Human CD4 positive T cells were isolated from cord blood using CD4 positive isolation kit from Dynal. Cells were activated with plate bound anti-CD3 and soluble anti-CD28 in presence (iTreg) or absence (Th0) of IL2, TGF beta and ATRA. The cells were harvested at 0, 0.5, 1, 2, 4, 6, 12, 24, 48, and 72 hours. Overall design: Comparing the gene expression in activated CD4+ cells and iTreg differentiated cells in human. 9 time points, 3 replicates for each time point.
Transcriptional Repressor HIC1 Contributes to Suppressive Function of Human Induced Regulatory T Cells.
Specimen part, SubjectView Samples
For most multigenic disorders, clinical manifestation (penetrance) and presentation (expressivity) are likely to be an outcome of genetic interaction between multiple susceptibility genes. Here, using gene knockouts in mice we evaluated genetic interaction between loss of Ret and loss of Sema3d, two Hirschsprung disease (HSCR) susceptibility genes. We intercrossed Ret and Sema3d double null heterozygotes to generate mice with the nine possible genotypes and assessed survival by counting various genotypes, myenteric plexus development by acetylcholinesterase (AchE) staining and embryonic day 12.5 (E12.5) gut transcriptome by RNA-sequencing. Survival rates of Ret wildtype, null heterozygote and null homozygote mice at E12.5, birth and weaning were not influenced by the genotypes at Sema3d locus and vice-versa. Loss of myenteric plexus was observed only in all Ret null homozygotes, irrespective of the genotypes at Sema3d locus, and Sema3d null heterozygote and homozygote mice had normal gut innervation. As compared to wildtype mice gut gene expression, loss of Ret in null homozygotes led to differential expression of ~300 genes, whereas loss of Sema3d in null homozygotes had no major consequence and there was no evidence supporting major interaction between the two genes influencing gut transcriptome. Overall, given the null alleles and phenotypic assays used, we did not find evidence for genetic interaction between Ret and Sema3d affecting survival, myenteric plexus formation or gut transcriptome. Overall design: poly-A RNA-seq in embryonic day 12.5 mouse gut from 3 wildtype males, 3 wildtype females, 3 Ret null homozyogote males, 3 Ret null homozyogote females, 3 Sema3d null homozyogote males, 3 Sema3d null homozyogote females, 3 Ret-Sema3d double null homozyogote males, 3 Ret-Sema3d double null homozyogote females
Testing the Ret and Sema3d genetic interaction in mouse enteric nervous system development.
Sex, Specimen part, Cell line, SubjectView Samples
Huntington’s disease (HD) symptoms are driven to a large extent by dysfunction of the basal ganglia circuitry. HD patients exhibit reduced striatal phoshodiesterase 10 (PDE10) levels. Using HD mouse models that exhibit reduced PDE10, we demonstrate the benefit of pharmacologic PDE10 inhibition to acutely correct basal ganglia circuitry deficits. PDE10 inhibition restored corticostriatal input and boosted cortically driven indirect pathway activity. Cyclic nucleotide signaling is impaired in HD models and PDE10 loss may represent a homeostatic adaptation to maintain signaling. Elevation of both cAMP and cGMP by PDE10 inhibition were required for rescue. Phosphoproteomic profiling of striatum in response to PDE10 inhibition highlighted plausible neural substrates responsible for the improvement. Early chronic PDE10 inhibition in Q175 mice showed improvements beyond those seen with acute administration after symptom onset, including partial reversal of striatal deregulated transcripts and the prevention of the emergence of HD neurophysiological deficits. Overall design: Transcriptional profiling of cortex and striatal tissue following chronic dosing of either vehicle or the PDE10A inhibitor PF-02545920 (0.32, 1 and 3.2 mg/kg po qd) in the Q175 homozygous knock-in mouse model of Huntington’s disease (dosing from 5-weeks to 9 months of age).
Phosphodiesterase 10A Inhibition Improves Cortico-Basal Ganglia Function in Huntington's Disease Models.
Sex, Age, Specimen part, Cell line, Treatment, SubjectView Samples
Total mRNA seq was perfomed on widtype and Ret null mouse embryonic gut at 2 stages of devlopment- E11.5 and E12.5 Overall design: Total RNA from 3 replicates each of wildtype and Ret null emryonic gut was converted to cDNA and run on HiSeq 2000 (75 bp paired end)
Enhancer Variants Synergistically Drive Dysfunction of a Gene Regulatory Network In Hirschsprung Disease.
Cell line, SubjectView Samples
Screening small molecules and drugs for activity to modulate alternative splicing, we found that amiloride, distinct from four other intracellular pH-affecting analogues, could normalize the splicing of BCL-X, HIPK3 and RON/MISTR1 transcripts in human hepatocellular carcinoma Huh-7 cells. To elucidate the underlying mechanisms, our proteomic analyses of amiloride-treated cells detected hypo-phosphorylation of splicing factor SF2/ASF and also decreased levels of SRp20 and two un-identified SR proteins. We further observed decreased phosphorylation of AKT, ERK1/2 and PP1, while increased phosphorylation of p38 and JNK, suggesting that amiloride treatment down-regulated kinases and up-regulated phosphatases in the signal pathways known to affect the splicing factor protein phosphorylation. The amiloride effects of splicing factor protein hypo-phosphorylation andnormalizedoncogenic RNA splicing were both abrogated by pre-treatment with a PP1 inhibitor. We then performed global exon array analysis of Huh-7 cells treated with amiloride for 24 hours. Using gene array chips (Affymetrix GeneChip Human Exon 1.0 ST Array of >518000 exons of 42974 genes) for exon array analysis (set parameters of correlation coefficient 0.7, splicing index -1.585 , and log2 ratio -1.585), we found that amiloride influenced the splicing patterns of 551 genes involving at least 584 exons, which included 495 known protein-coding genes involving 526 exons, many of which play key roles in functional networks of ion transport, extracellular matrix, cytoskeletons and genome maintenance. Cellular functional analyses revealed subsequent invasion and migration defects, cell cycle disruption, cytokinesis impairment, and lethal DNA degradation in amiloride-treated Huh-7 cells. This study thus provides mechanistic underpinnings for exploiting small molecule modulation of abnormal RNA splicing for cancer therapeutics.
Small molecule amiloride modulates oncogenic RNA alternative splicing to devitalize human cancer cells.
Cell lineView Samples
To elucidate biological processes underlying the keratocyte, fibroblast, and myofibroblast phenotypes of corneal stromal cells, the gene expression patterns of these primary cultures from mouse cornea were compared with those of the adult and 10-day postnatal mouse cornea.
Microarray studies reveal macrophage-like function of stromal keratocytes in the cornea.
No sample metadata fieldsView Samples
The cornea continues to mature after birth to develop a fully functional, refractive and protective barrier tissue. Here we investigated the complex biological events underlying this process by profiling global genome-wide gene expression patterns of the immature postnatal day 10 and seven week-old adult mouse cornea. The lens and tendon were included in the study to increase the specificity of genes identified as up regulated in the corneal samples. Notable similarities in gene expression between the cornea and the tendon were in the mesenchymal extracellular matrix collagen (types I, III, V, VI) and proteoglycan (lumican, decorin and biglycan) genes. Expression similarities in the cornea and lens were limited to certain epithelial genes and the crystallins. Approximately 76 genes were over expressed in the cornea samples that showed basal expression levels in the lens and tendon. Thirty-two of these were novel with no known functions in the cornea. These include genes with a potential role in protection against oxidative stress (Dhcr24, Cdo1, Akr1b7, Prdx6), inflammation (Ltb4dh, Wdr1), ion-transport (Pdzk1ip1, Slc12a2, Slc25a17) and transcription (Zfp36l3, Pdzk1ip1). Direct comparison of the cornea of two ages showed selective up regulation of 50 and 12 genes in the P10 and adult cornea, respectively. Of the up regulated P10 genes several encode extracellular matrix collagens and proteoglycans that are stable components of the adult cornea and their high transcriptional activity at P10 indicate a period of active corneal growth and matrix deposition in the young cornea. Much less is known about the genes selectively over expressed in the adult cornea; some relate to immune response and innervations (Npy), and possibly to electron transport (Cyp24a1, Cyp2f2) and others of yet unknown functions in the cornea (Rgs10, Psmb8, Xlr4)). This study detected expression of genes with known functions in the cornea, providing additional validation of the microarray experiments. Importantly, it identified several novel genes whose functions have not been investigated in the cornea.
Differential gene expression patterns of the developing and adult mouse cornea compared to the lens and tendon.
No sample metadata fieldsView Samples
The ability of high-risk neuroblastoma to survive unfavorable growth conditions and multimodal therapy is hypothesized to result from a phenomenon known as reversible adaptive plasticity (RAP). RAP is a novel phenomenon enabling neuroblastoma cells to transition between a proliferative anchorage dependent (AD) state and a slow growing anoikis-resistant anchorage independent (AI) state.
A mechanism linking Id2-TGFβ crosstalk to reversible adaptive plasticity in neuroblastoma.
Specimen partView Samples
Identification of genes and pathways relevant to Cervical cancer pathogenesis. The study also aimed at identifying probable mechanistic differences in the low and high HOTAIR expressing cervical cancers patients .
Bridging Links between Long Noncoding RNA HOTAIR and HPV Oncoprotein E7 in Cervical Cancer Pathogenesis.
Age, Specimen partView Samples
It is becoming better understood that radiation resistance in glioblastomas (GBMs) may be secondary to a self-renewing subpopulation of cells in the bulk tumor that form neurospheres in culture. This population has been referred to as Glioma stem cells (GSCs). One of the limitations regarding the use of GSCs is that these studies require fresh tumor biopsy samples obtained from patients, and can be extremely difficult to culture, propagate, and perform treatment-response assays. This report describes the generation of a self-renewing population of GSCs derived from commercially available U87 cells using NOD-SCID mice as carrier. The tumors were dissociated to obtain GSCs that demonstrate stem-like properties and high degree of chemo and radiation resistance. Pathological analysis of tumors obtained using GSCs exhibit all the histological hallmarks of human GBMs which is quite uncommon in GBM rodent models and hence could serve as a better model for pre-clinical study. We have shown that MGH87GSCs have an enhanced tumorogenicity than parental U87 and about 500 cells are sufficient to form tumors. To understand the transcriptome and accompanied proteome better, we explored the gene expression profiles of MGH87GSC and U87. We have shown that these GSCs are plastic like stem cells and can be directed towards a particular progeny within neural lineage by providing suitable growth factor. Our objective was to understand the genetic and biochemical mechanisms that control the self-renewal phenotype, asymmetric subdivision, chemo and radiation resistance and the role of the GSC niche in regulating the biological properties of GSC. Through this model we anticipate to devise therapeutic strategies to target this sub population of GSCs within GBMs to eradicate treatment resistance and tumor recurrence.
Cells isolated from residual intracranial tumors after treatment express iPSC genes and possess neural lineage differentiation plasticity.
Specimen part, Cell lineView Samples