Helenalin-mediated Post-transcriptional Regulation of p21(Cip1) Inhibits 3T3-L1 Preadipocyte Proliferation

Abstract: We have previously shown that post-transcriptional mechanisms involving the 26S proteasome regulate the cyclin-dependent kinase inhibitors (CKIs), p21(Cip1) and p27(Kip1) during preadipocyte proliferation. Earlier studies further demonstrated that the anti-inflammatory, anticarcinogenic phytochemical, helenalin is a potent inhibitor of periodic Skp2 accumulation, an Fbox protein mediating SCF E3 ligase ubiquitylation and degradation of both CKIs during S phase progression. Data presented here demonstrate that helenalin dose-dependently induced G1 arrest of synchronously replicating 3T3-L1 preadipocytes. This effect occurred in the absence of discernable indices of cell toxicity or apoptosis under the conditions used in this study. Our results demonstrate that helenalin markedly increased p21 protein accumulation in both densityarrested and proliferating preadipocytes in a dose-dependent manner. This increase in p21 protein abundance occurred without change in mRNA transcript demonstrating that posttranscriptional mechanisms were involved. This notion was further supported by the modest accumulation of polyubiquitylated p21 following treatment with helenalin suggesting that suppression of targeted p21 proteolysis by the 26S proteasome contributed to helenalin-mediated p21 accumulation. The increase in p21 protein was compartmentalized to the nucleus where p21 is known to inhibit cell cycle progression. Finally, helenalin increased protein-protein interactions between p21 and cyclin-dependent kinase 2 (Cdk2) which may account in part for the anti-proliferative effect in 3T3-L1 preadipocytes. Article: INTRODUCTION Helenalin is a naturally occurring phytochemical extracted from the aerial portion of the flowering plant, arnica montana L. Alcohol extracts containing this sesquiterpene lactone and its derivatives have been used in herbal medicine for many years to treat haematomas, sprains, rheumatic diseases, and superficial skin inflammation. Recently, helenalin has found principal use in cellular studies as a commercially available, potent inhibitor of IκB degradation and NF-κB transcriptional activity We have previously reported that p27 is regulated during S phase progression of replicating 3T3-L1 preadipocytes by post-transcriptional mechanisms involving ubiquitylation and targeted protein degradation by the 26S proteasome MATERIALS AND METHODS Materials Dulbecco's Modified Eagle's Medium (DMEM), calf bovine serum (CS), fetal bovine serum (FBS) and Trypsin-EDTA were purchased from Invitrogen. Antibodies used for immunoblotting were purchased as follows: p21 (Oncogene), Cyclin D1, α-tubulin, cleaved caspase-3, cleaved PARP, ubiquitin (Cell Signaling), GAPDH, Cyclin A (Santa Cruz), Nucleoporin p62, Cdk2, and Cdk4 (BD Biosciences). Polyclonal p21 antibody for immunoprecipitation was purchased from Santa Cruz Biotechnology. Helenalin was purchased from Biomol. Recombinant Protein-G beads for immunoprecipitation were from Pierce. Propidium Iodide and RNase A were purchased from Sigma. Polyvinylidene fluoride (PVDF) membrane was purchased from Millipore. Enhanced chemiluminescence (ECL) reagents, secondary antibodies and Nethylmaleimide (NEM) were from Pierce. Cell Culture and Induction of Differentiation Murine 3T3-L1 preadipocytes were propagated in DMEM supplemented with 10% CS until reaching density arrest at 2 days post-confluence. Cells were subsequently induced to differentiate with DMEM containing 10% FBS supplemented with 0.5mM 3-isobutyl-1-methyxanthine, 1μM dexamethasone, and 1.7μM insulin (MDI). Throughout the study, the term "post-MDI" refers to the time elapsed since the addition of the differentiation cocktail to the culture medium. Additionally, the term "time 0" refers to density-arrested, 2 days post-confluent cells immediately before the addition of MDI to the culture medium. All experiments were repeated 3-5 times to validate results and ensure reliability. Protein isolation Cells were washed with phosphate-buffered saline (PBS) and harvested in ice cold lysis buffer containing 0.1M Tris (pH 7.4), 150mM NaCl, 10% sodium dodecyl sulfate (SDS), 1% Triton X, 0.5% NP40, 1mM EDTA, 1mM EGTA, and 10mM NEM. Phosphatase inhibitors (20mM β-glycerophosphate, 10mM sodium fluoride and 2μM sodium orthovanadate) and protease inhibitors (0.3μM aprotinin, 21μM leupeptin, 1μM pepstatin, 50μM phenanthroline, 0.5uM phenylmethylsulfonyl fluoride) were freshly added to the lysis buffer immediately before use. Lysates were sonicated and centrifuged at 13,000 × g for 10 min at 4°C. Protein content was determined by bicinchoninic acid (BCA) procedures according to manufacturer's (Pierce) instructions. Immunoblotting Equal amounts of whole cell lysate protein were separated by SDS-PAGE electrophoresis. Protein samples were mixed with loading buffer containing 0.25M Tris, (pH 6.8), 4% SDS, 10% glycerol, 0.01% bromophenol blue, and 10% dithiothreitol, then heated at 80°C for 5 min prior to electrophoresis. Proteins were transferred to PVDF (Millipore), blocked in 4% milk and probed overnight at 4°C with specified primary antibodies. Membranes were subsequently probed for 1 hr at room temperature with secondary antibodies conjugated with horseradish peroxidase and results visualized with ECL using CL-XPosure film (Pierce). RNA Isolation and Analysis Total RNA was extracted from 3T3-L1 preadipocytes using the RNeasy Mini Kit according to manufacturer's (QIAGEN) instructions. Briefly, cells were lysed and homogenized using the provided QIAshredders. Column-bound RNA was DNase-treated and eluted with RNase-free water. Total RNA (1ug) from the cells was subjected to Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) using One-Step RT-PCR kit (QIAGEN).Reactions were carried out in the presence of buffer containing dNTP's (400μM), reverse transcriptase and DNA polymerase enzyme mix, and RNase inhibitor (10U). Gene specific primers designed for p21 (forward: 5'-GTCTGAGCGGCCTGAAGATT'3' and reverse: 5'-TCCTGACCCACAGCAGAAGA-3') were used at a final concentration of 0.6μM. Quantam RNA Classic II 18S primer/ competimer was used according to manufacturer's (Ambion) instruction as an internal standard in the RT-PCR reactions at a concentration of 0.6μM. RT-PCR products were electrophoresed in 1.5% agarose in the presence of ethidium bromide and analyzed on a Kodak Digital Imager. Flow Cytometry Cell cycle progression was assessed by flow cytometry as described previously Nuclear/Cytosolic Fractionation Cells were washed with PBS and incubated with ice cold isotonic buffer containing 20mM Tris, pH7.4, 125mM NaCl, 1mM EGTA, 1mM EDTA and 1mM MgCl 2 for 6 min on ice. The buffer was supplemented with fleshly prepared 0.1% NP40, and phosphatase/protease inhibitors as described above. Following detergent solubilization, cytosolic fractions were collected from the cell monolayers and clarified by centrifugation (13,000 × g for 5 min at 4°C). Intact nuclei were subsequently collected in PBS and gently pelleted by centrifugation at 300 × g for 3 min at 4°C. Nuclear proteins were extracted in ice cold buffer containing 20mM Tris (pH 7.4), 1% Triton X, 150mM NaCl, 1mM EDTA, 1mM EGTA supplemented with protease/phosphatase inhibitors and frozen at -80°C. Nuclear fraction were subsequently thawed on ice, sonicated, passed through a 21 gauge needle to shear DNA, and clarified by centrifugation at 13,000 × g for 10 min at 4°C. Immunoflourescence Cells, cultured on glass coverslips in 35mm plates, were washed with PBS, fixed with methanol free 3% Formaldehyde (Polysicences) for 20 min, permeabilized with 0.2% Triton X for 5 min, blocked for one hr with 3% BSA, and incubated overnight with primary antibody at 4°C. Coverslips were subsequently incubated with fluorescently-labeled secondary antibody, Alexa Fluor 488 anti-mouse (Molecular Probes) for 1 hr at room temperature, washed with PBS, mounted on glass slides using Antifade mounting solution (Molecular Probes) and visualized by confocal microscopy. Immunoprecipitation/Co-immunoprecipitation Cells were lysed with ice cold immunoprecipitation buffer containing 10mM Tris, 150mM NaCl, 1% TritonX, 0.5% NP-40, 1mM EDTA, 1mM EGTA and 10mM NEM as well as protease and phosphatase inhibitors as described above. Immunoprecipitations were performed using recombinant Protein G beads per manufacturer's (Pierce) instructions. Briefly, lysate (1000μg) was incubated with the appropriate antibody for 2 hrs at 4°C, mixed with beads, and incubated for an additional 2 hrs at 4°C. Immune complexes were collected by centrifugation at 2500 × g for 3 min at 4°C, washed 6 times with PBS, resuspended in loading buffer, and heated at 70°C for 3 min. Samples were centrifuged at 2500 × g for 3 min and supernatants resolved with SDS-PAGE, transferred to PVDF membranes and immunoblotted. Recombinant beads were also incubated with whole cell lysates in the absence of antibody to rule out non-specific binding of proteins to the beads. RESULTS Helenalin dose-dependently arrest preadipocytes during G1-phase of cell cycle progression independent of apoptosis Previous reports from our laboratory and others have demonstrated that density-arrested 3T3-L1 preadipocytes synchronously re-enter the cell cycle for a limited period of proliferation that precedes and is obligatory for adipocyte development Helenalin dose-dependently enhances p21 protein accumulation It is well established that cell cycle progression through the G1/S transition requires the timely decay of both p21 and p27. We have previously reported that helenalin inhibits p27 degradation through suppression of SCF E3 ligase activity responsible for polyubiquitylation targeting p27 for proteasomal degradation Helenalin induces p21 protein accumulation post-transcriptionally during density arrest and mid-G1 phase progression To determine the global mechanism linking helenalin and p21 regulation, total cell lysates and RNA were collected for comparative immunoblotting Helenalin modestly promotes the accumulation of ubiquitylated p21 To determine if helenalin increased p21 protein accumulation through posttranscriptional mechanisms involving ubiquitylation and targeted proteasomal degradation, density-arrested were treated with and without helenalin in the presence and absence of MDI for 12 hr. Cell lysates were harvested under non-denaturing conditions, immunoprecipitated with a polyclonal p21 antibody, and immunoblotted with both ubiquitin and p21 (monoclonal) antibodies. For comparative purposes, lysates were also analyzed from density-arrested cells following 6 hr exposure to the potent, specific 26S proteasome inhibitor, epoxomicin. To enhance accumulation of ubiquitin-conjugated substrates, these studies were conducted in the presence of NEM to suppress deubiquitylating enzyme activity. As illustrated in Helenalin increases p21 protein accumulation in the nucleus Subcellular protein localization is an important aspect governing p21 protein accumulation and function. To determine if helenalin leads to increased p21 compartmentalization, density-arrested cells were treated with or without helenalin either in presence or absence of MDI. Nuclear and cytosolic fractions were harvested 12 hr post-treatment and collected cell fractions were immunoblotted for p21. To confirm efficiency of fractionation, lysates were also immunoblotted for nucleoporin and α-tubulin as markers of nuclear and cytosolic fractions, respectively. As illustrated in Helenalin enhances protein interactions between p21 and Cdk2 It is well established that nuclear accumulation of p21 inhibits cell cycle progression Figure 6: Increased interaction between p21 and Cyclin D1, Cdk2, and Cdk4 following helenalin exposure (A) Density-arrested cells stimulated with and without MDI in the absence and presence of 3μM helenalin (HLN) for 12 hr were harvested, immunoprecipitated for p21 and immunoblotted for Cyclin D1, Cdk4, Cdk2 and p21. 10% input protein was immunoblotted and designated by asterisks. Helenalin does not inhibit mid-G1 accumulation of Cyclin D1 As shown above, helenalin increased protein-protein interaction between p21 and Cdk2, completely ablated the accumulation of Cyclin A known to occur during S phase progression, and increased the proportion of cells in G1 phase of the cell cycle following MDI stimulation. Each of these observations was consistent with the working model that p21 inhibition of Cdk2 activity suppressed G1/S phase transition. To further explore this premise, density-arrested cells were stimulated with and without MDI in the presence and absence of helenalin. Lysates were harvested at 12 hr post-treatment and cell cycle proteins immunoblotted as illustrated in Figure 7: Helenalin does not inhibit accumulation of Cyclin D1 at mid-G1 Total cell lysates were harvested from density-arrested 3T3-L1 preadipocytes at 0 hr and 12 hr following exposure to MDI in the absence and presence of 3μM helenalin (HLN) for 12 hr and immunoblotted as indicated. DISCUSSION Adipocyte hyperplasia, defined as the proliferation of preadipocytes and their subsequent differentiation into mature adipocytes, contributes to the development of obesity, a chronic disease that is reaching pandemic proportions in developed and developing nations Although helenalin has been shown to have anti-tumorigenic effects in cancer cells, the mechanisms by which helenalin controls cell proliferation are yet to be determined. Our study is the first to demonstrate, in any cell type, that helenalin treatment can lead to a dramatic accumulation of p21 protein. Furthermore, data presented here support a novel mechanism whereby helenalin-induced accumulation of p21 occurs through post-transcriptional processes independent of changes in mRNA transcript categorically ruling out stress-induced p53 activation and consequential changes in p21 gene expression. Data presented here demonstrate that helenalin promotes nuclear accumulation of p21 as well as modest of polyubiquitylated p21 in a manner similar to that observed following blockade of the 26S proteasome. While the precise mechanisms remains to be determined, data presented here support the premise that helenalin-mediated suppression of proteasomal activity, either direct or indirect, contributes to the dramatic accumulation of p21 in preadipocytes. We have previously reported that helenalin blocks mRNA and protein accumulation of Skp2, an F-box protein associated with the SCF E3 ligase that functions in p27 polyubiquitylation Helenalin is commercially available as a specific inhibitor of the NF-κB signaling pathway. NF-κB is a well studied transcription factor that regulates responses to inflammatory cytokines and its aberration leads to numerous chronic diseases. Helenalin inhibits the induction of NF-κB by multiple mechanisms, including alkylation of the p65 subunit which subsequently prevents DNA binding Other potential mechanisms by which helenalin could induce post-transcriptional p21 protein accumulation might involve protein-protein interactions that either restricts p21 from the nucleus where it is degraded as well as interactions that restrict p21 from proteolytic machinery within the nucleus. Selective compartmentalization is unlikely as we also demonstrate that helenalin increased nuclear, as opposed to cytosolic, p21 accumulation. Within the nucleus, others have demonstrated that p21 interaction with proliferating cell nuclear antigen (PCNA) protects p21 from proteasome-dependent degradation and promotes its protein levels Regulation of p21 protein accumulation represents a possible mechanism linking helenalin with G1 arrest. During mid-G1, p21 facilitates the assembly of Cyclin D1/Cdk4 complexes. However, under conditions of increased nuclear p21 accumulation, D-type cyclins become saturated In summary, this study provides novel data that demonstrate how a naturally occurring phytochemical, helenalin, inhibits preadipocyte proliferation. Investigation of the mechanisms responsible for G1 arrest in 3T3-L1 preadipocytes revealed that helenalin post-transcriptionally induces p21 accumulation, at least in part, by suppressing its ubiquitin-dependent and proteasome-dependent degradation. Moreover, the enhanced nuclear abundance of p21 and increased associations between p21 and Cdk2 in response to helenalin are consistent with mechanisms that suppress preadipocyte proliferation. ACKNOWLEDGMENTS We are grateful to Howard Green (Harvard Medical School) for the murine 3T3-L1 cell line

The kinematic identification of a thick stellar disc in M31

We present the first characterization of a thick disc component in the Andromeda galaxy (M31) using kinematic data from the DEIMOS multi-object spectrograph instrument on Keck II. Using 21 fields in the South West of the galaxy, we measure the lag of this component with respect to the thin disc, as well as the dispersion, metallicity and scale length of the component. We find an average lag between the two components of =46.0+/-3.9km/s. The velocity dispersion of the thick disc is sigma_{thick}=50.8+/-1.9km/s, greater than the value of dispersion we determine for the thin disc, sigma_{thin}=35.7+/-1.0km/s. The thick disc is more metal poor than the thin disc, with [Fe/H]_{spec}=-1.0+/-0.1 compared to [Fe/H]_{spec}=-0.7+/-0.05 for the thin disc. We measure a radial scale length of the thin and thick discs of h_r=7.3+/-1.0 kpc and h_r=8.0+/-1.2 kpc. From this, we infer scale heights for both discs of 1.1+/-0.2 kpc and 2.8+/-0.6 kpc, both of which are ~2--3 times larger than those observed in the Milky Way. We estimate a mass range for the thick disc component of 2.4x10^{10}Msun< M_{*,thick} <4.1x10^{10}Msun. This value provides a useful constraint on possible formation mechanisms, as any proposed method for forming a thick disc must be able to heat (or deposit) at least this amount of material.Comment: 22 pages, 17 figures. Minor revisions made to text following referee report. Accepted for publication in MNRA

The effectiveness of interventions to change six health behaviours: a review of reviews

Background: Several World Health Organisation reports over recent years have highlighted the high incidence of chronic diseases such as diabetes, coronary heart disease and cancer. Contributory factors include unhealthy diets, alcohol and tobacco use and sedentary lifestyles. This paper reports the findings of a review of reviews of behavioural change interventions to reduce unhealthy behaviours or promote healthy behaviours. We included six different health-related behaviours in the review: healthy eating, physical exercise, smoking, alcohol misuse, sexual risk taking (in young people) and illicit drug use. We excluded reviews which focussed on pharmacological treatments or those which required intensive treatments (e. g. for drug or alcohol dependency). Methods: The Cochrane Library, Database of Abstracts of Reviews of Effectiveness (DARE) and several Ovid databases were searched for systematic reviews of interventions for the six behaviours (updated search 2008). Two reviewers applied the inclusion criteria, extracted data and assessed the quality of the reviews. The results were discussed in a narrative synthesis. Results: We included 103 reviews published between 1995 and 2008. The focus of interventions varied, but those targeting specific individuals were generally designed to change an existing behaviour (e. g. cigarette smoking, alcohol misuse), whilst those aimed at the general population or groups such as school children were designed to promote positive behaviours (e. g. healthy eating). Almost 50% (n = 48) of the reviews focussed on smoking (either prevention or cessation). Interventions that were most effective across a range of health behaviours included physician advice or individual counselling, and workplace- and school-based activities. Mass media campaigns and legislative interventions also showed small to moderate effects in changing health behaviours. Generally, the evidence related to short-term effects rather than sustained/longer-term impact and there was a relative lack of evidence on how best to address inequalities. Conclusions: Despite limitations of the review of reviews approach, it is encouraging that there are interventions that are effective in achieving behavioural change. Further emphasis in both primary studies and secondary analysis (e.g. systematic reviews) should be placed on assessing the differential effectiveness of interventions across different population subgroups to ensure that health inequalities are addressed.</p

Elevated plasma CXCL12 alpha is associated with a poorer prognosis in pulmonary arterial hypertension.

Recent work in preclinical models suggests that signalling via the pro-angiogenic and proinflammatory cytokine, CXCL12 (SDF-1), plays an important pathogenic role in pulmonary hypertension (PH). The objective of this study was to establish whether circulating concentrations of CXCL12α were elevated in patients with PAH and related to mortalit

Measurement of the cross-section and charge asymmetry of WW bosons produced in proton-proton collisions at s=8\sqrt{s}=8 TeV with the ATLAS detector

This paper presents measurements of the $W^+ \rightarrow \mu^+\nu$ and $W^- \rightarrow \mu^-\nu$ cross-sections and the associated charge asymmetry as a function of the absolute pseudorapidity of the decay muon. The data were collected in proton--proton collisions at a centre-of-mass energy of 8 TeV with the ATLAS experiment at the LHC and correspond to a total integrated luminosity of 20.2~\mbox{fb^{-1}}. The precision of the cross-section measurements varies between 0.8% to 1.5% as a function of the pseudorapidity, excluding the 1.9% uncertainty on the integrated luminosity. The charge asymmetry is measured with an uncertainty between 0.002 and 0.003. The results are compared with predictions based on next-to-next-to-leading-order calculations with various parton distribution functions and have the sensitivity to discriminate between them.Comment: 38 pages in total, author list starting page 22, 5 figures, 4 tables, submitted to EPJC. All figures including auxiliary figures are available at https://atlas.web.cern.ch/Atlas/GROUPS/PHYSICS/PAPERS/STDM-2017-13

Search for chargino-neutralino production with mass splittings near the electroweak scale in three-lepton final states in √s=13 TeV pp collisions with the ATLAS detector

A search for supersymmetry through the pair production of electroweakinos with mass splittings near the electroweak scale and decaying via on-shell W and Z bosons is presented for a three-lepton final state. The analyzed proton-proton collision data taken at a center-of-mass energy of √s=13  TeV were collected between 2015 and 2018 by the ATLAS experiment at the Large Hadron Collider, corresponding to an integrated luminosity of 139  fb−1. A search, emulating the recursive jigsaw reconstruction technique with easily reproducible laboratory-frame variables, is performed. The two excesses observed in the 2015–2016 data recursive jigsaw analysis in the low-mass three-lepton phase space are reproduced. Results with the full data set are in agreement with the Standard Model expectations. They are interpreted to set exclusion limits at the 95% confidence level on simplified models of chargino-neutralino pair production for masses up to 345 GeV

Design and Synthesis of High Affinity Inhibitors of Plasmodium falciparum and Plasmodium vivax N-Myristoyltransferases Directed by Ligand Efficiency Dependent Lipophilicity (LELP)

N-Myristoyltransferase (NMT) is an essential eukaryotic enzyme and an attractive drug target in parasitic infections such as malaria. We have previously reported that 2-(3-(piperidin-4-yloxy)benzo[b]thiophen-2-yl)-5-((1,3,5-trimethyl-1H-pyrazol-4-yl)methyl)-1,3,4-oxadiazole (34c) is a high affinity inhibitor of both Plasmodium falciparum and P. vivax NMT and displays activity in vivo against a rodent malaria model. Here we describe the discovery of 34c through optimization of a previously described series. Development, guided by targeting a ligand efficiency dependent lipophilicity (LELP) score of less than 10, yielded a 100-fold increase in enzyme affinity and a 100-fold drop in lipophilicity with the addition of only two heavy atoms. 34c was found to be equipotent on chloroquine-sensitive and -resistant cell lines and on both blood and liver stage forms of the parasite. These data further validate NMT as an exciting drug target in malaria and support 34c as an attractive tool for further optimization

Search for new phenomena in final states with an energetic jet and large missing transverse momentum in pp collisions at √ s = 8 TeV with the ATLAS detector

Results of a search for new phenomena in final states with an energetic jet and large missing transverse momentum are reported. The search uses 20.3 fb−1 of √ s = 8 TeV data collected in 2012 with the ATLAS detector at the LHC. Events are required to have at least one jet with pT > 120 GeV and no leptons. Nine signal regions are considered with increasing missing transverse momentum requirements between Emiss T > 150 GeV and Emiss T > 700 GeV. Good agreement is observed between the number of events in data and Standard Model expectations. The results are translated into exclusion limits on models with either large extra spatial dimensions, pair production of weakly interacting dark matter candidates, or production of very light gravitinos in a gauge-mediated supersymmetric model. In addition, limits on the production of an invisibly decaying Higgs-like boson leading to similar topologies in the final state are presente

Hepatopulmonary syndrome in patients with chronic liver disease: role of pulse oximetry

BACKGROUND: Hepatopulmonary syndrome (HPS) is a rare complication of liver diseases of different etiologies and may indicate a poor prognosis. Therefore, a simple non-invasive screening method to detect HPS would be highly desirable. In this study pulse oximetry was evaluated to identify patients with HPS. METHODS: In 316 consecutive patients with liver cirrhosis (n = 245), chronic hepatitis (n = 69) or non-cirrhotic portal hypertension (n = 2) arterial oxygen saturation (SaO(2)) was determined using a pulse oximeter. In patients with SaO(2 )≤92% in supine position and/or a decrease of ≥4% after change from supine to upright position further diagnostic procedures were performed, including contrast-enhanced echocardiography and perfusion lung scan. RESULTS: Seventeen patients (5.4%) had a pathological SaO(2). Four patients (1.3%) had HPS. HPS patients had a significant lower mean SaO(2 )in supine (89.7%, SD 5.4 vs. 96.0%, SD 2.3; p = 0.003) and upright position (84.3%, SD 5.0 vs. 96.0%, SD 2.4; p = 0.001) and had a lower mean PaO(2 )(56.2 mm Hg, SD 15.2 vs. 71.2 mm Hg, SD 20.2; p = 0.02) as compared to patients without HPS. The mean ΔSaO(2 )(difference between supine and upright position) was 5.50 (SD 7) in HPS patients compared to non-HPS patients who showed no change (p = 0.001). There was a strong correlation between shunt volume and the SaO(2 )values (R = -0.94). CONCLUSION: Arterial SaO(2 )determination in supine and upright position is a useful non-invasive screening test for HPS and correlates well with the intrapulmonary shunt volume

Impact of Reference Gene Selection for Target Gene Normalization on Experimental Outcome Using Real-Time qRT-PCR in Adipocytes

Background: With the current rise in obesity-related morbidities, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) has become a widely used method for assessment of genes expressed and regulated by adipocytes. In order to measure accurate changes in relative gene expression and monitor intersample variability, normalization to endogenous control genes that do not change in relative expression is commonly used with qRT-PCR determinations. However, historical evidence has clearly demonstrated that the expression profiles of traditional control genes (e.g., b-actin, GAPDH, a-tubulin) are differentially regulated across multiple tissue types and experimental conditions. Methodology/Principal Findings: Therefore, we validated six commonly used endogenous control genes under diverse experimental conditions of inflammatory stress, oxidative stress, synchronous cell cycle progression and cellular differentiation in 3T3-L1 adipocytes using TaqMan qRT-PCR. Under each study condition, we further evaluated the impact of reference gene selection on experimental outcome using examples of target genes relevant to adipocyte function and differentiation. We demonstrate that multiple reference genes are regulated in a condition-specific manner that is not suitable for use in target gene normalization. Conclusion/Significance: Data are presented demonstrating that inappropriate reference gene selection can have profound influence on study conclusions ranging from divergent statistical outcome to inaccurate data interpretation of significan