Glycogen synthase kinases 3α and 3β in cardiac myocytes: regulation and consequences of their inhibition

Inhibition of glycogen synthase kinase 3β (GSK3β) as a consequence of its phosphorylation by protein kinase B/Akt (PKB/Akt) has been implicated in cardiac myocyte hypertrophy in response to endothelin-1 or phenylephrine. We examined the regulation of GSK3α (which we show to constitute a significant proportion of the myocyte GSK3 pool) and GSK3β in cardiac myocytes. Although endothelin increases phosphorylation of GSK3 and decreases its activity, the response is less than that induced by insulin (which does not promote cardiac myocyte hypertrophy). GSK3 phosphorylation induced by endothelin requires signalling through the extracellular signal-regulated kinase 1/2 (ERK1/2) cascade and not the PKB/Akt pathway, whereas the reverse is true for insulin. Cardiac myocyte hypertrophy involves changes in morphology, and in gene and protein expression. The potent GSK3 inhibitor 1-azakenpaullone increases myocyte area as a consequence of increased cell length whereas phenylephrine increases both length and width. Azakenpaullone or insulin promotes AP1 transcription factor binding to an AP1 consensus oligonucleotide, but this was significantly less than that induced by endothelin and derived principally from increased binding of JunB protein, the expression of which was increased. Azakenpaullone promotes significant changes in gene expression (assessed by Affymetrix microarrays), but the overall response is less than with endothelin and there is little overlap between the genes identified. Thus, although GSK3 may contribute to cardiac myocyte hypertrophy in some respects (and presumably plays an important role in myocyte metabolism), it does not appear to contribute as significantly to the response induced by endothelin as has been maintained

Notch/Delta signaling constrains reengineering of pro-T cells by PU.1

PU.1 is essential for early stages of mouse T cell development but antagonizes it if expressed constitutively. Two separable mechanisms are involved: attenuation and diversion. Dysregulated PU.1 expression inhibits pro-T cell survival, proliferation, and passage through β-selection by blocking essential T cell transcription factors, signaling molecules, and Rag gene expression, which expression of a rearranged T cell antigen receptor transgene cannot rescue. However, Bcl2 transgenic cells are protected from this attenuation and may even undergo β-selection, as shown by PU.1 transduction of defined subsets of Bcl2 transgenic fetal thymocytes with differentiation in OP9-DL1 and OP9 control cultures. The outcome of PU.1 expression in these cells depends on Notch/Delta signaling. PU.1 can efficiently divert thymocytes toward a myeloid-like state with multigene regulatory changes, but Notch/Delta signaling vetoes diversion. Gene expression analysis distinguishes sets of critical T lineage regulatory genes with different combinatorial responses to PU.1 and Notch/Delta signals, suggesting particular importance for inhibition of E proteins, Myb, and/or Gfi1 (growth factor independence 1) in diversion. However, Notch signaling only protects against diversion of cells that have undergone T lineage specification after Thy-1 and CD25 up-regulation. The results imply that in T cell precursors, Notch/Delta signaling normally acts to modulate and channel PU.1 transcriptional activities during the stages from T lineage specification until commitment

Constitutive Expression of PU.1 in Fetal Hematopoietic Progenitors Blocks T Cell Development at the Pro-T Cell Stage

AbstractThe essential hematopoietic transcription factor PU.1 is expressed in multipotent thymic precursors but downregulated during T lineage commitment. The significance of PU.1 downregulation was tested using retroviral vectors to force hematopoietic precursors to maintain PU.1 expression during differentiation in fetal thymic organ culture. PU.1 reduced thymocyte expansion and blocked development at the pro-T cell stage. PU.1-expressing cells could be rescued by switching to conditions permissive for macrophage development; thus, the inhibition depends on both lineage and developmental stage. An intact DNA binding domain was required for these effects. PU.1 expression can downregulate pre-Tα, Rag-1, and Rag-2 in a dose-dependent manner, and higher PU.1 levels induce Mac-1 and Id-2. Thus, downregulation of PU.1 is specifically required for progression in the T cell lineage

Subversion of T lineage commitment by PU.1 in a clonal cell line system

Specification of mammalian T lymphocytes involves prolonged developmental plasticity even after lineage-specific gene expression begins. Expression of transcription factor PU.1 may maintain some myeloid-like developmental alternatives until commitment. Commitment could reflect PU.1 shutoff, resistance to PU.1 effects, and/or imposition of a suicide penalty for diversion. Here, we describe subclones from the SCID.adh murine thymic lymphoma, adh.2C2 and adh.6D4, that represent a new tool for probing these mechanisms. PU.1 can induce many adh.2C2 cells to undergo diversion to a myeloid-like phenotype, in an all-or-none fashion with multiple, coordinate gene expression changes; adh.6D4 cells resist diversion, and most die. Diversion depends on the PU.1 Ets domain but not on known interactions in the PEST or Q-rich domains, although the Q-rich domain enhances diversion frequency. Protein kinase C/MAP kinase stimulation can make adh.6D4 cells permissive for diversion without protecting from suicide. These results show distinct roles for regulated cell death and another stimulation-sensitive function that establishes a threshold for diversion competence. PU.1 also diverts normal T-cell precursors from wild type or Bcl2-transgenic mice to a myeloid-like phenotype, upon transduction in short-term culture. The adh.2C2 and adh.6D4 clones thus provide an accessible system for defining mechanisms controlling developmental plasticity in early T-cell development

Genome-wide characterization of pancreatic adenocarcinoma patients using next generation sequencing

Pancreatic adenocarcinoma (PAC) is among the most lethal malignancies. While research has implicated multiple genes in disease pathogenesis, identification of therapeutic leads has been difficult and the majority of currently available therapies provide only marginal benefit. To address this issue, our goal was to genomically characterize individual PAC patients to understand the range of aberrations that are occurring in each tumor. Because our understanding of PAC tumorigenesis is limited, evaluation of separate cases may reveal aberrations, that are less common but may provide relevant information on the disease, or that may represent viable therapeutic targets for the patient. We used next generation sequencing to assess global somatic events across 3 PAC patients to characterize each patient and to identify potential targets. This study is the first to report whole genome sequencing (WGS) findings in paired tumor/normal samples collected from 3 separate PAC patients. We generated on average 132 billion mappable bases across all patients using WGS, and identified 142 somatic coding events including point mutations, insertion/deletions, and chromosomal copy number variants. We did not identify any significant somatic translocation events. We also performed RNA sequencing on 2 of these patients' tumors for which tumor RNA was available to evaluate expression changes that may be associated with somatic events, and generated over 100 million mapped reads for each patient. We further performed pathway analysis of all sequencing data to identify processes that may be the most heavily impacted from somatic and expression alterations. As expected, the KRAS signaling pathway was the most heavily impacted pathway (P<0.05), along with tumor-stroma interactions and tumor suppressive pathways. While sequencing of more patients is needed, the high resolution genomic and transcriptomic information we have acquired here provides valuable information on the molecular composition of PAC and helps to establish a foundation for improved therapeutic selection

Self-productivity and complementarities in human development : evidence from MARS

This paper investigates the role of self-productivity and home resources in capability formation from infancy to adolescence. In addition, we study the complementarities between basic cognitive, motor and noncognitive abilities and social as well as academic achievement. Our data are taken from the Mannheim Study of Children at Risk (MARS), an epidemiological cohort study following the long-term outcome of early risk factors. Results indicate that initial risk conditions cumulate and that differences in basic abilities increase during development. Self-productivity rises in the developmental process and complementarities are evident. Noncognitive abilities promote cognitive abilities and social achievement. There is remarkable stability in the distribution of the economic and socio-emotional home resources during the early life cycle. This is presumably a major reason for the evolution of inequality in human development

Genome-wide association and HLA fine-mapping studies identify risk loci and genetic pathways underlying allergic rhinitis

Allergic rhinitis is the most common clinical presentation of allergy, affecting 400 million people worldwide, with increasing incidence in westernized countries1,2. To elucidate the genetic architecture and understand the underlying disease mechanisms, we carried out a meta-analysis of allergic rhinitis in 59,762 cases and 152,358 controls of European ancestry and identified a total of 41 risk loci for allergic rhinitis, including 20 loci not previously associated with allergic rhinitis, which were confirmed in a replication phase of 60,720 cases and 618,527 controls. Functional annotation implicated genes involved in various immune pathways, and fine mapping of the HLA region suggested amino acid variants important for antigen binding. We further performed genome-wide association study (GWAS) analyses of allergic sensitization against inhalant allergens and nonallergic rhinitis, which suggested shared genetic mechanisms across rhinitis-related traits. Future studies of the identified loci and genes might identify novel targets for treatment and prevention of allergic rhinitis

The CD4+ T-cell transcriptome and serum IgE in asthma: IL17RB and the role of sex

<p>Abstract</p> <p>Background</p> <p>The relationships between total serum IgE levels and gene expression patterns in peripheral blood CD4+ T cells (in all subjects and within each sex specifically) are not known.</p> <p>Methods</p> <p>Peripheral blood CD4+ T cells from 223 participants from the Childhood Asthma Management Program (CAMP) with simultaneous measurement of IgE. Total RNA was isolated, and expression profiles were generated with Illumina HumanRef8 v2 BeadChip arrays. Modeling of the relationship between genome-wide gene transcript levels and IgE levels was performed in all subjects, and stratified by sex.</p> <p>Results</p> <p>Among all subjects, significant evidence for association between gene transcript abundance and IgE was identified for a single gene, the interleukin 17 receptor B (IL17RB), explaining 12% of the variance (r²) in IgE measurement (p value = 7 × 10^-7, 9 × 10^-3after adjustment for multiple testing). Sex stratified analyses revealed that the correlation between IL17RB and IgE was restricted to males only (r²= 0.19, p value = 8 × 10^-8; test for sex-interaction p < 0.05). Significant correlation between gene transcript abundance and IgE level was not found in females. Additionally we demonstrated substantial sex-specific differences in IgE when considering multi-gene models, and in canonical pathway analyses of IgE level.</p> <p>Conclusions</p> <p>Our results indicate that IL17RB may be the only gene expressed in CD4+ T cells whose transcript measurement is correlated with the variation in IgE level in asthmatics. These results provide further evidence sex may play a role in the genomic regulation of IgE.</p

Paired Tumor and Normal Whole Genome Sequencing of Metastatic Olfactory Neuroblastoma

Olfactory neuroblastoma (ONB) is a rare cancer of the sinonasal tract with little molecular characterization. We performed whole genome sequencing (WGS) on paired normal and tumor DNA from a patient with metastatic-ONB to identify the somatic alterations that might be drivers of tumorigenesis and/or metastatic progression.Genomic DNA was isolated from fresh frozen tissue from a metastatic lesion and whole blood, followed by WGS at >30X depth, alignment and mapping, and mutation analyses. Sanger sequencing was used to confirm selected mutations. Sixty-two somatic short nucleotide variants (SNVs) and five deletions were identified inside coding regions, each causing a non-synonymous DNA sequence change. We selected seven SNVs and validated them by Sanger sequencing. In the metastatic ONB samples collected several months prior to WGS, all seven mutations were present. However, in the original surgical resection specimen (prior to evidence of metastatic disease), mutations in KDR, MYC, SIN3B, and NLRC4 genes were not present, suggesting that these were acquired with disease progression and/or as a result of post-treatment effects.This work provides insight into the evolution of ONB cancer cells and provides a window into the more complex factors, including tumor clonality and multiple driver mutations

Single-cell analysis identifies cellular markers of the HIV permissive cell.

Cellular permissiveness to HIV infection is highly heterogeneous across individuals. Heterogeneity is also found across CD4+ T cells from the same individual, where only a fraction of cells gets infected. To explore the basis of permissiveness, we performed single-cell RNA-seq analysis of non-infected CD4+ T cells from high and low permissive individuals. Transcriptional heterogeneity translated in a continuum of cell states, driven by T-cell receptor-mediated cell activation and was strongly linked to permissiveness. Proteins expressed at the cell surface and displaying the highest correlation with T cell activation were tested as biomarkers of cellular permissiveness to HIV. FACS sorting using antibodies against several biomarkers of permissiveness led to an increase of HIV cellular infection rates. Top candidate biomarkers included CD25, a canonical activation marker. The combination of CD25 high expression with other candidate biomarkers led to the identification of CD298, CD63 and CD317 as the best biomarkers for permissiveness. CD25highCD298highCD63highCD317high cell population showed an enrichment of HIV-infection of up to 28 fold as compared to the unsorted cell population. The purified hyper-permissive cell subpopulation was characterized by a downregulation of interferon-induced genes and several known restriction factors. Single-cell RNA-seq analysis coupled with functional characterization of cell biomarkers provides signatures of the "HIV-permissive cell"