preclinical testing of strategies for cell replacement therapies in NHP.

A High-Throughput Method as a Diagnostic Tool for HIV Detection in Patient-Specific Induced Pluripotent Stem Cells Generated by Different Reprogramming Methods.

Induced pluripotent stem cells (iPSCs) provide a unique opportunity for generation of patient-specific cells for use in translational purposes. We aimed to compare iPSCs generated by different reprogramming methods regarding their reprogramming efficiency, pluripotency capacity, and the possibility to use high-throughput PCR-based methods for detection of human pathogenic viruses. iPSCs from skin fibroblasts (FB), peripheral blood mononuclear cells (PBMCs), or mesenchymal stem cells (MSCs) were generated by using three different reprogramming systems including chromosomal integrating and nonintegrating methods. Reprogramming efficiencies were in accordance with the literature, indicating that the parental cell type and the reprogramming method play a major role for the reprogramming efficiencies (FB: STEMCCA: 1.30 ± 0.18, Sendai virus: 1.37 ± 0.01, and episomal plasmids: 0.04 ± 0.02; PBMCs: Sendai virus: 0.002 ± 0.001, episomal plasmids: 0) but result in the same characteristics of pluripotency. We found the highest reprogramming efficiencies for MSC with 3.32 ± 1.2 by using episomal plasmids. Since GMP standard working procedures and screening units need virus contamination-free cell lines, we studied HIV-1 contamination in the generated iPSCs. We used the high-throughput cobas® 6800/8800 system, which is normally used for detection of HIV-1 in plasma of patients, and found that footprint-free reprogramming methods as episomal plasmids and Sendai virus are useful for the described virus detection method. This fast, cost-effective, robust, and reliable assay demonstrates the feasibility to use high-throughput PCR-based methods for detection of human pathogenic viruses in ps-iPSC lines that were generated with nongenome integrating reprogramming methods.

Catecholamine-Dependent ß-Adrenergic Signaling in a Pluripotent Stem Cell Model of Takotsubo Cardiomyopathy.

BACKGROUND: Takotsubo syndrome (TTS) is characterized by an acute left ventricular dysfunction and is associated with life-threating complications in the acute phase. The underlying disease mechanism in TTS is still unknown. A genetic basis has been suggested to be involved in the pathogenesis. OBJECTIVES: The aims of the study were to establish an in vitro induced pluripotent stem cell (iPSC) model of TTS, to test the hypothesis of altered ß-adrenergic signaling in TTS iPSC-cardiomyocytes (CMs), and to explore whether genetic susceptibility underlies the pathophysiology of TTS. METHODS: Somatic cells of patients with TTS and control subjects were reprogrammed to iPSCs and differentiated into CMs. Three-month-old CMs were subjected to catecholamine stimulation to simulate neurohumoral overstimulation. We investigated ß-adrenergic signaling and TTS cardiomyocyte function. RESULTS: Enhanced ß-adrenergic signaling in TTS-iPSC-CMs under catecholamine-induced stress increased expression of the cardiac stress marker NR4A1; cyclic adenosine monophosphate levels; and cyclic adenosine monophosphate-dependent protein kinase A-mediated hyperphosphorylation of RYR2-S2808, PLN-S16, TNI-S23/24, and Cav1.2-S1928, and leads to a reduced calcium time to transient 50% decay. These cellular catecholamine-dependent responses were mainly mediated by ß1-adrenoceptor signaling in TTS. Engineered heart muscles from TTS-iPSC-CMs showed an impaired force of contraction and a higher sensitivity to isoprenaline-stimulated inotropy compared with control subjects. In addition, altered electrical activity and increased lipid accumulation were detected in catecholamine-treated TTS-iPSC-CMs, and were confirmed by differentially expressed lipid transporters CD36 and CPT1C. Furthermore, we uncovered genetic variants in different key regulators of cardiac function. CONCLUSIONS: Enhanced ß-adrenergic signaling and higher sensitivity to catecholamine-induced toxicity were identified as mechanisms associated with the TTS phenotype. (International Takotsubo Registry [InterTAK Registry] [InterTAK]; NCT01947621).

"The usual suspects"- analysis of transcriptome sequences reveals deviating B gene activity in C. vulgaris bud bloomers.

BACKGROUND: The production of heather (Calluna vulgaris) in Germany is highly dependent on cultivars with mutated flower morphology, the so-called diplocalyx bud bloomers. So far, this unique flower type of C. vulgaris has not been reported in any other plant species. The flowers are characterised by an extremely extended flower attractiveness, since the flower buds remain closed throughout the complete flowering season. The flowers of C. vulgaris bud bloomers are male sterile, because the stamens are absent. Furthermore, petals are converted into sepals. Therefore the diplocalyx bud bloomer flowers consist of two whorls of sepals directly followed by the gynoecium. RESULTS: A broad comparison was undertaken to identify genes differentially expressed in the bud flowering phenotype and in the wild type of C. vulgaris. Transcriptome sequence reads were generated using 454 sequencing of two flower type specific cDNA libraries. In total, 360,000 sequence reads were obtained, assembled to 12,200 contigs, functionally mapped, and annotated. Transcript abundances were compared and 365 differentially expressed genes detected. Among these differentially expressed genes, Calluna vulgaris PISTILLATA (CvPI) which is the orthologue of the Arabidopsis B gene PISTILLATA (PI) was considered as the most promising candidate gene. Quantitative Reverse Transcription Polymerase Chain Reaction (qRT PCR) was performed to analyse the gene expression levels of two C. vulgaris B genes CvPI and Calluna vulgaris APETALA 3 (CvAP3) in both flower types. CvAP3 which is the orthologue of the Arabidopsis B gene APETALA 3 (AP3) turned out to be ectopically expressed in sepals of wild type and bud bloomer flowers. CvPI expression was proven to be reduced in the bud blooming flowers. CONCLUSIONS: Differential expression patterns of the B-class genes CvAP3 and CvPI were identified to cause the characteristic morphology of C. vulgaris flowers leading to the following hypotheses: ectopic expression of CvAP3 is a convincing explanation for the formation of a completely petaloid perianth in both flower types. In C. vulgaris, CvPI is essential for determination of petal and stamen identity. The characteristic transition of petals into sepals potentially depends on the observed deficiency of CvPI and CvAP3 expression in bud blooming flowers.

AFLP-based genetic mapping of the "bud-flowering" trait in heather (Calluna vulgaris).

BACKGROUND: Calluna vulgaris is one of the most important landscaping plants produced in Germany. Its enormous economic success is due to the prolonged flower attractiveness of mutants in flower morphology, the so-called bud-bloomers. In this study, we present the first genetic linkage map of C. vulgaris in which we mapped a locus of the economically highly desired trait "flower type". RESULTS: The map was constructed in JoinMap 4.1. using 535 AFLP markers from a single mapping population. A large fraction (40%) of markers showed distorted segregation. To test the effect of segregation distortion on linkage estimation, these markers were sorted regarding their segregation ratio and added in groups to the data set. The plausibility of group formation was evaluated by comparison of the "two-way pseudo-testcross" and the "integrated" mapping approach. Furthermore, regression mapping was compared to the multipoint-likelihood algorithm. The majority of maps constructed by different combinations of these methods consisted of eight linkage groups corresponding to the chromosome number of C. vulgaris. CONCLUSIONS: All maps confirmed the independent inheritance of the most important horticultural traits "flower type", "flower colour", and "leaf colour". An AFLP marker for the most important breeding target "flower type" was identified. The presented genetic map of C. vulgaris can now serve as a basis for further molecular marker selection and map-based cloning of the candidate gene encoding the unique flower architecture of C. vulgaris bud-bloomers.

Development and application of high-throughput amplified fragment length polymorphism technique in Calluna vulgaris (Ericaceae)

Calluna vulgaris is an important ornamental crop of the horticultural industry in Europe. In order to improve breeding of this species, especially of the most important trait of ‘bud-flowering', the implementation of molecular techniques that allow rapid, reproducible and efficient screening of whole segregating populations e.g. for molecular marker and mapping approaches is a requirement. We therefore aimed to introduce the powerful tool of amplified fragment length polymorphisms (AFLP®), a widely and successfully applied method, into our methodological assortment. As an essential prerequisite, the isolated DNA should be of adequate quality which is a common obstacle when dealing with woody species and their interfering secondary components/metabolites. The results of screening different and modified DNA isolation protocols are described. As the outcome of our evaluations of reaction conditions during the AFLP® procedure, we circumstantiate a functional protocol ranging from DNA extraction to visualization of AFLP® banding patterns for the woody crop C. vulgaris. This method is suitable for high throughput genetic applications and may even be transferable to other species. In addition, costs are reduced by reasonable reagents and multiplexing assays.

'Who's who' in two different flower types of Calluna vulgaris (Ericaceae): morphological and molecular analyses of flower organ identity.

BACKGROUND: The ornamental crop Calluna vulgaris is of increasing importance to the horticultural industry in the northern hemisphere due to a flower organ mutation: the flowers of the 'bud-flowering' phenotype remain closed i.e. as buds throughout the total flowering period and thereby maintain more colorful flowers for a longer period of time than the wild-type. This feature is accompanied and presumably caused by the complete lack of stamens. Descriptions of this botanical particularity are inconsistent and partially conflicting. In order to clarify basic questions of flower organ identity in general and stamen loss in detail, a study of the wild-type and the 'bud-flowering' flower type of C. vulgaris was initiated. RESULTS: Flowers were examined by macro- and microscopic techniques. Organ development was investigated comparatively in both the wild-type and the 'bud-flowering' type by histological analyses. Analysis of epidermal cell surface structure of vegetative tissues and perianth organs using scanning electron microscopy revealed that in wild-type flowers the outer whorls of colored organs may be identified as sepals, while the inner ones may be identified as petals. In the 'bud-flowering' type, two whorls of sepals are directly followed by the gynoecium. Both, petals and stamens, are completely missing in this flower type. The uppermost whorl of green leaves represents bracts in both flower types. In addition, two MADS-box genes (homologs of AP3/DEF and SEP1/2) were identified in C. vulgaris using RACE-PCR. Expression analysis by qRT-PCR was conducted for both genes in leaves, bracts, sepals and petals. These experiments revealed an expression pattern supporting the organ classification based on morphological characteristics. CONCLUSIONS: Organ identity in both wild-type and 'bud-flowering' C. vulgaris was clarified using a combination of microscopic and molecular methods. Our results for bract, sepal and petal organ identity are supported by the 'ABCDE model'. However, loss of stamens in the 'bud-flowering' phenotype is an exceptional flower organ modification that cannot be explained by modified spatial expression of known organ identity genes.

Implementation of a model for identifying Essentially Derived Varieties in vegetatively propagated Calluna vulgaris varieties.

BACKGROUND: Variety protection is of high relevance for the horticultural community and juridical cases have become more frequent in a globalized economy due to essential derivation of varieties. This applies equally to Calluna vulgaris, a vegetatively propagated species from the Ericaceae family that belongs to the top-selling pot plants in Europe. We therefore analyzed the genetic diversity of 74 selected varieties and genotypes of C. vulgaris and 3 of Erica spp. by means of RAPD and iSSR fingerprinting using 168 mono- and polymorphisms. The same data set was utilized to generate a system to reliably identify Essentially Derived Varieties (EDVs) in C. vulgaris, which was adapted from a method suggested for lettuce and barley. This system was developed, validated and used for selected tests of interest in C. vulgaris. RESULTS: As expected following personal communications with breeders, a very small genetic diversity became evident within C. vulgaris when investigated using our molecular methods. Thus, a dendrogram-based assay to detect Essentially Derived Varieties in this species is not suitable, although varieties are propagated vegetatively. In contrast, the system applied in lettuce, which itself applies pairwise comparisons using appropriate reference sets, proved functional with this species. CONCLUSION: The narrow gene pool detected in C. vulgaris may be the genetic basis for juridical conflicts between breeders. We successfully tested a methodology for identification of Essentially Derived Varieties in highly identical C. vulgaris genotypes and recommend this for future proof of essential derivation in C. vulgaris and other vegetatively propagated crops.