Anisotropic graphene domains are of significant interest since the electronic properties of pristine graphene strongly depend on its size, shape, and edge structures. In this work, considering that the growth of graphene domains is governable by the dynamics of the graphene-substrate interface during growth, we investigated the shape and defects of graphene domains grown on copper lattices with different indices by chemical vapor deposition of methane at either low pressure or atmospheric pressure. Computational modeling identified that the crystallographic orientation of copper strongly influences the shape of the graphene at low pressure, yet does not play a critical role at atmospheric pressure. Moreover, the defects that have been previously observed in the center of four-lobed graphene domains grown under low pressure conditions were demonstrated for the first time to be caused by a lattice mismatch between graphene and the copper substrate. For instance, graphene domains grown under LPCVD conditions typically has a dendritic four- or six-lobed structure, whereas under APCVD conditions hexagonal domains with six-fold symmetry are dominantly formed, with their edges macroscopically oriented parallel to the zigzag directionsIn this study, we investigated a correlation between the surface diffusion of carbon adatoms and the growth of anisotropic graphene domains synthesized using a CVD method. We varied the reaction pressure in CVD synthesis, which in turn caused variations in the growth mechanism and kinetics, and found that the crystallographic orientation of a copper substrate differently influences the shape and defects of graphene domains formed on it under LPCVD and APCVD conditions. We also calculated the energy barrier for surface diffusion of carbon adatoms on different Cu lattices, and propose a surface diffusion directed growth of anisotropic graphene domains on different Cu lattices with experimental observations.We synthesised graphene domains on Cu foil by CVD method under low and atmospheric pressures, using methane as the carbon precursor gas11−1) peak, and thus the ratio of the 2D (~2680 cm−1) to G (~1580 cm−1) peak intensity (I2D/IG) and D to G peak intensity (ID/IG) was determined for each of the graphene domains produced. Their spatial dependencies (Raman maps) are given in 2D/IG values are greater than 2.0. And, D/IG, in which the higher value under LPCVD (~0.4) than APCVD (~0.2) indicates a higher defects density in the former. This was attributed to the greater likelihood of Cu sublimation under low pressure, with recent reports suggesting that copper at 1000 °C in a vacuum has an evaporation rate approaching as high as 4 μm/h, while at higher pressures, sublimation of copper is suppressed18D/IG of ~0.7, with additional Raman maps of this region provided in the D/IG value in our case is higher than that of normal CVD graphene (~0.05). This is due to the fact that partial graphene growths have more Raman active edge19D/IG value of general graphene film in our system is in the Micro Raman maps were used to further identify the uniformity of the graphene domains produced. Typically, the presence of defects is indicated by the presence of an additional D  images were obtained and are shown in Computational modeling for the diffusion of carbon adatom on the Cu (001) and Cu (102) lattice orientations was used to further explore how the Cu lattice influences the shape of the four- and six-lobed graphene domains, respectively, at low pressure . In Fig.This change in the influence of the crystallographic orientation of Cu substrate on the anisotropic growth of graphene domains with reaction pressure is due to a variation in the pressure of carbon precursor gas, which provides an explanation as to why the Cu substrate does not always have the same effect. For example, in the case of LPCVD, carbon adatoms diffuse only along those directions with a low energy barrier, which these being determined by the crystallographic orientation of Cu substrate  lattice. That is, the defects produced by this symmetry mismatch provide an activation site for further reaction, with hydrogen in particular having been identified as both an activator for the formation of surface-bound carbon and an etching reagent that affects the shape of the graphene domainsIn summary, we have demonstrated that the crystallographic orientation of Cu substrate differently influences the shape and defects of graphene domains formed on it. Under low pressure, the growth of graphene domains is governed by the underlying Cu substrate, resulting in four- and six-lobed structures on Cu (001) and Cu (102) lattice, respectively. Experimental observations of such structures were corroborated by computational modeling, which identified the preferential diffusion route of carbon adatoms on each copper lattice. In contrast, hexagonal graphene domains were found to form over the entire Cu substrate under atmospheric pressure, suggesting that growth under these conditions is determined by the intrinsic symmetry in the atomic structure of the graphene. Based on these results, the holes and multilayered spots that have frequently observed in the center of four-lobed graphene domains in the past are considered to be due to defects originated from the lattice mismatch between the intrinsic six-fold symmetry of graphene and the preferential surface diffusion of carbon adatoms on Cu (001) lattice. We believe that our research on the surface diffusion directed graphene growth can greatly improve graphene-based engineering by allowing controlled alignment of individual graphene domains, in addition to providing greater insight into producing high-quality large area graphene for use in transparent conducting electrodes, sensors, and nano-electronic devices.2 pressure of 40 mTorr was kept with 2 sccm flow. Then, 35 sccm of CH4 was introduced in the tube for 10 minutes at a total pressure of 500 mTorr. After exposure to methane, the furnace was cooled to room temperature. The cooling rate was 20 °C/sec.Graphene was synthesized by copper-catalyzed chemical vapor deposition under low and atmospheric pressure, using methane as the carbon containing precursor gas. The Cu foil was loaded in the 1 inch quartz tube of tube furnace for annealing process. The tube was evacuated, back filled with hydrogen, and heated to 1000 °C. It was annealed for 1 h. For APCVD, a methane/argon gas mixture (50 ppm methane) of 300 sccm was allowed to flow across the copper at a temperature of 1000 °C, while maintaining 20 sccm hydrogen. The precursor supply time was 15 min during graphene growth. The sample was left to rapidly cool under a hydrogen and argon atmosphere. And, for LPCVD, the temperature was maintained to 1000 °C, and a H2/Si wafer for Raman spectroscopy. A thin layer of PMMA  was spin-coated on an as-synthesized sample at 3000 rpm for 1 min. Since both Cu surfaces were exposed to CH4, graphene was grown on both sides of the Cu foil. Excessive PMMA on the back side of PMMA coated graphene was removed by acetone. Subsequently, the sample was placed in a 0.1 M aqueous (NH4)2SO4 to etch off the Cu foil. Generally, the etching process runs overnight. After the Cu foil was completely etched away, graphene with PMMA coating was scooped out from the solution and transferred it into DI water for 10 min to wash away remaining etchants (done three times). Then, SiO2/Si wafer was dipped into water and the film was picked up. The PMMA was then removed with acetone and the sample was rinsed several times with DI water.After growth, graphene was transferred by a PMMA-assisted wet-transfer method on 300 nm SiOScanning electron microscopy (SEM) images of the synthesized graphene domains were obtained using a field-emission SEM system (JEOL 7600F) operated at acceleration voltages of 10 to 20 kV. Atomic force microscopy (AFM) and electron backscatter diffraction (EBSD) analyses were performed to identify the surface roughness and crystallographic nature of the Cu foil samples, respectively. Raman spectra of the synthesized graphene samples were obtained using a laboratory-made micro-Raman spectroscopy system; the system used the 514.5 nm line of an Ar ion laser as excitation source with a power of ~1 mW. The heating effect of the laser could be neglected at this power level. The laser beam was focused onto the graphene sample using a 50× microscope objective lens . The collected scattered light was dispersed using a Shamrock SR 303i spectrometer (1200 grooves/mm) and was detected using a charge-coupled device (CCD) detector.The obtained Cu crystal structures were optimized using density functional theory (DFT) calculations carried out with Dmol package included in materials studio. The calculation of total energy and electronic structure was followed by the geometry optimization using the generalized gradient approximation (GGA) with the Perdew–Burke–Ernzerhof (PBE) functional. The density functional semi-core pseudopotential (DSPP) was employed for the Cu atoms to take relativistic effects into account, while all electrons were considered for the carbon atoms. Further, using linear synchronous transit (LST) and quadratic synchronous transit (QST) methods, a new transition state (TS) search technique for stable configurations of carbon atom is developed, in which the stable configuration is independent on selected initial configurations. Double numerical atomic basis sets augmented with the polarization function (DNP) were used to describe the valence electrons. Two Cu surfaces were simulated using six layer with 54 atoms for (001) and eight layer with 72 atoms for Cu (102), respectively. Each system was sampled with nine (3×3 × 1) in-plane k-points and a vacuum of 10 Å.How to cite this article: Jung, D. H. et al. Surface Diffusion Directed Growth of Anisotropic Graphene Domains on Different Copper Lattices. Sci. Rep.6, 21136; doi: 10.1038/srep21136 (2016).


Dendropanax morbifera Léveille is used in herbal medicine as a cancer treatment. In this study, we investigated the effects of Dendropanax morbifera stem extract (DMS) on cadmium (Cd) excretion from the blood and kidney and brain tissues of rats exposed to cadmium, as well as the effects of DMS on oxidative stress and antioxidant levels in the hippocampus after Cd exposure.Seven-week-old Sprague-Dawley rats were exposed to 2 mg/kg of cadmium by intragastric gavage and were orally administered 100 mg/kg of DMS for 4 weeks. Animals were sacrificed and Cd determination was performed using inductively coupled plasma mass spectrometry. In addition, the effects of Cd and/or DMS on oxidative stress were assayed by measuring reactive oxygen species production, protein carbonyl modification, lipid peroxidation levels, and antioxidant levels in hippocampal homogenates.S-transferase (GST) activity in the hippocampus were significantly decreased after exposure to Cd, and administration of DMS significantly inhibited these Cd-induced changes.Exposure to Cd significantly increased Cd content in the blood, kidneys, and hippocampi. DMS treatment significantly reduced Cd content in the blood and kidneys, but not in the hippocampi. Exposure to Cd significantly increased reactive oxygen species production, protein carbonyl modification, lipid peroxidation, total sulfhydryl content, reduced glutathione content, and glutathione reductase activity. In contrast, Cu, Zn-superoxide dismutase (SOD1), catalase (CAT), glutathione peroxidase (GPx), and glutathione-S-transferase activities.These results indicate that DMS facilitates cadmium excretion from the kidneys, reduces cadmium-induced oxidative stress in the hippocampus, and modulates SOD1, CAT, GPx, and glutathione- Dendropanax morbifera Léveille is mainly distributed in southwestern Korea. This tree was initially used for the production of golden varnish [Dendropanax morbifera leaves have anti-complement, anti-diabetic, and anti-atherogenic activities [ varnish , but the varnish . The poltivities , 3–5.Cadmium (Cd) is one of the most harmful heavy metals in nature and it is released into the environment through mining and smelting. The main sources of Cd exposure include production of certain types of batteries, intake of contaminated food and water, and inhalation of tobacco smoke or polluted air , 7. Cd eDendropanax morbifera stem extract (DMS) decreases 2,2-diphenyl-1-picrylhydrazyl radical levels, and its antioxidant capacity is greater than that of other organ extracts from D. morbifera[D. morbifera, significantly decreases rotenone-induced generation of reactive oxygen species in SH-SY5Y cells and mitochondrial membrane potential depletion [Several approaches have been used to promote or facilitate the excretion of Cd from the body because it has a long biological half-life (approximately 30 years in humans) and readily accumulates in tissues . Debarkeorbifera. In addiepletion . HoweverMale Sprague-Dawley rats were purchased from Orient Bio Inc. . Rats were housed in a conventional animal facility at 23°C with 60% humidity, a 12 h/12 h light/dark cycle, and free access to food and tap water. The handling and care of the animals conformed to the guidelines established in order to comply with current international laws and policies , and were approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University (SNU-130522-1). All of the experiments were conducted with an effort to minimize the number of animals used and the suffering caused by the procedures used in the study.Dendropanax morbifera Léveille was purchased from a local market on Jeju Island in Korea. The plant was authenticated by two practitioners of traditional Asian medicine, and a voucher specimen was deposited with Egreen Co. Ltd. (deposition number: 2013-001). Stems from the plant samples (100 g) were chopped, blended, soaked in 2 L of 80% ethanol, and refluxed three times at 20°C for 2 h. The insoluble materials were removed by centrifugation at 10,000 × g for 30 min, and the resulting supernatant was concentrated and freeze-dried to yield a powder (yield: 4.7%). Before each experiment, the dried extract was dissolved in distilled and deionized water.Fresh n = 29 in each group): a control group, a group treated with 100 mg/kg DMS, a group treated with 2 mg/kg Cd, and a group treated with Cd and DMS. Cd and/or DMS were administered orally to 7-week-old rats once a day for 4 weeks.Cadmium chloride was obtained from Sigma-Aldrich . Animals were divided into 4 treatment groups (n = 7 in each group) were anesthetized with 100 mg/kg of Zoletil 50®  and the blood, hippocampi, and kidneys were extracted. Blood samples were allowed to clot, after which they were centrifuged for 30 min at 1,000 g to separate the serum. Hippocampi and kidneys were weighed in glass vessels, and tissues were digested by adding 3-8 mL of HNO3 at 130°C for 3 h, after which 2 mL of H2O2 was added and the samples were heated for 1 h. Serum and digested samples were transferred to polypropylene flasks for Cd determination. Cd determination was performed by using inductively coupled plasma mass spectrometry .To measure Cd concentration in the blood and brain and kidney tissues, control, DMS-, Cd-, and Cd-DMS-treated rats (n = 5 in each group) were assessed using the fluorescent probe 2′,7′-dichlorofluorescin diacetate (DCFH-DA) [The effects of Cd and/or DMS on ROS production in the hippocampus of control, DMS-, Cd-, and Cd-DMS-treated rats (DCFH-DA) . IntraceDCFH-DA) . MitochoDCFH-DA)  using bon = 5 in each group) were assessed by measuring malondialdehyde (MDA) formation using the Bioxytech MDA-586 kit . Briefly, bilateral hippocampi were collected using a surgical blade and the left part was homogenized in 20 mM PBS (pH 7.4) containing 5 mM butylated hydroxytoluene. After centrifugation of the homogenates at 3000 × g for 10 min at 4°C, the supernatants were collected. For each reaction, 10 μL of probucol and 640 μL of diluted R1 reagent (1:3 of methanol:N-methyl-2-phenylindole) were added and mixed with 150 μL of 12 N HCl. Each reaction was incubated at 45°C for 60 min and centrifuged at 10,000 × g for 10 min. The supernatant was collected and MDA formation was determined by measuring the absorbance at 586 nm. MDA data were normalized to the protein concentration of each sample.The effects of Cd and/or DMS on lipid peroxidation levels in control, DMS-, Cd-, and Cd-DMS-treated rats (n = 5 in each group) by the Levine method [3 L/mol cm), and results were expressed as nmol/mg of protein.To elucidate the effects of Cd and/or DMS on protein modification, carbonylation of proteins as a result of oxidative stress was determined in control, DMS-, Cd-, and Cd-DMS-treated rats (e method . The coln = 7 in each group). Briefly, the right part of the hippocampus (matching the right part used for measurement of lipid peroxidation) was homogenized in 10 mM Tris buffer containing 1 mM EDTA and 1 mM PMSF. The homogenates were centrifuged at 600 × g for 10 min, and then centrifuged at 13,000 × g for 20 min at 4°C.To elucidate the effects of Cd and/or DMS on Cu, Zn-superoxide dismutase 1 (SOD1), catalase (CAT), and glutathione peroxidase (GPx), the activity of these enzymes was measured in control, DMS-, Cd-, and Cd-DMS-treated rats . The gel was exposed to a fluorescent light source until the bands showed maximum resolution.SOD1 activity was measured by monitoring its capacity to inhibit the reduction of ferricytochrome ridovich . SOD1 acridovich . Briefly2O2 degradation in 10 mM of potassium phosphate buffer (pH 7.0) according to Aebi’s method [2O2 consumed per min and the specific activity is reported as units per mg of protein.CAT activity was assayed at 25°C by determining the rate of Hs method . An extit-butyl-hydroperoxide as a substrate according to the method of Maral et al. [GPx activity was assayed by measuring nicotinamide adenine dinucleotide phosphate (NADPH) oxidation with l et al. . Brieflyn = 5 in each group) were investigated using the tissue samples obtained for the measurement of protein carbonyl levels. Glutathione-S-transferase (GST) activity was determined spectrophotometrically using 1-chloro-2,4-dinitrobenzene as a substrate [The effects of Cd and/or DMS on glutathione-related enzymes in the hippocampus of control, DMS-, Cd-, and Cd-DMS-treated rats (ubstrate . Glutathubstrate .All data are expressed as mean ± standard error of the mean (SEM). To determine the effects of Cd and DMS, differences between the means were statistically analyzed using two-way analysis of variance (ANOVA) with repeated measures and Bonferroni’s post hoc test.The ICP-MS method was used to determine Cd concentrations in serum, kidney, and hippocampal tissues. Cd concentrations in serum, kidney, and hippocampal tissues were similar in the control and DMS-treated groups. In the Cd-treated group, Cd concentrations in the serum and kidney were significantly higher than those in the control group. In contrast, there was no significant difference in the Cd concentration in the hippocampus between the control and DMS-treated groups, although the level was increased 1.57-fold in the control group. In the Cd-DMS-treated group, Cd concentrations in the serum and kidneys were significantly lower than those in the Cd-treated group (Table DCF fluorescence levels were similar in the control and DMS-treated groups. DCF fluorescence was significantly increased 1.71-fold) in the Cd-treated group in comparison with that of the control group. However, the MDA level of the Cd-DMS-treated group was significantly decreased to 74.4% of that of the Cd-treated group  in comparison with that of the control group. The protein carbonyl level of the Cd-DMS-treated group was significantly decreased to 57.4% of that of the Cd-treated group Figure B.MDA levels in hippocampal homogenates were 25.8 and 25.4 nmol/mg protein in the control and DMS-treated groups, respectively. The MDA level of the Cd-treated group was 2.2-fold higher than that of the control group. However, the MDA level of the Cd-DMS-treated group was significantly lower (39.1 nmol/mg protein) than that of the Cd-treated group  level, and the activities of GR and GST were similar in the hippocampal homogenates. In the Cd-treated group, TSH level and GR activity were significantly decreased to 53.5% and 37.8% of those of the control group, respectively. In contrast, GST activity in the Cd-treated group was significantly increased to 147.7% of that of the control group. In the Cd-DMS-treated group, TSH level and GR activity were significantly increased to 172.9% and 174.7% of those of the control group, respectively. GST activity in the Cd-DMS-treated group was significantly decreased to 58.5% of that of the Cd-treated group (Figure Cd causes toxicity by disturbing the cellular homeostasis of essential metal ions such as copper and zinc. In addition, Cd affects antioxidant enzymes, which use copper and zinc, through oxidation or peroxidation in the brain . In contDendropanax morbifera Léveille significantly decreases rotenone-induced generation of reactive oxygen species in SH-SY5Y cells [Cd can affect the integrity and permeability of the vascular endothelium, penetrate the blood-brain barrier, and accumulate in the brain –28. In t5Y cells .In this study, we also determined the effects of DMS and/or Cd on the activity of SOD1, CAT, and GPx in the hippocampus because Cd has been shown to inhibit antioxidant enzymes –38. We fWe focused on glutathione-related enzymes because the increase in activity after DMS treatment in Cd-exposed rats was most prominent for GPx, among the antioxidative enzymes. Exposure to Cd significantly decreased GR activity, TSH level, and GSH level, while GST activity was significantly increased. The decrease of GR activity, TSH level, and GSH level after Cd exposure could be responsible for increased ROS levels and subsequent lipid peroxidation and protein carbonylation in the brain, because GSH can directly scavenge ROS or act as a substrate for GPx and GST in the detoxification of hydrogen peroxide . DMS admThe administration of DMS promotes the excretion of Cd from the kidney and ameliorates Cd-induced increases in ROS, lipid peroxidation, and protein carbonyl levels, which are modified by oxidative stress. In addition, DMS efficiently attenuates Cd-induced deficits in SOD1, CAT, GPx, and glutathione-related enzyme activities in the hippocampus.


Graphene, whose absorbance is approximately independent of wavelength, allows broadband light–matter interactions with ultrafast responses. The interband optical absorption of graphene can be saturated readily under strong excitation, thereby enabling scientists to exploit the photonic properties of graphene to realize ultrafast lasers. The evanescent field interaction scheme of the propagating light with graphene covered on a D-shaped fibre or microfibre has been employed extensively because of the nonblocking configuration. Obviously, most of the fibre surface is unused in these techniques. Here, we exploit a graphene-clad microfibre (GCM) saturable absorber in a mode-locked fibre laser for the generation of ultrafast pulses. The proposed all-surface technique can guarantee a higher efficiency of light–graphene interactions than the aforementioned techniques. Our GCM-based saturable absorber can generate ultrafast optical pulses within 1.5 μm. This saturable absorber is compatible with current fibre lasers and has many merits such as low saturation intensities, ultrafast recovery times, and wide wavelength ranges. The proposed saturable absorber will pave the way for graphene-based wideband photonics. Ultrafast lasers that produce pico- to femto-second optical pulses have many potential applications, such as in basic scientific research, materials processing, electronic components, metrology, telecommunication, and medicine1234678910111213et al. proposed using an ultrathin graphene sheet as an SA in an ultrafast fibre laseret al. incorporated graphene flakes into polyvinyl alcohol (PVA) and demonstrated the use of a graphene–PVA nanocomposite film as an SA1120212225Graphene, a single sheet of carbon atoms forming a honeycomb crystal lattice, exhibits a variety of exceptional electronic and photonic properties1516et al.To enhance the interaction of graphene with the evanescent field of propagating light, it is imperative to explore new operating schemes of graphene-based SAs with a nonblocking configuration. When a layer of graphene is wrapped around a microfibre , the allThe microfibre, with a minimum diameter of ~6 μm, is fabricated via a flame-brushing technique and integrated onto a hollow glass substrate, as shown in x0 is the position of the microfibre at the minimum diameter. The circles are the experimental data, which are fitted with a polynomial function of degree 4 (red solid curve). The polynomial equation that produces the best fit is D = 5.8465 − 0.02548 · Δx + 0.27112 · Δx2 − 4.54967 · 10−4 · Δx3 + 9.33575 · 10−4 · Δx4, where D is the diameter of the microfibre and Δx = x − x0 is the relative position of the microfibre. The minimum diameter of the GCM is approximately 6 μm. −1 exhibits a single Lorentzian feature with a narrow full width at half maximum (FWHM) of ~30.6 cm−1, and the intensity of the 2D band is much larger than that of the G band, with a 2D-to-G intensity ratio of ~3, as shown in 313234350), the nonsaturable absorption (αns), and the saturation intensity (Isat) are approximately 4.37%, 95.27%, and 1.51 GW/cm2, respectively.Pair/P) for the fundamental mode  along the diameter of the microfibre , respectively. The central wavelength of the guided wave is 1531.3 nm. The inset illustrates the cross-sectional intensity distribution in a 10-μm diameter microfibre. These figures were calculated using COMSOL. In the calculation, the peak power of the pulse wave is 200 W, which is consistent with the experimental results. One can see from D. For instance, the power densities are approximately 20, 3, and 0.038 MW/cm2 for D = 6, 10, and 30 μm, respectively. Therefore, the light–graphene interaction is enhanced significantly in the GCM via a tightly confined evanescent field guided along the surface of the microfibre. The energy fraction in the air, Pair/P, increases exponentially with decreasing D. For example, Pair/P ≈ 22.7% and 77.6% for D = 1 and 0.5 μm, respectively, as shown in A schematic of the GCM-based fibre laser is shown in λ, is 2.08 nm. The sidebands at 1527.28, 1528.81, and 1533.82 nm are typical of soliton-like pulse formations, which originate from intracavity periodic perturbations2-shaped fit. Assuming a sech2 temporal profile, the deconvolution yields a pulse duration of 1.21 ps. The time bandwidth product (TBP) is 0.322, which is near the value of the transform-limited sech2-shaped pulses. 6 contrast), indicating low-amplitude fluctuations and good mode-locking stabilityThe threshold pump power for CW lasing is approximately 5 mW. Self-starting mode-locking operation starts at the proper pump power when the polarization controller is adjusted appropriately. Once the stable laser is obtained, no further polarization controller adjustment is needed. D, is changed from ~1 to ~30 μm. We can see from D > 30 μm, the light–graphene interaction is so weak  that the mode locking fails. When D is less than 1 μm, a considerable fraction of the energy of the pulse is from the microfibre, in which case slight roughness on the surface of the microfibre can cause large losses. As a result, the loss that occurs in the GCM prevents the mode-locking operation of a laser.In the experiments, the average power of pulses in the laser cavity is ~0.45 mW, corresponding to a peak power of ~200 W. The experimental observations demonstrate that the proposed fibre laser can generate ultrafast pulses when the diameter of GCM, The experimental observations demonstrate that if the GCM-SA component is excluded from the proposed fibre laser, it fails to mode-lock; thus, the CW rather than ultrafast pulses is generated. Although the polarization effects can affect the characteristics of pulses, they hardly play such a role in emitting pulses. Both theoretical and experimental results indicate that the polarizer plays the key role in the nonlinear polarization rotation techniqueThe loss that occurs in the GCM is dependent on the diameter of the taper and the length of the graphene. In this work, the loss is approximately 1 dB. Although the taper fibre is not protected, the proposed laser can work well over the long term and deliver pulses consistently in our ultraclean laboratory. Outside the ultraclean laboratory environment, the taper fibre must be protected appropriately. Because the diameter of the taper is approximately 6 μm, the damage threshold is sufficiently high.3 solution for ~10 h. After the Cu is etched completely, transparent PMMA/graphene sheet floats on the surface of the solution. The PMMA/graphene sheet is then transferred into distilled water five times and is immersed for approximately 2 h each time to remove the remaining FeCl3 etchant. Subsequently, a microfibre, which is integrated onto a hollow glass substrate in advance, is placed under the centre of the PMMA/graphene sheet. When the microfibre is lifted together with the hollow substrate from the water, the PMMA/graphene spontaneously wraps around the microfibre , an autocorrelator, a 6-GHz oscilloscope, a radio-frequency (RF) analyser, and a 10-GHz photodetector are used to measure the laser output performance.How to cite this article: Liu, X. M. et al. Graphene-clad microfibre saturable absorber for ultrafast fibre lasers. Sci. Rep.6, 26024; doi: 10.1038/srep26024 (2016).


PD-L1 is an immune checkpoint molecule expressed by a variety of tumors, including ovarian, which binds to circulating PD-1 expressing effector T cells allowing for tumor escape from the immune system. PD-L1 blockade prevents PD-L1/PD-1 interaction and is currently explored as therapy of solid tumors. Ovarian cancer patients receive combination cisplatin/taxane chemotherapy as standard of care. Chemo-induced effects on tumor PD-L1 expression have been only partially addressed. We studied here the effect of platinum/taxane exposure on PD-L1 expression in vitro and in vivo.Human (OVCA 420 and OVCA432) and mouse (2F8) ovarian cancer cell lines were exposed to increasing doses of cisplatin and paclitaxel for different time periods. PD-L1 expression was analyzed with flow cytometry and Western blot. Through continuous exposure in vitro of mouse 2F8 ovarian cancer cells to increasing doses of cisplatin we have derived a new cisplatin-resistant line (2F8-Cis). In vivo, we have challenged n=37 mice IP with 0.8 million 2F8 cells. Tumor-bearing mice were treated with cisplatin, anti-PD-L1 antibody, both drugs, or isotype control every two weeks for three doses starting at day 14 post-inoculation. Tumor- and ascites-derived cancer cells were analyzed with flow cytometry.Exposure of OVCA420 and OVCA432 to cytotoxic doses of cisplatin or paclitaxel trigger PD-L1 up-regulation. Similarly, 2F8-Cis cells show increased cell surface PD-L1 compared to parental 2F8 cells, providing the rationale for combination therapy with PD-L1 blockade. In vivo treatment of mice with aggressive 2F8 tumors respond well to cisplatin and anti-PD-L1 individually with increased survival . At necropsy, anti-PD-L1 therapy significantly reduced tumor burden . Tumor cells cultured from cisplatin-only treated mice expressed higher levels of PD-L1, in line with our in vitro results. A higher percentage of PD-1 expressing cells were found amongst the tumor cells in these cultures versus cisplatin/anti-PD-L1 treated mice. Although high dose anti-PD-L1 immediately following cisplatin administration can control tumor burden (0.48 g), it does not significantly prolong survival (median 29 days). We are currently testing an alternative therapeutic schema exploring a lower anti-PD-L1 dose and a different timing post-chemo.Tumor cells upregulate PD-L1 in response to chemotherapy exposure and combination PD-L1 blockade in conjunction with chemotherapy effectively controls tumor burden. Optimization of timing and dosage for this combination therapy will likely increase its therapeutic benefit.


And we replaced the substrate with Ni/SiO2/Si and measured the thickness of Ni film by Rutherford backscattering spectrometry (RBS). Combined with suitable anneal conditions, these samples implanted by various small carbon clusters were made to grow graphene. Results from Raman spectrum reveal that few-layer graphene were obtained and discuss whether IG/I2D can contribute to explain the relationship between the number of graphene layers and cluster implantation dosage.Based on the extensive application of 2 × 1.7MV Tandetron accelerator, a low-energy cluster chamber has been built to explore for synthesizing graphene. Raman spectrum and atomic force microscopy (AFM) show that an amorphous carbon film in nanometer was deposited on the silicon by C For instance, the energetic ion-solid interaction was taken as a classic model to characterize some structure information of superconductor at room temperature or high K by projecting MeV ions to impact on superconductive targets [in situ characterization of these transients which can exhibit a clear physical image on changeable process of the structure was performed by the accelerator-transmission electron microscopy (TEM) interface system [In the past of several decades, ion beam analysis (IBA) based on low-energy accelerator has developed to be a comprehensive particle analytical discipline system -4. A fur targets . In ordee system ,8. For pe system ,10. In ae system .In Wuhan University, the double 1.7 MV Tandetron accelerator was inherited from Physical Institution of Chinese Academy of Sciences in 2004. After several important maintenances and upgrades of facility, some primary ion beam analysis with terminal voltage at 1.2 MV can be performed in a good state, such as Rutherford backscattering spectrometry (RBS), elastic recoil detection analysis (ERDA), and nuclear reaction analysis (NRA). Besides, we have developed some extensive applications, including accelerator-TEM interface system [As another kind of ultra-thin carbon film, graphene is a promising material which is probable to replace silicon integration technique due to its advanced and novel physical properties ,13. But −, C−, Si−, P−, Fe−, Cu−, and Au−[A source of negative ion by cesium sputtering (SNICS) can produce various negative ions from solid targets, such as B, and Au−,17, whic−) from SNICS at an energy of 20 keV are chosen for desirable targets by mass analyzer, then are decelerated to a few hundred electron volt or below 3 keV by the deceleration field after voltage scanner mounted on two aligned directions of X and Y-axis, finally to soft-land to the substrate. The current integrator is used for monitoring implantation dose simultaneously. To eliminate some impacts on the current integrator from high voltage at decelerated filed, an isolation transformer was introduced to guarantee safety. In addition, a rotated target holder  is about 5.10 nm for thin film, which indicates a great promise of preparing ultra-thin film under much lower energy ion implantation.Figure  edition . About 12/Si with about 300 nm Ni film deposited Ni atoms onto silica by e-beam evaporating.It is an essential purpose that we designed this low-energy cluster chamber for graphene preparation. In the process of exploring some effective methods, after depositing carbon films with the scale of several nanometers on the silicon, we selected suitable substrates to succeed in achieving few-layer graphene. Uninstalling the decelerated field, we selected small carbon cluster ions to inject to the substrate below 30 keV. The substrate Ni/SiO2+ which was produced by the double 1.7 MV tandem accelerator was collimated to the target with ion current of 5 nA and the round beam spot of 1.5 mm. The backscattered ions were detected by passivated implanted planar silicon (PIPS) detector with the resolution of 14 keV for α particle at 165°. The abscissa of spectra stands for channel numbers of multi-channel analyzer (MCA), which is proportional to the energy of scattered ions. A broad peak indicates that the surface edge of Ni is about channel 269 and the back edge is about channel 195. The channel difference of both edges is corresponding to the energy loss of projectile Li ions in Ni in correlation with the thickness of thin film. A straightforward route is simulating the trajectories of incident ions in matter. The red curve of this graph is simulation result from SIMNRA6.05 code, which is in coincidence with experimental data absolutely. The simulated results reveal that the areal density of Ni film is 2.1 × 1018 atoms/cm2, and a corresponding thickness is 227.3 nm in the case of the volume density at 8.9 g/cm3, which is thinner than the estimated value.The thickness of Ni film has influence on carbon segregation from inside up to the surface, so it is significant to evaluate the thickness of the substrate, and RBS spectra of the sample was carried out, as shown in Figure 15 cm−2 by ion implantation consisting of different cluster sizes at 20 keV. After implantation, these samples were annealed from room temperature to 900°C and dwell time was 60 min, then cooled down to room temperature naturally at 2.0 torr.With regard to depositing Ni film onto silica but not silicon substrate, it was reported that the silicon oxide at a thickness of 300 nm can enhance scattered signals of Raman resonance spectrum drastically because photon can evoke continuous interferences at the interface between Ni and silica . All the−1 is relative to the disorder and defects in the structures performing sp3 hybridization of carbon atoms, while sp2 hybridization induced by the in-plan optical phonon E2g near the first Brillouin Zone center is characterized as G peak at 1,580 cm−1[−1 of graphene is single and symmetrical to characterize monolayer. These samples were implanted with the same dosage of 8 × 1015 carbon atoms/cm2 at 20 keV by the different small carbon cluster sizes . Almost the three characteristic peak positions appear, and every peak position for different cluster sizes has also negligible shifts, as shown in Figure −1 and IG/I2D (the intensity ratio of G peak and 2D peak), which is the smaller and thinner film that can be obtained, were also evaluated to differentiate graphite and confirm the layers of graphene sheets [−1 in the spectra, corresponding to three and more layers. A visualized trend is observed that IG/I2D decreases as carbon cluster size increases, described in Figure 1 to C4, meanwhile larger clusters C4, C6, C8 are presenting a relatively gradual shrink. In the case of such low-energy ion implantation, light cluster can penetrate into deeper sites than heavy cluster in the substrate, which is dependent on the energy distribution of cascade collision between cluster and matter.Raman spectroscopy is always employed as one of the powerful non-destructive methods to identify graphene and determine the layer of graphene ,21. In t,580 cm−1. The 2D C1, C2, C, C6, C8.1 implantation instead of saving time. For example, implantation time is about 8.5 h for cluster C8 at 20 keV in this work, but the IG/I2D ratio is the smallest which indicates that the graphene quality is better than that in the other smaller cluster sizes.In previous studies, we have achieved some carbon cluster mass spectra at the different extractor voltages of sputtering ion source in this machine and paid much attention to investigate Raman characterizations of graphene by means of the carbon cluster ion implantation ,23. BecaE0 is cluster energy, and every atom of Cn cluster can be allocated as homogeneous energy of E0/n. Therefore, in comparison with C1, Cn (n > 1) has more sophisticated interactions with the substrate, involving in non-linear damage effect and atomic self-sputtering effect [15 cm−2 and 8 × 1015 cm−2 of carbon concentration on the surface are in agreement with monolayer and bilayer graphene [n (n > 10) under suitable energy is more likely to develop this process. But we have to take the atomic self-sputtering effect and more sophisticated cluster-matter interaction into consideration. More investigations are probable to promote the nucleation mechanism of graphene including ion-matter interaction, crystal quality of the substrate, anneal temperature, and other details about growth conditions.Simply, g effect ,25. Durig effect , no mattg effect ,15,20,214 cluster ions to the silicon at 14 keV, and the RMS is about 5.10 nm. Another substrates Ni/SiO2/Si whose thickness was measured at 227.3 nm by RBS were implanted with the same carbon atoms at 8 × 1015 cm−2 by several kinds of small clusters Cn ; after annealing, Raman spectra indicate that few-layer graphene was prepared successfully. And the ratio IG/I2D shows that the number of graphene layers cannot be controlled by implantation dosage purely but are associated with carbon atoms precipitation and segregation from inside to the surface grain boundaries of the substrate during thermal treatment. From ultra-thin carbon film to graphene by means of the similar cluster ion implantation technique, it is conductive for cluster implantation of light elements to develop low-energy shallow ion implantation in semiconductor industry.We have developed a low-energy cluster chamber on the base of extensive application for the double 1.7 MV Tandetron accelerator, which was used to explore for graphene synthesis. In our previous work, a kind of amorphous ultra-thin carbon film was fabricated by projecting CAFM: atomic force microscopy; ERDA: elastic recoil detection analysis; IBA: ion beam analysis; MCA: multi-channel analyzer; NRA: nuclear reaction analysis; RBS: Rutherford backscattering spectrometry; RMS: root mean square roughness; TEM: transmission electron microscopy.The authors declare that they have no competing interests.ZW designed parts of the experiments and sample preparations and drafted the manuscript. DF is the corresponding author and provided a great help for experimental designs. Other co-authors took part in sample preparation and characterizations and discussed the results. All authors have read and approved the final manuscript.


Tξ in the graphene layer.We report on gate-tuned locality of superconductivity-induced phase-coherent magnetoconductance oscillations in a graphene-based Andreev interferometer, consisting of a T-shaped graphene bar in contact with a superconducting Al loop. The conductance oscillations arose from the flux change through the superconducting Al loop, with gate-dependent Fraunhofer-type modulation of the envelope. We confirm a transitional change in the character of the pair coherence, between local and nonlocal, in the same device as the effective length-to-width ratio of the device was modulated by tuning the pair-coherence length  W) superconductor–normal-conductor–superconductor (SNS) proximity junction1x1 on the interface N/S1 is governed by sum of phase-coherent contributions from positions x2 along the opposite interface N/S2, located within the pair coherence length Tξ from x1. For an S1-to-S2 spacing L1 sufficiently shorter than Tξ, however, the pair transport depends on the phase difference between the same lateral positions (x1 = x2) only, revealing the local nature of pair coherenceCarrier transport in a mesoscopic system is governed by quantum mechanical interference over the spatial phase-coherence range where the interfering phase of carrier waves is determined by the nonlocal response to electromagnetic fields. This nonlocal nature also emerges for the transport of superconducting pair carriers in a laterally extended (width h/2e). The critical current periodicity of h/e was observed, employing separate extended Josephson junctions of channel length different L1/W ratiosdifferent physical geometryL1 and W of a junction changeFully nonlocal Josephson pair coupling is predicted to show twice the magnetic flux periodicity of the junction critical current for local pair interference4φl (> L1 > Tξ). In contrast to studies in the Josephson configuration, in existing pair-coherence studies using Andreev interferometry, the nonlocal character has been commonly observed1213Tξ marginally close to L1/2. To that end, we adopted an extended S-graphene-S junction consisting of a graphene normal-conductor (N) layer16Tξ as adopted in studies of graphene-based Josephson phenomena192021222324In an SNS proximity junction, the pair coherence can be examined more closely by employing the Andreev interferometry67891011121314Tξ in highly doped range (near the CNP) in graphene. We continuously adjusted the character of the superconductivity-induced pair coherence in a GAI by changing the effective aspect ratio of the junction along with gate-tuning Tξ in the graphene layer. All three GAI devices that we examined exhibited similar features and we present below the data for one particular device. Accurate tracing of the pair-coherence characteristics, in this study, was possible by utilising the highly stable gate tunability of both the carrier density and the corresponding Tξ in graphene. The Andreev interferometry adopting graphene offers a convenient and unique platform to investigate the locality of the pair coherence effect.Andreev interferometers adopted in this work consisted of an extended SNS junction with the graphene N layer. We observed Fraunhofer-like magnetoconductance (MC) modulation; with the local  character for a long (short) h/2e threading the area of loop and (ii) Fraunhofer-type envelop variations of the rapid conductance oscillations, attributed to the magnetic flux threading the closed carrier path in the phase-coherent region of the TGBh/2e  and h/e  along with the gate-dependent variation of Tξ. This cannot be due to a change in the flux focusing because the physical geometry of the junction did not vary with gating. The gradual shift of the field periodicity is a consequence of tuning the locality of the superconductivity-induced phase coherence represented by Tξ in a GAI.The GAI consisted of a T-shaped graphene bar (TGB) and an open Al superconducting loop, with the two ends of the Al loop in direct contact with the two arms of the TGB see . In the 2/Vs for a backgate voltage bgV of 50 V at a base temperature of 50 mK. The carrier motion in the TGB was diffusive  of the left (right) G/Al interface, while each attains the phase of the superconducting order at x1(2). These Andreev-reflected partial waves propagate back to P in a diffusive way to be recombined. The phase value at x1(2) can be expressed as ϕ10(20) is the phase at the center of the left (right) G/Al interface and x-axis direction.The superconductivity-mediated phase coherence of the carriers was established in the TGB between the two G/Al lateral interfaces. Suppose that an electron is scattered at position P in graphene into two electron-like partial waves. These waves are then Andreev-reflected as hole-like partial waves at different points ϕ = ϕ1 − ϕ2 = 2πΦ/Φ0, where Φ0 = h/2e is the flux quantum and Φ is the magnetic flux threading the area, A1 (≈13.3 μm2), enclosed by the centers of the Al-loop wires connected to the left and right G/Al lateral interfaces .The phase difference between the ends of the superconducting Al loop was controlled by an external magnetic field with fluxes threading loops (i) and (ii); this induced the above conductance oscillations with a modulated envelope. The total phase difference over loop (i) was Δterfaces . The areG as a function of the magnetic field B for bgV = 50 V at T = 50 mK, which resulted from combined contributions of the superconductivity-induced conductance enhancement and the conductance oscillation ΔG below the critical field of Al, cB = 10.5 mT. The conductance enhancement defined by (SG − NG)/NG was a consequence of the penetration of the Al superconducting order into the graphene at the G/Al interfaces. Here, SG and NG represent the differential conductance of the graphene in contact with the Al ring in the superconducting and normal states, respectively. The maximum of (SG − NG)/NG was ~ 0.18, where G (B = 0 mT) and G (B = 12 mT) were used as SG and NG, respectively. The phase coherence of the GAI over the length L2 was represented by the conductance oscillation of ΔG(B) . ΔG(B) consisted of fast h/2e oscillations in B in loop (i) was 0.158 mT, which is in good agreement with the value of 0.155 mT estimated from the GAI geometry  is about an order of magnitude larger than a previous report for a GAIG (~ 0.3e2/h), we obtained similar Fraunhofer behaviour (not shown) as the device with the large value of ΔG.etry see . In Fig.G oscillation in G(B) in h/2e, h/4e, and h/6e oscillations, which led to the field dependence of ΔG as G(B) [solid curve in i.e., the constructive interference of hole (electron) carriers implying the diffusion  of carriers with an increase (a decrease) of the conductance.The ΔR (≡ 1/G) as a function of bgV, where bgV = −19 V is the CNP. ΔG in graphene increased with the electron- or hole-like carrier density by positive or negative gating away from the CNP. The oscillation periods (ΔB) of the ΔG envelope for bgV = 50, −10, −19, −30, and −50 V were 2.85, ~5, ~6, ~5, and 2.95 mT, respectively. The significant variation in ΔB with the carrier density was caused by the change in the spatial distribution of the e–h phase coherence  at the G/Al lateral interfaces (described below), rather than by the change in A2. The weak gate dependence of the phase-coherent TGB area did not induce a significant change in A2  is the effective temperature of the carriers, and Fv is the Fermi velocity in graphene. Tξ was estimated using the relationship ΔG (B = 0) ~ exp(−L2/Tξ); ~320 nm for bgV = 50 V and decreased as bgV approached the CNP [see G (B = 0) remained finite at ~0.1e2/h at the CNP due to e–h puddle-induced fluctuations of the carrier density, which kept Tξ finite on the scale of the e–h puddle-induced density fluctuations . Assuming processes involving only a single Andreev reflection from each G/Al interface, the conductance oscillation is given byf can be approximated by a Gaussian-type weighting factor as x1 in a G/Al interface and x2 in the opposite-side interface. The parameter α simply controls the range of interference. For 2Tξ ≈ L1, the overlap of Tξ between x1 and x2 is weighted at x1 ≈ x2, thus, the magnitude of the e–h phase coherence at the overlapped region of Tξ is much larger than that at the other region and the corresponding effective range of interference is also localised , f ≈ δ(x1 − x2), which is 1 for x1 = x2 and 0 otherwise. For this local pair coherence, ΔG is simplified  becomes almost constant for all combinations of , leading to nonlocal pair coherence. In this case, an Andreev-reflected hole at a point x1 of the left G/Al interface interferes, with equal probability, with an Andreev-reflected hole at any value of point x2 of the right interface, and vice versaG is given by . Here, as ΔG is proportional to the square of the sine function, ΔG has a zero phase value for any magnetic field B  modulation has recently been observed in S-graphene-S-type Andreev interferometersB-field-dependent phase relationship. It was accomplished by varying the effective L1/W ratio continuously with fixed physical geometry of the devices along with changing the backgate voltages and the resulting Tξ in a given device. The local behaviour was confirmed by fine tuning Tξ marginally close to L1/2 in the highly doped range of graphene, while the nonlocal behaviour was obtained for Tξ much shorter than L1 close to the CNP of graphene. The close examination of the pair coherence characteristics was made possible using the highly stable and effective gate tunability of both the carrier concentration and the corresponding pair coherence length in graphene. GAI provides a convenient and unique platform to investigate the locality of the pair coherence.The locality of the pair coherence in a normal conductor in contact with superconductors is at the core of studies on the Josephson coupling in mesoscopic scales. We report the first Andreev-interferometry observation of the local behaviour of the pair coherence as well as the common nonlocal behaviour and a continuous tuning between them using GAIs, which was confirmed by the flux period of the Fraunhofer-type conductance variations and the contrasting L1 between the two G/Al lateral interfaces was 550 nm. The spacing L2 between the voltage leads B and C was ~0.8 μm. The Al loop was prepared by in-situ sequential deposition of a Ti/Al/Au (7 nm/70 nm/5 nm thick) tri-layer onto the prepatterned e-beam resist. The Ti and Au layers improved the contact at the G/Al interfaces and protected the Al surface from oxidation, respectively. The high transparency of the G/Al interfaces was confirmed by the almost vanishing (<0.1 Ω) contact resistance. With the interferometer mounted in a dilution fridge , a bias current I was injected into the interferometer in a symmetrical manner between leads A and D. The voltage difference V was monitored between leads B and C [A monolayer graphene sheet was exfoliated from a thin graphite flake onto a heavily electron-doped Si substrate capped with a 300-nm-thick oxidation layer. The Si substrate was used as a backgate to modulate the carrier density and types in the graphene. An open-square Al loop of 400 nm in width was directly coupled to the graphene by electron (e)-beam patterning, e-gun deposition, and the lift-off technique. The graphene layer was patterned using e-beam lithography and oxygen-plasma etching into a T-shaped structure having a linewidth of 500 nm. The spacing  B and C  using a M.K., D.J. and H.-J.L. designed the experiments. M.K. prepared the samples and performed measurements. D.J. and G.-H.L. assisted in measurements. M.K., G.-H.L., Y.-S.S. and H.-J.L. analysed the data, H.-W.L. provided the theoretical consultation and H.-J.L. supervised the project. M.K., Y.-S.S. and H.-J.L. wrote the manuscript. All authors contributed to the discussion and reviewed the manuscript.Tuning Locality of Pair Coherence in Graphene-based Andreev Interferometers


Contextual elements have significant impact on uptake of health care innovations. While existing conceptual frameworks in implementation science suggest contextual elements interact with each other, little research has described how this might look in practice. To bridge this gap, this study identifies the interconnected patterns among contextual elements that influence uptake of an anticoagulation clinic improvement initiative.a priori approach based on the Promoting Action on Research Implementation in Health Services (PARIHS) model and an emergent thematic analysis.We completed 51 semi-structured interviews and ethnographic observations across five case study sites involved in an evidence-based practice (EBP) quality improvement initiative. We analyzed data in NVivo 10 using an Key contextual elements, such as leadership, teamwork, and communication, interacted with each other in contributing to site-level uptake of the EBP, often yielding results that could not be predicted by looking at just one of these elements alone. Sites with context conducive to change in these areas predictably had high uptake, while sites with uniformly weak contextual elements had low uptake. Most sites presented a mixed picture, with contextual elements being strongly supportive of change in some areas and weak or moderate in others. In some cases, we found that sites with strong context in at least one area only needed to have adequate context in other areas to yield high uptake. At other sites, weak context in just one area had the potential to contribute to low uptake, despite countervailing strengths. Even a site with positive views of EBPs could not succeed when context was weak.Interrelationships among different contextual elements can act as barriers to uptake at some sites and as facilitators at others. Accounting for interconnections among elements enables PARIHS to more fully describe the determinants of successful implementation as they operate in real-world settings. The uptake of evidence-based practices (EBPs) and quality improvement (QI) interventions can be notoriously challenging. The effort to implement EBPs entails attention not only to designing an appropriate intervention, but also to the dynamic organizational context in which the intervention is to be implemented. Established implementation science (IS) work on barriers and facilitators to uptake has proven useful in real-world implementation efforts, especially in regard to identifying individual contextual elements and noting ways these singular contextual components influence the implementation of health care innovations -6.Implementation researchers anticipate that when a majority of contextual elements are conducive to change, implementation is usually successful. In contrast, when a majority of contextual elements are not conducive to change, implementation is not successful. However, in many cases where EBPs or QI projects are implemented, there is a more mixed, intermediate picture that can produce interesting, yet unpredictable, patterns of uptake. Our study identifies and describes a range of such situations at five sites with a “mixed” picture of contextual elements. Therefore in this study we discuss more broadly how interactions of contextual elements impact QI projects. Since little is known about these interactions , the preWe used the Promoting Action on Research Implementation in Health Services (PARIHS) model prospectively to implement a large anticoagulation improvement project. PARIHS states that successful implementation (SI) is a function (f) of the nature and type of evidence (E), the qualities of the context (C) where the evidence is being employed and the way the process is facilitated (F) or as Kitson et al. represent it, SI = f  . EvidencContext is dynamic, multivalent, and highly variable in organizational life. Key work on PARIHS recognizes the dynamic relationship among elements and sub-elements and therefore PARIHS contains within it an understanding that the same intervention will likely vary widely in different settings ,10. HowePARIHS elements are often described on a continuum which ranks their “strength” in regard to support for the intervention from low to high . This asEvidence is the stakeholder’s perceptions of the quality and validity of the evidence supporting the intervention, in this case the ACC treatment algorithm, the dashboard that measures TTR and work processes that increase standardization. In this paper, a leader refers to those in positions of management such as Pharmacy Service Line Chiefs and middle managers . Leadership is an element of Context in PARIHS. In this study, leadership has three dimensions, where it: (1) describes the activities (such as supporting effective teamwork) and attitudes taken by leaders (those in management positions) to respond to the ACCII as documented by the research and implementation teams, (2) includes the perceptions staff have of their leadership’s actions and behaviors in response to the ACCII, and (3) refers to the way leaders represent themselves and their responses to the ACCII in interviews. Teamwork is a contextual factor that is embedded within the PARIHS constructs of culture and leadership. In this study, teamwork has two dimensions: (1) the ethnographically observed behaviors and actions that indicate whether a cooperative, coordinated effort by the ACC team is present and the extent of its presence in regard to the ACCII, and (2) the perception ACC staff have of the working relationships among their fellow ACC staff and the general staff attitude toward the ACCII. Communication is a contextual factor that is embedded in the PARIHS contextual constructs of Receptive Context, Leadership and Evaluation and Feedback. For this project, communication is understood as the imparting of information and meaning through speech, technology, body language, and signs. Communication also has several dimensions, referring to (1) communication from leadership to ACC team about the ACCII, and (2) the quality and strength of intra-ACC team communication about ACCII and about ACC care.Recognizing that contextual elements are multidimensional, we characterize each element here. interaction to be a relationship in which two or more contextual elements may affect one another, as opposed to referring to an interaction among people. These effects are not unidirectional but rather multi-directional. Interplay can be understood as reciprocal action, interaction and the mutual influencing that occurs among contextual elements. The present article is an attempt to capture and understand these additional dimensions of how contextual features may work to promote SI.However, characterizing contextual elements only in terms of their relative strength (conducive or not conducive to change) relative to SI, while useful, is not sufficient. In fact, contextual elements can have mixed effects, or their effects can vary over time. In this study we do rank the strength of contextual elements  was introduced in a Veterans Integrated Service Network (VISN), based in the northeastern United States. The ACCII’s focus is all pharmacy-run anticoagulation clinics and all anticoagulation clinical staff in the VISN, encompassing eight medical centers and their affiliated outpatient clinics.The most commonly used anticoagulant in the United States is warfarin, also known by the brand name Coumadin . While wTo improve anticoagulation care, clinical pharmacists in the VISN have been asked to adopt a dosing algorithm, an EBP, which has been shown to improve anticoagulation control and reduce rates of patient complications ,17. SecoThe ACCII is delivered by two overlapping teams. The core facilitation team at the VISN level consists of a clinical pharmacist with training in facilitation, anticoagulation care and QI, an industrial engineer, and an executive level leader, who is a QI specialist. This team is delivering and facilitating the intervention to each medical center and all satellite ACCs. The research team is studying the entire process of implementation, aggregating data on the implementation, and feeding back information to the core team to assist in tailoring and facilitating uptake. After each site visit, the research team quickly processed interim data to identify any barriers to SI which the research team then fed back to the core implementation team for tailoring their facilitation efforts. The extended team involves both the core facilitation team and research team.The present study focused on five ACC sites. Our cross-site comparison combined a case-oriented approach and a variable-oriented approach  to betteData was collected in the latter half of 2012 and early part of 2013 (October 2012 – January 2013) and analyzed in 2013 (February – May 2013). All frontline anticoagulation pharmacy staff in the VISN was eligible to participate in the study of the ACCII by the research team. We specifically requested participation from clinical pharmacists, pharmacy technicians and medical clerks who worked a majority and/or a regular amount of time in the anticoagulation clinic. The purposive sample included pharmacy administrators, clinical pharmacists, nurses, as well as support staff . Fifty-oTwo trained qualitative observers shadowed clinic staff as they engaged in delivery of care including but not limited to, work processes, job tasks and roles, and social dynamics at each ACCII site for at least an entire day and often two days. Observations were collected in the form of field notes. Field notes are written records made during site visits or immediately thereafter. The Institutional Review Board (IRB) of the Bedford VA Medical Center approved the study.a priori coding scheme guided by PARIHS to identify information on evidence, context and facilitation was developed. We also used an iterative thematic analysis so that we could capture any interesting relationships, patterns, surprises, and inconsistencies among people and within and across sites  for data management, coding and analysis. An ss sites . Two resiability . ObservaMembers of the qualitative team met regularly to review coded texts and resolve discrepancies through consensus as well as identify and resolve within site and cross-site variation in themes. The qualitative team also used the same process to resolve any discrepancies in our definitions and characterizations contextual elements at each site and our characterizations of the strength of contextual elements in regard to the ACCII Table  and our The degree of evidence-based practice (EBP) uptake varied across sites and we found interaction among contextual elements was a major factor influencing uptake. In this section, we provide site summaries which describe the state of contextual elements in site-specific case study tables Tables  and 10. Our findings demonstrate how interaction among contextual elements, like teamwork, communication and leadership, led to what seemed to us to be relatively consistent patterns with regard to the impact on uptake. In the context of our implementation effort, we ranked the contextual elements as strong, medium and weak Table  in termsWe observed three general patterns of interaction among contextual elements and evidence. First, belief in the evidence, while necessary for uptake, was not sufficient. Second, we observed that while belief in evidence was important, a site needed at least one other contextual element to be strongly favorable, be it leadership, teamwork, or communication, for uptake to occur. In contrast, if a site had unfavorable context in all three of areas, successful uptake was unlikely to occur. Third, we observed that when belief in the evidence was limited, even noteworthy strengths in other areas only served to reinforce the determination to resist any change to embrace the EBP. Details about these interaction patterns are found below.“We have very strong communication…We try to be very thorough in our notes so that the next person picking it up will know exactly where it was left off, what the story is…. We use (Microsoft) Communicator. We use the phone and we use the spreadsheet to share info…and I think that we’re really good about talking to each other about difficult cases and that kind of helps us to make the best clinical decisions that we can. …we work really well as a team…we talk a lot and we communicate about what’s going on”.One of our most striking findings was that belief in and comfort with the evidence, while important, was not by itself a determining factor in ACCII uptake. Sites A Table  and B T perceive“We are looking to optimize the process that we use… I think it’s a fabulous idea. I’m looking for ways that we can improve… so you are less likely to make mistakes or worry about making mistakes … what would be wonderful is if we could figure out who’s doing a better job with that kind of thing and how can we implement it here”.Site A already had a culture of improvement, as seen in the following quote:Having a new leader who needed time to adjust and learn on the job was not a barrier to change for Site A, because of their strong teamwork and communication.“…It’s really individual specific…For some folks it’s…really good. Others, there’s a chronic lack of communication and lack of…intuition or empathy, you know, as far as what other people’s schedules are and… [this] impacts other people’s function”.Site B had generally adequate teamwork and communication, which was not without its weaknesses however. One staff member remarked of team work and communication at Site B,“[The middle manager] is so organized… [The manger] is wonderful”.It was through the enthusiastic leadership of a new middle manager that Site B had high levels of uptake in the ACCII. One staff person remarked,“[MM] is just so helpful and…she really understands the issues that we face and, I mean, she is obviously on board with anticoag project…”Another stated:Teamwork and communication was mediocre at Site B, but in this case it was strong leadership, and a team willing to be led, that changed the contextual interplay dynamics and enabled the site to become a site with high uptake. In this case, the interaction among contextual elements that varied in strength nevertheless led to high uptake. Here again, we see that at least a moderate belief in the evidence, combined with at least one strong contextual factor  were sufficient to enable successful implementation to occur.“…I’m kind of willing to give it a shot. But …I think we might have some other pharmacists here that might want to use the clinical judgment card pretty frequently on that one”.At Site C Table , many el“…We’re such a good team, we really work together to help everybody and I think when you respect the person that you’re working with and that feeling is mutual I think it makes the clinic run a lot smoother. You know, I think patients are satisfied and… We really do work together as a team”.They were referring to their clinical leader. Teamwork and communication were strong at this Site C. One pharmacist stated:“… if I have a question…Like, procedural stuff a lot of times I’ll ask…. If I have a quick question about…dosing or something like that, a lot of times I’ll ask [the middle manager]”.Staff made it clear that they felt their leader was very much a member of the ACC team. A newer staff member notes that intra-team communication was great and that communication with their leader was excellent as well. In fact this staff member noted,Staff felt comfort with and respect for their middle manager. However, this site remained at a mid-level of uptake initially in part because of this leader’s lukewarm response to the EBP, resulting in only partial fidelity to the ACCII. Site C illustrates the pattern where if belief in the evidence is lukewarm, even strengths in other areas cannot entirely compensate enough to improve uptake.At other sites, there was an open distrust of EBPs; we observed that uptake was limited in proportion to the extent of the distrust. At Site D Table , distrus“…to me you can’t categorize every patient into that algorithm. They don’t all fit and a lot of times it’s a gut feeling that you need to change a dose”. A participant at Site D referred to anticoagulation care as an “art” more than a “science,” indicating a discomfort with the EBP. This pharmacist explains,“… [We] know that we can depend on each other and anyone’ll back you up and help you out”. Another noted“We all [have]… a similar philosophy, a similar training and a similar mindset, so I think our team play [emphasis in interview] works well…”In the context of such dismissal of evidence, the strong teamwork and communication present at Site D only served to reinforce resistance to the effort to implement ACCII. In regard to teamwork, one team member stated,“the most resistant to change”. In discussing why there was resistance to change, there was a celebration of this team’s closeness. For example, in the past, there had been an effort to standardize one aspect of ACC practice at this site. This local QI effort met with no success at Site D as seen in almost identical quotes by two Site D team members,“Well we didn’t want to use that [the proposed QI tool]…and I don’t remember actually my own feelings about it but I remember that you know as a team we kind of shut it down…we decided to just stay with our way….”And another staff member noted that they anticipated that their site would be, “We didn’t want to use it [the proposed QI tool]…as a team we… said no”.Another staff member said,“I don’t keep my ear that close to the ground so, I just sit back and wait for official things to come…Just [heard from leadership] that it’s… [ACCII] coming…It’s [ACCI] in the works…”Communication and teamwork worked together to prevent uptake of a local QI project. Leadership engagement and communication about the fact that the ACCII was being implemented had not even reached all staff when the research team began interviewing. A staff person said,Since leadership had a “hands-off” approach to the running of the ACC at Site D, staff felt that participation in the ACCII was not required and therefore they either did not participate or participated reluctantly and infrequently. At Site D Table , the res3“…I have seen a change in people’s attitudes towards…using the algorithm. I like it. [It] takes a little bit more time when we’re using [the algorithm] because we’re kind of dosing a little bit differently than we had in the past…and you kinda have to think things through a little bit more which isn’t a bad thing”.At Site E Table , we foun“It [teamwork] is awful …There are certain people that will not be in a team, no matter how hard you try…I would love to have a team atmosphere and be able to count on everybody and feel like there’s trust and that we’re providing the best care, but we do not have that here”.However, other contextual factors were notably weak. Teamwork at Site E is described by a staff member as,“…the other thing is communication…we don’t have staff meetings. We…just get emails…but nothing is really discussed among the group”.The feeling was that poor intra-team relations and communication impacts work performance. Furthermore communication between leadership and the ACC staff also impacted staff attitude toward their teamwork, their work and their attitude toward change. A Site E pharmacist reported,;“I think the hardest part of our job is communication…. the volume of our workload is very high so that’s challenging. We have so many patients that walk right in our door. …I think another challenge is the lack of communication among the pharmacists. With the clinical staff [some] have meetings with our [leadership] …they knew about all these…changes, however it doesn’t get trickled down to the rest of the clinic… we’re not [the entire staff] informed as to what’s happening”.This was reinforced by another staff member“…looking at what can we do better. But anyone above him, we don’t see, we don’t hear from”. The following quote illustrated general staff attitude in this area:“…People here want to always make an impression, okay? Especially the people in management… They come around when there’s the photo op, I always like to say… On a daily basis, forget it, okay? That’s the way it is, you know”.As to leadership, the picture is generally one of frustration. Some staff note that that the middle manager is, Here, we see that even a site with a positive view of the evidence cannot succeed when context was very weak.Evidence alone was not sole determining factor in uptake.A site needed at least one and possibly two contextual elements to be strong  for uptake to occur. If a site was relatively weak in all three of these, uptake would not occur, despite strong belief in the evidence.If belief in the evidence was limited, strengths in other areas often did not work in favor of uptake, but rather served to reinforce the status quo (i.e. resistance to change).Theoretical frameworks in implementation science and related fields generally state that several contextual elements contribute to successful implementation, defined as full and high-fidelity uptake of an EBP. While interactions among these contextual elements have been tacitly assumed to occur, they have rarely been described in detail. Our study examined five sites as examples of how contextual elements may interact with each other and with perceptions of the evidence to promote or inhibit adoption of an evidence-based practice. We observed that no single shortcoming served as a “fatal flaw,” but that three general interaction patterns emerged from our data that mapped with successful or unsuccessful uptake of the ACCII.With the recent emphasis on theory-driven implementation studies, theory has seemed to diverge regarding whether to focus on the key role of individuals in uptake of interventions, or whether to shift attention to the roles of teamwork and social networks . In partIf there is a contextual element that is particularly strong, this strength may compensate for weaker areas, as was seen with Site A Table . Site A Many conceptual frameworks present taxonomies of contextual elements which can be used prospectively to guide a theory-driven implementation, to tailor interventions to address perceived needs on an ongoing basis, and to analyze implementation retrospectively. In our study, PARIHS was used prospectively as part of the overall study design which mapped out how the research team would study the implementation as it rolled out, developed and spread. Specifically, the research team drew on PARIHS to construct the interview guide and to develop the code book for the interviews and ethnographic observations. However, in the course of collecting and analyzing our data we realized that we were encountering a limitation in our conceptual model. PARIHS did not provide us a way to describe and the interplay among contextual elements, nor did it represent how contextual interrelationships influenced initial rates of uptake. In this study, we learned that interactions among contextual elements can produce a synergy which magnifies contextual effects and moves them in certain patterns, some of which we were able to characterize here.We operationalized the patterns we observed in the qualitative data by feeding the information back to the ACCII facilitation team and working with them to tailor the intervention to be site specific. Some of this tailoring took the form of doing some targeted capacity building and development of contextual elements at sites to enhance uptake. For example, there were acts of facilitation such as teamwork development, encouraging and facilitating better communication within and across sites about the ACCII, and reaching out to leadership to enlist support for the intervention that came directly out of the research team’s rapid site assessment. Additionally there were increased efforts in algorithm presentations, journal clubs and other venues to educate ACC staff about the scientific basis of the algorithm and the importance of its use, especially where belief in the evidence seemed to be the weakest link.As a concrete example of the value added by our approach to analyzing not only contextual elements, but also their interaction, the research team observed that teamwork was likely to be the “weak link” at Sites D and E. The team was “weak” at Site D – with regard to our intervention – because they worked so well together and mutually reinforced each other’s views of the limited value placed ACCII. In this case, team cohesion created resistance that negatively impacted implementation of the intervention. The team at site E was weak because they did not know how to work toward a common goal, they did not support one another and they did not have established means of communicating about common goals because they did not hold regular meetings. Our assessment of teamwork as the key factor driving successful implementation at these sites was in part predicated on our understanding that “unlocking” teamwork would also enable other aspects of context to improve. Informed by this observation, both the research team and the facilitation-implementation team concentrated on team-building activities at Site E, and on trying to convince team members at Site D regarding the evidence for the algorithm, as well as reaching out to the Site D leadership to educate them. Based on our interim findings, our efforts seem to be making a difference at both Sites D and E, and indeed focusing on teamwork first at both sites  did enable other aspects of context to improve subsequently. This serves as a concrete example of how our interactional approach to data analysis also helped us tailor the intervention more effectively.Quality improvement (QI) does not rest on the intervention itself but often on the context in which the intervention takes place. For example, one recent study examined how contextual elements interacted in the setting of a single-site intervention to reduce surgery cancellations among 20 staff . In thatOur study’s cross-site analysis suggests that the relationship between evidence, contextual factors and uptake is a very mixed picture but that there are common patterns among interactional data that influence uptake Tables  and 3. FThis study has some limitations. Most importantly, interactional data can be challenging to identify and discuss as they are based on social actions and actors who are always changing. We added ethnographic observations, which allowed for a more detailed understanding of interactional data, to ameliorate this limitation.Interrelationships among different contextual elements can act as barriers to uptake at some sites and as facilitators at others. Accounting for interconnections among elements enables PARIHS to more fully describe the determinants of successful implementation as they operate in real-world settings. Our study is an important start to better understand these interactions, which have been proposed and assumed, but rarely described in detail. This approach to understanding the interaction of contextual elements also has the potential to help inform a more effective approach to tailoring the intervention to promote successful implementation.


PGK1 promoter (PPGK1) from the methylotrophic yeast Pichia pastoris. The L1 gene was cloned under regulation of PPGK1 into pPGKΔ3 expression vector to achieve intracellular expression. In parallel, secretion of the L1 protein was obtained through the use of an alternative vector called pPGKΔ3α, in which a codon optimized α-factor signal sequence was inserted. We devised a work-flow based on the detection of the L1 protein by dot blot, colony blot, and western blot to classify the positive clones. Finally, intracellular HPV VLPs assembly was demonstrated for the first time in yeast cells. This study opens up perspectives for the establishment of an innovative platform for the production of HPV VLPs or other viral antigens for vaccination purposes, based on constitutive expression in P. pastoris.The human papillomavirus (HPV) L1 major capsid protein, which forms the basis of the currently available vaccines against cervical cancer, self-assembles into virus-like particles (VLPs) when expressed heterologously. We report the development of a biotechnology platform for HPV16 L1 protein expression based on the constitutive  HPVs are a large family of dsDNA viruses that cause benign warts and malignant tumors. Persistent infection by HPV imposes a huge burden on health services worldwide owing to its links with cancer of the vagina, vulva, penis, anus, tongue, and, in particular, uterine cervix, which is the most serious outcome . About 7 Saccharomyces cerevisiae and baculovirus-infected cells, respectively. Although these vaccines are highly effective and safe  was routinely used as a host for cloning and plasmid manipulations. This strain was cultured at 37°C in LB medium  supplied with appropriate antibiotics. P. pastoris X-33 strain (wild-type) used in this study was purchased from Invitrogen. The yeast cells were grown at 30°C on YPD medium  and YPDS  supplemented with 100 μg/mL zeocin (Invitrogen) when necessary.TheAll the molecular cloning techniques were carried out as previously described . Restric P. pastoris. Restriction sites for XhoI and NotI were added to the flanking regions of the L1 gene so that the cloning could be directed into the expression vector. The gene was synthesized by Epoch Biosciences , and cloned into pBSK plasmid. The resulting construct called pBSK/L1 was amplified in E. coli DH5α. L1 gene was released from pBSK after double-digestion with XhoI and NotI and employed for creating the expression vectors.A DNA sequence encoding the HPV16 L1 protein (Gen Bank access number GI: 27752860) was designed with codons optimized for expression inGK1 promoter from P. pastoris for constitutive expression of the L1 protein. The original 2 kb PPGK1 sequence described by de Almeida et al. [PGK1 sequence was used to generate the pPGKΔ3 expression vector for intracellular expression. Additionally, a pPGKΔ3α expression vector carries a codon-optimized S. cerevisiaeα-factor signal sequence (α-MF) cloned downstream to PPGK1 to drive recombinant protein secretion. Both PPGK1-based vectors contain the zeocin resistance gene Sh ble for positive selection of E. coli and P. pastoris recombinants, as well as a C-terminal polyhistidine (6xHis) tag for detection of the fusion protein by immunoblot assays. The L1 gene previously digested with XhoI and NotI was cloned into pPGKΔ3α and pPGKΔ3 expression vectors digested with the same enzymes, and the resulting vectors were called pPGKΔ3α/L1 and pPGKΔ3/L1, respectively. The construction of the vectors was confirmed by restriction digestion, PCR and DNA sequencing with specific primers for both L1  and PPGK1 (5′ TCATAGTTCATCCCTCTCTCC 3′) sequences.Two expression vectors were constructed with the Pa et al.  was redua et al. . The res PGK1 locus of P. pastoris was achieved after linearization of the 5 μg PPGK1-based expression vectors with SacI [ P. pastoris electrocompetent cells were prepared and transformed, as described elsewhere [ P. pastoris/pPGKΔ3α/L1 or P. pastoris/pPGKΔ3/L1, depending on the vector that was used.Stable integration into theith SacI . P. pastlsewhere . In thisμg/mL zeocin, the transformants were subjected to a general procedure for the selection of clones containing multiple copies of the expression cassette, called Posttransformational Vector Amplification (PTVA) process [μg/mL).After selection on agar plates containing YPD supplemented with 100  process . For thiμg/mL zeocin were subjected to a general screening for selection of recombinant strains expressing high levels of HPV L1 protein. The screening was carried out as follows: (i) P. pastoris/pPGKΔ3α/L1 clones were cultivated in agar plates and subsequently subjected to colony blot for detection of HPV L1 protein with the CamVir anti-HPV16 L1 monoclonal antibody ; (ii) In contrast, P. pastoris/pPGKΔ3/L1 clones were subsequently grown in deep-well plates to detect HPV L1 protein by dot blot with the anti-L1 monoclonal antibody.Multicopy clones resistant to 1000  P. pastoris/pPGKΔ3α/L1 multicopy clones in YPD agar plates for 3 days at 30°C, the PVDF membranes were cut as discs and left standing with the surface colonies for 3 hours at 28°C. Colonies on the master plate could be replicated by placing the PVDF discs on a fresh agar plate when desired. The PVDF discs were washed with tris-buffered saline (TBS) containing 0.05% Tween 20 (TBST) to remove adhering cells. The membranes were blocked by incubation in TBST, supplemented with 5% nonfat milk for 1 hour at room temperature, and then incubated overnight at 4°C with anti-L1 antibody properly diluted (1 : 1000) in the blocking solution. PVDF discs were washed three times with TBST for 10 min and then incubated for 1 hour at room temperature with peroxidase-conjugated goat anti-mouse immunoglobulin  diluted 1 : 3000 in the blocking solution. Chemiluminescence reaction was detected with an ECL kit .In the case of colony blot, the protocol described by Goodnough et al.  was modi P. pastoris/pPGKΔ3/L1 multicopy clones were first grown in a deep-well plate containing YPD medium for 3 days at 30°C. The plate was shaken at 300 rpm and P. pastoris cells were harvested by centrifugation at 3000 rpm for recovery of the pellets. Preparation of yeast extracts was performed with an “alkaline lysis” procedure as previously described [β-mercaptoethanol) and heated to 90°C for 10 min and the lysate was brought to neutral pH. To improve solubilization, the lysate was heated again for 10 min and mixed with loading buffer . The PVDF membranes were cut in a proper way and inserted into a dot blotter apparatus. Protein transfer to the membranes was carried out with 100 μL of the lysate for 1 hour and, thereafter, the immunoblot proceeded as already described for the colony blot. The HPV16 L1 protein that was episomally expressed in P. pastoris [For the dot blot assay,escribed . Brieflypastoris  was usedP. pastoris multicopy clones were selected for baffled-flask cultivation in accordance with the highest detected level for the HPV L1 protein. P. pastoris/pPGKΔ3α/L1 clones were first preinoculated in 5 mL of YPD to achieve the secretion of the L1 protein and grown at 28–30°C in a shaking incubator (250–300 rpm) until the cultures reached an OD600 of ~10 (24 hours). Following this, the cells were inoculated in 20 mL of YPD for 3 days and aliquots were taken for analysis at periodic intervals. These samples were centrifuged at 12000 g for 2 min and the supernatants were stored for further analysis. For the intracellular expression of the L1 protein, P. pastoris/pPGKΔ3/L1 clones were subjected to the same protocol but the cell pellets were stored instead of the supernatants.β-mercaptoethanol, 0.1% bromophenol blue) was then added to the washed pellet (corresponding to 100x concentration) and this mix was resolved on 15% SDS PAGE after the samples had been heated for 10 min at 75°C. Polyacrylamide gel was either stained with Coomassie brilliant blue  or transferred to PVDF membranes using a V20-SDB semidry protein transfer apparatus . Immunoblot was performed as described in Aliquots of supernatants were submitted to precipitation with trichloroacetic acid (TCA) (Sigma-Aldrich) to a final concentration of 10% to allow the secretory production of the HPV L1 protein to be analyzed. After concentration, the supernatant was discarded, and the protein pellet was resuspended again in 100% acetone to remove residual TCA. A final volume of SDS-loading buffer , according to Cregg et al. . This crAt the end of the induction course, both the cells and culture supernatants were subjected to absorption into carbon-coated grids, as recommended by Falcón et al. . The gri P. pastoris PGK1 promoter which was originally described and employed for secretion of α-amylase from Bacillus subtilis [ PGK1 promoter sequence containing ~400 bp was used to allow integration of the expression vector via homologous recombination, as well as a codon-optimized α-factor from S. cerevisiae to drive the secretion of recombinant proteins  together with the detection of the L1 protein expressed episomally in P. pastoris cytosol ensured the specificity of the reaction.Compared withanalyzed . Multicoanalyzed . Hence,  Figures . The lac P. pastoris secretes few proteins into the medium [It is worth noting that this practical and rapid workflow provides a strategy to screen well-expressing clones through cultivation under nonoptimal conditions—with regard to culture volume, shaking, and aeration, as is the case for deep-well and agar plate cultures—among a considerable high number of colonies. The colony blot assay has an important feature, particularly in the case of PGK-based clones that secrete L1 protein, which is the dispensable use of liquid media. Sincee medium , 30, 43, P. pastoris was first reported by Bazan et al. [ S. cerevisiae. Despite being innovative and important means of demonstrating the feasibility of Pichia in producing HPV VLPs, the work carried out by Bazan and colleagues, employed a nonintegrative system. As the authors (and other studies) point out, the use of episomal plasmids is not recommended for industrial purposes, since they have been associated with the generation of recombinant strains that are genetically unstable and have two undesirable outcomes: (i) instability in the heterologous expression levels; (ii) continuous antibiotic selection which is needed to maintain the expression plasmids [ P. pastoris by employing AOX1-based integrative vectors [Expression of the major capsid protein of HPV16 inn et al. . Accordiplasmids , 26, 44. vectors , 23, 24. P. pastoris strains present three distinct phenotypes: (i) the wild-type phenotype designated as methanol utilization plus (Mut+) and characterized by the presence of a functional copy of the alcohol oxidase 1 gene (AOX1) which is responsible for 85% of the utilization of methanol by the alcohol oxidase enzyme; (ii) methanol utilization slow (Muts) phenotype, characterized by the absence of the AOX1 gene and presence of the AOX2 gene, which is about 97% homologous to AOX1 but much less expressed; (iii) methanol utilization minus (Mut−) phenotype, where both AOX genes are absent. Hence, while Muts strains show a poor growth on methanol medium, the Mut+ strains have a greater growth rate and Mut− strains, in contrast, are unable to grow on methanol as the sole carbon source. Interestingly, Cregg et al. [ P. pastoris Mut+strains expressed 10-fold less HBsAg than Muts strains, which suggests that consumption of methanol at high levels, coupled with a rapid growth rate, may not lead to efficient HBsAg assembly [AOX1 induction are nonoptimal for the production of HBV and HPV VLPs. Finally, even though recombinant protein expression in Pichia Muts strain could circumvent the deficiencies observed for the generation of VLPs in Mut+ strains, the slower methanol utilization phenotype requires long fermentation times to reach peak product concentrations. Once induced, the entire culture cannot be used to start a new culture [With regard to the metabolization of methanol,g et al.  showed tassembly . In addiassembly , althoug culture .AOX1-based system, we constitutively expressed the L1 protein through PPGK1 in the presence of glucose and by employing an easier cultivation/expression schedule in shake flasks, since L1 expression occurred together with the cell growth and the laborious control of the methanol/inducer levels was dispensable  status by the FDA (Food and Drug Administration) and has been recognized as an efficient vehicle for the delivery of viral antigens when administered by an oral and intramuscular route [The assembly of HBV and HCV core proteins into VLPs has been previously reported in yeast , 51. Howar route . Furtherar route . P. pastoris recombinant strains described here provides evidence of electrodense structures with estimated diameters of 55 nm, as expected for the HPV VLPs (Figures  P. pastoris strains that had been transformed by the parental vectors ( P. pastoris through immunoelectron microscopy were hindered by the retention of cell membrane components in the architecture of the HCV core particles which has an envelope-like structure [Electron microscopy analysis of Figures  and in a Figures  and insi Figures  (either  vectors . We alsotructure . Accordi P. pastoris recombinant strains. It has been shown that assembly of HPV VLPs can be achieved with neutral pH, high ionic strength, and relatively low concentrations of reducing agents [ P. pastoris growth progresses in unbuffered medium, the pH drops to 3 or below [We attempted to characterize assembled HPV VLPs in the culture media fromg agents . Conversor below . It can or below , 48–51.  P. pastoris PGK1 promoter for the production of HPV16 VLPs. Constitutive expression of the HPV16 L1 capsid protein was efficiently achieved for the first time through an easier production schedule than is the case when an AOX1 inducible promoter is employed. In addition, an optimized α-MF secretion signal downstream to PPGK1 provided a prompt secretion of L1 protein in the culture media. Although further experiments are needed to determine which production strategy is more effective for HPV L1 protein production, the data outlined here underline the efficiency of a PGK1-based platform as a heterologous expression system and support its employment for the production of HPV VLPs for vaccination purposes. Moreover, our preliminary data showing intracellular VLP assembly open up perspectives for the employment of P. pastoris cells that can express HPV L1 protein as a live attenuated vaccine. To the best of our knowledge, this is the first time that the expression of HPV L1 protein was achieved in P. pastoris through an IP-free biotechnology platform. The discovery of alternatives to biotechnology platforms for producing an efficient and cost-effective vaccine has the potential both to offer greater protection—since not all the HPV genotypes are covered by the current vaccines—and to allow the vaccine to be widely disseminated.The data presented in this work demonstrate the functionality of a biotechnology platform based on