Amino acids (AA) are one of the key nutrients that regulate cell proliferation and casein synthesis in cow mammary epithelial cells (CMEC), but the mechanism of this regulation is not yet clear. In this study, the effect of SESN2 on AA-mediated cell proliferation and casein synthesis in CMEC was assessed. After 12 h of AA starvation, CMECs were cultured in the absence of all AA (AA−), in the presences of only essential AA (EAA+), or of all AA (AA+). Cell proliferation, casein expression, and activation of the mammalian target of rapamycin complex 1 (mTORC1) pathway were increased; but SESN2 expression was decreased in response to increased EAA or AA supply. Overexpressing or inhibiting SESN2 demonstrated that cell proliferation, casein expression, and activation of the mTORC1 pathway were all controlled by SESN2 expression. Furthermore, the increase in cell proliferation, casein expression, and activation of the mTORC1 pathway in response to AA supply was inhibited by overexpressing SESN2, and those effects were reversed by inhibiting SESN2. These results indicate that SESN2 is an important inhibitor of mTORC1 in CMEC blocking AA-mediated cell proliferation and casein synthesis. Among the nutrients, amino acids (AA) are the most important as they are not only the building blocks of protein synthesis but also the regulators of cell proliferation and casein synthesis in mammalian epithelial cells7.Proliferation of cow mammary epithelial cells (CMEC) and casein synthesis by those cells are regulated by hormones , nutrients (e.g. glucose and amino acids) and environmental stress (e.g. heat stress)9. mTORC1 is the main regulatory factor in the pathway, and it is composed of mTOR, G protein β subunit-like protein (GβL), regulatory associated protein of mammalian target of rapamycin (Raptor), proline-rich Akt substrate of 40 kDa (PRAS40), and Deptor10. When AA are sufficient, mTORC1 is stimulated by an unknown signaling pathway and moves to the lysosomal surface from an undefined location, causing mTOR to be phosphorylated. Phosphorylated mTOR activates the downstream molecules, ribosomal protein S6 kinase 1 (S6K1) and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1), which promotes participation in the translation process and protein synthesis12. The downstream actions of mTORC1 have been well characterized, but the mechanism of AA action on mTORC1 is poorly understood15.The main signaling pathway that mediates AA-induced cell proliferation and protein synthesis is the mammalian target of rapamycin complex 1 (mTORC1) pathway18. Previous reports have shown that SESN2 can suppress reactive oxygen species arising from oxidative stress through its antioxidant function19. In addition to its antioxidant activity, SESN2 can activate adenosine monophosphate-activated protein kinase (AMPK), subsequently inhibiting the activation of mTORC121. In human cells (mainly HELA and human embryonic kidney (HEK) 293 cells), SESN2 protein was found to respond to AA depletion  resulting in negative effects on the mTORC1 pathway. It has been reported that sestrins control mTORC1 signaling by inhibiting Rag GTPase25. Kimball et al. reported that sestrins/sestrin2 negatively regulated the amino-acid-sensing pathway upstream of mTORC1 by interacting with GTPase-activating proteins toward Rags 2 (GATOR2)26.Sestrins are a family of highly conserved, stress-inducible, metabolic regulators. In mammals, there are three members of the family: sestrin1 (SESN1), sestrin2 (SESN2) and sestrin3 (SESN3), of which, SESN2 is the most importantIn this study, we investigated the function of SESN2 in the process of cell proliferation and casein synthesis associated with AA-mediated stimulation of the mTORC1 pathway in CMEC. We hypothesized that SESN2 mediated AA responses through inhibition of mTORC1 activation.P < 0.05) for essential AA (EAA+) and AA+ groups as compared to the AA- group. Treatment with EAA+ or AA+ also increased (P < 0.05) alpha S1 casein (CSN1S1), alpha S2 casein (CSN1S2), beta casein (CSN2), kappa casein (CSN3), mTOR, p-mTOR, Raptor, S6K1 and p-S6K1 protein expression, and the ratio of p-S6K1 to S6K1, p-mTOR to mTOR  when cells were treated with EAA+ or AA+  compared to the AA- group, but the responses were much smaller than for the EAA+ group  when SESN2 expression was inhibited, and the reverse when it was overexpressed  or overexpressed (SESN2 GO). The null transfection group (B) was used as blank control; the negative control siRNA transfected group (NC) was used as negative control; and the empty plasmid transfected group (EV) was used as empty plasmid control. Cell growth and cell cycle were analyzed. The results showed that cell growth Fig.  and the sed Fig. .Figure 2P < 0.05) when SESN2 was inhibited  by SESN2 overexpression    Fig.  in cells05) Fig.  in cellsP < 0.05) by inhibition of SESN2  by SESN2 overexpression    by SESN2 inhibition  expression of p-mTOR, Raptor, S6K1 and p-S6K1, as well as the ratio of p-S6K1 to S6K1, p-mTOR to mTOR; and the increase was inhibited (P < 0.05) by SESN2 overexpression  the expression of SESN2 and up regulated (P < 0.05) the mTORC1 signaling pathway  expression of SESN2 but up regulated (P < 0.05) the mTORC1 signaling pathway  the expression of SESN2 or the mTORC1 signaling pathway  Fig.  in cells05) Fig.  in cellsion Fig. . AA stimion Fig. . The indP < 0.05)   by AA starvation, and this decrease was rescued (P < 0.05) by SESN2 inhibition  by SESN2 overexpression  Fig.  in cells05) Fig.  in cellsion Fig. . Lysosomion Fig. .Figure 5P < 0.05) and the proportion of cells in the G2 and S stages decreased (P < 0.05) when mTOR gene expression was inhibited. mTOR inhibition also prevented (P < 0.05) cell growth and G2 and S stage changes caused by SESN2 inhibition  expression of CSN1S1, CSN1S2, CSN2 and CSN3, and inhibition of SESN2 increased each; however, inhibition of mTOR blocked (P < 0.05) the SESN2 responses  T1R1/T1R340; PB1-containing kinase MEEK3/p38δ/p62/E3-ubiquitin ligase TRAF641; and Sestrins/GATOR2/GATOR125. We have shown herein that in CMEC, the expression of SESN2 was significantly decreased in response to EAA or AA supply  cells. These results support SESN2 as the mechanism of regulation of AA effects on the mTORC1 pathway in CMEC.The activation of mTORC1 is important for cell growth and protein synthesis, but how AA affect mTORC1 is still uncertain. A number of independent reports have identified several potential pieces of the mechanism, but a clear picture has not yet emerged. They include AA activation of mTORC1 via protein phosphatase 2 oligomer containing the PR61ε-targeting subunit (PP2AT61ε), ply Fig. , which i42. Previous studies have shown that the expression of SESN2 was induced by DNA damage, oxidative stress and glucose starvation44. Expression of SESN2 was stimulated by genotoxic stress via tumor suppressor p5345 in HEK293 and MCF-7 cells; by glucose starvation through activation of transcription factor 4 (ATF4) and nuclear factor (erythroid-derived 2)-like 2 (NRF2) transcription factors46 in human lung adenocarcinoma H1299 and H460 cell lines and MEF; and by AA deprivation acting through GCN2, ATF4 in MEF cells47. In this study, we showed that the expression of SESN2 and GCN2 was significantly decreased in response to increased EAA or AA supply , is a member of the sestrins familyply Fig. , suggest49. However, recent studies demonstrated that SESN2 regulates mTORC1 in mouse embryonic fibroblast (MEF), HEK-293T, and Hela cells50 by binding to GATOR2, which disrupts the GATOR1/GATOR2 complex thus releasing GATOR1 to inhibit mTORC125. Kimball et al. reported that SESN2 regulates the mTORC1 pathway depending on its phosphorylation state which is driven by Leu deprivation, and no other EAA26. Control can also be exerted through changes in SESN2 gene expression. Ye et al. and Wolfson et al. showed that Ile, lysine, Val, Gln and Met regulate the expression of SESN2 which, in turn, inhibits the mTORC1 pathway47. In this study, we found that Leu, Ile, Val, Met and Gln independently regulated the mTORC1 pathway through changes in expression of SESN2  play an important metabolic regulation role in mammals. Early studies concluded that SESN2 inhibited the mTORC1 pathway through AMPK-mTORC1 signalingSN2 Fig. . These fThe current work cannot differentiate between the above alternatively proposed upstream mechanisms; however, it clearly demonstrates that SESN2 is involved in mediating AA effects on mTORC1 and that SESN2 lies upstream of mTORC1. It also extends prior observations in other species and cell types to CMEC. Cell proliferation, casein synthesis and activation of the mTORC1 pathway were all significantly increased with SENS2 inhibition and significantly decreased with SESN2 overexpression Figs  and 5B. Because AA act as both a substrate and a regulator of protein synthesis, it is of interest to discern the effects of each mechanism. From Figs et al. and Arriola et al. observed independent responses to multiple EAA51; however, preliminary screenings of NEAA, using a bovine tissue slice model, did not show any responses to the NEAA53. The current results provide evidence that the NEAA should be screened for regulatory activity and possibly included in models of AA effects on mammary function54. This is an important finding as it potentially adds considerable complexity to the system where the response surface may have as many as 20 dimensions, thus making parameterization of the system exceedingly difficult.The results also demonstrate that NEAA are impacting SESN2 with subsequent effects on mTORC1 signaling, cellular growth rates, and casein synthesis. Appuhamy In summary, the present work shows that SESN2 is an important negative regulatory factor which was induced by EAA and NEAA starvation, and it negatively regulates AA-mediated cell proliferation and casein synthesis via inhibition of the mTORC1 pathway in CMEC.All experiments involving dairy cattle were conducted according to the principles of the Chinese Academy of Agricultural Sciences Animal Care and Use Committee , which approved study protocols.3 pieces using sterile techniques. The explants were plated into 25 cm2 flasks  coated with 5 mg/ml sterile rat tail collagen type II (diluted with 0.006 M acetic acid)  and cultured with DMEM/F12 media  containing 10% fetal bovine serum (FBS), 10,000U of penicillin, 10,000U of streptomycin, and 25 mg/mL of amphotericin B in an incubator at 37 °C in and atmosphere of 5% CO2. At 80% confluence, the explants were removed from the culture. Epithelial and fibroblast cells were segregated using 0.25% trypsin and 0.15% trypsin plus 0.02% ethylenediaminetetraacetic acid (EDTA)  as previously described55 yielding primary cow mammary epithelial cells (CMEC).Mammary gland tissue was dissected from the udder of mid-lactation healthy Holstein dairy cow and cut into 1 mm5 cells per well and cultured with DMEM/12 containing 10% FBS. When they reached 80% confluence, cells were starved of AA  for 12 h, and then treated with DMEM/F12 media without FBS and devoid of all AA (AA−), containing only essential AA  at normal DMEM/F12 concentrations (EAA+), or complete DMEM/F12 (AA+) for 6 h. For cell growth tests, treatment duration was 24 h. The concentrations of each EAA in the EAA+ treatment were those of normal DMEM/F12 medium. All AA used in the study were obtained from Sigma . The main effects of AA were also assessed with overexpression (SESN2 GO) or inhibition of SESN2 (si-SESN2) yielding the additional treatments of AA−/si-SESN2, EAA+/SESN2 GO, AA+/SESN2GO, si-mTOR and si-SESN2/si-mTOR.For the experiments, CMEC were plated into 6 well plates at a density of 1.0 × 104 cells/ml and cultured with DMEM/12 containing 10% FBS. After 12 h, media were switched to an AA and FBS free form followed by treatment media  for 24 h. At the end of the treatment period, MTT (5 mg/mL diluted in PBS) was added to the media at a final concentration of 0. 5 mg/mL and the cells were cultured for an additional 4 h. The media were then discarded, and 150 μl of DMSO  was added to each well. The plates were shaken for 10 minutes at room temperature, and the absorbance at 450 nm was determined using an automated enzyme-linked immunosorbent assay (ELISA) reader 56. The relative growth rate of the treated cells as compared to the control cells (AA− group) was reported. This experiment was repeated with three different batches of cells.Cell growth was analyzed with 3--dimethylthiahiazo (-z-y1)-3, 5-di-phenytetrazoliumromide (MTT) . CMEC were plated in 96 well plates at a density of 5.0 × 1057. The CMEC were plated into 6 well plates and treated as described for cell growth analysis, but the density of plated cells was 1.0 × 105 cells per well. Cells were digested with 0.25% trypsin-0.02% EDTA, harvested, washed 3 times with PBS , fixed overnight with ice-cold 70% ethanol at 4 °C, and washed 3 times with PBS followed by incubation with 1 mL RNase A  for 0.5 h at 37 °C, and 3 additional washes with PBS. The cells were then incubated with 1 mL propidium iodide  for 0.5 h at 37 °C in a darkened environment, washed 3 times with PBS, and re-suspended in PBS. The cell cycle was analyzed using FACS Calibur . The resulting data were analyzed using Modfit software58.The cell cycle was analyzed by flow cytometry (FC) as previously reported2O, pH 7.5) containing 5% skim milk (M/V) for 1.5 h at room temperature (about 25 °C), and then incubated with primary antibody (diluted with TBST containing 5% skim milk) for 1.5 h at room temperature or overnight at 4 °C. The membrane was washed 3 times (5 min/time) with TBST and then incubated with HRP-conjugated secondary antibody  for 1 h at room temperature. The membrane was washed 3 times (10 min/time) with TBST, and the chemiluminescence of HRP was visualized with Super ECL Plus . Protein band intensity was analyzed with ImageJ2X software. The β-actin was use as the reference protein. The primary antibodies used were the following: anti-CSN1S1 , anti-CSN1S2 , anti-CSN2 , anti-CSN3 , anti-β-actin , anti-mTOR , anti-p-mTOR (Ser2481) , anti-Raptor , anti-S6K1 , anti-p-S6K1 (Thr389) , anti-GCN2  and anti-SESN2 .Protein expression was analyzed by Western blotting (WB) analysis. Treated cells were harvested with a lysis buffer , and total protein concentrations were analyzed using a BCA Protein Assay Kit . About 30 μg of total protein was separated on a 10% SDS-PAGE gel and transferred onto a polyvinylidene fluoride (PVDF) membrane  using 200 mA of constant electrical current. The membrane was blocked with TBST buffer , and the cDNA was synthesized with M-MLV reverse transcriptase . The CDS area of SESN2 was amplified with PCR. The SESN2 primers (GenBank: BT021909.1) were the following: Forward: 5′-CGGAATTCCACACCATGATCGTTGCGG-3′, the EcoR I site is underlined; and Reverse: 5′-GAAGATCTCAGGTGAGTAAATGGGCTTCC-3′, the Bgl II site is underlined. The PCR product was sequenced  and then subcloned into the MCS of eukaryotic expression vector pCMV-C-Flag . The plasmid will be subsequently refered to as SESN2-Flag.Plasmid construction was performed as previously reported5 cells per well, and at about 70% confluence, the medium was changed with OPTI-MEM I medium . SESN2-Flag vector and pCMV-C-Flag empty vector were transfected with Lipofectamine 2000 transfection reagent  according to the manufacturer’s instructions. Briefly, for cells of each well to be transfected, 5 ìg DNA plasmid and 10 μl Lipofectamine 2000 transfection reagent were diluted into 250 ìl OPTI-MEM I medium, respectively. After incubating for 5 min at room temperature, the diluted DNA plasmid and Lipofectamine 2000 transfection reagent were mixed, and incubated for 20 min at room temperature. Then the mixture was added to well containing cells. After 6 h, the OPTI-MEM I media were switched to DMEM/12 media containing 10% FBS.For the SESN2 GO, the transfection of SESN2-Flag was performed. The CMEC were plated into 6 well plates a density of 1.0 × 10The specific siRNA of genes indicated in this experiment and the negative control siRNA were synthesized . The si-SESN2 was transfected using Lipofectamine 2000 transfection reagent according to the manufacturer’s instructions. The operation process was the as same as that of SESN2-Flag DNA plasmid transfection, but the amount of siRNA and Lipofectamine 2000 transfection reagent were 100 pM and 10 μl per well, respectively. The siRNA sequences used in this study are shown in Table 5 cells per well and cultured with DMEM/12 containing 10% FBS for 12 h. The cells were treated for 6 h and used for lysosomal localization analysis of mTOR in the following manner. Media were removed. Cells were washed 3 times with PBS, fixed with ice-cold methanol at 4 °C for 10 min, and washed 3 times (5 min/time) with TBST. Cells were blocked with a blocking buffer  for 1 h at 37 °C and then incubated with an anti-mTOR rabbit polyclonal primary antibody  and an anti-LAMP2 goat polyclonal primary antibody  for 1 h at 37 °C. Cells were subsequently washed 3 times (5 min/time) with TBST and incubated with mouse-anti-rabbit alexa fluor 488-conjugated secondary antibody  and mouse-anti-goat alexa fluor 647-conjugated secondary antibody  for 45 min at 37 °C in the dark. The cells were washed 3 times (5 min/time) with TBST, and incubated with 4′,6-diamidino-2-phenylindole (DAPI)  for 10 min at 37 °C in a dark environment, and then washed 3 times (10 min/time) with TBST. The cells were fixed on the slides with antifade mounting medium  and observed with laser scanning confocal microscopy . Co-localization was analyzed with ImageJ2X software, and at least 10 treated cells in each co-localization were used to analyze the index of co-localization.The CMEC were plated on cover slips in 6 well plates with the concentration of 1.0 × 10P < 0.05. Data were presented as LSM ± standard error of the means (SEM). All LSM represented at least three independent experiments.Date were analyzed using the Proc Mixed procedure of SAS . The fixed effect was treatment for all models. For growth rate measurements which were conducted across 3 cell preparations, cell batch was included as a random effect. For all other measurements, plate was included as a random effect. Least squares means (LSM) of treatments were compared using Tukey’s multiple comparisons procedure, and the effects were considered significant at a probability value of Supplementary Information


This study explores clinical outcome in cytochrome P450 2C19 (CYP2C19)-related poor metaboliser patients treated with either clopidogrel or prasugrel after percutaneous coronary intervention (PCI) and investigates whether this could be cost-effective.n = 10, 31 %) compared with the poormetaboliser group treated with prasugrel  (p = 0.003). Costs per gained quality-adjusted life years (QALY) were estimated, showing thatgenotype-guided post-PCI treatment with prasugrel could be cost-effective with less than € 10,000 per gained QALY.This single-centre, observational study included 3260 patients scheduled for elective PCI between October 2010and June 2013 and followed for adverse cardiovascular events until October 2014. Post PCI, CYP2C19 poor metaboliserpatients were treated with clopidogrel or prasugrel, in addition to aspirin. In total, 32 poor metabolisers weretreated with clopidogrel and 41 with prasugrel. The number of adverse cardiovascular events, defined as death fromcardiovascular cause, myocardial infarction, stent thrombosis, every second visit to the catheterisation room forre-PCI in the same artery, or stroke, within 1.5 years of PCI, was significantly higher in the CYP2C19 poormetaboliser group treated with clopidogrel (This study provides evidence that for CYP2C19-related poor metabolisers prasugrel may be more effective than clopidogrel to prevent major adverse cardiovascular events after PCI and this approach could be cost-effective. Over the last decade, dual antiplatelet therapy with aspirin and clopidogrel has been considered the gold standard therapy for patients undergoing elective percutaneous coronary interventions (PCI) in preventing major adverse cardiovascular events (MACE) . HoweverClopidogrel is a pro-drug that requires biotransformation to generate an active metabolite involving cytochrome P450 (CYP) enzymes . ClopidoTherefore, in September 2010 Catharina Hospital started to determine the CYP2C19 genotype of patients scheduled for elective PCI in order to identify CYP2C19 poor metabolisers. At first, poor metabolisers were treated with clopidogrel. After the FDA boxed warning was placed, prasugrel was prescribed instead of clopidogrel to the poor metabolisers, with the goal to reduce the number of MACE. In this paper the results of this therapy change are presented. In addition, the cost-effectiveness of genotype-guided post-PCI treatment is discussed.http://circ.ahajournals.org/content/115/17/2344). For verification of medication prescribed after PCI, the medical file was consulted or the pharmacy or general practitioner was contacted.Between September 2010 and June 2013, patients who were scheduled for elective PCI at Catharina Hospital inEindhoven were enrolled in this study. Patients with ST-segment elevation myocardial infarction (STEMI) who receivedprimary PCI were not included. CYP2C19 genotyping was performed for all patients. Initially, all patients were treatedwith a start-up dose of clopidogrel (300 mg) and, post-PCI, clopidogrel (75 mg daily) in addition to aspirin (80 mgdaily) was prescribed for at least one year. But since the FDA boxed warning, gradually only prasugrel (10 mg daily)instead of clopidogrel was prescribed to CYP2C19 poor metabolisers within 1 to 5 days of PCI, starting in September2011. Thereby, two groups of CYP2C19 poor metabolisers were studied, one group treated with clopidogrel and anotherwith prasugrel. As stent thrombosis was shown by autopsy to be the cause of death 492 days after PCI for one of thedeceased patients in our study, all CYP2C19 poor metabolisers were followed for at least 1.5 years after PCI, the lastpatient until October 2014. For all CYP2C19 poor metabolisers clinical baseline characteristics were recorded and themedical history was examined for cardiovascular events. An adverse cardiovascular event was defined as death froma cardiovascular cause, myocardial infarction, stent thrombosis, every second visit to the catheterisation room forre-PCI in the same artery, or stroke. Stent thrombosis was defined according to Academic Research Consortium and a semi-automatic extraction device, the Magnapure (Roche). CYP2C19 genotyping was performed with real-time polymerase chain reaction and melting temperature analysis using the Lightcycler hybridisation DNA master mix (Roche Diagnostics Inc.) and specific primers and probes for detection of the CYP2C19*2 and *3 alleles and the *17 allele (TIP-MOLBIOL and Sigma-proligo) on the Lightcycler 480 (Roche). Based on the genotypes, patients with CYP2C19 wild-type were phenotyped as extensive metabolisers, CYP2C19*2 heterozygote and CYP2C19*3 heterozygote patients as intermediate metabolisers, CYP2C19*2 homozygotes as poor metabolisers and CYP2C19*17 homozygotes as ultra-rapid metabolisers. The CYP2C19*17 heterozygote patients were phenotyped as extensive metabolisers and CYP2C19*2*17 as intermediate metabolisers.P-values were evaluated by a Chi-squared or Fisher’s exact test using the SPSS statistics software (IBM), version 19 for statistical analyses.For the baseline characteristics, variables with a Gaussian distribution were presented as mean ± standard deviation. The cost-effectiveness analysis was based on the low-discrimination scenario of Kazi and colleagues, includiA total of 3260 patients were enrolled in this study of which 2293 were wild-type, 932 were heterozygotes and 89 homozygotes for the CYP2C19*2 allele. In addition, 6 patients were heterozygotes for the CYP2C19*3 allele, 1121 were heterozygotes for the CYP2C19*17 allele and 175 patients were homozygotes for the CYP2C19*17 allele. This results in allele frequencies of 17, 0.1 and 22.5 % for CYP2C19*2, CYP2C19*3 and CYP2C19*17, respectively, which is in agreement with the allele frequency of a normal Caucasian population .p < 0.001 for all events and p = 0.003 for events within 1.5 years of PCI.Although all patients were scheduled for PCI, only 71 poor metabolisers (CYP2C19*2 homozygote) (80 %) underwent PCI with stent placement. These 71 poor metabolisers were subdivided based on the antiplatelet drug used. Initially, 32 poor metaboliser patients were treated with clopidogrel, 35 with prasugrel, 2 with ticagrelor and for 2 patients the drug used could not be confirmed  , 8 in the clopidogrel group and 1 in the prasugrel group , but still higher than for extensive metaboliser or ultra-rapid metaboliser patients (0.8 %) , the pharmaceutical expenses for six different therapy options for the Catharina study population were compared .In the Netherlands, the mean ‘willingness to pay’ is estimated to be about € 65,000 per QALY based on the Dutch EQ-5D tariffs and visual analogue scale valuations , 20. OurIn addition to the high-risk CYP2C19 poor metaboliser patients, also the intermediate metabolisers are at increased risk for MACE . FollowiAlthough being in line with the literature , 8, the This study provides evidence that for CYP2C19-related poor metabolisers prasugrel may be more effective than clopidogrel to prevent major adverse cardiovascular events after PCI and this approach could be cost-effective.


Notably we demonstrated that one of these RAB proteins, RAB9, competes with the catalytic subunit PPP2CA in binding to PPP2R1A. This competitive association has an important role in controlling the PP2A catalytic activity, which is compromised in several solid tumors and leukemias.Protein phosphatase 2A (PP2A) is one of the most abundant serine–threonine phosphatases in mammalian cells. PP2A is a hetero-trimeric holoenzyme participating in a variety of physiological processes whose deregulation is often associated to cancer. The specificity and activity of this phosphatase is tightly modulated by a family of regulatory B subunits that dock the catalytic subunit to the substrates. Here we characterize a novel and unconventional molecular mechanism controlling the activity of the tumor suppressor PP2A. By applying a mass spectrometry-based interactomics approach, we identified novel PP2A interacting proteins. Unexpectedly we found that a significant number of RAB proteins associate with the PP2A scaffold subunit (PPP2R1A), but not with the catalytic subunit (PPP2CA). Such interactions occur  Protein phosphatases act in concert with kinases to fine-tune signaling events by modulating the level of phosphorylated serine, threonine and tyrosine residues1Besides recapitulating most of the known PP2A interactors, we found that only the scaffold subunit, and not the catalytic nor the regulatory ones, interacts with a significant number of RAB family members. RAB GTPases (Ras-related in brain) belong to the RAS superfamily of small GTPases and play a prominent role in controlling vesicle trafficking from the donor compartments to the acceptor onesHere we report that RAB8 and RAB9 proteins interact with the PP2A scaffold subunit, PPP2R1A, in a GTP independent manner. This interaction impairs the assembly of the PP2A holoenzyme, which consequently is inactivated. Our results are consistent with a model whereby some specific members of the RAB family play a crucial role in selectively inhibiting the PP2A tumor suppressor in specific subcellular compartments.Protein-protein interactions play a pivotal role in defining the function of PP2A, one of the most abundant serine/threonine phosphatase implicated in cancer development. In order to investigate the PP2A interactome, we have recently exploited an immunoprecipitation assay combined with mass spectrometry (MS)-based proteomic analysis to investigate the PP2A interactome in HeLa cells. The PP2A holoenzyme protein interaction network has been investigated using transient expression and affinity purification of SF-TAG constructs of the PP2A subunits combined with MS-based proteomic analysis, as previously describedAmong the subset of RAB family members identified as PPP2R1A interactors , we deciRAB8 mediates constitutive biosynthetic trafficking from the trans-Golgi network (TGN) to the plasma membrane, is involved in the regulation of GLUT4 vesicle translocation and in the exocytosis of a matrix metalloprotease involved in cell invasion9in vivo or in vitro. As shown in As shown in To support the functional relevance of the observed interactions we looked at the colocalization of PPP2R1A with RAB7, as negative control, and RAB8 and RAB9 at endogenous level by performing an immunofluorescence assay. HeLa cells, chosen for their morphology, were stained with antibodies recognizing PPP2R1A and RAB7 or RAB8 or RAB9 proteins, and then analyzed by confocal microscopy. As shown in 3in situ Proximity Ligation Assay (PLA). This technique allows monitoring interactions of endogenous proteins directly in individual cells at single-molecule resolutionNext we examined the close association of PPP2R1A with RAB7, as negative control, RAB8 and RAB9 at endogenous levels using the in situ in cells.Taken together these observations demonstrate that PPP2R1A co-localizes with RAB8 and RAB9 in specific subcellular compartment and physically interacts with them We next aimed at mapping the contact regions on the PPP2R1A-RAB complex. 190 peptides, 13 amino acids long and spanning the entire PPP2R1A amino acid sequence, were synthesized on two nitrocellulose membranes that were incubated respectively with GST-RAB9 and GST, as negative control . The spoIn a complementary experiment we mapped the RAB9 peptides showing affinity for PPP2R1A. The RAB9 amino acid sequence was synthesized as 13 amino acid long peptides spotted onto two nitrocellulose membranes. One membrane was incubated with GST-PPP2R1A and the other with GST, as negative control. As shown in Next we tested whether the scaffold subunit can discriminate the active (GTP bound) from the inactive (GDP bound) conformation of RAB proteins. To this aim, we analyzed the PPP2R1A association with RAB9 and other two RABs (RAB1A and RAB2A), which were previously identified by our MS-based pull-down approach, in the presence of GTPγS or GDP. As shown in in vitro result is also supported by co-immunoprecipitation assays, demonstrating that the over-expression of PPP2CA significantly decreases in vivo the association of PPP2R1A with RAB9  impair the activity of the phosphatase by disrupting the PPP2R1A-PPP2CA interaction and have been identified in different human cancers andin vitro and in vivo.The association of PPP2R1A with RAB8 and RAB9 was further verified by PLAs and confocal microscopy at endogenous protein concentrations. These experiments demonstrate that both the PPP2R1A-RAB8 and PPP2R1A-RAB9 complexes occur Since the activity and specificity of the PP2A phosphatase depends on the association of the catalytic and the scaffold subunitHere we propose that the disruption of the PPP2CA-PPP2R1A dimer by the competition with other regulatory proteins represent a novel molecular mechanism to regulate the catalytic activity of the PP2A holoenzyme in different subcellular compartments . The modAnti-FLAG and anti-Flag M1 agarose beads were from Sigma; anti-PPP2R1A and anti-Rab8, anti-Rab9 and Rab7 antibodies were from Cell Signaling. Anti-PPP2CA from Millipore, Inc. Peroxidase-conjugated anti-rabbit, anti-mouse and anti-goat secondary antibodies were from Jackson ImmunoResearch. Antibodies for immunofluorescence: anti-Rab8 and anti Rab9 were from Cell Signaling, anti-PPP2R1A was from Santa Cruz, anti-mouse Alexa Fluor-555-conjugated secondary antibody and anti-rabbit Alexa Fluor 488-conjugated secondary antibody were from Invitrogen. PPP2CA and PPP2R3C encoding plasmids were purchased from OpenBiosystem. PPP2R1A construct was kindly provided by Marc Vidal. RAB-GST cDNAs were kindly provided by Prof. FukudaCells were maintained in a humidified atmosphere at 37 °C and 5% CO2 in Dulbecco’s modified Eagle’s medium (Invitrogen), supplemented with 10% fetal bovine serum (Sigma) and 0.1% penicillin/streptomycin (Invitrogen). SILAC experiments were performed as previously describedFor one step Strep purifications, SF-TAP tagged proteins and associated protein complexes were purified essentially as described earlier24HeLa cells were lysed as described previously. The whole cell lysates were incubated with anti-Flag antibody conjugated to Sepharose beads over-night at 4 °C. The beads were washed with lysis buffer, and the immunoprecipitated proteins were separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and immunoblotted with antibodies. The immunoreactions were visualized using ECL detection system (Amersham Biosciences).After 24 h of transfection, confluent HeLa cells were washed with ice-cold PBS and lysed in RIPA buffer  supplemented with 1 mm pervanadate, 1 mm NaF, protease inhibitor mixture 200× (Sigma), inhibitor phosphatase mixture I and II 100× (Sigma). The samples were kept on ice for 30 min and centrifuged at 15,000 rpm at 4 °C for 30 min. The supernatant was collected, and the total amount of protein was determined by Bradford colorimetric assay (Bio-Rad). The whole cell lysates were incubated with 50 μg of the indicated GST fusion protein at 4 °C for 1 h. Thus, glutathione-Sepharose 4B beads were blocked by incubating with 3% bovine serum albumin with rocking at 4 °C for 1 h, and then after centrifugation for 3 min at 4000 × g, at 4 °C, the dry beads were bound to lysates mixed with GST fusion proteins at 4 °C for 1 h. The supernatant was discarded by centrifugation, and the beads were washed six times with lysis buffer for 3 min at 4000 × g, at 4 °C, and then the dry beads were resuspended in SDS sample buffer, boiled and analyzed by SDS-PAGE and Western blotting on nitrocellulose membrane. GST beads coupled with approximately 5 μg of the purified GST-Rabs were incubated for 20 min at 4 °C with 50 mm HEPES-KOH, pH 7.2, 150 mm NaCl, and 2.5 mm EGTA, and then MgCl2 and GTPγS (or GDP) were added to the solution at the final concentration of 10 mm and 0.5 mm (or 1 mm), respectively. The GST-Rab beads were incubated for 1 h at 4 °C with 400 μL of the cell lysates in 50 mm HEPES-KOH, pH 7.2, 150 mm NaCl, 1 mm MgCl2, 1% Triton-X-100, and protease inhibitors in the presence of 0.5 mm GTPγS (or 1 mm GDP)Peptides of 13 amino acids were synthesized according to the standard solid phase synthesis protocols (Frank 2002), using an automatic spot synthesizer . The synthesis was carried out on amino-PEG membranes (Intavis). Peptides cover the whole sequences of Rab9 and PPPR1A, with a pass of 3 amino acids. The membranes were washed with PBS 1X and incubated in blocking buffer (BSA 5% PBS 1X) for 2 h at 40 C. The membranes were then incubated with 10 mg/mL dialyzed recombinant GST-Rab9 or GST-PPPR1A in blocking buffer overnight at 4 °C. After washing three times for 10 min with PBS, the anti-GST antibody was added 1/1000 in blocking buffer for 2 h at 4 °C. After three washings with PBS (10 min each), a peroxidase labeled anti-goat mAb was added 1/5000 in blocking buffer and incubated for 1.5 h at room temperature, followed by washing three times with PBS. Quantification of peptide bound domain was carried out using a chemo-luminescence substrate and the LAS-3000 instrument  instrument.HeLa cells, grown on glass coverslips, were fixed in 4% paraformaldehyde, treated with 0.1 M glycine and then permeabilized with 0.1% Triton X-100 in PBS. Cells were incubated in blocking solution , labeled with primary antibodies against the protein of interest in PBS-Triton 0,1%-BSA 10%, and lastly incubated with the specific secondary antibodies. Nuclei were stained with 4,6-diamidino-2-phenylindole  in PBS. Coverslips were mounted with Fluoromount Aqueous Mounting Medium (SIGMA) for observation by epifluorescence microscope. Single-plane images were acquired with a confocal laser scanner microscope . Quantitative analysis of the extent of colocalization was performed by mesuring Mander’s Coefficient through the JACoP pluginin situ PLA was performed according to manufacturer’s protocol , as previously describedHeLa cells were grown on glass coverslips, fixed in 4% paraformaldehyde, permeabilized with 0.1% TritonX-100-PBS1, incubated in blocking solution  for 1 h and then labeled with primary antibodies targeting PPP2R1A and Rab8 or Rab9 proteins. For the negative controls cells were labeled with only one of the two primary antibodies. Duolink 400 ng of PPP2R1A in Na2CO3 50 mM were immobilized on each microwell of an ELISA plate, 0/N at 4C. After 30 min in blocking buffer (BSA 5% in PBS1x), PPP2R1A was incubated with 100 μL of 0.4 μM Rab9 or 100 μL of 0.4 μM Rab9 plus increasing amounts of PPP2CA (from 0.03 to 0.3 μM). Wells were washed 10 times with PBS1× 0.5% Tween. Anti Rab9 (Santa Cruz) was added 1:1000 in blocking buffer for 2 h, 4 °C. After ten wash in PBS1× 0.5% Tween, a secondary antibody POD-conjugated was added for 1 h at 4 C. After washes, the binding was revealed by adding 100 μl ABTS (A3219 Sigma) and measuring optical density at 405 nm.How to cite this article: Sacco, F. et al. A subset of RAB proteins modulates PP2A phosphatase activity. Sci. Rep.6, 32857; doi: 10.1038/srep32857 (2016).


Ovarian cancer is often termed a silent killer due to the late onset of symptoms. Whilst patients initially respond to chemotherapy, they rapidly develop chemo-resistance. Oncolytic adenoviruses (OAds) are promising anti-cancer agents engineered to “hijack” the unique molecular machinery of cancer cells enabling tumour-selective viral replication. This allows spread to adjacent cells and amplification of oncolysis within the tumour. OAds represent an excellent opportunity for ovarian cancer therapy via intra-peritoneal delivery, however the efficacy of OAds thus far is limited. Here, we evaluate chromatin (histone) modification in chemo-resistant cells and its relationship to Ad efficacy (wild-type or oncolytic Ad). In contrast to cisplatin-sensitive A2780 cells that show an efficient reduction of cell viability by Ad in the presence of cisplatin, cisplatin-resistant A2780/cp70 cells show diminishing Ad-mediated reduction of cell viability with escalating doses of cisplatin. Histone deacetylase (HDAC)-2 and to a lesser extent HDAC1 were up-regulated in cisplatin-resistant but not cisplatin-sensitive cells. Cisplatin-resistant cells treated with a pan-HDAC inhibitor trichostatin A (TsA) significantly enhanced Ad-mediated reduction of cell viability in the presence of cisplatin. Cells treated with TsA alone did not reduce cell viability suggesting these findings are Ad-dependent. Thus, we identify HDAC inhibition as a potential means to sensitise cisplatin-resistant ovarian cancer cells to virotherapies, an observation that may offer improved outcomes for patients with late stage, chemotherapy-resistant ovarian cancer. HDAC antibodies  against human HDAC1, -2 and -3 antibodies were purchased from Abcam . HDAC2 siRNAs were purchased from Qiagen.Cisplatin was obtained from the Velindre Cancer Centre, Cardiff. Trichostatin A (TsA) was purchased from Sigma-Aldrich. Both reagents were suspended in complete incubation media supplemented with 2% fetal calf serum. The CellTiter 96Ethics permission for the collection of ascites was granted through a Wales Cancer Bank application for biomaterials, reference WCB 14/004. All patients gave written informed consent for the use of their samples, prior to collection.Ad5WT was generated by AdZ homologous recombineering as previously described . dl24 wa2. The resulting primary cultures were passaged when cells had reached confluence. All reagents were purchased from Gibco or Thermo Scientific .Ascites samples were collected from patients with ovarian cancer at the Velindre Cancer Centre, Cardiff, UK and anonymously coded (EOC003 and EOC009). Ascites was stored at 4° C immediately after collection and processed within 24 h. Approximately 400 mL of ascites was centrifuged at 1000 rpm for 5 min to separate primary EOC cells from the fluid. The supernatant was stored at –70° C for subsequent use with autologous tumour cells. Red blood cell lysis buffer  was added to the pellet according to the manufacturer`s instructions, where appropriate. Tumour cell pellets were frozen in 10% DMSO and 90% autologous supernatant (passage 0). A further 100 mL of ascites was used to generate primary EOC cultures, by separating into 20 mL aliquots and adding to 20 mL of complete (RPMI 1640) medium, supplemented with 10% (v/v) fetal calf serum (FCS), 200 µM glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin and 10% (v/v) autologous ascitic fluid supernatant. Cells were maintained at 37° C and 5% COSKOV3 cells (human ovarian adenocarcinoma derived from ascites) were originally obtained from the American Type Culture Collection (ATCC). A2780 parental (cisplatin-sensitive) human ovarian carcinoma and A2780/cp70 (cisplatin-resistant) human ovarian carcinoma cells were a kind gift from Dr Alwyn Dart, Cardiff University. Cells were cultured as described above and passed a mycoplasma test in November 2016.5 cells per well in a 96-well plate. Cells were washed in 200 µl of wash buffer (PBS/1% BSA) and incubated with 100 µl of wash buffer containing 1:500 of mouse anti-human monoclonal antibody against CAR  or mouse IgG control antibody  for 1 hour on ice. Cells were washed three times and incubated with a 1:500 dilution of goat anti-mouse Alexa Fluor 647 antibody  for 30 min on ice. Cells were fixed in 4% paraformaldehyde for 20 min at 4° C. 2 × 104 gated events were acquired in channel FL-4 on a BD Accuri C6  flow cytometer and data analysed in BD Accuri C6 software version 1.0.264.21 . hCAR expression was analysed by flow cytometry relative to IgG isotype expression.EOC, SKOV3, A2780 and A2780/cp70 cells were seeded at a density of 1.5 × 104 cells/well in 100 µl of RPMI media supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were incubated in humidified boxes at 5% CO2, 37° C. After 24 h, cells were infected with Ad at an MOI of 0–10  and cultured as described above. Cell viability (% relative to uninfected cells) was measured by MTS assay. In brief, 20 µl of CellTiter 96® AQueous One Solution Cell Proliferation Assay reagent was added to 100 µl of culture medium, followed by incubation at 5% CO2, 37° C for 2 h. Plates were read at 490 nM using a multimode plate reader . Cell viability for cells infected with Ad was calculated as a percentage of uninfected cells (control). All values were corrected for background absorbance from the incubation media. All experiments were performed in triplicate. Data are expressed as the mean ± SEM.Cells were seeded in 96-well plates at a density of 2 × 104 cells/well in RPMI media supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were incubated in humidified chambers at 37° C, 5% CO2. After 24 h, cells were infected with Ad at 100 MOI in RPMI media supplemented with 2% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. At 2 h post-infection, cisplatin was added to cells at doses of 0.01, 1, 10 and 20 µM. Cisplatin only cells were treated with 20 µM cisplatin. Cell viability was measured at 72 h post-infection by MTS assay as described above. Cell viability for each Ad infected cell in the absence and presence of cisplatin was calculated as a percentage of uninfected cells (no Ad or cisplatin). Data was corrected for background absorbance from the incubation media. Experiments were performed in triplicate. Data are expressed as the mean ± SEM from 3 independent experiments. Statistics were performed using the Dunnett`s multiple comparison test. P values ≤ 0.05 are considered statistically significant and calculated to determine differences between adenovirus infected cells and adenovirus infected cells in the presence of cisplatin.Cells were seeded in 96-well plates at a density of 2 × 104 cells/well in a 96-well plate. After 24 hours, cells were infected with a luciferase expressing dl24 Ad at doses of 1000, 5000 and 10 000 virus particles (vp) per cell in a total volume of 100 µl of serum-free medium and incubated at 5% CO2, 37° C for 3 hours. The medium was removed and replaced with 200 μl of complete medium (RPMI 1640 medium supplemented with 10% (v/v) fetal calf serum, 100 U/mL penicillin, 100 µg/mL streptomycin) and cultured for an additional 45 hours. Cells were lysed in 1 × Cell Culture Lysis Buffer  and frozen at –70° C. The cells were thawed and 20 µl of cells mixed with 100 µl of luciferase assay reagent in a white 96-well plate. Luciferase activity in relative light units (RLU) was measured immediately using a multimode plate reader . Samples were normalised for total protein content, as measured by bicinchoninic acid (BCA) assay in RLU/mg protein. 2 × 104 gated events were acquired in channel FL-1 on a BD Accuri C6 using the plate reader as described above.Assays were performed as previously described . In brie5 cells per well, fixed with 4% paraformaldehyde, permeabilised with 0.1% Triton-X100 in PBS and blocked with 10% normal goat serum. Cells were incubated with rabbit anti-HDAC1, anti-HDAC2 and anti-HDAC3 antibodies (1:50)  and detected with Alexa Fluor 488 goat anti-rabbit secondary antibody (1:2000) . 2 × 104 gated events were acquired in channel FL-1 on a BD Accuri C6 using the plate reader as described above. HDAC expression was determined relative to the IgG isotype expression.A2780 and A2780cp/70 cells were seeded at a density of 1 × 104 cells per well in a 96-well plate in complete incubation media in the absence of antibiotics. 24 h after seeding, cells were transfected with 6 pmol HDAC2 siRNAs, All Stars negative control (scrambled) or AbI 1 5 (positive control) (Qiagen) and 0.3 µl Lipofectamine reagent per well in OptiMEM media (in triplicate). Transfection control wells contained nuclease-free water and OptiMEM media only. Cells were incubated in a humidified box at 37° C, 5% CO2. For cisplatin wells, cells were incubated for 2 h then dosed with 20 µM cisplatin. Cell viability was measured at 48 h by MTS assay and calculated as a percentage of non-transfected cells. All values were corrected for background absorbance from the incubation media. Experiments were performed in triplicate. Data is expressed as the mean ± SEM.A2780/cp70 (cisplatin-resistant cells) were seeded at 10 × 10Cells were lysed in 1% SDS lysis buffer . Each triplicate wells were pooled. 10 µl of each sample were used for a BCA assay. Samples were freeze thawed and diluted as appropriate with water to 10 µg. Samples were centrifuged at full speed and the same volume of 2 X Laemmli buffer  added to each sample. Samples were boiled at 95° C for 10 mins. Samples were loaded onto a 10% pre-cast Bis-Tris SDS-PAGE gel and electrophoresed at 200 V for 1 h. Gels were transferred at 20 V for 1 h (semi-dry transfer) to a nitrocellulose membrane. Membranes were blocked in 1 X TBST, 2% BSA at 37° C, room temperature (RT) 1 h. The siRNA membrane was incubated with rabbit anti-HDAC2 antibody (ab32117) (1:2000) in 1 X TBST, 2% BSA and the positive control siRNA AbI 1 membrane was incubated in mouse anti-ABI antibody (1:200) in 1 X TBST, 2% BSA overnight at 4° C. Membranes were washed 3 × 10 min in 1 X TBST. Anti-rabbit HRP and anti-mouse HRP (1:2000) in 1 X TBST, 2% BSA was added to each membrane respectively and incubated at RT for 1 h. Membranes were washed 3 × 10 min in 1 X TBST. Membranes were incubated with West Pico chemiluminescent reagent and immediately imaged. For actin loading control, membranes were stripped (Restore Western Blot Stripping buffer) for 15 min at RT and washed with 1 X TBST and incubated with rabbit anti-actin antibody (1:2000) in 1 X TBST, 2% BSA for 1 h at RT. Membranes were washed 3 × 10 min in 1X TBST. Membranes were incubated with anti-rabbit HRP antibody (1:2000) in 1X TBST, 2% BSA for 1 h at RT. Membranes were washed 3 × in 1 X TBST and incubated with West Pico. Membranes were imaged immediately.4 cells/well in RPMI media supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin in 96-well plates incubated in humidified boxes at 37° C, 5% CO2. Cells were treated with cisplatin at either 100, 50, 10 or 1 µM and TsA at either 2 µM, 1 µM or 600 nM as described above. Combinations of each dose of cisplatin and TsA were also tested. Cells were harvested at 72 h. Cell viability was measured by MTS assay (as described above) and calculated as a percentage of untreated cells (no cisplatin or TsA). All values were corrected for background absorbance from the incubation media. Experiments were performed in triplicate. Data are expressed as the mean ± SEM.A2780 and A2780/cp70 cells were seeded at 2 × 104 cells/well in complete media containing 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin in 96-well plates incubated in humidified boxes at 37° C, 5% CO2. After 24 h, cells were infected with Ad5WT, dl24 or control Ad at 100 MOI in RPMI media supplemented with 2% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. At 2 h post-infection, 20 µM cisplatin and either 300 nM or 600 nM TsA [P values ≤ 0.05 were considered statistically significant and calculated to determine differences in cell viability between Ad infected cells in the presence of cisplatin and adenovirus infected cells in the presence of cisplatin and TsA.A2780 and A2780cp/70 cells were seeded at 2 × 100 nM TsA  was adde4 cells / well in RPMI media supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were incubated in humidified boxes at 37° C, 5% CO2. After 24 h, cells were infected with Ad5WT or dl24 at 100 MOI in RPMI media supplemented with 2% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. At 2 h post-infection, 20 µM cisplatin and/or 600 nM of TsA and 2.5% ascitic fluid containing highly neutralising antibodies to Ad5 (patient OAS001) [A2780 and A2780cp/70 cells were seeded in 96-well plates at a density of 2 × 10 OAS001)  was adde4 cells per well (day 0). After 24 h (day 1), cells were treated with TsA (600 nM). Cells were maintained at 37° C, 5% CO2 in a humidified chamber. At 24 h post-treatment, cells were stained for CAR expression. Primary antibodies were mouse IgG isotype control antibody  (1:200) or mouse anti-hCAR antibody  (1:500) for 1 h at 4° C followed by detection with anti-mouse Alexa Fluor 647  (1:500) for 45 min at 4° C. Cells were fixed in 4% paraformaldehyde for 20 min at 4° C. CAR expression was analysed by flow cytometry relative to IgG isotype expression using the BD Accuri C6 as described above.Cells were seeded at a density of 2 × 10Data are presented as experiments performed in triplicate. Analysis of variance (ANOVA) and the Dunnett’s or Sidak`s multiple comparisons post hoc test was performed when three or more groups of data were analysed. All analyses and graphs were created in GraphPad Prism version 6.03 .


When these composites were used as electrode material in lithium-ion batteries, a reversible capacity of 711.2 mAh·g−1 at a current density of 500 mA·g−1 after 400 cycles was obtained. The result indicated that Fe2O3/COOH-MWCNT composite is a potential anode material for lithium-ion batteries.Metall oxides have been proven to be potential candidates for the anode material of lithium-ion batteries (LIBs) because they offer high theoretical capacities, and are environmentally friendly and widely available. However, the low electronic conductivity and severe irreversible lithium storage have hindered a practical application. Herein, we employed ethanolamine as precursor to prepare Fe When used as anode material for LIBs, these nanoparticles demonstrated a capacity of 911 mAh·g−1 at a current density of 200 mA·g−1 even after 50 cycles. As the current density was increased to 10 A·g−1, a discharge capacity of 424 mAh·g−1 was obtained. However, the capacity of this material declined gradually with increased number of cycles due to the low conductivity of Fe2O3. Liang et al. [2O3 microspheres by using sodium citrate as surfactant. A reversible discharge capacity of 489.5 mAh·g−1 was obtained at a current density of 100 mA·g−1 of up to 50 cycles. The specific capacity of the synthesized α-Fe2O3 microspheres was much better than that of commercial graphite (372 mAh·g−1) although further improvement in cyclic stability was needed [Various methods have been reported for the synthesis of Feo et al.  reported2O3 is to fabricate Fe2O3/carbon nanotube (CNT), Fe2O3/graphene or Fe2O3/graphene/CNT composites, which have been demonstrated to have improved cycling performance [2O3 nanoparticles inside CNTs, which reduced the volume change of Fe2O3 nanoparticles during charge/discharge. A highly reversible conversion reaction between Fe0 and Fe3+ (Fe2O3) during lithiation/delithiation can also be observed. The synthesized Fe2O3/CNT has successfully overcome the shortcoming of low electrical conductivity of Fe2O3. Zhou et al. [2O3@GS by using a simple spray drying method, which significantly improved the capacity of Fe2O3 albeit with a low cycling performance. Ye et al. [2O3, which demonstrated an excellent capacity retention of 777 mAh·g−1 at a current density of 100 mA·g−1 after 30 cycles. Wang et al. [2O3/GCNTs via a hydrothermal synthesis method and subsequent thermal reduction. The synthesized Fe2O3/GCNTs exhibited a high reversible capacity of 716 mAh·g−1 at 50 mA·g−1 after 120 cycles. Nevertheless, these materials demonstrated low capacity at high current densities.An effective method to improve the electrical conductivity of Feformance –25. All formance –22, highformance  successfu et al.  has fabre et al.  applied g et al.  synthesi2O3/COOH-MWCNT composites through a simple hydrothermal synthesis. Hydrothermal syntheses are frequently used to obtain composite oxides with uniform particle size distribution. The synthesized material can effectively buffer volume change caused by charge and discharge; and improve the electrical conductivity of the electrode [In this paper, we have used ethanolamine as precursor to prepare Felectrode ,29–34.3·6H2O, 2.1624 g, ≥99.0%, Sinopharm Chemical Reagent Co, Ltd) and ethanolamine were each dissolved separately 100 mL distilled water and stirred for 30 min to obtain uniform solutions. Following that, the iron(III) chloride solution was added dropwise into the ethanolamine solution. The solution was then kept at room temperature for three days to obtain orange metal hydroxide solution.The composite was synthesized via a simple hydrothermal method. Firstly, iron(III) chloride hexahydrate . The mixture was magnetically stirred for 12 h. Finally, the mixture was transferred into teflon-lined stainless steel autoclave, and kept at 150 °C for 24 h. The autoclave was then cooled to room temperature. The obtained precipitates were filtered, washed with distilled water for several times and kept in 30 mL of DMF solution to remove ethanolamine. Finally, the obtained black solid powder was washed with ethanol solution for several times and fully dried in a vacuum oven at 80 °C for 10 h. Details of the synthesis process of the Fe2O3/COOH-MWCNT were characterized using X-ray diffractometry , scanning electron microscopy , transmission electron microscopy  and thermogravimetric analysis .The as-prepared FeN-methyl-2-pyrrolidone to form a uniform slurry that was and stirred for 6 h. The slurry was spread on to the surface of a copper foil using a medical blade. The casted film was heated at 110 °C for 12 h in a vacuum oven to evaporate the residual solvent. After cooling down to room temperature, the casted film was cut into small round shape (average diameter of 14 mm) and used as anode. Metallic lithium was used as counter electrode as well as reference electrode. The cells separator was Celgard 2400 polypropylene film. Electrolyte was prepared by dissolving 1 M lithium hexafluorophosphate (LiPF6) in a mixed solution of fluoroethylene carbonate/ethyl methyl carbonate/dimethyl carbonate . Coin cells (2032 type) were assembled inside an argon filled glove box with a moisture and oxygen levels of less than 0.1 ppm. The assembled cells were kept at room temperature for 12 h before electrochemical performance testWorking electrodes were fabricated by a standard slurry casting procedure. The as-prepared anode material (70 wt %), conductive carbon black  and polyvinylidene fluoride  were mixed in +). The cyclic voltammetry measurements were conducted in the same potential window at a scanning rate of 0.05 mV·s−1. Electrochemical impedance spectroscopy was carried out at a frequency range 10 mHz to 1 MHz with an AC amplitude of 10 mV. Both CV and EIS measurements were carried out on an electrochemical workstation (Ametek 1470E)Electrochemical performance of the assembled cells were then tested by galvanostatic charge/discharge measurements, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Charge/discharge tests of the assembled cells were carried out using a commercial battery test system  at a constant current in the potential range of 0.01–3.00 V , indicating the well-crystalline structure of the as-prepared Fe2O3 nanoparticles. The black spectrum refers to carbon nanotubes, and the peak at 26° is the characteristic peak of carbon nanotubes. No obvious impurity peak was observed, indicating that high crystallinity and purity of the synthesized materials. 2O3 composites, which consist of Fe2O3 nanoparticles (ca. 100–200 nm) evenly distributed within the MWCNT networks. Such networks are advantageous for ionic migration. 2O3/MWCNT composite. Three distinct elements  can be observed. All the elements were uniformly distributed. The aforementioned results demonstrated that Fe2O3 nanoparticles were evenly distributed over the COOH-MWCNT frameworks.2O3/COOH-MWCNT composite material was further examined by TEM and HRTEM. The results are presented in 2O3 particles with a size of around 200 nm are evenly distributed between COOH-MWCNTs, which is in good agreement with the SEM result. 2O3 nanoparticles are formed on larger Fe2O3 nanoparticles. 2O3 nanoparticles. Smaller secondary particles are clearly observed. The measured (110) lattice spacing is 0.25 nm, which is in agreement with [The structure of the as-prepared Feent with .2O3/COOH-MWCNT composites was measured using a thermogravimetric analyzer. Thermogravimetric analysis (TGA) was carried out in oxygen by heating the samples to 700 °C at a heating rate of 10 °C·min−1  film and the decomposition of electrolyte [OH-MWCNT ,d. Figurctrolyte –10. The 2O3/COOH-MWCNT composites. The first discharge curve of cells exhibited an apparent plateau at around 0.8 V. During recharging, there was a reversible oxidation from Fe0 to Fe3+ with a plateau at 1.5–1.9 V. Both plateaus are typical features for Fe2O3 materials (Fe2O3 + 6Li+ + 6e− 2Fe + 3Li2O) [−1 and 1250 mAh·g−1, respectively, with a coulombic efficiency of 68%. A large irreversible capacity loss of 400 mAh·g−1 might be related to formation of SEI and other side reactions. In the second cycle, the potential plateaus were detected at 1.5–1.7 and 0.9–1.1 V, which are consistent with previously reported results [+ 3Li2O) ,29,36. T2O3/COOH-MWCNT composites was measured and the results were displayed in −1. In the first charge and discharge process, two anodic peaks at about 1.7 V and 1.2 V were observed, which is consistent with the charge and discharge curves observed in 2O3/COOH-MWCNT composites (3+ into Fe0 [0 to Fe3+). The peak at about 0.8 V also shifted to a more positive potential of 0.86 V due to polarization [In order to measure electrochemical activity and oxidation/reduction potential, cyclic voltammograms of Femposites , there iinto Fe0 26,32,37.rization ,29. Afte2O3/COOH-MWCNT composite and commercial Fe2O3 at a current density of 500 mA·g−1. A rising trend after an initial capacity drop can be observed for the Fe2O3/COOH-MWCNT composite. This is a common phenomenon in transition metal oxide anode materials. It can be ascribed to the penetration of the electrolyte and gradual exposure of the active sites [2O3/COOH-MWCNT composite stabilized at 711.2 mAh·g−1 at a current of 500 mA·g−1 following 400 cycles, which is superior to that of COOH-MWCNT and commercial Fe2O3. The Fe2O3/COOH-MWCNT composite showed excellent cycle stability due to the high surface area and flexibility of carbon nanotubes, which is capable of reducing the volume change of metal oxide during charging and discharging [2O3 followed by addition into GO suspension, which resulted in uneven dispersion of Fe2O3 in the GO suspension and reduced cycling performance. Different from the previous works by Liu et al. [2O3 particles onto the carbon nanotubes.ve sites –40. The charging . In compcharging , our syncharging  and Zhoucharging  reportedu et al.  and Zhouu et al. , the met2O3/COOH-MWCNT composite. A highly symmetric pattern was obtained . As the current density increased to 1000 mA·g−1, the discharge capacity can reach 650 mAh·g−1. When the current density dropped to 100 mA·g−1, the capacity returned to 920 mAh·g−1, with a coulombic efficiency as high as 99%, showing a good high-rate discharge ability and cycle stability. It can be inferred that COOH-MWCNTs play an important role to maintain structural integrity and to improve the electrical conductivity of the hybrid structure. In comparison to Yu et al. [−1 at a current of 500 mA·g−1 after 400 cycles. Meanwhile, Yu et al. [−1 at a current of 500 mA·g−1. The good electrochemical cycle performance is probably due to the high capacity of Fe2O3 and good conductivity of COOH-MWCNTs.u et al.  and Yu eu et al. , the hydu et al.  reported+ storage performance of the composites in the frequency region from 100 MHz to 0.01 Hz at room temperature. As shown in 2O3/COOH-MWCNT composites, indicating a good charge transfer in the electrode. The resistance after the 400th cycle demonstrated a slight increase, indicating that the composites can keep a high capacity and a good cycle stability.EIS experiments were carried out to estimate the enhanced Li2O3/COOH-MWCNT composite material using ethanolamine and iron chloride hexahydrate as precursors. Fe2O3 nanoparticles with secondary structures were evenly distributed in the COOH-MWCNT network. A reversible capacity of around 711.2 mAh·g−1 was maintained at a current density of 500 mA·g−1 after 400 cycles, which is much higher than that of COOH-MWCNT composite and commercial Fe2O3. The result indicated that Fe2O3/COOH-MWCNT composite can be a potential anode material for lithium-ion batteries.In summary, we have successfully fabricated an Fe


Collagen 1A1 (COL1A1), RNA-binding and pre-mRNA Processing Factor (PRPF40A), and Uncoupling Protein 2 (UCP2) were identified as downstream effectors of cytoglobin (CYGB), which was shown implicated in tumour biology. Although these three genes have been previously associated with cancer, little is known about their status in lung malignancies.Hereby, we investigated the expression and promoter methylation of COL1A1, PRPF40A, and UCP2 in 156 non-small cell lung cancer (NSCLC) and adjacent normal tissues.p < 1 × 10−4), while UCP2 exhibits a trend of upregulation (p = 0.066). Only COL1A1 promoter revealed hypermethylation in NSCLCs (36%), which was particularly evident in squamous cell carcinomas (p = 0.024) and in the tumours with moderate-to-good differentiation (p = 0.01). Transcript level of COL1A1, as well as PRPF40A and UCP2, exhibited striking association (p ≤ 0.001) with the expression of hypoxia markers. In addition, we demonstrate in lung cancer cell lines exposed to hypoxia or oxidative stress that COL1A1 transcription significantly responds to oxygen depletion, while other genes showed the modest upregulation in stress conditions.We demonstrate that COL1A1 and PRPF40A mRNAs are significantly overexpressed in NSCLC (In conclusion, our data revealed that COL1A1, UCP2, and PRPF40A are novel players implicated in the complex network of hypoxia response in NSCLC. Lung cancer is among the most prevalent neoplastic diseases accounting for the highest mortality in both genders worldwide  as a putative tumour suppressor gene in lung  and adenocarcinoma (N = 70). With regard to the pathological stage, tumours were split into the following groups: T1 (N = 11), T2 (N = 125), T3 (N = 15), and T4 (N = 5), while according to differentiation status, the group breakdown was: poor (N = 48) and moderate/good (N = 107). Nodal metastasis occurred in 75 cases, while 80 patients had metastasis-free nodes. The follow-up data included 12 alive and 57 dead patients. The tumour samples were snap frozen and grossly micro-dissected to ascertain more than 80% tumour tissue. The normal adjacent tissue was resected at least 5 cm from the tumour mass. All the methylation and expression values were included in the analysis, even in the absence of normal, methylation, or expression counterpart data for the given patient. Overall, we utilised RNA from 128 normal and 146 NSCLC tissues and DNA from 26 normal and 91 NSCLC tissues.NSCLC and adjacent normal tissues were collected from 156 patients operated between 1995 and 2005 at the Liverpool Heart and Chest Hospital, UK. All patients submitted informed consent, and the study was approved by the local ethics committee. The patient group [described in  with 10% FBS. For hypoxic studies, the cells were cultured at 1% O2 for 48h, while oxidative stress was achieved with 300 µM H2O2 treatment for 24 h. H2O2 concentration was chosen as an IC50 of CALU1 and H358 according to our survival curve.Adenocarcinoma (H358) and SqCLC (CALU1) lung cancer cell lines (ATCC) were maintained at 37 °C/5% CO®Plus Mastermix (Qiagen) according to manufacturer’s protocol with 85 ng bisulfite-converted DNA and 0.16 µM primers. The following PCR primers (MWG Operon) were used: COL1A1_F GGAGAGAAGGTAAATGGAAG, COL1A1_R-biotinylated AACCTAACCCCAACCCTA, PRPF40A_F GAGTAGAGAATAGAGAGGATTTG, PRPF40A_R-biotinylated TAATCAAAACCCAAAAAAC, UCP2_F-biotinylated GTTTTGGGATTGATTGTT, and UCP2_R CAAAACTAAAACCAAACTCAC. For pyrosequencing reaction, 0.33 µM sequencing primer was used: COL1A1_S (GAGAAGGTAAATGGAAGA), PRPF40A_S (ATAGAGAGGATTTGGA), and UCP2_S (AAACTAAAACCAAACTC).DNA extraction, bisulfite conversion, and pyrosequencing analyses were performed as described  on Genios plate reader (Tecan) according to manufacturer’s instructions. The luminescence values were normalised to the total protein level as assessed with the DC Biorad Assay (Biorad).Non-parametric tests were used for statistical analyses  as 1-sample Kolmogorov–Smirnov test showed skewed distribution of continuous variables. Pairwise comparisons between normal and NSCLC samples were performed with Wilcoxon test. The differences for continuous variables among independent cohorts within clinical parameters were determined with the Kruskal–Wallis and Mann–Whitney tests. Bivariate correlation was probed with Spearman’s test. Bonferroni correction was applied to adjust for multiple comparisons. Patients’ survival was calculated with the log rank test.N = 132) when compared to adjacent normal tissues     than in SqCLCs . Apart from that no other associations were observed between COL1A1, PRPF40A or UCP2 mRNA expression and patients’ gender, age, survival, smoking history, TNM classification, and tumour histology and differentiation.Our comparative qPCR analysis showed abundant COL1A1 overexpression in tumours . The samples selected for the latter analysis featured clinicopathological profile representative of all tumour samples utilised in this study (Table N = 56) than in adenocarcinomas  in comparison with poorly differentiated NSCLCs  , HIF1α , and VEGFa . The hypoxia dependence was observed as well in the case of COL1A1 , HIF1α  and, to a lesser extent, with VEGFa . Similar relationships were seen in the case of UCP2 , PRPF40A and UCP2 , while the weakest between COL1A1 and UCP2 .Next, we evaluated interrelationships between COL1A1, PRPF40A, UCP2, and hypoxia markers , whose expression profiles were previously reported , but not in H358 . PRPF40A and UCP2 expression little changed at 1% O2, as only CALU1 exhibited modest upregulation of UCP2 . Similarly, oxidative stress did not evoke significant changes in the expression of COL1A1, UCP2, and PRPF40A genes. This lack of responsiveness to stress conditions was not caused by CpG methylation, as the promoters of COL1A1, UCP2, and PRPF40A showed low methylation level (MtI < 10%) in both cell lines  (Creighton et al. 2 level by COL1A1, UCP2, and PRPF40A is dependent on cellular context.Despite the clustering of the three genes with hypoxia markers in tissues, in vitro hypoxia had differential effect on their expression. The statistical correlation between various genes might reflect similar, but unrelated expression patterns. These patterns could manifest the specificity of pathologic transcriptome rather than the activation of a particular signalling pathway. From this point of view, lack of responsiveness in the cells incubated at low OWhile UCP2 overexpression might contribute to Warburg effect and promote tumourigenic phenotype (Ayyasamy et al.


Lactobacillus curvatus KG6, L. curvatus MRS6, and Lactobacillus sakei FAM18311 were sequenced and assembled using PacBio single-molecule real-time (SMRT) technology. The strains were isolated from Swiss fermented meat products. Circular chromosomes were of 1.98 Mbp (KG6), 2.11 Mbp (MRS6), and 1.95 Mbp (FAM18311), with a G+C content of 41.3 to 42.0%.The genomes of  Lactobacillus curvatus and Lactobacillus sakei are closely related species (–L. curvatus KG6 was isolated on de Man-Rogosa-Sharpe (MRS) agar  from a salami-type fermented meat product purchased at the retail level in Switzerland in 1999, and it showed phenotypic novobiocin resistance (L. curvatus MRS6 is a tyramine producer that was isolated at 106 CFU ml−1 on MRS agar (Biolife) from the traditional Swiss fermented sausage “salsiz” made of deer meat purchased at the retail level in Switzerland in 2016 . L. sakei FAM18311 is part of the FAM strain collection of Agroscope Liebefeld-Posieux  originating from a fermented meat product . The genomes were annotated using the NCBI Prokaryotic Genome Annotation Pipeline. Ori-Finder 2 was used to predict the chromosomal origin of replication (An overnight culture was used to propagate the strains under anaerobic conditions at 37°C in MRS broth (Biolife) for DNA isolation. The Wizard genomic DNA purification kit  was used for DNA isolation, including modifications as previously described  and a G+C content of 41.3%. Plasmids pFAM18311_1 and pFAM18311_2 harbored 90 CDSs and 29 CDSs, with G+C contents of 34.5% and 40.4%, respectively. KG6 harbored a circular chromosome of 1,985,155 bp and a plasmid pKG6_1 of 17,609 bp, with G+C contents of 42.0% and 44.9%, respectively. The chromosome of KG6 and its plasmid pKG6_1 featured 1,970 and 17 predicted CDSs, respectively. MRS6 harbored a circular chromosome of 2,114,309 bp, with a G+C content of 41.7% and 1,975 predicted CDSs. MRS6 was previously found to produce tyramine . The presumptive operon encoding tyramine production was located around the tyrosine decarboxylase-encoding gene (CG419_06595). CP020459 to CP020461 (FAM18311), CP022474 (MRS6), and CP022475 and CP022476 (KG6). The versions described in this paper are the first versions, CP020459.1 to CP020461.1 and CP022474.1 to CP022476.1.This whole-genome project has been deposited at DDBJ/ENA/GenBank under the accession numbers