From Stem Cells to Populations—Using hiPSC, Next-Generation Sequencing, and GWAS to Explore the Genetic and Molecular Mechanisms of Congenital Heart Defects

Congenital heart defects (CHD) are developmental malformations affecting the heart and the great vessels. Early heart development requires temporally regulated crosstalk between multiple cell types, signaling pathways, and mechanical forces of early blood flow. While both genetic and environmental factors have been recognized to be involved, identifying causal genes in non-syndromic CHD has been difficult. While variants following Mendelian inheritance have been identified by linkage analysis in a few families with multiple affected members, the inheritance pattern in most familial cases is complex, with reduced penetrance and variable expressivity. Furthermore, most non-syndromic CHD are sporadic. Improved sequencing technologies and large biobank collections have enabled genome-wide association studies (GWAS) in non-syndromic CHD. The ability to generate human to create human induced pluripotent stem cells (hiPSC) and further differentiate them to organotypic cells enables further exploration of genotype–phenotype correlations in patient-derived cells. Here we review how these technologies can be used in unraveling the genetics and molecular mechanisms of heart development

From Stem Cells to Populations—Using hiPSC, Next-Generation Sequencing, and GWAS to Explore the Genetic and Molecular Mechanisms of Congenital Heart Defects

Congenital heart defects (CHD) are developmental malformations affecting the heart and the great vessels. Early heart development requires temporally regulated crosstalk between multiple cell types, signaling pathways, and mechanical forces of early blood flow. While both genetic and environmental factors have been recognized to be involved, identifying causal genes in non-syndromic CHD has been difficult. While variants following Mendelian inheritance have been identified by linkage analysis in a few families with multiple affected members, the inheritance pattern in most familial cases is complex, with reduced penetrance and variable expressivity. Furthermore, most non-syndromic CHD are sporadic. Improved sequencing technologies and large biobank collections have enabled genome-wide association studies (GWAS) in non-syndromic CHD. The ability to generate human to create human induced pluripotent stem cells (hiPSC) and further differentiate them to organotypic cells enables further exploration of genotype–phenotype correlations in patient-derived cells. Here we review how these technologies can be used in unraveling the genetics and molecular mechanisms of heart development.Peer reviewe

Genetic profiling of the interactions between soft rot Pectobacterium species and plants

The interactions between phytopathogenic bacteria and their host plants can be characterized as an intricate web of signals and appropriate responses. Phytopathogenic soft rot bacteria occur globally, causing disease in Solanum tuberosum (potato) and other tubular staple foods in both the field and storage. One widely studied soft rot bacterium is Pectobacterium wasabiae, which has been identified in Eutrema wasabi (wasabi) plants in Japan and in potatoes in Finland. Generally, the interactions between this type of bacterium and host plants are characterized by maceration of plant tissue, due to the actions of secreted plant cell wall degrading enzymes (PCWDE), and the induction of phytohormone dependent defenses in the plants. The maceration of plant tissue involves the release of pectic oligogalacturonides (OGs) from plant cell walls. OGs have been identified as important signaling compounds, inducing the expression of a variety of defense-related genes. As the bacterial infection advances, the bacteria coordinate the production of virulence factors by utilizing regulatory proteins that modulate the transcriptome. Transcriptomic analyses have been used extensively in past studies to identify regulatory networks and signaling pathways, and these studies have provided insights into the processes underlying plant-pathogen interactions. The novel scientific results of this dissertation are derived from a combination of transcriptomic, genomic, genetic, and phenotypic analyses. This study analyzed various aspects of plant-pathogen interactions. The central bacterial model used was P. wasabiae, and the model plant of interest was Arabidopsis thaliana. This study characterized the genome of P. wasabiae via sequencing and bioinformatics analysis. Various virulence associated genes and operons, such as two distinct type 6 secretion systems, were identified and annotated. The bacterium was found to in fact be more related to P. wasabiae than Pectobacterium carotovorum, which the strain originally had been named after. Furthermore, a combination of functional genetics and transcriptomic methods, such as reverse transcription quantitative PCR (RT-qPCR) and microarrays, were used to determine the regulons controlled by the proteins ExpA and RsmA in P. wasabiae. These two proteins have been identified as important for the virulence of several γ-proteobacterial pathogens. This study analyzed the regulons via the use of three mutants: expA, rsmA, and an expA rsmA double mutant (DM). Overlapping and independently regulated targets were identified between ExpA and RsmA. Phenotypic assays for motility, growth, PCWDE activity, and virulence confirmed the transcriptomic data for the mutant strains. Novel findings included reduction of swimming motility in agar medium for P. wasabiae expA and rsmA mutants. In addition, the DM exhibited enhanced virulence and fitness in planta compared to either single mutant. Via analysis of transcriptomic data, a subset of genes was identified as affected in expression by an expA mutation independently of the presence of rsmA. The relatively unexplored role of short OGs (with a degree of polymerization (DP) less than 10) in damage-associated molecular pattern (DAMP) signaling in A. thaliana was characterized in this study. Comparative gene expression profiling based on RNA sequencing and RT-qPCR was performed on RNA harvested from plants treated with short OGs or with a mock suspension. Phenotypic assays confirmed the gene expression data. In a meta-data analysis, the resulting RNA sequencing and RT-qPCR data were compared with gene expression data from previous studies, in which long OGs (DP more than 10) were used to treat plants. This work demonstrated that short and long OGs induce genes and genesets associated with pathogen defense and phytohormone signaling, whereas reducing plant growth and development. The transcriptomic data of this study suggests that plant treatment with a mixture of short or long OGs yields a more pronounced and varied modulation of global gene expression, compared to treatment with only trimeric OGs. The regulation of the virulence of P. wasabiae, and the DAMP signaling triggered by plant cell wall damage in A. thaliana, are elements of the interactions between the plant and pathogen. The studies presented in this dissertation provide novel information about these two biological processes and highlights their connection.Interaktionerna mellan fytopatogena bakterier och deras värdväxter kan karaktäriseras som ett komplicerat nätverk av signaler och lämpliga svar. Fytopatogena blötrötebakterier förekommer globalt, och orsakar sjukdom i Solanum tuberosum (potatis) och andra knölväxter för basföda i både fält och lagringrum. En väldigt studerad blötrötebakterie är Pectobacterium wasabiae, som blivit identifierad i Eutrema wasabi (wasabi) växter i Japan och i potatis i Finland. Generellt så är interaktionerna mellan denna typ av bakterie och värdväxter karaktäriserade av nedbrytning utav växtvävnad, på grund av växtcellväggsnedbrytande enzymer, samt induktionen av fytohormonberoende försvar hos växterna. Nedbrytningen av växtvävnader involverar utsläpp av pektiska oligogalakturonider (OGs) från växtcellväggarna. OGs har identifierats som viktiga signalföreningar, som inducerar uttrycket av en varieté av försvarsrelaterade gener. Medan bakterieinfektionen avancerar koordinerar bakterierna produktionen av virulensfaktorer genom användandet av regleringsproteiner som modulerar transkriptomet. Transkriptomiska analyser har använts i stor utsträckning i tidigare studier för att identifiera regleringsnätverk och signaleringsvägar i växter, och dessa studier har gett insikt i anpassning och infektionsprocesser hos både växter och bakterier. De nya vetenskapliga resultaten i denna avhandling är härledda från en kombination av transkriptomiska, genomiska och genetiska analyser. Denna studie karaktäriserar olika aspekter av växt-patogen interaktioner. Den centrala bakteriemodellen som använts var P. wasabiae, och modellväxten av intresse var Arabidopsis thaliana. I fallet för P. wasabiae, så användes en kombination av funktionell genetik och transkriptomiska metoder, såsom omvänd transkription kvantitative polymeras kedjereaction (RT-qPCR) och mikroarrayer, för att identifiera regulonen kontrollerade av ExpA och RsmA. Dessa två proteiner har identifierats som viktiga för virulensen för flera γ-proteobakteriella patogener. Denna studie jämförde regulonen via användandet av tre mutanter: expA, rsmA och en expA rsmA dubbelmutant (DM). Transkriptomanalyser identifierade överlappande och självständigt reglerade mål mellan ExpA och RsmA. Fenotypiska prov för motilitet, tillväxt, enzymaktivitet och virulens bekräftade den transkriptomiska datan för mutantstammarna. Nya upptäckter inkluderade reduktionen av simmningsmotilitet i agarmedium och överlevnad in planta för P. wasabiae expA och rsmA mutanter. Dessutom, en grupp gener identifierades via analys av transkriptomikdata som påverkade i expression av en expA mutation oberoende av närvaro av RsmA. Den relativt outforskade rollen för korta OGs (med en polymeriseringsgrad (DP) under 10) inom skadeassocierade molekylära mönster (DAMP) signalering i A. thaliana karaktäriserades i denna studie. Komparativ genuttrycksprofilering baserat på RNA-sekvensering och RT-qPCR utfördes på RNA som skördats från växter som behandlats med korta OGs eller en falskbehandling. Fenotyptest bekräftade genexpressionsdatan. Vid en metadataanalys jämfördes den resulterande datan från RNA-sekvensering och RT-qPCR med genexpressionsdata från tidigare studier, där långa OGs (DP över 10) använts för att behandla växter. Detta arbete demonstrerade att korta OGs, liksom långa OGs, inducerar gener associerade med patogenförsvar och fytohormonsignalering. Vidare, korta och långa OGs påvisades nedreglera gener associerade med växttillväxt och utveckling. Transkriptomikdatan i denna studie föreslår även att växtbehandling med en blandning av korta OGs samt långa OGs har en stor och varierad inverkan, jämfört med behandling med endast trimera OGs. Regleringen av virulens hos P. wasabiae och DAMP signaleringen som utlöses av växtcellväggsskada i A. thaliana är element av interaktionerna mellan växt och fytopatogen. Studierna i denna avhandling tillhandhåller ny information om dessa två biologiska processer och markerar deras samband

Mendelian randomization highlights insomnia as a risk factor for pain diagnoses

Study Objective: Insomnia has been linked to acute and chronic pain conditions; however, it is unclear whether such relationships are causal. Recently, a large number of genetic variants have been discovered for both insomnia and pain through genome-wide association studies (GWASs) providing a unique opportunity to examine the evidence for causal relationships through the use of the Mendelian randomization paradigm. Methods: To elucidate the causality between insomnia and pain, we performed bidirectional Mendelian randomization analysis in FinnGen, where clinically diagnosed ICD-10 categories of pain had been evaluated. In addition, we used measures of self-reported insomnia symptoms. We used endpoints for pain in the FinnGen Release 5 (R5) (N = 218,379), and a non-overlapping sample for insomnia (UK Biobank (UKBB) and 23andMe, N = 1,331,010 or UKBB alone N = 453,379). We assessed the robustness of results through conventional Mendelian randomization sensitivity analyses. Results: Genetic liability to insomnia symptoms increased the odds of reporting pain (odds ratio (OR) [95% confidence interval (CI)] = 1.47 [1.38-1.58], p = 4.12 x 10(-28)). Manifested pain had a small effect on increased risk for insomnia (OR [95% CI] = 1.04 [1.01-1.07], p < 0.05). Results were consistent in sensitivity analyses. Conclusions: Our findings support a bidirectional causal relationship between insomnia and pain. These data support a further clinical investigation into the utility of insomnia treatment as a strategy for pain management and vice versa.Peer reviewe

LysM Proteins Regulate Fungal Development and Contribute to Hyphal Protection and Biocontrol Traits in Clonostachys rosea

Lysin motif (LysM) modules are approximately 50 amino acids long and bind to peptidoglycan, chitin and its derivatives. Certain LysM proteins in plant pathogenic and entomopathogenic fungi are shown to scavenge chitin oligosaccharides and thereby dampen host defense reactions. Other LysM proteins can protect the fungal cell wall against hydrolytic enzymes. In this study, we investigated the biological function of LysM proteins in the mycoparasitic fungus Clonostachys rosea. The C. rosea genome contained three genes coding for LysM-containing proteins and gene expression analysis revealed that lysm1 and lysm2 were induced during mycoparasitic interaction with Fusarium graminearum and during colonization of wheat roots. Lysm1 was suppressed in germinating conidia, while lysm2 was induced during growth in chitin or peptidoglycan-containing medium. Deletion of lysm1 and lysm2 resulted in mutants with increased levels of conidiation and conidial germination, but reduced ability to control plant diseases caused by F. graminearum and Botrytis cinerea. The Delta lysm2 strain showed a distinct, accelerated mycelial disintegration phenotype accompanied by reduced biomass production and hyphal protection against hydrolytic enzymes including chitinases, suggesting a role of LYSM2 in hyphal protection against chitinases. The Delta lysm2 and Delta lysm1 Delta lysm2 strains displayed reduced ability to colonize wheat roots, while only Delta lysm1 Delta lysm2 failed to suppress expression of the wheat defense response genes PR1 and PR4. Based on our data, we propose a role of LYSM1 as a regulator of fungal development and of LYSM2 in cell wall protection against endogenous hydrolytic enzymes, while both are required to suppress plant defense responses. Our findings expand the understanding of the role of LysM proteins in fungal-fungal interactions and biocontrol

Short oligogalacturonides induce pathogen resistance-associated gene expression in Arabidopsis thaliana

Background Oligogalacturonides (OGs) are important components of damage-associated molecular pattern (DAMP) signaling and influence growth regulation in plants. Recent studies have focused on the impact of long OGs (degree of polymerization (DP) from 10–15), demonstrating the induction of plant defense signaling resulting in enhanced defenses to necrotrophic pathogens. To clarify the role of trimers (trimeric OGs, DP3) in DAMP signaling and their impact on plant growth regulation, we performed a transcriptomic analysis through the RNA sequencing of Arabidopsis thaliana exposed to trimers. Results The transcriptomic data from trimer-treated Arabidopsis seedlings indicate a clear activation of genes involved in defense signaling, phytohormone signaling and a down-regulation of genes involved in processes related to growth regulation and development. This is further accompanied with improved defenses against necrotrophic pathogens triggered by the trimer treatment, indicating that short OGs have a clear impact on plant responses, similar to those described for long OGs. Conclusions Our results demonstrate that trimers are indeed active elicitors of plant defenses. This is clearly indicated by the up-regulation of genes associated with plant defense signaling, accompanied with improved defenses against necrotrophic pathogens. Moreover, trimers simultaneously trigger a clear down-regulation of genes and gene sets associated with growth and development, leading to stunted seedling growth in Arabidopsis. Keywords Plant signaling Arabidopsis thaliana Oligogalacturonides OG Trimers Transcriptomics Defense induction Growth inhibition Disease resistance Pectobacterium carotovorum Botrytis cinereaPeer reviewe

Short oligogalacturonides induce pathogen resistance-associated gene expression in Arabidopsis thaliana

Background Oligogalacturonides (OGs) are important components of damage-associated molecular pattern (DAMP) signaling and influence growth regulation in plants. Recent studies have focused on the impact of long OGs (degree of polymerization (DP) from 10–15), demonstrating the induction of plant defense signaling resulting in enhanced defenses to necrotrophic pathogens. To clarify the role of trimers (trimeric OGs, DP3) in DAMP signaling and their impact on plant growth regulation, we performed a transcriptomic analysis through the RNA sequencing of Arabidopsis thaliana exposed to trimers. Results The transcriptomic data from trimer-treated Arabidopsis seedlings indicate a clear activation of genes involved in defense signaling, phytohormone signaling and a down-regulation of genes involved in processes related to growth regulation and development. This is further accompanied with improved defenses against necrotrophic pathogens triggered by the trimer treatment, indicating that short OGs have a clear impact on plant responses, similar to those described for long OGs. Conclusions Our results demonstrate that trimers are indeed active elicitors of plant defenses. This is clearly indicated by the up-regulation of genes associated with plant defense signaling, accompanied with improved defenses against necrotrophic pathogens. Moreover, trimers simultaneously trigger a clear down-regulation of genes and gene sets associated with growth and development, leading to stunted seedling growth in Arabidopsis. Keywords Plant signaling Arabidopsis thaliana Oligogalacturonides OG Trimers Transcriptomics Defense induction Growth inhibition Disease resistance Pectobacterium carotovorum Botrytis cinereaPeer reviewe