The activated T cells can be suppressed by programed death-1 (PD-1) axis through low affinity interaction between PD-1 and PD-ligand 1 (PD-L1) in solution or on antigen presenting cells. In clinic, the concentration of soluble PD-L1 in peripheral blood negatively correlates with cancer prognosis. However, there is little information about the relation between the affinity of PD-1/PD-L1 interaction and the suppressive capacity of PD-1 axis. In this study, we analyzed inhibitory roles of high affinity soluble human PD-L1 (hPD-L1) variants, which were generated with directed molecular evolution. Resultant two clones L3C7-hPD-L1 and L3B3-hPD-L1 showed over 20 folds greater affinity than that of native hPD-L1. We found that L3B3-hPD-L1 and L3C7-hPD-L1 could compete with an anti-PD-1 antibody (EH12.1) for binding to hPD-1. More importantly, although native soluble hPD-L1 can induce suppressive effects on activated T cells, we found L3B3-hPD-L1 and L3C7-hPD-L1 attenuated the strength of PD-1 axis for suppressing the proliferation and interferon γ (IFN-γ) secretion of PBMC. In conclusion, our data provide direct evidence in which immune checkpoint receptor-ligand interactive strength can alter the the suppressive function, in particular, the suppressive capacity of PD-1 axis could be decreased with enhanced affinity of soluble PD-L1 and PD-1 interaction. Our study might provide a new direction for manipulating immune checkpoints. PDCD1 gene in human, which was upregulated in a T-cell hybridoma undergoing apoptotic cell death [PD-L1 gene encodes a putative type I transmembrane protein of 290 amino acids, which consists of an extracellular region, a hydrophobic transmembrane domain and a cytoplasmic tail of 30 amino acids [As retaining the function of induced long-lasting immune responses, particularly against infectious diseases and tumor, T cells are the essential and critical component in adaptive immunity. Once activated, however, T cells begin to express other receptors, which include those receptors of negative co-stimulatory molecules such as programmed cell death protein 1 (PD-1) that suppresses the immune response [ll death , 3. Overll death -6. PD-1 ll death . Two PD-ll death -10. PD-Lll death , 12. Theno acids .During T-cell activation, up-regulated PD-1 is translocated to dynamic T-cell receptor microclusters and accumulates at the signaling central supramolecular activation cluster (c-SMAC) . Src homKd of ∼8.2 µM), which is significantly weaker than that of CTLA-4/B7-1 complexes (Kd of ∼0.2 µM) and PD-1/PD-L2 complexes (Kd of ∼2.3 µM) [As critical regulators of T cell biology, the potent inhibitory signaling is mediated by interaction of PD-1 to PD-L1 (∼2.3 µM) , 25. It ∼2.3 µM) . However∼2.3 µM) -28. Desp∼2.3 µM) , no repo∼2.3 µM) . We are In this study, in order to explore how T cell status was regulated by the high affinity interaction of PD-1/PD-L1, we utilized directed molecular evolution and phage-display to obtain soluble hPD-L1 variants that bound to PD-1 with enhanced affinity. We found that the interaction between PD-1 and high affinity hPD-L1 variants could modulate the inhibition of TCR-CD3 complex signal. Our study would provide evidence for manipulating the immune-check point signals of the adaptive immune response through changing the biophysical characters of the receptor/ligand interactions.After dialyzing, refolded hPD-1 biotin fusion was purified by loading the dialysate to anion QHP column followed by elusion with sodium gradient. Non-reducing SDS-PAGE analysis revealed a single band, and the protein was collected at the conductivity of 0.55 mS/cm to 3.98 mS/cm (data not shown), and Figure Biotinylated hPD-1 was produced with biotin and BirA biotin ligase, and purified with gel-filtration column. The void volume time of the single band of biotinylated hPD-1 was at 18 min from the column (data not shown). Biotinylated hPD-1 could be separated from BirA biotin ligase by gel-filtration chromatography and no impurity was detected in a coomassie-stained SDS-PAGE gel Figure . Non-red-2 µM of the positive control antibody  [8 unique oligonucleotide sequences (data not shown), which were about 3-fold of the theoretical library diversity. After three rounds of bio-panning, polyclonal phage ELISA showed multiple clones having high readings, which indicated that the selection was successful, and L2 and L3 appeared to have higher signals than that of L1 and L4  , we rand 8.2 µM) . Using t 8.2 µM)  as the r4 Figure . We sequ4 Figure . Sequenc4 Figure .in vitro for three days, the dialysates containing soluble hPD-L1, L3B3-hPD-L1 or L3C7-hPD-L1 were loaded to anion QHP column respectively. Non-reducing SDS-PAGE analysis revealed a single band of hPD-L1, L3B3-hPD-L1 and L3C7-hPD-L1 were present respectively at the third peak (data not shown). The proteins were pooled and concentrated, and further purified with gel-filtration chromatography. Non-reducing SDS-PAGE analysis revealed a single band  assays at room temperature. For equilibrium analysis of affinity, increasing amounts of hPD-L1, L3B3-hPD-L1 and L3C7-hPD-L1 were injected respectively over immobilized biotinylated hPD-1. During the association phase, the bindings to hPD-1 of hPD-L1, L3B3-hPD-L1 and L3C7-hPD-L1 all reached equilibrium quickly. However, in the disassociation phase, hPD-L1 had a quicker off-rate in comparison with that of L3B3-hPD-L1 and L3C7-hPD-L1, and the kinetics of L3B3-hPD-L1 and L3C7-hPD-L1 were similar. The Kd values of 0.4994 µM, 0.75 µM and 17.66 µM were obtained for the binding of L3B3-hPD-L1, L3C7-hPD-L1 and hPD-L1 to hPD-1 respectively using a 1:1 binding model. The hPD-L1 variants L3B3-hPD-L1 and L3C7-hPD-L1 had ∼35 and ∼24 folds greater affinity for binding to hPD-1 in comparison to the hPD-L1/hPD-1 interaction  to bind hPD-1 on the cells. Our results showed that the affinity enhanced hPD-L1 variants L3B3-hPD-L1 and L3C7-hPD-L1 could inhibit the anti-hPD-1 antibody for binding to hPD-1 in a dose dependent manner, and the ICM Figure  and 2.3 M Figure  respectiM Figure . These rIt has been reported that the interaction between PD-L1.Ig and PD-1 inhibits anti-CD3-mediated T cell proliferation . In ordeIt is well established that PD-1 axis can suppress activated T cell to secrete IFN-γ . The effKd of ∼8.2 µM) in comparison to that of negative regulator CTLA-4 and B7-1 (Kd of ∼0.2 µM). On the other hand, the inhibition pathway of PD-1 provides opportunities to develop strategies to evade host immune surveillance for cancer and chronic infections.The activation and proliferation of T cells are necessary components of effective acquired immune responses. The T cell activation efficiency needs proper regulatory measures to inhibit immune hyper-activation, which leads to autoimmune diseases. The regulation of T cell activation and proliferation relate to the coordinated interaction of multiple signaling pathways. The PD-1 and PD-L1 interaction of PD-1 axis negatively regulates the T cell responses and maintains tolerance to self-antigens and limits inappropriate immune activities with much low affinity  revealed the contact interface involving total 14 residues of hPD-L1 . There iin vivo and in vitro previously, and we are trying to analyze the potential change of the biological function of high affinity hPD-L1 variants for suppressing T cell activation. Previous studies have demonstrated that PD-1 deficient mice developed autoimmune diseases [in vivo facts confirm the role of PD-1 axis in immune suppression. However, because different in vitro conditions used, the role of PD-L1 in the regulation of T cell responses seems to be complicated and is still under debate. Hideto Tamura et al. reported mPD-L1.Ig promoted the proliferation of T cells stimulated with a suboptimal dose of anti-mouse CD3 antibody, whilst both reagents were pre-coated on the 96 well plates [in vitro effect of the soluble hPD-L1 binding to T cell surface PD-1 conforms to the in vivo fashion of PD-1-axis function. In contrast our results demonstrated that L3B3-hPD-L1 and L3C7-hPD-L1 significantly attenuated the inhibition of PD-1 axis to the proliferation and IFN-γ secretion of PBMC.The biological functions of PD-L1 have been analyzed diseases , and PD-diseases , 18, 20.diseases . These il plates . Gordon l plates . Davide l plates . Haidongl plates . The difl plates . HoweverSH2D1A, which was thought to act as a negative regulator of immune cell activation, leaded to X-linked lymphoproliferative syndrome (XLP) [+ T cell model [Considering potential molecular mechanism of the reduced inhibition, we find that the cytoplasmic tail of CD150 has a paired of tyrosine-based motifs (TxYxxV/I), which bind to SH2-containing inositol phosphatase (SHIP) depended on the presence of the small adapter SH2D1A (SH2-domain-containing gene 7A), otherwise it will bind to SHP-2. The motif (TxYxxV/I) was designated as ITSM . The preme (XLP) , in contme (XLP) . This wame (XLP) . The cytme (XLP)  and a prll model  suggestell model . In ordeKd of 17.66 µM, which was resulted mainly from a quick off-rate , and ligated into the pET28a vector  which had a biotin tag gene for encoding tagged proteins and predigested by the identical restriction enzymes. The genes of hPD-L1 variants obtained from hPD-L1 phage libraries were amplified by PCR from the pGZ196 vector (in house construct), and cloned into the pET28a vector. The ligated products were transformed routinely into the competent E. coli strain BL21 (DE3) . Successful clones were confirmed with DNA sequencing.Cloning primers and synthetic genes encoding 33-150 residues of hPD-1 (NCBI: NM_005018.2) and 19-229 residues of human PD-L1 (GenBank: AY254342.1) were ordered from GenScript  for the expression in Transformed cells containing pET28a constructs were grown in Luria-Bertani (LB) medium supplemented with 50 µg/ml kanamycin overnight at 37 °C, and the proteins were expressed as inclusion bodies by induction for 4 h with 1 mM isopropylthiogalactosidase (IPTG) at the next day. The collection of cells and purification of inclusion bodies were executed as previously described . The purAfter filtration through a 0.45 µm membrane, the dialysis mixture was loaded to an anion QHP column ; bound proteins were eluted by sodium gradients of achieving 100% 1 M NaCl in 80 min produced with buffer of 10 mM Tris-HCl and 1 M NaCl, and collected and checked with SDS-PAGE at each peak. Refolded proteins were pooled and concentrated by ultrafiltration. For hPD-L1 and hPD-L1 variants, proteins were further loaded to Superdex™ 75 10/300GL gel filtration column  with PBS at pH 7.2; hPD-1 protein samples containing biotin tag were buffer exchanged with 10 mM Tris-HCl pH 8.0 for further process as following methods.The hPD-1 protein with biotin tag was biotinylated with a kit from Avidity  according to the manufacturer’s instructions. Briefly, the components of biotinylated reaction were hPD-1 with biotin tag as the substrate for BirA biotin ligase. The working concentration of protein substrate was adjusted to 40 µM at the final reaction mixture, which contained diluted 10-fold Biomix-A and Biomix-B, and 2.5 µg of BirA biotin ligase (per 10 nmol substrate) and incubated for 40 min at 30 °C. The completed reaction mixture was concentrated by ultrafiltration, and loaded to Superdex G-75 gel filtration column with PBS pH 7.2. The purity of biotinylated hPD-1 was determined by SDS-PAGE. The percentage of biotinylation of hPD-1 was evaluated using following methods. Streptavidin (SA) and biotinylated hPD-1 were mixed at 1:2, 1:4, and 1:8 molar ratios and incubated for 30 min at 37 °C, then the mixture was analyzed by SDS-PAGE to show the amount of the binding of biotinylated hPD-1 to SA.7. The PD-L1 variant fragments were cloned to fuse at 5’-end of the M13 gene three in pGZ196 vector at Nco I/Not I sites. After ligation, the DNA were purified and concentrated with a DNA purification kit  according to the manufacturer’s instructions. We electroporated 200 µl of TG1 competent cells  with 800 ng of pGZ196: hPD-L1 constructed DNA to make libraries.Seventeen PD-1 contact residues in hPD-L1 were determined using crystal structure of the mPD-1(mouse PD-1)/hPD-L1 (human PD-L1) complex (Protein Data Bank (PDB): 3SBW). In order to investigate if there was an optimal contact, identified contact residues were randomized with degenerate codons of NNK and overlapping PCR to build libraries, which were cloned into a phage display vector pGZ196 with the primers listed in Table E. Coli cells, followed by spreading on TYE plates supplemented with 100 µg/ml ampicillin and 2% glucose and growing overnight at 37 °C in an incubator.High affinity hPD-L1 variants were selected by bio-panning with the phage libraries on immobilized hPD-1 receptor. The selection methods were similar as those described previously . Briefly8 of M13 helper phages and incubated for 1 h at 37 °C. The cells were pelleted and re-suspended in 2×TY medium supplemented with 100 µg/ml ampicillin, then grown shaking overnight at 30 °C. The supernatant containing phages were obtained by spun down the cells at 1800 g for 10 min at 4 °C, and was used for screening high affinity binders.After the last round of selection, we assessed individual ampicillin-resistant colonies as described previously . BrieflyTo screen binders, we captured biotinylated hPD-1 on 96-well Nunc MaxiSorp plates pre-coated with streptavidin and non-specific protein binding surface was blocked with blocking buffer (PBS containing 3% skimmed milk) for 1 h. Phage supernatant pre-mixed with the blocking buffer was added to the 96-well plate at the same volume used for the biotinylated hPD-1 coated on the plate. The hPD-1 binding by phage displayed hPD-L1 variants was detected with horseradish-peroxidase (HRP)-conjugated anti-M13 antibody , and the antibody binding signals were revealed with TMB reaction buffer and read at 450 nm.The affinities of selected binders were ranked with inhibitive phage ELISA. Briefly, the phage displaying hPD-L1 variants were incubated with 1.6 µM non-biotinylated hPD-1 for 1 h before adding to the 96-well plate pre-treated as above. Binding phage remaining in the solution was detected as above.kon: association constant; koff: dissociation constant; Kd = koff/kon).The affinity was determined by Biacore T200 . A CM5 BIAcore chip was coated with streptavidin using amine coupling, then biotinylated hPD-1 was captured on the active channel. After blocking non-specific binding surface with 50 mM biotin, both the reference and active channels were injected hPD-L1 and its variants sequentially at various concentrations, and a channel injected with buffer alone was used as a blank control. The affinities were determined with the method of single-cycle kinetics. The data was fitted with a 1:1 binding model using Biacore T200 evaluation software to obtain the kinetics constants  at room temperature for 30 min. For indirect immunofluorescence, the cells were incubated with escalated concentrations  of biotinylated hPD-1 at room temperature for 30 min. The cells were washed twice with FACS buffer (PBS containing 2% FBS), and further incubated with SA-PE (BD Pharmingen) at room temperature for 30 min. SA-PE and/or anti-mouse IgG1 κ-PE-Cy™7 (BD Pharmingen) were used to stain the same number of A375 cells at the same conditions as negative controls for the flow cytometric analysis. Fluorescence was evaluated by FACS analysis using BD Accuri™ C6 within 4 h, and the data was analyzed using FlowJo 7.6 software .The A375 cells were purchased from the American Type Culture Collection , and grown in Dulbecco’s modified Eagle’s medium  supplemented with 10% heat-inactivated fetal bovine serum , and maintained at 37 °C in a humidified 5% COPeripheral blood from anonymous healthy donors were obtained from the Guangzhou blood center, and peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque gradient centrifugation.5 /well pre-stained PBMCs were cultured in 96-well flat-bottomed plates in a volume of 200 µl RPMI-1640 containing 10% FBS. The PBMCs were stimulated by adding the combination of OKT3  and anti-CD28 Ab  at the final concentrations of 50 ng/ml and 30 ng/ml respectively, and assays were carried out in the presence or absence (as a comparable control) of hPD-L1 , L3B3-hPD-L1 (30 µg/ml) or L3C7-hPD-L1 (30 µg/ml) respectively. The culture was incubated at 37 °C in a humidified 5% CO2 incubator for 6 days for duration of the whole assay. On day 3, recombinant human interleukin-2 (R&D systems) was added to a working concentration of 50 IU/ml.For proliferation assays, PBMCs were pre-stained with the final concentration of 1 µM carboxyfluorescein diacetate succinimidyl ester  as described previously , and 4×1For IFN-γ secretion assays, PBMCs without pre-stained were stimulated as above. The cells were added with 0.13 µl of BD GolgiStop™ per well and incubated for the last 6 hours of 48 h activation assays. Cells were collected and fixed/permeabilized with Cytofix/Cytoperm solution (BD Pharmingen) according to the manufacturer’s instructions, and stained with anti-human IFN-γ-PE (BD Pharmingen). Fluorescence was evaluated and the data was analyzed as above.6 cells/ml) were stimulated with 10 ng/ml phorbol 12-myristate 13-acetate  and 1 µg/ml ionomycin  in a volume of 200 µl RPMI-1640 containing 10% FBS at 37 °C in a humidified 5% CO2 incubator for 24 h. Cells were collected and washed twice with FACS buffer, and added anti-human PD-1-PE (BD Pharmingen) and hPD-L1, L3B3-hPD-L1 and L3C7-hPD-L1 respectively at serial concentrations  in the same time, and incubated in room temperature for 1 h. Samples added with anti-human PD-1-PE (BD Pharmingen) and anti-mouse IgG1κ-PE (BD Pharmingen) were considered as positive control and negative control respectively. Fluorescence was evaluated and the data was analyzed as above.To prepare activated T cells, freshly isolated human PBMCs . Percent suppression was calculated using the formula: percent T-cells responding in unsuppressed condition minus percent T-cells responding in suppressed condition divided by percent T-cells responding in unsuppressed condition×100. The data was expressed as the mean ± SEM. Unpaired student’s


The total reflectance of chemically-derived graphene on pyramidal Si is ~12% at the wavelength of 550 nm which is remarkably reduced compared with that of reduced graphene oxide on planar Si. By modifying the density and distribution of reduced graphene oxide flakes on Si, the power conversion efficiency of 5.20% is achieved. Additionally, the simulated absorbance of different-thick graphene is implemented to optimize the performance of graphene/pyramidal Si devices. The fabrication technique for rGO-based devices has the merits of simplicity, large scale, high throughput and low cost, which is a new starting point in the direction of graphene-based material for the applications of next generation optoelectronics.In the study, the chemically-derived reduced graphene oxide flakes on the pyramidal Si substrate to construct the heterojunction solar cells  Additionally, a PCE of ~8.6% was performed by using bis(trifluoromethanesulfonyl)-amide[((CF3SO2)2NH)] (TFSA) doped graphene. Unfortunately, the graphene films are typically grown by chemical vapor deposition (CVD) under high temperature and high vacuum conditionDeveloping cost-effective devices for energy storage and conversion are the major and urgent issue to overcome the global warming and climate change. Consequently, seeking a low-cost and high-efficient alternative structure in the form of heterojunction solar cells is in accordance with the roadmap of solar cells13567S) of GO/planar Si, GO/pyramidal Si, rGO/planar Si and rGO/pyramidal Si is measured by Hall effect measurement system. To confirm the quality of rGO, the Raman spectra are carried out with a laser excitation of 514 nm. The Fourier transform infrared spectroscopy (FTIR) measurement of GO and rGO are also taken to demonstrate the oxygen related groups derived from the intensive oxidation are eliminated. The reflectance and absorbance spectra of planar and pyramidal Si with rGO are characterized to prove the antireflection effect of the textured structure. The parameters of solar cells are measured by a Keithley 2400 source meter with an air mass (AM) 1.5 filter to generate simulated AM 1.5 G illumination. By optimizing the uniformity of rGO flakes on pyramidal Si, the PCE of 5.20% is achieved which shows a significant improvement compared with the rGO/planar Si solar cell of 2.96% in PCE. Additionally, the absorbance spectra of different-thickness graphene on pyramidal Si and the electric field intensity distribution are simulated by finite-differentiate time-domain (FDTD) method. By controlling the effective thickness of graphene flakes, the absorbance is modified, leading to an optimized performance of graphene-based devices. This research provides a technique for the fabrication of large-scale rGO films on textured silicon via cost-effective spin-coating process which gives an insight for the future applications of rGO optoelectronic devices.To improve the performance of rGO/Si solar cells, the formation of structured Si is one of the promising key to enhance the efficiency because the reflection of Si would be reduced and then the light path length can be increased. In this work, the chemically-derived rGO flakes spin-coated on planar and pyramidal Si substrates to construct rGO/Si heterojunction solar cells are realized. Morphologies of the planar and pyramidal Si solar cells with rGO flakes are observed by scanning electron microscopy (SEM) and atomic force microscopy (AFM). To ensure the rGO is reduced from graphene oxide (GO) and the resistivity of rGO on textured Si is similar to that of rGO on planar Si and, the sheet resistance  solution and then square-based pyramids on the crystalline Si wafer with a (100) orientation were formed due to anisotropic wet etching. The samples were then rinsed in deionized (DI) water, cleaned in hydrochloric acid (HCl) and hydrofluoric acid (HF) followed by rinsed in DI water. Prior to spin-coating graphene flakes on pyramidal Si, Al film and Ag bars served as bottom contact electrodes were deposited on Si. Subsequently, the native oxide film was removed using a diluted HF. The samples were then exposed to ambient air for oxygen passivation of dangling bonds to reduce surface states. To deposit graphene flakes suspension on top of solar cells, the chemically-derived GO and rGO was fabricated for mass production in the future. First, the GO was obtained by the modified Hummers’ methods15We deposited rGO on pyramidal n-Si wafer To demonstrate the rGO is reduced from GO and ensure the resistivity of rGO on different substrate is similar, the sheet resistance of GO/planar Si, GO/pyramidal Si, rGO/planar Si and rGO/pyramidal Si is measured. The sheet resistance of GO/planar Si and GO/pyramidal Si is around 4256 ~ 5534 Ω/square, while the sheet resistance of rGO/planar Si and rGO/pyramidal Si is significantly reduced to 88 ~ 300 Ω/square. The sheet resistance of GO either on planar or pyramidal Si substrate is expected to be higher than that of rGO17−1, G band at 1591 cm−1 and almost no 2D-band, respectively. After the reduction, the Raman spectrum of rGO flakes is characterized by D-band at 1351 cm−1, G band at 1587 cm−1 and 2D-band at 2691 cm−1, respectively. The G band is ascribed to the Stokes Raman scattering with the E2g phonon of sp2 C atoms and the presented D band is attributed from a breathing mode of ҡ-point photons of A1g symmetry19−1), the G band of the rGO sample is up-shifted due to the presence of isolated double bonds that resonate at the higher frequencies. In addition, the G band at 1587 cm−1 is close to that of pure graphite21−1 compared with 2D-band of GO also indicates the achievement of reduction. The decrease of D/G ratio after the reduction demonstrates that there are less oxygen related groups in rGO23The Raman spectrum of solution-processed GO and rGO flakes cast on the pyramidal Si with a 514 nm laser are measured to characterize the reduction degree of GO and the quality of rGO. The Ram−1, the stretching vibration band of C=O at 1731 cm−1, showing that there are abundant oxygen containing groups inserted into carbon skeleton. After the reduction, the intensities of the FTIR peaks related to the oxygen containing groups is reduced obviously and there is an appearance of a new peak at 1563 cm−1 owing to the skeletal vibration of graphene sheets, which reveals that these functional groups derived from the oxidation have been eliminated.To determine the antireflection effect of the textured Si, the reflectance  and abso25OC) of 0.41 V, a JSC of 18.26 mA/cm2 and a fill factor (FF) of 39.61%, which corresponds to a PCE of 2.96%. The parameters of VOC, JSC, FF and PCE for rGO/pyramidal Si are 0.44 V, 23.30 mA/cm2, 50.78% and 5.20%, respectively. The increase in VOC and JSC of rGO/pyramidal Si solar cell are resulted from the efficient antireflection effect and enhanced light path length, boosting the PCE significantly. In addition, this improvement can also be attributed to the lower series resistance and higher shunt resistance because of fewer wrinkle and stack of rGO on the surface of pyramidal Si compared with planar Si. Previous work on high-efficiency graphene/textured Si Schottky junction solar cells showed that the nanowire structure may induce severe recombination, leading to lower FF and PCF of the solar cellsThe chemically-derived rGO with high optical bandgap is beneficial for costly heterojunction solar cells. The photovoltaic parameters of the rGO/pyramidal and rGO/planar Si solar cells are extracted by the measurement of current density-voltage (J-V) curves under AM 1.5G illumination. As the light incident on the sample, the generated carriers are separated by the built-in electric field at the graphene/Si interface and the holes are pulled to the graphene. As seen in To further examine the spectral response of rGO on pyramidal and planar Si substrates, the external quantum efficiency (EQE) of graphene on pyramidal and planar Si substrates are characterized . Planar For a better study of the mechanism of graphene heterojunction solar cells, a two-dimensional FDTD simulation of the graphene/pyramidal Si structure under illumination is performed to calculate the absorption spectra  and the In conclusion, the field of graphene/Si heterojunction solar cells has rapidly developed to become the next-generation photovoltaics. To overcome the limited graphene size and the complicated transferring process, we propose a simple approach for constructing large-scale rGO on pyramidal Si through a cost-effective solution process. The pyramidal Si exhibits the superior antireflection effect, showing a substantial reduction in the reflectance of rGO/pyramidal Si compared with rGO on the planar Si substrate. Attributed to the uniform distribution of rGO on pyramidal Si, the performance of solar cell shows a promising PCE of 5.20%. Additionally, the simulated absorbance of different-thick graphene on pyramidal Si and the electric field intensity distribution are implanted with FDTD method to further optimize the characteristics of graphene/Si structure. The proposed approach and the analysis of solution-processed rGO flakes on large-scale and textured substrates have immense potential for graphene-based devices integrated into the future industrial production.How to cite this article: Lee, W.-C. et al. Fabrication and Analysis of Chemically-Derived Graphene/Pyramidal Si Heterojunction Solar Cells. Sci. Rep.7, 46478; doi: 10.1038/srep46478 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Biogenic volatile organic compounds (BVOCs) affect atmospheric chemistry, climate and regional air quality in terrestrial and marine atmospheres. Although isoprene is a major BVOC produced in vascular plants, and marine phototrophs release dimethyl sulfide (DMS), lakes have been widely ignored for their production. Here we demonstrate that oligotrophic Lake Constance, a model for north temperate deep lakes, emits both volatiles to the atmosphere. Depth profiles indicated that highest concentrations of isoprene and DMS were associated with the chlorophyll maximum, suggesting that their production is closely linked to phototrophic processes. Significant correlations of the concentration patterns with taxon-specific fluorescence data, and measurements from algal cultures confirmed the phototrophic production of isoprene and DMS. Diurnal fluctuations in lake isoprene suggested an unrecognised physiological role in environmental acclimation similar to the antioxidant function of isoprene that has been suggested for marine biota. Flux estimations demonstrated that lakes are a currently undocumented source of DMS and isoprene to the atmosphere. Lakes may be of increasing importance for their contribution of isoprene and DMS to the atmosphere in the arctic zone where lake area coverage is high but terrestrial sources of BVOCs are small. Although lakes are recognised as hot-spots for CO2 exchange and the release of methane3, freshwater biomes have received little attention for their total contribution to the atmospheric BVOC burden. Here, we demonstrate a flux of isoprene  and DMS ((CH3)2S) out of Lake Constance and suggest that oligotrophic lakes can be a source of these BVOCs to the overlying atmosphere. Our findings are of particular importance for our understanding of BVOC emissions at night and suggest that lakes may sustain a substantial flux to the atmosphere at high latitudes where lake area density is exceptionally high but terrestrial emission very low.Surface-to-atmosphere emissions of reactive BVOCs control the atmosphere’s oxidation capacity and secondary aerosol formation. These aerosols contribute considerably to the formation of particles affecting biogeochemical cycling, atmospheric processes, climate, and regional air quality in terrestrial6. In contrast, this gas is unreported in lakes despite the demonstration that heterotrophic bacteria7, marine cyanobacteria, phytoplankton and seaweeds also produce isoprene8. Two biosynthesis pathways exist for isoprene that result in isopentenyl diphosphate, the universal isoprenoid precursor. They are named after their key intermediate metabolites, mevalonate (MVA) and 2-C-methyl-d-erythritol 4-phosphate (MEP). Under low light heterotrophic growth conditions, several freshwater eukaryotic microalgae and a cyanobacterium differentially expressed one or both pathways9 but the production of isoprene by freshwater biota is undocumented and not represented in Earth system models.Isoprene comprises about a third of all BVOCs in the terrestrial atmosphere and is recognised for its function in the physiological acclimation in vascular plants10 and various physiological and ecological functions have been attributed to the production of this BVOC from its cellular precursor dimethylsulfoniopropionate (DMSP) in algae and bacteria11. These include cryoprotection, an overflow mechanism under unbalanced algal growth, as grazing deterrents, an antioxidant system that quenches reactive oxygen species10 or as chemical cues12. Molecular genetic evidence for various DMSP catabolic pathways that produce DMS exists for bacteria, fungi and algae14. DMS is also produced by trees and soils15 and in freshwater systems17. However, eutrophic lakes are suggested to be a minor source of DMS-sulfur to the atmosphere during periods of stratification since increased concentrations are associated with the anoxic hypolimnion16, likely as a result of microbial biomethylation of hydrogen sulfide17.Marine environments are a predominant source of DMS23 and such information has facilitated the estimation of the source strength of these climate-active BVOCs to the atmosphere25. A transect study from the North to South Atlantic21 indicated that isoprene and DMS do not correlate with concentrations of chlorophyll-a (chl-a) but positively correlate with the concentration of 19′-hexanoyloxyfucoxanthin, an accessory pigment occurring in the primarily marine haptophyte and some dinoflagellate algae, in areas characterised by low nitrogen concentrations. Limited information exists on the production and flux of DMS from lakes and freshwater sediments27 but similar data for isoprene is lacking. This shortage of ecosystem observations precludes the accurate estimation of global gas fluxes15.Concentrations and production rates of isoprene and DMS have previously been reported for estuarine and marine environments28. Isoprene and DMS are produced in response to oxidative stress from, for example, high light and temperature conditions in terrestrial plants (isoprene:29), phytoplankton  and air exposure in corals (DMS:32). Further evidence suggests that the strong relationship between isoprene and photoprotective carotenoids in marine phytoplankton could relate to a photoprotective function33 and that marine phytoplankton use DMS and/or isoprene to mitigate ROS-induced metabolic damage under sublethal environmental stresses6. Hence, it is possible that the production of these BVOCs also assists with physiological acclimation to environmental conditions in freshwater phytoplankton. To date this has been largely unexplored.BVOCs have important roles for the physiology of producers and consumers in aquatic food websThis study investigated the concentrations of isoprene and DMS in Lake Constance , 9.0 m on 16 July (4.3 µg L−1) and 4.6 and 8.3 m on 23 July (both 7.4 µg L−1). The majority of biomass from the surface to the chl-a maxima had optical characteristics of chromophytes  and chlorophytes (31 to 50%). Linear regression analysis indicated a significant positive correlation between BVOC and total chl-a concentrations , and between isoprene with chromophytes . Cryptophyte- and cyanobacteria-derived chl-a abundance was relatively low  and did not correlate significantly with trace gas concentrations (P > 0.05). The fluorescence data provide a basic indication that the production of both BVOCs is relatively wide-spread across the different algal taxonomic groups.Our weekly depth profiles showed a typical distribution of temperature and phytoplankton pigments in stratified lakes during summer with increasing stratification from 9–23 July 2013. We observed relatively high concentrations of isoprene (183 to 722 pM) and DMS (185 to 377 pM) associated with phototrophic processes in the epilimnion, which progressively deepened from approximately 4.5 to 8.3 m  to isoprene only , DMS only  or both isoprene and DMS production (Scenedesmus obliquus). This suggests that cryptophytes and cyanobacteria may have contributed to DMS and isoprene production in the lake despite the low abundance indicated by the optical profiler.The importance of phototrophic processes for the production of isoprene and DMS was confirmed by screening unialgal phytoplankton cultures of ten algal species from four algal classes. These measurements represent net rates resulting from the interplay between gross production and gross consumption processes in the alga and associated microbiota. After normalisation of our data to chl-es Table , we founes Table  that ran34, and terrestrial environments have no or negligible isoprene in the atmosphere during the night35. Aqueous isoprene level was significantly lower in the morning than in the afternoon , constraining the transfer of gases into the atmosphere during our investigation. Isoprene flux was relatively small at the beginning (07:47 h: 0.8 nmol m−2 h−1) and towards the end of our diurnal study (21:29 h: 1.2 nmol m−2 h−1). Highest fluxes were observed between 12:22 h and 15:43 h (11.1 to 14.6 nmol m−2 h−1). Isoprene fluxes were likely similar at Sites 1 and 2 since they showed similar surface concentrations  and were driven by the diurnal variation in wind speed that directly affects the gas-transfer velocity used in our calculations. Using chl-a concentrations for the epilimnion on 23 July (48 mg m−2 or mean of 6.2 ± 1.36 µg chl-a L−1 from surface to 8.3 m depth), we can further calculate a biomass-normalised maximum isoprene flux of 304 nmol [g chl-a]−1 h−1. Flux of DMS showed a similar pattern to isoprene and ranged from 0.8 nmol m−2 h−1 at 07:47 h to a maximum of 12.3 nmol m−2 h−1 (256 nmol [g chl-a]−1 h−1) at 13:34 h to 1.6 nmol m−2 h−1 at 21:29 h.Using air and water temperatures, and wind speeds Fig. , the con−2 s−1 35. Using a conservative estimate of the leaf area index (5.5 m2 m−2 for beech and oak)36, this hourly flux equates to 10 to 19 µmol m−2 h−1 based on the one-sided leaf surface area. Our data on atmospheric isoprene concentrations were similar  but flux from the lake (14.6 nmol m−2 h−1) was substantially lower than the flux from terrestrial vegetation. We also normalised the isoprene flux to depth-integrated chl-a for the lake epilimnion on 23 July, compared this with chl-a normalised terrestrial fluxes and find that these are about 80 to 160-fold higher than fluxes from the lake.We first compared isoprene concentrations and fluxes from the lake with measurements from a temperate mixed-deciduous forest of beech (48%), oak (44%) and birch (8%) at a location 416 km to the north-northeast of Lake Constance in July 2003. This is an example for a high isoprene-producing terrestrial environment in the northern European temperate zone where atmospheric isoprene concentrations ranged from near zero at night to about 3 ppb around noon indicating mean hourly fluxes from the terrestrial vegetation of 1 to 2 µg m37. Fluxes in Lake Constance are similar to typical fluxes from arctic tundra vegetation (9 to 39 nmol m−2 h−1)39. This raises the question whether arctic lakes may be a substantial source of isoprene to the local atmosphere, provided their production and resulting flux is similar to that of Lake Constance. Pelagic mean chl-a concentration in arctic lakes ranges from 0.3 to 5.6 µg chl-a L−1 40 but the typically shallow arctic lakes have large parts of the benthic sediments located in the euphotic zone. This provides a surface for growth of attached algae resulting in substantial epilithic chl-a concentrations (258 to 458 mg m−2) that generate 28 to 77% of total primary production in six arctic lakes41. Although pelagic chl-a was higher in Lake Constance (6.2 ± 1.36 µg chl-a L−1), its morphometry suggests that epilithic primary production was small and restricted to the immediate shoreline. Furthermore, the taxonomic composition of arctic and subarctic lakes is similar to oligotrophic temperate lakes with frequent domination by diatoms and chlorophytes, cryptophytes only temporally important in the seasonal succession and a low abundance of cyanobacteria45. This generally matches the taxonomic composition of oligotrophic Lake Constance based on the fluorescence characteristics from the optical profiler that showed a high abundance of chromophytes (including diatoms) and chlorophytes, and a lower abundance of cryptophytes and cyanobacteria  of 10 to 50%47, the relative importance of lakes in the release of isoprene to the atmosphere may exceed that of terrestrial sources in the arctic where lake area is large and terrestrial inputs are small. This suggests that, relative to terrestrial sources, lakes in cold-temperate and subarctic climates could add substantially to the local atmospheric isoprene burden.We then compared our measured fluxes with examples from low isoprene-producing terrestrial environments. The arctic tundra is relatively poorly vegetated and only 17–20% of plant species from cold environments produce isoprene48, we calculated an overall mean marine concentration of 30 pM . Typical marine fluxes range from 2.8 nmol m−2 h−1 in the Southern North Sea19 to 313 nmol m−2 h−1 in the Southern Indian Ocean49. These fluxes are strongly controlled by wind speed owing to the relatively small isoprene concentrations in the marine atmosphere and its relatively high concentrations in seawater34. In comparison to the marine examples, isoprene concentration in Lake Constance was higher and ranged from 183 to 722 pM in the epilimnion. Even at the relatively low wind speed during our study, high concentrations resulted in a substantial flux (maximum of 14.6 nmol m−2 h−1 around noon). Hence, similar to the marine example, Lake Constance was an important source of isoprene to the local atmosphere with an extrapolated emission of 59 moles (4 kg) of isoprene on the day of our diurnal measurements alone. Since aqueous isoprene concentrations can build up during periods of low wind speed when loss due to water-to-air transfer is limited, Lake Constance also provides a reservoir of isoprene. It is further possible that, depending on wind conditions, isoprene flux can be sustained into the night-time as indicated by the increased flux from the lake when light intensity was relatively low and wind speed temporarily increased from 19:00 to 20:30 h , and calculated an initial flux of 49.0 nmol m−2 h−1. Hence, the lake likely acts as an important source of night-time isoprene when terrestrial production ceases due to the strong light and temperature dependency of biological isoprene production4. This night-time release is unrecognised but of particular relevance since day-time isoprene is predominantly and rapidly oxidised (lifetime of few hours) by the light-generated hydroxyl radical (•OH), whilst isoprene emitted during the night will be mostly rapidly oxidised by the typically 100-fold more abundant nitrate radical . This should then impact the type, yield and fate of the isoprene-nitrates formed locally and consequently the NOX recycling, ozone and particle formation that may affect polluted urban atmospheres in the vicinity of lakes50.We then assessed our data against measurements from marine environments. Using recent reviews on marine isoprene0 h Fig.  and wind51 and impact on the night-time chemistry of the NO3 radical50. The transfer of DMS-sulfur into the atmosphere is estimated at 19.6 Tg per year25 which equates to a flux of 193 nmol m−2 h−1. As expected, the flux of DMS from Lake Constance (maximum of 12.3 nmol m−2 h−1) was lower than the marine flux and similar to the earlier estimates from Lake Kinneret that showed an estimated DMS-flux of 0.1 mmol m−2 month−1 52 and the mean flux from 10 Canadian lakes (7.1 nmol m−2 h−1) that, extrapolated to the Canadian boreal region, sustains an important 83% of biogenic sulfur in the atmosphere27.DMS is the largest natural source of sulfur in the remote marine atmosphere and, similar to isoprene, may play some role in the formation and growth of atmospheric aerosola]−1 h−1 and three produced DMS at 3.5 to 21.1 nmol [g chl-a]−1 h−1. The isoprene production rates in the algal cultures were lower than the calculated lake flux after normalisation to chl-a biomass (304 nmol [g chl-a]−1 h−1). This could indicate that important isoprene-producing taxa were excluded from our screening or that environmental conditions  can significantly affect isoprene production rates in freshwater algae. This supports the idea that light-stress may drive the production of freshwater isoprene since it is linked to photoprotection in marine algae33. Our data agree with net-production rates in 21 marine algal strains from 7 taxonomic groups that varied by two orders of magnitude between strains (30 to 1340 nmol [g chl-a]−1 h−1)8. This suggests that the physiological processes involved in the production of isoprene are fundamentally similar between marine and freshwater environments.Five of the phytoplankton cultures showed net-production rates for isoprene ranging from 12.6 to 174.3 nmol −1 h−1. This is about 4 times higher than the DMS-production rate in our culture of the freshwater cyanobacterium Aphanizomenon flos-aquae but 21-times higher than in the chlorophyte Chlorella vulgaris. Marine dinoflagellates are among the highest producers of DMSP and DMS55. For example, the dinoflagellate symbiont Symbiodinium sp. produces DMS at 20 to 107 µmol [g chl-a]−1 h−156, at least three orders of magnitude higher than the freshwater phytoplankton in our study.As far as we are aware, surprisingly little information on the rates of DMS production in algal cultures is available in the literature. The high DMS-producing marine haptophyte 60. Freshwater algae are recognised as a rich source of volatiles that are documented for their effects on drinking water quality61, and used as directional cues to find food in freshwater gastropods62, hence can affect food web structure and function12. It is timely and important to address the ecological and physiological relevance of isoprene and DMS in freshwater environments and assess their roles in the infochemistry and structuring of freshwater food webs.It is likely that isoprene and DMS are of ecological importance2), deep (zmax = 252 m), warm-monomictic, oligotrophic lake in south-western Germany at the northern fringe of the Alps  and included samples from the surface (0 m) and from a maximum depth of 60 m. This probe has been shown to resolve the distribution of the four different taxonomic groups of chromophytes , chlorophytes, cryptophytes, and cyanobacteria in laboratory cultures63 and lakes64 so that their abundances can be recorded based on fluorescence characteristics. For the quantification of discrete chlorophyll-a (chl-a) and organic carbon, samples were filtered immediately onto glass-fibre filters  and stored in a cool box before freezing filters at −20 °C at the Institute for subsequent analysis. For trace gas analysis, water was filled bubble-free into 250 mL gas-tight Winkler bottles (acid-washed and rinsed with ultrapure water prior to sampling) with a short length of silicone rubber tubing allowing for copious overflow before bottle closure. Samples were taken in analytical replicates (n = 3) and stored in a cool box equipped with several ice-packs before analysis of trace gases (n = 2 to 3) commenced ~1 hour after sampling.Water was collected from 6 depths (surface to 60 m) using a Niskin sampler at Site 1 at approximately 11:00 h on 9, 16 and 23 July 2013. Depths for discrete samples were selected based on −1 into the cryo-focussing apparatus to trap trace gases from the atmosphere as described below.Water was collected bubble-free using an inverted aspirator approximately every 1.5 h at Site 2 between 06:25 and 21:29 h on 25 July 2013. Water was transferred into gas-tight bottles as described above and analysis of trace gases commenced about 10 min later. Air samples were taken from outside the institute located in a rural setting approximately 80 m from the lake shore with an air intake at 7 m above the lake level by sucking air through a 10 m long 1/8 inch (3.2 mm) OD Teflon tube using a vacuum pump. Air was flushed for 10 min at 80 mL minAlgal cultures were aerated with compressed and filtered (0.2 µm pore size) air and grown under constant growth conditions using culture media depending on the cultures’ specific requirements Table . The cul0) and one bottle was immediately sacrificed for the quantification of isoprene and DMS. The other bottle was incubated under culture growth conditions and gases quantified at t1 after approximately 4 h. This was repeated twice using a staggered protocol resulting in 3 bottles each quantified for gases at t0 and t1. Treatments with significant difference to the controls  were considered for further analysis by subtracting control production rates and normalisation to culture chl-a and particulate organic carbon (POC) concentrations. It is important to note that previous incubation experiments with filtered seawater suggest that isoprene can also be produced at very low rates by photochemical processes with the bulk of this production controlled by ultraviolet light65. However, these experiments were affected by the presence of bacteria that could potentially lead to isoprene production from dissolved organic carbon. Furthermore, since we used borosilicate bottles and light derived from fluorescent tubes in our experiments photochemical production of isoprene was likely negligible during the incubations but small photochemical isoprene production may have added to the biological production processes at the lake surface.On the day of the experiment, duplicate glass bottles were filled with algal medium (controls) or culture (treatment) at time zero (ta determination after wet extraction in ethanol66. Particulate organic carbon (POC) was quantified with an EuroEA3000 elemental analyser  loaded with aliquots of the algal suspensions were used for photometric chl-67 while using best practices for sampling and storage68. Calibration stocks for aqueous measurements of isoprene and DMS were volumetrically prepared, and a commercially-sourced isoprene gas standard was used for the calibration of atmospheric isoprene measurements. For method details see Supporting Information.Gas chromatography with flame ionisation detection combined with a purpose-built purge-and-trap system for the cryogenic enrichment of BVOCs was used for the analysis of isoprene and DMS following established protocolsCw) and air (Ca) together with water temperature, air temperature and wind speeds measured at the Meteorological Station Konstanz , where k is the wind speed-dependent gas transfer velocity (cm hr−1)69, adjusted to the in situ Schmidt number70, and Hc is the Henry’s Law constant for isoprene (1.3 × 10−2 M atm−1)71. DMS flux calculations used the same approach and wind speed-based parametrisation of gas transfer velocity, but assumed Ca = 0 as atmospheric DMS levels were below the level of detection.Concentrations of isoprene in water  concentrations of 400 mg m−2 and chl-a/chl-b ratios in oak of 3.473.To compare the water-to-air flux with terrestrial flux estimates based on area or chl-Commercial software  was used for peak integration and data retrieval. We confirmed that test assumptions were met before conducting statistical analyses  in MS Excel version 14.The datasets generated and analysed during the current study are available from the corresponding author on reasonable request.Supplementary Information


Objective: Hypoxic exposure can be used as a therapeutic tool by inducing various cardiovascular, neuromuscular, and metabolic adaptations. Hypoxic conditioning strategies have been evaluated in patients with chronic diseases using either sustained (SH) or intermittent (IH) hypoxic sessions. Whether hypoxic conditioning via SH or IH may induce different physiological responses remains to be elucidated.Methods: Fourteen healthy active subjects  performed two interventions in a single blind, randomized cross-over design, starting with either 3 x SH (48 h apart), or 3 x IH (48 h apart), separated by a 2 week washout period. SH sessions consisted of breathing a gas mixture with reduced inspiratory oxygen fraction (FiO2), continuously adjusted to reach arterial oxygen saturations (SpO2) of 70–80% for 1 h. IH sessions consisted of 5 min with reduced FiO2 (SpO2 = 70–80%), followed by 3-min normoxia, repeated seven times. During the first (S1) and third (S3) sessions of each hypoxic intervention, cardiorespiratory parameters, and muscle and pre-frontal cortex oxygenation (near infrared spectroscopy) were assessed continuously.Results: Minute ventilation increased significantly during IH sessions (+2 ± 2 L·min−1) while heart rate increased during both SH (+11 ± 4 bpm) and IH (+13 ± 5 bpm) sessions. Arterial blood pressure increased during all hypoxic sessions, although baseline normoxic systolic blood pressure was reduced from S1 to S3 in IH only (−8 ± 11 mmHg). Muscle oxygenation decreased significantly during S3 but not S1, for both hypoxic interventions ; pre-frontal oxygenation decreased in S1 and S3, and to a greater extent in SH vs. IH (−13 ± 3% vs. −6 ± 6%). Heart rate variability indices indicated a significantly larger increase in sympathetic activity in SH vs. IH . From S1 to S3, further reduction in heart rate variability was observed in SH  while heart rate variability increased in IH (SDNN and RMSSD increase).Conclusions: These results showed significant differences in heart rate variability, blood pressure, and tissue oxygenation changes during short-term SH vs. IH conditioning interventions. Heart rate variability may provide useful information about the early adaptations induced by such intervention. Although chronic hypoxia is known to be an aggravating factor in several cardiovascular and respiratory diseases, hypoxic exposure may also be of significant benefits to one's cardiorespiratory status as demonstrated in animal models  < 0.08–0.10 and arterial oxygen saturation (SpO2) <70%, with short cycles <2 min], with intermediate doses  and patterns (intermittent hypoxia with >5-min cycles or sustained hypoxia) of hypoxia that can have positive cardiovascular and metabolic effects , the hypoxia-induced reduction in heart rate variability disappears almost immediately  in order to determine the most efficient strategy and to identify physiological biomarkers able to provide early assessment of each hypoxic conditioning program, with emphasis on distinguishing protective vs. deleterious responses. We hypothesized that IH conditioning would induce larger cardiorespiratory  benefits due to repetitive deoxygenation-reoxygenation cycles compared to SH conditioning.−2) were included in the present study after routine medical assessment consisting of a short clinical examination, a 12-lead electrocardiogram and a respiratory function test. All subjects were non-smokers and had no history of cardiorespiratory or neuromuscular disease. They lived at sea level  and were not exposed to altitude >1000 m over the past 3 months. Subjects were engaged 1–3 times a week in low- to moderate-intensity endurance activities such as walking, jogging, or cycling. They were instructed to keep their diet, hydration status, and physical activity routine identical throughout the study. Subjects refrained from physical exercise the days prior to the tests, abstained from drinking caffeinated beverages on test days, and had their last meal at least 2 h prior to the tests. The study was approved by the local ethics committee (CPP Sud-Est V) and performed according to the Declaration of Helsinki. Subjects were fully informed of the procedure and risks involved and gave their written consent prior to all assessments.Fourteen  active, healthy volunteers  to assess the early effects of this intervention , all participants performed first a full familiarization trial in normoxia including sitting and breathing quietly through a facemask, with continuous electrocardiogram and blood pressure measurements. Then, they were randomized (using block randomization) into either the sustained hypoxia (SH) or intermittent hypoxia (IH) condition. After performing the first hypoxic condition (three ~1-h sessions separated by 48 h), all subjects completed the second hypoxic condition (three ~1-h sessions separated by 48 h) after a minimum washout period of 15 days. Cardiorespiratory and NIRS signals were continuously recorded on session 1 (S1), and session 3 (S3) of each hypoxic condition (described below). On session 2, only SpO2 was individually adjusted to reach the targeted SpO2 of 70–80% in both hypoxic conditioning interventions . FiO2 from 0.21 to 0.10 over 10 min. Afterwards, FiO2 was automatically adjusted to reach 70–80% SpO2. This target SpO2 level was sustained until 55 min of hypoxic exposure. There was a final normoxic recovery phase where participants breathed ambient air for an additional 10 min, for a total testing duration of 70 min.SH sessions started with an initial phase of 5 min breathing normoxic, ambient air for baseline data collection. Then, the hypoxic phase began with a progressive decrease of FiO2 to reach the target SpO2of 70–80%) and 3-min normoxic breathing. The initial 5-min hypoxia was performed with a FiO2 of 0.10 for all subjects, and FiO2 was individually adjusted for the subsequent 5-min hypoxic phases in order to reach the target SpO2. After the seven hypoxia–normoxia cycles, 7 min of normoxic recovery were performed for a total session duration of 68 min.IH sessions consisted of a 5 min baseline breathing normoxic, ambient air, followed by seven cycles of 5-min hypoxia . Gas analyzers and volume transducers were calibrated prior to each test with a 3-L syringe and references gases, respectively, according to manufacturer's instructions. Blood pressure was measured on the dominant arm twice at each time point with a digital pressure monitor system . ECG was continuously recorded using a 2-lead ECG at a sampling rate of 1000 Hz .biceps brachialis using a 4-cm interoptode distance. Left pre-frontal cortex hemodynamics were assessed between Fp1 and F3 locations according to the international 10–20 EEG system with 3.5-cm inter-optode distance. The probe holders were secured to the skin with double-sided tape and maintained with Velcro bands. Data were recorded continuously at 10 Hz and filtered with a 2-s width moving Gaussian smoothing algorithm before analysis.Oxy[HbO2]-, deoxy[HHb]-, and total[HbTot]-hemoglobin concentration changes and tissue oxygenation index (TSI) were estimated continuously throughout testing sessions over multiple sites using a two-wavelength (780 and 850 nm) multichannel, continuous wave NIRS system . Muscle hemodynamics were assessed at the middle of right ECG data was transferred to the QuickReader software (Holter supplies) and R–R intervals were analyzed by means of the Kubios HRV Analysis Software 2.2 , gas exchanges, heart rate, SpO2, and NIRS parameters were averaged over the last 30 s of the baseline normoxic phase, of each 5-min periods in hypoxia and of the normoxic recovery phase. During IH sessions, the same parameters were averaged over the last 30 s of the baseline normoxic phase, of each 5-min hypoxic, and 3-min normoxic periods, and of the normoxic recovery phase. Arterial blood pressure was calculated as the average of two measurements at each of these time points. Heart rate variability parameters were calculated over the 5-min baseline normoxic phase, every 5 min during the hypoxic phase of SH sessions and during every 5-min hypoxic phases of IH sessions, and over the last 5 min of the normoxic recovery phase.During SH sessions, minute ventilation . Post-hoc Tukey's tests were applied to determine a difference between two mean values if the ANOVA revealed a significant main effect or interaction effect. For all statistical analyses, a two-tailed alpha level of 0.05 was used as the cut-off for significance. All data are presented as mean values ± SD within text and tables.All statistical procedures were completed on Statistica version 10 . Normality of distribution and homogeneity of variances of the main variables were confirmed using a Shapiro-Wilk normality test and the Levene's test, respectively. For each hypoxic condition (SH and IH), in order to assess changes over time within the sessions and changes from S1 to S3, a two-way ANOVA (time × session) with repeated measures was performed for each dependent variable . In order to assess differences between hypoxic conditions (SH vs. IH), a three-way ANOVA (time × session × condition) with repeated measures was performed for each dependent variable, using four time points only [baseline normoxia, min 25 (mid-session) and 60 (end-session) in SH and min 26 (mid-session) and 58 (end-session) in IH, and normoxic recovery). These four time points were selected in order to compare SH and IH under similar hypoxemic conditions, i.e., at mid and end hypoxic phase, when SpOSubjects reported no significant discomfort and no adverse effect was observed during all SH and IH sessions.2 and SpO2 during the hypoxic conditioning sessions are presented in Figure 2 was significantly reduced from baseline normoxic values throughout SH sessions  and during the hypoxic phases of IH sessions . FiO2 reduction magnitude was similar between S1 and S3 in both conditions  and was significantly larger at mid- and end-session in IH vs. SH  and during the hypoxic phases of IH sessions . SpO2 reduction  was similar between S1 and S3 in both conditions  and was significantly larger at the end of the session in SH vs. IH  but not during SH sessions . PetCO2 decreased significantly from baseline normoxic values during SH sessions both in S1 and S3  and during the hypoxic phases of IH sessions both in S1 and S3 . VE and PetCO2 changes did not differ between SH and IH at mid- and end-session  and during the hypoxic phases of IH sessions  both in S1 and S3. Heart rate increase was similar in SH and IH  while it increased during IH in S3 only . Baseline normoxic SBP was significantly reduced at S3 compared to S1 in IH (p = 0.009). Diastolic blood pressure (DBP) increased significantly from baseline normoxic values during SH and IH both in S1 and S3 . Changes in SBP and DBP did not differ between SH and IH conditions  and increased significantly in IH during both S1 and S3 . SDNN in IH was significantly larger during S3 compared to S1 . SDNN was significantly lower in SH compared to IH  and IH  sessions. PNN50 in SH decreased to a greater extent during S3 compared to S1 . PNN50 was significantly lower in SH compared to IH  and during both S1 and S3 in IH  sessions. RMSSD in SH decreased to a greater extent during S3 compared to S1  while in IH it was larger during S3 compared to S1 . RMSSD was significantly lower in SH compared to IH  and IH . HF decreased significantly during both S1 and S3 in SH  and IH  sessions. LF/HF increased significantly during both S1 and S3 in SH  but not in IH . LF/HF did not differ significantly between SH and IH  and IH  sessions. Muscle TSI reduction was similar between SH and IH conditions  but not HbO2  nor HbTot  showed larger changes in S3 compared to S1 in both SH and IH conditions. HbO2 reduction  and HHb increase  were larger during SH compared to IH conditions  and IH  sessions. Pre-frontal cortex HbO2 significantly decreased  whereas HHb significantly increased  during both S1 and S3 in SH and IH. Pre-frontal cortex HbTot did not change over time both in SH  and IH . Pre-frontal cortex TSI  and HbO2  reduction as well as HHb increase  were larger during SH compared to IH conditions .Changes in pre-frontal cortex TSI, HbOThe present study aimed to assess the acute and short-term (3 sessions) cardiorespiratory and tissue oxygenation responses to two hypoxic conditioning strategies, i.e., SH and IH, in order to determine their respective effects and potential physiological biomarkers able to detect early adaptations. During SH and IH sessions (acute effects), hyperventilation, tachycardia, increased blood pressure, and reduced heart rate variability were observed. SH sessions were associated with larger reduction in heart rate variability and greater muscle and prefrontal cortex deoxygenation compared to IH sessions. From S1 to S3 (short-term adaptations), IH reduced normoxic SBP and increased heart rate variability, SH sessions induced further reduction in heart rate variability, while both SH and IH sessions induced larger muscle deoxygenation. Hence, these results emphasize differences between SH and IH conditioning strategies regarding cardiovascular and tissue oxygenation responses and suggest some early adaptations after three hypoxic conditioning sessions only.E was observed during the 5-min hypoxic phases of IH sessions but not during SH sessions .Acute hypoxic exposure is also known to induce tachycardia as observed during both SH and IH sessions Figure , due to 2 (2–3% lower at mid- and end-session) in SH compared to IH sessions (Table 2 ~ 85%) vs. 2 h of IH (~2-min cycles with minimum SpO2 ~ 85%). The more severe reduction of heart rate variability during SH compared to IH observed in the present , possibly due to oxidative stress associated with repetitive deoxygenation–reoxygenation cycles , the present study demonstrates that (i) SH induces larger reductions in muscle (larger HbO2 and HHb but not TSI changes) and even more pre-frontal cortex  oxygenation compared to IH sessions ns Table . Of notens Table , 8, sugg2 = 0.12 . Several studies also suggested that in patients with hypertension, hypoxic conditioning programs can significantly reduce blood pressure [see for a review . Hence, the present study suggests that even in healthy young subjects with normal blood pressure and no cardiovascular risk factor, early improvements in blood pressure can occur after only three IH conditioning sessions. Improvements in blood pressure after hypoxic conditioning may be due to changes in the autonomic function (with augmented parasympathetic tone), increased endothelial NO production and enhanced vasoactive factors such as vascular endothelial growth factor  compared to SH  in young healthy subjects, i.e., an increase in nitric oxide synthases expression in circulating lymphocytes negatively correlated with blood pressure after three IH conditioning sessions only.While SH induced similar increase in blood pressure during S1 and S3, a significant increase in SBP was observed in IH during S3 only, due to a reduction in baseline normoxic value compared to S1 Figure  reported2 = 0.10–4 min FiO2 = 0.21  may require several sessions to be induced. The reasons for this reduction in muscle oxygenation triggered after several hypoxic conditioning sessions remain to be determined but larger muscle sympathetic nerve activity as observed by Lusina et al.  as usually performed to induce health benefits. The present study focused however on acute differences and early adaptations to hypoxic conditioning strategies in order to provide potential physiological biomarkers able to monitor the benefits and potential deleterious consequences of these hypoxic conditioning interventions. Females were included in the present study without taking account of potential effects of menstrual cycles on hypoxic responses. Although recent results indicate that the menstrual cycle may not affect hypoxic ventilatory responses for instance  and the potential physiological biomarkers that could be used during the first sessions in order to detect early adaptations. During SH sessions, larger homeostasis disruption seemed to occur compared to IH sessions, at least when considering the larger reduction in heart rate variability and greater tissue deoxygenation in SH compared to IH sessions Tables , 3. AlthThe present results showed significant differences in heart rate variability and tissue  oxygenation changes during acute SH vs. IH exposures in healthy young subjects. In addition, after three hypoxic conditioning sessions, IH only induced significant reduction in normoxic SBP while SH and IH induced opposite effects  on heart rate variability. Hence this study indicates that the pattern of hypoxic exposure  during hypoxic conditioning intervention should be carefully selected and suggests that heart rate variability may provide useful information about the early adaptations induced by such intervention.SC, PF, SD, and SV contributed to the conception and design of the work; SC, AB, SM, SB, PF, SD, and SV contributed to data acquisition, analysis, and interpretation; SC, AB, SM, SB, PF, SD, and SV drafted the work and revised it critically; SC, AB, SM, SB, PF, SD, and SV approved the final version to be published; SC, AB, SM, SB, PF, SD, and SV agree to be accountable for all aspects of the work and ensure that questions related to the accuracy and integrity of all parts of the work are appropriately investigated and resolved.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.


Two kinds of humidity-induced, bendable smart clothing have been designed to reversibly adapt their thermal insulation functionality. The first design mimics the pores in human skin, in which pre-cut flaps open to produce pores in Nafion sheets when humidity increases, as might occur during human sweating thus permitting air flow and reducing both the humidity level and the apparent temperature. Like the smart human sweating pores, the flaps can close automatically after the perspiration to keep the wearer warm. The second design involves thickness adjustable clothes by inserting the bent polymer sheets between two fabrics. As the humidity increases, the sheets become thinner, thus reducing the gap between the two fabrics to reduce the thermal insulation. The insulation layer can recover its original thickness upon humidity reduction to restore its warmth-preservation function. Such humidity sensitive smart polymer materials can be utilized to adjust personal comfort, and be effective in reducing energy consumption for building heating or cooling with numerous smart design. Around 48% of the building energy consumption in the United States is attributed to temperature control using heating, ventilation and air conditioning (HVAC) systems12357Toward this end, there is a tremendous effort to develop smart clothes which can automatically adapt to ambient temperatures, for example, using wearable electronic textiles91012141517143H). Upon humidity increase, Nafion can rapidly exchange H+ with water and formed a large number of water transport channels, leading to microphase separation between the hydrophobic back bone and the hydrophilic side chain groups and consequently the swelling242930In contrast to the insufficient temperature-based response of SMPs, a humidity-based shape change, which might be activated by human sweat, can be substantially more effective21222425273-). Nafion can easily exchange the H+ with water and form numerous water transport channels, leading to rapid swell to achieve the equilibrium. As the outer surface is exposed to the ambient environment with a lower humidity, the inner side of the Nafion flap swells more than the outer portion, creating a differential expansion that causes the flaps to bend upward and open up the pores. The steepness of the flex increases with the magnitude of the humidity gradient across the film, and is bounded by the maximal absorption and expansion levels of the material. To mimic human sweat pore functionality, Nafion films with flaps are designed as shown in The wetting and swelling behavior of Nafion film with a droplet of water on it is shown in Nafion-based humidity sensitive sheets are washable and possess long-term usability and reversibility. 3) as used in the simulation. The deformation contours of the flap  and the arch  matched very well with the simulation. As shown in To understand the mechanical response of above designs, finite element analysis (FEA) was conducted by using the ABAQUS standard package. 3 can fully flatten the arch , but for 10 mm sized arch, the needed humidity to fully flatten the arch is doubled. This can also attribute to the store elastic energy curve shown in As shown in In summary, we have designed two kinds of Nafion-based smart clothing structures triggered by humidity change, which can quickly and reversibly change its porosity or thermal insulation in response to an individual’s perspiration level. A sweating pore mimicking design consisting of a flap array structure on a Nafion sheet has been shown to respond to a humidity gradient and automatically opens or closes to regulate air flow through the pores, thereby achieving humidity and temperature control. A double layered, thickness-changeable structure with Nafion ribbons as an insert was also designed and demonstrated to adjust the air gap and alter the thermal insulation between two fabric layers. Both designs are sensitive to humidity and temperature changes and have been successfully demonstrated for reversible operations over numerous cycles. These human skin-mimicking structures work in an adaptive and repeatable manner. If designed into textile geometry, these humidity sensitive polymer structures will extend a wearer’s thermal comfort range and can contribute to energy saving of building HVAC systems.Nafion ® N117 with the thickness of 0.183 mm was purchased from DuPont. The specimens were dried and annealed at 130 °C before using.A rectangular array of elongated 2 mm × 10 mm flaps was cut into the film (100 mm × 100 mm) via razor blade with 4 mm horizontal pitch and 15 mm vertical pitch. (2) Nafion ribbons (10 mm × 40 mm) for the thickness adjustment design were molded into an arched shape with a height of 15 mm at temperature higher than its glass transition temperature (130 °C).3, which is one of the smallest commercialized humidity sensors to guarantee the accuracy of the targeted location.The humidity and temperature measurements were carried out using a digital humidity sensor SHTC1 (RH/T) (Sensirion Co. Ltd.), which can measure humidity and temperature simultaneously. The accuracy tolerance for its relative humidity and temperature measurement is ± 3%RH and ± 0.3 °C, respectively. The resolution is 0.01%RH and 0.01 °C, respectively. The dimension of the sensor is 2 × 2 × 0.75 mmAll the angle measurements were done by taking pictures with a camera with its lens positioning perpendicular to the substrate or the film. The pictures were then loaded into a photography software to draw a tangent line and measure its angle with the substrate or the original film. For each reported data point, the pictures were collected from at least five samples, and each picture was measured 5 times to get the average value to minimize the measurement error.The Nafion film was cut into 20 mm × 20 mm squares and immersed into water for 3 min and then dried at 70 °C repeatedly. The volume expansion ratio was obtained by measuring the new length, width and thickness of the film every 10 cycles and divide their product with the original one.The dynamic thermo-mechanical properties of Nafion film were carried out using a dynamic mechanical analyzer  with a heating rate of 10 °C/min and frequency of 2 Hz. The dimensions of the samples were 5.5 mm × 2.8 mm. Different samples were measured for different cycle times.Mechanical response finite element analysis (FEA) was conducted by using ABAQUS standard package. To replicate the confinement of film on the flap, one unit of the flap was modeled with 2 mm wide fringes at the root. All boundary displacements were fixed to zero at the root of the flap. In order to save computational cost, the arch structure was modeled to be 2-mm-wide half structure, with both symmetric boundary conditions along width directions and at the middle span of the arch. The bottom of the arch was set to be rolling-free in the horizontal direction. For the flap structure, displacement in X and Y direction were recorded at node A; for the arch structure, the displacement in X direction was recorded at F, and Y direction was recorded at D. For both designs, the strain was recorded at three different locations, on both top and bottom sides of the structure as A, B, C and D, E, F respectively for each design.3. The humidity of vapor was linearly increased on the bottom of the structure until reaching 92% relative humidity at 37 °C, which is equivalent to an absolute humidity 40 g/m3. According to Fick’s law, under steady state, a linear distribution of moisture along film thickness direction was assumed.Moisture boundary condition was applied at both top and bottom sides of these two structures. On top of the structure. An ambient humidity was applied, with 70% relative humidity at 23 °C, which is equivalent to an absolute humidity of 14 g/m3). According to mesh sensitivity analysis, the computational results converged when mesh size reduced to a size of 50 mm, leading to four layers of mesh along the thickness direction.Three different cases were calculated, with the length or height of model to be 100%, 75 and 50% of the original size. Linear-elastic model was adopted in the simulation. The elastic modulus and Poisson’s ratio were taken to be 275 MPa and 0.487, respectively. The isotropic uniaxial expansion ratio was set to be 0.002/.Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Arabidopsis thaliana, there are 127,854 introns in the nuclear genes; 99.23% of them are less than 1 kb, and only 16 introns are annotated to be larger than 5 kb, which are extremely large introns (ELI) in Arabidopsis. To learn whether these introns are true introns or not and how large introns could be in Arabidopsis, RT-PCR analysis of genes containing these ELIs were carried out. The results indicated that some of these putative introns are indeed ELIs. These ELIs are mainly composed of transposons or transposable elements (TE), excepting one, whose counterparts are also very long in diverse plant species. Thus, this study confirms the existence of introns larger than 5 kb or even 10 kb in Arabidopsis.Most of the eukaryotic genes contain introns, which are removed from the pre-RNA during RNA processing. In contrast to the introns in animals, which are usually several kilo base pairs (kb), those in plants generally are very small, which are mostly from dozens of base pairs (bp) to a few hundred bp. According to annotation version 10.0 of the genome of  Introns are a common and important feature of eukaryotic genes. It is the nucleotide sequence within a gene that is spliced out during the maturation of RNA. The splicing of RNA is a part of RNA processing pathway after the transcription and before the translation. Through RNA splicing, introns are removed and the exons are ligated to form the mature RNA product. Introns are found in the genes of most organisms and many viruses and exist in a wide range of genes, including protein coding messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA) . IntronsArabidopsis thaliana and maize indicated that their introns are substantially shorter than those in humans. The mean and median lengths of introns are 168 and 100 bp in Arabidopsis [Physcomitrella patens is 311 bp [Schizosaccharomyces pombe (93 bp on average), Drosophila melanogaster (564 bp on average), and Caenorhabditis elegans (467 bp on average) are also substantially shorter [The lengths of introns vary widely among different organisms and different genes. Generally, the sizes of introns in animal genes are larger than those in plant genes. For example, human genes tend to have small exons separated by long introns with the mean and median size of 3356 and 1023 bp, respectively [bidopsis . In maizArabidopsis have a length of several kb, which are very large and rare for Arabidopsis. Therefore, we wanted to know how large the introns in Arabidopsis can be. In this study, we systematically analyzed the sizes of introns in the genome of A. thaliana. We found that although 99.23% of the introns are shorter than 1 kb, a very small part of the introns are annotated or predicted to be larger than 5 kb, and in very rare case, introns are annotated to be larger than 10 kb. Because these introns are extremely large in Arabidopsis, we wanted to investigate whether they are true introns or not. The existence of some of them was validated by reverse transcription PCR (RT-PCR) analysis and DNA sequencing in this study, and sequences of these introns were also analyzed to reveal their origin and evolution. The implications of the results are also discussed.In our studies, we found that some of the introns in A. thaliana and the related information were retrieved from the BLAST data sets at TAIR website . Statistical analysis of the length of the introns was carried out via Microsoft Excel. Gene structure analysis of Arabidopsis genes was done manually and assisted with the information from TAIR and SALK T-DNA express . Primers from the exons before and after the annotated large intron were designed for RT-PCR analysis. Multiple protein sequence alignments were done with CLUSTAL W. To investigate the intron information in other species, BLAST against the genome sequences with Arabidopsis protein sequences was carried out with NCBI tBLASTN (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The lengths of the introns were calculated manually based on the boundary of intron/exon defined by the RT-PCR product and compared with the genomic sequence.Sequences of introns in A. thaliana plants used in this study were Columbia-0 (Col-0) and Landsberg (Ler) ecotypes. Seeds were sowed in soil or on Murashige and Skoog (MS) plates and then placed in the growth chamber under long-day conditions (16 h light/8 h of darkness) at 22 °C. Leaves of four-week-old plants grown in soil, floral tissues, and one-week-old seedlings grown on MS plates were used for RNA isolation.AT3G60961, AT1G58602, AT3G05410, and AT5G13250 were amplified with primers indicated in Total RNA was extracted by the RNeasy Plant Kit . For RT-PCR analysis, total RNA was reverse-transcribed with RevertAid First Strand cDNA Synthesis Kit . Complementary DNA (cDNA) of Arabidopsis genome, there are 127,854 introns in the nuclear genome. After analyzing the lengths of all the introns, we found that a large part (48.93%) of the introns is below 100 bp, and 50.30% of the introns are between 100 and 999 bp  of the total introns. Introns larger than 3 kb are very rare, with only 58 in the nuclear genome. There are 16 introns annotated to be larger than 5 kb, which we labeled as extremely large introns (ELI) in Arabidopsis in this study . It has multiple alternative splice variants. The second intron of AT3G05410.2 is 5748 bp long, and was validated by our RT-PCR analysis. This intron is mainly composed of AT3TE06550, which belongs to non-LTR retrotransposon family (LINE). The protein AT3G05410 and its closest homolog in Arabidopsis, AT3G55330, have an identity of 25% and an E value of 5 × 10−10 when a BLAST search was done at TAIR. However, it was shown to have an identity of 86% and an E value of 2 × 10−172 with its homolog in Raphanus sativus in a BLAST search at the National Center for Biotechnology Information (NCBI). This suggests AT3G05410 is a single copy gene in the genome of Arabidopsis and it is an essential gene for photosynthesis. The exon after this intron is not included in AT3G05410.1. However, the coding sequence of this exon is well conserved in different plant species  domain. FAF belongs toa family of proteins that regulate the size of the plant shoot meristem by modulating the CLV3-WUS feedback loop. Similar to At2G34100, AT5G22090 was annotated to have two splice variants that encode the same protein sequences. AT5G22090.2 has a large intron of 5081 bp , or full of TEs. It is well known that the genome of nsposons ,5,8. TraArabidopsis and other plants are usually small (shorter than a few hundred base pairs) [Introns in the genome of e pairs) ,5,6,9. Ie pairs) . Our rese pairs) ,12,13. IArabidopsis and other plant species [Arabidopsis . These mutants were once seen as null alleles, although it is conceivable that some of the insertions in the introns could abolish the splicing, our study also suggests that some of the introns with insertions of several kb length could be spliced out with little or no problem.Our results show that the insertion of transposons in the introns of some genes might have little or no effect on the splicing of the pre-mRNA. Mutants caused by transfer-DNA (T-DNA) and transposon insertions are widely used in the study of  species ,15,16,17