The chemical vapor deposition (CVD) method to obtain tailored graphene as a transparent and flexible gas barrier has been developed. By separating nucleation step from growth, we could reduce early graphene nucleation density and thus induce better stitching between domain boundaries in the second growth step. Furthermore, two step growth in conjunction with electrochemical polishing of Cu foils achieved large graphene domains and improved graphene quality with minimized defects. The performance of resulting graphene as a gas barrier was superior to the graphene obtained by one-step growth on polished or unpolished Cu foils. The CVD graphene reported here could open up the possibility for exploring graphene-based gas barrier due to the minimized density of defect area. As a consequence, unfavorable gas and aerosol molecules can be transferred through these defects, causing local corrosive change in the surface and degradation of the protection ability of the graphene18212223242526272812et al.For practical applications as protective barrier, preparation of large area, defect-free graphene and a suitable method for its integration on polymeric or other substrates are important requirements. Among many preparation methods, chemical vapor deposition (CVD) is the most effective method to reliably produce high-quality, wafer-scale monolayer graphene films. By virtue of this, CVD-grown graphene has been investigated as a protective barrier to prevent transport of gas molecular between the environment and the arbitrary surface such as polymers or metal layer9101112131415161284/H2 mixture which act as nucleation seeds and can influence the gas barrier properties of graphene26Here, we use a combined approach of electro-chemical polishing (ECP) and two-step growth methods to improve the gas barrier property of graphene without an increase in the layer numbers or additional fabrication process. ECP is known as a highly effective step to reduce the surface roughness of Cu foil and remove the impurity particles4 to H2 flow ratio, total pressure, temperature, and Cu foil treatments are crucial parameters that influence the graphene domain shape21222324252, twice or four times larger than the graphene domains obtained by one-step growth on ECP and unpolished Cu foils, respectively, as illustrated in 6/cm2 in the case of two-step growth on ECP Cu. It is well known that steps, wrinkles, defects, and grain boundaries in Cu foils have much lower nucleation barriers than flat surface. These could act as nucleation sites, triggering the formation of graphene nuclei and eventually give rise to small single-crystal graphene domains. On the other hand, ECP process reduces the surface roughness as well as defects and impurities that subsist on Cu foils, leading to the suppression of nucleation sites and hence reduced domain density of graphene. In addition, during the early stage of two-step growth, the low CH4 concentration also mitigates the graphene nucleation and provides space for the growth of large graphene domains. As a result, two-step growth of graphene on ECP Cu foil gives large-size single crystalline graphene domains with a reduced domain density.The initial stage of growth is critical to obtain large size domains and high quality graphene layer. In this study, three different graphene layers are grown as follows: The first graphene was grown on as-received Cu foil without any treatment by one step growth (1step-w/o ECP), the second graphene was grown on electrochemically polished Cu foil by one step growth (1step-ECP) and the third graphene was grown on ECP Cu foil by two step growth (2step-ECP). 21222/Si substrate. −1 and 2690 cm−1, respectively. However, the G-band position is found to be blue shifted compared to the value 1582 cm−1 reported in the literaturep-type doping of graphene caused by the redox reaction between oxygen from air and adsorbed water at the surface of SiO2/Si substrate having hydrophilic property−1 and the intensity ratio of 2D to G greater than 2. Also, the so-called defects or disorder-induced D-band peak is seen around 1350 cm−1 at about half of the frequency of the 2D band with negligible intensity.To investigate the quality and number of graphene layers, Raman measurements were performed on representative graphene films after being transferred on to SiOEven though the transmittance of 2step-ECP graphene films shows no difference compared with that of 1step-w/o ECP or the 1step-ECP graphene films, sheet resistance change reflects graphene quality rather than thickness. In 30352-day, which is 57.9% less than that of the value of 1.58 ± 0.029 g/m2-day measured for the bare PET. −14, 5.806 × 10−14 and 4.224 × 10−14 mols−1 m−1atm−1, respectively. It is evident that the lowest WVTR and P values are obtained for monolayer graphene formed by two-step growth on ECP Cu foil without any additional treatment or control of graphene number.4)2S2O8], is dropped onto graphene synthesized on Cu foils. After waiting for about 5 sec, the dropped etchant is removed through rinsing process with deionized water and subsequently the surface is examined by atomic force microscope (AFM), as shown in −7 ± 3.3 × 10−6/cm2, 2.3 × 10−7 ± 2.8 × 10−6/cm2, and 2.2 × 10−6 ± 6.0 × 10−5/cm2, respectively. It is evident that the graphene grown by two-step method on ECP Cu has the lowest etch pit density, signifying that the two-step growth method reduces the defect density to a great extent compared to conventional one-step growth. PET) and etch pit density and etched to the total area ratio (Δ) obtained by a commercial image processor  from corresponding AFM images. The Etch pit density shows a linear decrease from the first sample (1step-w/o ECP) to the third one (2step-ECP), which is in accordance with the SEM results shown in Though Raman analysis can well assess the defect distribution and quality of graphene through the G-to-D band intensity ratio, it is hard to measure the spatial distribution of defects that spread across over large area. To give a further insight into the structural quality of graphene on large scale, a film-induced frustrated etching (FIFE) is carried out, which is a simple and facile method to observe the structural defects spread on large area of grapheneThe solution of the Fick’s second law of diffusion is used to fit the measured WVTR, by assuming the water vapor concentration is close to zero at the exit side of the film as given belowP0 = Jd, P = Jsd = SD, J and Js are the water vapor molar flux at time t and at steady state, d is the sample thickness, P is permeability, D is diffusivity, and S is solubility. If the whole area of PET film is fully covered by impermeable graphene layer, the permeability should be zero. However, the CVD graphene layer consists of defect-holes through which water molecules may diffuse. The water molecules in the PET film may diffuse out through the defect-holes. The surface defect-hole density, ψ, can be defined as the ratio between the defect-hole area and the total area. Inside of the defect-holes, the inhibition of water diffusion by graphene layer may be negligible, when the sizes of the holes are much larger than those of the water molecules and the thickness of hole is much smaller than the mean free path of water molecules. In this case, the permeability can be simply expressed aswhere Peff , PG and P° represent the permeability of graphene layer/PET film, bare graphene film, and pure PET film, respectively. However, PG is not zero but a tiny value due to undetected defects, and thus the equation can be approximated in the form.because graphene without defects can be assumed as an impermeable membrane in the simple model where B is a correction constant arising from undetected defects by the FIFE. It is reasonable to assume that the Δ is proportional to ψ such as ψ = AΔ. Then the reduced permeability can be related to Δ as follows;where −14 ± 2.205 × 10−16, respectively. Thus, the ψ value of graphene grown by 2step-ECP on PET is found to be 0.624 ± 0.0081. This result indicates that the formation of graphene layer on PET is an effective way to improve the water vapor barrier property of the polymer film. If barrier performances of graphene are further improved by optimizing growth conditions, it can be used as transparent and flexible gas barrier in display devices.where A is an arbitrary constant. The permeability of graphenes on PET with Δ examined in In conclusion, a combination of electrochemical polishing and two-step growth is demonstrated to obtain high-quality monolayer graphene by low pressure CVD. This method allows achieving large size graphene domains by controlling the nucleation density of graphene on Cu foils, a crucial starting step for the realization of high-quality graphene. The characteristics of the graphene were compared to that of graphene produced by one-step growth on both unpolished and electrochemically polished Cu foils. Two-step growth on electrochemically polished Cu also led to improved permeability property. This is attributed to the decrease in structural defects and suppression of nucleation of graphene at the initial stage. A simple and accurate model has been proposed to examine the barrier property of CVD monolayer graphene. Our study also brings into picture that the effective permeability of CVD monolayer graphene is proportional to defect area rather than defect density.3PO4:H2O acid solution at a constant voltage of 2.0 V for 12 min using Cu plate as cathode and Cu foil (80 mm × 80 mm) as working electrode.ECP was performed in diluted 3H2 gas was flown through the reactor at 15 standard cubic centimeters per minute (sccm) during the temperature ramp-up up to 1030 °C. Then, the Cu foils were annealed for 50 min. In the one-step growth, CH4 of 13 sccm and H2 of 15 sccm were supplied at 1030 °C for 23 min under a growth pressure of 0.160 Torr. In the case of two-step growth, at first, large size graphene domains were formed on Cu foil in a mixed CH4/H2 (5 sccm/100 sccm) ambient for 1 min at 1030 °C under a growth pressure of 0.365 Torr. This is the optimal condition for obtaining uniform and large domains of graphene, as shown in the 4 and growth time were increased to 13 sccm and 8 min, respectively, while maintaining the H2 flow rate and temperature unchanged. Finally, the samples were cooled down rapidly to room temperature with a cooling rate of 30 °C min−1 at 15 sccm of H2 flow by moving the furnace out of the substrate region.Large-scale graphene layer studied in this work was synthesized on 35-μm-thick Cu foils (Nippon Mining) by low pressure chemical vapor deposition (LPCVD). The Cu foil was then set into a 2 inch quartz tube and heated by split-tube furnace. Meanwhile, quartz tube was pumped down to 0.072 Torr, and H2 plasma etching. Then, the Cu foil with PMMA-covered graphene was dipped in 0.1 M Ammonium Persulfate [(NH4)2S2O8] solution for 4 h to etch the Cu foil. Thereafter, PMMA/graphene layer was transferred onto various substrates, such as 300 nm SiO2/Si substrate, PET film, and glass, to investigate the opto-electrical and gas barrier properties. The PMMA was removed by using acetone and the sample was annealed in an Ar/H2 gas mixture at 400 °C for 3 h to eradicate remained PMMA residues.Polymethyl methacrylate (PMMA) was spin-coated onto graphene surface at 4200 rpm for 50 s to protect and transfer the graphene layer onto other substrates of interest. Prior to graphene transfer, graphene unintentionally formed on the back side of the Cu foil was removed by O4)2S2O8], was dropped on to graphene synthesized on Cu foils. After 5 sec, the dropped etchant was removed by rinsing with deionized water.Commercial copper etchant, ammonium persulfate [ was used to evaluate the surface morphology of the graphene samples studied in this work. The surface topography of graphene/Cu foils after FIFE test was probed by atomic force microscope  in tapping mode. The Raman spectra were recorded using 514 nm-line of an Ar ion laser as an excitation source. In order to observe the change in electrical conductivity as a function of graphene domain size, the sheet resistance was measured by Van der Pauw method using Hall measurement system (Lakeshore 7500 serises).How to cite this article: Seo, T. H. et al. Tailored CVD graphene coating as a transparent and flexible gas barrier. Sci. Rep.6, 24143; doi: 10.1038/srep24143 (2016).


Recent clinical studies show that tyrosine kinase inhibitors slow the rate of lung function decline and decrease the number of acute exacerbations in patients with Idiopathic Pulmonary Fibrosis (IPF). However, in the murine bleomycin model of fibrosis, not all tyrosine kinase signaling is detrimental. Exogenous ligands Fibroblast Growth Factor (FGF) 7 and 10 improve murine lung repair and increase survival after injury via tyrosine kinase FGF receptor 2b-signaling. Therefore, the level and location of FGF/FGFR expression as well as the exogenous effect of the most highly expressed FGFR2b ligand, FGF1, was analyzed on human lung fibroblasts.FGF ligand and receptor expression was evaluated in donor and IPF whole lung homogenates using western blotting and qPCR. Immunohistochemistry for FGF1 and FGFR1/2/3/4 were performed on human lung tissue. Lastly, the effects of FGF1, a potent, multi-FGFR ligand, were studied on primary cultures of IPF and non-IPF donor fibroblasts. Western blots for pro-fibrotic markers, proliferation, FACS for apoptosis, transwell assays and MetaMorph analyses on cell cultures were performed.Whole lung homogenate analyses revealed decreased FGFR b-isoform expression, and an increase in FGFR c-isoform expression. Of the FGFR2b-ligands, FGF1 was the most significantly increased in IPF patients; downstream targets of FGF-signaling, p-ERK1/2 and p-AKT were also increased. Immunohistochemistry revealed FGF1 co-localization within basal cell sheets, myofibroblast foci, and Surfactant protein-C positive alveolar epithelial type-II cells as well as co-localization with FGFR1, FGFR2, FGFR3, FGFR4 and myofibroblasts expressing the migratory marker Fascin. Both alone and in the presence of heparin, FGF1 led to increased MAPK-signaling in primary lung fibroblasts. While smooth muscle actin was unchanged, heparin + FGF1 decreased collagen production in IPF fibroblasts. In addition, FGF1 + heparin increased apoptosis and cell migration. The FGFR inhibitor (PD173074) attenuated these effects.Strong expression of FGF1/FGFRs in pathogenic regions of IPF suggest that aberrant FGF1-FGFR signaling is increased in IPF patients and may contribute to the pathogenesis of lung fibrosis by supporting fibroblast migration and increased MAPK-signaling.The online version of this article (doi:10.1186/s12931-015-0242-2) contains supplementary material, which is available to authorized users. Idiopathic pulmonary fibrosis (IPF) is a rare interstitial lung disease of unknown origin, with prevalence rates ranging from 2-4/10000 , and theBoth Fgf7 and Fgf10 are mesenchymal-derived growth factors that signal in a paracrine manner to bind with high affinity to epithelial expressed Fgfr2 b-isoform . OverexpFgf/Fgfr-binding is stabilized by heparin and transduced by a phosphorylation cascade, mediated by fibroblast growth factor receptor substrate (Frs2)  which acUntil now, studies investigating the activity of heparin + FGF1 in the context of lung fibrosis have been carried out exclusively on cell lines. Treatment of lung fibroblast cell-line N12, with heparin + FGF1 had no impact on proliferation but induced apoptosis and decreased smooth muscle actin production . MoreoveThe study protocol for tissue donation was approved by the Ethics Committee of the Justus-Liebig-University School of Medicine (AZ 111/08). Informed consent was obtained from each individual patient or next of kin.n = 36) or donors (n = 15) were obtained during transplantation at the Department of Cardiothoracic Surgery, University of Vienna, Austria or University Hospital Giessen-Marburg, Giessen, Germany and processed as previously described [Lung homogenates from IPF n = 6 or donoLoading buffer was added to protein samples from cell extracts (5 % SDS in bromophenol blue and β-mercaptoethanol) denatured for 5 min at 95 °C and cooled on ice. At least 10 μg of sample was loaded on a 10 % polyacrylamide gel and run at 25 mA for 2 h then transferred to a polyvinylidene fluoride (PVDF) membrane (Amersham) by semi-dry electro-blotting  for 90 min. The membrane was blocked with 5 % milk in TBS-blocking buffer at RT for 1 h followed by 4 °C incubation with primary antibody overnight. Membranes were washed in 1X TBST buffer four times and incubated with HRP-labeled secondary antibody at RT for 1 h followed by washing with 1X TBS-T. Bands were detected by ECL  treatment, followed by exposure of the membrane. . cDNA was diluted to 20 ng/μL. Primers were designed to span introns using Roche Applied Sciences online Assay Design Tool. Sybr Green Master Mix (Applied Biosciences 4309155) was used for qPCR with a Roche LightCycler480 machine. Samples were run in triplicates using 3. cDNA wHuman lungs were placed in 4 % (w/v) paraformaldehyde for 12-24 h, and processed for paraffin embedding. 3 μm sections were cut and mounted on slides . Paraffin-embedded tissue sections of donor and IPF-lungs were deparaffinized in xylene and rehydrated in graded alcohol. Antigens were retrieved by microwave antigen retrieval (800 W); in 10 mmol/L freshly prepared citrate buffer (pH 6.0) for 5 min. For immunostaining, the streptavidin-biotin-alkaline phosphatase (AP) - or the streptavidin-biotin- horseradish peroxidase (HRP) method was used with the ZytoChem-Plus AP Kit (Fast Red), Broad Spectrum  according to the manufacturer’s protocol. Sections were counterstained with hemalaun  and mounted in Glycergel (DakoCytomation). Control sections were treated with 2 % BSA in PBS alone or with rabbit or mouse primary antibody isotype control  to determine the specificity of the staining. Lung tissue sections were scanned with a Mirax Desk slide-scanning device , and examined histo-pathologically at 50×, 100×, 200×, 400×. IHC for mentioned antibodies was performed in at least 8 IPF and 5 control-lung samples. A complete list of antibodies and dilutions is provided in the online supplement.2 flask and grown out at 37 °C, 5 % CO2 for up to one month with weekly media changes according to a previously published protocol [5 cm lung cubic biopsies of human lung tissue were washed in PBS and cut into small pieces in growing culture medium: Dulbecco's Modified Eagle's Medium (DMEM) with 10 % Fetal calf serum (FCS) 1 % glutamine, and 1 % Penicillin-Streptomycin. Pieces were seeded initially in a large 75 cmprotocol . No enzyThe affymetrix APC annexin V and propidium iodide staining kit was used to perform an apoptosis assay according to manufacturers instructions (eBioscience #88-8007-74) on an Accuri C6 flow cytometer (BD Biosciences). Briefly, cells were washed twice in cold PBS, then trypsinized at 37 °C for 5 min. 2 wells of fibroblasts grown in 6-well plates were gently collected, combined and pelleted. Pellets were washed in PBS, followed by binding buffer, and incubated first with kit antibody, then propidium iodide in the dark for 10 min. At least 30,000 cells were counted per FACS experiment. Gating was established based on plots of propidium iodide alone and annexin V alone.Primary lung fibroblasts were starved for 24 h and seeded  in the upper chamber of the transwell  containing serum free DMEM F-12 medium and the lower chambers with various experimental conditions. The system was incubated at 37 °C, 5 % CO2 to allow the migration of cells through the membrane (8.0 μm). After 16 h, media was removed using a gentle suction and cells were washed with 1X PBS, fixed with methanol and stained with crystal violet. Next, the transwell was swabbed to remove the non-migrated cells and the total number of migrated cells was quantified using phase contrast microscope.2 and 37 °C environment. Random regions were marked, and Leica Live Imaging Software snapped a photo of these regions every 5 min for 20 h. LIF files were exported and analyzed in Leica MetaMorph software version 1.5.0. The average total distance traveled of at least 6 random cells per group were tracked and recorded.Fibroblasts from at least 3 biologically unique samples were seeded at low density, in a 24-well-plate, starved 24 h, and underwent various treatments. The plate was set on a motorized stage in a 5 % COt-Test was performed on the log-transformed value of the qPCR fold changes. For western blots, t-tests were performed on the probit values. One-way ANOVAs with Dunnet’s test (untreated groups served as controls) were performed on Transwell and MetaMorph data.A Student’s , FGF7, and FGF10. A significant increase in FGF1 was observed in IPF patients  and Collagen 1a1 (COL1A1) transcripts  of donor lungs  which assemble into ‘sheets’,  Fig. 3,,3, b1,2.Keratin 5-staining was also used to identify bronchial epithelium of IPF lungs Fig. ; 1,2. InGiven the strong expression of FGF1 and FGFR1/2/3/4 in regions of usual interstitial FF, as well as their co-localization with the migratory marker Fascin, and the myofibroblast marker α-SMA, the effect of exogenous FGF1 on IPF lung fibroblasts was addressed.IPF and non-IPF, donor fibroblasts  were harvested and cultured as previously described . Cells wHeparin + FGF1 treatment of IPF fibroblasts resulted in a trend towards decreased collagen production by IPF fibroblasts while no effect was observed on non-IPF fibroblasts Fig. 5,d.Fig. 5The trend towards decreased collagen production in IPF fibroblasts treated with heparin + FGF1 may be in part regulated via activation of p-ERK1/2 signaling. The p-ERK1/2 signal was significantly increased in both FGF1 alone and FGF1 + heparin treated IPF fibroblasts Fig. ,f,g. TheIn summary, the FGFR inhibitor PD173074 efficiently mitigated both endogenous and exogenous FGFR mediated p-ERK signaling. Furthermore, FGF1 + heparin did not significantly attenuate α-SMA production nor did it significantly regulate Fascin. However, COL1a1 production trended towards a decrease in this group. Due to the substantial activation of p-ERK signaling following heparin + FGF1 treatment, whether proliferation and apoptosis were influenced was tested next.Flow cytometry was performed on treated donor and IPF fibroblasts for Annexin V and propidium iodide. In accordance with previous reports , 28, theTo further investigate potential functional effects of exogenous FGF1 + heparin on primary lung donor and IPF fibroblasts, both transwell migration experiments and MetaMorph analyses of cell cultures were performed. The FGFR inhibitor alone (0.1 μM) did not inhibit migration through the transwell Fig. . The addMetaMorph analyses of low-density fibroblasts cultures revealed that heparin + FGF1 stimulated IPF fibroblasts to travel longer distances than non-stimulated fibroblasts Fig. . In thisIn summary, heparin + FGF1 stimulated migration of both IPF and donor fibroblasts and this effect could be attenuated by the simultaneous addition of FGFR inhibitor.In the rodent bleomycin-model, enhanced Fgfr2b-signaling on alveolar epithelial cells via the exogenous application or induction of FGF10 or FGF7, conferred increased survival and reduced lung fibrosis . On the FGF1 emerged as the most highly expressed FGFR2b-binding ligand in IPF lung homogenates. FGF1 is expressed by both mesenchymal and epithelial cell types in the lung  and bindIn this study, FGF1 and FGFR1/2/3 were increased at the protein level in whole lung homogenates taken from end-stage IPF patients compared to non-IPF lung homogenates. IHC analyses revealed robust expression of FGF1 in regions of irregular lung architecture particular to IPF, including: basal cell sheets or basal cells of hyperplastic bronchioles Fig.  and SMA+Former in vitro studies suggest that FGF1 may have an anti-fibrotic effect on lung fibroblasts. For example, heparin + FGF1 was found to decrease smooth muscle actin production and had a pro-apoptotic effect on a normal lung fibroblast cell line . In addiFGF1 and FGFR co-localization with the motility and invasion marker Fascin was observed in usual interstitial fibroblastic foci. In addition, a preliminary scratch assay experiment revealed that FGF1 + heparin treated IPF fibroblasts closed a scratched area of confluent fibroblasts faster than untreated cells (data not shown). Although Fascin expression was not observed to be significantly regulated by heparin + FGF1, the failure of heparin + FGF1 to reduce SMA expression, the preliminary scratch assay data, the absence of FGF1 from condensed regions of smooth muscle cells, as well as previous studies indicating that FGF1 influences cell migration , was ratThe addition of heparin + FGF1 stimulated significant migration of both IPF and non-IPF fibroblasts through a transwell filter compared to starved, untreated cells. When heparin + FGF1 were added in the presence of the FGFR inhibitor, this effect was mitigated. Interestingly, while the addition of FGF1 alone or heparin alone did not stimulate migration in donor fibroblasts, these conditions stimulated migration in IPF fibroblasts, but to a lesser extent than heparin + FGF1 together. These results suggest that IPF fibroblasts may be primed to receive chemotactic signals. Faster migration by IPF fibroblasts may also be due to enhanced p-ERK1/2 signaling as stronger activation of p-ERK1/2 by FGF1 alone and heparin + FGF1 was also observed in IPF fibroblasts compared to donor, non-IPF fibroblasts used in this study.In addition, MetaMorph analyses of low-density cultures of fibroblasts revealed that heparin + FGF1 stimulated IPF fibroblasts to travel longer distances while no effect was observed in non-IPF, donor fibroblasts. Unlike the transwell experiments, addition of the inhibitor alone reduced overall distance travelled in both donor and IPF fibroblasts compared to untreated cells. However, as in the transwell experiments, the FGFR inhibitor efficiently attenuated the effect of exogenous heparin + FGF1 in IPF fibroblasts when added simultaneously. Lastly, unlike the transwell assay, FGF1 alone had no effect on IPF fibroblasts, while heparin alone reduced distance travelled of IPF fibroblasts. The discrepancies between the transwell and live imaging experiments may be due the lack of a gradient formation in the 24-well plates used for live imaging. In summary, MetaMorph analyses of live imaging experiments support the conclusion that the migration of IPF fibroblasts was enhanced following exposure to heparin + FGF1.In summary, this study described strong expression of FGF1 and FGFR1/2/3/4 receptors in pathogenic areas of IPF lungs and identified FGF1-FGFRs as potential contributors to increased MAPK-activity in IPF. Though IPF lung fibroblasts responded to heparin + FGF1 treatment by attenuating COL1a1 expression and increased apoptosis, increased p-ERK1/2 signaling along with enhanced cell migration was also observed reflecting a potentially dual nature of FGF1/FGFR in the context of lung fibrosis. Though tyrosine kinase inhibitors have recently been approved for the treatment of IPF, given the multi-faceted nature of FGF1-FGFR signaling, further studies should be designed to identify targets of growth factor signaling that mediate specific cellular functions such as fibroblast apoptosis and migration.


Patients were prospectively followed up for 40 months. Receiver operating characteristics curve analysis determined cut-off points associated with mortality, and Cox proportional hazard ratio (HR) were conducted. Thresholds for increased mortality risk in IPF patients were determined as IPAQ ≤ 417 metabolic equivalent task (METS)-min/week, p = 0.004 , p = 0.027), and ∆SpO2 ≥ 10%, p = 0.002, , p = 0.025). This study demonstrated a significant association of PA and ED with mortality in IPF patients. The findings emphasize the clinical importance of PA and ED assessments to aid in IPF risk stratification, prognosis prediction, and in providing early appropriate treatments, such as pulmonary rehabilitation, PA consultation, oxygen supplementation, and lung transplantation referral. These results underscore that even low levels of PA corresponding to 100–105 min/week were associated with a reduced mortality risk and better survival in IPF.Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease that manifests in hypoxemia, inactivity, and poor prognosis. This study aimed to assess the prognostic role of physical activity (PA) and exertional desaturation (ED) with mortality in IPF. At baseline, 34 IPF patients (68 (50–81) years) were interviewed using the International Physical Activity Questionnaire (IPAQ), and SpO Exey in IPF ,4. In ady in IPF . Worldwiy in IPF ,8,9. Mory in IPF . Neverthy in IPF ,12.2 < 88% may be different for patients with various resting saturation levels (with and without resting hypoxemia), and possibly has less prognostic sensitivity for all spectrums of disease severities. Secondly, physical inactivity is a well-established, independent risk factor for mortality in the general population [2 pre- to post-6MWT (∆SpO2)) with mortality among patients with IPF.In reviewing the literature addressing IPF, we detected some clinically important gaps related to levels of physical activity (PA) and exertional desaturation (ED) with mortality risk. Firstly, the previous proposed exertional nadir of absolute SpOpulation  as well pulation . NonetheThis observational prospective follow-up study was conducted at the Pulmonary Institute, Rabin Medical Center, Beilinson Hospital, Petach-Tikva, Israel. The study protocol was approved by the Ethics Committee of the hospital and was registered with clinicaltrials.gov NCT01499745). The study was conducted in accordance with the Declaration of Helsinki and written informed consent was obtained from each subject prior to participation. Patients were included if they were diagnosed with IPF according to the accepted clinical-radiological criteria of the ATS/ERS and were clinically stable in previous 3–6 months [499745. Tn = 34) were used to determine the prognostic predictors of survival. Subjects were tested as described below and followed up for 40 months from baseline:In the current study, subjects from our previous exercise training study  were fol600 METS-min/week (150 min/week * 4METS of moderate intensity PA) was considered a threshold for meeting the general PA recommendation [Self-report 7-day short form International Physical Activity Questionnaire (IPAQ) was used for all patients ,23. Eachendation ,26,27,282%; Pulse Oximeter 2500 30 EM, Nonim Medical, Minneapolis, MN, USA) were obtained prior and immediately after the test [2% pre- to post-6MWT (∆SpO2%).The 6MWT was conducted according to ATS guidelines in a 35-meter corridor at the pulmonary unit within the hospital . Borg dythe test . Oxygen Pulmonary function tests, including spirometry total lung capacity, maximal voluntary ventilation, and diffusion capacity for carbon monoxide were performed according to standard techniques and ATS/ERS guidelines  ,32. All p < 0.05. Demographics, clinical, and physiological data of the participants are presented as median and (range). Categorical variables are presented in absolute values and (%) of group. Receiver operating characteristics (ROC) curve analysis for cut-off points’ ˃70% sensitivity and ˃70% specificity to detect mortality was performed for IPAQ and ∆SpO2. A multivariate Cox proportional hazard model using significant cut-off points determined from ROC was analyzed for IPAQ and ∆SpO2. The model was adjusted for gender, age, forced vital capacity %predicted, diffusion capacity for carbon monoxide %predicted, and Prednisone usage. Kaplan–Meir curves were used for survival analysis. Subjects who underwent lung transplants were considered as fatalities in the statistical analysis, as has been reported previously [2) with 6 min walking distance (6MWD) [The statistical analyses were conducted using SPSS v.23 software . The significance level was set at eviously ,35. Exple (6MWD) . PA and The study population consisted of 34 IPF subjects from the Pulmonary Institute who were recruited for the pulmonary rehabilitation study . All sub2 were determined as prognostic predictors using ROC analysis , p = 0.027 and ∆SpO2 ≥ 10%, p = 0.002, HR; 23.3, CI 95% (1.5–365), p = 0.025.  but not for ED and 6MWD . Mortality was more prevalent among subjects with IPAQ ≤ 417 METS-min/week and ∆SpO2 ≥ 10% . Two of the 11 patients were at end-stage IPF and underwent lung transplants, and were considered as fatalities in the analysis. In the current study, significant cut-off points for IPAQ and ∆SpOanalysis . Patient= 0.025. . ExploraO2 ≥ 10% .2 ≥ 10% in 6MWT were significantly associated with mortality in patients with IPF  between PA and 6MWD, which strengthens the findings, since the latter is a well-established prognostic marker in IPF [This prospective follow-up study sought to evaluate the prognostic utility of PA and ED among IPF patients. We found that PA levels ≤417 METS-min/week and ∆SpOwith IPF . In thisr in IPF ,38,39. T2 < 88% during 6MWT as a strong prognostic marker in IPF [2 levels. In this regard, desaturation of 10% or more during 6MWT was significantly associated with mortality, which overcomes the variation in resting SpO2 levels across patients. Furthermore, the findings demonstrated for the first time in an IPF group that PA levels assessed by IPAQ are associated with mortality, and even low activity levels  revealed a significant reduced risk for death [2 ≥ 10%) that can enhance the overall risk stratification and add important information in the process of decision making and treatment options for patients with IPF, which further studies using large sample sizes should explore more extensively. The results of the current study provide novel evidence on the importance of PA and ED for survival in IPF. These findings are consistent with a previous large cohort study showing mortality risk reduction with only 15 min of daily PA in a general population . Our finr in IPF ,42. Howeor death ,43,44. Tor death ,46. MorePossible mechanistic explanations for these findings can be related to the fact that physical inactivity is a well-established all-cause cardiovascular and respiratory mortality risk factor ,47,48,492 ≥ 10% is a good sensitive threshold for detecting high risk IPF patients, and as such may be considered for implementation in clinical and research settings, due to the low cost, ease of assessment, and practical availability. However, despite the strong association between PA and ED with mortality observed in the current study, given the multifactorial manifestation of the disease and the diversity of predictors, we believe that prognosis should be based on a combination of predictors rather than on a single variable, and future trials should address this issue using large cohorts and multivariate analyses.ED was also clearly demonstrated as a significant risk factor for death and was associated with more severe conditions of IPF disease ,4. HigheA few possible limitations in our study are related to the small sample size and the level of IPF severity among patients ; 68% predicted), thus caution should be taken with generalization of our findings. Despite these limitations, the study has a number of strengths: collecting data prospectively, analyses using various statistical methods for the detection of sensitive thresholds, and calculating the risk for mortality and survival analysis. Moreover, the study found novel, sensitive, and practical indicators that can be easily implemented in clinical practice. The number of participants in our cohort is comparable to a previous study of PA and prognosis in IPF ,23,24,522 ≥ 10% in 6MWT were significantly associated with increased risk of mortality. This study strengthens the evidence for assessing PA and ED for the detection of high-risk IPF patients and aids in addressing appropriate treatments, such as PA consultation, pulmonary rehabilitation programs, oxygen supplementation, and referral for lung transplantation. The findings also underscore that even low levels of PA seem to have prognostic benefits for IPF patients. We suggest considering physical inactivity as a new independent modifiable prognostic risk factor for IPF. Further research with large cohorts is warranted to encourage implementation and the establishment of PA assessments in standard clinical practice.In summary, this prospective follow-up study originates new, simple, and practical prognostic cut-off points of PA levels and ED in IPF subjects. The results demonstrated that PA levels ≤417 METS-min/week and ∆SpO


Drosophila embryo. Smaug has two identified direct target mRNAs that it differentially regulates: nanos and Hsp83. Smaug represses the translation of nanos mRNA but has only a modest effect on its stability, whereas it destabilizes Hsp83 mRNA but has no detectable effect on Hsp83 translation. Smaug is required to destabilize more than one thousand mRNAs in the early embryo, but whether these transcripts represent direct targets of Smaug is unclear and the extent of Smaug-mediated translational repression is unknown.Smaug is an RNA-binding protein that induces the degradation and represses the translation of mRNAs in the early To gain a panoramic view of Smaug function in the early embryo, we identified mRNAs that are bound to Smaug using RNA co-immunoprecipitation followed by hybridization to DNA microarrays. We also identified mRNAs that are translationally repressed by Smaug using polysome gradients and microarrays. Comparison of the bound mRNAs to those that are translationally repressed by Smaug and those that require Smaug for their degradation suggests that a large fraction of Smaug’s target mRNAs are both translationally repressed and degraded by Smaug. Smaug directly regulates components of the TRiC/CCT chaperonin, the proteasome regulatory particle and lipid droplets, as well as many metabolic enzymes, including several glycolytic enzymes.Drosophila embryo, and has unanticipated functions in control of protein folding and degradation, lipid droplet function and metabolism.Smaug plays a direct and global role in regulating the translation and stability of a large fraction of the mRNAs in the early  Drosophila, Caenorhabditis elegans, zebrafish and mouse embryos have highlighted the substantial role that cytoplasmic post-transcriptional regulation plays in early embryos [Post-transcriptional regulatory mechanisms that function in the cytoplasm to control mRNA translation, stability and subcellular localization play essential roles in a wide variety of biological processes. While these types of controls function in a variety of cell types, they are particularly prevalent during early metazoan development where mRNAs synthesized from the mother’s genome direct the early stages of embryogenesis . Indeed, embryos -13.cis-acting elements that represent binding sites for microRNAs (miRNAs) or RNA-binding proteins. For example, miRNAs induce degradation of specific transcripts in both zebrafish and Drosophila[Drosophila embryo [During early embryogenesis, regulation of specific transcripts is achieved through rosophila,10. Simia embryo . Smaug ia embryo -18. SRE a embryo ,19-22.Drosophila Smaug can recruit the Cup protein to an mRNA and Cup in turn interacts with the cap-binding protein eIF4E [Upon binding to target mRNAs Smaug family members repress translation and/or induce transcript decay through their ability to recruit various factors to a transcript -18,23,24in eIF4E . The Cupin eIF4E . Typicalin eIF4E . In contin eIF4E .Smaug can also remove an mRNA’s poly(A) tail through its ability to recruit the CCR4/NOT deadenylase -31. In tnanos and Hsp83. Smaug represses nanos translation through two SREs in the nanos 3′ untranslated region (UTR) whereas loss of Smaug has only a modest effect on nanos mRNA stability [Hsp83 mRNA through eight SREs in the Hsp83 open reading frame, while having no detectable effect on Hsp83 translation [Smaug has two well-characterized target mRNAs, tability -16,28,33nslation . Thus, Snanos and Hsp83 mRNAs are localized to the posterior of the embryo and Smaug’s regulation of these two transcripts is intimately associated with their localization. nanos mRNA is inefficiently localized to the posterior and nanos mRNA that escapes the localization machinery is found distributed throughout the bulk of the embryo where it is translationally repressed by Smaug [nanos mRNA localized to the posterior is not repressed by Smaug and Nanos protein expression is thus restricted to the posterior of the embryo. Hsp83 mRNA is uniformly distributed in early embryos and, as embryogenesis proceeds, Smaug degrades Hsp83 mRNA in the bulk cytoplasm of the embryo while transcripts at the posterior of the embryo are protected [Hsp83 mRNA to the posterior of the embryo.by Smaug -16,34,35rotected ,31,36,37nanos and Hsp83 mRNA, Smaug is likely to regulate the expression of a large number of mRNAs in the early embryo through direct binding. For example, genome-wide experiments have shown that embryos collected from homozygous-mutant smaug females show stabilization of approximately 1,000 transcripts [smaug mutant embryos also show cell-cycle defects associated with a failure of DNA replication checkpoint activation and they also fail to undergo zygotic genome activation [nanos or Hsp83, this is consistent with a role for Smaug in regulation of the expression of additional mRNAs.In addition to nscripts . In additivation ,15. As nTo elucidate the global functions of Smaug in early embryos we employed two genome-wide approaches: 1) RNA co-immunoprecipitations followed by microarray analysis (RIP-Chip) to identify mRNAs that are bound by Smaug and 2) polysome gradients coupled to microarrays to identify targets of Smaug-mediated translational repression. Our data suggest that Smaug directly regulates the expression of a large number of mRNAs in the early embryo. Comparison of Smaug-bound mRNAs to those that are translationally repressed by Smaug (identified in this study), and those that are degraded in a Smaug-dependent manner  suggest nanos and Hsp83.To identify Smaug’s target mRNAs on a genome-wide scale we used RIP-Chip. Extracts, prepared from 0- to 3-hour-old wild-type embryos, were immunoprecipitated with an anti-Smaug antibody (hereafter denoted as Smaug RIPs) while immunoprecipitations using non-immune serum served as a negative control (hereafter denoted as control RIPs). Genes that were not expressed or were expressed at low levels in starting crude extracts were removed from further analysis and Significance Analysis of Microarrays (SAM)  was thennanos and Hsp83, were enriched in Smaug RIPs compared to control RIPs. In contrast, four mRNAs that, based on our RIP-Chip data, are not bound by Smaug showed little or no enrichment  to assay the enrichment of specific mRNAs in Smaug RIPs compared to control RIPs. Twelve selected mRNAs from the RIP-Chip target list with FDRs <5%, including t Figure .Smaug is a multifunctional regulator that is capable of both repressing translation and inducing the degradation of target mRNAs. To complement our identification of the targets of Smaug-mediated mRNA decay  and our 2-transformed version of this ratio. We removed genes from the polysome data that were not expressed or were expressed at only low levels. We also omitted the data from pool 2 in the TI calculation as it represents a mixed population of translated and translationally repressed mRNAs. We note that inclusion of pool 2 in the TI calculation has little effect on the calculated TI  as the logP < 3 × 10-16), indicating an excellent correlation between the two data sets.We then compared the TI for each gene in wild-type embryos to previously published polysome/microarray data from similarly staged wild-type embryos . In thatsmaug mothers (hereafter denoted as ‘smaug-mutant embryos’). We then compared the TI for each expressed gene in wild-type and smaug-mutant embryos  for the bulk of mRNAs present in smaug-mutant embryos , consistent with the fact that the majority of the mRNAs that are present in pools 3 and 4 of our gradients are indeed polysome-associated. Similarly, we also saw a significant decrease in the TI  for the 342 genes that are targets of Smaug translational repression (FDR <5%), consistent with the fact that, in smaug-mutant embryos, these mRNAs are highly associated with polysomes.To confirm that the increase in TI was indeed the result of the recruitment of mRNAs onto polysomes, smaug mutants. This suggests that a substantial subset of the genes with >5% FDR are potential targets of Smaug-mediated translational repression. Since SAM corrects for an average change in TI, if a large proportion of transcripts were in fact translationally repressed by Smaug, SAM would over-correct, thereby increasing the number of false negatives. To further evaluate the extent of Smaug-mediated translational repression we generated lists of genes that encode mRNAs that are unlikely to be bound by Smaug and are, therefore, unlikely to be targets of Smaug-mediated translational repression and then assessed their behavior in the polysome-gradient microarray experiments. We did this by identifying the 250, 500 and 1,000 genes whose mRNAs showed the lowest fold-enrichment in Smaug RIPs versus control RIPs. A comparison of the TI for each of these genes in wild-type and smaug-mutant embryos showed a distribution with little bias towards an increase in TI in the smaug mutant, confirming that few are likely to be targets of Smaug-mediated translational repression . This trend is highly significant .In addition to those genes that meet an FDR of <5%  for each of the top three sets of bound transcripts, respectively  as compared with the 250, 500 and 1,000 genes whose mRNAs were unlikely to be bound by Smaug . This analysis showed a peak change of TI in N and bottom N Smaug-binders , respectively. Based on this analysis, we estimated that 3,135, 3,094, or 2,728 are likely to be translationally repressed by Smaug using the distributions for N = 250, 500, or 1,000, respectively . We conclude that Smaug represses the translation of approximately 3,000 mRNAs in early embryos, representing about half of the 5,886 genes whose expression we detected in the polysome-microarray data set.To estimate the actual number of genes that are translationally repressed by Smaug, we deconvolved the distribution of TI changes for all genes Figure  to estim0-3 (where N is any base) [Smaug binds to and regulates its target mRNAs through SRE stem-loop structures and, as such, we would expect that mRNAs bound by Smaug as well as mRNAs translationally repressed by Smaug would be enriched for these stem-loops. The consensus sequence for the SRE loop is CNGGNny base) ,20. The ny base) . Thus, l0-4 . Our method assigned a probability for each potential SRE within a transcript based on the likelihood that it would fold into a stem-loop structure where the loop matches the CNGGN0-4 consensus. For each mRNA, an SRE score was then calculated as the sum of the probabilities for each SRE within that mRNA [smaug-mutant and wild type for translational repression) did not have higher SRE scores than the median for the bound or repressed mRNAs with FDR <5%.To ask whether SREs are predictive of Smaug binding and translational repression we searched all expressed genes in the RIP-Chip and polysome-microarray datasets for stem-loops with the loop sequence CNGGNhat mRNA . Strikin0-4 loops along with six altered stem-loops. The altered structures contained changes in the invariant nucleotides in the CNGGN0-4 loop that are predicted to lower their affinity for the Smaug RNA-binding domain. We found that the bona fide SRE was a significantly better predictor of both Smaug binding and Smaug-mediated translational repression than any of the altered stem-loops  and Smaug-mediated translational repression  than mRNAs whose decay is not regulated by Smaug. Using these two measures we also found a genome-wide correlation between Smaug binding and Smaug-mediated translational repression . Similar results were obtained for Smaug-mediated translational repression . Together these data suggest that a large fraction of the mRNAs that require Smaug for their degradation and/or translational repression that were scored as unbound in the RIP-Chip experiments are nonetheless directly bound by Smaug.The comparisons from Figure et al. . The founanos mRNA’s SREs are found in the 3′ UTR [Hsp83 mRNA’s SREs are found in the open reading frame [The e 3′ UTR -16 and tng frame ,31, raisnanos and Hsp83, whose mRNAs are localized to the posterior. Of these 44 genes, 38 are regulated by Smaug at the level of mRNA stability and/or translation 15), Replication Protein A 70 (RpA-70), Regulator of cyclin A1 (Rca1), Cyclin E (CycE), Minichromosome maintenance 3 (Mcm3), CDC45L, mutagen-sensitive 201 (mus201) and Msh6. Of these 32 genes, 29 are regulated by Smaug at the level of mRNA stability and/or translation , Nucleosome assembly protein 1 (Nap1), Nucleosome remodeling factor - 38kD (Nurf-38), anti-silencing factor 1 (asf1), Caf1-180, Caf1-105, and vig2. Of these 25 genes, 24 are regulated by Smaug at the level of mRNA stability and/or translation . These analyses suggest several previously unrecognized roles for Smaug in the early embryo  that are members of the Chaperonin Cpn60/TCP-1 family as defined by the Interpro database and are involved in protein folding. The last four of these proteins are subunits of the eukaryotic chaperonin TCP1-ring complex (TRiC), also known as the chaperonin containing TCP-1 (CCT), which consists of two rings composed of eight different subunits . ConsistRegulatory particle triple-A ATPase 3 (Rpt3), Regulatory particle triple-A ATPase 5 (Rpt5), Regulatory particle non-ATPase 1 (Rpn1), Regulatory particle non-ATPase 2 (Rpn2), Regulatory particle non-ATPase 7 (Rpn7), Regulatory particle non-ATPase 9 (Rpn9), Regulatory particle non-ATPase 10 (Rpn10), Regulatory particle non-ATPase 11 (Rpn11) and Regulatory particle non-ATPase 13 (Rpn13)). In contrast, Smaug does not interact with any of the mRNAs that encode the 20S core particle proteins. In addition, Smaug interacts with mRNAs that encode proteins involved in other aspects of the ubiquitin-proteasome system (Ubiquitin activating enzyme 1 (Uba1), Ubiquitin fusion-degradation 1-like (Ufd1-like), TER94 and CG9588). Consistent with a role for Smaug in control of the ubiquitin-proteasome system, 12 out of these 13 mRNAs  term ‘ubiquitin proteasome pathway’. The ubiquitin proteasome system plays a vital part in a variety of cellular processes through its role in the degradation of target proteins. This mechanism involves the post-translational addition of multiple ubiquitin moieties onto a protein, which, in turn, target the protein for proteasomal degradation . The 26SDrosophila fat-body tissue or from Drosophila embryos [Third, Smaug might play a role in regulating lipid storage and/or mobilization since the GO term ‘lipid droplet’ is enriched in the Smaug-bound mRNAs. Lipid droplets are ubiquitous organelles that are found in a wide range of organisms from bacteria to humans. They consist of a neutral-lipid core composed of triacylglycerols and sterol esters surrounded by a phospholipid monolayer, and they serve as storage sites for energy, sterols and membrane precursors . Several embryos ,57. CompHexokinase A (Hex-A), Phosphoglycerate kinase (Pgk), Phosphoglucose isomerase (Pgi) and both genes encoding Glyceraldehyde 3 phosphate dehydrogenase (GAPDH1 and GAPDH2)) and our data indicated that all nine are regulated by Smaug at the level of stability and/or translation repression  and Triose phosphate isomerase (Tpi) have FDRs in the RIP-Chip data of 5.15% and 6.08%, respectively, and both are targets of Smaug-mediated transcript degradation and translational repression  and Pyruvate kinase (Pyk) are regulated by Smaug at the level of stability and/or translation. In summary, our data suggest that 8 of the 10 glycolytic enzymes may be regulated by Smaug.Fourth, a direct role for Smaug in regulation of metabolism is suggested by the enrichment for terms such as the SwissProt keywords ‘oxidoreductase’ and ‘NAD’ and the GO terms ‘oxidation reduction’ and ‘cofactor binding’ within Smaug-bound mRNAs. Together these lists comprise a total of 37 metabolic enzymes that function in a wide variety of pathways, including fatty acid metabolism, pyruvate metabolism, amino acid metabolism, the citric acid cycle and oxidative phosphorylation. Our data suggested that 28 out of 37 of these genes are regulated by Smaug at the level of mRNA stability and/or translation 12, and Bicaudal C. Rpn7 is a proteasome regulatory particle subunit and was selected because of the observed enrichment for GO terms related to ‘proteasome regulatory particle’. Likewise, because of enrichment for the GO term ‘glucose metabolic process’ and the KEGG pathway ‘glycolysis/gluconeogenesis’, we assayed hexokinase, the first enzyme in glycolysis, and phosphofructokinase, which represents a critical point of regulation [smaug-mutant embryos [Drosophila oogenesis [smaug-mutant embryos could disrupt normal patterns of post-transcriptional regulation.To assess the role of Smaug in regulating the expression of the new target mRNAs, we selected five for further analysis: gulation ,59 and cgulation . Thus, S embryos . Bicaudaogenesis . Thus, Bsmaug-mutant embryos versus wild-type ones 12, Bicaudal C; Figure  Figures  and 10, Drosophila SRE consensus is more restricted than previously thought [Here we have used genome-wide approaches to identify mRNAs that are bound by Smaug and those that are translationally repressed by Smaug. Our results show that the presence of SREs is predictive of both binding and translational repression and, consistent with previous work on the yeast and human Smaug homologs ,45-48, i thought . Integra thought  has led nanos and Hsp83, are differentially regulated by Smaug [nanos and Hsp83, Smaug both translationally represses and degrades a large fraction of its target mRNAs. We hypothesize that the extent to which Smaug regulates the translational repression and/or destabilization of its targets is likely to be a consequence of additional cis-elements within target mRNAs. For example, the Hsp83 3′ UTR contains a translational enhancer that may mitigate Smaug-mediated translational repression [nanos mRNA observed in the absence of Smaug suggests that additional cis-elements within the nanos transcript function in its destabilization.Previous work has firmly established that Smaug can both repress translation and induce degradation of target mRNAs. However, Smaug’s two well-characterized target transcripts, by Smaug ,28,31,33pression . SimilarSmaug functions in the localization and regulation of its target mRNAs at the posterior of the embryo ,31,34-37nanos and Hsp83 mRNA is the fact that transcripts found at the posterior of the embryo escape Smaug regulation. The molecular mechanisms that underlie this spatial regulation of Smaug function are not understood, but Oskar protein has been implicated in blocking Smaug function at the posterior and has been shown to physically interact with Smaug [torso mRNA carrying the first 96 nucleotides of the nanos mRNA’s 3′ UTR, which includes one of the nanos SREs, is repressed at both the anterior and posterior of the embryo [torso mRNA carrying the first 185 nucleotides of the nanos 3′ UTR, which contains both nanos SREs, is repressed at the anterior but is expressed at the posterior [cis-elements mapping within nucleotides 97 to 185 of the nanos 3′ UTR that localize nanos transcripts to the germ plasm [nanos mRNA expression in the germ plasm. Our identification of several dozen posterior-localized, Smaug-bound transcripts should facilitate identification of any additional cis-elements.A critical aspect of Smaug’s role in the regulation of th Smaug ,15,36,63th Smaug ,64. Thise embryo . In addiosterior . Taken trm plasm  and/or aOur analysis of the mRNAs that are bound by Smaug has identified a number of mRNAs that encode proteins that are involved in cell-cycle control and transcriptional regulation. Mis-regulation of one or more of these mRNAs could underlie the cell-cycle and transcriptional defects that occur in the absence of Smaug. Our data also suggest that Smaug has several new and unanticipated biological functions, including control of protein folding and degradation, lipid droplet function and basic metabolism.smaug mutants.Our data suggest that Smaug downregulates the expression of 9 of the 19 subunits of the proteasome regulatory particle and 4 out of the 8 that encode the TRiC/CCT complex. In addition, three of the four remaining TRiC/CCT mRNAs and eight of the remaining ten proteasome regulatory particle mRNAs require Smaug for their degradation and/or translational repression .Lipid droplets are storage sites of triacylglycerols, hydrolysis of which yields fatty acids that can be metabolized for energy or serve as a source of membrane precursors. Thus, lipid droplets could function as the source of membrane precursors that are required during blastoderm cellularization, a process during which plasma membrane invaginates around the syncytial nuclei that are found at the embryo’s periphery. A role for Smaug in regulating lipid droplet function is intriguing as Drosophila embryos but are rapidly degraded [Drosophila, but also in frog and mammalian early embryos [Our data also suggest a widespread role for Smaug in regulating metabolism in the early embryo, including a role for Smaug in down-regulation of glycolysis. Previous work has suggested that maternal mRNAs encoding the glycolytic enzymes are present in early degraded -75. Glyc embryos ,77 but tOur data are consistent with Smaug directly regulating a large number of mRNAs in early embryos through translational repression and/or transcript degradation. This raises the question as to whether all of these repressive interactions are biologically important.trans-acting factors that control mRNA translation and/or stability will regulate a large number of transcripts and, as such, the same concepts should apply.In one model only a subset of Smaug’s targets are biologically relevant because the extent of downregulation by Smaug varies in a target-dependent manner. For the biologically relevant target transcripts, Smaug would effectively turn off their expression while, for the others, Smaug would reduce their expression insufficiently to have an effect on their biological function. A similar type of model has been suggested for repression mediated by individual miRNAs, which, as in the case of Smaug, regulate the expression of a large number of transcripts . Given tsmaug-mutant embryos.An alternative, but not mutually exclusive, model is that factors like Smaug, which repress the expression of a large number of mRNAs, do so in order to limit the total levels of available mRNA within a cell. This reduction could result from both Smaug-directed degradation of transcripts and/or Smaug-mediated translational repression, the former eliminating the mRNAs and the latter removing them from the pool of available mRNAs. In this model, Smaug would function to control the competition among transcripts for limiting cellular components, such as the translation machinery. We note, however, that our data do not support this model - at least in regard to the translation machinery - as we fail to see a decrease in the translation of mRNAs that are not bound by Smaug in Drosophila embryogenesis, nuclei are transcriptionally silent and development is driven by mRNAs synthesized by the mother and deposited into the egg during oogenesis. Subsequently, one- to two-thirds of these maternal mRNAs are degraded [A third model to explain the biological significance of the regulation of a large number of mRNAs by a single factor relates to a requirement for large-scale changes in a cell’s function. Under such a circumstance one might expect that the expression of a large number of mRNAs must be translationally repressed and/or degraded while a new group of genes is activated. For example, during the first 2 to 3 hours of degraded ,9,12 - tdegraded ,37 or ardegraded . Thus, SDrosophila embryo and a significant fraction of these mRNAs are both translationally repressed and destabilized. Smaug plays a major role in controlling the expression of mRNAs that are localized to the posterior of the embryo and regulates a diverse set of processes, including metabolism, lipid droplet function, protein folding and protein stability.Smaug directly regulates the expression of a large number of mRNAs in the early w1118 stock maintained in a large-scale Drosophila culture. smaug mutant alleles included smaug1[smaug47. The smaug47 allele was generated via imprecise excision of a P-element (GE21229) using standard methods [GE21229 is inserted 2,499 bp 5′ of the smaug start codon and 20 bp downsteam of the transcriptional start site of the smaug-RB isoform. All isoforms are defined as described at [smaug1 allele showed homozygous maternal effect lethality [smaug30 and smaug47, showed deletions removing large portions of the smaug gene, but not affecting the neighboring upstream and downstream genes - CG5087 and CG5280, respectively. Sequencing revealed that the smaug30 allele is a 4,514 bp deletion of the smaug gene beginning 2,480 bp 5′ of and ending 2,034 bp 3′ of the smaug start codon. Sequencing also showed that this allele retains 933 bp of the P-element. This deletion removes 2,020 of 2,997 bp of the open reading frame of smaug RA, RB, RC, and RE isoforms. The smaug47 allele is a 5,542 bp deletion beginning 2,483 bp 5′ of and ending 3,059 bp 3′ of the smaug start codon. This deletion leaves 39 bp of the open reading frame in the smaug RA, RB, RC, and RE isoforms.Wild-type flies consisted of the ed smaug1 and smau methods . GE21229ribed at . The oriethality  and we r2, 1 mM dithiothreitol (DTT), 1× protease inhibitor cocktail ). Extracts were centrifuged for 10 minutes at 4°C, and the supernatant was supplemented with 9 M urea to a final concentration of 2 M. Protein A beads were pre-incubated with either guinea pig anti-Smaug antibody [Embryos collected at 0 to 3 hours post-egglaying were dechorionated with 50% bleach and homogenized in a minimal volume of RIP lysis buffer  benzenesulfonyl fluoride hydrochloride (AEBSF), 2 μg/ml leupeptin, 2 mM benzamidine, 2 μg/ml pepstatin A). Lysed samples were diluted 1 in 12.5 in polysome lysis buffer and 30% triton was added to a final concentration of 1% and then spun at 6,000xg for 10 minutes and the resulting supernatant was diluted in polysome lysis buffer supplemented with 1% Triton to an A260 of 12.5.Embryos laid by wild-type or 2, 500 mM NaCl, 50 mM Tris pH 7.5 was created using a BioComp Model 117 Gradient Mate gradient maker  using a rotation angle of 80.5° and a rotation speed of 18 rpm for 1 minute and 58 seconds. After chilling the polysome gradient on ice, 400 μl of diluted embryo extract was loaded onto the top of the gradient, which was then spun at 36,000 rpm in a Beckman SW 41 Ti rotor for 2.5 hours. The gradients were then separated into four pools . A fixed amount of exogenous in vitro transcribed Arabidopsis spike-in RNAs was then added to each pool. Our microarrays contain probes that allow for the detection of these RNAs allowing for subsequent data normalization. We added 20% SDS, 0.5 M EDTA and 20 mg/ml proteinase K to each fraction to final concentrations of 0.8%, 0.01 M and 0.128 mg/ml, respectively, and then incubated them for 30 minutes at room temperature. Glycogen was then added to a final concentration of 80 μg/ml and samples were ethanol precipitated overnight and the resulting pellet was washed with 75% ethanol and resuspended in phenol-saturated water. Following two phenol-chloroform extractions, samples were precipitated by the addition of 7.5 M LiCl to a final concentration of 1.5 M and an overnight incubation at 4°C. The resulting pellet was washed with 75% ethanol, resuspended in water and ethanol precipitated in the presence of 80 μg/ml of glycogen and 0.3 M sodium acetate. The precipitate was then washed with 75% ethanol and resuspended in water. The integrity of RNA in each pool was confirmed via northern blots, which were probed for nanos mRNA . The lysed samples were split in half and cycloheximide was added to one sample to a final concentration of 0.5 mg/ml and puromycin was added to the other sample to a final concentration of 2 mM. Samples were left on ice for 20 minutes and then incubated at 30°C for 10 minutes. Both samples were then diluted 1 in 12.5 with polysome lysis buffer supplemented with either puromycin or cycloheximide and 30% triton was added to a final concentration of 1%. The samples were then spun at 6,000xg for 10 minutes and the supernatant was diluted with polysome lysis buffer supplemented with either puromycin or cycloheximide to give an A260 of 12.5 and these diluted samples were then fractionated as described above.For experiments that utilized puromycin embryos were lysed in puromycin lysis buffer  that contain probes for Arabidopsis spike-in RNAs (see below). Microarrays were scanned using Genepix Pro software on a Molecular Devices  GenePix 4000B or 4300A scanner and quantified using Nimblescan.RNA samples from RIP experiments were used to prepare single-stranded cDNA using anchored oligo(dT) primers and the Canadian iewed at . Anchore2 and the log2 using ‘two class unpaired analysis’ in SAM (FDR <5%).RIP microarrays were normalized using the Robust Multi-array Average (RMA) quantile method and transcripts that were expressed at levels significantly above background in total RNA collected 0 to 3 hours post-egglaying were determined using ‘one class unpaired analysis’ in SAM and transcripts with an FDR >5% were excluded from further analysis of the RIP data. mRNAs that were reproducibly enriched in Smaug RIPs versus control RIPs were then identified by comparing the logArabidopsis spike-in RNAs. The hybridization signals from the spike-in RNAs were utilized by applying a linear transformation to each sample with the parameters, a and b, determined by fitting the linear function Y = aX + b using the spike-in signal, where X is the expression level of the spike-in RNAs in a specific sample, and Y is the mean expression level of the spike-in RNAs across all the samples. The genes significantly expressed in wild-type or smaug-mutant embryos in each of pools 1, 2, 3 and 4 were separately determined using ‘one class unpaired analysis’ in SAM (FDR <5%). We defined the genes significantly expressed in the wild-type and smaug-mutant embryos as the union of the significantly expressed genes from the four fractions derived from that genotype. We then compared these two lists and defined their intersection as the list of genes significantly expressed in both wild-type and smaug-mutant embryos, and restricted all the following analysis to the genes on this list. To determine the list of genes with different polysome association in wild-type and smaug mutants, we compared the geometric mean of the expression level in pools 3 and 4  in wild-type and smaug-mutant embryos, using ‘two class unpaired analysis’ in SAM.Polysome microarrays were normalized using the RMA quantile method. We further normalized the data using . Levels of RpL32 mRNA in each immunoprecipitated sample were used to normalize the levels of the experimental mRNA in that sample.cDNA was synthesized using SuperScript II reverse transcriptase (Invitrogen) and random primers according to the manufacturer’s instructions. Quantitative PCR reactions were carried out using the BioRad  Real-time PCR system as per the manufacturer’s instructionssmaug-mutant and wild-type embryo samples for the top N and bottom N Smaug-binders  was calculated using the R (version 2.14.1) algorithm sm.density in the sm package (version 2.2-4.1). The sm.density algorithm provided smoothed density estimates for 100 values of change in TI for the top and bottom N binders, with the 100 values calculated by the sm.density algorithm with each smoothed density estimate.The fraction of genes expected to have changed in TI in N and bottom N Smaug-binders . First, for each gene, the density of its change in TI under the positive and negative distributions as defined by N top and bottom binders, respectively, was set to be equal to that of the closest grid point higher than the change in TI. We then estimated the probability that a gene was a positive by taking the ratio of its density under the positive distribution and the sum of its densities under the positive and negative distributions. This procedure was repeated for each of our three sets of positive and negative distributions to give us three different sets of probabilities. For each of these three sets of probabilities, we estimated the expected number of Smaug targets for that set by summing the ‘positive probabilities’ for all genes.For every gene expressed in our polysome gradient experiments, the probability that it was a positive target  was estimated using the top 0-4 on a non-specific stem. First, we performed an initial scan using RNAplfold (version 2.0.7) [et al.[We used a two-step procedure to computationally predict SRE stem/loops carrying the loop sequence CNGGNn 2.0.7)  with the) [et al.. Potenti) [et al. routine et al.[These analyses were carried out as described in Laver et al..Antibodies against Rpn7 , Su(z)12  and Bicasmaug-mutant embryos were collected from females homozygous for the smaug47 allele, while 'wild-type' embryos were collected from females homozygous for the smaug47 allele that were also homozygous for a genomic smaug rescue transgene that was inserted at the attP40 site on the second chromosome by Genetic Services  using PhiC31 integrase-mediated transgenesis [smaug transgene, which rescues the smaug mutant phenotype, is a modified version of a previously generated smaug rescue construct [For enzyme assays sgenesis . The smaonstruct  that exp2) and assayed in extraction buffer supplemented with 16.5 mM ATP, 20 mM beta-NADP and 0.67 M glucose. Hexokinase catalytic activity was measured by adding Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase  dissolved at a concentration of 300 IU/ml in extraction buffer. The production of beta-NADPH was monitored at 340 nm in a Thermo SPECTRONIC spectrophotometer. Experiments were conducted with an amount of embryo extract that was in the linear range of the assay and enzyme activity was normalized to protein concentrations in each homogenate measured using the Bradford assay (BioRad). Enzyme activity was calculated using the formula: Units/mg protein = ΔA340/minute ÷ 6.22 × mg enzyme/ml reaction mixture, as described by Worthington [For the hexokinase assay, embryos were homogenized in extraction buffer , which converts fructose-6-phosphate and ATP to fructose-diphosphate and ADP. The final product, NADH, reduces a colorless probe to a colored product with strong absorbance at 450 nm. The absorbance was measured with a TECAN INFINITE m200 microplate reader. Experiments were conducted with an amount of embryo extract that was in the linear range of the assay and enzyme activity was normalized to protein concentration.The data reported in this study have been deposited in NCBI’s GEO . The RIPAEBSF: 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride; AGO: Argonaute; bp: Base pair; CCT: Chaperonin containing TCP-1; DTT: Dithiothreitol; FDR: False discovery rate; GEO: Gene Expression Omnibus; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; miRNA: MicroRNA; RIP: RNA co-immunoprecipitation; RIP-Chip: RNA co-immunoprecipitations followed by microarray analysis; RMA: Robust Multi-array Average; RT-qPCR: Reverse transcription followed by quantitative polymerase chain reaction; SAM: Significance Analysis of Microarrays; SRE: Smaug recognition element; TI: Translation index; TRiC: TCP1-ring complex; UTR: Untranslated region.The authors declare that they have no competing interests.smaug30 and smaug47 alleles by MHKC; mRNA localization enrichment analysis by JDL; enzyme assays by ZY and construction of the smaug rescue transgene by NUS. CAS wrote the first draft of the manuscript with input from LC and JGD and the manuscript was revised by CAS and HDL with input from all other authors. All authors read and approved the final manuscript.HDL and CAS conceived of the project and designed its overall goals. LC and JDL are co-supervised by HDL and CAS; JGD and MHKC by CAS; XL by QM and HDL; ZY and NUS by HDL. All experiments and analyses were carried out by the indicated trainees under the supervision of their respective supervisors. All microarray experiments were conducted with advice of JTW; RIP-Chip and RT-qPCR validation was carried out by LC; polysome gradient-microarrays by JGD; RIP-Chip data analysis by LC with the assistance of XL; polysome gradient-microarray data were normalized by XL and JGD; subsequent polysome gradient-microarray data analyses and SRE-enrichment analyses were carried out by JGD with suggestions from QM; comparisons of binding, translation and stability data sets were carried out by JGD and LC; GO term analysis by LC; generation of the A table listing replicate-to-replicate comparisons of transcript microarray signal intensities from RIP-Chip experiments.Click here for fileA table listing the genes that encode Smaug-bound mRNAs .Click here for fileA figure showing northern analysis of the distribution of 18S ribosomal RNA in polysome gradients run with or without EDTA.Click here for fileComparison of wild-type TIs calculated with and without data from pool 2.Click here for fileComparison of the change in TIs in smaug-mutant versus wild type with and without data from pool 2.Click here for fileTables listing the replicate-to-replicate comparisons of transcript microarray signal intensities from wild-type polysome gradients, smaug-mutant polysome gradients, and smaug-mutant polysome gradients with or without puromycin.Click here for fileA table listing the genes that encode mRNAs that are translationally repressed by Smaug .Click here for fileA figure comparing the TIs of the bottom 250, 500 and 1,000 Smaug binders in wild-type and smaug-mutant embryos.Click here for fileA figure showing the kernel density plots comparing the change in TI in smaug-mutant versus wild-type embryos of the top and bottom 250, 500 and 1,000 Smaug binders.Click here for fileA figure showing the FDR-based rank of genes from the polysome gradient-microarrays.Click here for fileThe simultaneous overlap between the Smaug-bound mRNAs and those mRNAs that are regulated by Smaug at the level of translational repression and degradation.Click here for fileCumulative density plots that show the SRE scores for the 5′ UTR, open reading frame and 3′ UTR of Smaug-target mRNAs that are translationally repressed, degraded and both repressed and degraded.Click here for fileA table listing the Fly-FISH degradation categories and localization patterns enriched among Smaug-bound mRNAs.Click here for fileA figure showing the Fly-FISH degradation categories and localization patterns enriched among Smaug-bound mRNAs.Click here for fileA table listing the genes that encode Smaug-bound mRNAs that are localized to the posterior of the embryo.Click here for fileA table listing the genes that encode Smaug-bound mRNAs that are annotated as having roles in cell cycle, checkpoint response and/or response to DNA damage.Click here for fileA table listing the genes that encode Smaug-bound mRNAs annotated as having roles in transcription and/or chromatin.Click here for fileA table listing the genes that encode Smaug-bound mRNAs encoding proteins in the Chaperonin Cpn60/TCP-1 family.Click here for fileA table listing the genes that encode Smaug-bound mRNAs encoding proteins found in the proteasome regulatory particle and the ubiquitin proteasome pathway.Click here for fileA table listing the genes that encode Smaug-bound mRNAs encoding proteins associated with lipid droplets.Click here for fileA table listing the genes that encode Smaug-bound mRNAs encoding metabolic enzymes.Click here for fileA table listing the genes that encode Smaug-bound mRNAs encoding enzymes involved in glycolysis and related pathways.Click here for fileA northern blot that assesses the integrity of nanos mRNA in polysome gradient pools.Click here for file


According to the Yerkes–Dodson's law, the relationship between arousal and performance resembles an inverted U-shape curve with a moderate level of arousal being associated with an optimal level of performance  and are better at distinguishing the target stimuli from the distractors (α), in comparison with the low arousal condition. In addition, we have found that as the level of participants' arousal increases the capacity of their visual short-term memory (K) tends to decrease  signal to the visual cortex via reciprocal neural connections, thus ensuring that a threatening object in the visual field receives a greater allocation of attentional processing resources (e.g., Sugase et al., If arousal is capable of increasing attentional selectivity independently of any emotional manipulation, then why do van Steenbergen and colleagues find a difference between the positive and negative conditions in their study? (Previous studies by, e.g., McNamara and Fisch, In principle, then, arousal and attentional selectivity can be separated, although arousal serves to increase the effects of attentional selectivity. We propose that a general increase in cortical arousal modulated via brain arousal systems such as the RAS is sufficient to cause a narrowing of the attentional focus in line with the Easterbrook hypothesis. On top of this, however, the appearance of threatening stimuli in an otherwise neutral environment (cf. McNamara and Fisch, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Solanum lycopersicum) plants subjected to water stress as a model system, we standardized a sensitive and accurate protocol for detecting and quantifying DMSP pool sizes, and potential DMS emissions, in cryoextracted leaves. The method relies on the determination of DMS free and from DMSP pools before and after the alkaline hydrolysis via Headspace-Solid Phase Micro Extraction-Gas Chromatography-Mass Spectrometry (HS-SPME-GC-MS). We found a significant (2.5 time) increase of DMSP content in water-stressed leaves reflecting clear stress to the photosynthetic apparatus. We hypothesize that increased DMSP, and in turn DMS, in water-stressed leaves are produced by carbon sources other than direct photosynthesis, and function to protect plants either osmotically or as antioxidants. Finally, our results suggest that SPME is a powerful and suitable technique for the detection and quantification of biogenic gasses in trace amounts.Dimethylsulfoniopropionate (DMSP) and dimethyl sulphide (DMS) are compounds found mainly in marine phytoplankton and in some halophytic plants. DMS is a globally important biogenic volatile in regulating of global sulfur cycle and planetary albedo, whereas DMSP is involved in the maintenance of plant-environment homeostasis. Plants emit minute amounts of DMS compared to marine phytoplankton and there is a need for hypersensitive analytic techniques to enable its quantification in plants. Solid Phase Micro Extraction from Head Space (HS-SPME) is a simple, rapid, solvent-free and cost-effective extraction mode, which can be easily hyphenated with GC-MS for the analysis of volatile organic compounds. Using tomato ( Biogenic DMS (dimethyl sulfide), a compound mainly emitted by marine environment, is a significant natural source of tropospheric sulfur . DMS is Figure 1). Because methionine synthesis requires nitrogen and sulfur, it was shown that nitrogen availability affected the concentration of DMSP , the crop plant Saccharum (sugarcane), the wild mesophyte W. biflora, a moderately salt-tolerant species, and seven tropical species are reported to emit DMS  and unsuitability of sulfur containing volatiles for thermal desorption techniques  have hinSolanum lycopersicum), using HS-SPME-GC-MS, showed that tomato leaves emitted DMS upon alkaline hydrolysis of its leaf extracts (data not shown). Tomato is an important model species for VOCs analysis  were germinated in 0.7 dm3 pots filled with a mixture of peat:sand (1:1). Plants were grown in a growth chamber under controlled conditions , regularly watered to full pot capacity and fertilized once a week in order to supply mineral nutrients at free-access rates until the day of the imposition of water stress (potweight). Then, part of the plants (n = 4) was water-stressed by withholding water, whereas the other part (n = 4) was daily watered to pot capacity. As indicator of soil water availability, the FTSW /, where Dailypotweight is the weight of the water-stressed plants recorded during the water stress cycle and Finalpotweight is the “pot weight” at which stomatal conductance approached zero. DMSP analysis was performed at FTSW of 30% (FTSW30), when stomatal conductance (gs) decreased on average about 50% of the value FTSW100 plants.Seeds of a dwarf variety of r stress . The expthe FTSW  was calca fluorescence measurements were performed using a Li-6400 IRGA . A portion of a leaf was enclosed in a 2 cm2 cuvette, and exposed to a photosynthetic photon flux density of 700 μmol m-2 s-1, at a temperature of 25°C and with the relative humidity of the air within the cuvette ranging between 45 and 55%. By infrared gas analysis, steady-state net photosynthesis (A) and gs were measured. By chlorophyll fluorescence [a sensitive indicator of PSII photochemistry (= ΔF/Fs/Fs)/ and by comparing retention times and mass spectrum with those of an injected authentic standard, DMS standard was purchased by Sigma-Aldrich (Milano) and its assay was ≥98%.An Agilent 7820 GC-chromatograph equipped with a 5977A MSD with EI ionization operating at 70 eV was used for analysis. A three-phase DVB/Carboxen/PDMS 75-μm 2 cm SPME fiber  was exposed in the headspace of the vials at 40°C for 10 min for VOCs sampling. DMS has a boiling temperature of 37.34°C, so we used 40°C as SPME incubation temperature to favor the partitioning of DMS on the headspace for fiber’s uptake. The best extraction time was established to be 10 min, which afforded DMS uptake without causing fiber’s saturation phenomena as established in preliminary experiments. A Gerstel MPS2 XL autosampler equipped with a magnetic transportation adapter and a temperature controlled agitator (250 rpm with on/cycles of 10 s) was used for ensuring consistent SPME extraction conditions. This device ensured homogeneous sample mixing and favored the partitioning of VOCs into the headspace during SPME extraction. Chromatographic conditions were: column HP-Innowax ; injection temperature 250°C, splitless mode, oven program 40° for 1 min, rate of 5°C minFigure 2). Control analyses on specimens before hydrolysis, indicated that the amounts of free DMS eventually present was negligible as compared to DMS after hydrolysis . On the other hand, a comparison of the “free DMS” between well-watered and water-stressed plants revealed that “free DMS” in water-stressed leaves was significantly higher compared to that of well-watered leaves, albeit the amounts were negligible as compared to the DMS recovered after alkaline hydrolysis . The limit of detection (LOD), the limit of quantification (LOQ) were calculated as reported by The DMS standard was also used to quantify DMSP with a calibration curve in the range of 50–2000 pg  .Physiological data and DMS variations were tested using student’s A, ϕPSII and qP showed a slight, non-significant decline in well-watered plants (FTSW100), but gs (the indicator used to assess stress level) remained stable at FTSW100 (data not shown). As expected . Stomatal conductance decreased from 0.20 ± 0.20 to 0.09 ± 0.01 mmol s-1  , whereas A dropped from 11 ± 0.86 to the value of 4.9 ± 0.64 μmol m-2s-1 when the FTSW of water-stressed S. lycopersicum pots reached 30% compared to FTSW100 . Similarly, at FTSW30, ϕPSII  and qP  had a reduction of approximately 54 and 60%, respectively, compared to FTSW100 plants.During the time course of the experiment expected , all the-2), was determined by using the calibration curve of DMS .We have developed a sensitive and reliable methodology to detect DMSP and DMS in higher plants. The DMSP amount, expressed on a leaf area basis , and statistical analysis performed showed significant differences between the two treatments with a P < 0.01 .The DMSP content retrieved in FTSWIn general, DMS emission by plants has been elusive, and only occasionally reported . EmissioSpartina alterniflora  and 1025.29 (±96.27) pg, respectively. Consequently, the calibration curve obtained by SPME technique allowed estimating DMSP content in S. lycopersicum leaves with an accuracy, calculated in the highest levels of calibration range, of 97.7% (Table 1). SPME DVB/Carboxen/PDMS fibers efficiently captured DMS from the headspace vials at the selected experimental conditions allowing good reproducibility of results. A possible limitation of this technical approach is its application for quantitative analysis, because some phenomena  may occur along with the experiments and, as a consequence, alter the linearity of calibration response. On the other hand, fiber saturation phenomena can be eventually corrected by using suitable internal standards . This may of course represent a problem, if the amounts of DMS in samples are close to the LOD (32.2 pg), which is not our case. As shown in Table 1, at the lowest calibration levels, the accuracy of the methodology was not as good (49.7%) as at highest calibration levels (97.7%), despite an high precision along the of calibration range.The LOD, the LOQ, the accuracy and the precision of the calibration curve were reported in tandards . As an aThe Headspace-Solid Phase Microextraction (HS-SPME) technique used for assessing both DMSP content and the potential emission of DMS in tomato resulted highly effective in the detection and quantification of these compounds. DMSP and DMS are mostly found in marine ecosystems and in halophytic plants, where they serve a protective function against different abiotic stresses. In our knowledge, this study is the first to report DMSP and DMS in higher plants such as tomato. Using this novel, powerful methodology, we found a strong positive effect of water stress on DMSP content in tomato leaves experiencing an inhibition of their photosynthetic apparatus. These results suggest a potential functional role for DMSP and for its volatile product DMS in terrestrial vascular plants.SC, LC, and MC designed the research. SC and LC performed the experiment; SC, LC, and SK analyzed data; All authors contributed to write the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The plasma growth enlarged, over one hour, the nuclei to graphene sheets larger than one hundred nm2 in area. Characterization by electron and X-ray diffraction, high-resolution transmission electron microscopy images provide evidence for the presence of monolayer graphene sheets.In this paper, we demonstrate the seeded growth of graphene under a plasma chemical vapor deposition condition. First, we fabricate graphene nanopowders (~5 nm) by ball-milling commercial multi-wall carbon nanotubes. The graphene nanoparticles were subsequently subject to a direct current plasma generated in a 100 Torr 10%CH Graphene layers, as a result of weak van der Waals attraction, cause the material to adopt a stacked multilayer configuration24527910111291011121316Recently we have shown that multi-wall carbon nanotubes (MWNTs) are composed of AA′ stacked graphene helices where width is of the order of a few nanometers18XRD analysis of the samples provides more specific information as to the structural changes of the MWNTs following milling. The data showed that 002, 020, 004, and 200 peaks of orthorhombic AA′ graphene stacked MWNTs2 in area when the nanographene seeds were subject to a direct current plasma, as shown in the HRTEM image in 582 exhibiting layer edges are evident in The GNPs grew to form graphene plates of hundred nm45The broad 002 peak with the d-value of 3.60 Å of the milled samples  indicateμm (see Methods). The intensity ratio of 2D peak to G peak, I2D/IG, is about 0.62, and this value is similar to those for the graphene samples grown on Co or Ni (see Table 1 of Ref. The intersections (black arrows) in We propose a mechanism for the plasma growth of graphene via the lateral growth of the zigzag lines of graphene which subtend angles of 120°  with twenty steel balls (each 2 g) and processed by a conventional (Spex) milling apparatus for 1 ~ 6 hours. For the novel CVD growth, the purified GNPs which were prepared on a molybdenum substrate were subjected to a direct current plasma generated at 100 Torr of a 10%CH4-90%H2 gas mixture for I hour. The temperature of the substrate was kept at 1,200°C.Commercial MWNTs , prepared by VCCVD technique, were used as a starting material for the fabrication of graphene nanopowders (GNPs) to be served as nuclei for the seeded plasma growth of graphene. Diameters and lengths of the MWNT samples were 10–20 nm and 3–20 μm.The milled samples, i.e., GNPs were confirmed by HRTEM observation as well as X-ray diffraction pattern analysis. The GNPs including metal particles originated from the steel balls were purified through a solution treatment where the samples are dispersed in ethyl alcohol with sonication and the metal impurities are removed by magnets. HRTEM observation for grown graphene sheets were performed by a JEM-2100F operating at 200 kV and a FEI Titan Cubed with aberration corrector and a monochrometer operating at 80 kV. Raman analysis was performed by a Renishaw In-Via Raman Microscope with laser excitation of 532 nm and spot size of 1–2 J.L. designed the experiments and performed data analysis. S.L., Y.P. and K.L. carried out the experiments. Y.K. worked on XRD analysis of the materials. J.K. and B.M. worked on HRTEM observation. J.L. and P.J. co-wrote the paper.