Notably, mOS conditions resulted in increased levels of the BACE1 C-terminal cleavage product of APP, β-CTF. Subcellular fractionation techniques showed that mOS caused a major rearrangement of BACE1 localization from light to denser fractions, resulting in an increased distribution of BACE1 in fractions containing APP and markers for trans-Golgi network and early endosomes. Collectively, these data demonstrate that mOS does not modify BACE1 expression but alters BACE1 subcellular compartmentalization to favour the amyloidogenic processing of APP, and thus offer new insight in the early molecular events of AD pathogenesis.BACE1 is responsible for β-secretase cleavage of the amyloid precursor protein (APP), which represents the first step in the production of amyloid β (Aβ) peptides. Previous reports, by us and others, have indicated that the levels of BACE1 protein and activity are increased in the brain cortex of patients with Alzheimer’s disease (AD). The association between oxidative stress (OS) and AD has prompted investigations that support the potentiation of BACE1 expression and enzymatic activity by OS. Here, we have established conditions to analyse the effects of mild, non-lethal OS on BACE1 in primary neuronal cultures, independently from apoptotic mechanisms that were shown to impair BACE1 turnover. Six-hour treatment of mouse primary cortical cells with 10–40 µM hydrogen peroxide did not significantly compromise cell viability but it did produce mild oxidative stress (mOS), as shown by the increased levels of reactive radical species and activation of p38 stress kinase. The endogenous levels of BACE1 mRNA and protein were not significantly altered in these conditions, whereas a toxic H By mediating β-secretase cleavage of the amyloid precursor protein (APP) it initiates production of amyloid β (Aβ) peptides A variety of stress factors can induce BACE1 expression in cellular and animal models. These include oxidative stress 2O2 and 4-hydroxynonenal increased BACE1 protein and mRNA levels 2O2Previous experimental studies have investigated the effect of oxidative agents on BACE1 expression. Using a cellular luciferase reporter system, Tong et al demonstrated that BACE1 gene expression could be induced by hydrogen peroxide In the present study, we have examined alterations in endogenous BACE1 expression in response to mild levels of OS by subjecting mouse primary cortical neurons to non-toxic levels of oxidants. We have established mild oxidative stress (mOS) conditions, wherein oxidant treatment significantly increased levels of intracellular free radicals and activation of stress kinase without compromising cell viability. We showed that mOS treatment did not alter BACE1 levels. Notably, we found that mOS triggered a change in BACE1 compartmentalization that increases its colocalisation. with APP and its amyloidogenic processing.All tissue culture reagents were purchased from Life Technologies, Mulgrave VIC, Australia. Chemicals were of tissue culture grade and were obtained from Sigma-Aldrich, Sydney, Australia, unless otherwise stated.3. 1.2 mM NaH2PO4. 1.2 mM MgCl2. 2.5 mM CaCl2, pH 7.2) and incubated in a shaking water-bath for 20 min at 37°C to dissociate the connective tissue. Then, 0.008% w/v DNase I ) and 0.026% w/v soybean trypsin inhibitor  in 10 mL Krebs solution were added to neutralize trypsin. Fragmentation of the tissue precipitate was hastened by gentle inversion followed by centrifugation at 259×g  for 3 min. The supernatant was aspirated and the cell pellet resuspended in 1 mL of DNase I/SBTI supplemented Krebs solution by passing thirty times through a blunt filtered tip. The resulting single cell suspension was diluted out in Krebs buffer solution and centrifuged to sediment the cells. The supernatant was aspirated and the cells resuspended in plating medium  and counted using a haemocytometer. The primary cortical cells were seeded onto poly-D-Lysine coated tissue culture dishes and cultured for up to 4 hours. Then, the plating medium was aspirated and the cells maintained in Neurobasal medium supplemented with 0.2 mM L-Glutamine, 0.01 mg/mL Gentamicin and B27 Supplement.Briefly, cortices from C57BL/6 embryonic mice at day 14 were removed, dissected free of meninges, dissociated in 0.025% (w/v) trypsin in Krebs Buffer  at a final concentration of 10, 20, or 40 µM for 6–7 h. Working H2O2 concentrations were prepared in culture medium supplemented with B27 Minus Antioxidants in dark 15 mL tubes to minimise oxidant degradation by light. Control untreated cells were cultured in parallel.Mouse primary cortical cells were plated in 12-well poly-D-Lysine-coated tissue culture plates at a density of 8×10The 3--2,5-diphenyltetrazolium bromide (MTT) solution was prepared in sterile PBS at a concentration of 5 mg/mL and diluted 100 times in the culture medium. MTT was added into conditioned medium 30 min before the end of oxidative stress treatments; the cells were returned to the incubator until formazan purple coloration became visible. The conditioned medium was aspirated and the cells were lysed in DMSO for measuring absorbance at 540 nm using a Wallac VICTOR™ 3 Multilabel Counter . Cell viability was calculated as the ratio of absorption value for treated versus control cells and expressed as a percentage.After treatment, cells were washed with PBS and incubated in culture medium supplemented with 50 µM 2′,7′-dichlorofluorescin-diacetate (DCFH-DA) for 40 min at 37°C. Next, the cells were washed with PBS and lysed in 0.1 M Tris-HCl pH 7.5 supplemented with protease inhibitor cocktail  and 1% Triton X-100. Dichlorofluorescein (DCF) fluorescence was measured at 485/535 nm in a Wallac VICTOR™ 3 Multilabel Counter. The fluorescence values were normalised to the protein concentration in cell lysates, as determined by the Bicinchoninic assay , and the data expressed relative to untreated cells.Cells were lysed using RIPA buffer , P8340 protease inhibitor cocktail, and 0.05 mg/mL DNase I (Roche)). The lysate was centrifuged at 14,000×g for 5 min at 4°C and protein concentration was determined by BCA.Samples (20 µg protein) were denatured in Laemmli sample buffer . For BACE1 detection, samples were heated at 55°C for 10 min, whereas for analysis of other proteins, samples were heated at 95°C for 5 min. Proteins were separated 8.5% Tris-Glycine gels using a Mini Protean III system  and electrophoresed at 40 mA/gel in 25 mM Tris, 192 mM Glycine, 0.1% SDS buffer, followed by transfer to nitrocellulose membrane (Bio-Rad) for 60 min at 600 mA in 25 mM Tris, 200 mM Glycine buffer containing 20% methanol. The 10–20% Novex Tris-Tricine gels (Life Technologies) were chosen to separate proteins of molecular weight lower than 40 kDa and were used following the manufacturer’s protocol. For APP-CTF detection, blots were boiled in PBS for 5 min before blocking. Western blot antibodies were as follows: p38 MAP kinase and phospho-specific p38 MAP kinase antibodies, BACE1 rabbit monoclonal D10E5, full-length (pro-) caspase-3, ), transferrin receptor (Life Technologies), GGA3, TGN38 and EEA1 . APP mouse monoclonal 22C11 and C-terminal rabbit polyclonal 369 were gifts from Prof K. Beyreuther and Prof S. Gandy, respectively. The blots were developed using West Dura chemiluminescence reagent  and signals captured with a LAS-3000 Imager (FUJIFILM). For reprobing membranes, antibodies were stripped by incubating the blots at 55°C for 15 min in 100 mM 2-mercaptoethanol (Bio-Rad), 2% SDS, 63.5 mM Tris-HCl pH 6.7, before repeating the immunoblotting procedure with another antibody.6 cells per dish and cultured for six days (6 DIV) before treatment. Three dishes were subjected to H2O2 treatment, and three other dishes were left untreated for use as controls. The cells from three identical dishes were pooled, resuspended in homogenisation buffer  and lysed with a Dounce homogeniser, followed with 5 passages through a 25-gauge needle. The homogenate was centrifuged at 1,200×g for 15 min, at 4°C. The supernatant was adjusted to 1.4 M sucrose. Sucrose gradients were constructed in 17 mL ultracentrifuge tubes by pipetting successively, 2 mL of 2 M sucrose, 2 mL of the 1.4 M sucrose mixture, 3.4 mL of 1.2 M sucrose, and 9.5 mL of 0.8 M sucrose. The tubes were centrifuged for 16 h, at 100,000×g, at 4°C, in a SW32.1 rotor in an L8-80M ultracentrifuge (Beckman Coulter). Fractions were collected and stored at −80°C until use. Fractions were concentrated by methanol precipitation Mouse primary cortical cells were grown on 100 mm diameter poly-D-Lysine-coated dishes at a density of 10×10260/280) using a NanoDrop 3300 fluorospectrometer (Thermo Scientific). The ratio obtained for RNA samples was ∼2. The RNA samples were distributed into aliquots and stored at −80°C. RNA was reverse-transcribed using the High-Capacity cDNA Reverse Transcription kit , which primed first strand synthesis with random primers. The reaction consisted of 2 µL 10× Reverse Transcription buffer, 4 mM dNTP mix, random primers, 50 U MultiScribe™ Reverse Transcriptase in a total volume of 10 µL plus 2 µg RNA in 10 µL nuclease-free water . The reactions were placed in a Mastercycler thermal cycler (Eppendorf), programmed for cycles at 25°C for 10 min, 37°C for 120 min and 85°C for 5 min. TaqMan® Gene Expression Assays (Applied Biosystems) were used for detecting the mouse BACE1 transcript (Assay ID Mm00478664_m1), and the mRNA transcripts of the house keeping genes actin (Assay ID Mm00607939_s1) and β2-macroglobulin (Assay ID Mm00437762_m1). In each 20 µL TaqMan® reaction, a volume containing 1 ng of cDNA (A260/280 ∼1.8) was mixed with 1 µL 20X TaqMan® Gene Expression Assay, 10 µL 2X TaqMan® Gene Expression Master Mix and nuclease-free water. Control reactions substituting cDNA with nuclease-free water were set up concurrently. Quantitative real-time PCR was performed using the Rotor-Gene™ 6000 (Corbett Life Science) with the conditions as follows: 50°C for 2 min proceeded by 95°C for 10 min, then 40 cycles of 15 s at 95°C and 60 s at 60°C. Data were analysed with Rotor-Gene 6000 Series software version 1.7 to determine the threshold cycle (CT), which used no cDNA template control reactions to establish the background signal of the assays. The 2-ΔΔCT comparative method was used to determine the fold differences of BACE1 gene expression between treated and untreated cells Cells were lysed using TRIzol reagent (Life Technologies) and RNA prepared according to the manufacturer’s protocol. The RNA pellet was air-dried for 10 min, before dissolution in 12 µL of DNase- and RNase-free water (Life Technologies) with incubation for 10 min at 57°C. RNA concentration and purity was assessed by measuring the 260 nm to 280 nm absorbance ratio  in PBS, before successive 1 h incubation with each primary antibody and Alexa Fluor-labelled secondary antibodies (Invitrogen), diluted in blocking buffer. Nuclei staining was carried out by incubation with 300 nM DAPI  in PBS for 5 min. Cells were visualized using a Leica (model DM IRB) inverted microscope, and images captured with a Zeiss Axiocam HRc camera. Images were superimposed using the Axio Vision software (Carl Zeiss) for assessment of colocalisation.Mouse primary cortical cells were plated at a density of 8×10post-hoc analysis using the Tukey’s HSD (honestly significant difference) test or, when the Levene’s test indicated unequal variances (p<0.05), with the Games-Howell test. Error bars on the graphs correspond to the standard deviation.All experiments were performed at least three times, either with duplicates or triplicates. Statistical analyses were carried out using the SPSS statistics software version 17 (IBM). Comparison of the means was performed by One-way ANOVA (significant when p<0.05), followed by 2O2 (p<0.001) increase in cell death after treatment with 50 µM H2O2, and 85%±4% (p<0.001) increased cell death after treatment with 100 µM H2O2. Therefore, a lower range of H2O2 concentrations was trialled and treatment of primary cortical cultures with 10–40 µM H2O2 for 6 h did not cause any significant decrease in cell viability , 27%±15% after treatment with 20 µM H2O2(p = 0.024), and 63%±26% after treatment with 40 µM H2O2(p = 0.007). As a significant accumulation of intracellular free radicals would only occur when endogenous antioxidants have been overwhelmed, the MTT and DCF data demonstrated that 10–40 µM concentrations of H2O2 could induce mOS in viable mouse primary cortical cells.Next, the levels of intracellular reactive oxygen species were determined using the 2′,7′-dichlororofluorescein (DCF) assay to confirm that the cells were undergoing oxidative stress 2O2 , with a 2O2 were undergoing stress, p38 stress kinase activation was examined by immunoblotting with a specific phospho-p38 antibody. The p38 mitogen-activated protein (MAP) kinase belong to a family of serine/threonine protein kinases that play an important role in stress-activated pathways and become phosphorylated in response to stress stimuli 2O2 contained detectable levels of phosphorylated p38 kinase , 20 µM H2O2  and 40 µM H2O2   . These d2O2 did not significantly alter BACE1 protein levels (2O2 ((p = 0.04).Next, BACE1 protein levels were analysed by immunoblotting with D10E5 BACE1 C-terminal antibody. BACE1 was detected as the expected 70 kDa signal . Resultsn levels . In contels  was performed to assess possible changes in BACE1 mRNA levels in mouse primary cortical cells subjected to mOS. RNA was extracted with TRIzol, its purity checked, and it was used for reverse-transcription to generate cDNA for real-time PCR analysis using TaqMan gene expression assays. Actin and β2-macroglobulin (B2M) transcripts were chosen as the housekeeping gene controls. The mouse BACE1 TaqMan probe targets the junction between exons 2 and 3 of the BACE1 transcript, therefore signals generated by qPCR would not result from genomic DNA contamination. An increasing trend in BACE1 mRNA levels was observed in cells treated with Hlin mRNA .2O2 treatment, and was not consistently observed in all experiments. In agreement, no statistically significant change in GGA3 levels occurred in response to H2O2 treatment  could be due to non-specific signals or to the APP-like proteins and fragments, as the cytosolic domain is conserved among the APP family of proteins. A trend for decreases in full length APP . Attempts to analyse changes in Aβ secretion in the cell conditioned media were unsuccessful, as the levels produced by mouse primary neurons are very low and were below the detection threshold of our sandwich ELISA that is less effective at measuring mouse Aβ than its human counterpart.We examined levels of the APP C-terminal product of BACE1 cleavage, β-CTF (C99) by immunoblotting with the polyclonal 369 antibody, which was raised against the whole cytosolic domain of APP ngth APP  and α-CTngth APP  was obsereatment , with a Since mOS caused no change in either BACE1 mRNA or protein levels, but increased β-CTF, we reasoned that an alteration in the subcellular distribution of BACE1 might be promoting APP processing. Indeed, it was previously reported that oxidative stress treatment of NT2 neurons increased BACE1 localisation in the Golgi apparatus 2O2 did not markedly change BACE1 subcellular localisation, as overlap with the early endosomal marker was preserved , which contained transferrin receptor, EEA1, and TGN38  . These r2O2, which suggested a cellular response to promote the amyloidogenic processing of APP. Subcellular fractionation demonstrated a shift in the distribution of BACE1 protein from fractions of light density towards fractions containing APP and markers of the trans-Golgi network (TGN38) and early endosome (EEA1)  . A previ2O2 seems to induce the redistribution of BACE1 from these light membrane vesicles or raft domains to facilitate its interaction with APP in endosomal and TGN membranes.The intricate and regulated intracellular trafficking of BACE1 has been mostly studied in overexpressing systems Click here for additional data file.Figure S2DCF assay of cortical cells treated with SIN-1 and paraquat. DCF fluorescence of cell lysate was normalised for protein concentration. Experiments were performed three times. The results show a significant increase in oxidative radicals in cells treated with 2.5, 5, and 10 µM SIN-1 (A) and in cells treated with 200 and 400 µM paraquat (B), indicating that the cells experienced oxidative stress.(TIF)Click here for additional data file.Figure S3Immunoblot of phosphorylated p38 and total p38 in cortical cells treated with SIN-1 and paraquat. Western blot was carried out with 20 µg of cell lysates. Blots for SIN-1 and paraquat treatments are displayed in (A) and (C), respectively. These are representative of three separate experiments. The results indicate that the ratio of phospho-p38/total p38 was elevated in cells treated with 5 and 10 µM SIN-1 (B), supporting activation of p38 stress kinase. Treatment with 400 µM paraquat also caused a significant increase in phospho-p38/total p38 ratio (D).(TIF)Click here for additional data file.Figure S4Analysis of BACE1 expression in primary cortical cells treated with SIN-1 and paraquat. Western blot was carried out with 20 µg of cell lysates using anti-BACE1 CT D10E5 antibody A. Representative immunoblot for SIN-1 treatment C. Representative immunoblot for paraquat treatment, respectively. Blots were re-probed for actin. B. and D. Densitometry analysis of blots for SIN-1 and paraquat treatments. The data represent averages of three individual experiments. BACE1 signal density was normalised to actin. No statistical difference was found between treated and control cells.(TIF)Click here for additional data file.Figure S5Immunoblot of caspase-3 and GGA3 in cortical cells treated with SIN-1 and paraquat. 20 µg of lysate was loaded per lane. A and D. Blots of caspase-3 and GGA3 for cells treated with SIN-1 and paraquat, respectively. B. Densitometry analysis of full-length pro-caspase-3 normalized to actin, for cells treated with SIN-1 (N = 3). C. Densitometry analysis of 75 kDa GGA3 signal normalized to actin, for cells treated with SIN-1 (N = 3). E. Densitometry analysis of full-length pro-caspase-3 normalized to actin, for cells treated with paraquat (N = 3). F. Densitometry analysis of 75 kDa GGA3 signal normalized to actin, for cells treated with paraquat (N = 3). There was significant change in pro-caspase 3 and GGA3 levels after either treatment with SIN-1 or paraquat at the indicated concentrations, suggesting that the cells were not undergoing apoptosis.(TIF)Click here for additional data file.Figure S6Immunofluorescence analysis of mouse primary cortical neurons treated with SIN-1 and paraquat. The left panel shows cells treated with SIN-1 and control. The right panel shows cells treated with paraquat and control. (a) and (b) show immunolabelling of BACE1 as punctate perinuclear staining . (c) and (d) show detection of early endosomal marker EEA1. (e) and (f) are the merged images of BACE1 and EEA1. Partial colocalisation of BACE1 and EEA1 was observed in untreated cells (e), and this was increased in the cells treated with SIN-1  or with paraquat . Bar = 20 µm.(TIF)Click here for additional data file.Methods S1(DOCX)Click here for additional data file.


G-protein coupled receptors are a diverse group that are the target of over 50% of marketed drugs. Activation of these receptors results in the exchange of bound GDP for GTP in the Gα subunit of the heterotrimeric G-protein. The Gα subunit dissociates from the β/γ subunits and both proceed to affect downstream signaling targets. The signal terminates by the hydrolysis of GTP to GDP and is temporally regulated by Regulators of G-protein Signaling (RGS) proteins that act as GTPase Activating Proteins (GAPs). This makes RGS proteins potentially desirable targets for “tuning” the effects of current therapies as well as developing novel pharmacotherapies. Current methods for evaluating RGS activity depend on laborious and/or expensive techniques. In this study we developed a simple and inexpensive assay for the steady state analysis of RGS protein GAP activity, using RGS4, RGS8 and RGS17 as models. Additionally, we report the use of RGS4 as a model for high throughput assay development. After initial setup, this assay can be conducted in a highly parallel fashion with a read time of less than 8 minutes for a 1536-well plate. The assay exhibited a robust Z-factor of 0.6 in a 1536-well plate. We conducted a pilot screen for inhibitors using a small, 2320 compound library. From this screen, 13 compounds were identified as compounds for further analysis. The successful development of this assay for high-throughput screening provides a low cost, high speed, simple method for assessing RGS protein activity. G-protein coupled receptors (GPCRs) are a diverse group of seven transmembrane-spanning receptors that represent targets for over 50% of drugs available on the market 32P labeled GTP in single turnover or steady-state assays 32P assays provides a measure of RGS activity on GTPase activity, they are technically challenging, even in low throughput benchtop experiments which involve the use of radioactivity and required careful timing for reproducible results Due to their important role in regulating GPCR signaling, RGS proteins represent intriguing targets for drug development. In developing high-throughput screening (HTS) assays for RGS targets, methods have emerged for the targeting of the RGS-Gα protein-protein interaction, such as flow cytometry, Alpha Screen, fluorescence polarization, and time-resolved fluorescence resonance energy transfer i1) that results in a marked reduction in the intrinsic GTPase activity of the Gα subunit while maintaining sensitivity to the GAP activity of RGS proteins i1, allows for a ∼25 fold increase in koff(GDP) while maintaining normal GTPase activity The limitations of these approaches have driven our group, and others, to develop simple, non-radioactive assays to measure RGS protein GAP function. Early work focused on the development of entire receptor/protein complexes contained within phospholipid vesicles In this study, we developed a malachite-based assay to measure GAP activity of a variety of RGS proteins. RGS4 was selected as the pilot RGS for this assay due to the results of recent RGS4 HTS campaigns and the availability of a small collection of control compounds Tobacco etch virus (TEV) protease was expressed and purified as a His-tagged protein in E. coli, BL21-pRIL , in the pRK793 vector as previously described by the Waugh lab Rat RGS4, sharing 97% sequence identity with human RGS4, and the cysteine to alanine mutant were expressed as fusion proteins of maltose binding protein (MBP), a 10× His tag, and a TEV protease recognition site fused to the N-terminus of an RGS4 construct containing amino acids 51–205, in the vector pMALC2H10T in BL21-DE3 E. coli  600 nm of 2.0 and cultured for 16 h at 18°C. Pellet was lysed, centrifuged, and filtered as described above except in RGS8 Buffer . Samples were loaded onto a Ni-NTA column , 3 mL for every 1 L media, and washed with RGS8 Buffer supplemented with 25 mM imidazole. The protein was eluted using 200 mM imidazole and fractions were analyzed by SDS-PAGE gel. Fractions containing RGS8>95% purity were pooled and protein concentration was determined by 280 nm absorbance as accomplished above.Human RGS8 expression and purification was performed similar to other RGS8 purifications previously reported Human RGS17 was expressed and purified as a His-tagged protein in E. coli BL21-DE3 , in the pET28 vector as previously described i1  rate-altered variant described in literature, was expressed in BL21-DE3 E. coli, grown in TB media, as a 6×His labeled protein in the pQE80 vector 600 nm of 1.0, with 100 µM IPTG at 30°C for 16 h. Pellets were lysed, centrifuged, and filtered as described above, but in Gαi Buffer . The sample was first loaded onto a Ni-NTA column , containing 3 mL of resin for every 1 L of media. The column was first washed with Gαi Buffer supplemented with 25 mM imidazole. Gαi was then eluted from the column with Gαi buffer supplemented with 300 mM imidazole. After analysis by SDS-PAGE gel, fractions that contained Gαi were pooled and dialyzed overnight against Gαi Dialysis Buffer . The sample was then loaded onto a Q-sepharose column  and eluted along a salt gradient from 50 mM NaCl to 1 M NaCl in Gαi Buffer. The resulting peaks were then analyzed by SDS-PAGE for fractions containing >99% Gαi. The purified Gαi was then assayed for activity utilizing the [35S]GTPγS binding assay Human Gαo was expressed in LB media as a fusion protein of glutathione-S-transferase (GST), 6× His, and Gαo, in pQLinkGD vector. Expression was induced, at OD600 nm of 0.5, with 100 µM IPTG at 30°C for 16 h. Pellets were lysed, centrifuged, and filtered as described above, but in Gαo Buffer phosphine). The protein was first purified over a nickel charged resin column, 1 mL resin for every 1 L culture. Prior to elution, the column was washed with 20 mM imidazole to clear weak binding contaminants from the sample. The fusion protein was eluted with 250 mM imidazole. Fractions were collected and analyzed by SDS-PAGE gel. Fractions containing the protein of interest were pooled and loaded onto glutathione sepharose column , 1.5 mL resin for every 1 L culture. The protein was then eluted with 1 mM free glutathione and analyzed by SDS-PAGE gel. Fractions containing >99% pure protein were pooled for activity determination. The purified Gαo was then assayed for activity utilizing the [35S]GTPγS binding assay Rat Gα3PO4 at pH 7.5 as the negative control.Stock solutions of each of the 3 components of the developing solution were prepared, which are stable for long-term storage 2, 0.01% lubrol) into a clear 384-well plate  using a MultiDrop dispenser . The second component dispensed was 10 µL of a 4× stock of RGS4, typically 200 nM to 1.6 µM with the target final concentration of 50 nM to 400 nM, diluted in MGB. After a 30 min incubation, 10 µL of the third component, 4× stock of Gαi diluted in MGB, was dispensed . After a minimum of 5 min incubation, 10 µL of the fourth component, 4× GTP diluted in MGB, was added at 10 minute intervals from 1–110 minutes. The 0 min time point was excluded due to amount of time required to proceed from GTP addition to quenching with DS. 4× GTP concentrations varied between 0.2 mM and 2.4 mM, with a target final concentration of 50 µM to 600 µM. To terminate the reaction, 10 µL of DS was added to each well using a Microlab Star liquid handling robot , to achieve a final ratio 4∶1 (sample:developing solution). Following the spin, the plate was incubated for 25 min before being read at 642 nm for absorbance using an EnVision plate reader . RGS8 was evaluated similarly to as described for RGS4, with 4× stock concentrations from 20 nM and 800 nM. For each time-course, corresponding GTP only wells were included to account for spontaneous hydrolysis of GTP over time.The malachite green assay involves 5 components, with a 1 min spin at 100×g between each addition. For time-course experiments, the first component was 10 µL Malachite Green Assay Buffer . For the compound library, the diverse set of known biologically active compounds, The Spectrum Library , was chosen. Each component was dispensed as 1.8 µL samples into each well using a FlexDrop . To develop the plates, 1.8 µL of DS were stamped in quadrants using the Microlab Star liquid handling robot. After a 25 min incubation, the plates were analyzed using an EnVision plate reader  at 642 nm absorbance.Chemical labeling of RGS4 was performed using biotinamidohexanoic acid N-hydroxy succinimide ester . The reaction was carried out at a molar ratio of 3∶1 (label/protein) for 3 h at 4°C in 50 mM HEPES at pH 8 and 100 mM NaCl, similar to as previously described o. The final concentrations for RGS4 and compound will be 20 nM and 40 µM respectively.To prepare RGS4 for analysis using the ALPHA-Screen assay, RGS4 constructs were first labeled in a 1440 µL sample, diluted in Assay Buffer , containing 60 nM RGS4, 14.4 µL streptavidin ALPHA-Screen beads . The sample was then incubated for 30 min, on ice, prior to dilution with AB to 2880 µL. In duplicate, 20 µL of each compound at 120 µM was plated across a white 384-well plate . 20 µL RGS4 was then plated into each well and the samples were incubated at 19°C for 30 min prior to the addition of GST-Gαo was prepared for the assay by creating a 1440 µL labeling reaction, diluted in AB, containing 3 nM GST-Gαo, 10 µM GDP, and 14.4 µL anti-GST ALPHA-Screen Beads. The sample was incubated for 30 min on ice. A 40 µL sample was then removed and diluted with 40 µL AB; this is positive control. The remaining 1400 µL is then diluted with 1400 µL AB supplemented with AMF  to a final volume of 2800 µL. 20 µL of each sample was then dispensed into the each well. The final concentration of the GST-Gαo will be 0.5 nM.GST-Gαo and biotinylated Δ51-RGS4, the plates were incubated at 19°C for 1 hr prior to reading using the Synergy 2 plate reader.Following the addition of both GST-Gα50 values for each compound were determined by fitting the data to a sigmoidal curve, which was used to calculate the IC50 value.Data was analyzed using Prism analysis software . Initial malachite green assay optimization was accomplished by comparing the fit of both straight line and hyperbolic functions. The fit that mostly closely resembled the data was used to represent the data. IC3PO4 at pH 7.5 developed for 50 min with 10 µL DS yielded an intense signal peak at 642 nm, with a secondary peak at 436 nm  of the screened library. ALPHA Screen was utilized as an orthogonal assay to confirm each of the remaining compounds as hits, 4−. This orthogonal assay eliminated 15 compounds, leaving 30 compounds or 1.3% of the total compounds screened. Finally, compounds were challenged against the RGS4(Δ7) mutant in the malachite green phosphate detection assay, with the desire of eliminating thiol-modifiers similar to those previously discovered in HTS campaigns against RGS4 Initial hits were confirmed by single point malachite green assay at 40 µM compound. Of the initial 59 compounds, 7 compounds fell within 3 standard deviations of the negative control, 50 of 126 µM and 454 µM against the RGS4(WT) and RGS4(Δ7) respectively. The most potent compound, UI-1590 (50 of 724 nM against RGS4(WT) and an IC50 of 88 µM against RGS4(Δ7). Finally, two structurally similar compounds, UI-1907 , respectively. Against the RGS4(Δ7) mutant, the compounds had IC50 values of 51 µM and 181 µM, respectively. Each of the hit compounds were far less potent against the RGS4(Δ7) mutant than RGS4(WT), similar to what has been reported in literature The activity of each of the 13 remaining compounds was assayed by generating concentration-response curves against RGS4 as well as the RGS4(Δ7) mutant.  UI-1590 , had an  UI-1907  and UI-2 UI-1907 , had IC54−, which mimics the transition state of GTP bound to a Gα subunit 4− generates an RGS-Gα protein:protein interaction with approximately 50-fold higher than basal affinity RGS proteins are interesting targets due to their role in modulating G-protein signaling. Previous work identifying inhibitors of R4 family RGS proteins have centered on the disruption of the high affinity RGS – Gα interaction observed in the presence of AlFThe initial setup for the assay, for each 1536-well plate, was 1.5 h, which includes incubation steps for the production of free phosphate, allowing the assay to be conducted in highly parallel fashion. Using a colorimetric dye for readout is straightforward and can be accomplished on the simplest of plate readers in absorbance mode. Speed is also essential, and the total read time for each 1536-well plate was only 8 minutes, though this is plate-reader dependent. Perhaps most important is that this assay ameliorates a major concern in high throughput screening – the presence of library compounds that may absorb at a wavelength critical for the assay’s readout. In the case of this malachite green assay, the primary wavelength for the absorption read of the assay is at 642 nm, however, a secondary peak is also present at 436 nm, which provides a second readout to help discriminate compounds that may interfere with the primary readout at 642 nm. The absorbance at 436 nm is lower intensity than that at 642 nm, however it is quite usable as a secondary, confirmatory readout – and one that can be run on the same sample as the primary read .i, generated by hydrolysis of GTP, available to complex with malachite green and increase absorbance at 642 nm. An initial triage included the exclusion of hit compounds that interfered with the assay by directly inhibiting the chemical reactions of the assay readout or inhibiting Gαi itself reduced this hit rate to approximately 2.0%. 7 compounds failed to inhibit RGS4 greater than 3 standard deviations from the negative control in the initial hit confirmation assay using 40 µM compound. 1 compound was found to interfere with the malachite green assay directly, as shown when challenged in an assay containing only 50 µM PO4, (greater than 3 standard deviations from the negative control). Finally, an additional 5 compounds were found to inhibit the intrinsic GTPase activity (greater than 3 standard deviations from the negative control) of the Gαi subunit alone. A second, confirmatory screen of the initial hit compounds was performed using an orthogonal assay, ALPHA Screen , further reduced this to a hit rate of 1.3% After careful characterization of the constraints of the assay itself, we moved to a small-scale, proof-of-concept screen using a small molecule library of 2320 compounds , summarized in 50 values from 20–30 µM, UI-1775, UI-2144, UI-2202, and UI-2249 represent some of the most potent compounds reported for the RGS4(Δ7) mutant in vivoEach of the compounds demonstrates inhibition of RGS4. Some of the more potent compounds identified, such as UI-587 and UI-662, contain covalent cysteine and free amine chemical functionalities similar to those that have been discovered in other screens against RGS4 32P liberation assays represent the only well used method for determining the effect of RGS proteins on the rate of GTP hydrolysis. This malachite green assay is capable of almost completely replacing that assay due to its ease of use as well as cost. The mutant used in this assay, Gαi1, is capable of being used with a variety of RGS proteins beyond the R4 family, such as the RZ family q, R183C functions very similarly to the mutation R178M in Gαi1i2, R179C, also ablates intrinsic GTPase activity t, which increases the exchange rate by 150 times The development of this assay provides a new method for evaluating RGS proteins and their interactions with G-proteins. Steady-state analysis of RGS activity will allow for more accessible interpretations of compound effects on RGS G-protein interactions. i allows for a simple counter screen to remove compounds that interfere with the assay by direct inhibition of the Gαi construct. This assay has the potential to expand to encompass a variety of RGS protein families and increase the number of available tools to study this interesting family of proteins.In conclusion, we developed a simple, easy to use, and inexpensive assay for the evaluation of the GAP activity of a variety of RGS proteins. This study shows that this colorimetric assay is both robust and readily miniaturized for HTS application. The dual absorbance peak of the assay, 642 nm and 436 nm, allows for an in well counter-screen to include compounds that may have been lost due to absorbance at the primary reading wavelength. The slow but detectable intrinsic GTPase rate of the mutant GαFigure S13PO4.Wavelength Scan of 10 µM Na Using 10 µM Na3PO4 as a control, a wavelength scan of the absorbance of the system was evaluated to determine the optimal wavelength for detection. Two peaks were detected with local maxima at 436 nm and 642 nm.(TIFF)Click here for additional data file.Figure S2Peak Evaluation. The two selected peaks, 642 nm and 436 nm, were evaluated using ½ dilutions of Na3PO4 from 50 µM to 0.4 µM. The peak at 642 nm had a three fold greater response to Na3PO4 than the peak at 436 nm at equivalent concentrations.(TIFF)Click here for additional data file.


Cranial base growth plates are important centers of longitudinal growth in the skull and are responsible for the proper anterior placement of the face and the stimulation of normal cranial vault development. We report that the presphenoidal synchondrosis (PSS), a midline growth plate of the cranial base, closes in the DBA/2J mouse strain but not in other common inbred strains. We investigated the genetics of PSS closure in DBA/2J mice by evaluating F1, F1 backcross, and/or F1 intercross offspring from matings with C57BL/6J and DBA/1J mice, whose PSS remain open. We observed that PSS closure is genetically determined, but not inherited as a simple Mendelian trait. Employing a genome-wide SNP array, we identified a region on chromosome 11 in the C57BL/6J strain that affected the frequency of PSS closure in F1 backcross and F1 intercross offspring. The equivalent region in the DBA/1J strain did not affect PSS closure in F1 intercross offspring. We conclude that PSS closure in the DBA/2J strain is complex and modified by different loci when outcrossed with C57BL/6J and DBA/1J mice.The online version of this article (doi:10.1007/s00335-012-9437-8) contains supplementary material, which is available to authorized users. The synchondroses of the mammalian cranial base are important centers of longitudinal growth in the skull, playing a critical role in both the proper anterior placement of the face and the development of a normal cranial vault Bjork . The midStudies in humans and mice have identified genes and pathways that affect growth and maintenance of synchondroses. In humans, mutations in TWIST and in FGF signaling pathway components have been identified in patients with craniosynostosis .This work was approved by the Institutional Animal Care and Use Committees at Children’s Hospital Boston and Harvard School of Dental Medicine. Inbred mice of the DBA/2J (stock number 000671), DBA/1J (000670), and C57BL/6J (000664) strains were obtained from The Jackson Laboratory , P15, P21, P25, P40, P50, P61, P66, P80, and P180 (≥3/age). In addition, 82 DBA/2J mice 180 days or older were examined. The cranial bases of F1, F1 intercross, and F1 backcross offspring were examined either at the time of weaning (P21) or at P35.N = 3 per time point and strain), decalcified, dehydrated, cleared in xylene, and then mounted in paraffin. Seven-micron sagittal sections were obtained through the PSS and SOS. Slides were cleared in xylene, rehydrated, soaked in Gill Modified Hematoxylin Harlecohematoxylin (Harleco), Clarifier 2 (Richard-Allan Scientific), Bluing reagent (Richard-Allan Scientific), and Eosin Y (Richard-Allan Scientific), followed by dehydration and a final clearing in xylene. Slides were mounted with coverslips, viewed on a Leica DMLb microscope, and imaged using the OpenLab software suite. Measurement of the length of the PSS was performed digitally using the ImageJ software suite: for each animal, a measurement was taken spanning the midline of the PSS in two adjacent sections, giving a total of six measurements per mouse line per time point.Crania from DBA/2J and C57BL/6J mice were recovered at P1, P3, P5, P10, and P15 . After washing twice in 95 % EtOH and soaking in 2 % KOH in water for 3 h, bone was stained in 1 % KOH, 7.5 mg/ml Alizarin red S (Sigma) in water overnight. Specimens were destained in 20 % glycerol, 1 % KOH in water for 2 days and then put in 20 % glycerol in water, changed daily for 5 days. Finally, crania were placed in 20 % glycerol, 20 % EtOH overnight and then stored and photographed in 50 % glycerol, 50 % EtOH.2 analysis. SNPs yielding χ2-derived p values <5 × 10−5  were considered significant. Chromosome 11 microsatellite markers D11Mit62 (5.78 cM), D11Mit78 (10.44 cM), D11Mit109 (23.57 cM), and D11Mit111 (31.97 cM) were genotyped in 67 additional F2 offspring that had closed PSS and in more than 102 F2 offspring that had open PSS. The microsatellites were also genotyped in F1 backcross offspring that had closed PSS (N = 43) and open PSS (N = 38). DNA for PCR-based genotyping was obtained from tail snips using the HotSHOT protocol  following the manufacturer’s protocol. Whole-genome SNP genotyping was performed at the University of Toronto using the Illumina Bead Array mouse genotyping platform , which contains 874 informative SNPs between the DBA/2J and C57BL/6J strains. Because no common region of homozygosity was identified among the F1 intercross offspring that had PSS closure, we analyzed each SNP for deviation from the expected 1:2:1 distribution by χGenomic DNA from two female DBA/1J mice was extracted using the Qiagen DNeasy Blood and Tissue Kit and equal amounts of DNA were then pooled. Similarly, equal amounts of DNA from nine P35 F2 progeny with open PSS from the DBA/1J × DBA/2J cross were pooled. For each DNA pool, 4 μg of DNA was sheared to an average size of 200 bp using Adaptive Focused Acoustics following the manufacturer’s protocol . The DNA fragments were then blunt-ended, 5′ phosphorylated, A-tailed, and ligated to adaptors as previously described . The DBA/2J aligned sequence was downloaded from the Welcome Trust Sanger Institute (http://www.sanger.ac.uk/). The known SNPs between the DBA/1J and DBA/2J strains were downloaded from the Mouse Phenome Database (http://phenome.jax.org/). SNPs imputed to differ between DBA/1J and DBA/2J were downloaded from the Mouse HapMap Imputed Genotype Resource (http://mouse.cs.ucla.edu/mousehapmap/beta/index.html/). Custom perl scripts were used to determine whether the known and imputed SNPs were supported by the DBA/1J and DBA/2J whole genome sequencing (WGS). Only sites covered by at least one read in each strain were considered, and for sites covered by more than one read, only homozygous sites were considered. To identify novel SNPs, variant calling was performed using SAMtools/BCFtools  using BWA  from several commonly used inbred mouse strains , we noticed that the PSS remained patent in every strain except for DBA/2J . By P5, there was a loss of distinct growth plate morphology in the PSS of DBA/2J mice; hypertrophic chondrocytes spanned the entire ventral surface of the synchondrosis , this was not the case for the F1 intercross offspring (p = 0.00031), suggesting that the genetic control of the PSS closure trait is complex.We crossed DBA/2J and C57BL/6J mice to determine whether PSS closure was heritable and, if so, to see if it was due to a single locus. Since we could see evidence of PSS closure in DBA/2J mice when their cranial bases were examined at P21, we decided to score all offspring at this age. Offspring were considered affected if under the dissecting microscope we observed superior protrusion of the PSS cartilage, angular deformity of the PSS, or unilateral or bilateral bony bridging. We performed crosses using male and female mice from both strains to control for parent-of-origin effects. None of the F1 offspring exhibited PSS closure (0/67). When we intercrossed F1 animals to produce F2 offspring, 17.9 % (86/481) had PSS closure. When we backcrossed F1 animals to DBA/2J mice, 52.9 % (45/85) of offspring had PSS closure. Although the observed segregation pattern of the backcross offspring did not differ significantly from the pattern expected for autosomal recessive inheritance (D11Mit78 at 10.44 cM (located at 17.8 Mb on chromosome 11 in the mouse reference genome), exhibited the greatest enrichment in offspring with PSS closure. DBA/2J alleles were also enriched in offspring with closed PSS from the F1 × DBA/2J backcross  had PSS closure. When we examined F1 offspring at P35, 90 % (19/21) exhibited PSS closure. This result suggests that alleles inherited from the DBA/1J strain alter the timing of PSS closure. Consistent with this hypothesis, when we performed an F1 intercross and examined F2 offspring at P35, we observed PSS closure in 63 % (15/24) of the offspring.If a single locus in the DBA/1J strain was responsible for influencing PSS closure, F2 offspring with open PSS at P35 should be enriched for DBA/1J alleles at that locus. To identify genomic regions enriched for DBA/1J alleles, we performed low-coverage WGS on pooled DNA from the nine P35 F2 offspring that had an open PSS. Since the DBA/1J strain has not been fully sequenced, we also performed WGS on pooled DNA from two adult DBA/1J females. We obtained 76 million and 96 million 100-bp Illumina single-end reads, which resulted in 2.3- and 2.9-fold genome coverage when aligned to the reference genome (mm9), for DBA/1J and the F2 offspring, respectively. We compared our DBA/1J sequence to the publicly available DBA/2J sequence, which had also been aligned to the reference genome  in humans, which is expressed on bone tumor cells of mesenchymal origin or sarcomas  on mouse chromosome 11. In humans, the intrasphenoidal synchondrosis corresponds to the PSS in mice. Closure of the intrasphenoidal synchondrosis normally occurs shortly after birth. Therefore, the DBA/2J strain, whose PSS also closes shortly after birth, may be a useful model for understanding molecular mechanisms that contribute to normal synchondrosis closure in humans.Below is the link to the electronic supplementary material.Supplementary Fig. 1(PDF 6160 kb)Supplementary Fig. 2(PDF 1836 kb)Supplementary Table 1(TXT 234 kb)Supplementary Table 2(TXT 3 kb)Supplementary Table 3(TXT 8558 kb)Supplementary Table 4(TXT 860 kb)Supplementary Table 5(TXT 176 kb)Supplementary Table 6(TXT 54 kb)Supplementary Table 7(TXT 2 kb)


Bisphosphonates interfere with the mevalonate pathway and inhibit the prenylation of small GTP-binding proteins such as ras and rap. We hypothesized that zoledronic acid would synergistically inhibit T24 bladder cancer cell growth in combination with fluvastatin and paclitaxel.Increasing doses of fluvastatin, zoledronic acid, and paclitaxel were investigated as single agents and in combination, and synergistic interactions were evaluated by the Chou-Talalay method. Western blots were used to assess effects on signal transduction pathways.50 values of 2.67 ± 0.61 μM for fluvastatin and 5.35 ± 1.35 μM for zoledronic acid after 72 hours treatment. Geranylgeranyl pyrophosphate and farnesyl pyrophosphate was able to block, in part, this inhibitory activity. The combinations of zoledronic acid and paclitaxel, zoledronic acid and fluvastatin, and fluvastatin and paclitaxel were all synergistic. Both fluvastatin and zoledronic acid inhibited Ras and Rap prenylation, and the phosphorylation of ERK1/2 and AKT. The degree of inhibition of phosphorylation of these key signaling transduction pathways appears to closely correlate with their synergistic interactions.Growth of T24 was significantly inhibited with ICZoledronic acid enhances fluvastatin and paclitaxel activity against T24 in a synergistic manner and this is mediated largely by inhibition of both the Ras/Raf/MEK/ERK and PI3K/AKT signaling pathways via isoprenylation inhibition. Recently, it has been shown that nitrogen-containing bisphosphonates such as zoledronic acid inhibit farnesyldiphosphate synthase . We hypo2.2.1.50 2.67 ± 0.61 μM at 72 h) than to zoledronic acid (IC50 5.35 ± 1.35 μM at 72 h) (Treatment of T24 cells with increasing concentrations of fluvastatin (0.1 to 100 μM) or zoledronic acid (0.1 to 100 μM) for 24–72 hours caused dose and time-dependent cell growth inhibition. T24 cells were slightly more sensitive to fluvastatin (ICat 72 h) .2.2.In these experiments, fluvastatin (3μM) and zoledronic acid (10μM) inhibited cell proliferation by 44 ± 6.2%, and 71 ± 2%, respectively . Geranyl2.3.At lower concentrations of fluvastatin (0.5–1.5 μM), zoledronic acid (0.5–3 μM), and paclitaxel (below 2 nM) additive growth inhibition was achieved (CI = 0.9–1.1), whereas 5–10 μM of zoledronic acid and 1.5–3 μM fluvastatin combined with 4–8 nM of paclitaxel produced greater synergistic effects (combination indexes ranging from 0.3 to 0.6)  was dissolved in ddH2O and fluvastatin was dissolved in DMSO; 100 mM of stock solutions were stored in −20 °C, and were diluted in growth medium before use. Both zoledronic acid and fluvastatin were kindly provided by Novartis Pharmaceuticals Australia. Paclitaxel (Sigma) was dissolved in DMSO and stored in 4 °C as a stock solution. Geranylgeranyl pyrophosphate and Farnesyl pyrophosphate were both purchased from Sigma and stored in −20 °C; Anti-RAP1A antibody, anti-® actin and horseradish peroxidase (HRP) -linked anti-goat antibody were purchased from Santa Cruz Biotechnology (U.S.). Anti-RAP1A antibody can bind to the nongeranylgeranylated form RAP1 [Human bladder cancer cell line T24 cells were grown in RPMI medium (Sigma), supplemented with 10% of fetal calf serum (GIBCOBRL), 100 U/mL of penicillin and 100 μg/mL of streptomycin (GIBCOBRL), at a constant temperature of 37 °C in a humidified atmosphere with 5% COorm RAP1 . Anti-ph3.2.Cell growth inhibition was determined by the sulforhodamine B colorimetric assay (SRB) . Briefly3.3.T24 cells were seeded in 96-well plates at 1000/well for 24 hours, then treated with the drugs (3 μM fluvastatin or 10 μM zoledronic acid) alone. Farneysl pyrophosphate and geranylgeranyl pyrophosphate (10 μM each) were then added within 60 minutes, and growth inhibition was assessed by SRB assay as above.3.4.Drug combination effects were analyzed using “CalcuSyn Windows Software for Dose Effect Analysis” developed by Chou and Talalay . BrieflyX)1 is the dose of drug-1 required to produce x percent effect alone and (D)1 is the dose of drug-1 required to produce the same x percent effect in combination with drug-2; similarly, (DX)2 is the dose of drug-2 required to produce x percent effect alone and (D)2 is the dose of drug-2 required to produce the same x percent effect in combination with drug-1. CI < 1, =1, and >1 indicates synergism, additive effect, and antagonism, respectively.Where (D3.5.3 (2 mM) and PMSF (1 mM). Lysates were sonicated in an ice water bath for 20 seconds; the supernatant containing protein was separated from the insoluble material by centrifugation at 10,000rpm for 10 minutes at 4 °C. The total protein in each sample was then quantified using Bicinchoninic Acid Kit (Sigma) by following the protocol described. Samples containing equal amounts of protein were mixed with loading buffer and boiled for 5 minutes. Proteins were then separated by SDS-PAGE, and the proteins on the gels were transferred to PVDF-Membrane (Amersham Biosciences) and incubated with different antibodies. After incubation with HRP-conjugated secondary antibodies, the primary antibody binding was visualized with Western Lightning™ Chemiluminescence . Density of the bands were quantified by scanning with Gel Doc 200 (Bio-Rad) and normalized by β-actin as loading control (density of the sample /density of β-actin); P-AKT and P-ERK expression levels were further normalized by density of the band of the non-drug treated control. All Western blots were repeated at least once, and similar results were confirmed.Cells were allowed to reach 70% confluence in RPMI containing 10% of FCS for 24 hours, before drugs were added in serum-free medium for another 24 hours. Cells were then washed with cold PBS buffer and lysed in buffer containing 50 mM Tris-HCI (pH7.5), 150 mM NaCI (150 mM), EGTA (1 mM), EDTA (2 mM), 2.5%(V/V) glycerol, NaF (10 mM), 0.2%(V/V) Triton X-100, 0.3% (V/V) NP40, aprotinin (5 μg/mL), Leupeptin (10 μg/mL), pepstatin (10 μg/mL), activated NaVO3.6.Each experiment was performed at least three times, and all values are represented as the means ± SD of triplicates. Student's t-test was used to determine the statistical significance of the results. Values of p < 0.05 were considered statistically significant.4.In conclusion, our paper demonstrates a number of novel observations of the mechanism of the antitumor activity of zoledronic acid on T24 cells. We show that zoledronic acid inhibits isoprenylation, and that this can be rescued by GGPP, but not by FPP. Further, isoprenylation inhibition inhibits the function of Ras, and this results in reduced phosphorylation of downstream targets such as ERK and AKT. The antiproliferative and anti-Ras function of zoledronic acid can be improved by the addition of fluvastatin and paclitaxel in a synergistic manner. The mechanism of this synergy appears to be largely due to drug effects on two major signaling transduction pathways, Ras, and AKT.


Both nuclear receptor subfamily 2 group F member 1 (NR2F1) and microRNAs (miRNAs) have been shown to play critical roles in the developing and functional inner ear. Based on previous studies suggesting interplay between NR2F1 and miRNAs, we investigated the coregulation between NR2F1 and miRNAs to better understand the regulatory mechanisms of inner ear development and functional maturation.–/–Nr2f1) mice compared to wild-type littermates but is unchanged in the brain. Based on this, we performed chromatin-immunoprecipitation followed by qRT-PCR and confirmed that NR2F1 directly binds and regulates both miR-140 and Klf9invivo. Furthermore, we performed luciferase reporter assay and showed that miR-140 mimic directly regulates KLF9-3’UTR, thereby establishing and validating an example coregulatory network involving NR2F1, miR-140, and Klf9.Using a bioinformatic approach, we identified 11 potential miRNAs that might coregulate target genes with NR2F1 and analyzed their targets and potential roles in physiology and disease. We selected 6 miRNAs to analyze using quantitative real-time (qRT) -PCR and found that miR-140 is significantly down-regulated by 4.5-fold (P=0.004) in the inner ear of NR2F1 knockout (Klf9 in the inner ear and we propose the existence of many such coregulatory networks important for both inner ear development and function.We have described and experimentally validated a novel tissue-dependent coregulatory network for NR2F1, miR-140, and  Nr2f1–/–) which impacts neurogenesis, axonal guidance, neocortical patterning, morphogenesis, and patterning of the cochlear sensory epithelium and the inner and outer hair cells in the organ of Corti [Nr2f1 locus, implicating haploinsufficiency of NR2F1 as a cause for a 4-year-old child’s deafness, dysmorphism and developmental delay [Nuclear receptors are a large family of cellular regulators that function as activators, repressors and silencers of transcription when bound by specific ligands or cofactors ,2. They of Corti –10. A real delay .in vivo, Montemayor et al. developed a methodology to identify NR2F1 target genes by intersecting gene expression profiling, computational binding site queries, and evolutionary conservation data [cold spots within the critical genomic segments that regulate gene expression [In an effort to define transcription factor binding sites and target genes ion data . This appression . Using tpression . C. elegans. They have diverse expressive patterns and regulate a variety of areas, including embryologic development, physiology, pathophysiology, and growth, development, progression, and metastasis in cancer [MicroRNAs were discovered as small temporal RNAs that regulate developmental transitions in n cancer –17. Currn cancer . In human cancer . This lan cancer ,16. ThesDicer is required for survival of inner ear hair cells in mice, and a loss-of-function results in aberrant hair bundle formation, stereocilia defects, disrupted inner ear morphogenesis, and impaired hearing function [MicroRNAs have a specific role in the developing inner ear and hearing, where many miRNA expression profiles change both temporally and spatially during embryogenesis and post-natal maturation and are conserved through evolution –22. The function ,24.Klf9). This is the first description of such a pathway and delineates the regulatory actions of a circuit involving the non-steroidal nuclear receptor transcription factor and miRNAs.Given the apparent roles of NR2F1 and miRNAs in the ontogenesis of the inner ear, and more notably the functional deficits caused when these regulators are mutated or deleted, we sought to discover the genetic coregulatory networks of NR2F1 and miRNAs. We hypothesized that knowing if, how, and when gene regulatory pathways intersect could shed light on the current incomplete knowledge of the regulatory mechanisms of inner ear development and functional maturation , and perThe Institutional Animal Care and Use Committee of Baylor College of Medicine approved all studies involving the use of animals as mandated by the United States federal government. http://genome.ucsc.edu) with the NR2F1 binding site custom track supplied by Montemayor et al. [http://www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5), TargetScan 6.0 (http://www.targetscan.org), microRNA.org (http://www.microrna.org/microrna/home.do), and PITA (http://genie.weizmann.ac.il/pubs/mir07/mir07_dyn_data.html) [In order to find the overlap of any miRNA gene with NR2F1 target genes, the human genome (GRCh37/hg19) and mouse genome (NCBI37/mm9) builds were viewed using the UCSC genome browser   . The preta.html) .http://www.microrna.org/microrna/home.do) was used to determine binding sites within the translated regions of the genes while TargetScan 6.2 (http://www.targetscan.org) [http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk/index.html) [MicroRNA.org (can.org)  and miRWex.html)  were useThe Database for Annotation, Visualization and Integrated Discovery (DAVID) ,36 queryNr2f1–/– (–/–) and WT (+/+) littermates were placed in RNA Later (Ambion) after dissection, stored at 4°C overnight and then at -20°C until the genotype was determined [Inner ears of P0 termined . Total RReverse transcription and real-time PCR of mRNAs were carried out using 0.25μg of RNA, nuclease-free water, 0.5μl of Oligo(dT), 10μl 5x First-Strand buffer, 4μl of 10mM dNTP, 5μl of 0.1M DTT, 1μl ribonuclease inhibitor (Invitrogen), and 2μl SuperScript II (Invitrogen). Reverse transcription was performed at 42°C for 1h before heating to 70°C for 15 min then returning to 4°C. Real-time PCR was performed in triplicates in 96-well plates in Mastercycler ep realplex  using the following program: 1) 50°C for 2min, 2) 95°C for 2min, 3) 45 cycles of 95°C for 15s, 60°C for 1min, and 4) melting curve ramp. 5’-ctcaactggtgtcgtggagtcggcaattcagttgagaatttggt-3’ and 5’- acactccagctgggaaacaaacaaaca-3’. Briefly, 0.25μg of RNA for each sample was incubated for 5 minutes at 65°C with nuclease-free water and 0.5μl of 10mM dNTP mix in a final volume of 12.65μl and returned to ice for at least 2 minutes. A mix of 4μl 5X First-Strand buffer, 2μl 0.1M DTT, 0.1μl ribonuclease inhibitor (Invitrogen), 0.25μl SuperScript II RT (Invitrogen) and 1μl of 1μM stem-loop primer was added for each reaction. Reverse transcription was performed using the following program: 1) 16°C for 30min, 2) 45 cycles at 30°C for 30s, 42°C for 30s, and 50°C for 1s, and 3) 85°C for 5min. For real-time PCR, 2μl of the reverse transcribed samples were mixed with 1μl each of the forward and reverse primers (1μM), 4μl of Express SYBR Green (Invitrogen) and 12μl nuclease-free water. The real-time PCR was performed in triplicates in 96-well plates in Mastercycler ep realplex  using the following program: 1) 95°C for 5min, 2) 45 cycles of 95°C for 5s, 60°C for 10s, 72°C for 1s, 3) melting curve ramp from 65°C to 95°C at 0.1°C per second. Reverse transcription and real-time PCR of microRNAs were carried out using a protocol modified from Varkonyi-Gasic et al. using stem-loop primers and SYBR Green without any commercial miRNA detection kit . All steT (cycle number) method [S7 in Expression levels of both mRNA and miRNAs were compared using the relative C) method  after noCerebral cortex was harvested from C57Bl/6 mice and processed by chromatin crosslinking and immunoprecipitation as described in the Active Motif ChIP-IT Express Kit . Briefly, 150 mg of cortex was minced and cross-linked with 1% formaldehyde at room temperature with gentle shaking for 12 min. Glycine was added  to quench the formaldehyde by incubating for 5 min with gentle shaking. The tissue pieces were then dounce homogenized  in 2 ml of ice-cold PBS supplemented with 10 ul proteinase inhibitor cocktail  and 10 ul phenylmethylsulfonyl fluoride  to obtain a single-cell slurry which were pelleted at 660xg at 4°C for 5 min. Cells were resuspended in 2 ml Lysis Buffer supplemented with PIC and PMSF, incubated for 30 min on ice before being dounce-homogenized again  to release the nuclei, which were then pelleted with centrifugation for 10 min at 660xg rpm at 4°C. Chromatin fragmentation was performed in shearing buffer using a 450 Branson Sonifier  and the sheared chromatin was collected at 20,000xg for 15 min at 4°C, after which the supernatant was stored at -80°C. Chromatin-immunoprecipitation (ChIP) was performed at 4°C overnight with antibodies against NR2F1  and histone H3K9-Ac  while control immunoprecipitations (IPs) were performed with mouse IgG . Twenty microliters of magnetic beads (Active Motif), 2 ug of antibody, and the chromatin samples were rotated overnight at 4°C in 200 ul volumes each. The next day, beads were washed twice with 800 ul ChIP Buffer 1 and four times with 800 ul ChIP Buffer 2. The beads were then resuspended in Elution Buffer and rotated for 15 min at room temperature. Fifty microliters of Reverse-Cross-linking Buffer were added and the supernatant transferred to a fresh tube. ChIP and input samples were denatured at 95°C for 15 min in a BioRad thermocycler before the proteins were digested with 2 μl Proteinase K (0.5ug/ul) for 1 hour at 37°C. Genomic DNA was isolated using Qiagen PCR Kit  and used for RT-PCR as described above.KLF9 3’ untranslated region (UTR) and an empty luciferase reporter construct were purchased from SwitchGear Genomics . Putative miR-140 binding sites were identical with Klf9 3’UTR. miR-140 mimic and a non-targeting scramble miR mimic were purchased from Life Technologies  and used at a final concentration of 30 nM. Transfection and luciferase assay were carried out according to the manufacturer’s published protocol [A luciferase reporter construct containing a 3304-bp fragment of the protocol , using HData are presented as mean ±SEM. Statistical analysis was performed using two-tailed Student’s t-test with α=0.05. Klf9 gene, which contains the gene encoding miR-1192, was previously validated to be up-regulated and inner ear of Nr2f1–/– mice [Yipf3 and Ak1 genes, respectively. A multi-step approach was used to discover miRNAs that might participate to coregulate NR2F1 target genes. A list of putative NR2F1 target genes identified by Montemayor et al.  was used–/– mice . Beyond Next, we investigated recent articles that identified miRNAs in the developing embryonic mouse inner ear –24,30,44Nr2f1–/– mice, as quantified by microarray expression profiling and/or validated by qRT-PCR [Table S1 in Nr2f1–/– [To determine the relative potential for coregulation of target genes by NR2F1 and the miRNAs, we generated a list of the total number of genes targeted by the 11 miRNAs, as well as the subset of target genes whose expression was changed in the  qRT-PCR  , which suggests a feedback regulatory system may be possible. Other interesting observations include that Pdgfrα, a gene known for its role in both development and oncogenesis [Psen1 and Nefl [S3 in We next selected the genes targeted by at least 3 miRNAs for an in-depth analysis. This group of 28 genes, along with the miRNAs by which they are targeted, and the relative change in expression in the r2f1–/–  is shownogenesis ,53, was and Nefl ,55. Analand Nefl ,36 queryand Nefl  and the and Nefl , revealeTable S4 in S5 in Multiple binding sites for a miRNA on a target mRNA might indicate greater regulatory control by that miRNA, whereas binding motifs shared by multiple miRNAs may indicate a lack of specificity in regulation . Thus, wCrym and Snai2 were both targeted by miR-96 and miR-181b; Gjb2 was targeted by miR-140 and miR-183 , the Serial Analysis of Gene Expression (SAGE) and the literature ,38, and Table S2 in Table S6 in Crabp1, Fabp7, Rbp1) involve cellular transport of fatty acids and hormones in metabolic process, including forebrain development, which is defective in Nr2f1–/–. Cluster 2 genes  involve RNA binding and metabolic processes and post-translational regulation of biological processes. The 10 genes in clusters 3-5 involve steroid, lipid and cholesterol biosynthesis, cation transport and chromatin assembly and organization, which are key processes during development. Cluster 6 has 20 genes that explicitly involve transcription, and include Nr2f1 and Klf9. Clusters 7 through 9 have lower enrichment scores but involves skeletal development, nucleotide binding, cell adhesion, tube development and morphogenesis, the endomembrane system and energy regulation, many functions of which are defective in Nr2f1–/– [Using the Gene Ontology (GO) Gene Functional Classification analysis and the DAVID database ,36, we dr2f1–/– .Nr2f1–/– mice using qRT-PCR. Of note, miR-140, -181b and -191 each have a putative NR2F1 binding site close to the proximal portion of their genes and since miR-181b is found in a family cluster we also included miR-181a and -181c, which may be coregulated and share similar gene targets. MicroRNA-140 has 4 co-targeted genes with NR2F1 amongst the validated expression changes in the Nr2f1–/– mice and is predicted to target NR2F1 itself (Table 1 & S1 in Klf9 gene which is up-regulated in the inner ear of Nr2f1–/– mouse [Nr2f1–/– inner ear by 4.5-fold (P=0.004) and 1.7-fold (P=0.046) compared with wildtype (WT), respectively, while the other 4 miRNAs were not significantly down-regulated   but was unchanged in the cerebral cortex of Nr2f1–/– mice  discovery of a mechanism by which NR2F1 regulates direct target gene expressions; 2) identification of specific miRNAs, such as miR-140, that co-regulate and alter NR2F1 target gene expressions, such as Klf9; 3) delineation of a feedback coregulation that occurs between NR2F1 and miR-140; and 4) identification of a tissue-dependent coregulatory network involving NR2F1, miR-140 and Klf9.The orphan nuclear receptor NR2F1, located on chromosome 5q14, and its homolog NR2F2, are the most evolutionarily conserved nuclear receptors across all species . NR2F1 imiR-140 gene, located on chromosome 16q22, is most notable for its role in cartilage development and maintenance [Nr2f1 expression during inner ear development [miR-140 by NR2F1. Indeed, we found miR-140 transcripts were significantly down-regulated by 4.5-fold in Nr2f1–/– newborn inner ears but remained unchanged in the cerebral cortex  are expressed in the developing inner ear and adult brain  during neurogenesis, there are other tissues where miR-140, NR2F1, and Klf9 functionally interact, but the mechanisms of their interactions have not yet been delineated. Indeed, both KLF9 and NR2F1 have a role in lipid processing [Klf9 are both regulated in the brain by estrogen-receptor modulation [Klf9 opposes estrogen hormonal action by repressing estrogen receptor (ERα) expression and activity [Klf9 is important for estrogen action in the inner ear. t factor , has a rt factor , and is t factor . Since aocessing ,77, and dulation ,78. In tdulation –81. Klf9activity –84. ERα activity . The comKlf9. Klf9 is regulated by thyroid hormone and its nuclear receptor TRβ, which are essential for normal development and functional maturation of hearing [Klf9) [Notch1 expression and its downstream signaling which promotes differentiation and inhibits glioblastoma neurosphere growth [Klf9 and miR-140 are consistent with the co-regulatory network proposed  ,88. Addie growth . Indeed,e growth . Cumulatproposed 6. Our fKlf9 by a regulatory network involving miR-140 and NR2F1, our bioinformatics search revealed at least 20 other common targets shared by miR-140 and NR2F1, four of which have been validated to have a change of expression in the Nr2f1–/– mice [Table S1 in Klf9 is only one of these genes, which according to this model is transactivated at the transcriptional level by NR2F1 but repressed at the post-transcriptional level by miR-140 in order to achieve a balanced expression level. The remaining common targets are also potential miR-140 and NR2F1 co-regulated genes. In addition to this focused regulation of –/– mice   were targeted by multiple miRNA, which may indicate a higher level of control over expression 1B. PdgfNr2f1, Crym, Snai2, Gjb2) [Pdgfrα, Eln, Jun) [Nr2f1, Notch1, Jag 1, Nptx1, Runx1) [S4 & S5 in Nr2f1–/– mice have inner ear developmental defects [Klf9 in the inner ear compared to the cortex of Nr2f1–/– mice given the site-specific regulation of miR-140 and NR2F1. Nor is it unexpected that defects in the cochlear duct, organ of Corti, and hair cells can occur in animals with impaired NR2F1 or miRNA function [Furthermore, these coregulatory networks may have clinical significance. The genes targeted by both NR2F1 and the 11 miRNAs that we identified bioinformatically have previously been implicated in hearing loss  –94 and cln, Jun) ,95–98, a, Runx1) ,8,22,99, defects ,8,22,99. defects  and is d defects . It is nfunction ,21,27. TKlf9. We also uncovered several other potential pathways between NR2F1, miRNAs, and genes important for development and disease. These targets and pathways require further studies and validation in order to advance our understanding of ontogenesis and provide novel strategies for protecting or regenerating hair cells and sensory neurons in patients with hearing loss.In conclusion, we have demonstrated that NR2F1 and the 11-selected miRNA may play an important or indispensable role in ontogenesis and development in general. As a proof-of-principle, we experimentally demonstrated the coregulatory mechanisms and feedback loops between NR2F1, miR-140, and File S1Supporting Information File containing Tables S1 to S7:Table S1. Number of predicted miRNAs and NR2F1 inner ear targets.Table S2. Genes targeted by at least 3 of the 11 selected miRNAs.Table S3. Representative list of genes targeted by multiple miRNAs and their associated neurological and other diseases based on the Genetic Association Database.Table S4. List of genes targeted by at least 3 of the 11 select miRNAs and the respective miRNA binding sites in their mRNA 3’ untranslated region based on TargetScan 6.2.Table S5. Genes targeted by at least 3 of 11 selected miRNAs and the respective miRNA binding sites based on miRanda analysis from microRNA.org.Table S6. Functional classification of the genes targeted by miRNAs and NR2F1.Table S7. List of primers for real-time RT-PCR.(DOCX)Click here for additional data file.


Accumulation of β-amyloid (Aβ) and cholinergic deficiency are two prominent features of Alzheimer’s disease (AD). To understand how Aβ-induced dysfunction of the nicotinic system may contribute to cognitive impairment in AD, we examined the effect of Aβ on nicotinic regulation of synaptic transmission and neuronal excitability in prefrontal cortex (PFC), a brain region critical for cognitive processes.We found that activation of nicotinic acetylcholine receptors (nAChRs) with nicotine increased the inhibitory postsynaptic currents recorded in PFC pyramidal neurons, which was associated with the nicotine-induced increase in the excitability of PFC layer I GABAergic interneurons. Both effects of nicotine were disrupted by Aβ. However, Aβ did not impair nicotinic regulation of excitatory neurotransmission in PFC interneurons. The nicotinic effect on synaptic inhibition was also lost in transgenic mice with five familial Alzheimer’s disease mutations. Inhibiting PKC attenuated nicotinic regulation of inhibitory, but not excitatory, neurotransmission.Our study suggests that Aβ selectively impairs nicotinic regulation of inhibitory inputs to PFC pyramidal neurons, which might be due to its interference with PKC activation. Thus, in the PFC circuits of AD, the balance between inhibition and excitation under the control of nAChRs may be disturbed by Aβ. Aβ plays a critical role in the pathogenesis of Alzheimer’s disease (AD) -4. EarliIn addition to Aβ accumulation, another hallmark of AD is a selective loss of cholinergic neurons in basal forebrain , and theIt has been suggested that nAChRs are involved in regulating the integrated circuit activity between interneurons and projection neurons ,21. We hA receptors . Nicotine 5 μM, ,23) was  was 23])s Figure A. In thes Figure D.To identify the possible receptor subtypes that mediate the nicotinic effect, we applied nAChR antagonists to PFC slices. As shown in Figure To assess whether nicotinic enhancement of sIPSC is due to nAChR-induced increase in the excitability of PFC GABAergic interneurons, we examined the effect of nicotine on action potential firing of interneurons in PFC. Since layer I of neocortex contains exclusively GABAergic interneurons with extensive axonal plexus innervating neurons in deep layers -26, we fAs shown in Figure We also assessed whether nicotine affects the excitability of deep layer neurons. We found that nicotine did not cause a significant increase in the firing rate or resting potential of layer V fast-spiking interneurons (n = 5). Nicotine also failed to show any significant effect on the firing rate of layer V pyramidal neurons (n = 5).Given the involvement of cholinergic hypofunction in AD, we next examined whether Aβ could alter the nicotinic regulation of inhibitory synaptic transmission in PFC pyramidal neurons. Aβ oligomers were generated as we described and characterized before . PFC sliin vivo, we examined the transgenic mice with five familial Alzheimer’s disease mutations (5xFAD), which rapidly recapitulate major features of AD amyloid pathology. These mice start to accumulate intraneuronal Aβ42 at ~1.5 months of age within neuron soma and neurites and amyloid deposition reaches a very large burden in deep cortical layers   was a ge3, 3 KCl, 5 MgCl2, 1.25 NaH2PO4, 2 CaCl2, and 10 glucose, pH 7.4, 300 mOsm) bubbled with 95% O2 and 5% CO2.All experiments were performed under the supervision of State University of New York at Buffalo Animal Care Committee. Sprague Dawley rats (3–5 weeks old) were anesthetized with 2-bromo-2-chloro-1,1,1-trifluoroethane (1 ml/100 g) inhalation before decapitation ,58. Brai2, 1 EGTA, 30 N-methyl-d-glucamine (NMG), 5 MgATP, 0.5 Na2GTP and 12 phosphocreatine, pH 7.2–7.3, 270–280 mOsm. CNQX or DNQX (20 μM) and APV (40 μM) were added to ACSF to block AMPA and NMDA receptors. For sEPSC recording, the internal solution was composed of (in mM): 130 Cs-methanesulfonate, 10 HEPES, 10 CsCl, 4 NaCl, 1 MgCl2, 1 EGTA, 5 NMG, 5 MgATP, and 0.5 Na2GTP and 12 phosphocreatine, pH 7.2, 275–290 mOsm. Bicuculline (10 μM) was added to ACSF to block GABAA receptors. Cells were visualized with a 40x water-immersion lens and illuminated with near infrared (IR) light, and the image was detected with an IR-sensitive CCD camera. A Multiclamp 700A amplifier (Axon Instruments) was used for the recording. Tight seals (2–10 GΩ) from visualized neurons were obtained by applying negative pressure. The membrane was disrupted with additional suction, and the whole-cell configuration was obtained. The access resistances were 13–18 MΩ. The membrane potential was held at −70 mV.Voltage clamp recording of synaptic currents in slices was performed as described previously ,59. The 2SO4, 60 NMG, 40 HEPES, 4 MgCl2, 0.5 BAPTA, 12 phosphocreatine, 2 Na2ATP and 0.2 Na3GTP, pH 7.2–7.3, 265–270 mOsm. The firing rate of each neuron was averaged from 10 consecutive traces under each condition.Mini Analysis program  was used to analyze synaptic activity. Individual synaptic events with fast onset and exponential decay kinetics were captured with threshold detectors in Mini Analysis software. All quantitative measurements were taken 4–6 min after drug application. Spontaneous IPSC or EPSC recordings of 3 min under each condition were used for obtaining cumulative distribution plots of the amplitudes and inter-event intervals. To measure cell excitability, the whole-cell current-clamp technique ,59 was ut-tests. Experiments with more than two groups were subjected to one-way ANOVA, followed by post hoc Tukey tests.Statistical comparisons of the synaptic currents were made using the Kolmogorov-Smirnov (K-S) test. Experiments with two groups were analyzed statistically using unpaired Student’s Numerical values were expressed as mean ± SEM.The authors declare that they have no competing interests.GJC and ZX performed experiments and analyzed data. ZY designed experiments and wrote the manuscript. All authors read and approved the final manuscript.


The harmful effect of hypocapnia on the neonatal brain emphasizes the importance of monitoring arterial carbon dioxide tension (PaCO2). Transcutaneous monitoring of carbon dioxide (tcPCO2) reduces the need for arterial blood sampling. Drawbacks are high electrode temperature causing risks of skin burning. The aim was to determine the accuracy and precision of tcPCO2 at reduced electrode temperature.Forty newborns (GA 24.9-41.7) were included. Two tc-monitors were applied . Arterial blood gas sampling and monitoring of tcPCO2-level at different electrode temperatures was done simultaneously . Difference of PaCO2-tcPCO2 was expressed as a percentage of the mean.2. Only mild transient erythema was observed.Mean PaCO2 was 5.8kPa . Bias (PaCO2 -tcPCO2) increased from 5% at 44°C to 17% at 39°C, but did not differ significantly between 41°C and 40°C. The precision of the tcPCO2 at each temperature ranged from +7-10%. After correction for the temperature-dependent over-reading, we found increasing PaCO2 — tcPCO2 difference with increasing PaCO2, approx. 2% pr. kPa increase of COA lower electrode temperature in tcPCO2-monitoring increases systematic overreading of the tc-electrode. However, in very preterm babies, monitoring at 40°C or 41°C is possible provided a bias correction of 12-15% is applied. The harmful effects of hyperventilation and hypocapnia on the neonatal brain emphasize the importance of a stable arterial carbon dioxide tension (PaCO2) especially in mechanically ventilated newborns . Transcu2. Drawbacks of the high electrode temperature are risk of skin burns  and median birth weight 1822 g  .n = 11), asphyxia (n = 1), meconium aspiration syndrome (n = 1), necrotizing enterocolitis (n = 1), congenital heart disease (n = 3), oesophageal atresia (n = 1), extracorporeal membrane oxygenation (n = 1) and no diagnosis specified (n = 2).Diagnoses were manifold: Prematurity (n = 20) were preterm, however other diagnoses included pneumothorax (n = 4), rhesus immunization (n = 1), persistent fetal circulation (n = 1) and lung bleeding (n = 1).In the preterm group, the median gestational age was 28.9 weeks  and median birth weight 1006 g  . All infn = 1) and ‘apnoea, bradycardia and desaturation of prematurity’ (n = 1), while in the preterm group four infants had remarks: ‘restless’ (n = 2), ‘increasing oxygen demand’ (n = 2), ‘apnoea, bradycardia and desaturation of prematurity’ (n = 4).No infants were excluded due to instability, since this was not a part of the protocol. However, if there were any events that potentially could influence results, it was remarked. In the GA mixed group, two infants had remarks: ‘restless’ (n = 220) was normally distributed  and was not associated with electrode temperature (p = 0.9). The repeatability of arterial pCO2 measurements (n = 19) was 0.07 ± 0.15 kPa.Arterial pCO2 , at 40°C 73% (44/60 measurements), at 41°C 68% (27/40 measurements), at 42°C 90% (18/20 measurements) and at 44°C 94% had a bias below 1 kPa (75/80 measurements).The mean PaCO2 - tcPCO2 difference (bias) increased from 5% at 44°C to 17% at 39°C, but did not differ significantly between 41°C and 40°C (median 14.8% vs. 11.8%)  & 3. TheAll outliers were above the 95% confidence interval. These outliers occurred primarily at low electrode temperature. Sequence of electrode temperatures, including preheating of the skin to 44°C, had no influence on precision. We did not find any significant electrode drift after each session by control measurement using the calibration gas. No skin lesions apart from mild transient erythema were observed.The aim of this study was to investigate how reduction of the electrode temperature influences accuracy and precision in tcPCO2 measurements in newborn infants. Lower electrode temperature reduces the risk of skin burning and allows longer periods of continued monitoring .The bias of tcPCO2 using the normal electrode temperature of 44°C was approximately 5%. We had set the metabolic constant at 0.53 kPa (4 mmHg), which appears to be too low for our population.Lower electrode temperatures were associated with higher bias, but at 41°C the bias was still typically less than 1 kPa. Precision was unchanged. We therefore conclude that it is reasonable to monitor tcPCO2 with a temperature of 41°C, particularly if the bias of 12-15% is accounted for. However, changing the electrode temperature requires an increased clinical awareness. With measurements close to the normal PaCO2 range an error of 1 kPa will have no great consequences, however readings in the subnormal range of PaCO2 can have a significant impact on cerebral perfusion. On our ward, we recommend an arterial blood gas if there is any doubt of the actual level of PaCO2 and in particular if the tcPCO2 is out of the normal range. At 41°C, the tcPO2 cannot be relied upon, because the capillary bed will not be sufficiently arterialized. The best procedure would be to deactivate the pO2 readout of the TCM4. In most situations, oxygen treatment of newborns can be sufficiently monitored by pulseoximetry.2 at 42°C and even 37°C [Our results agree reasonably with previous studies. A study in neonates found a good correlation between tcPCO2 and arterial COven 37°C . The corven 37°C . Furtherven 37°C . In adulven 37°C . The TOS2 changes the pH of the electrolyte solution and the pH changes are proportional to the logarithm of pCO2-changes. Since tissue heating increases tissue pCO2 by 4.5%/°C and some CO2 is produced locally, the tcPCO2 is higher than the arterial pCO2 and corrections for this are included in the monitor. For optimal use, the electrolyte and membrane must be changed every week, and the sensor must also be calibrated whenever a new patient is monitored. This was done meticulously for our two experimental monitors and furthermore control measurements using the calibration gas were done after measurements in all infants. If out of the acceptable range, recalibration and/or change of membrane was done. Therefore we cannot explain the pCO2 dependency of the bias. Combined with the upwards skew of the distribution of transcutaneous-arterial pCO2 difference, it meant that occasionally tcPCO2 overestimated significantly, and this occurred more often at high arterial tcPCO2. In practical terms this means that a very high tcPCO2 must be used with care. Safe practice would be taking a blood sample before significant clinical action, e.g. intubation for mechanical ventilation.Surprisingly, in our study the bias was pCO2-dependent. The transcutaneous electrode uses an electrochemical principle: tcPCO2 is measured by determining the pH of an electrolyte layer separated from the skin by a highly permeable membrane. Diffusing COIn conclusion, a lower transcutaneous electrode temperature increases the systematic overreading of tcPCO2. Monitoring at 40 or 41°C, however, appears reasonable since the typical bias is less than 1 kPa. In some cases with high arterial pCO2 the overreading was clinically significant and care should be taken that this does not have undue clinical consequences.