Trnp1 regulates expansion and folding of the mammalian cerebral cortex by control of radial glial fate.

Evolution of the mammalian brain encompassed a remarkable increase in size of the cerebral cortex, which includes tangential and radial expansion. However, the mechanisms underlying these key features are still largely unknown. Here, we identified the DNA-associated protein Trnp1 as a regulator of cerebral cortex expansion in both of these dimensions. Gain- and loss-of-function experiments in the mouse cerebral cortex in vivo demonstrate that high Trnp1 levels promote neural stem cell self-renewal and tangential expansion. In contrast, lower levels promote radial expansion, with a potent increase of the number of intermediate progenitors and basal radial glial cells leading to folding of the otherwise smooth murine cerebral cortex. Remarkably, TRNP1 expression levels exhibit regional differences in the cerebral cortex of human fetuses, anticipating radial or tangential expansion. Thus, the dynamic regulation of Trnp1 is critical to control tangential and radial expansion of the cerebral cortex in mammals.

Inceptor counteracts insulin signalling in ß-cells to control glycaemia.

Resistance to insulin and insulin-like growth factor 1 (IGF1) in pancreatic ß-cells causes overt diabetes in mice; thus, therapies that sensitize ß-cells to insulin may protect patients with diabetes against ß-cell failure1-3. Here we identify an inhibitor of insulin receptor (INSR) and IGF1 receptor (IGF1R) signalling in mouse ß-cells, which we name the insulin inhibitory receptor (inceptor; encoded by the gene Iir). Inceptor contains an extracellular cysteine-rich domain with similarities to INSR and IGF1R4, and a mannose 6-phosphate receptor domain that is also found in the IGF2 receptor (IGF2R)5. Knockout mice that lack inceptor (Iir-/-) exhibit signs of hyperinsulinaemia and hypoglycaemia, and die within a few hours of birth. Molecular and cellular analyses of embryonic and postnatal pancreases from Iir-/- mice showed an increase in the activation of INSR-IGF1R in Iir-/- pancreatic tissue, resulting in an increase in the proliferation and mass of ß-cells. Similarly, inducible ß-cell-specific Iir-/- knockout in adult mice and in ex vivo islets led to an increase in the activation of INSR-IGF1R and increased proliferation of ß-cells, resulting in improved glucose tolerance in vivo. Mechanistically, inceptor interacts with INSR-IGF1R to facilitate clathrin-mediated endocytosis for receptor desensitization. Blocking this physical interaction using monoclonal antibodies against the extracellular domain of inceptor resulted in the retention of inceptor and INSR at the plasma membrane to sustain the activation of INSR-IGF1R in ß-cells. Together, our findings show that inceptor shields insulin-producing ß-cells from constitutive pathway activation, and identify inceptor as a potential molecular target for INSR-IGF1R sensitization and diabetes therapy.

Inhibition of LTßR signalling activates WNT-induced regeneration in lung.

Lymphotoxin ß-receptor (LTßR) signalling promotes lymphoid neogenesis and the development of tertiary lymphoid structures1,2, which are associated with severe chronic inflammatory diseases that span several organ systems3-6. How LTßR signalling drives chronic tissue damage particularly in the lung, the mechanism(s) that regulate this process, and whether LTßR blockade might be of therapeutic value have remained unclear. Here we demonstrate increased expression of LTßR ligands in adaptive and innate immune cells, enhanced non-canonical NF-κB signalling, and enriched LTßR target gene expression in lung epithelial cells from patients with smoking-associated chronic obstructive pulmonary disease (COPD) and from mice chronically exposed to cigarette smoke. Therapeutic inhibition of LTßR signalling in young and aged mice disrupted smoking-related inducible bronchus-associated lymphoid tissue, induced regeneration of lung tissue, and reverted airway fibrosis and systemic muscle wasting. Mechanistically, blockade of LTßR signalling dampened epithelial non-canonical activation of NF-κB, reduced TGFß signalling in airways, and induced regeneration by preventing epithelial cell death and activating WNT/ß-catenin signalling in alveolar epithelial progenitor cells. These findings suggest that inhibition of LTßR signalling represents a viable therapeutic option that combines prevention of tertiary lymphoid structures1 and inhibition of apoptosis with tissue-regenerative strategies.

Insights into energy balance dysregulation from a mouse model of methylmalonic aciduria.

Inherited disorders of mitochondrial metabolism, including isolated methylmalonic aciduria, present unique challenges to energetic homeostasis by disrupting energy-producing pathways. To better understand global responses to energy shortage, we investigated a hemizygous mouse model of methylmalonyl-CoA mutase (Mmut)-type methylmalonic aciduria. We found Mmut mutant mice to have reduced appetite, energy expenditure and body mass compared with littermate controls, along with a relative reduction in lean mass but increase in fat mass. Brown adipose tissue showed a process of whitening, in line with lower body surface temperature and lesser ability to cope with cold challenge. Mutant mice had dysregulated plasma glucose, delayed glucose clearance and a lesser ability to regulate energy sources when switching from the fed to fasted state, while liver investigations indicated metabolite accumulation and altered expression of peroxisome proliferator-activated receptor and Fgf21-controlled pathways. Together, these shed light on the mechanisms and adaptations behind energy imbalance in methylmalonic aciduria and provide insight into metabolic responses to chronic energy shortage, which may have important implications for disease understanding and patient management.

COPD basal cells are primed towards secretory to multiciliated cell imbalance driving increased resilience to environmental stressors.

INTRODUCTION: Environmental pollutants injure the mucociliary elevator, thereby provoking disease progression in chronic obstructive pulmonary disease (COPD). Epithelial resilience mechanisms to environmental nanoparticles in health and disease are poorly characterised. METHODS: We delineated the impact of prevalent pollutants such as carbon and zinc oxide nanoparticles, on cellular function and progeny in primary human bronchial epithelial cells (pHBECs) from end-stage COPD (COPD-IV, n=4), early disease (COPD-II, n=3) and pulmonary healthy individuals (n=4). After nanoparticle exposure of pHBECs at air-liquid interface, cell cultures were characterised by functional assays, transcriptome and protein analysis, complemented by single-cell analysis in serial samples of pHBEC cultures focusing on basal cell differentiation. RESULTS: COPD-IV was characterised by a prosecretory phenotype (twofold increase in MUC5AC+) at the expense of the multiciliated epithelium (threefold reduction in Ac-Tub+), resulting in an increased resilience towards particle-induced cell damage (fivefold reduction in transepithelial electrical resistance), as exemplified by environmentally abundant doses of zinc oxide nanoparticles. Exposure of COPD-II cultures to cigarette smoke extract provoked the COPD-IV characteristic, prosecretory phenotype. Time-resolved single-cell transcriptomics revealed an underlying COPD-IV unique basal cell state characterised by a twofold increase in KRT5+ (P=0.018) and LAMB3+ (P=0.050) expression, as well as a significant activation of Wnt-specific (P=0.014) and Notch-specific (P=0.021) genes, especially in precursors of suprabasal and secretory cells. CONCLUSION: We identified COPD stage-specific gene alterations in basal cells that affect the cellular composition of the bronchial elevator and may control disease-specific epithelial resilience mechanisms in response to environmental nanoparticles. The identified phenomena likely inform treatment and prevention strategies.

Sfrp1 inhibits lung fibroblast invasion during transition to injury-induced myofibroblasts.

BACKGROUND: Fibroblast-to-myofibroblast conversion is a major driver of tissue remodelling in organ fibrosis. Distinct lineages of fibroblasts support homeostatic tissue niche functions, yet their specific activation states and phenotypic trajectories during injury and repair have remained unclear. METHODS: We combined spatial transcriptomics, multiplexed immunostainings, longitudinal single-cell RNA-sequencing and genetic lineage tracing to study fibroblast fates during mouse lung regeneration. Our findings were validated in idiopathic pulmonary fibrosis patient tissues in situ as well as in cell differentiation and invasion assays using patient lung fibroblasts. Cell differentiation and invasion assays established a function of SFRP1 in regulating human lung fibroblast invasion in response to transforming growth factor (TGF)ß1. MEASUREMENTS AND MAIN RESULTS: We discovered a transitional fibroblast state characterised by high Sfrp1 expression, derived from both Tcf21-Cre lineage positive and negative cells. Sfrp1 + cells appeared early after injury in peribronchiolar, adventitial and alveolar locations and preceded the emergence of myofibroblasts. We identified lineage-specific paracrine signals and inferred converging transcriptional trajectories towards Sfrp1 + transitional fibroblasts and Cthrc1 + myofibroblasts. TGFß1 downregulated SFRP1 in noninvasive transitional cells and induced their switch to an invasive CTHRC1+ myofibroblast identity. Finally, using loss-of-function studies we showed that SFRP1 modulates TGFß1-induced fibroblast invasion and RHOA pathway activity. CONCLUSIONS: Our study reveals the convergence of spatially and transcriptionally distinct fibroblast lineages into transcriptionally uniform myofibroblasts and identifies SFRP1 as a modulator of TGFß1-driven fibroblast phenotypes in fibrogenesis. These findings are relevant in the context of therapeutic interventions that aim at limiting or reversing fibroblast foci formation.

Assessment of wood smoke induced pulmonary toxicity in normal- and chronic bronchitis-like bronchial and alveolar lung mucosa models at air-liquid interface.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) has the highest increased risk due to household air pollution arising from biomass fuel burning. However, knowledge on COPD patho-mechanisms is mainly limited to tobacco smoke exposure. In this study, a repeated direct wood smoke (WS) exposure was performed using normal- (bro-ALI) and chronic bronchitis-like bronchial (bro-ALI-CB), and alveolar (alv-ALI) lung mucosa models at air-liquid interface (ALI) to assess broad toxicological end points. METHODS: The bro-ALI and bro-ALI-CB models were developed using human primary bronchial epithelial cells and the alv-ALI model was developed using a representative type-II pneumocyte cell line. The lung models were exposed to WS (10 min/exposure; 5-exposures over 3-days; n = 6-7 independent experiments). Sham exposed samples served as control. WS composition was analyzed following passive sampling. Cytotoxicity, total cellular reactive oxygen species (ROS) and stress responsive NFkB were assessed by flow cytometry. WS exposure induced changes in gene expression were evaluated by RNA-seq (p ≤ 0.01) followed by pathway enrichment analysis. Secreted levels of proinflammatory cytokines were assessed in the basal media. Non-parametric statistical analysis was performed. RESULTS: 147 unique compounds were annotated in WS of which 42 compounds have inhalation toxicity (9 very high). WS exposure resulted in significantly increased ROS in bro-ALI (11.2%) and bro-ALI-CB (25.7%) along with correspondingly increased NFkB levels (bro-ALI: 35.6%; bro-ALI-CB: 18.1%). A total of 1262 (817-up and 445-down), 329 (141-up and 188-down), and 102 (33-up and 69-down) genes were differentially regulated in the WS-exposed bro-ALI, bro-ALI-CB, and alv-ALI models respectively. The enriched pathways included the terms acute phase response, mitochondrial dysfunction, inflammation, oxidative stress, NFkB, ROS, xenobiotic metabolism of AHR, and chronic respiratory disorder. The enrichment of the 'cilium' related genes was predominant in the WS-exposed bro-ALI (180-up and 7-down). The pathways primary ciliary dyskinesia, ciliopathy, and ciliary movement were enriched in both WS-exposed bro-ALI and bro-ALI-CB. Interleukin-6 and tumor necrosis factor-α were reduced (p < 0.05) in WS-exposed bro-ALI and bro-ALI-CB. CONCLUSION: Findings of this study indicate differential response to WS-exposure in different lung regions and in chronic bronchitis, a condition commonly associated with COPD. Further, the data suggests ciliopathy as a candidate pathway in relation to WS-exposure.

A novel stem cell type at the basal side of the subventricular zone maintains adult neurogenesis.

According to the current consensus, murine neural stem cells (NSCs) apically contacting the lateral ventricle generate differentiated progenitors by rare asymmetric divisions or by relocating to the basal side of the ventricular-subventricular zone (V-SVZ). Both processes will ultimately lead to the generation of adult-born olfactory bulb (OB) interneurons. In contrast to this view, we here find that adult-born OB interneurons largely derive from an additional NSC-type resident in the basal V-SVZ. Despite being both capable of self-renewal and long-term quiescence, apical and basal NSCs differ in Nestin expression, primary cilia extension and frequency of cell division. The expression of Notch-related genes also differs between the two NSC groups, and Notch activation is greatest in apical NSCs. Apical downregulation of Notch-effector Hes1 decreases Notch activation while increasing proliferation across the niche and neurogenesis from apical NSCs. Underscoring their different roles in neurogenesis, lactation-dependent increase in neurogenesis is paralleled by extra activation of basal but not apical NSCs. Thus, basal NSCs support OB neurogenesis, whereas apical NSCs impart Notch-mediated lateral inhibition across the V-SVZ.

Redox state and altered pyruvate metabolism contribute to a dose-dependent metformin-induced lactate production of human myotubes.

Metformin-induced glycolysis and lactate production can lead to acidosis as a life-threatening side effect, but slight increases in blood lactate levels in a physiological range were also reported in metformin-treated patients. However, how metformin increases systemic lactate concentrations is only partly understood. Because human skeletal muscle has a high capacity to produce lactate, the aim was to elucidate the dose-dependent regulation of metformin-induced lactate production and the potential contribution of skeletal muscle to blood lactate levels under metformin treatment. This was examined by using metformin treatment (16-776 µM) of primary human myotubes and by 17 days of metformin treatment in humans. As from 78 µM, metformin induced lactate production and secretion and glucose consumption. Investigating the cellular redox state by mitochondrial respirometry, we found metformin to inhibit the respiratory chain complex I (776 µM, P < 0.01) along with decreasing the [NAD+]:[NADH] ratio (776 µM, P < 0.001). RNA sequencing and phospho-immunoblot data indicate inhibition of pyruvate oxidation mediated through phosphorylation of the pyruvate dehydrogenase (PDH) complex (39 µM, P < 0.01). On the other hand, in human skeletal muscle, phosphorylation of PDH was not altered by metformin. Nonetheless, blood lactate levels were increased under metformin treatment (P < 0.05). In conclusion, the findings suggest that metformin-induced inhibition of pyruvate oxidation combined with altered cellular redox state shifts the equilibrium of the lactate dehydrogenase (LDH) reaction leading to a dose-dependent lactate production in primary human myotubes.NEW & NOTEWORTHY Metformin shifts the equilibrium of lactate dehydrogenase (LDH) reaction by low dose-induced phosphorylation of pyruvate dehydrogenase (PDH) resulting in inhibition of pyruvate oxidation and high dose-induced increase in NADH, which explains the dose-dependent lactate production of differentiated human skeletal muscle cells.

Acute and long-term exercise adaptation of adipose tissue and skeletal muscle in humans: a matched transcriptomics approach after 8-week training-intervention.

BACKGROUND: Exercise exerts many health benefits by directly inducing molecular alterations in physically utilized skeletal muscle. Molecular adaptations of subcutaneous adipose tissue (SCAT) might also contribute to the prevention of metabolic diseases. AIM: To characterize the response of human SCAT based on changes in transcripts and mitochondrial respiration to acute and repeated bouts of exercise in comparison to skeletal muscle. METHODS: Sedentary participants (27 ± 4 yrs) with overweight or obesity underwent 8-week supervised endurance exercise 3×1h/week at 80% VO2peak. Before, 60 min after the first and last exercise bout and 5 days post intervention, biopsies were taken for transcriptomic analyses and high-resolution respirometry (n = 14, 8 female/6 male). RESULTS: In SCAT, we found 37 acutely regulated transcripts (FC > 1.2, FDR < 10%) after the first exercise bout compared to 394, respectively, in skeletal muscle. Regulation of only 5 transcripts overlapped between tissues highlighting their differential response. Upstream and enrichment analyses revealed reduced transcripts of lipid uptake, storage and lipogenesis directly after exercise in SCAT and point to ß-adrenergic regulation as potential major driver. The data also suggest an exercise-induced modulation of the circadian clock in SCAT. Neither term was associated with transcriptomic changes in skeletal muscle. No evidence for beigeing/browning was found in SCAT along with unchanged respiration. CONCLUSIONS: Adipose tissue responds completely distinct from adaptations of skeletal muscle to exercise. The acute and repeated reduction in transcripts of lipid storage and lipogenesis, interconnected with a modulated circadian rhythm, can counteract metabolic syndrome progression toward diabetes.

Identification of proliferative and mature ß-cells in the islets of Langerhans.

Insulin-dependent diabetes is a complex multifactorial disorder characterized by loss or dysfunction of ß-cells. Pancreatic ß-cells differ in size, glucose responsiveness, insulin secretion and precursor cell potential; understanding the mechanisms that underlie this functional heterogeneity might make it possible to develop new regenerative approaches. Here we show that Fltp (also known as Flattop and Cfap126), a Wnt/planar cell polarity (PCP) effector and reporter gene acts as a marker gene that subdivides endocrine cells into two subpopulations and distinguishes proliferation-competent from mature ß-cells with distinct molecular, physiological and ultrastructural features. Genetic lineage tracing revealed that endocrine subpopulations from Fltp-negative and -positive lineages react differently to physiological and pathological changes. The expression of Fltp increases when endocrine cells cluster together to form polarized and mature 3D islet mini-organs. We show that 3D architecture and Wnt/PCP ligands are sufficient to trigger ß-cell maturation. By contrast, the Wnt/PCP effector Fltp is not necessary for ß-cell development, proliferation or maturation. We conclude that 3D architecture and Wnt/PCP signalling underlie functional ß-cell heterogeneity and induce ß-cell maturation. The identification of Fltp as a marker for endocrine subpopulations sheds light on the molecular underpinnings of islet cell heterogeneity and plasticity and might enable targeting of endocrine subpopulations for the regeneration of functional ß-cell mass in diabetic patients.

HAND2 is a novel obesity-linked adipogenic transcription factor regulated by glucocorticoid signalling.

AIMS/HYPOTHESIS: Adipocytes are critical cornerstones of energy metabolism. While obesity-induced adipocyte dysfunction is associated with insulin resistance and systemic metabolic disturbances, adipogenesis, the formation of new adipocytes and healthy adipose tissue expansion are associated with metabolic benefits. Understanding the molecular mechanisms governing adipogenesis is of great clinical potential to efficiently restore metabolic health in obesity. Here we investigate the role of heart and neural crest derivatives-expressed 2 (HAND2) in adipogenesis. METHODS: Human white adipose tissue (WAT) was collected from two cross-sectional studies of 318 and 96 individuals. In vitro, for mechanistic experiments we used primary adipocytes from humans and mice as well as human multipotent adipose-derived stem (hMADS) cells. Gene silencing was performed using siRNA or genetic inactivation in primary adipocytes from loxP and or tamoxifen-inducible Cre-ERT2 mouse models with Cre-encoding mRNA or tamoxifen, respectively. Adipogenesis and adipocyte metabolism were measured by Oil Red O staining, quantitative PCR (qPCR), microarray, glucose uptake assay, western blot and lipolysis assay. A combinatorial RNA sequencing (RNAseq) and ChIP qPCR approach was used to identify target genes regulated by HAND2. In vivo, we created a conditional adipocyte Hand2 deletion mouse model using Cre under control of the Adipoq promoter (Hand2AdipoqCre) and performed a large panel of metabolic tests. RESULTS: We found that HAND2 is an obesity-linked white adipocyte transcription factor regulated by glucocorticoids that was necessary but insufficient for adipocyte differentiation in vitro. In a large cohort of humans, WAT HAND2 expression was correlated to BMI. The HAND2 gene was enriched in white adipocytes compared with brown, induced early in differentiation and responded to dexamethasone (DEX), a typical glucocorticoid receptor (GR, encoded by NR3C1) agonist. Silencing of NR3C1 in hMADS cells or deletion of GR in a transgenic conditional mouse model results in diminished HAND2 expression, establishing that adipocyte HAND2 is regulated by glucocorticoids via GR in vitro and in vivo. Furthermore, we identified gene clusters indirectly regulated by the GR-HAND2 pathway. Interestingly, silencing of HAND2 impaired adipocyte differentiation in hMADS and primary mouse adipocytes. However, a conditional adipocyte Hand2 deletion mouse model using Cre under control of the Adipoq promoter did not mirror these effects on adipose tissue differentiation, indicating that HAND2 was required at stages prior to Adipoq expression. CONCLUSIONS/INTERPRETATION: In summary, our study identifies HAND2 as a novel obesity-linked adipocyte transcription factor, highlighting new mechanisms of GR-dependent adipogenesis in humans and mice. DATA AVAILABILITY: Array data have been submitted to the GEO database at NCBI (GSE148699).

miR-34a is upregulated in AIP-mutated somatotropinomas and promotes octreotide resistance.

Pituitary adenomas (PAs) are intracranial tumors associated with significant morbidity due to hormonal dysregulation, mass effects and have a heavy treatment burden. Growth hormone (GH)-secreting PAs (somatotropinomas) cause acromegaly-gigantism. Genetic forms of somatotropinomas due to germline AIP mutations (AIPmut+) have an early onset and are aggressive and resistant to treatment with somatostatin analogs (SSAs), including octreotide. The molecular underpinnings of these clinical features remain unclear. We investigated the role of miRNA dysregulation in AIPmut+ vs AIPmut- PA samples by array analysis. miR-34a and miR-145 were highly expressed in AIPmut+ vs AIPmut- somatotropinomas. Ectopic expression of AIPmut (p.R271W) in Aip-/- mouse embryonic fibroblasts (MEFs) upregulated miR-34a and miR-145, establishing a causal link between AIPmut and miRNA expression. In PA cells (GH3), miR-34a overexpression promoted proliferation, clonogenicity, migration and suppressed apoptosis, whereas miR-145 moderately affected proliferation and apoptosis. Moreover, high miR-34a expression increased intracellular cAMP, a critical mitogenic factor in PAs. Crucially, high miR-34a expression significantly blunted octreotide-mediated GH inhibition and antiproliferative effects. miR-34a directly targets Gnai2 encoding Gαi2, a G protein subunit inhibiting cAMP production. Accordingly, Gαi2 levels were significantly lower in AIPmut+ vs AIPmut- PA. Taken together, somatotropinomas with AIP mutations overexpress miR-34a, which in turn downregulates Gαi2 expression, increases cAMP concentration and ultimately promotes cell growth. Upregulation of miR-34a also impairs the hormonal and antiproliferative response of PA cells to octreotide. Thus, miR-34a is a novel downstream target of mutant AIP that promotes a cellular phenotype mirroring the aggressive clinical features of AIPmut+ acromegaly.

Nutrition and its role in epigenetic inheritance of obesity and diabetes across generations.

Nutritional constraints including not only caloric restriction or protein deficiency, but also energy-dense diets affect metabolic health and frequently lead to obesity and insulin resistance, as well as glucose intolerance and type 2 diabetes. The effects of these environmental factors are often mediated via epigenetic modifiers that target the expression of metabolic genes. More recently, it was discovered that such parentally acquired metabolic changes can alter the metabolic health of the filial and grand-filial generations. In mammals, this epigenetic inheritance can either follow an intergenerational or transgenerational mode of inheritance. In the case of intergenerational inheritance, epimutations established in gametes persist through the first round of epigenetic reprogramming occurring during preimplantation development. For transgenerational inheritance, epimutations persist additionally throughout the reprogramming that occurs during germ cell development later in embryogenesis. Differentially expressed transcripts, genomic cytosine methylations, and several chemical modifications of histones are prime candidates for tangible marks which may serve as epimutations in inter- and transgenerational inheritance and which are currently being investigated experimentally. We review, here, the current literature in support of epigenetic inheritance of metabolic traits caused by nutritional constraints and potential mechanisms in man and in rodent model systems.

A comprehensive and comparative phenotypic analysis of the collaborative founder strains identifies new and known phenotypes.

The collaborative cross (CC) is a large panel of mouse-inbred lines derived from eight founder strains (NOD/ShiLtJ, NZO/HILtJ, A/J, C57BL/6J, 129S1/SvImJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ). Here, we performed a comprehensive and comparative phenotyping screening to identify phenotypic differences and similarities between the eight founder strains. In total, more than 300 parameters including allergy, behavior, cardiovascular, clinical blood chemistry, dysmorphology, bone and cartilage, energy metabolism, eye and vision, immunology, lung function, neurology, nociception, and pathology were analyzed; in most traits from sixteen females and sixteen males. We identified over 270 parameters that were significantly different between strains. This study highlights the value of the founder and CC strains for phenotype-genotype associations of many genetic traits that are highly relevant to human diseases. All data described here are publicly available from the mouse phenome database for analyses and downloads.

DEUS: an R package for accurate small RNA profiling based on differential expression of unique sequences.

SUMMARY: Despite their fundamental role in various biological processes, the analysis of small RNA sequencing data remains a challenging task. Major obstacles arise when short RNA sequences map to multiple locations in the genome, align to regions that are not annotated or underwent post-transcriptional changes which hamper accurate mapping. In order to tackle these issues, we present a novel profiling strategy that circumvents the need for read mapping to a reference genome by utilizing the actual read sequences to determine expression intensities. After differential expression analysis of individual sequence counts, significant sequences are annotated against user defined feature databases and clustered by sequence similarity. This strategy enables a more comprehensive and concise representation of small RNA populations without any data loss or data distortion. AVAILABILITY AND IMPLEMENTATION: Code and documentation of our R package at http://ibis.helmholtz-muenchen.de/deus/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Murine tissue factor disulfide mutation causes a bleeding phenotype with sex specific organ pathology and lethality.

Tissue factor is highly expressed in sub-endothelial tissue. The extracellular allosteric disulfide bond Cys186-Cys209 of human tissue factor shows high evolutionary conservation and in vitro evidence suggests that it significantly contributes to tissue factor procoagulant activity. To investigate the role of this allosteric disulfide bond in vivo, we generated a C213G mutant tissue factor mouse by replacing Cys213 of the corresponding disulfide Cys190-Cys213 in murine tissue factor. A bleeding phenotype was prominent in homozygous C213G tissue factor mice. Pre-natal lethality of 1/3rd of homozygous offspring was observed between E9.5 and E14.5 associated with placental hemorrhages. After birth, homozygous mice suffered from bleedings in different organs and reduced survival. Homozygous C213G tissue factor male mice showed higher incidence of lung bleedings and lower survival rates than females. In both sexes, C213G mutation evoked a reduced protein expression (about 10-fold) and severely reduced pro-coagulant activity (about 1000-fold). Protein glycosylation was impaired and cell membrane exposure decreased in macrophages in vivo. Single housing of homozygous C213G tissue factor males reduced the occurrence of severe bleeding and significantly improved survival, suggesting that inter-male aggressiveness might significantly account for the sex differences. These experiments show that the tissue factor allosteric disulfide bond is of crucial importance for normal in vivo expression, post-translational processing and activity of murine tissue factor. Although C213G tissue factor mice do not display the severe embryonic lethality of tissue factor knock-out mice, their postnatal bleeding phenotype emphasizes the importance of fully functional tissue factor for hemostasis.

Correction: From Dynamic Expression Patterns to Boundary Formation in the Presomitic Mesoderm.

[This corrects the article DOI: 10.1371/journal.pcbi.1002586.].