GAPDH mRNA Research Articles

Activation of the Leukotriene B4 Receptor 2-Reactive Oxygen Species (BLT2-ROS) Cascade following Detachment Confers Anoikis Resistance in Prostate Cancer Cells

The majority of prostate cancer-related deaths are associated with advanced and metastatic malignancies. Although anoikis resistance has been recognized as one of the hallmarks of metastatic prostate malignancies, the molecular events that cause anoikis resistance are poorly understood. In this study, we found that the detachment of PC-3 prostate cancer cells caused a time-dependent increase in the expression level of the leukotriene B4 receptor-2 (BLT2) and that BLT2 played a critical role in establishing anoikis resistance in these cells. Blocking BLT2 with the pharmacological inhibitor LY255283 or with RNAi knockdown clearly abolished anoikis resistance and resulted in severe apoptotic death. Additionally, we demonstrated that the activation of NADPH oxidase (NOX) and subsequent generation of reactive oxygen species (ROS) were downstream of BLT2 signaling and led to the activation of NF-κB, thus establishing anoikis resistance during cell detachment. Furthermore, we observed that the ectopic expression of BLT2 in normal prostate PWR-1E cells rendered the cells resistant to anoikis and apparently diminished apoptotic cell death following detachment. Taken together, our results suggest that BLT2-NOX-ROS-NF-κB cascade induction during detachment confers a novel mechanism of anoikis resistance in prostate cancer cells and potentially contributes to prostate cancer progression.

Validation of Suitable Reference Genes for Expression Studies in Different Pilocarpine-Induced Models of Mesial Temporal Lobe Epilepsy

It is well recognized that the reference gene in a RT-qPCR should be properly validated to ensure that gene expression is unaffected by the experimental condition. We investigated eight potential reference genes in two different pilocarpine PILO-models of mesial temporal lobe epilepsy (MTLE) performing a stability expression analysis using geNorm, NormFinder and BestKepeer softwares. Then, as a validation strategy, we conducted a relative expression analysis of the Gfap gene. Our results indicate that in the systemic PILO-model Actb, Gapdh, Rplp1, Tubb2a and Polr1a mRNAs were highly stable in hippocampus of rats from all experimental and control groups, whereas Gusb revealed to be the most variable one. In fact, we observed that using Gusb for normalization, the relative mRNA levels of the Gfap gene differed from those obtained with stable genes. On the contrary, in the intrahippocampal PILO-model, all softwares included Gusb as a stable gene, whereas B2m was indicated as the worst candidate gene. The results obtained for the other reference genes were comparable to those observed for the systemic Pilo-model. The validation of these data by the analysis of the relative expression of Gfap showed that the upregulation of the Gfap gene in the hippocampus of rats sacrificed 24 hours after status epilepticus (SE) was undetected only when B2m was used as the normalizer. These findings emphasize that a gene that is stable in one pathology model may not be stable in a different experimental condition related to the same pathology and therefore, the choice of reference genes depends on study design.

Differential response of IVP, parthenogenetic and nuclear transfer derived bovine embryos upon environmental heat stress-implications for expression of autosomal and X-linked genes

The present study was conducted to examine the metabolic activities and expression differences of X-linked and autosomal genes upon exposure to heat stress using different types of bovine embryos. Embryos from in vitro fertilized (IVF), parthenogenetic (PA) and nuclear transfer (NT) were given heat treatment at morula stages at 40°C for 6 hrs. Treated embryos were studied for metabolic activities by quantification of G6PD-activity through Brilliant Cresyl Blue (BCB) staining and were used for X-linked and autosomal gene expression studies. Quantification of G6PD, PGK1, XIST as sex linked and SOX, BAX, OCT4, HSP70.1 as autosomal genes and GAPDH (endogenous control) mRNAs in each cDNA sample from heat treated and untreated embryos were assessed by quantitative PCR. Results of the present study indicate sexually dimorphic expression of selected X-linked and autosomal genes upon exposure to heat stress at morula stage in IVF, PA and NT embryos. Moreover, our study showed that female IVP derived embryos show highly decreased levels of XIST after heat shock. That could be one of the reasons for reduced in vitro developmental competence of female bovine embryos since XIST is the responsible gene for X-chromosome inactivation. Any disturbance would result in dysregulation of all X-Chromosomal encoded genes. DOI: http://dx.doi.org/10.3329/bjas.v42i1.15759 Bang. J. Anim. Sci. 2013. 42 (1): 1-10

Gene expression analysis in Merkel cell polyomavirus‐positive non‐small cell lung cancer from Japanese patients

We read with great interest the recent article by Lasithiotaki et al. entitled “The presence of Merkel cell polyomavirus is associated with deregulated expression of BRAF and Bcl-2 genes in non-small cell lung cancer.”1 This very interesting report gives a new insight into the currently important issue of the infectious causes of cancer. The analysis presented in this study was based on 110 patients with non-small cell lung cancer (NSCLC) in Crete, Greece. The authors found that 9.1% (10/110) of NSCLC specimens were positive for the presence of Merkel cell polyomavirus (MCPyV), identified by polymerase chain reaction (PCR). The article also provided interesting data on the expression patterns of seven cellular genes in the MCPyV-positive NSCLC specimens by comparing 6 MCPyV-positive and 10 MCPyV-negative samples. Around the same time, we reported on our analysis of 112 Japanese NSCLC patients with an MCPyV detection rate of 17.9% (20/112).2 The viral prevalence rate was higher in these Japanese NSCLC patients than in the Greek patients reported by Lasithiotaki et al. In our study, MCPyV was detected more frequently in never smokers than in smokers, although Lasithiotaki et al. showed a positive trend for smoking status. Recent studies have indicated that the increase in the incidence of lung cancer in never smokers is higher in East Asia, including Japan, than in the United States and European countries.3-5 Thus, the incidence of lung cancer in never smokers seems to vary between geographic locations or ethnic groups; the higher prevalence in Asia may account for the discordance between our study and that by Lasithiotaki et al. Japanese ethnicity compared with Caucasian ethnicity and never-smoker status are independent favorable prognostic factors for overall survival in NSCLC patients.6 In addition, Asians and Caucasians may have different genetic susceptibilities to lung cancer, as evidenced by gene polymorphisms and genome-wide association studies. For example, East Asian patients with NSCLC have a much higher prevalence of mutation of the “epidermal growth factor receptor” gene and a lower prevalence of mutation of the K-ras gene. These clinical and genetic differences between East Asians and Caucasians prompted us to investigate the gene-expression patterns in MCPyV-positive NSCLC samples from Japanese patients and to compare the results with those reported by Lasithiotaki et al. Our study included 27 surgically resected frozen tumors from Japanese NSCLC patients, including 12 squamous cell carcinomas (SCCs) and 15 adenocarcinomas. Nine samples (four SCCs and five adenocarcinomas) were positive for MCPyV and 18 samples (8 SCCs and 10 adenocarcinomas) were negative. Total RNA was extracted with the High Pure RNA Tissue Kit (Roche Diagnostics, Tokyo, Japan). The extracted RNA was treated with DNase to avoid amplification of DNA. One microgram of RNA was reverse transcribed using the SuperScript III First-Strand Synthesis System (Life Technologies, Tokyo, Japan). The transcript levels of the K-ras, BRAF, RKIP, p53, RB1, Bcl-2, and Bax genes were measured using the StepOne Plus real-time PCR system with SYBER Green PCR Master Mix (Life Technologies). Primer sequences and PCR conditions were the same as those used by Lasithiotaki et al. All reactions were run in triplicate. mRNA of the housekeeping gene GAPDH was used as an internal control to normalize mRNA expression levels. Relative gene expression was calculated by the 2–ΔΔCt method, as described.1, 7 The gene expression levels were first evaluated by the F-test to determine whether they followed a normal distribution pattern. Depending on the results, the Aspin–Welch's t test or Student's t test was used to compare the gene expression levels between MCPyV-positive and MCPyV-negative samples. A p-value less than 0.05 was considered significant. This study was approved by the Ethics Committee of Kochi Medical School, Kochi University. As shown in Table 1, the transcript levels of the K-ras, BRAF, RKIP, p53, RB1, Bcl-2, and Bax genes did not differ statistically between MCPyV-positive and MCPyV-negative NSCLC samples. However, we found a trend toward higher expression levels of the Bcl-2 gene in MCPyV-positive samples than in negative samples (p = 0.073). This trend was noticeable in MCPyV-positive adenocarcinoma (p = 0.012). Considering the antiapoptotic effect of the Bcl-2 gene, it is conceivable that this proto-oncogene might be associated, at least partially, with the pathogenesis of MCPyV-infected NSCLC in a subset of Japanese patients. Our results did not match exactly the findings of Lasithiotaki et al., who found downregulation of the Bcl-2 gene and upregulation of the BRAF gene in MCPyV-positive patients. These two reports clearly illustrate the need for further worldwide large-scale studies to elaborate on possible ethnic differences in the genetic pathogenecity of MCPyV-associated NSCLC. Yours sincerely, Yumiko Hashida, Masayuki Imajoh, and Masanori Daibata

AB0289 Can cd11c expression successfully predict response to etanercept in rheumatoid arthritis patients?

BackgroundThe treatment of rheumatoid arthritis (RA) with biologic drugs such as anti-tumor necrosis factor alpha (TNF) has significantly improved the clinical outcome in patients; however, 30-40% of patients fail to...

Spdef Null Mice Lack Conjunctival Goblet Cells and Provide a Model of Dry Eye

Goblet cell numbers decrease within the conjunctival epithelium in drying and cicatrizing ocular surface diseases. Factors regulating goblet cell differentiation in conjunctival epithelium are unknown. Recent data indicate that the transcription factor SAM-pointed domain epithelial-specific transcription factor (Spdef) is essential for goblet cell differentiation in tracheobronchial and gastrointestinal epithelium of mice. Using Spdef(-/-) mice, we determined that Spdef is required for conjunctival goblet cell differentiation and that Spdef(-/-) mice, which lack conjunctival goblet cells, have significantly increased corneal surface fluorescein staining and tear volume, a phenotype consistent with dry eye. Microarray analysis of conjunctival epithelium in Spdef(-/-) mice revealed down-regulation of goblet cell-specific genes (Muc5ac, Tff1, Gcnt3). Up-regulated genes included epithelial cell differentiation/keratinization genes (Sprr2h, Tgm1) and proinflammatory genes (Il1-α, Il-1β, Tnf-α), all of which are up-regulated in dry eye. Interestingly, four Wnt pathway genes were down-regulated. SPDEF expression was significantly decreased in the conjunctival epithelium of Sjögren syndrome patients with dry eye and decreased goblet cell mucin expression. These data demonstrate that Spdef is required for conjunctival goblet cell differentiation and down-regulation of SPDEF may play a role in human dry eye with goblet cell loss. Spdef(-/-) mice have an ocular surface phenotype similar to that in moderate dry eye, providing a new, more convenient model for the disease.

Abstract 4914: ABCB5 expression in colorectal cancer circulating tumor cells correlates with metastatic disease progression.

Abstract Targeted personalized treatment approaches are critical for the development of successful therapies for advanced colorectal cancer (CRC). In this regard, circulating tumor cells (CTC) are of foremost interest as potential predictors of metastatic disease progression and patient therapeutic response. We have previously demonstrated that ATP-binding cassette member B5 (ABCB5) is expressed by cancer stem cells (CSC) in several human malignancies, including CRC, and that ABCB5 identifies therapy-refractory tumor subpopulations in CRC patients that correlate with disease progression. In order to evaluate the potential role of ABCB5 as a clinically relevant CTC marker, we utilized a human-to-mouse xenograft model system to examine the relationship of ABCB5-positive CRC-CTC to CRC metastasis. Human CRC tumor cells were grafted subcutaneously into immunocompromized NSG mice. 7 weeks after xenotransplantation, peripheral blood mononuclear cells and lungs were evaluated by qPCR for expression of human ABCB5 and GAPDH mRNAs. Expression analysis of these specimens revealed a significant positive correlation between peripheral blood human ABCB5 mRNA expression and aggregate metastatic disease load as determined by human GAPDH mRNA expression levels in lungs (r=0.8994, P=0.0147). Remarkably, no significant correlation existed between peripheral blood human GAPDH mRNA expression and metastatic disease load, indicating a unique and essential role of ABCB5-positive CSC-CTC in colorectal cancer metastatic disease progression. Significantly increased peripheral blood ABCB5 expression was also observed in a clinical series of UICC stages I-III colorectal cancer patients (n=105) compared to healthy controls (P<0.001). Thus, our results identify the CSC antigen ABCB5 as a novel molecular marker of metastasis-associated CRC-CTC and point to potential utility of ABCB5 as a prognostic indicator and predictor of therapeutic response in patients with advanced colorectal cancer. Citation Format: Qin Guo, Jie Ma, Brian J. Wilson, Martin Gasser, Ana Maria Waaga-Gasser, Markus H. Frank, Natasha Y. Frank. ABCB5 expression in colorectal cancer circulating tumor cells correlates with metastatic disease progression. [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 4914. doi:10.1158/1538-7445.AM2013-4914

Human IL10 Gene Repression by Rev-erbα Ameliorates Mycobacterium tuberculosis Clearance

Nuclear receptors modulate macrophage effector functions, which are imperative for clearance or survival of mycobacterial infection. The adopted orphan nuclear receptor Rev-erbα is a constitutive transcriptional repressor as it lacks AF2 domain and was earlier shown to be present in macrophages. In the present study, we highlight the differences in the relative subcellular localization of Rev-erbα in monocytes and macrophages. The nuclear localization of Rev-erbα in macrophages is subsequent to monocyte differentiation. Expression analysis of Rev-erbα elucidated it to be considerably more expressed in M1 phenotype in comparison with M2. Rev-erbα overexpression augments antimycobacterial properties of macrophage by keeping IL10 in a basal repressed state. Further, promoter analysis revealed that IL10 promoter harbors a Rev-erbα binding site exclusive to humans and higher order primates and not mouse, demonstrating a species barrier in its functionality. This direct gene repression is mediated by recruitment of co-repressors NCoR and HDAC3. In addition, our data elucidate that its overexpression reduced the survival of intracellular pathogen Mycobacterium tuberculosis by enhancing phagosome lysosome maturation, an event resulting from IL10 repression. Thus, these findings suggest that Rev-erbα bestows protection against mycobacterial infection by direct gene repression of IL10 and thus provide a novel target in modulating macrophage microbicidal properties.

Protein Phosphatase 2A Catalytic Subunit α Plays a MyD88-Dependent, Central Role in the Gene-Specific Regulation of Endotoxin Tolerance

MyD88, the intracellular adaptor of most TLRs, mediates either proinflammatory or immunosuppressive signaling that contributes to chronic inflammation-associated diseases. Although gene-specific chromatin modifications regulate inflammation, the role of MyD88 signaling in establishing such epigenetic landscapes under different inflammatory states remains elusive. Using quantitative proteomics to enumerate the inflammation-phenotypic constituents of the MyD88 interactome, we found that in endotoxin-tolerant macrophages, protein phosphatase 2A catalytic subunit α (PP2Ac) enhances its association with MyD88 and is constitutively activated. Knockdown of PP2Ac prevents suppression of proinflammatory genes and resistance to apoptosis. Through site-specific dephosphorylation, constitutively active PP2Ac disrupts the signal-promoting TLR4-MyD88 complex and broadly suppresses the activities of multiple proinflammatory/proapoptotic pathways as well, shifting proinflammatory MyD88 signaling to a prosurvival mode. Constitutively active PP2Ac translocated with MyD88 into the nuclei of tolerant macrophages establishes the immunosuppressive pattern of chromatin modifications and represses chromatin remodeling to selectively silence proinflammatory genes, coordinating the MyD88-dependent inflammation control at both signaling and epigenetic levels under endotoxin-tolerant conditions.

MUC1 and toll-like receptor-2 expression in burning mouth syndrome and oral lichen planus

ObjectiveExpression levels of MUC1 and TLR-2 were evaluated in burning mouth syndrome (BMS) patients and compared with those of controls and oral lichen planus (OLP) patients. The relationships between the expression levels of MUC1 and TLR-2 and levels of salivary pro-inflammatory cytokines were also investigated. DesignTen female BMS and ten female OLP patients were included. Ten female age-matched volunteers served as controls. RNA was isolated from stimulated whole saliva samples. Real-time PCR was used to quantify MUC1 and TLR-2 mRNA levels relative to β-actin and GAPDH mRNA levels. The clarified supernatants of saliva samples were used to measure IL-1β, IL-6, IL-8, and TNF-α levels. The level of blood contamination in saliva samples was also determined. ResultsThere were significant increases in MUC1 transcripts in BMS patients compared with OLP patients (1.766-fold) as well as controls (1.840-fold). There was no significant difference in TLR-2 expression among the groups. The OLP patients showed significantly higher levels of IL-6 and blood contamination in saliva than other groups. The levels of MUC1 or TLR-2 expression did not correlate significantly with the levels of cytokines or blood contamination in saliva. ConclusionsMUC1 may play a role in the development and/or progression of BMS.

The origins of glucosylsphingosine in Gaucher disease

p For nearly forty years, the accumulation of GlcCer and GlcSph has been demonstrated in affected tissues of Gaucher patients - both in the viscera and CNS. Despite its lower abundance when compared to GlcCer, GlcSph has been shown to be cytotoxic to cells at very low concentrations. Moreover, clinical evidence supports that elevated levels of GlcSph play a role in Gaucher disease pathology. i a scrambled siRNA control. Results : Cells incubated with CBE had increased levels of GlcCer. In the absence of CBE, cells had very low levels of GlcSph, but GlcSph levels increased significantly when cultured in the presence of CBE. However, cells incubated with ASAH1 siRNA showed a significant decrease (~50%) in GlcSph formation. No decrease in GlcSph levels were seen with scrambled or GAPDH siRNAs. Both GAPDH and ASAH1 mRNA levels were reduced by >90% when respective siRNA oligos were transfected into the cells. For lipid levels, columns represent mean + SD

Разработка нового метода детектирования экспрессии генов с помощью количественной ОТ-ПЦР единичных клеток без выделения РНК в сочетании с лазерной захватывающей микродиссекцией

Laser capture microdissection (LCM) combined with real time RT-PCR represents a powerful method for analyzing gene expression levels in selected cell types. To avoid degradation of RNA by endogenous and exogenous RNases and to ensure selection of target cells only, we designed a protocol in which the inactivation of exogenous and endogenous RNases by RNaseZap and RNA later is combined with immunofluorescent labeling, and labeled cells in sections used for subsequent LCM and real time RT-PCR. Immunolabelled neurons were captured onto the caps of RNase-free microcentrifuge tubes using LCM and lysed with 3% NP-40 without prior RNA purification. Subsequent reverse transcription for cDNA synthesis was performed in situ on the cap surface to avoid mRNA loss due to transfer between tubes. Applying this protocol, we determined immunoglobulin G (IgG), nerve growth factor (NGF) and GAPDH mRNA levels into small numbers of captured neurons with real time RT-PCR. Thus, this novel method combining LCM with real time RT-PCR without RNA purification appears to be an effective tool for the analysis of cell-specific target gene transcription in small numbers of cells isolated from RNA later-treated tissues.

An Efficient Method for Isolation of Hantaan Virus through Serial Passages in Suckling Mice

Objective: Hantaan virus (HTNV) is one of the main etiologic agents for hemorrhagic fever with renal syndrome in China. However, it is very difficult to isolate the virus from its original host, which hampers the viral characterization. This study describes an efficient method for isolating HTNV in suckling mice. Methods: Hantavirus-infected Apodemus agrarius were screened by quantitative real-time PCR. The homogenates of one positive rodent lung tissue were inoculated into suckling mice for virus propagation through serial passages. Results: During the three passages in suckling mice, the number of viral RNA copies/nanogram of GAPDH mRNA increased significantly ranging from 477 to 7,278 and 46 to 4,898 in the tissues of brain and lung, respectively. Hantaviral antigens could be detected by indirect immunofluorescence assay and around 100-nm virion-like structures were also observed in brain tissue by transmission electron microscopy. No nucleotide exchange was found except for one in the 3′-non-coding domain of S segment when comparing the complete genome sequences from hantavirus in the first and the third passages. Conclusion: These results suggest inoculation of suckling mice with suspected hantavirus-infected rodent samples is an efficient method for isolation and maintenance of HTNV.

Impairment of HuR-Mediated FOS mRNA Stabilization in Granulocytes From Myelodysplastic Syndrome Patients.

Abstract 2805Infection is a major cause of death in patients with myelodysplastic syndromes (MDS). Although qualitative and quantitative granulocyte defects have been described, the underlying molecular basis of granulocyte dysfunction in MDS is largely unknown. Recently, we found that FOS mRNA elevation following translation inhibition was significantly smaller in MDS granulocytes than in healthy granulocytes (Shikama et al, Br J Haematol 154, 525–527, 2011). FOS is an immediate early gene that is rapidly expressed in response to various stimuli and cellular stresses, and possesses an AU-rich element (ARE) in its mRNA 3’UTR, to which mRNA stability-regulating proteins, such as HuR, bind. The aim of this study was to clarify the cause of impaired FOS induction following translation inhibition in MDS.We first confirmed impaired FOS induction in MDS by comparing 17 patients with MDS (refractory anemia) with 17 age-matched healthy controls. Granulocyte fractions, enriched to >90% neutrophils, were cultured with 200 ng/mL of the translation inhibitor emetine at 37 °C for 30 min. FOS mRNA increased 6.2±1.1-fold in controls, vs. 3.5±0.8-fold in MDS (p<0.01). In contrast, when 5 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF) or 100 ng/mL lipopolysaccharide (LPS) was added instead of emetine, there were no significant differences in the FOS mRNA elevation between controls and MDS (GM-CSF: 2.4±0.5-fold vs. 2.3±1.3-fold, LPS: 2.2±0.9-fold vs. 2.4±1.1-fold), suggesting that FOS mRNA induction by a translation inhibitor was specifically impaired in MDS granulocytes.To study the mechanisms of FOS mRNA elevation by emetine, we next examined the involvement of (1) MAPK signaling pathways, (2) FOS transcription and mRNA stabilization, and (3) ARE-binding protein HuR, using healthy granulocytes. Inhibition of MAPK p38, which completely blocked the FOS-upregulating effect of LPS, reduced FOS elevation by emetine to nearly 50%. Neither inhibition of JNK nor ERK altered the effects of emetine. Thus, emetine increases FOS mRNA via MAPK p38-dependent and MAPK-independent mechanisms. Emetine treatment for 30 min increased nascent FOS mRNA 3.5±0.8-fold, which was reduced to 1.1±0.8-fold by MAPK p38 inhibition (p<0.01). When transcription was inhibited, FOS mRNA decayed to 19.2±9.1% of the initial level in 45 min. In the presence of emetine, 78.1±23.5% of FOS mRNA remained at 45 min, and was not affected by p38 inhibition. These data indicate that FOS mRNA elevation by emetine results from both upregulation of transcription via MAPK p38 and p38-independent mRNA stabilization. RNA-protein complex immunoprecipitation showed that 2.5±1.2-fold more FOS mRNA 3’UTR coprecipitated with HuR in emetine-treated cells than in untreated cells (p<0.05). GAPDH mRNA 3’UTR without ARE was not detected regardless of emetine treatment. Although HL60 cells treated with HuR and control siRNA showed similar FOS mRNA decay rates in the absence of emetine, emetine treatment led to a significantly lower FOS mRNA at 45 min in HuR-reduced cells (48.0±12.8%) than in control cells (77.4±13.9%, p<0.05). Thus, emetine stabilizes FOS mRNA via upregulated interaction of HuR with FOS mRNA.Finally, we investigated which process was impaired in MDS, p38-mediated FOS transcription, or HuR-mediated mRNA stabilization. The increase of FOS transcription in MDS (2.0±0.3-fold) did not significantly differ from that in controls (2.4±0.3-fold). When FOS transcription was blocked by a MAPK p38 inhibitor, FOS mRNA increase by emetine was significantly smaller in MDS (2.9±0.7-fold) than in controls (4.4±1.5-fold, p<0.05), suggesting that the aberrant FOS induction in MDS was not due to impaired FOS transcription. FOS mRNA, which decayed similarly in MDS and control cells in the absence of emetine, remained 41.9±24.6% and 61.0±19.9% in MDS and control granulocytes, respectively, at 45 min in the presence of emetine (p<0.05).In conclusion, the aberrant FOS mRNA induction by emetine in MDS results from the impairment of HuR-mediated FOS mRNA stabilization. Since nearly 15% of the transcriptome in mammalian cells is targeted by HuR, these results may provide new insights into the functional defects in MDS. Disclosures:No relevant conflicts of interest to declare.

Matrigel plug assay: evaluation of the angiogenic response by reverse transcription-quantitative PCR

The subcutaneous Matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic molecules. However, quantification of the angiogenic response in the plug remains a problematic task. Here we report a simple, rapid, unbiased and reverse transcription-quantitative PCR (RT-qPCR) method to investigate the angiogenic process occurring in the Matrigel plug in response to fibroblast growth factor-2 (FGF2). To this purpose, a fixed amount of human cells were added to harvested plugs at the end of the in vivo experimentation as an external cell tracer. Then, mRNA levels of the pan-endothelial cell markers murine CD31 and vascular endothelial-cadherin were measured by species-specific RT-qPCR analysis of the total RNA and data were normalized for human GAPDH or β-actin mRNA levels. RT-qPCR was used also to measure the levels of expression in the plug of various angiogenesis/inflammation-related genes. The procedure allows the simultaneous, quantitative evaluation of the newly-formed endothelium and of non-endothelial/inflammatory components of the cellular infiltrate in the Matrigel implant, as well as the expression of genes involved in the modulation of the angiogenesis process. Also, the method consents the quantitative assessment of the effect of local or systemic administration of anti-angiogenic compounds on the neovascular response triggered by FGF2.

Increased Death of Adipose Cells, a Path to Release Cell‐Free DNA Into Systemic Circulation of Obese Women

Remodeling of adipose tissue is required to support the expansion of adipose mass. In obesity, an increased death of adipocytes contributes to the accelerated cellular turnover. We have shown that obesity in pregnancy is associated with metabolic and immune alterations in the adipose tissue. In this study we characterized the mechanisms responsible for increased death of adipose cells of pregnant obese women and its functional consequences. We postulated that a higher turnover of dead cells in white adipose tissue of obese women would translate into release of cell free DNA (cfDNA) into their systemic circulation.Increase in adipose mass of obese compared to lean women results from a lesser number of hypertrophic adipocytes and an accumulation of macrophages in the stromal vascular fraction (SVF). The adipocytes of obese displayed enhanced necrosis with a loss of perilipin staining at the plasma membrane. Apoptosis was prominent in SVF cells with an increased expression of caspase 9 and caspase 3 and a higher rate of TUNEL positive CD68 macrophages in obese vs lean. Whereas circulating fetal cfDNA concentrations were not changed, there was a 2-fold increase in circulating GAPDH cfDNA and adipose tissue GAPDH mRNA in obese women. The maternal systemic GAPDH cfDNA was positively correlated with BMI and gestational weight gain. These data suggest that the active remodeling of adipose tissue of obese pregnant women results in an increased release of cfDNA of maternal origin into the circulation.

Modulation of lipid metabolism with the overexpression of NPC1L1 in mouse liver

Niemann-Pick C1-like 1 protein (NPC1L1), a transporter crucial in intestinal cholesterol absorption, is expressed in human liver but not in murine liver. To elucidate the role of hepatic NPC1L1 on lipid metabolism, we overexpressed NPC1L1 in murine liver utilizing adenovirus-mediated gene transfer. C57BL/6 mice, fed on normal chow with or without ezetimibe, were injected with NPC1L1 adenovirus (L1-mice) or control virus (Null-mice), and lipid analyses were performed five days after the injection. The plasma cholesterol levels increased in L1-mice, and FPLC analyses revealed increased cholesterol contents in large HDL lipoprotein fractions. These fractions, which showed α-mobility on agarose electrophoresis, were rich in apoE and free cholesterol. These lipoprotein changes were partially inhibited by ezetimibe treatment and were not observed in apoE-deficient mice. In addition, plasma and VLDL triglyceride (TG) levels decreased in L1-mice. The expression of microsomal triglyceride transfer protein (MTP) was markedly decreased in L1-mice, accompanied by the reduced protein levels of forkhead box protein O1 (FoxO1). These changes were not observed in mice with increased hepatic de novo cholesterol synthesis. These data demonstrate that cholesterol absorbed through NPC1L1 plays a distinct role in cellular and plasma lipid metabolism, such as the appearance of apoE-rich lipoproteins and the diminished VLDL-TG secretion.

Histological evidence: housekeeping genes beta-actin and GAPDH are of limited value for normalization of gene expression

Housekeeping genes are widely used as internal controls for gene expression normalization for western blotting, northern blotting, RT-PCR, etc. They are generally thought to be expressed in all cells of the organism at similar levels because it is assumed that these genes are required for the maintenance of basic cellular function as constitutive genes. However, real-time RT-PCR experiments revealed that their expression may vary depending on the developmental stage, type of tissue examined, experimental condition, and so on. To date, no histological data on their expression are available for embryonic development. In the present study, we compared the histological expression profile of two commonly used housekeeping genes, GAPDH and beta-actin, in the developing chicken embryo by using section and whole mount in situ hybridization supplemented by RT-PCR. Our results show that neither GAPDH mRNA nor beta-actin mRNA is expressed in all cell types or tissues at high levels. Strikingly, expression levels are very low in some organs. Moreover, the two genes show partially complementary expression patterns in the liver, the vascular system and the digestive tract. For example, GAPDH is more strongly expressed in the liver than beta-actin, but at lower levels in the arteries. Vice versa, beta-actin is more strongly expressed in the gizzard than GAPDH, but it is almost absent from cardiac muscle cells. Researchers should consider these histological results when using GAPGD and beta-actin for gene expression normalization in their experiments.

Conservation of a Triple-Helix-Forming RNA Stability Element in Noncoding and Genomic RNAs of Diverse Viruses

Abundant expression of the long noncoding (lnc) PAN (polyadenylated nuclear) RNA by the human oncogenic gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) depends on a cis-element called the expression and nuclear retention element (ENE). The ENE upregulates PAN RNA by inhibiting its rapid nuclear decay through triple-helix formation with the poly(A) tail. Using structure-based bioinformatics, we identified six ENE-like elements in evolutionarily diverse viral genomes. Five are in double-stranded DNA viruses, including mammalian herpesviruses, insect polydnaviruses, and a protist mimivirus. One is in an insect picorna-like positive-strand RNA virus, suggesting that the ENE can counteract cytoplasmic as well as nuclear RNA decay pathways. Functionality of four of the ENEs was demonstrated by increased accumulation of an intronless polyadenylated reporter transcript in human cells. Identification of these ENEs enabled the discovery of PAN RNA homologs in two additional gammaherpesviruses, RRV and EHV2. Our findings demonstrate that searching for structural elements can lead to rapid identification of lncRNAs.

NAD+-dependent Sirtuin 1 and 6 Proteins Coordinate a Switch from Glucose to Fatty Acid Oxidation during the Acute Inflammatory Response

The early initiation phase of acute inflammation is anabolic and primarily requires glycolysis with reduced mitochondrial glucose oxidation for energy, whereas the later adaptation phase is catabolic and primarily requires fatty acid oxidation for energy. We reported previously that switching from the early to the late acute inflammatory response following TLR4 stimulation depends on NAD(+) activation of deacetylase sirtuin 1 (SirT1). Here, we tested whether NAD(+) sensing by sirtuins couples metabolic polarity with the acute inflammatory response. We found in TLR4-stimulated THP-1 promonocytes that SirT1 and SirT 6 support a switch from increased glycolysis to increased fatty acid oxidation as early inflammation converts to late inflammation. Glycolysis enhancement required hypoxia-inducing factor-1α to up-regulate glucose transporter Glut1, phospho-fructose kinase, and pyruvate dehydrogenase kinase 1, which interrupted pyruvate dehydrogenase and reduced mitochondrial glucose oxidation. The shift to late acute inflammation and elevated fatty acid oxidation required peroxisome proliferator-activated receptor γ coactivators PGC-1α and β to increase external membrane CD36 and fatty acid mitochondrial transporter carnitine palmitoyl transferase 1. Metabolic coupling between early and late responses also required NAD(+) production from nicotinamide phosphoryltransferase (Nampt) and activation of SirT6 to reduce glycolysis and SirT1 to increase fatty oxidation. We confirmed similar shifts in metabolic polarity during the late immunosuppressed stage of human sepsis blood leukocytes and murine sepsis splenocytes. We conclude that NAD(+)-dependent bioenergy shifts link metabolism with the early and late stages of acute inflammation.