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Research Highlights The brain undergoes extensive development during late childhood and early adolescence. Cortical thinning is a prominent feature of this development, and some researchers have suggested that differences in cortical thickness may be related to internalizing symptoms, which typically increase during the same period. However, research has yielded inconclusive results. We utilized a new method that estimates the combined effect of individual differences in vertex‐wise cortical thickness on internalizing symptoms. This approach allows for many small effects to be distributed across the cortex and avoids the necessity of correcting for multiple tests. Using a sample of 8763 children aged 8.9 to 11.1 from the ABCD study, we decomposed the total variation in caregiver‐reported internalizing symptoms into differences in cortical thickness, additive genetics, and shared family environmental factors and unique environmental factors. Our results indicated that individual differences in cortical thickness accounted for less than 0.5% of the variation in internalizing symptoms. In contrast, the analysis revealed a substantial effect of additive genetics and family environmental factors on the different components of internalizing symptoms, ranging from 06% to 48% and from 0% to 34%, respectively. Overall, while this study found a minimal association between cortical thickness and internalizing symptoms, additive genetics, and familial environmental factors appear to be of importance for describing differences in internalizing symptoms in late childhood. We utilized a new method for modelling the total contribution of vertex‐wise individual differences in cortical thickness to internalizing symptoms in late childhood. The total contribution of individual differences in cortical thickness accounted for <0.5% of the variance in internalizing symptoms. Additive genetics and shared family environmental variation accounted for 17% and 34% of the variance in internalizing symptoms, respectively. Our results suggest that cortical thickness is not an important indicator for internalizing symptoms in childhood, whereas genetic and environmental differences have a substantial impact. [ABSTRACT FROM AUTHOR]

Abstract: In a road simulator study, a significant source of sub-micrometer fine particles produced by the road–tire interface was observed. Since the particle size distribution and source strength is dependent on the type of tire used, it is likely that these particles largely originate from the tires, and not the road pavement. The particles consisted most likely of mineral oils from the softening filler and fragments of the carbon-reinforcing filler material (soot agglomerates). This identification was based on transmission electron microscopy studies of collected ultrafine wear particles and on-line thermal treatment using a thermodesorber. The mean particle number diameters were between 15–50nm, similar to those found in light duty vehicle (LDV) tail-pipe exhaust. A simple box model approach was used to estimate emission factors in the size interval 15–700nm. The emission factors increased with increasing vehicle speed, and varied between 3.7×1011 and 3.2×1012 particlesvehicle−1 km−1 at speeds of 50 and 70kmh−1. This corresponds to between 0.1–1% of tail-pipe emissions in real-world emission studies at similar speeds from a fleet of LDV with 95% gasoline and 5% diesel-fueled cars. The emission factors for particles originating from the road–tire interface were, however, similar in magnitude to particle number emission factors from liquefied petroleum gas-powered vehicles derived in test bench studies in Australia 2005. Thus the road–tire interface may be a significant contributor to particle emissions from ultraclean vehicles. [Copyright &y& Elsevier]

Background: Recent technological advances opened the opportunity to simultaneously study gene expression for thousands of individual cells on a genome-wide scale. The experimental accessibility of such single-cell RNA sequencing (scRNAseq) approaches allowed gaining insights into the cell type composition of heterogeneous tissue samples of animal model systems and emerging models alike. A major prerequisite for a successful application of the method is the dissociation of complex tissues into individual cells, which often requires large amounts of input material and harsh mechanical, chemical and temperature conditions. However, the availability of tissue material may be limited for small animals, specific organs, certain developmental stages or if samples need to be acquired from collected specimens. Therefore, we evaluated different dissociation protocols to obtain single cells from small tissue samples of Drosophila melanogaster eye-antennal imaginal discs. Results: We show that a combination of mechanical and chemical dissociation resulted in sufficient high-quality cells. As an alternative, we tested protocols for the isolation of single nuclei, which turned out to be highly efficient for fresh and frozen tissue samples. Eventually, we performed scRNAseq and single-nuclei RNA sequencing (snRNAseq) to show that the best protocols for both methods successfully identified relevant cell types. At the same time, snRNAseq resulted in less artificial gene expression that is caused by rather harsh dissociation conditions needed to obtain single cells for scRNAseq. A direct comparison of scRNAseq and snRNAseq data revealed that both datasets share biologically relevant genes among the most variable genes, and we showed differences in the relative contribution of the two approaches to identified cell types. Conclusion: We present two dissociation protocols that allow isolating single cells and single nuclei, respectively, from low input material. Both protocols resulted in extraction of high-quality RNA for subsequent scRNAseq or snRNAseq applications. If tissue availability is limited, we recommend the snRNAseq procedure of fresh or frozen tissue samples as it is perfectly suited to obtain thorough insights into cellular diversity of complex tissue. [ABSTRACT FROM AUTHOR]

Cognitive functions and psychopathology develop in parallel in childhood and adolescence, but the temporal dynamics of their associations are poorly understood. The present study sought to elucidate the intertwined development of decision-making processes and attention problems using longitudinal data from late childhood (9–10 years) to mid-adolescence (11–13 years) from the Adolescent Brain Cognitive Development (ABCD) Study (n = 8918). We utilised hierarchical drift-diffusion modelling of behavioural data from the stop-signal task, parent-reported attention problems from the Child Behavior Checklist (CBCL), and multigroup univariate and bivariate latent change score models. The results showed faster drift rate was associated with lower levels of inattention at baseline, as well as a greater reduction of inattention over time. Moreover, baseline drift rate negatively predicted change in attention problems in females, and baseline attention problems negatively predicted change in drift rate. Neither response caution (decision threshold) nor encoding- and responding processes (non-decision time) were significantly associated with attention problems. There were no significant sex differences in the associations between decision-making processes and attention problems. The study supports previous findings of reduced evidence accumulation in attention problems and additionally shows that development of this aspect of decision-making plays a role in developmental changes in attention problems in youth. [ABSTRACT FROM AUTHOR]

Background: A child's socioeconomic environment can shape central aspects of their life, including vulnerability to mental disorders. Negative environmental influences in youth may interfere with the extensive and dynamic brain development occurring at this time. Indeed, there are numerous yet diverging reports of associations between parental socioeconomic status (SES) and child cortical brain morphometry. Most of these studies have used single metric‐ or unimodal analyses of standard cortical morphometry that downplay the probable scenario where numerous biological pathways in sum account for SES‐related cortical differences in youth. Methods: To comprehensively capture such variability, using data from 9758 children aged 8.9–11.1 years from the ABCD Study®, we employed linked independent component analysis (LICA) and fused vertex‐wise cortical thickness, surface area, curvature and grey‐/white‐matter contrast (GWC). LICA revealed 70 uni‐ and multimodal components. We then assessed the linear relationships between parental education, parental income and each of the cortical components, controlling for age, sex, genetic ancestry, and family relatedness. We also assessed whether cortical structure moderated the negative relationships between parental SES and child general psychopathology. Results: Parental education and income were both associated with larger surface area and higher GWC globally, in addition to local increases in surface area and to a lesser extent bidirectional GWC and cortical thickness patterns. The negative relation between parental income and child psychopathology were attenuated in children with a multimodal pattern of larger frontal‐ and smaller occipital surface area, and lower medial occipital thickness and GWC. Conclusion: Structural brain MRI is sensitive to SES diversity in childhood, with GWC emerging as a particularly relevant marker together with surface area. In low‐income families, having a more developed cortex across MRI metrics, appears beneficial for mental health. [ABSTRACT FROM AUTHOR]

A subpopulation of deeply quiescent, so-called dormant hematopoietic stem cells (dHSCs) resides at the top of the hematopoietic hierarchy and serves as a reserve pool for HSCs. The state of dormancy protects the HSC pool from exhaustion throughout life; however, excessive dormancy may prevent an efficient response to hematological stresses. Despite the significance of dHSCs, the mechanisms maintaining their dormancy remain elusive. Here, we identify CD38 as a novel and broadly applicable surface marker for the enrichment of murine dHSCs. We demonstrate that cyclic adenosine diphosphate ribose (cADPR), the product of CD38 cyclase activity, regulates the expression of the transcription factor c-Fos by increasing the release of Ca2+ from the endoplasmic reticulum (ER). Subsequently, we uncover that c-Fos induces the expression of the cell cycle inhibitor p57Kip2 to drive HSC dormancy. Moreover, we found that CD38 ecto-enzymatic activity at the neighboring CD38-positive cells can promote human HSC quiescence. Together, CD38/cADPR/Ca2+/c-Fos/p57Kip2 axis maintains HSC dormancy. Pharmacological manipulations of this pathway can provide new strategies to improve the success of stem cell transplantation and blood regeneration after injury or disease. Dormant hematopoietic stem cells (dHSC) represent a stem cell reserve that is important for hematopoiesis throughout life. In this study, CD38 is identified as a surface marker and novel regulator of mouse HSCs that promotes their dormancy. Interestingly, while human HSCs lack CD38 expression, CD38 expression in neighboring cells similarly promotes human HSC quiescence. [ABSTRACT FROM AUTHOR]

Background: Focal adhesion signaling involving receptor tyrosine kinases (RTK) and integrins co-controls cancer cell survival and therapy resistance. However, co-dependencies between these receptors and therapeutically exploitable vulnerabilities remain largely elusive in HPV-negative head and neck squamous cell carcinoma (HNSCC). Methods: The cytotoxic and radiochemosensitizing potential of targeting 10 RTK and β1 integrin was determined in up to 20 3D matrix-grown HNSCC cell models followed by drug screening and patient-derived organoid validation. RNA sequencing and protein-based biochemical assays were performed for molecular characterization. Bioinformatically identified transcriptomic signatures were applied to patient cohorts. Results: Fibroblast growth factor receptor (FGFR 1–4) targeting exhibited the strongest cytotoxic and radiosensitizing effects as monotherapy and combined with β1 integrin inhibition, exceeding the efficacy of the other RTK studied. Pharmacological pan-FGFR inhibition elicited responses ranging from cytotoxicity/radiochemosensitization to resistance/radiation protection. RNA sequence analysis revealed a mesenchymal-to-epithelial transition (MET) in sensitive cell models, whereas resistant cell models exhibited a partial epithelial-to-mesenchymal transition (EMT). Accordingly, inhibition of EMT-associated kinases such as EGFR caused reduced adaptive resistance and enhanced (radio)sensitization to FGFR inhibition cell model- and organoid-dependently. Transferring the EMT-associated transcriptomic profiles to HNSCC patient cohorts not only demonstrated their prognostic value but also provided a conclusive validation of the presence of EGFR-related vulnerabilities that can be strategically exploited for therapeutic interventions. Conclusions: This study demonstrates that pan-FGFR inhibition elicits a beneficial radiochemosensitizing and a detrimental radioprotective potential in HNSCC cell models. Adaptive EMT-associated resistance appears to be of clinical importance, and we provide effective molecular approaches to exploit this therapeutically. [ABSTRACT FROM AUTHOR]

Selective isolation of DNA is crucial for applications in biology, bionanotechnology, clinical diagnostics and forensics. We herein report a smart methanol-responsive polymer (MeRPy) that can be programmed to bind and separate single- as well as double-stranded DNA targets. Captured targets are quickly isolated and released back into solution by denaturation (sequence-agnostic) or toehold-mediated strand displacement (sequence-selective). The latter mode allows 99.8% efficient removal of unwanted sequences and 79% recovery of highly pure target sequences. We applied MeRPy for the depletion of insulin, glucagon, and transthyretin cDNA from clinical next-generation sequencing (NGS) libraries. This step improved the data quality for low-abundance transcripts in expression profiles of pancreatic tissues. Its low cost, scalability, high stability and ease of use make MeRPy suitable for diverse applications in research and clinical laboratories, including enhancement of NGS libraries, extraction of DNA from biological samples, preparative-scale DNA isolations, and sorting of DNA-labeled non-nucleic acid targets. Krieg et al. describe a methanol responsive polymer that can capture complementary DNA using grafted oligonucleotides. They successfully demonstrate its efficacy with simultaneous and sequence-specific isolation of three target genes (cDNA) from clinical NGS libraries with high efficiency. This method is fast, effective, scalable, modular, and versatile. [ABSTRACT FROM AUTHOR]

In the lesioned zebrafish retina, Müller glia produce multipotent retinal progenitors that generate all retinal neurons, replacing lost cell types. To study the molecular mechanisms linking Müller glia reactivity to progenitor production and neuronal differentiation, we used single-cell RNA sequencing of Müller glia, progenitors and regenerated progeny from uninjured and light-lesioned retinae. We discover an injury-induced Müller glia differentiation trajectory that leads into a cell population with a hybrid identity expressing marker genes of Müller glia and progenitors. A glial self-renewal and a neurogenic trajectory depart from the hybrid cell population. We further observe that neurogenic progenitors progressively differentiate to generate retinal ganglion cells first and bipolar cells last, similar to the events observed during retinal development. Our work provides a comprehensive description of Müller glia and progenitor transcriptional changes and fate decisions in the regenerating retina, which are key to tailor cell differentiation and replacement therapies for retinal dystrophies in humans. [ABSTRACT FROM AUTHOR]

MsLDR-creator is a free web service to design assays for the new DNA methylation detection method msLDR. The service provides the user with all necessary information about the oligonucleotides required for the measurement of a given CpG within a sequence of interest. The parameters are calculated by the nearest neighbour approach to achieve optimal behaviour during the experimental procedure. In addition, to guarantee a good start using msLDR, further information, like protocols and hints and tricks, are provided. [ABSTRACT FROM AUTHOR]

The hypothalamus-pituitary- adrenal (HPA) axis is activated in response to inflammation leading to increased production of anti-inflammatory glucocorticoids by the adrenal cortex, thereby representing an endogenous feedback loop. However, severe inflammation reduces the responsiveness of the adrenal gland to adrenocorticotropic hormone (ACTH), although the underlying mechanisms are poorly understood. Here, we show by transcriptomic, proteomic, and metabolomic analyses that LPS-induced systemic inflammation triggers profound metabolic changes in steroidogenic adrenocortical cells, including downregulation of the TCA cycle and oxidative phosphorylation, in mice. Inflammation disrupts the TCA cycle at the level of succinate dehydrogenase (SDH), leading to succinate accumulation and disturbed steroidogenesis. Mechanistically, IL-1β reduces SDHB expression through upregulation of DNA methyltransferase 1 (DNMT1) and methylation of the SDHB promoter. Consequently, increased succinate levels impair oxidative phosphorylation and ATP synthesis and enhance ROS production, leading to reduced steroidogenesis. Together, we demonstrate that the IL-1β- DNMT1-SDHB- succinate axis disrupts steroidogenesis. Our findings not only provide a mechanistic explanation for adrenal dysfunction in severe inflammation, but also offer a potential target for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Left–right asymmetry is an important organizing feature of the healthy brain that may be altered in schizophrenia, but most studies have used relatively small samples and heterogeneous approaches, resulting in equivocal findings. We carried out the largest case–control study of structural brain asymmetries in schizophrenia, with MRI data from 5,080 affected individuals and 6,015 controls across 46 datasets, using a single image analysis protocol. Asymmetry indexes were calculated for global and regional cortical thickness, surface area, and subcortical volume measures. Differences of asymmetry were calculated between affected individuals and controls per dataset, and effect sizes were meta-analyzed across datasets. Small average case–control differences were observed for thickness asymmetries of the rostral anterior cingulate and the middle temporal gyrus, both driven by thinner left-hemispheric cortices in schizophrenia. Analyses of these asymmetries with respect to the use of antipsychotic medication and other clinical variables did not show any significant associations. Assessment of age-and sex-specific effects revealed a stronger average leftward asymmetry of pallidum volume between older cases and controls. Case–control differences in a multivariate context were assessed in a subset of the data (N = 2,029), which revealed that 7% of the variance across all structural asymmetries was explained by case–control status. Subtle case–control differences of brain macrostructural asymmetry may reflect differences at the molecular, cytoarchitectonic, or circuit levels that have functional relevance for the disorder. Reduced left middle temporal cortical thickness is consistent with altered left-hemisphere language network organization in schizophrenia. [ABSTRACT FROM AUTHOR]

Though electrical stimulation is used as a therapeutic approach to treat retinal and spinal injuries, many protective mechanisms at cellular level have not been elucidated. We performed a detailed analysis of cellular events in blue light (Li) stressed 661W cells, which were subjected to direct current electric field (EF) stimulation. Our findings revealed that EF stimulation induced protective effects in 661W cells from Li-induced stress by multiple defense mechanisms, such as increase in mitochondrial activity, gain in mitochondrial potential, increase in superoxide levels, and the activation of unfolded protein response (UPR) pathways, all leading to an enhanced cell viability and decreased DNA damage. Here, our genetic screen results revealed the UPR pathway to be a promising target to ameliorate Li-induced stress by EF stimulation. Thus, our study is important for a knowledgeable transfer of EF stimulation into clinical application. [ABSTRACT FROM AUTHOR]

[Display omitted] • Previously published mitogenomes included heterospecific sequence information. • Previous phylogenies suffered from further sources of error such as flawed taxonomy. • Genera Montifringilla and Pyrgilauda are reciprocally monophyletic. • Afghan Pyrgilauda theresae originated from a Pliocene out-of-Tibet dispersal. • European and Asian lineages of M. nivalis separated roughly 2 million years ago. Historical specimens from museum collections provide a valuable source of material also from remote areas or regions of conflict that are not easily accessible to scientists today. With this study, we are providing a taxon-complete phylogeny of snowfinches using historical DNA from whole skins of an endemic species from Afghanistan, the Afghan snowfinch, Pyrgilauda theresae. To resolve the strong conflict between previous phylogenetic hypotheses, we generated novel mitogenome sequences for selected taxa and genome-wide SNP data using ddRAD sequencing for all extant snowfinch species endemic to the Qinghai-Tibet Plateau (QTP) and for an extended intraspecific sampling of the sole Central and Western Palearctic snowfinch species (Montifringilla nivalis). Our phylogenetic reconstructions unanimously refuted the previously suggested paraphyly of genus Pyrgilauda. Misplacement of one species-level taxon (Onychostruthus tazcanowskii) in previous snowfinch phylogenies was undoubtedly inferred from chimeric mitogenomes that included heterospecific sequence information. Furthermore, comparison of novel and previously generated sequence data showed that the presumed sister-group relationship between M. nivalis and the QTP endemic M. henrici was suggested based on flawed taxonomy. Our phylogenetic reconstructions based on genome-wide SNP data and on mitogenomes were largely congruent and supported reciprocal monophyly of genera Montifringilla and Pyrgilauda with monotypic Onychostruthus being sister to the latter. The Afghan endemic P. theresae likely originated from a rather ancient Pliocene out-of-Tibet dispersal probably from a common ancestor with P. ruficollis. Our extended trans -Palearctic sampling for the white-winged snowfinch, M. nivalis , confirmed strong lineage divergence between an Asian and a European clade dated to 1.5 – 2.7 million years ago (mya). Genome-wide SNP data suggested subtle divergence among European samples from the Alps and from the Cantabrian mountains. [ABSTRACT FROM AUTHOR]

Deceptive flowers, unlike in mutualistic pollination systems, mislead their pollinators by advertising rewards which ultimately are not provided. Although our understanding of deceptive pollination systems increased in recent years, the attractive signals and deceptive strategies in the majority of species remain unknown. This is also true for the genus Aristolochia, famous for its deceptive and fly-pollinated trap flowers. Representatives of this genus were generally assumed to be oviposition-site mimics, imitating vertebrate carrion or mushrooms. However, recent studies found a broader spectrum of strategies, including kleptomyiophily and imitation of invertebrate carrion. A different deceptive strategy is presented here for the western Mediterranean Aristolochia baetica L. We found that this species is mostly pollinated by drosophilid flies (Drosophilidae, mostly Drosophila spp.), which typically feed on fermenting fruit infested by yeasts. The flowers of A. baetica emitted mostly typical yeast volatiles, predominantly the aliphatic compounds acetoin and 2,3-butandiol, and derived acetates, as well as the aromatic compound 2-phenylethanol. Analyses of the absolute configurations of the chiral volatiles revealed weakly (acetoin, 2,3-butanediol) to strongly (mono- and diacetates) biased stereoisomer-ratios. Electrophysiological (GC-EAD) experiments and lab bioassays demonstrated that most of the floral volatiles, although not all stereoisomers of chiral compounds, were physiologically active and attractive in drosophilid pollinators; a synthetic mixture thereof successfully attracted them in field and lab bioassays. We conclude that A. baetica chemically mimics yeast fermentation to deceive its pollinators. This deceptive strategy (scent chemistry, pollinators, trapping function) is also known from more distantly related plants, such as Arum palaestinum Boiss. (Araceae) and Ceropegia spp. (Apocynaceae), suggesting convergent evolution. In contrast to other studies working on floral scents in plants imitating breeding sites, the present study considered the absolute configuration of chiral compounds. [Display omitted] • Main pollinators of deceptive Aristolochia baetica are drosophilid flies. • The flowers release a scent resembling yeast-fermenting fruits. • Main compounds are acetoin, 2,3-butanediol monoacetate and related chemicals. • Some but not all of the chiral compounds are dominated by a single stereoisomer. • Most compounds but not all stereoisomers were biologically active in pollinators. [ABSTRACT FROM AUTHOR]

Background: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a dismal prognosis. Although combined treatment with gemcitabine and albumin-bound paclitaxel has improved the prognosis of PDAC, both intrinsic and acquired chemoresistance remain as severe hurtles towards improved prognosis. Thus, new therapeutic targets and innovative strategies are urgently needed. Methods: In this study, we used the KPC mouse model-derived PDAC cell line TB32047 to perform kinome-wide CRISPR-Cas9 loss-of-function screening. Next-generation sequencing and MAGeCK-VISPR analysis were performed to identify candidate genes. We then conducted cell viability, clonogenic, and apoptosis assays and evaluated the synergistic therapeutic effects of cyclin-dependent kinase 7 (CDK7) depletion or inhibition with gemcitabine (GEM) and paclitaxel (PTX) in a murine orthotopic pancreatic cancer model. For mechanistic studies, we performed genome enrichment analysis (GSEA) and Western blotting to identify and verify the pathways that render PDAC sensitive to GEM/PTX therapy. Results: We identified several cell cycle checkpoint kinases and DNA damage-related kinases as targets for overcoming chemoresistance. Among them, CDK7 ranked highly in both screenings. We demonstrated that both gene knockout and pharmacological inhibition of CDK7 by THZ1 result in cell cycle arrest, apoptosis induction, and DNA damage at least predominantly through the STAT3-MCL1-CHK1 axis. Furthermore, THZ1 synergized with GEM and PTX in vitro and in vivo, resulting in enhanced antitumor effects. Conclusions: Our findings support the application of CRISPR-Cas9 screening in identifying novel therapeutic targets and suggest new strategies for overcoming chemoresistance in pancreatic cancer. [ABSTRACT FROM AUTHOR]

Bipolar disorder (BD) is associated with cortical and subcortical structural brain abnormalities. It is unclear whether such alterations progressively change over time, and how this is related to the number of mood episodes. To address this question, we analyzed a large and diverse international sample with longitudinal magnetic resonance imaging (MRI) and clinical data to examine structural brain changes over time in BD. Longitudinal structural MRI and clinical data from the ENIGMA (Enhancing Neuro Imaging Genetics through Meta Analysis) BD Working Group, including 307 patients with BD and 925 healthy control subjects, were collected from 14 sites worldwide. Male and female participants, aged 40 ± 17 years, underwent MRI at 2 time points. Cortical thickness, surface area, and subcortical volumes were estimated using FreeSurfer. Annualized change rates for each imaging phenotype were compared between patients with BD and healthy control subjects. Within patients, we related brain change rates to the number of mood episodes between time points and tested for effects of demographic and clinical variables. Compared with healthy control subjects, patients with BD showed faster enlargement of ventricular volumes and slower thinning of the fusiform and parahippocampal cortex (0.18 < d < 0.22). More (hypo)manic episodes were associated with faster cortical thinning, primarily in the prefrontal cortex. In the hitherto largest longitudinal MRI study on BD, we did not detect accelerated cortical thinning but noted faster ventricular enlargements in BD. However, abnormal frontocortical thinning was observed in association with frequent manic episodes. Our study yields insights into disease progression in BD and highlights the importance of mania prevention in BD treatment. [ABSTRACT FROM AUTHOR]

Epigenetic mechanisms are gatekeepers for the gene expression patterns that establish and maintain cellular identity in mammalian development, stem cells and adult homeostasis. Amongst many epigenetic marks, methylation of histone 3 lysine 4 (H3K4) is one of the most widely conserved and occupies a central position in gene expression. Mixed lineage leukemia 1 (MLL1/KMT2A) is the founding mammalian H3K4 methyltransferase. It was discovered as the causative mutation in early onset leukemia and subsequently found to be required for the establishment of definitive hematopoiesis and the maintenance of adult hematopoietic stem cells. Despite wide expression, the roles of MLL1 in non-hematopoietic tissues remain largely unexplored. To bypass hematopoietic lethality, we used bone marrow transplantation and conditional mutagenesis to discover that the most overt phenotype in adult Mll1-mutant mice is intestinal failure. MLL1 is expressed in intestinal stem cells (ISCs) and transit amplifying (TA) cells but not in the villus. Loss of MLL1 is accompanied by loss of ISCs and a differentiation bias towards the secretory lineage with increased numbers and enlargement of goblet cells. Expression profiling of sorted ISCs revealed that MLL1 is required to promote expression of several definitive intestinal transcription factors including Pitx1, Pitx2, Foxa1, Gata4, Zfp503 and Onecut2, as well as the H3K27me3 binder, Bahcc1. These results were recapitulated using conditional mutagenesis in intestinal organoids. The stem cell niche in the crypt includes ISCs in close association with Paneth cells. Loss of MLL1 from ISCs promoted transcriptional changes in Paneth cells involving metabolic and stress responses. Here we add ISCs to the MLL1 repertoire and observe that all known functions of MLL1 relate to the properties of somatic stem cells, thereby highlighting the suggestion that MLL1 is a master somatic stem cell regulator. Author summary: The ability of stem cells to replenish cellular lineages is critical for the maintenance and integrity of postnatal life. In mammals, the blood and the intestinal lining are two of the highest turnover tissues and both are anchored by stem cells. For blood, hematopoeitic stem cells require MLL1 (mixed lineage leukemia), which is an epigenetic regulator that methylates histone 3 on lysine 4 (H3K4). MLL1 was discovered because it is mutated in about 10% of all human leukemias prominently including early onset childhood leukemias. In this study we show that MLL1 is also required for maintenance of intestinal stem cells (ISCs). MLL1 is expressed in the intestinal crypt, which harbors the ISC niche and transit amplifying cells but is not expressed in the intestinal villi that line the gut. Conditional mutagenesis of Mll1 in adult mice rapidly leads to intestinal failure, wasting and death. ISCs are lost and the niche is perturbed. Loss of ISC identity can be recapitulated by conditional mutagenesis of Mll1 in intestinal organoids. In ISCs, MLL1 is required to maintain the expression of several DNA binding transcription factors that are implicated in regulating ISC cellular identity. Hence we report that two mammalian tissues with very high turnover depend upon MLL1 to maintain their respective stem cells. Together with other indications, MLL1 appears to be specifically related to adult stem cell properties. [ABSTRACT FROM AUTHOR]

Most of the commercial apple cultivars are highly susceptible to fire blight, which is the most devastating bacterial disease affecting pome fruits. Resistance to fire blight is described especially in wild Malus accessions such as M. × robusta 5 (Mr5), but the molecular basis of host resistance response to the pathogen Erwinia amylovora is still largely unknown. The bacterial effector protein AvrRpt2EA was found to be the key determinant of resistance response in Mr5. A wild type E. amylovora strain and the corresponding avrRpt2EA deletion mutant were used for inoculation of Mr5 to induce resistance or susceptible response, respectively. By comparison of the transcriptome of both responses, 211 differentially expressed genes (DEGs) were identified. We found that heat-shock response including heat-shock proteins (HSPs) and heat-shock transcription factors (HSFs) are activated in apple specifically in the susceptible response, independent of AvrRpt2EA. Further analysis on the expression progress of 81 DEGs by high-throughput real-time qPCR resulted in the identification of genes that were activated after inoculation with E. amylovora. Hence, a potential role of these genes in the resistance to the pathogen is postulated, including genes coding for enzymes involved in formation of flavonoids and terpenoids, ribosome-inactivating enzymes (RIPs) and a squamosa promoter binding-like (SPL) transcription factor. [ABSTRACT FROM AUTHOR]

The article presents a study related to Odontoblast TRPC5 channels signal cold pain in teeth. It provides concrete functional evidence that equipping odontoblasts with the cold-sensor TRPC5 expands traditional odontoblast functions and renders unknown integral cellular component of the dental cold sensing system.

Glioblastoma (GBM) is the most common and malignant brain tumor in adults. Genomic and epigenomic alterations of multiple cancer-driving genes are frequent in GBM. To identify molecular alterations associated with epigenetic aberrations, we performed whole exome sequencing-based analysis of DNA copy number variations in 55 adult patients with IDH -wild-type GBM. Beside mutations in common GBM driver genes such as TERT p (76%), TP53 (22%) and PTEN (20%), 67% of patients were affected by amplifications of genes associated with RTK/Rb/p53 cell signaling, including EGFR (45%), CDK4 (13%), and MDM2/4 (both 7%). The minimal deleted region at chromosome 10 was detected at the DNA demethylase TET1 (93%), mainly due to a loss-of-heterozygosity of complete chromosome 10 (53%) or by a mono-allelic microdeletion at 10q21.3 (7%). In addition, bi-allelic TET1 deletions, detected in 18 patients (33%), frequently co-occurred with EGFR amplification and were associated with low levels of TET1 mRNA expression, pointing at loss of TET1 activity. Bi-allelic TET1 loss was not associated with global concentrations of 5-hydroxymethylcytosine, indicating a site-specific effect of TET1 for DNA (de)methylation. Focal amplification of EGFR positively correlated with overall mutational burden, tumor size, and poor long-term survival. Bi-allelic TET1 loss was not an independent prognostic factor, but significantly associated with poor survival in patients with concomitant EGFR amplification. Rates of genomic TET1 deletion were significantly lower in a cohort of IDH1 -mutated patients. Despite the relevance of TET1 for DNA demethylation and as potential therapeutic target, a frequent genomic loss of TET1 has not previously been reported in GBM. [ABSTRACT FROM AUTHOR]

Background: In recent years, therapies with CD4+CD25highFoxP3+ regulatory T cells (Tregs) have been successfully tested in many clinical trials. The important issue regarding the use of this treatment in autoimmune conditions remains the specificity toward particular antigen, as because of epitope spread, there are usually multiple causative autoantigens to be regulated in such conditions. Methods: Here we show a method of generation of Tregs enriched with antigen-reactive clones that potentially covers the majority of such autoantigens. In our research, Tregs were expanded with anti-CD28 and anti-CD154 antibodies and autologous monocytes and loaded with a model peptide, such as whole insulin or insulin β chain peptide 9–23. The cells were then sorted into cells recognizing the presented antigen. The reactivity was verified with functional assays in which Tregs suppressed proliferation or interferon gamma production of autologous effector T cells (polyclonal and antigen-specific) used as responders challenged with the model peptide. Finally, we analyzed clonotype distribution and TRAV gene usage in the specific Tregs. Results: Altogether, the applied technique had a good yield and allowed us to obtain a Treg product enriched with a specific subset, as confirmed in the functional tests. The product consisted of many clones; nevertheless, the content of these clones was different from that found in polyclonal or unspecific Tregs. Conclusions: The presented technique might be used to generate populations of Tregs enriched with cells reactive to any given peptide, which can be used as a cellular therapy medicinal product in antigen-targeted therapies. [ABSTRACT FROM AUTHOR]

Methylation of histone 3 lysine 4 (H3K4) is a major epigenetic system associated with gene expression. In mammals there are six H3K4 methyltransferases related to yeast Set1 and fly Trithorax, including two orthologs of fly Trithorax-related: MLL3 and MLL4. Exome sequencing has documented high frequencies of MLL3 and MLL4 mutations in many types of human cancer. Despite this emerging importance, the requirements of these paralogs in mammalian development have only been incompletely reported. Here, we examined the null phenotypes to establish that MLL3 is first required for lung maturation, whereas MLL4 is first required for migration of the anterior visceral endoderm that initiates gastrulation in the mouse. This collective cell migration is preceded by a columnar-to-squamous transition in visceral endoderm cells that depends on MLL4. Furthermore, Mll4 mutants display incompletely penetrant, sexdistorted, embryonic haploinsufficiency and adult heterozygous mutants show aspects of Kabuki syndrome, indicating that MLL4 action, unlike MLL3, is dosage dependent. The highly specific and discordant functions of these paralogs in mouse development argues against their action as general enhancer factors. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are short (~22 nt) single-stranded non-coding RNAs that regulate gene expression at the post-transcriptional level. Over recent years, many studies have extensively characterized the involvement of miRNA-mediated regulation in neurogenesis and brain development. However, a comprehensive catalog of cortical miRNAs expressed in a cell-specific manner in progenitor types of the developing mammalian cortex is still missing. Overcoming this limitation, here we exploited a double reporter mouse line previously validated by our group to allow the identification of the transcriptional signature of neurogenic commitment and provide the field with the complete atlas of miRNA expression in proliferating neural stem cells, neurogenic progenitors and newborn neurons during corticogenesis. By extending the currently known list of miRNAs expressed in the mouse brain by over twofold, our study highlights the power of cell type-specific analyses for the detection of transcripts that would otherwise be diluted out when studying bulk tissues. We further exploited our data by predicting putative miRNAs and validated the power of our approach by providing evidence for the involvement of miR-486 in brain development. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are short (~22 nt) single-stranded non-coding RNAs that regulate gene expression at the post-transcriptional level. Over the past years, many studies have extensively characterized the involvement of miRNA-mediated regulation in neurogenesis and brain development. However, a comprehensive catalog of cortical miRNAs cell-specifically expressed in progenitor types of the developing mammalian cortex is still missing. Overcoming this limitation, here we exploited a double reporter mouse line previously validated by our group to allow the identification of the transcriptional signature to neurogenic commitment and provide the field with the complete atlas of miRNAs expression in proliferating neural stem cells, neurogenic progenitors and newborn neurons during corticogenesis. By extending the currently known list of miRNAs expressed in the mouse brain by over two fold, our study highlights the power of cell type-specific analyses for the detection of transcripts that would otherwise be diluted out when studying bulk tissues. We further exploited our data by predicting putative novel miRNAs and validated the power of our approach by providing novel evidence for the involvement of miR-486 as a novel player in brain development. [ABSTRACT FROM AUTHOR]

Zebrafish display widespread and pronounced adult neurogenesis, which is fundamental for their regeneration capability after central nervous system injury. However, the cellular identity and the biological properties of adult newborn neurons are elusive for most brain areas. Here, we have used short-term lineage tracing of radial glia progeny to prospectively isolate newborn neurons from the her4.1+ radial glia lineage in the homeostatic adult forebrain. Transcriptome analysis of radial glia, newborn neurons and mature neurons using single cell sequencing identified distinct transcriptional profiles, including novel markers for each population. Specifically, we detected two separate newborn neuron types, which showed diversity of cell fate commitment and location. Further analyses showed that these cell types are homologous to neurogenic cells in the mammalian brain, identified neurogenic commitment in proliferating radial glia and indicated that glutamatergic projection neurons are generated in the adult zebrafish telencephalon. Thus, we prospectively isolated adult newborn neurons from the adult zebrafish forebrain, identified markers for newborn and mature neurons in the adult brain, and revealed intrinsic heterogeneity among adult newborn neurons and their homology with mammalian adult neurogenic cell types. [ABSTRACT FROM AUTHOR]

The presence of progenitor or stem cells in the adult pancreas and their potential involvement in homeostasis and cancer development remain unresolved issues. Here, we show that mouse centroacinar cells can be identified and isolated by virtue of the mitochondrial enzyme Aldh1b1 that they uniquely express. These cells are necessary and sufficient for the formation of self-renewing adult pancreatic organoids in an Aldh1b1-dependent manner. Aldh1b1-expressing centroacinar cells are largely quiescent, self-renew, and, as shown by genetic lineage tracing, contribute to all 3 pancreatic lineages in the adult organ under homeostatic conditions. Single-cell RNA sequencing analysis of these cells identified a progenitor cell population, established its molecular signature, and determined distinct differentiation pathways to early progenitors. A distinct feature of these progenitor cells is the preferential expression of small GTPases, including Kras, suggesting that they might be susceptible to Kras-driven oncogenic transformation. This finding and the overexpression of Aldh1b1 in human and mouse pancreatic cancers, driven by activated Kras, prompted us to examine the involvement of Aldh1b1 in oncogenesis. We demonstrated genetically that ablation of Aldh1b1 completely abrogates tumor development in a mouse model of KrasG12D-induced pancreatic cancer. [ABSTRACT FROM AUTHOR]

Axolotl limb regeneration is accompanied by the transient induction of cellular senescence within the blastema, the structure that nucleates regeneration. The precise role of this blastemal senescent cell (bSC) population, however, remains unknown. Here, through a combination of gain- and loss-of-function assays, we elucidate the functions and molecular features of cellular senescence in vivo. We demonstrate that cellular senescence plays a positive role during axolotl regeneration by creating a pro-proliferative niche that supports progenitor cell expansion and blastema outgrowth. Senescent cells impact their microenvironment via Wnt pathway modulation. Further, we identify a link between Wnt signaling and senescence induction and propose that bSC-derived Wnt signals facilitate the proliferation of neighboring cells in part by preventing their induction into senescence. This work defines the roles of cellular senescence in the regeneration of complex structures. [Display omitted] • Senescent cells comprise a pro-regenerative niche during axolotl limb regrowth • Senescent cells promote progenitor proliferation via the modulation of WNT signaling • WNT signaling prevents blastema progenitors from undergoing senescence • The regenerative senescence program is distinct from DNA-damage-induced senescence Yu et al. address the features, heterogeneity, and functions of senescent cells that arise during axolotl limb regeneration, showing that senescent cells form a niche that sustains limb progenitor proliferation in a short-range manner via WNT signaling. This work defines the roles of cellular senescence in the regeneration of complex structures. [ABSTRACT FROM AUTHOR]

Synthesis of renin in renal renin-producing cells (RPCs) is controlled via the intracellular messenger cAMP. Interference with cAMP-mediated signaling by inducible knockout of Gs-alpha (Gsα) in RPCs of adult mice resulted in a complex adverse kidney phenotype. Therein, glomerular endothelial damage was most striking. In this study, we investigated whether Gsα knockout leads to a loss of RPCs, which itself may contribute to the endothelial injury. We compared the kidney phenotype of three RPC-specific conditional mouse lines during continuous induction of recombination. Mice expressing red fluorescent reporter protein tdTomato (tdT) in RPCs served as controls. tdT was also expressed in RPCs of the other two strains used, namely with RPC-specific Gsα knockout (Gsα mice) or with RPC-specific diphtheria toxin A expression (DTA mice, in which the RPCs should be diminished). Using immunohistological analysis, we found that RPCs decreased by 82% in the kidneys of Gsα mice as compared with controls. However, the number of tdT-positive cells was similar in the two strains, demonstrating that after Gsα knockout, the RPCs persist as renin-negative descendants. In contrast, both renin-positive and tdT-labeled cells decreased by 80% in DTA mice suggesting effective RPC ablation. Only Gsα mice displayed dysregulated endothelial cell marker expression indicating glomerular endothelial damage. In addition, a robust induction of genes involved in tissue remodelling with microvascular damage was identified in tdT-labeled RPCs isolated from Gsα mice. We concluded that Gsα/renin double-negative RPC progeny essentially contributes for the development of glomerular endothelial damage in our Gsα-deficient mice. [ABSTRACT FROM AUTHOR]

Cancer cells can switch between signaling pathways to regulate growth under different conditions. In the tumor microenvironment, this likely helps them evade therapies that target specific pathways. We must identify all possible states and utilize them in drug screening programs. One such state is characterized by expression of the transcription factor Hairy and Enhancer of Split 3 (HES3) and sensitivity to HES3 knockdown, and it can be modeled in vitro. Here, we cultured 3 primary human brain cancer cell lines under 3 different culture conditions that maintain low, medium, and high HES3 expression and characterized gene regulation and mechanical phenotype in these states. We assessed gene expression regulation following HES3 knockdown in the HES3-high conditions. We then employed a commonly used human brain tumor cell line to screen Food and Drug Administration (FDA)-approved compounds that specifically target the HES3-high state. We report that cells from multiple patients behave similarly when placed under distinct culture conditions. We identified 37 FDA-approved compounds that specifically kill cancer cells in the high-HES3-expression conditions. Our work reveals a novel signaling state in cancer, biomarkers, a strategy to identify treatments against it, and a set of. [ABSTRACT FROM AUTHOR]

Deadwood is an important structural component in forest ecosystems and plays a significant role in global carbon and nutrient cycling. Relatively little is known about the formation and decomposition of CWD by microbial communities in situ and about the factors controlling the associated processes. In this study, we intensively analyzed the molecular fungal community composition and species richness in relation to extracellular enzyme activity and differences in decomposing sapwood and heartwood of 13 temperate tree species (four coniferous and nine deciduous species, log diameter 30–40 cm and 4 m long) in an artificial experiment involving placing the logs on the forest soil for six years. We observed strong differences in the molecular fungal community composition and richness among the 13 tree species, and specifically between deciduous and coniferous wood, but unexpectedly no difference was found between sapwood and heartwood. Fungal species richness correlated positively with wood extractives and negatively with fungal biomass. A distinct fungal community secreting lignocellulolytic key enzymes seemed to dominate the decomposition of the logs in this specific phase. In particular, the relative sequence abundance of basidiomycetous species of the Meruliaceae (e.g. Bjerkandera adusta) correlated with ligninolytic manganese peroxidase activity. Moreover, this study reveals abundant white-rot causing Basidiomycota and soft-rot causing Ascomycota during this phase of wood decomposition. [ABSTRACT FROM AUTHOR]

Summary: Deadwood represents an important structural component of forest ecosystems, where it provides diverse niches for saproxylic biota. Although wood‐inhabiting prokaryotes are involved in its degradation, knowledge about their diversity and the drivers of community structure is scarce. To explore the effect of deadwood substrate on microbial distribution, the present study focuses on the microbial communities of deadwood logs from 13 different tree species investigated using an amplicon based deep‐sequencing analysis. Sapwood and heartwood communities were analysed separately and linked to various relevant wood physico‐chemical parameters. Overall, Proteobacteria, Acidobacteria and Actinobacteria represented the most dominant phyla. Microbial OTU richness and community structure differed significantly between tree species and between sapwood and heartwood. These differences were more pronounced for heartwood than for sapwood. The pH value and water content were the most important drivers in both wood compartments. Overall, investigating numerous tree species and two compartments provided a remarkably comprehensive view of microbial diversity in deadwood. [ABSTRACT FROM AUTHOR]

The regenerative capacity of hematopoietic stem cells (HSCs) is limited by the accumulation of DNA damage. Conditional mutagenesis of the histone 3 lysine 4 (H3K4) methyltransferase, Setd1a, revealed that it is required for the expression of DNA damage recognition and repair pathways in HSCs. Specific deletion of Setd1a in adult long-term (LT) HSCs is compatible with adult life and has little effect on the maintenance of phenotypic LT-HSCs in the bone marrow. However, SETD1A-deficient LT-HSCs lose their transcriptional cellular identity, accompanied by loss of their proliferative capacity and stem cell function under replicative stress in situ and after transplantation. In response to inflammatory stimulation, SETD1A protects HSCs and progenitors from activation-induced attrition in vivo. The comprehensive regulation of DNA damage responses by SETD1A in HSCs is clearly distinct from the key roles played by other epigenetic regulators, including the major leukemogenic H3K4 methyltransferase MLL1, or MLL5, indicating that HSC identity and function is supported by cooperative specificities within an epigenetic framework. [ABSTRACT FROM AUTHOR]

Background: Due to the predominant usage of short-read sequencing to date, most bacterial genome sequences reported in the last years remain at the draft level. This precludes certain types of analyses, such as the in-depth analysis of genome plasticity. Results: Here we report the finalized genome sequence of the environmental strain Aeromonas salmonicida subsp. pectinolytica 34mel, for which only a draft genome with 253 contigs is currently available. Successful completion of the transposon-rich genome critically depended on the PacBio long read sequencing technology. Using finalized genome sequences of A. salmonicida subsp. pectinolytica and other Aeromonads, we report the detailed analysis of the transposon composition of these bacterial species. Mobilome evolution is exemplified by a complex transposon, which has shifted from pathogenicity-related to environmental-related gene content in A. salmonicida subsp. pectinolytica 34mel. Conclusion: Obtaining the complete, circular genome of A. salmonicida subsp. pectinolytica allowed us to perform an in-depth analysis of its mobilome. We demonstrate the mobilome-dependent evolution of this strain's genetic profile from pathogenic to environmental [ABSTRACT FROM AUTHOR]

Genetic barcodes are increasingly used to track individual cells and to quantitatively assess their clonal contributions over time. Although barcode quantification relies entirely on counting sequencing reads, detailed studies about the method's accuracy are still limited. We report on a systematic investigation of the relation between barcode abundance and resulting read counts after amplification and sequencing using cell-mixtures that contain barcodes with known frequencies ('miniBulks'). We evaluated the influence of protocol modifications to identify potential sources of error and elucidate possible limitations of the quantification approach. Based on these findings we designed an advanced barcode construct (BC32) to improved barcode calling and quantification, and to ensure a sensitive detection of even highly diluted barcodes. Our results emphasize the importance of using curated barcode libraries to obtain interpretable quantitative data and underline the need for rigorous analyses of any utilized barcode library in terms of reliability and reproducibility. [ABSTRACT FROM AUTHOR]

Germ cell development involves major reprogramming of the epigenome to prime the zygote for totipotency. Histone 3 lysine 4 (H3K4) methylations are universal epigenetic marks mediated in mammals by six H3K4 methyltransferases related to fly Trithorax, including two yeast Set1 orthologs: Setd1a and Setd1b. Whereas Setd1a plays no role in oogenesis, we report that Setd1b deficiency causes female sterility in mice. Oocyte-specific Gdf9-iCre conditional knockout (Setd1bGdf9cKO) ovaries developthrough all stages; however, follicular loss accumulated with age and unfertilized metaphase II (MII) oocytesexhibited irregularitiesof thezonapellucida andmeioticspindle. Most Setd1bGdf9cKO zygotes remained in the pronuclear stage and displayed polyspermy in the perivitelline space. Expression profiling of Setd1bGdf9 cKO MII oocytes revealed (1) that Setd1b promotes the expression of the major oocyte transcription factors including Obox1, 2, 5, 7, Meis2 and Sall4; and (2) twice as many mRNAs were upregulated than downregulated, suggesting that Setd1b also promotes the expression of negative regulators of oocyte development with multiple Zfp-KRAB factors implicated. Together, these findings indicate that Setd1b serves as maternal effect gene through regulation of the oocyte gene expression program. ) [ABSTRACT FROM AUTHOR]

Background: Clonal competition in cancer describes the process in which the progeny of a cell clone supersedes or succumbs to other competing clones due to differences in their functional characteristics, mostly based on subsequently acquired mutations. Even though the patterns of those mutations are well explored in many tumors, the dynamical process of clonal selection is underexposed. Methods: We studied the dynamics of clonal competition in a BcrAbl-induced leukemia using a γ-retroviral vector library encoding the oncogene in conjunction with genetic barcodes. To this end, we studied the growth dynamics of transduced cells on the clonal level both in vitro and in vivo in transplanted mice. Results: While we detected moderate changes in clonal abundancies in vitro, we observed monoclonal leukemias in 6/30 mice after transplantation, which intriguingly were caused by only two different BcrAbl clones. To analyze the success of these clones, we applied a mathematical model of hematopoietic tissue maintenance, which indicated that a differential engraftment capacity of these two dominant clones provides a possible explanation of our observations. These findings were further supported by additional transplantation experiments and increased BcrAbl transcript levels in both clones. Conclusion: Our findings show that clonal competition is not an absolute process based on mutations, but highly dependent on selection mechanisms in a given environmental context. [ABSTRACT FROM AUTHOR]

Tudor containing protein 6 (TDRD6) is a male germ line-specific protein essential for chromatoid body (ChB) structure, elongated spermatid development and male fertility. Here we show that in meiotic prophase I spermatocytes TDRD6 interacts with the key protein arginine methyl transferase PRMT5, which supports splicing. TDRD6 also associates with spliceosomal core protein SmB in the absence of RNA and in an arginine methylation dependent manner. In Tdrd6-/- diplotene spermatocytes PRMT5 association with SmB and arginine dimethylation of SmB are much reduced. TDRD6 deficiency impairs the assembly of spliceosomes, which feature 3.5-fold increased levels of U5 snRNPs. In the nucleus, these deficiencies in spliceosome maturation correlate with decreased numbers of SMN-positive bodies and Cajal bodies involved in nuclear snRNP maturation. Transcriptome analysis of TDRD6-deficient diplotene spermatocytes revealed high numbers of splicing defects such as aberrant usage of intron and exons as well as aberrant representation of splice junctions. Together, this study demonstrates a novel function of TDRD6 in spliceosome maturation and mRNA splicing in prophase I spermatocytes. [ABSTRACT FROM AUTHOR]

During development, progenitor expansion, lineage allocation, and implementation of differentiation programs need to be tightly coordinated so that different cell types are generated in the correct numbers for appropriate tissue size and function. Pancreatic dysfunction results in some of the most debilitating and fatal diseases, including pancreatic cancer and diabetes. Several transcription factors regulating pancreas lineage specification have been identified, and Notch signalling has been implicated in lineage allocation, but it remains unclear how these processes are coordinated. Using a combination of genetic approaches, organotypic cultures of embryonic pancreata, and genomics, we found that sphingosine-1-phosphate (S1p), signalling through the G protein coupled receptor (GPCR) S1pr2, plays a key role in pancreas development linking lineage allocation and specification. S1pr2 signalling promotes progenitor survival as well as acinar and endocrine specification. S1pr2-mediated stabilisation of the yes-associated protein (YAP) is essential for endocrine specification, thus linking a regulator of progenitor growth with specification. YAP stabilisation and endocrine cell specification rely on Gαi subunits, revealing an unexpected specificity of selected GPCR intracellular signalling components. Finally, we found that S1pr2 signalling posttranscriptionally attenuates Notch signalling levels, thus regulating lineage allocation. Both S1pr2-mediated YAP stabilisation and Notch attenuation are necessary for the specification of the endocrine lineage. These findings identify S1p signalling as a novel key pathway coordinating cell survival, lineage allocation, and specification and linking these processes by regulating YAP levels and Notch signalling. Understanding lineage allocation and specification in the pancreas will shed light in the origins of pancreatic diseases and may suggest novel therapeutic approaches. [ABSTRACT FROM AUTHOR]

Psychiatric disorders are highly heritable and polygenic, and many have their peak onset in late childhood and adolescence, a period of tremendous changes. Although the neurodevelopmental antecedents of mental illness are widely acknowledged, research in youth population cohorts is still scarce, preventing our progress towards the early characterization of these disorders. We included 7,124 children (9–11 years old) from the Adolescent Brain and Cognitive Development Study to map the associations of structural and diffusion brain imaging with common genetic variants and polygenic scores for psychiatric disorders and educational attainment. We used principal component analysis to derive imaging components, and calculated their heritability. We then assessed the relationship of imaging components with genetic and clinical psychiatric risk with univariate models and Canonical correlation analysis (CCA). Most imaging components had moderate heritability. Univariate models showed limited evidence and small associations of polygenic scores with brain structure at this age. CCA revealed two significant modes of covariation. The first mode linked higher polygenic scores for educational attainment with less externalizing problems and larger surface area. The second mode related higher polygenic scores for schizophrenia, bipolar disorder, and autism spectrum disorder to higher global cortical thickness, smaller white matter volumes of the fornix and cingulum, larger medial occipital surface area and smaller surface area of lateral and medial temporal regions. While cross-validation suggested limited generalizability, our results highlight the potential of multivariate models to better understand the transdiagnostic and distributed relationships between mental health and brain structure in late childhood. [ABSTRACT FROM AUTHOR]

Defects of the intracellular enzyme 3' repair exonuclease 1 (Trex1) cause the rare autoimmune condition Aicardi-Goutières syndrome and are associated with systemic lupus erythematosus. Trex1-/- mice develop type I IFN-driven autoimmunity, resulting from activation of the cytoplasmic DNA sensor cyclic GMP-AMP synthase by a nucleic acid substrate of Trex1 that remains unknown. To identify cell types responsible for initiation of autoimmunity, we generated conditional Trex1 knockout mice. Loss of Trex1 in dendritic cells was sufficient to cause IFN release and autoimmunity, whereas Trex1-deficient keratinocytes and microglia produced IFN but did not induce inflammation. In contrast, B cells, cardiomyocytes, neurons, and astrocytes did not show any detectable response to the inactivation of Trex1. Thus, individual cell types differentially respond to the loss of Trex1, and Trex1 expression in dendritic cells is essential to prevent breakdown of self-tolerance ensuing from aberrant detection of endogenous DNA. [ABSTRACT FROM AUTHOR]

Chromatoid bodies (CBs) are spermiogenesis-specific organelles of largely unknown function. CBs harbor various RNA species, RNA-associated proteins and proteins of the tudor domain family like TDRD6, which is required for a proper CB architecture. Proteome analysis of purified CBs revealed components of the nonsense-mediated mRNA decay (NMD) machinery including UPF1. TDRD6 is essential for UPF1 localization to CBs, for UPF1-UPF2 and UPF1-MVH interactions. Upon removal of TDRD6, the association of several mRNAs with UPF1 and UPF2 is disturbed, and the long 3’ UTR-stimulated but not the downstream exon-exon junction triggered pathway of NMD is impaired. Reduced association of the long 3’ UTR mRNAs with UPF1 and UPF2 correlates with increased stability and enhanced translational activity. Thus, we identified TDRD6 within CBs as required for mRNA degradation, specifically the extended 3’ UTR-triggered NMD pathway, and provide evidence for the requirement of NMD in spermiogenesis. This function depends on TDRD6-promoted assembly of mRNA and decay enzymes in CBs. [ABSTRACT FROM AUTHOR]

Acute myeloid leukemia (AML) is characterized by a marked genetic heterogeneity, which complicates the development of novel therapeutics. The delineation of pathways essential within an individual patient's mutational background might overcome this limitation and facilitate personalized treatment. We report the results of a large-scale lentiviral loss-of-function RNA-interference-(RNAi) screen in primary leukemic cells. Stringent validation identified 6 genes (BNIPL1, ROCK1, RPS13, STK3, SNX27, WDHD1) whose knockdown impaired growth and viability of the cells. Dependence on these genes was not caused by mutation or overexpression, and although some of the candidates seemed to be rather patient specific, others were essential in cells isolated from other AML patients. In addition to the phenotype observed after ROCK1 knockdown, treatment with the approved ROCK inhibitor fasudil resulted in increased apoptosis and decreased viability of primary AML cells. In contrast to observations in some other malignancies, ROCK1 inhibition did not foster growth of immature malignant progenitors but was also toxic to this cell fraction in feeder coculture and xenotransplantion experiments, indicating a distinct effect of ROCK1 inhibition on leukemic progenitors. We conclude that large-scale RNAi screens in primary patient-derived cells are feasible and can complement other methods for personalized cancer therapies, such as expression and mutation profiling. [ABSTRACT FROM AUTHOR]

Major efforts are invested to characterize the factors controlling the proliferation of neural stem cells. During mammalian corticogenesis, our group has identified a small pool of genes that are transiently downregulated in the switch of neural stem cells to neurogenic division and reinduced in newborn neurons. Among these switch genes, we found Tox, a transcription factor with hitherto uncharacterized roles in the nervous system. Here, we investigated the role of Tox in corticogenesis by characterizing its expression at the tissue, cellular and temporal level. We found that Tox is regulated by calcineurin/Nfat signalling. Moreover, we combined DNA adenine methyltransferase identification (DamID) with deep sequencing to characterize the chromatin binding properties of Tox including its motif and downstream transcriptional targets including Sox2, Tbr2, Prox1 and other key factors. Finally, we manipulated Tox in the developing brain and validated its multiple roles in promoting neural stem cell proliferation and neurite outgrowth of newborn neurons. Our data provide a valuable resource to study the role of Tox in other tissues and highlight a novel key player in brain development. [ABSTRACT FROM AUTHOR]

The article focuses on an analysis of the transcriptomes of progenitor subpopulations using a cell polarity-based approach to isolate them in developing human and mouse neocortex. It states 56 genes were identified that preferentially expressed in human apical and basal radial glia that house orthologs lacked with the gene ARHGAP11B having the highest degree of expression. It comments on the role human-specific gene ARHGAP11B may have contributed in human neocortex evolutionary expansion.

Autoreactive CD4+ T cells are an essential feature of type 1 diabetes mellitus. We applied single-cell TCR α- and β-chain sequencing to peripheral blood GAD65-specific CD4+ T cells, and TCR α-chain next-generation sequencing to bulk memory CD4+ T cells to provide insight into TCR diversity in autoimmune diabetes mellitus. TCRs obtained for 1650 GAD65-specific CD4+ T cells isolated from GAD65 proliferation assays and/or GAD65 557I tetramer staining in 6 patients and 10 islet autoantibody-positive children showed large diversity with 1003 different TCRs identified. TRAV and TRBV gene usage was broad, and the TRBV5.1 gene was most prominent within the GAD65 557I tetramer+ cells. Limited overlap (<5%) was observed between TCRs of GAD65-proliferating and GAD65 557I tetramer+ CD4+ T cells. Few TCRs were repeatedly found in GAD65-specific cells at different time points from individual patients, and none was seen in more than one subject. However, single chains were often shared between patients and used in combination with different second chains. Next-generation sequencing revealed a wide frequency range (<0.00001-1.62%) of TCR α-chains corresponding to GAD65-specific T cells. The findings support minor selection of genes and TCRs for GAD65-specific T cells, but fail to provide strong support for TCR-targeted therapies. [ABSTRACT FROM AUTHOR]

Transcriptome analysis of somatic stem cells and their progeny is fundamental to identify new factors controlling proliferation versus differentiation during tissue formation. Here, we generated a combinatorial, fluorescent reporter mouse line to isolate proliferating neural stem cells, differentiating progenitors and newborn neurons that coexist as intermingled cell populations during brain development. Transcriptome sequencing revealed numerous novel long non-coding (lnc)RNAs and uncharacterized protein-coding transcripts identifying the signature of neurogenic commitment. Importantly, most lncRNAs overlapped neurogenic genes and shared with them a nearly identical expression pattern suggesting that lncRNAs control corticogenesis by tuning the expression of nearby cell fate determinants. We assessed the power of our approach by manipulating lncRNAs and protein-coding transcripts with no function in corticogenesis reported to date. This led to several evident phenotypes in neurogenic commitment and neuronal survival, indicating that our study provides a remarkably high number of uncharacterized transcripts with hitherto unsuspected roles in brain development. Finally, we focussed on one lncRNA, Miat, whose manipulation was found to trigger pleiotropic effects on brain development and aberrant splicing of Wnt7b. Hence, our study suggests that lncRNA-mediated alternative splicing of cell fate determinants controls stem-cell commitment during neurogenesis. [ABSTRACT FROM AUTHOR]

10x Genomics is a highly accessible single cell RNA sequencing platform that allows for simultaneous gene expression analysis and identification of receptor chain combinations in cells of the adaptive immune system. Here, we asked whether the gene and receptor expression measurements in peripheral blood mononuclear cells (PBMC) are influenced by technical, cell freezing, FACS-processing, and day to day biological variation. No differentially expressed gene was observed between 1. triplicates aliquots taken from the same vial of frozen PBMC; 2. triplicate vials of frozen PBMC; and 3. triplicate aliquots taken from the same vial of frozen PBMC and processed separately for FACS staining and sorting of different PBMC populations. A small number of differentially expressed genes were observed between PBMC sampled, isolated and frozen from the same donor on different days, and these differences were more pronounced in the memory B cells than other cell populations. T cell receptors were recovered in all replicates when at least 5 cells per clonotype were identified. These findings show high reproducibility of 10x Genomics single cell RNA sequencing data in the immune cell context. [ABSTRACT FROM AUTHOR]