Affymetrix GeneChip Mouse Gene Research Articles

Metabolic trade‐off and adipose tissue role in Neonatal Sepsis

BackgroundNeonatal sepsis contributes substantially to neonatal morbidity and mortality. The activation of an inflammatory response by the host triggers a significant metabolic challenge in which mobilization of stored energy is redistributed. Metabolic costs of immunity are high and physiological trade‐offs that neonates suffering from sepsis undergoes can determine the disease tolerance. Adipose tissue (AT) is a highly dynamic endocrine organ that partakes inter‐organs communication and regulates metabolism. In this study, we investigated whether immunity activation due to distinct TLRs ligands in neonatal sepsis promoted an energetic trade‐off and if the AT depots promoted tissue tolerance.MethodsPostnatal day 5 (PD5) CD1 mice were I.P. treated either with saline (Control), LPS, or Pam3CSK4 (PAM). Body weight and temperature were accessed prior treatments and after 3 hours. Oxygen and CO2 consumption was assessed through Comprehensive Lab Animal Monitoring System (CLAMS). Following euthanasia, blood was collected and subcutaneous white AT (WAT) and brown AT (BAT) depots were dissected out and submitted to microarray analysis utilizing Clariom™ S Assay mouse and the Affymetrix GeneChip Mouse Gene ST 2.0 arrays. Blood glucose was assessed using a glucometer.ResultsThe activation of inflammation promoted either by LPS or PAM resulted in significantly reduced total body weight due to the treatment only after 3 hours. We asked if this effect was due to the growth or nutritional status, we found the control pups kept with the mom increased body weight, whereas the pups fasting did not, as expected. However, these changes were not observed in the TLR4 and TLR1/2 ligands treated pups, in which, the bodyweight was decreased independently of them being fasted or not. The cutaneous temperature remained similar in the control group, while LPS induced a loss of ‐1.5°C degrees post‐treatment, and only ‐0.5°C variation was observed in the PAM treated pups. A reduction in VO2 and VCO2 consumption was stronger in LPS than in PAM treated pups, in which they started to increase these levels after 90 minutes, indicating that TLR4 induced energy conservation also in neonates. LPS and PAM induced diminished blood glucose levels compared to control in fasting pups, and only in LPS in fed pups. Next, we evaluated the contribution of AT to lower metabolic rate and promote energy conservation. Microarray analysis showed LPS induced a total of 103 up‐regulated genes, and 238 due to PAM in BAT. Enrichment analysis of this depot was related to Collagen‐containing extracellular matrix, and Inflammatory response in LPS treated pups, while mRNA metabolic process was highlighted in pups treated with PAM. Moreover, in the WAT, LPS promoted enrichment of inflammatory responses along with lipid storage, fatty acid‐binding and lipid metabolism, whereas PAM induced several pathways related to lipid storage, catabolism, and temperature homeostasis.ConclusionsCollectively, these data highlight the physiological trade‐offs occurring between immunity and energy suggesting that the hypometabolic‐hypothermic state is inducing energy conservation. AT depots are promoting fatty acid oxidation and perhaps mediating this energetic trade‐off. Thus, these physiological outcomes demonstrate the importance of AT regulation over the course of neonatal sepsis.

Dysregulation of breathing control through distinct molecular mechanisms of the brainstem cell population in neonatal sepsis

Introduction Neonatal sepsis is triggered by a dysregulated immune response leading to systemic inflammation and life-threatening organ dysfunction. The systemic alterations and activation of immune cells will impact the activity of neural circuits, neural and non-neuronal cell populations, including those in the brainstem involved in breathing rhythm generation and pattern. Elucidating the extent to which inflammation modulates the CNS control of breathing has eluded us due to the lack of appropriate physiological maneuvers capable of interrogating specific brainstem circuits in the neonatal period. We perform novel respiratory testing in newborn pups with unrestrained plethysmography using distinct TLR ligands to test the hypothesis that distinct forms of systemic inflammation modulate brainstem circuits in different ways. Our goal was to investigate how different systemic inflammatory insults at the neonatal period affect the brainstem by determining the contribution of neurons, astrocytes, and microglia in the breathing plasticity response. Methods CD1 mice were I.P. treated at postnatal day 5 (PD5) with LPS, or Pam3CSK4(PAM), or saline (control). Using a whole-body plethysmography system, we assessed brainstem arousal reflex. Briefly, unrestrained pups were placed inside of the chamber individually, and submitted to a room air (RA) exposure for 06 min, followed by 01 min of a gas mixture of 97%N2/3%CO2 (anoxia) repeated 05 times. Ultrasonic vocalization recordings, a measure of brainstem-spinal circuit prolonged-forced expiration, were obtained with the Avisoft SASLab Pro software. Bulk brainstem and gene expression analysis of isolated neurons, astrocytes, and microglia were performed with Clariom™ S Assay mouse and Affymetrix GeneChip Mouse Gene ST 2.0 arrays. Differential gene expression was analyzed using a cutoff of 2-fold change and P-value of 0.01 and t-test in comparison to the control. Results Respiratory frequency during the baseline phase was 176 BPM in the control group, while was founded lower in the LPS and PAM group. Tidal volume (TVb) and minute ventilation (MVb) were also diminished in both groups compared to the control. The gasp latency, or time to recover after each anoxic episode, was prolonged impairing the autoresuscitation, especially, in the PAM treated group. Vocalization function was also affected by inflammation presence resulting in a reduced number of calls per min. A robust gene modulation in the brainstem bulk of PD5 pups was caused in both inflammatory insults. The enrichment analysis showed activation of T helper cells whereas CIBERSORT analysis indicated enrichment of Treg and B cells memory. The molecular pathways activated in neurons, astrocytes, and microglia were founded distinct and specific to the TLR ligand while astrocytes exhibited a lack of responsiveness caused by PAM. Conclusions We conclude that TRLs ligands activate specific neural mechanisms that will detrimentally affect ventilatory response and the ability to autoresuscitate via a central mechanism in the course of neonatal sepsis.

Identification of long non-coding RNA and mRNA expression in βΒ2-crystallin knockout mice.

βΒ2-crystallin (CRYBB2) is expressed at an increased level in the postnatal lens cortex and is associated with cataracts. Improved understanding of the underlying biology of cataracts is likely to be critical for the development of early detection strategies and new therapeutics. The present study aimed to identify long non-coding RNAs (lncRNAs) and mRNAs associated with CRYBB2 knockdown (KO)-induced cataracts. RNAs from 3 non-treated mice and 3 CRYBB2 KO mice were analyzed using the Affymetrix GeneChip Mouse Gene 2.0 ST array. A total of 149 lncRNAs and 803 mRNAs were identified to have upregulated expression, including Snora73b, Klk1b22 and Rnu3a, while the expression levels of 180 lncRNAs and 732 mRNAs were downregulated in CRYBB2 KO mice, including Snord82, Snhg9 and Foxn3. This lncRNA and mRNA expression profile of mice with CRYBB2 KO provides a basis for studying the genetic mechanisms of cataract progression.

The effect of maternal chromium status on lipid metabolism in female elderly mice offspring and involved molecular mechanism.

Maternal malnutrition leads to the incidence of metabolic diseases in offspring. The purpose of this project was to examine whether maternal low chromium could disturb normal lipid metabolism in offspring, altering adipose cell differentiation and leading to the incidence of lipid metabolism diseases, including metabolic syndrome and obesity. Female C57BL mice were given a control diet (CD) or a low chromium diet (LCD) during the gestational and lactation periods. After weaning, offspring was fed with CD or LCD. The female offspring were assessed at 32 weeks of age. Fresh adipose samples from CD–CD group and LCD–CD group were collected. Genome mRNA were analysed using Affymetrix GeneChip Mouse Gene 2.0 ST Whole Transcript-based array. Differentially expressed genes (DEGs) were analysed based on gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis database. Maternal low chromium irreversibly increased offspring body weight, fat-pad weight, serum triglyceride (TG) and TNF-α. Eighty five genes increased and 109 genes reduced in the offspring adipose of the maternal low chromium group. According to KEGG pathway and String analyses, the PPAR signalling pathway may be the key controlled pathway related to the effect of maternal low chromium on female offspring. Maternal chromium status have long-term effects of lipid metabolism in female mice offspring. Normalizing offspring diet can not reverse these effects. The potential underlying mechanisms are the disturbance of the PPAR signalling pathway in adipose tissue.

Subversion of Pkca and Pkcbii Upregulation Play an Essential Role in Chronic Lymphocytic Leukaemia Development, Inducing Constitutive B Cell Receptor-Mediated Signals

B cell receptor (BCR)-mediated signals orchestrate key events during the life cycle of B lymphocytes enabling normal B cell development and maturation. Chronic lymphocytic leukaemia (CLL), an incurable malignancy of mature B cells, displays deregulated BCR-mediated signalling, the intensity of which correlates disease pathogenesis. As such, signals generated upon BCR engagement represent promising targets for novel therapies. Of note, the expression profile of selected protein kinase C (PKC) isoforms, which link proximal BCR mediated signals with downstream pathways, exhibit altered expression patterns in CLL cells: upregulation of PKCβII, PKCε, PKCζ and downregulation of PKCα and PKCβI compared with normal B cells. We previously demonstrated that introduction of a kinase inactive PKCα (PKCα-KR) construct in mouse lymphoid progenitor cells resulted in development of a CLL-like disease both in vitro and in vivo. This model resembles the more aggressive subset of CLL, exhibiting an upregulation of ZAP-70 and elevated ERK-MAPK-mTOR signalling resulting in enhanced proliferation and increased tumor load in the lymphoid organs. Interestingly, reduced PKCα function resulted in PKCβII upregulation, a key pathogenic feature of CLL. Inhibition of PKCβII in these cells with enzastaurin resulted in cell cycle arrest in vitro, reduced tumour load and elevated apoptosis in vivo indicating that PKCβII plays a vital role in maintaining cell survival in our model. To further elucidate the role of PKCβ in leukaemogenesis, we have performed sequential prkcb knockdown (KD) and PKCα-KR expression in the lymphoid progenitor cells. prkcb KD resulted in reduced proliferation and survival concurrent with reduced expression of leukaemic markers (CD23 and CD5) compared to control cells indicating that prkcb plays an essential role in initiation of leukaemogenesis in our model. To identify targets responsible for the regulation for prkcb transcription we performed global gene analysis (Affymetrix GeneChip mouse gene 1.0 ST) comparing MIEV (empty vector control) and PKCα-KR transduced cells at successive time-points mapping critical stages of B-cell transformation, pre- and post PKCβII upregulation. MetaCoreTM analysis revealed that upon upregulation of PKCβII, the BCR-mediated signalling pathway was significantly upregulated in PKCα-KR expressing cells. At the protein level, key hubs proximal to the BCR - Lyn, Btk and Akt - were upregulated, indicating constitutive activation of BCR signalling in the CLL-like PKCα-KR expressing cells. Additionally, proliferative signals downstream of the BCR (mTOR, NF-kB and c-myc) were also upregulated. Treatment of PKCα-KR expressing cells with the Btk inhibitor Ibrutinib (PCI-32765), which has recently been approved for the treatment of CLL, reduced cellular proliferation and inhibited phosphorylation of BtkY223, AktS473 and S6S235/236. Overall, we demonstrate that PKCβII expression is essential for leukaemogenesis and identify key signalling pathways that drive the initiation/development of CLL in the PKCα-KR model. DisclosuresNo relevant conflicts of interest to declare.

Abstract 523: Regulation of Pancreatic β-cell Gene Expression and Function by ABCA1 and ABCG1

Background: Loss-of-function mutations in the ABCA1 gene cause beta cell dysfunction in humans. Insulin secretion is impaired and glucose metabolism is adversely affected in mice with conditional deletion of ABCA1 in beta cells and in ABCG1 knockout mice. These effects are exacerbated in mice with conditional beta cell ABCA1 deletion plus global ABCG1 deficiency. To identify key beta cell pathways regulated by ABCA1 and ABCG1, we generated mice with beta cell-specific deletion of ABCA1 and ABCG1 (ABCA1 β-cell-/- /ABCG1 βcell-/- DKO mice). Aim: Identification of genes in key beta cell metabolic and signal transduction pathways that are regulated by ABCA1 and ABCG1. Methods: Islets were isolated from 16 week old chow fed ABCA1 β-cell-/- /ABCG1 β-cell-/- DKO mice. mRNA was extracted and subjected to Affymetrix GeneChip Mouse Gene ST 2.0 analysis, followed by Partek Genomics Suite and Ingeunity Pathway analysis. Islet cholesterol levels were determined by HPLC. Plasma and HDL cholesterol levels were measured enzymatically. Results: Total plasma cholesterol and HDL cholesterol levels in ABCA1 β-cell-/- /ABCG1 β-cell-/- DKO and Abca1fl/flAbcg1f/fl (control) mice were comparable. Islet cholesterol levels in the ABCA1 β-cell-/- /ABCG1 β-cell-/- DKO mice were increased relative to control (19.2±1.9 vs 8.7±1.0 μg cholesterol/μg protein, p<0.05). A total of 4216 genes were differentially expressed in the ABCA1 β-cell-/- /ABCG1 β-cell-/- DKO mouse islets compared to islets from control mice. Using more stringent criteria (1.2-fold change, p <0.05), 2617 down-regulated and 1599 up-regulated genes were identified in ABCA1 β-cell-/- /ABCG1 β-cell-/- DKO islets compared to control islets. Functional enrichment analysis of the differentially expressed genes revealed that ABCA1 and ABCG1 regulate pathways involved in cholesterol metabolism ( Hmgcr, Insing1, Ldlr, Srebf2 and ApoB ), insulin signaling/glucose metabolism ( Akt1, Fasn, Pik3r1, Irs1, Slc2a1 and Slc2a2 ) and inflammation ( Gbp11, Sftpd ) in beta cells. Differential expression of these genes was validated by real-time PCR. Conclusions: ABCA1 and ABCG1 regulate islet cholesterol levels as well as genes involved in insulin signaling, inflammation and glucose and cholesterol metabolism in beta cells.

Identification of genes with altered expression in male and female Schlager hypertensive mice.

BackgroundNumerous studies have shown sex differences in the onset and severity of hypertension. Despite these sex-differences the majority of animal studies are carried out in males. This study investigated expression changes in both male and female hypertensive mouse kidneys to identify common mechanisms that may be involved in the development of hypertension.MethodsThe Schlager hypertensive mouse model (BPH/2J) and its normotensive control (BPN/3J) were used in this study. Radiotelemetry was performed on 12 to 13 week old BPH/2J and BPN/3J male and female animals. Affymetrix GeneChip Mouse Gene 1.0 ST Arrays were performed in kidney tissue from 12 week old BPH/2J and BPN/3J male and female mice (n = 6/group). Genes that were differentially expressed in both male and female datasets were validated using qPCR.ResultsSystolic arterial pressure and heart rate was significantly higher in BPH/2J mice compared with BPN/3J mice in both males and females. Microarray analysis identified 153 differentially expressed genes that were common between males and females (70 upregulated and 83 downregulated). We validated 15 genes by qPCR. Genes involved in sympathetic activity (Hdc, Cndp2), vascular ageing (Edn3), and telomere maintenance (Mcm6) were identified as being differentially expressed between BPH/2J and BPN/3J comparisons. Many of these genes also exhibited expression differences between males and females within a strain.ConclusionsThis study utilised data from both male and female animals to identify a number of genes that may be involved in the development of hypertension. We show that female data can be used to refine candidate genes and pathways, as well as highlight potential mechanisms to explain the differences in prevalence and severity of disease between men and women.Electronic supplementary materialThe online version of this article (doi:10.1186/s12881-014-0101-x) contains supplementary material, which is available to authorized users.

Renal Tissue Thawed for 30 Minutes Is Still Suitable for Gene Expression Analysis

Some biosamples obtained from biobanks may go through thawing before processing. We aim to evaluate the effects of thawing at room temperature for different time periods on gene expression analysis. A time course study with four time points was conducted to investigate the expression profiling on 10 thawed normal mice renal tissue samples through Affymetrix GeneChip mouse gene 2.0 st array. Microarray results were validated by quantitative real time polymerase chain reactions (qPCR) on 6 candidate reference genes and 11 target genes. Additionally, we used geNorm plus and NormFinder to identify the most stably expressed reference genes over time. The results showed RNA degraded more after longer incubation at room temperature. However, microarray results showed only 240 genes (0.91%) altered significantly in response to thawing at room temperature. The signal of majority altered probe sets decreased with thawing time, and the crossing point (Cp) values of all candidate reference genes correlated positively with the thawing time (p<0.05). The combination of B2M, ACTB and PPIA was identified as the best choice for qPCR normalization. We found most target genes were stable by using this normalization method. However, serious gene quantification errors were resulted from improper reference genes. In conclusion, thirty minutes of thawing at room temperature has a limited impact on microarray and qPCR analysis, gene expression variations due to RNA degradation in early period after thawing can be largely reduced by proper normalization.

Overexpression of miR-9 in mast cells is associated with invasive behavior and spontaneous metastasis

BackgroundWhile microRNA (miRNA) expression is known to be altered in a variety of human malignancies contributing to cancer development and progression, the potential role of miRNA dysregulation in malignant mast cell disease has not been previously explored. The purpose of this study was to investigate the potential contribution of miRNA dysregulation to the biology of canine mast cell tumors (MCTs), a well-established spontaneous model of malignant mast cell disease.MethodsWe evaluated the miRNA expression profiles from biologically low-grade and biologically high-grade primary canine MCTs using real-time PCR-based TaqMan Low Density miRNA Arrays and performed real-time PCR to evaluate miR-9 expression in primary canine MCTs, malignant mast cell lines, and normal bone marrow-derived mast cells (BMMCs). Mouse mast cell lines and BMMCs were transduced with empty or pre-miR-9 expressing lentiviral constructs and cell proliferation, caspase 3/7 activity, and invasion were assessed. Transcriptional profiling of cells overexpressing miR-9 was performed using Affymetrix GeneChip Mouse Gene 2.0 ST arrays and real-time PCR was performed to validate changes in mRNA expression.ResultsOur data demonstrate that unique miRNA expression profiles correlate with the biological behavior of primary canine MCTs and that miR-9 expression is increased in biologically high grade canine MCTs and malignant cell lines compared to biologically low grade tumors and normal canine BMMCs. In transformed mouse malignant mast cell lines expressing either wild-type (C57) or activating (P815) KIT mutations and mouse BMMCs, miR-9 overexpression significantly enhanced invasion but had no effect on cell proliferation or apoptosis. Transcriptional profiling of normal mouse BMMCs and P815 cells possessing enforced miR-9 expression demonstrated dysregulation of several genes, including upregulation of CMA1, a protease involved in activation of matrix metalloproteases and extracellular matrix remodeling.ConclusionsOur findings demonstrate that unique miRNA expression profiles correlate with the biological behavior of canine MCTs. Furthermore, dysregulation of miR-9 is associated with MCT metastasis potentially through the induction of an invasive phenotype, identifying a potentially novel pathway for therapeutic intervention.

Upregulation of APP, ADAM10 and ADAM17 in the Denervated Mouse Dentate Gyrus

The disintegrin and metalloproteinases ADAM10 and ADAM17 are regarded as the most important α-secretases involved in the physiological processing of amyloid precursor protein (APP) in brain. Since it has been suggested that processing of APP by α-secretases could be involved in the reorganization of the brain following injury, we studied mRNA expression of the two α-secretases Adam10 and Adam17, the ß-secretase Bace1, and the App-gene family (App, Aplp1, Aplp2) in the dentate gyrus of the mouse following entorhinal denervation. Using laser microdissection, tissue was harvested from the outer molecular layer and the granule cell layer of the denervated dentate gyrus. Expression levels of candidate genes were assessed using Affymetrix GeneChip Mouse Gene 1.0 ST arrays and reverse transcription-quantitative PCR, revealing an upregulation of Adam10 mRNA and Adam17 mRNA in the denervated outer molecular layer and an upregulation of Adam10 mRNA and App mRNA in the dentate granule cell layer. Immunolabeling for ADAM10 or ADAM17 in combination with markers for astro- and microglia revealed an increased labeling of ADAM10 and ADAM17 in the denervated outer molecular layer that was associated with reactive astrocytes but not with microglia. Collectively, these data show that denervation affects the expression level of APP and its two most important α-secretases. This suggests that APP-processing could be shifted towards the non-amyloidogenic pathway in denervated areas of the brain and, thus, towards the formation of neuroprotective APP cleavage products, such as APPsα.

EPAB is required for expression of cumulus cell markers of oocyte and embryo viability

ObjectiveSeveral cumulus cell transcripts have been identified as potential non-invasive biomarkers of human embryo development and implantation. Embryonic poly(A) binding protein (EPAB) is expressed exclusively in Xenopus and mouse oocytes and early embryos, and is required for oocyte maturation. Importantly, although EPAB is not expressed in cumulus cells, cumulus expansion and ovulation are also impaired in Epab-/- mice. We therefore asked whether the oocyte-specific EPAB is required for the optimal expression of cumulus cell-biomarkers of embryo viability.DesignExperimental study.Materials and MethodsCumulus cells were collected from Epab-/- and Epab+/+ mice 4 hours after hCG injection and three pooled samples were generated for each group. Microarrays were performed for each of the six samples using Affymetrix GeneChip Mouse Gene 1.0 ST Array platform. Probe level expression values were extracted and background adjustment, inter-array quantile normalization and probe set summarization were performed according to the Robust Multi-Chip Average algorithm.Results663 genes were up-regulated and 1205 genes were down-regulated in Epab-/- mice. The expression levels of 47 target genes that were previously identified as potential biomarkers of human embryo viability were cross-analyzed in Epab driven mouse cumulus microarrays. Among those, 13 were found to be differentially expressed in Epab-/- mice, and confirmed by qRT-PCR. These transcripts were previously shown to be associated with oocyte maturation (HAS2, PTGS2), fertilization (HAS2, GREM1, PTGS2, PTX3), early cleavage (HSPB1, TNFAIP6, CCND2), blastocyst development (PTGS2, SDC4), pregnancy (HIST1H4C, CAMK1D, SDC4), and live birth (PTGS2, CAMK1D).ConclusionCandidate cumulus cell marker genes of oocyte and embryo viability identified in human demonstrated altered expression in Epab-deficient female mice where diverse regulations remain to be further investigated. ObjectiveSeveral cumulus cell transcripts have been identified as potential non-invasive biomarkers of human embryo development and implantation. Embryonic poly(A) binding protein (EPAB) is expressed exclusively in Xenopus and mouse oocytes and early embryos, and is required for oocyte maturation. Importantly, although EPAB is not expressed in cumulus cells, cumulus expansion and ovulation are also impaired in Epab-/- mice. We therefore asked whether the oocyte-specific EPAB is required for the optimal expression of cumulus cell-biomarkers of embryo viability. Several cumulus cell transcripts have been identified as potential non-invasive biomarkers of human embryo development and implantation. Embryonic poly(A) binding protein (EPAB) is expressed exclusively in Xenopus and mouse oocytes and early embryos, and is required for oocyte maturation. Importantly, although EPAB is not expressed in cumulus cells, cumulus expansion and ovulation are also impaired in Epab-/- mice. We therefore asked whether the oocyte-specific EPAB is required for the optimal expression of cumulus cell-biomarkers of embryo viability. DesignExperimental study. Experimental study. Materials and MethodsCumulus cells were collected from Epab-/- and Epab+/+ mice 4 hours after hCG injection and three pooled samples were generated for each group. Microarrays were performed for each of the six samples using Affymetrix GeneChip Mouse Gene 1.0 ST Array platform. Probe level expression values were extracted and background adjustment, inter-array quantile normalization and probe set summarization were performed according to the Robust Multi-Chip Average algorithm. Cumulus cells were collected from Epab-/- and Epab+/+ mice 4 hours after hCG injection and three pooled samples were generated for each group. Microarrays were performed for each of the six samples using Affymetrix GeneChip Mouse Gene 1.0 ST Array platform. Probe level expression values were extracted and background adjustment, inter-array quantile normalization and probe set summarization were performed according to the Robust Multi-Chip Average algorithm. Results663 genes were up-regulated and 1205 genes were down-regulated in Epab-/- mice. The expression levels of 47 target genes that were previously identified as potential biomarkers of human embryo viability were cross-analyzed in Epab driven mouse cumulus microarrays. Among those, 13 were found to be differentially expressed in Epab-/- mice, and confirmed by qRT-PCR. These transcripts were previously shown to be associated with oocyte maturation (HAS2, PTGS2), fertilization (HAS2, GREM1, PTGS2, PTX3), early cleavage (HSPB1, TNFAIP6, CCND2), blastocyst development (PTGS2, SDC4), pregnancy (HIST1H4C, CAMK1D, SDC4), and live birth (PTGS2, CAMK1D). 663 genes were up-regulated and 1205 genes were down-regulated in Epab-/- mice. The expression levels of 47 target genes that were previously identified as potential biomarkers of human embryo viability were cross-analyzed in Epab driven mouse cumulus microarrays. Among those, 13 were found to be differentially expressed in Epab-/- mice, and confirmed by qRT-PCR. These transcripts were previously shown to be associated with oocyte maturation (HAS2, PTGS2), fertilization (HAS2, GREM1, PTGS2, PTX3), early cleavage (HSPB1, TNFAIP6, CCND2), blastocyst development (PTGS2, SDC4), pregnancy (HIST1H4C, CAMK1D, SDC4), and live birth (PTGS2, CAMK1D). ConclusionCandidate cumulus cell marker genes of oocyte and embryo viability identified in human demonstrated altered expression in Epab-deficient female mice where diverse regulations remain to be further investigated. Candidate cumulus cell marker genes of oocyte and embryo viability identified in human demonstrated altered expression in Epab-deficient female mice where diverse regulations remain to be further investigated.

Transcriptome Analysis Identifies Regulators of Hematopoietic Stem and Progenitor Cells

SummaryHematopoietic stem cells (HSCs) maintain blood homeostasis and are the functional units of bone marrow transplantation. To improve the molecular understanding of HSCs and their proximal progenitors, we performed transcriptome analysis within the context of the ImmGen Consortium data set. Gene sets that define steady-state and mobilized HSCs, as well as hematopoietic stem and progenitor cells (HSPCs), were determined. Genes involved in transcriptional regulation, including a group of putative transcriptional repressors, were identified in multipotent progenitors and HSCs. Proximal promoter analyses combined with ImmGen module analysis identified candidate regulators of HSCs. Enforced expression of one predicted regulator, Hlf, in diverse HSPC subsets led to extensive self-renewal activity ex vivo. These analyses reveal unique insights into the mechanisms that control the core properties of HSPCs.

Abstract 5547: Exploration of selective small molecule compounds as caspase inhibitors for treatment of oral mucositis caused by cancer therapy.

Abstract Oral mucositis is major and painful side effect, and often a dose limiting factor in head and neck cancer therapy. Our objective is to develop small molecule caspase inhibitors to inhibit apoptosis for cytoprotection of normal cells from harmful side effects of cancer therapy. We previously used the Bionet 37,500 compound library and successfully completed High Throughput Screening (HTS) against caspase-1, 3, and 9 and got 43 hit compounds using the fluorogenic substrates DEVD-AMC and LEHD-AMC. In this study, the 43 hit compounds were screened for cytoprotection activity against staurosporine-induced cell death in NIH3T3 cells by Cell Tito-glo cell viability assay. 19 compounds were found to have significant protective effects. We also confirmed that compound #23 and #27 selected from the above 19 compounds protect NIH3T3 cells from staurosporine-induced caspase activation using cell-based Apo-one Caspase-3/7 activity assay. To explore the molecular mechanisms of the protective effect, a comprehensive phospho and signaling proteomic based study was performed using Phospho Explorer (containing 1328 antibodies) and Signaling Explorer (containing 1358 antibodies) antibody microarray. The results showed that compound#23 or #27 alone treatment induces increase of phosphorylation levels of a spectrum of proteins involved in cell survival signaling including Lyn, LIMK1, IKK-γ, IkB-α, p38 MAPK, NFkB-p65, EGFR and AKT1S. When we compare compound #23 or #27 plus staurosporine group with staurosporine alone group, we discover elevation of phosphorylation of some signaling proteins (c-met, FGFR1, NFAT4, MEK-2 and NFkB-p65) that are linked to cell proliferation and differentiation. In comparison to staurosporine alone treatment, co-treatment of compound #23 or #27 with staurosporine up-regulated the expression levels of some cellular signaling protein targets: HSP90A, RAB40B, FGF22, and CLIP. In addition, our DNA microarray analysis using Affymetrix GeneChip Mouse Gene 1.0ST which covers 26, 166 coding transcripts showed significant expression alterations of many apoptosis and signaling related genes such as Htra2 (Omi), Diablo (Smac), caspase-12 and Tuba1b in staurosporine and compound #23 or #27 plus staurosporine treated samples. Furthermore, compounds # 23 and #27 were also shown to protect human oral keratinocytes (HOK) cells from radiation insult using radiation colony formation assay. Currently, Cal27 and Fadu head and neck cancer cell lines and human normal HOK and HPLF cells are being tested in parallel in terms of cell viability, and apoptosis with anticancer drug Doxorubicin and Etoposide treatments with or without caspase inhibitors. Our study may lead to the discovery of applicable therapeutic drugs to combat oral mucositis caused by radiotherapy, chemotherapy, or combined chemo/radiotherapy. Citation Format: Jianghong Wu, Jie Qian, Lihui Lu, Haiching Ma. Exploration of selective small molecule compounds as caspase inhibitors for treatment of oral mucositis caused by cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5547. doi:10.1158/1538-7445.AM2013-5547

MTOR complex 2 (mTORC2) regulation of hepatic gene expression

mTORC2 has been connected to various disorders, including diabetes and cancer. It is required for insulin‐mediated suppression of hepatic glucose production. Our aim was to examine the role of mTORC2 in the regulation of liver metabolism. Control mice and RKO mice (liver‐specific knockout of Rictor, an mTORC2 constituent) were fasted overnight, fed for 3 hr, then sacrificed. Liver RNA (n = 4 per group) was analyzed using Affymetrix Genechip Mouse Gene 1.0 ST arrays. Gene Set Enrichment Analysis (GSEA) revealed upregulation of three categories of genes in the RKO mice: ribosomal proteins, TCA cycle/oxidative phosphorylation, and components of the 26S proteosome. GSEA results were consistent using both KEGG and Reactome datasets. Ingenuity Pathway Analysis (IPA) showed gene function categories relating to carbohydrate and lipid metabolism. In addition, three transcription factors were significantly altered in the RKO mice. FoxO1 was activated relative to the controls while CTNNB1 and NR1L3 were inactivated. Our results indicate that mTORC2 has broad effects on hepatic gene expression. mTORC2 is linked, in particular, to carbohydrate, lipid and mitochondrial energy metabolism. It also appears to regulate genes involved in ribosome and proteosome biogenesis. Supported by grants from NIH/NIA (DWL), the American Federation of Aging Research (DMS), Howard Hughes Medical Institute (DMS), and NIH/NICHD (PAG).

Effect of Acute Stressor and Serotonin Transporter Genotype on Amygdala First Wave Transcriptome in Mice

The most prominent brain region evaluating the significance of external stimuli immediately after their onset is the amygdala. Stimuli evaluated as being stressful actuate a number of physiological processes as an immediate stress response. Variation in the serotonin transporter gene has been associated with increased anxiety- and depression-like behavior, altered stress reactivity and adaptation, and pathophysiology of stress-related disorders. In this study the instant reactions to an acute stressor were measured in a serotonin transporter knockout mouse model. Mice lacking the serotonin transporter were verified to be more anxious than their wild-type conspecifics. Genome-wide gene expression changes in the amygdala were measured after the mice were subjected to control condition or to an acute stressor of one minute exposure to water. The dissection of amygdalae and stabilization of RNA was conducted within nine minutes after the onset of the stressor. This extremely short protocol allowed for analysis of first wave primary response genes, typically induced within five to ten minutes of stimulation, and was performed using Affymetrix GeneChip Mouse Gene 1.0 ST Arrays. RNA profiling revealed a largely new set of differentially expressed primary response genes between the conditions acute stress and control that differed distinctly between wild-type and knockout mice. Consequently, functional categorization and pathway analysis indicated genes related to neuroplasticity and adaptation in wild-types whereas knockouts were characterized by impaired plasticity and genes more related to chronic stress and pathophysiology. Our study therefore disclosed different coping styles dependent on serotonin transporter genotype even directly after the onset of stress and accentuates the role of the serotonergic system in processing stressors and threat in the amygdala. Moreover, several of the first wave primary response genes that we found might provide promising targets for future therapeutic interventions of stress-related disorders also in humans.

Retinoblastoma treatment: impact of the glycolytic inhibitor 2-deoxy-d-glucose on molecular genomics expression in LHBETATAG retinal tumors

PurposeThe purpose of this study was to evaluate the effect of 2-deoxy-D-glucose (2-DG) on the spatial distribution of the genetic expression of key elements involved in angiogenesis, hypoxia, cellular metabolism, and apoptosis in LHBETATAG retinal tumors.MethodsThe right eye of each LHBETATAG transgenic mouse (n = 24) was treated with either two or six subconjunctival injections of 2-DG (500 mg/kg) or saline control at 16 weeks of age. A gene expression array analysis was performed on five different intratumoral regions (apex, center, base, anterior-lateral, and posterior-lateral) using Affymetrix GeneChip Mouse Gene 1.0 ST arrays. To test for treatment effects of each probe within each region, a two-way analysis of variance was used.ResultsSignificant differences between treatment groups (ie, 0, 2, and 6 injections) were found as well as differences among the five retinal tumor regions evaluated (P < 0.01). More than 100 genes were observed to be dysregulated by ≥2-fold difference in expression between the three treatment groups, and their dysregulation varied across the five regions assayed. Several genes involved in pathways important for tumor cell growth (ie, angiogenesis, hypoxia, cellular metabolism, and apoptosis) were identified.Conclusions2-DG was found to significantly alter the gene expression in LHBETATAG retinal tumor cells according to their location within the tumor as well as the treatment schedule. 2-DG’s effects on genetic expression found here correlate with previous reported results on varied processes involved in its in vitro and in vivo activity in inhibiting tumor cell growth.

Abstract 3865: Regional and temporal variations in gene expression and vasculature during retinoblastoma tumorigenesis and its impact on ocular treatment

Abstract Purpose: The aims of this study were to evaluate the hypothesis that LHBETATAG retinoblastoma tumors exhibit regional and temporal variations in gene expression and vascular development in retinoblastoma. Methods: To evaluate intratumoral gene expression, LHBETATAG mice aged 12, 16, and 20 weeks were euthanized (n=9). Specimens were taken from 5 tumor areas (apex, anterior lateral, center, base, and posterior lateral). Samples were hybridized with an Affymetrix GeneChip Mouse Gene ST 1.0 arrays. To examine the spatial distribution of new and mature blood vessels, immunohistochemical analyses were conducted on enucleated human (n=10) and LHBETATAG retinal tumors from 16-week-old mice (n=11). Results: There were significant temporal differences in gene expression for LHBETATAG retinoblastoma tumors (p&amp;lt;10-8). Genes were identified with a greater than 2.5-fold difference in expression across the three time points with variation in expression depending on region. At p&amp;lt;0.01 and log2 fold change &amp;gt; 2.5 there were significant changes in gene expression for 190 genes apically, 84 genes anterolaterally, 126 genes posteriorly, 56 genes centrally, and 134 genes at the base. Differentially expressed genes were analyzed with significant involvement in angiogenesis (p&amp;lt;10-40, 4 pathways), hypoxia (p&amp;lt;10-7, 140 pathways), and cellular metabolism (p&amp;lt;10-8). Additionally, there were significant spatial changes in the distribution of blood vessels in human and LHBETATAG retinal tumors (p&amp;lt;0.01), which correlated with the gene expression related to angiogenesis. The density of new blood vessels was higher in the periphery of the tumor than in the center (p=0.021) in human retinoblastoma tumors. This finding was mostly attributed to the distribution of large-caliber vessels (i.e., new blood vessels were higher in the periphery for large [p=0.050]- and medium [p=0.032]-caliber vessels; and mature vessels were higher in the center for large-caliber vessels [p=0.032]). In this small series, vessel maturation did not correlate with risk for metastasis. Similar results were observed in LHBETATAG retinal tumors. The percentage of large-caliber new blood vessels was higher in the periphery than in the center (p=0.038). Conclusions: There are significant temporal and regional variations in the gene expression and vasculature of retinoblastoma. Differentially expressed genes overlap with angiogenesis, hypoxia, and cellular metabolism. There is a heterogeneous vessel population in advanced retinoblastoma disease. These findings provide further understanding of the mechanisms involved in tumor progression, and emphasize that tumor biopsies cannot be used for prognostication secondary to tumor regionalization. Finally, optimally-timed adjuvant therapies that target critical components of identified pathways may potentially enhance retinoblastoma tumor control. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3865. doi:10.1158/1538-7445.AM2011-3865

Global identification of the genes and pathways differentially expressed in hypothalamus in early and established neurogenic hypertension

The hypothalamus has an important etiological role in the onset and maintenance of hypertension and stress responses in the Schlager high blood pressure (BP) (BPH/2J) mouse, a genetic model of neurogenic hypertension. Using Affymetrix GeneChip Mouse Gene 1.0 ST Arrays we identified 1,019 hypothalamic genes whose expression differed between 6 wk old BPH/2J and normal BP (BPN/3J) strains, and 466 for 26 wk old mice. Of these, 459 were in 21 mouse BP quantitative trait loci. We validated 46 genes by qPCR. Gene changes that would increase sympathetic outflow at both ages were: Dynll1 encoding dynein light chain LC8-type 1, which physically destabilizes neuronal nitric oxide synthase, decreasing neuronal nitric oxide, and Hcrt encoding hypocretin and Npsr1 encoding neuropeptide S receptor 1, each involved in sympathetic response to stress. At both ages we identified genes for inflammation, such as CC-chemokine ligand 19 (Ccl19), and oxidative stress. Via reactive oxygen species generation, these could contribute to oxidative damage. Other genes identified could be responding to such perturbations. Atp2b1, the major gene from genome-wide association studies of BP variation, was underexpressed in the early phase. Comparison of profiles of young and adult BPH/2J mice, after adjusting for maturation genes, pointed to the proopiomelanocortin-α gene (Pomc) and neuropeptide Y gene (Npy), among others, as potentially causative. The present study has identified a diversity of genes and possible mechanisms involved in hypertension etiology and maintenance in the hypothalamus of BPH/2J mice, highlighting both common and divergent processes in each phase of the condition.

Changes of gene expression profiles in the cervical spinal cord by acupuncture in an MPTP-intoxicated mouse model: Microarray analysis

It has been shown that acupuncture at acupoints GB34 and LR3 inhibits the degeneration of nigrostriatal neurons in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease. The degeneration of spinal cord was reported to be induced in the MPTP-treated pre-symptomatic mouse. In this study, the gene expression profile changes following acupuncture at the acupoints were investigated in the cervical spinal cord of an MPTP-induced parkinsonism model using a whole transcript array (Affymetrix GeneChip mouse gene 1.0 ST array). It was shown that 8 of the probes up-regulated in MPTP, as compared to the control, were down-regulated after acupuncture at the acupoints. Of these 8 probes, 6 probes (4 annotated genes in 6 probes: Ctla2a, EG383229, Ppbp and Ube2l6) were exclusively down-regulated by acupuncture at the specific acupoints except for 2 probes as these 2 probes were commonly down-regulated by acupuncture at both the acupoints and the non-acupoints. In addition, 11 of the probes down-regulated in MPTP, as compared to the control, were up-regulated by acupuncture at the acupoints. Of these 11 probes, 10 probes (5 annotated genes in 10 probes: EG665033, ENSMUSG00000055323, Obox6, Pbp2 and Tmem150) were exclusively up-regulated by acupuncture at the specific acupoints except for the Fut11 because the Fut11 was commonly up-regulated by acupuncture at both the acupoints and the non-acupoints. The expression levels of the representative genes in the microarray were validated by real-time RT-PCR. These data suggest that the expression of these exclusively regulated 16 probes (9 genes) may be, at least in part, affected by acupuncture at the acupoints in the cervical spinal cord which can be damaged by MPTP intoxication.

Neuroprotective Changes of Striatal Degeneration-Related Gene Expression by Acupuncture in an MPTP Mouse Model of Parkinsonism: Microarray Analysis

Acupuncture at acupoints GB34 and LR3 has been reported to inhibit nigrostriatal degeneration in Parkinsonism models, yet the genes related to this preventive effect of acupuncture on the nigrostriatal dopaminergic system remain elusive. This study investigated gene expression profile changes in the striatal region of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinsonism models after acupuncture at the acupoints GB34 and LR3 using a whole transcript genechip microarray (Affymetrix genechip mouse gene 1.0 ST array). It was confirmed that acupuncture at these acupoints could inhibit the decrease of tyrosine hydroxylase and dopamine transporter in the nigrostriatal region of the MPTP model while acupuncture at the non-acupoints could not counteract this decrease. Genechip gene array analysis (fold change cutoff 1.3 and P < 0.05) showed that 12 of the 69 probes up-regulated in MPTP when compared to the control were down-regulated by acupuncture at the acupoints. Of these 12 probes, 11 probes (nine annotated genes) were exclusively down-regulated by acupuncture only at the acupoints; the Gfral gene was excluded because it was commonly down-regulated by acupuncture at both the acupoints and the non-acupoints. In addition, 28 of the 189 probes down-regulated in MPTP when compared to the control were up-regulated by acupuncture at the acupoints. Of these 28 probes, 19 probes (seven annotated genes) were exclusively up-regulated by acupuncture only at the acupoints while nine probes were commonly up-regulated by acupuncture at both the acupoints and the non-acupoints. The regulation patterns of representative genes in real-time RT-PCR correlated with those of the genes in the microarray. These results suggest that the 30 probes (16 annotated genes), which are affected by MPTP and acupuncture only at the acupoints, are responsible for exerting in the striatal regions the inhibitory effect of acupuncture at the acupoints on MPTP-induced striatal degeneration.