Immature human engineered heart tissues engraft in a guinea pig chronic injury model.

Engineered heart tissue (EHT) transplantation represents an innovative, regenerative approach for heart failure patients. Late preclinical trials are underway, and a first clinical trial started recently. Preceding studies revealed functional recovery after implantation of in vitro-matured EHT in the subacute stage, whereas transplantation in a chronic injury setting was less efficient. When transplanting matured EHTs, we noticed that cardiomyocytes undergo a dedifferentiation step before eventually forming structured grafts. Therefore, we wanted to evaluate whether immature EHT (EHTIm) patches can be used for transplantation. Chronic myocardial injury was induced in a guinea pig model. EHTIm (15×106 cells) were transplanted within hours after casting. Cryo-injury led to large transmural scars amounting to 26% of the left ventricle. Grafts remuscularized 9% of the scar area on average. Echocardiographic analysis showed some evidence of improvement of left-ventricular function after EHTIm transplantation. In a small translational proof-of-concept study, human scale EHTIm patches (4.5×108 cells) were epicardially implanted on healthy pig hearts (n=2). In summary, we provide evidence that transplantation of EHTIm patches, i.e. without precultivation, is feasible, with similar engraftment results to those obtained using matured EHT.

Missense variants in NOX1 and p22phox in a case of very-early-onset inflammatory bowel disease are functionally linked to NOD2.

Whole-genome and whole-exome sequencing of individual patients allow the study of rare and potentially causative genetic variation. In this study, we sequenced DNA of a trio comprising a boy with very-early-onset inflammatory bowel disease (veoIBD) and his unaffected parents. We identified a rare, X-linked missense variant in the NAPDH oxidase NOX1 gene (c.C721T, p.R241C) in heterozygous state in the mother and in hemizygous state in the patient. We discovered that, in addition, the patient was homozygous for a common missense variant in the CYBA gene (c.T214C, p.Y72H). CYBA encodes the p22phox protein, a cofactor for NOX1. Functional assays revealed reduced cellular ROS generation and antibacterial capacity of NOX1 and p22phox variants in intestinal epithelial cells. Moreover, the identified NADPH oxidase complex variants affected NOD2-mediated immune responses, and p22phox was identified as a novel NOD2 interactor. In conclusion, we detected missense variants in a veoIBD patient that disrupt the host response to bacterial challenges and reduce protective innate immune signaling via NOD2. We assume that the patient's individual genetic makeup favored disturbed intestinal mucosal barrier function.

Impact of high risk thrombophilia status on recurrence among children and adults with VTE: An observational multicenter cohort study.

BACKGROUND: Antithrombin [AT]-, protein C [PC]- or protein S [PS]-deficiency [D] constitutes a major risk factor for venous thromboembolism [VTE]. Primary study objective was to evaluate if the clinical presentation at first VTE onset differs between children and adults and to compare the individual recurrence risk among patients with respect to age at onset and their thrombophilia status ATD, PCD or PSD. METHODS/PATIENTS/RESULTS: In 137 of 688 consecutively enrolled pediatric and adult VTE patients we calculated the absolute risk of VTE recurrence and event-free-survival adjusted for thrombophilia and positive family VTE history. At first VTE children manifested i) with a lower rate of pulmonary embolism, ii) a higher rate of cerebral vascular events or multiple VTEs, and iii) showed a higher proportion of unprovoked VTE compared to adolescents and adults. Adult patients reported more often a positive VTE history compared to younger study participants. The adjusted odds of recurrence in adults was 2.05 compared to children. CONCLUSION: At disease manifestation children and adults differ with respect to i) thrombotic locations, ii) percentage of unprovoked versus provoked VTE, and iii) different rates of positive VTE family histories. Furthermore, adults showed a two-fold increase risk of VTE recurrence compared to children.

Impact of high-risk thrombophilia status on recurrence among children with a first non-central-venous-catheter-associated VTE: an observational multicentre cohort study.

Deficiency of antithrombin (AT), protein C (PC) or protein S (PS) constitutes a major risk factor for venous thromboembolism (VTE). Individuals at high risk for recurrence who benefit from screening need to be identified. The primary study objective was to determine the individual recurrence risk among children with a first non-central-venous-catheter-associated VTE with respect to their thrombophilia status and to evaluate if the clinical presentation at first VTE onset differs between children with AT, PC or PS deficiency versus no thrombophilia. We calculated the absolute risk of VTE recurrence and event-free-survival adjusted for thrombophilia, age, sex and positive family VTE history in 161 consecutively enrolled paediatric VTE patients. The presence of a deficiency relative to no thrombophilia was evaluated as a potential predictor of recurrence. Predictors for recurrence were AT deficiency (hazard ratio/95% CI: 6·5/2·46-17·2) and female gender (2·6/1·1-6·35). The annual recurrence rates (95% CIs) were 5·4% (2·6-10) in AT-deficient children, 1·3% (0·3-3·8) in patients with PC deficiency, 0·7% (0·08-2·4) in the PS-deficient cohort and 0·9% (0·4-1·8) in patients with no thrombophilia. Positive family VTE history or combined thrombophilias did not predict recurrence. Given the overall annual incidence rate of recurrence of 1·5% we suggest screening for AT deficiency in children with VTE.

Role of protein S deficiency in children with venous thromboembolism. An observational international cohort study.

Venous thromboembolism [TE] is a multifactorial disease, and protein S deficiency [PSD] constitutes a major risk factor. In the present study the prevalence of PSD and the clinical presentation at TE onset in a cohort of children is reported. In 367 unselected paediatric patients with TE (age 0.1-18 years) recruited between July 1996 and December 2013, a comprehensive thrombophilia screening was performed along with recording of anamnestic data. Thirty of 367 paediatric patients (8.2 %) derived from 27 families had PSD. Mean age at first TE onset was 14.5 years (range 0.1 to 18). Thrombotic locations were cerebral veins (n=8), calf vein TE (n=3) deep veins (DVT) of the leg (n=12), DVT & pulmonary embolism (n=5) and intra-cardiac veins (n=1) or purpura fulminans (n=1). PSD co-occurred with the factor 5 mutation at rs6025 or the homozygous factor 2 susceptibility variant at rs1799963 in one case each. The Heerlen polymorphism detected in five children presented with milder PSD. In 18 patients (60 %) a concomitant risk factor for TE was identified. A second TE event within primarily healthy siblings occurred in three of 27 PSD families (11.0 %). In this cohort of children with symptomatic TE, the prevalence of PSD adjusted for family status was 7.4 %. Given its clinical implication for patients and family members, thrombophilia testing should be performed and the benefit of medical or educational interventions should be evaluated in this high-risk population.

Clinical and laboratory characteristics of children with venous thromboembolism and protein C-deficiency: an observational Israeli-German cohort study.

Venous thromboembolism [TE] is a multifactorial disease and protein C deficiency [PCD] constitutes a major risk factor. In the present study the prevalence of PCD and the clinical presentation at TE onset, including neonatal purpura fulminans, in a cohort of children are reported. In 367 unselected children (0·1-19 years) recruited between July 1996 and December 2013, a comprehensive thrombophilia screening was performed along with recording of anamnestic data. Twenty-five of 338 children (7·4%) had PCD. Mean age at first TE onset was 10 years (range 0·1-18). Leading thromboembolic manifestations were neonatal purpura fulminans (n = 5), TE of cerebral veins (n = 3), stroke (n = 2) deep veinthrombosis (DVT) of the leg (n = 10), DVT & pulmonary embolism (n = 2) and DVT & pelvic veins (n = 3). Concomitant risk factors for TE were identified in 12 patients, whereas 13 children spontaneously developed TE. A positive family history of DVT was found in 10 children. In this unselected cohort of paediatric patients with symptomatic TE the overall prevalence of PCD was 7·4%; 1·5% presented with neonatal purpura fulminans. Given its clinical implication for patients and family members, thrombophilia testing should be performed and the benefit of medical or educational interventions should be evaluated in this high-risk population.

Apomictic and sexual germline development differ with respect to cell cycle, transcriptional, hormonal and epigenetic regulation.

Seeds of flowering plants can be formed sexually or asexually through apomixis. Apomixis occurs in about 400 species and is of great interest for agriculture as it produces clonal offspring. It differs from sexual reproduction in three major aspects: (1) While the sexual megaspore mother cell (MMC) undergoes meiosis, the apomictic initial cell (AIC) omits or aborts meiosis (apomeiosis); (2) the unreduced egg cell of apomicts forms an embryo without fertilization (parthenogenesis); and (3) the formation of functional endosperm requires specific developmental adaptations. Currently, our knowledge about the gene regulatory programs underlying apomixis is scarce. We used the apomict Boechera gunnisoniana, a close relative of Arabidopsis thaliana, to investigate the transcriptional basis underlying apomeiosis and parthenogenesis. Here, we present the first comprehensive reference transcriptome for reproductive development in an apomict. To compare sexual and apomictic development at the cellular level, we used laser-assisted microdissection combined with microarray and RNA-Seq analyses. Conservation of enriched gene ontologies between the AIC and the MMC likely reflects functions of importance to germline initiation, illustrating the close developmental relationship of sexuality and apomixis. However, several regulatory pathways differ between sexual and apomictic germlines, including cell cycle control, hormonal pathways, epigenetic and transcriptional regulation. Enrichment of specific signal transduction pathways are a feature of the apomictic germline, as is spermidine metabolism, which is associated with somatic embryogenesis in various plants. Our study provides a comprehensive reference dataset for apomictic development and yields important new insights into the transcriptional basis underlying apomixis in relation to sexual reproduction.

Regulation of polyp-to-jellyfish transition in Aurelia aurita.

BACKGROUND: The life cycle of scyphozoan cnidarians alternates between sessile asexual polyps and pelagic medusa. Transition from one life form to another is triggered by environmental signals, but the molecular cascades involved in the drastic morphological and physiological changes remain unknown. RESULTS: We show in the moon jelly Aurelia aurita that the molecular machinery controlling transition of the sessile polyp into a free-swimming jellyfish consists of two parts. One is conserved and relies on retinoic acid signaling. The second, novel part is based on secreted proteins that are strongly upregulated prior to metamorphosis in response to the seasonal temperature changes. One of these proteins functions as a temperature-sensitive "timer" and encodes the precursor of the strobilation hormone of Aurelia. CONCLUSIONS: Our findings uncover the molecule framework controlling the polyp-to-jellyfish transition in a basal metazoan and provide insights into the evolution of complex life cycles in the animal kingdom.

Janus--a comprehensive tool investigating the two faces of transcription.

MOTIVATION: Protocols to generate strand-specific transcriptomes with next-generation sequencing platforms have been used by the scientific community roughly since 2008. Strand-specific reads allow for detection of antisense events and a higher resolution of expression profiles enabling extension of current transcript annotations. However, applications making use of this strandedness information are still scarce. RESULTS: Here we present a tool (Janus), which focuses on the identification of transcriptional active regions in antisense orientation to known and novel transcribed elements of the genome. Janus can compare the antisense events of multiple samples and assigns scores to identify mutual expression of either transcript in a sense/antisense pair, which could hint to regulatory mechanisms. Janus is able to make use of single-nucleotide variant (SNV) and methylation data, if available, and reports the sense to antisense ratio of regions in the vicinity of the identified genetic and epigenetic variation. Janus interrogates positions of heterozygous SNVs to identify strand-specific allelic imbalance. AVAILABILITY: Janus is written in C/C++ and freely available at http://www.ikmb.uni-kiel.de/janus/janus.html under terms of GNU General Public License, for both, Linux and Windows 64×. Although the binaries will work without additional downloads, the software depends on bamtools (https://github.com/pezmaster31/bamtools) for compilation. A detailed tutorial section is included in the first section of the supplemental material and included as brief readme.txt in the tutorial archive. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

FoxO is a critical regulator of stem cell maintenance in immortal Hydra.

Hydra's unlimited life span has long attracted attention from natural scientists. The reason for that phenomenon is the indefinite self-renewal capacity of its stem cells. The underlying molecular mechanisms have yet to be explored. Here, by comparing the transcriptomes of Hydra's stem cells followed by functional analysis using transgenic polyps, we identified the transcription factor forkhead box O (FoxO) as one of the critical drivers of this continuous self-renewal. foxO overexpression increased interstitial stem cell and progenitor cell proliferation and activated stem cell genes in terminally differentiated somatic cells. foxO down-regulation led to an increase in the number of terminally differentiated cells, resulting in a drastically reduced population growth rate. In addition, it caused down-regulation of stem cell genes and antimicrobial peptide (AMP) expression. These findings contribute to a molecular understanding of Hydra's immortality, indicate an evolutionarily conserved role of FoxO in controlling longevity from Hydra to humans, and have implications for understanding cellular aging.

Genome and low-iron response of an oceanic diatom adapted to chronic iron limitation.

BACKGROUND: Biogeochemical elemental cycling is driven by primary production of biomass via phototrophic phytoplankton growth, with 40% of marine productivity being assigned to diatoms. Phytoplankton growth is widely limited by the availability of iron, an essential component of the photosynthetic apparatus. The oceanic diatom Thalassiosira oceanica shows a remarkable tolerance to low-iron conditions and was chosen as a model for deciphering the cellular response upon shortage of this essential micronutrient. RESULTS: The combined efforts in genomics, transcriptomics and proteomics reveal an unexpected metabolic flexibility in response to iron availability for T. oceanica CCMP1005. The complex response comprises cellular retrenchment as well as remodeling of bioenergetic pathways, where the abundance of iron-rich photosynthetic proteins is lowered, whereas iron-rich mitochondrial proteins are preserved. As a consequence of iron deprivation, the photosynthetic machinery undergoes a remodeling to adjust the light energy utilization with the overall decrease in photosynthetic electron transfer complexes. CONCLUSIONS: Beneficial adaptations to low-iron environments include strategies to lower the cellular iron requirements and to enhance iron uptake. A novel contribution enhancing iron economy of phototrophic growth is observed with the iron-regulated substitution of three metal-containing fructose-bisphosphate aldolases involved in metabolic conversion of carbohydrates for enzymes that do not contain metals. Further, our data identify candidate components of a high-affinity iron-uptake system, with several of the involved genes and domains originating from duplication events. A high genomic plasticity, as seen from the fraction of genes acquired through horizontal gene transfer, provides the platform for these complex adaptations to a low-iron world.

Validation of treatment strategies for enterohaemorrhagic Escherichia coli O104:H4 induced haemolytic uraemic syndrome: case-control study.

OBJECTIVE: To evaluate the effect of different treatment strategies on enterohaemorrhagic Escherichia coli O104:H4 induced haemolytic uraemic syndrome. DESIGN: Multicentre retrospective case-control study. SETTING: 23 hospitals in northern Germany. PARTICIPANTS: 298 adults with enterohaemorrhagic E coli induced haemolytic uraemic syndrome. MAIN OUTCOME MEASURES: Dialysis, seizures, mechanical ventilation, abdominal surgery owing to perforation of the bowel or bowel necrosis, and death. RESULTS: 160 of the 298 patients (54%) temporarily required dialysis, with only three needing treatment long term. 37 patients (12%) had seizures, 54 (18%) required mechanical ventilation, and 12 (4%) died. No clear benefit was found from use of plasmapheresis or plasmapheresis with glucocorticoids. 67 of the patients were treated with eculizumab, a monoclonal antibody directed against the complement cascade. No short term benefit was detected that could be attributed to this treatment. 52 patients in one centre that used a strategy of aggressive treatment with combined antibiotics had fewer seizures (2% v 15%, P = 0.03), fewer deaths (0% v 5%, p = 0.029), required no abdominal surgery, and excreted E coli for a shorter duration. CONCLUSIONS: Enterohaemorrhagic E coli induced haemolytic uraemic syndrome is a severe self limiting acute condition. Our findings question the benefit of eculizumab and of plasmapheresis with or without glucocorticoids. Patients with established haemolytic uraemic syndrome seemed to benefit from antibiotic treatment and this should be investigated in a controlled trial.

Molecular signatures of the three stem cell lineages in hydra and the emergence of stem cell function at the base of multicellularity.

How distinct stem cell populations originate and whether there is a clear stem cell "genetic signature" remain poorly understood. Understanding the evolution of stem cells requires molecular profiling of stem cells in an animal at a basal phylogenetic position. In this study, using transgenic Hydra polyps, we reveal for each of the three stem cell populations a specific signature set of transcriptions factors and of genes playing key roles in cell type-specific function and interlineage communication. Our data show that principal functions of stem cell genes, such as maintenance of stemness and control of stem cell self-renewal and differentiation, arose very early in metazoan evolution. They are corroborating the view that stem cell types shared common, multifunctional ancestors, which achieved complexity through a stepwise segregation of function in daughter cells.

A powerful method for transcriptional profiling of specific cell types in eukaryotes: laser-assisted microdissection and RNA sequencing.

The acquisition of distinct cell fates is central to the development of multicellular organisms and is largely mediated by gene expression patterns specific to individual cells and tissues. A spatially and temporally resolved analysis of gene expression facilitates the elucidation of transcriptional networks linked to cellular identity and function. We present an approach that allows cell type-specific transcriptional profiling of distinct target cells, which are rare and difficult to access, with unprecedented sensitivity and resolution. We combined laser-assisted microdissection (LAM), linear amplification starting from <1 ng of total RNA, and RNA-sequencing (RNA-Seq). As a model we used the central cell of the Arabidopsis thaliana female gametophyte, one of the female gametes harbored in the reproductive organs of the flower. We estimated the number of expressed genes to be more than twice the number reported previously in a study using LAM and ATH1 microarrays, and identified several classes of genes that were systematically underrepresented in the transcriptome measured with the ATH1 microarray. Among them are many genes that are likely to be important for developmental processes and specific cellular functions. In addition, we identified several intergenic regions, which are likely to be transcribed, and describe a considerable fraction of reads mapping to introns and regions flanking annotated loci, which may represent alternative transcript isoforms. Finally, we performed a de novo assembly of the transcriptome and show that the method is suitable for studying individual cell types of organisms lacking reference sequence information, demonstrating that this approach can be applied to most eukaryotic organisms.

Transcriptomic resilience to global warming in the seagrass Zostera marina, a marine foundation species.

Large-scale transcription profiling via direct cDNA sequencing provides important insights as to how foundation species cope with increasing climatic extremes predicted under global warming. Species distributed along a thermal cline, such as the ecologically important seagrass Zostera marina, provide an opportunity to assess temperature effects on gene expression as a function of their long-term adaptation to heat stress. We exposed a southern and northern European population of Zostera marina from contrasting thermal environments to a realistic heat wave in a common-stress garden. In a fully crossed experiment, eight cDNA libraries, each comprising ~125 000 reads, were obtained during and after a simulated heat wave, along with nonstressed control treatments. Although gene-expression patterns during stress were similar in both populations and were dominated by classical heat-shock proteins, transcription profiles diverged after the heat wave. Gene-expression patterns in southern genotypes returned to control values immediately, but genotypes from the northern site failed to recover and revealed the induction of genes involved in protein degradation, indicating failed metabolic compensation to high sea-surface temperature. We conclude that the return of gene-expression patterns during recovery provides critical information on thermal adaptation in aquatic habitats under climatic stress. As a unifying concept for ecological genomics, we propose transcriptomic resilience, analogous to ecological resilience, as an important measure to predict the tolerance of individuals and hence the fate of local populations in the face of global warming.

A tissue-specific landscape of sense/antisense transcription in the mouse intestine.

BACKGROUND: The intestinal mucosa is characterized by complex metabolic and immunological processes driven highly dynamic gene expression programs. With the advent of next generation sequencing and its utilization for the analysis of the RNA sequence space, the level of detail on the global architecture of the transcriptome reached a new order of magnitude compared to microarrays. RESULTS: We report the ultra-deep characterization of the polyadenylated transcriptome in two closely related, yet distinct regions of the mouse intestinal tract (small intestine and colon). We assessed tissue-specific transcriptomal architecture and the presence of novel transcriptionally active regions (nTARs). In the first step, signatures of 20,541 NCBI RefSeq transcripts could be identified in the intestine (74.1% of annotated genes), thereof 16,742 are common in both tissues. Although the majority of reads could be linked to annotated genes, 27,543 nTARs not consistent with current gene annotations in RefSeq or ENSEMBL were identified. By use of a second independent strand-specific RNA-Seq protocol, 20,966 of these nTARs were confirmed, most of them in vicinity of known genes. We further categorized our findings by their relative adjacency to described exonic elements and investigated regional differences of novel transcribed elements in small intestine and colon. CONCLUSIONS: The current study demonstrates the complexity of an archetypal mammalian intestinal mRNA transcriptome in high resolution and identifies novel transcriptionally active regions at strand-specific, single base resolution. Our analysis for the first time shows a strand-specific comparative picture of nTARs in two tissues and represents a resource for further investigating the transcriptional processes that contribute to tissue identity.

Maternal epigenetic pathways control parental contributions to Arabidopsis early embryogenesis.

Defining the contributions and interactions of paternal and maternal genomes during embryo development is critical to understand the fundamental processes involved in hybrid vigor, hybrid sterility, and reproductive isolation. To determine the parental contributions and their regulation during Arabidopsis embryogenesis, we combined deep-sequencing-based RNA profiling and genetic analyses. At the 2-4 cell stage there is a strong, genome-wide dominance of maternal transcripts, although transcripts are contributed by both parental genomes. At the globular stage the relative paternal contribution is higher, largely due to a gradual activation of the paternal genome. We identified two antagonistic maternal pathways that control these parental contributions. Paternal alleles are initially downregulated by the chromatin siRNA pathway, linked to DNA and histone methylation, whereas transcriptional activation requires maternal activity of the histone chaperone complex CAF1. Our results define maternal epigenetic pathways controlling the parental contributions in plant embryos, which are distinct from those regulating genomic imprinting.

Defining the origins of the NOD-like receptor system at the base of animal evolution.

Distinguishing self from nonself and the onset of defense effector mechanisms upon recognition of pathogens are essential for the survival of all life forms in the animal kingdom. The family of nucleotide -binding and oligomeriszation domain-like receptors (NLRs) was first identified in vertebrates and comprises a group of pivotal sensor protein of the innate immune system for microbial cell wall components or danger signals. Here, we provide first evidence that early diverging metazoans have large and complex NLR repertoires. The cnidarian NACHT/NB-ARC genes include novel combinations of domains, and the number of one specific type (NB-ARC and tetratricopeptide repeat containing) in Hydra is particularly large. We characterize the transcript structure and expression patterns of a selected HyNLR, HyNLR type 1 and describe putative interaction partners. In a heterologous expression system, we show induced proximity recruitment of an effector caspase (HyDD-Caspase) to the HyNLR type 1 protein upon oligomerization indicating a potential role of caspase activation downstream of NLR activation in Hydra. These results add substantially to our understanding of the ancestral innate immune repertoire as well as providing the first insights into putative cytoplasmic defense mechanisms at the base of animal evolution.