Despite developing differently, the tumors have similar: blood perfusion, oxygen consumption, and vascular surface area parameters, whereas the number of blood vessels is nearly doubled in the tumor arising from normoxia cultured cells. Interestingly, tumor oxygen tension, measured using 19F-oximetry, showed that the normoxia grown cells led to tumors characterized by mild hypoxic environment (approximately 4%) conditions, whilst the hypoxia grown cells led to tumors characterized by physioxic environment (approximately 6%) conditions. This reversal in oxygen concentration may be responsible for the apparent paradoxical growth profiles.Growing tumor cell lines, such as U87-MG glioma cells, under mild hypoxia (3% O Indeed, the fate of the cells after stereotactic inoculation in the mouse brain appears heterogeneous. Kathagen et al. [2), which grew faster as compared to its culture under atmospheric oxygen tension (21%). The same fate was reported by Bourseau-Guilmain et al. [2) conditions upon implantation in mice. This latest observation was also done with the established U87-MG cell line [Over the past years, the issue of the ‘appropriate’ oxygen tension to culture glioma cell lines or glioma stem cells has emerged as an important issue as the phenotype of the cells can be radically modified with oxygenation . Such chn et al.  describen et al.  for one ell line .in vivo characterization of a U87-MG tumor model initially expanded under mild (3%) or atmospheric oxygen (21%) conditions, in terms of blood perfusion and oxygenation. Data were obtained using magnetic resonance (MR) imaging, respectively; arterial spin labelling (ASL) [19F-oximetry) using dedicated oxygen nanosensors [Currently, the impact of oxygen tension concentration during cell culture, and the physiology of the subsequent brain tumor, is not extensively documented. With the present study, we are reporting the ng (ASL)  and fluoosensors .® HS15 and Lipoïd (surfactants) were precisely weighed: mPFCE = 5 g, mKolliphor = 2.115 g, mLipoïd = 0.188 g, mWater = 5 g and mNaCl = 0.5 g. A four step process was performed to obtain PFCE-LNCs: to dissolve the Lipoïd within the initial solution, the temperature was increased from standing room temperature to 95°C, where after a 1 minute sonication, using a Microson XL2007 sonication probe set at 10 W  was performed, prior to three heating and cooling cycles from 25°C to 95°C. Upon the final cooling, once the temperature reached 70°C, a rapid cold dilution was performed by adding 3.5 mL of cold (4°C) milli-Q water.Perfluoro-15-crown-5-ether (PFCE) loaded lipid nanocapsules (LNCs) (thus PFCE-LNC) were prepared as previously described using a phase-inversion process ,17. Brie® S75-3 (soybean lecithin– 69% phosphatidylcholine and 10% phosphatidylethanolamine) and Kolliphor® HS15 were supplied by Lipoïd GmbH  and BASF , respectively. Perfluoro-15-crown-5-ether (C10F20O5) was provided by Chemos GmbH . NaCl was purchased from Prolabo . Deionized water was obtained from a Milli-Q plus® system .Lipoïd2, and either 3% or 21% O2.All experiments were performed with cells between passages 12 and 15. The U87-MG cell line was provided by the ATCC  and grown in Dulbecco’s modified Eagle’s medium high glucose  supplemented with 10% fetal calf serum (FCS)  and 1% antibiotic solution  at 37°C in a humidified incubator under an atmosphere containing 5% CO®  and Clorketam®  anesthesia, mice were fixed in a stereotaxic holder and placed on a heating pad to maintain the appropriate physiological temperature. Through a 1mm drilled hole in the skull , a 5 μL suspension of 7.5 x 104 U87-MG glioma cells, grown at either 3 or 21% O2, were injected over a 10-minute period into the caudate putamen of the right hemisphere. After surgery, mice received a single 15μg/kg subcutaneous injection of Vetergesic®  and were monitored daily for mobility and grooming until their participation in the imaging protocol. Two groups of 18 mice were inoculated with cells grown at either 3 or 21% O2. Each group was subdivided into two; 6 mice were dedicated to tumor blood flow measurements and histological analysis and 12 mice for tumor oxygenation measurements.Animal care and use were in strict accordance with the regulations of the French Ministry of Agriculture and approved by the Pays de la Loire Ethics in Animal Experimentation Committee under project number 01858.03. The manuscript does not contain patient data. Brain tumors were induced via the stereotaxic inoculation of U87-MG human glioma cells in 7–8 week-old female nude mice  as previously described [1H cryoprobe and a rapid acquisition with relaxation enhancement (RARE) sequence  = 21.3 ms; acceleration factor = 4; FOV = 2×2 cm; matrix 256 × 256; nine contiguous slices of 0.5 mm, Nex = 1). Volumes were calculated from manually drawn region of interest. The tumor volume growth curves were subsequently fitted by the method of least squares with an exponential function and the time constant of the exponential converted into a doubling time value [MR imaging was performed using a 7T scanner  equipped with BGA12S gradient system (675mT/m). Animal body temperature was maintained throughout the experiment by hot water circulation in an animal bed. During the MR protocol, mice were anesthetized with 0.5% of isoflurane and respiration was monitored. Tumor volume was assessed over time using a me value . Tumor pme value . A serie1H/19F birdcage. The geometrical parameters were fixed at FOV = 2 x 2 cm, matrix 64 x 36 zero-filled to 64 x 64, slice thickness = 4 mm, and one average was performed. A Half-Fourier Acquisition Single-Shot Turbo Spin-Echo (HASTE) sequence  with 16 inversion times ranging between 30 ms to 3500 ms was used to produce T1 maps which were converted into tumoral pO2 as previously reported [When the tumors were approximately 10 μL in volume, the mice underwent an intra-tumoral stereotactic injection of 8 μL PFCE-LNCs, using a convection-enhanced delivery protocol . The flu2 was assessed on anaesthetized mice (0.5% of isoflurane) at rest, i.e. animals breathing air, as well as after a 20 minute challenge with O2. Upon switching back to air, tumor pO2 was measured over time, and the profile of the pO2 change was then fitted with a mono-exponential function whose time constant mainly reflected tumor oxygen consumption [POsumption .). At th2 initially grown U87-MG) were analyzed under a fluorescence microscope . The numbers of CD31 positive vessels and Ki 67 positive cells were counted using the MetaView computerized image-analysis system for three brain cryosections per mouse. Two-four fields/cryosection, at × 200 magnification, were randomly chosen in the tumor. The results are expressed as the mean number of positively stained vessels/mm2 for each group ±IC95%.Brain cryosections were air-dried, rehydrated in phosphate buffered saline (PBS) and fixed for 10 min in 4% paraformaldehyde (PFA), pH 7.4, at 4°C. To block non-specific binding, the sections were incubated in PBS containing 4% bovine serum albumin (BSA) and 10% normal goat serum. The sections were incubated overnight at 4°C with isotype controls and primary antibodies against endothelial cells  and proliferative cells . Primary antibodies were detected using biotinylated secondary antibody and the signal amplified using streptavidin—fluorescein (streptavidin-FITC) . Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) . Cryosections from 3 mice of each group (3% or 21% ORNeasy Mini Kit (Qiagen). Briefly, 1 μg of RNA was reverse-transcribed into complementary DNA using random primers and Superscript II according to the manufacturer’s recommendations . Quantitative real-time PCR was performed using the sybrgreen Master mix (Thermo Fisher Scientific) and the LightCycler 480 (Roche Diagnostics) according to the manufacturer’s instructions. Primer pairs used for each transcript were specifically designed using Primer Blast software. The relative abundance of mRNA levels was calculated using the 2−ΔΔCt method for each gene using hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) gene Ct values for normalization. Five mRNA levels were evaluated: endothelial growth factor A (VEGFA) involved in angiogenesis, the carbonic anhydrase 9 (CA9) and pyruvate dehydrogenase kinase 1 (PDK1) involved in metabolic reprogramming, of urokinase receptor (UPAR) and matrix metalloproteinase 2 (MMP2) involved in invasion and metastasis.Total RNAs were extracted from U87-MG glioblastoma tumors using the t-test. Differences were considered significant if the p value was < 0.05.All quantitative data are represented as mean± standard error of the mean, and the statistical significance was determined using in vivo [2, respectively.Growing U87-MG cells under mild hypoxic conditions, prior to cell implantation within mice brain, impacts subsequent growth in vivo . In the 2) was 7.1 ± 0.5 μL and 7.5 ± 0.5 for the normoxic group (21% O2). Under those circumstances, the measured TBF was not significantly different with values of TBF3% = 69 ± 4 mL/min/100g and TBF21% = 68 ± 5 mL/min/100g for each respective groups (p = 0.79).A fast and non-invasive characterization of the tumor perfusion can be gained using ASL-MR imaging. As shown in 3% = 660 ± 51 vessels/mm² versus n21% = 919 ± 35 vessels/mm2 (p < 0.001). Notably, the vessels tended to be larger in the 3% group (p = 0.03), with an average surface area of S3% 48 ± 5 μm2 versus S21% 33 ± 2 μm2 in the 21% group , carbonic anhydrase 9 (CA9), pyruvate dehydrogenase kinase (PDK1), urokinase receptor (UPAR), matrix metalloproteinase 2 (MMP2), vimentin (VIM) or octamer binding protein 4 (OCT4), an over-expression in the 3% and 21% tumors was measured in comparison to the contralateral brain. However, the comparison between the two groups of tumors only showed a limited 3-fold over-expression in CA9 and a 2-fold over-expression in PKD1, in the 21% O2 tumor group as compared to the 3% O2.Nevertheless, despite the absence of difference between both groups in terms of TBF, the histological analysis of the brain tumors showed that the number of vessels was significantly reduced in the 3% group, i.e. n1% group . Thus th2 value measured at rest, i.e. isoflurane anesthesia with spontaneous air breathing, within the mice bearing U87-MG brain tumor initially cultured under 3% pO2 was equal to pO2-Air3% = 47 ± 3 mmHg (n = 12). In the 21% group, the value is significantly reduced (p < 0.01) as pO2-Air21% = 33 ± 3 mmHg (n = 12)  as values are k3% = 0.35 ± 0.10 min-1 and k21% = 0.42 ± 0.13 min-1.In order to assess the tumoral oxygenation, an intratumoral injection of perfluorocarbon-loaded nanocapsules was performed as previously described . The pO2(n = 12) . After 2in vitro expansion prior to implantation in living systems, is currently under debate with respect to the pertinence of the models used [2) or under normoxia (21% O2) led to significant modifications in tumor growth [2 was also measured using specially designed fluorinated nanosensors [The impact of cell phenotype changes, induced by oxygen tension during els used ,22, and els used ,23. In tels used –26, the r growth . This obosensors .2 group; the TBF of the tumor sized matched groups at ca. 7–8 μL was not significantly different. Indeed, TBF were measured at TBF3% = 69 ± 4 mL/min/100g and TBF21% = 68 ± 5 mL/min/100g (p = 0.79). Nevertheless, the invasive histological analysis of the tumors showed significant microscopic differences between the two groups. As shown in 3% = 46 ± 5 μm2 versus S21% = 33 ± 2 μm2 in the 21% group (p = 0.03), but the number of vessels is significantly reduced n3% = 660 ± 51 vessels/mm2 versus n21% = 919 ± 35 vessels/mm2 (p < 0.001), leading to similar vascular surface area, ca. 3%, possibly explaining why the TBF between the two groups are not dissimilar. However, the vessel density results appear contradictory according to our previous work regarding the in vitro evaluation of the hypoxia impact on the cell phenotype [in vitro mild hypoxia led to the stabilization of HIF-1 and subsequently the over-expression of VEGF-A. As a consequence, we would have expected better angiogenesis in tumors arising from cells initially grown under mild hypoxia. However, a hypothesis involving the oxygen pressure in the host tissue where the cells were implanted, i.e. the rodent striatum, could be proposed. Indeed, a recent report has shown that average oxygen saturation reaches a value of 67% in the rat cortex and 59% in the rat striatum [2) and 44 mmHg (5.7% O2), respectively, using the Hill equation, wherein n = 2.6 and P50 = 38 mmHg [2 drives the tumoral cells phenotype from the cell inoculation and as well as throughout the tumor development. As a consequence, we have measured the tumoral pO2 using 19F-oximetry with specially designed fluorine loaded-nanocapsules [2 (2-Air3% = 47 ± 3 mmHg (ca. 6.2% O2) versus pO2-Air21% = 33 ± 3 mmHg (ca. 4.3% O2) (p < 0.01). Moreover, this difference in pO2 cannot be attributed to a larger use of oxygen in the 21% tumor group as time constant for this process was defined within an oxygen challenge [21% = 0.42 ± 0.13 min-1 versus k3% = 0.35 ± 0.10 min-1, p > 0.4). Taken all together, the faster growth of the 21% group may result from the tumoral cells creating a local environment, characterized by a lower pO2 and transferring the cells into a ‘deeper mild hypoxic’ environment which promotes tumor growth [It is now well established that, irrespective of the type of the solid tumor explored, the tumor blood flow (TBF) is dependent on tumor size ,28. In thenotype . Indeed,striatum . These v 38 mmHg  and Caco 38 mmHg . Howevercapsules . Prior asules [2 , within r growth , regardlFollowing this descriptive study of the macroscopic differences observed relating to hypoxic environments, the next step will be to define the exact biological mechanism(s) and the dynamics which underlays these paradoxical growth profiles as well as a systemic exploration of others glioblastoma cell lines.2) or normoxia (21% O2) leads to a peculiar phenotype, with over-expression of VEGF-A which should facilitate the growth of hypoxic cells in vivo. However, a paradoxical phenomenon is observed as normoxia grown cells gave rise to tumors faster than the mild hypoxia grown cells. Tumor oxygen tension measured using 19F-oximetry showed that in the former, mild hypoxic conditions are reached as tumors are growing whereas, for the latter, physioxic conditions are observed with tumor growth and may explain the paradoxical growth profile.Growing tumor cell lines such as U87-MG glioma cells under mild hypoxia (3% O


Kv4.3 co-expression also increased the protein expression and promoted the membrane localization of hERG. Western blot showed Kv4.3 increased hERG expression by Hsp70. hERG and Kv4.3 co-localized and co-immunoprecipitated in cultured 293 T cells, indicating physical interactions between hERG and Kv4.3 proteins in vitro. In addition, Hsp70 interacted with hERG and Kv4.3 respectively, and formed complexes with hERG and Kv4.3. The α subunit of Ito Kv4.3 can interact with and modify the localization of the α subunit of IKr hERG, thus providing potentially novel insights into the molecular mechanism of the malignant ventricular arrhythmia in heart failure.This study examines the interaction between hERG and Kv4.3. The functional interaction between hERG and Kv4.3, expressed in a heterologous cell line, was studied using patch clamp techniques, western blot, immunofluorescence, and co-immunoprecipitation. Co-expression of Kv4.3 with hERG increased hERG current density (tail current after a step to +10 mV: 26 ± 3  More specifically, the transient outward potassium current (Ito) is responsible for the early repolarization (phase 1) of the cardiac action potential2, while the rapid (IKr) and slow (IKs) delayed rectifier potassium currents are responsible for the rapid repolarization phase (phase 3)4. First proposed by Roden in 19985, “repolarization reserve” describes that the inhibition of a current, for example IKr, will not lead to failure of repolarization. Instead, another current, IKs, which remains activated, will act and prevent excessive repolarization delays8. This compensatory potential is lost when IKs is inhibited simultaneously, thus enhancing the effects of IKr inhibition and increasing the consequent risk of excessive repolarization delay and arrhythmias9. The “repolarization reserve” was then revised10 to that the loss of a single K+ current will not completely impair repolarization  because of redundancy of K+ currents; when one K+ current is dysfunctional, the other K+ current will increase to compensate so that the changes in action potential duration (APD) are minimized. This compensation requires delicate interaction between the different K+ currents.Cardiac repolarization is driven by potassium (K+ channel (Kv channel) consists of four pore-forming (α) subunits that contain the voltage sensor and ion selectivity filter and accessory regulating (β) subunits12. For example, the co-assembly of α (KvLQT1) with β (minK) subunits forms IKs channels, while the co-assembly of α subunits encoded by the human ether a-go-go-related gene (hERG)13 and β subunits encoded by the KCNE2 gene product minK-related peptide 1 (MiRP1)15 are responsible for the formation of IKr, although the contribution of MiRP1 to native IKr remains uncertain. There is a consensus that the pore-forming α subunits of the Kv4 subfamily, Kv4.3 (KCND3), encodes Ito channels16. However, β subunits have also been proposed to be part of myocardial Ito channels19. Delpon et al. suggested that the co-association of β subunits of MiRP1 with α subunits of the Kv4.3 contribute to the function of Ito channels19.A classic voltage-gated K+ currents interacted with each other through α-α subunits or α-β subunits. It is well established that the interaction between IKr and IKs is patho-physiologically relevant20. The prolongation of APD because of a reduction in IKr favors the activation of IKs, which prevents an excessive repolarization delay, and thus suggests potential interactions between the α subunits of IKr and IKs. In 2004, Ehrlich et al. demonstrated that the α subunit of IKs KvLQT1 could interact with the α subunit of IKr hERG to enhance hERG current densities and increase hERG-protein membrane localization20. Wu et al. also suggested that the α subunits of Ito Kv4.3 interact with the β subunits of IKr MiRP1, forming α-β subunits interaction, which is critical for the normal function of the native Ito channel complex in a human heart21.Studies show that different K23. hERG and Kv4.3 mRNAs, and protein expression levels decrease24. IKr current and Ito current densities are reduced26. However, it has not been reported if the α subunit of IKr hERG interacts with the α subunit of Ito Kv4.3 during heart failure. Therefore, in light of the “repolarization reserve” theory, our present study aim to investigate the interactions between the α subunits of IKr and Ito using heterologous cell line co-transfected with hERG and Kv4.3.During heart failure, electrophysiological and structural remodeling changes occurhERG, Fig. hERG densities, compared with cells transfected with hERG alone. Step Figure tep Fig.  and tailtep Fig.  IhERG de1/2 −13.89 ± 3.77675 versus −13.26 ± 1.27843 mV, n = 15 and 20, respectively, p = NS), , , Fig. , and in S), Fig. . The actS), Fig. . HoweverS), Fig. . The timhERG alone showed no significant difference when compared with cells co-expressed with hERG and Kv4.3 Fig. .hERG, we next determine whether the reversed could also be true; i.e., whether expression of hERG alters Ito currents. Figure to because the currents were significantly reduced by 5 nM 4-AP , , , Fig. . SimilarS), Fig. .Figure 2The western blot showed that co-transfection of Kv4.3 increased the protein levels of the fully glycosylated mature hERG (155 kDa), but not the core-glycosylated, immature form (135 kDa). However, the protein expression of Kv4.3 was not altered by the co-transfection of hERG Fig. . Confoca27, we therefore hypothesized that Kv4.3 increased the hERG protein expression via upregulation of Hsp70. To test the hypothesis, we examined the expression of hERG in cells transfected with both hERG and Kv4.3, in the presence or absence of Hsp70 siRNA or VER155008, a novel adenosine-derived inhibitor of Hsp7029. The results showed that downregulation of Hsp70 using siRNA ; the fully glycosylated mature protein is observable as a 155 kDa band and represents hERG localizes either in the Golgi apparatus or on the cell surface33. The comparison of the intensity of these two bands provides a useful details regarding hERG trafficking. We found that the mature form of hERG (155 kDa) increased after the cells were co-transfected with Kv4.3. However, since transient transfection was used in our experiments, majority of the protein may still be within intracellular milieu (immature). These results were further verified by confocal imaging. In the presence of Kv4.3, more hERG protein localized on the surface of the cells, thus suggesting that Kv4.3 enhances hERG trafficking.Under physiological conditions, the wild type hERG exhibits two bands on Western blot analysis; this represents different stages of asparagine (Asn)-linked glycosylationet al. showed that the cytosolic chaperones Hsp70 interacted with hERG during maturation27. The Hsp70 family, including stress-induced Hsp70 and constitutively expressed heat shock cognate protein (Hsc)70, was shown to interact with the core-glycosylated form of hERG34 and increases the levels of mature form of hERG27. Based on aforementioned studies, we hypothesize that Hsp70 may be involved in the maturation of hERG induced by Kv4.3. Indeed, we found that Hsp70 protein expressions were increased in cells co-transfected with hERG and K4.3. The upregulation of hERG expression by Kv4.3 is dependent on Hsp70, as Hsp70 siRNA could prevent the Kv4.3-induced hERG expression. Similar result was obtained when Hsp70 inhibitor, VER155008, was used. These results suggest that Kv4.3 enhances hERG protein trafficking through Hsp70.Ficker Since the confocal image demonstrated that hERG and Kv4.3 co-localized on cell surface, we further investigated the interaction between hERG and Kv4.3 using immunoprecipitation. The result showed that hERG and Kv4.3 bind to each other and form a complex. Both mature and non-mature forms of hERG co-immunoprecipitated with Kv4.3.20. KvLQT1 could also interact with hERG at the C-terminal cytoplasmic tails of hERG that contained cyclic nucleotide binding domain20. MiRP1 is also believed to interact with hERG at the C-terminal region36. It is known that long-QT syndrome type 2 (LQT2) mutations in the C-terminal tail affect trafficking of hERG from the ER to the cell surface39. A number of the C-terminal interacting proteins such as GM130 and 14–3–3 have been identified in playing potential roles in the trafficking of hERG to the cell surface42. However, in our study, we did not determine which domain of hERG interacted with Kv4.3.Interactions between hERG and potential α and β subunits have been suggested. For instances, KvLQT1 was shown to interact with hERG to modulate hERG localization and current densities27. Therefore, we tested if Hsp70 formed complexes with hERG and Kv4.3, and if Hsp70 mediated the interactions between hERG and Kv4.3. Our data demonstrated that both Kv4.3 and hERG interacted with Hsp70, suggesting that Hsp70 may form complexes with hERG and Kv4.3. However, although downregulation of Hsp70 decreased hERG protein expression in cells co-transfected with Kv4.3 and hERG, Hsp70 did not play a critical role in the complex formation between hERG and Kv4.3. These data indicate that Hsp70 increases hERG protein expression and forms complexes with hERG via two independent mechanisms.We observed that Kv4.3 interacted with hERG and increased hERG expression through Hsp70. It has been reported that hERG formed complexes with Hsp70Hsp90 only interacted with Kv4.3, and not with hERG when expressed alone Fig. . HoweverThere are a few limitations in the present study. In all experiments, hERG and Kv4.3 were transiently transfected in cells; therefore, the immature form of hERG had higher band intensity than the mature form. Our studies were performed primarily in HEK293T cells. It remained to be determined whether the interactions that we observed were specific to this cell line. The association between hERG and Kv4.3 was not verified in cardiac myocytes or animal model. Therefore caution should be taken to extrapolate the findings to cardiac myocytes because cardiac myocytes express many other ion channels which may increase or decrease the interaction between hERG and Kv4.3.Kr hERG and Ito Kv4.3. Kv4.3 modulates hERG current density by promoting hERG protein expression and localization via Hsp70. Kv4.3 and hERG co-localize with each other, and form complexes with Hsp70 in HEK293T cells. The physical and functional interactions between the hERG and Kv4.3 enrich the theory of “repolarization reserve”.In conclusion, our finding reveals the interaction between the α subunits of I2 in DMEM medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 100 units/ml penicillin, and 100 µg/ml streptomycin (Invitrogen). HEK293T cells were transfected transiently or stably with these constructs using a lipofectamine method as described previously20. After the transient transfection, HEK293T cells were studied at 48 hours. When the transiently transfected cells were used for patch clamp experiments, CD8 cDNA was co-transfected to be used as a reporter gene (Invitrogen). CD8 (1 µg) was co-transfected with hERG cDNA (1 µg) or Kv4.3 cDNA (1 µg). CD8-positive cells were identified using Dynabeads . Cells were harvested between 48–72 hours after the transfection.hERG cDNA and Kv4.3 cDNA were provided by Dr. Silvia G. Priori . Human embryonic kidney 293 (HEK293T) were cultured at 37 °C with 5% CO2 2, MgCl2 1, HEPES 10 and glucose 5, adjusted to pH 7.4 with NaOH. The current was recorded by the whole cell patch-clamp technique using a MultiClamp 700B amplifier . All signals were acquired at 5 kHz . Patch pipettes were pulled from borosilicate glass on a P-97 horizontal puller . The electrodes had a resistance of 2–3 MΩ. In order to record the K+ currents, pipettes were filled with (in mmol/L): K-aspartame acid 140, MgATP 4, MgCl2 1, EGTA 10, GTP 0.1, HEPES 10 and adjusted to pH 7.3 with KOH.The HEK293T cells were bathed in the solution containing (in mmol/L) NaCl 140, KCl 4, CaClto was elicited by a 300 ms depolarizing pulse from a holding potential of −80 mV to a testing potential of +70 mV and a conditioning test of −40 mV for 25 ms to eliminate the sodium current. This current was blocked by 5 nM 4-aminopyridine (4-AP). IhERG was investigated by applying voltage commands of 5000 ms duration from a holding potential of −80 mV to a range of test potentials from −70 mV to +50 mV, followed by a repolarizing step to −40 mV for 3000 ms to elicit tail currents. This current was significantly inhibited after exposure to 5 nM dofetilide. Average I/V relationships were used to compare current densities following the normalization of current traces to individual cell capacitance measurements. Steady-state I/V relationships were measured at the end of the test pulse.IFor immunofluorescent studies, transiently transfected HEK293T cells were grown on glass bottom cell culture dishes for 24 hours. Cells were then fixed (10 min) with 5% paraformaldehyde (Sigma), washed 3 times (5 min) with a phosphate-buffered saline, blocked with a 3% bovine serum albumin (Sigma), and then permeabilized with 0.1% Triton X-100 (Sigma) for 10 minutes. Cells were incubated overnight (4 °C) with primary antibodies  at 1:800 dilution followed by three 5-minutes washes with phosphate-buffered saline. Next, cells were incubated with Alexa Fluor 555 donkey anti-rabbit secondary antibody (Invitrogen) for 45 minutes at room temperature. After being washed, the cells were treated with 4′6-diamidino-2-phenylindole  for 5 minutes and analyzed under a confocal microscope.Whole cell proteins from HEK293T cells that expressed various channels were used for analysis. Proteins were separated on 8% or 12% SDS-polyacrylamide electrophoresis gels, transferred onto nitrocellulose membrane, and blocked for 1 hour with 5% nonfat milk. The blots were incubated with the primary antibody for 24 hours at 4 °C and then incubated with a horseradish peroxidase-conjugated secondary antibody. GAPDH expression was used as loading controls. The blots were visualized with Fujifilm using the ECL detection kit . For immunoprecipitation, a whole cell protein (0.5 mg) or cell surface protein was incubated with the appropriate primary antibody overnight at 4 °C and then precipitated with protein A/G plus agarose beads (Santa Cruz) for 4 hours at 4 °C. The beads were washed three times with an ice-cold radio immune precipitation assay lysis buffer, resuspended in 2 mL sample buffer, and boiled for 5 minutes. The samples were centrifuged at 20,000 g for 5 minutes, and the supernatants were collected and analyzed using Western blot.The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.


Raphidocelis subcapitata were studied in bacteria-free cultures exposed to single and mixture treatments at different concentrations for 96 h. The results showed that R. subcapitata exhibited a rapid and strong ability to remove E2 and DES in both single and mixture treatments by biodegradation. At the end of 96 h, the removal percentage of single E2 and DES achieved 82.0%, 80.4%, 74.6% and 89.9%, 73.4%, 54.1% in 0.1, 0.5, and 1.5 mg·L−1, respectively. With the exception of the 0.1 mg·L−1 treatment at 96 h, the removal capacity of E2 was more efficient than that of DES by R. subcapitata. Furthermore, the removal percentage of mixture E2 and DES achieved 88.5%, 82.9%, 84.3% and 87.2%, 71.8%, 51.1% in 0.1, 0.5, and 1.5 mg·L−1, respectively. The removal percentage of mixed E2 was significantly higher than that of the single E2. The presence of DES could accelerate the removal of E2 from the mixture treatments in equal concentrations. In addition, the removal was mainly attributed to the biodegradation or biotransformation process by the microalgae cells rather than simple sorption and accumulation in the cells. The microalgae R. subcapitata demonstrated a high capability for the removal of the E2 and DES indicating future prospects for its application.Natural steroidal and synthetic non-steroidal estrogens such as 17β-estradiol (E2) and diethylstilbestrol (DES) have been found in natural water, which can potentially endanger public health and aquatic ecosystems. The removal and biodegradation of E2 and DES by  The for1 in DES ,10,11,12els [−1) . Moreoveels [−1) .Chlorella species have been reported to remove inorganic nutrients, heavy metals, and toxic organic contaminants from wastewater with low cost and high efficiency [Chlorella vulgaris has been reported to remove EE2 and E1, and Scenedesmus dimorphus has also been used to remove E1, E2, E3, and 17α-estradiol in batch culture [Chlamydomonas reinhardtii presented high adsorption percentages of 86% and 71% for E2 and EE2. Nearly half of the removal was attributed to biodegradation processes, while the rest of the removal was due to adsorption [The removal and biodegradation of organic contaminants by microalgae have been reported. Green microalgae such as ficiency . Some grficiency . For ins culture ,19. Chlasorption . Furthersorption . Moreovesorption . In natuReports have shown that bacteria live freely attached to the algal surface or as intracellular algal co-existing in microalgae cultures . InteracSelenastrum capricornutum, Pseudokirchneriella subcapitata and Raphidocelis subcapitata are the same species named in different periods [R. subcapitata have been reported to remove and degrade phenanthrene (PHE), fluoranthene (FLA), and pyrene (PYR) using monooxygenase and dioxygenase enzymatic pathways [cis-dihydrodiols by a dioxygenase enzyme system [R. subcapitata has also been reported [R. subcapitata exposure on 17β-estradiol (E2) and diethylstilbestrol (DES) and to examine the natural steroidal estrogen and synthetic non-steroidal estrogen removal and biodegradation capacity of R. subcapitata including single and mixture treatments. periods . The spe periods . R. subcpathways . It has e system  and to re system . The abireported . Thus, iRaphidocelis subcapitata was obtained from the Algae Culture Collection of the Department of Ecology in Jinan University . The algae were cultivated with a BG11 medium in a homoeothermic incubator  at 60 μmol·m−1·s−1, a diurnal cycle of 12 h light and 12 h dark and a temperature 25 ± 1 °C. The components of the BG11 medium were as follows: NaNO3 1.5 g·L−1, K2HPO4 40 mg·L−1, CaCl2·2H2O 36 mg·L−1, MgSO4·7H2O 75 mg·L−1, NaHCO3 20 mg·L−1, citric acid 6 mg·L−1, and ferric ammonium citrate 6 mg·L−1. The trace metal solution contained H3BO3·2.86 mg·L−1, MnCl2·4H2O 1.81 mg·L−1, ZnSO4·7H2O 222 mg·L−1, Na2MoO4·2H2O 390 mg·L−1, CuSO4·5H2O 79 mg·L−1, and Co(NO3)2·6H2O 49.4 mg·L−1 [4 mg·L−1 .R. subcapitata cultures (100 mL) were filtered with a 10 µm pore size membrane. The algal cells were suspended in a 50 mL BG11 medium which was autoclaved at 121 °C for 15 min before centrifuging  and washed three times. The washed cells were suspended in a 50 mL sterile BG11 medium and treated with 0.005% Tween-80 and 0.1 M EDTA for 1 h at 20 °C. Next, lysozyme (0.5 mg·mL−1) was added for further treatment (10 min at 20 °C) before sodium dodecyl sulfate  was added into the culture environment. After 10 min of SDS treatment time, the algal cells were centrifuged  and washed twice to remove the lysozyme and SDS, and then suspended in a 50 mL BG11 medium. A cocktail of antibiotics including 100 µg·mL−1 penicillin and 50 µg·mL−1 kanamycin was added to the cultures. The treated R. subcapitata was cultured in the incubator at 20 ± 1 °C with a diurnal cycle of 12 h light and 12 h dark for 7 days. An assessment for bacterial presence was carried out after sub culturing three times to remove the antibiotics. The axenic status of R. subcapitata was confirmed after sub culturing three times in a sterile BG11 medium without antibiotics. A 4′,6-diamidino-2-phenylindole (DAPI) stain was added into samples at a final concentration of 4 µg·L−1 and then cultured in the dark for 15 min at 4 °C. Cells were filtrated with black polycarbonate membranes . Bacterial presence was immediately examined by epifluorescence microscopy  using oil immersion.Based on a previous report , mid-exp−1. An initial growth culture was prepared 5 days before the tests to ensure the population was in the exponential growth phase at the beginning of the test [−1 of E2 and 0, 0.1, 0.3, 0.5, 0.9, and 1.5 mg·L−1 of DES. The mixed E2 and DES treatment was 0, 0.1, 0.3, 0.5, 1.5, and 3 mg·L−1, and the mass concentration rate of E2:DES was 1:1 based on a previous report [5 cells·mL−1. All flasks were randomly arranged in racks. To avoid microalgae sedimentation, all flasks were hand-mixed twice per day during the test. Treatment with an equivalent amount of acetone  was included as a control. After 24, 48, 72, and 96 h of exposure time, 2 mL of each sample were fixed in 0.5% of methanal to further measure the algal density by counting with a Neubauer’s chamber  under microscope .17β-Estradiol (E2) and diethylstilbestrol (DES), purchased from Sigma-Aldrich  were dissolved in acetone and the dissolved form was added to a stock solution at a concentration of 1 g·Lthe test . All sols report . The iniFor the cell dry weight measurement, the pore-size GF/F glass-fiber filter  was dried overnight in an oven  at 60 °C until a constant weight was reached. A 20 mL culture was filtered through the filters. The filters with algal cells were dried overnight in an oven at the same conditions above until a constant weight was reached. The difference between the final weight and the weight before filtration was the dry weight of the algal cells.50  was calculated using linear regressions of the inhibition rate and expressed in terms of the estrogen concentration (mg·L−1).According to the Organization for Economic Co-operation and Development (OECD) (2006) , the 96 −1 was the internal surrogate standard, as described by Wang et al. [N,O-bis(trimethylsilyl)trifluoroacetamide  as the derivatization in a water bath at 70 °C for 1 h. Then, the reaction system was dried with a gentle nitrogen stream at 25 °C and dissolved in 100 μL of ethyl acetate for further analysis by gas chromatography-mass spectrometry (GC-MS).Immediately after the sampling timepoint, microalgal cells were separated from 5 mL medium by centrifugation at 5000 × rpm for 15 min at 4 °C. E2 and DES in the aqueous supernatant were extracted twice with ethyl acetate (25 mL) by liquid-liquid microextraction (DLLME), and 4-nonylphenol (4-NP) at a final concentration of 1 mg Lg et al. , with soFor every estrogens concentration, blank groups without algae were set up to evaluate the variation in estrogens concentration under abiotic conditions. The microalgal cells from the above section were washed with 5 mL ethyl acetate and shaken for 60 s, then the E2 and DES contained in the solution was considered as the surface adsorbed estrogens and then extracted with DLLME as described above and analyzed with GC-MS.2SO4, the microalgae cells obtained from the above section were mixed with ethyl acetate (4 mL) and sonicated for 30 min, then, the sample was centrifuged for 5 min at 5000 rpm. The cell pellets were extracted three times and the solvent fractions were combined for further analysis with GC-MS. Based on the measured concentrations, the removal efficiency (R) and biodegradation percentage (BDP) of estrogens by the algal biomass were calculated as previously described with minor modifications according to the following equations:After adding the appropriate amount of anhydrous NaR is the dissolved estrogens removal rate (percent); iC and fC are the initial and final concentrations (mg·L−1) of estrogens in the solution, respectively, and:iC is the initial concentration in the solution; rC is the residual concentration in the solution; AL is the concentration of abiotic losses (mg·L−1); dC is the concentration (mg·g−1) dry weight of estrogens adsorbed on the cell wall; cC is the concentration (mg·g−1 dry weight) of estrogens accumulated in the algal cells; and aW is the dry weight of algal biomass expressed in g·L−1 [in g·L−1 .−1. The GC injector port was held isothermally at 280 °C. The mass selective detector (MSD) was operated both in scan and selected ion monitoring (SIM) mode. The GC column temperature program was as follows: the initial oven temperature was set at 100 °C and increased to 300 °C at a rate of 10 °C·min−1 with a holding of 3 min at 300 °C. The split less injection was 1 µL and solvent delay was 6 min. The limit of quantification for both E2 and DES was 0.01 µg·L−1. The results obtained were compared to those obtained from a control group without estrogens.The analysis and quantification of estrogens in the extract were determined using a GC-MS (Agilent 7890/5975CMSD) with HP-5MS fused silica capillary column . The carrier gas was helium and set at a constant flow rate of 1 mL·minp < 0.05 and were described as “significant”. Data are presented as the mean ± standard deviation (mean ± SD) unless otherwise stated.Statistical analysis was carried out using the SPSS16.0 package . One Way-ANOVA followed by the Duncan test was used to check the significance of the treatments. The significance of difference among the E2 and DES in single and mixture treatments at the same concentration was comparatively analyzed with a one-way ANOVA. Levels of significance used were R. subcapitata exposure to estrogens is shown in R. subcapitata exposure to single and mixtures of E2 and DES are shown in R. subcapitata growth, excluding the possibility of the solvent to have caused toxic effects on algae. Results showed a negative effect with the test concentrations of DES. Growth of R. subcapitata was significantly reduced at 1.5 mg·L−1 after 48 h exposure with a reduction of 56.6%. After 72 h exposure, the growth had a strong decrease at 0.3–1.5 mg·L−1 concentration treatments. At the end of 96 h, growth of R. subcapitata at 0.3–1.5 mg·L−1 concentration treatments were further decreased and the reduction of 1.5 mg·L−1 concentration treatments was 61.5%. Compared with the DES treatments, the growth of R. subcapitata exposed to E2 were reduced gradually. Cell abundance had a significant decrease for 96 h exposure at 0.8–3.2 mg·L−1 concentrations. When the microalgae cells were exposed to mixtures of estrogen (1:1) for 48 h, the growth was obviously decreased at a 3 mg·L−1 concentration with the reduction was 43.4%. With the exposure time increasing to 72 and 96 h, significant decreases in growth were also observed in mixture treatments at 0.3–3 mg·L−1 concentrations. The 96 h EC50 of −1 for 96 h exposure, indicating that abiotic losses was negligible and would not influence the evaluation of the capacity of R. subcapitata in the removal of contaminants . After the 96 h treatment, the residual amount of E2 was more than DES at 7.7%. In the 0.5 and 1.5 mg·L−1 treatments, the residual amount of E2 was much less than DES at 24 h, 48 h and 96 h . The amount of estrogen uptake included that adsorbed onto the cell surfaces (extracellular) and absorbed into the cells (intracellular). The amounts of extracellular E2 and DES were increased with concentrations of exposure during 96 h. The amount of extracellular E2 was less than DES except the 0.1 mg·L−1 concentration treatment at 96 h. Intracellular estrogens were also increased with exposure concentrations. The amount of intracellular E2 was less than that of DES at the 0.1 mg·L−1 treatment and became more significant at 0.5 mg·L−1 treatments. With the exception of the 0.1 mg·L−1 treatment, R. subcapitata could remove more of E2 than DES and more DES were uptaken by the microalgae than that of E2.After exposure in single estrogens for 96 h, the residual, intracellular, and extracellular E2 and DES contents of −1 concentration. While the amounts of E2 remaining in the medium were reduced more than DES in the 0.5 and 1.5 mg·L−1 treatments at the end of 24 h, 48 h, and 96 h . The amount of extracellular E2 was less obvious than DES in all concentrations except the 0.1 mg·L−1 treatment at the first 24 h. Furthermore, the intracellular E2 in the mixture treatments were also less than DES and the statistical significance were observed other than that in 0.1 mg·L−1 treatment at the first 24 h.The residual and uptake of mixed estrogens under different treatments after 96 h exposure are shown in −1 concentration. The concentrations of both extra- and intra-cellular E2 were also less than that of DES in the single and mixed. Moreover, the residual amounts of E2 in the mixed treatments were less than that of E2 in the single treatment after 96 h. According to R. subcapitata for removing estrogens. R. subcapitata in different treatments with 0.1, 0.5, 1.5 mg·L−1 concentrations at the end of 96 h. Significance of difference among the E2 and DES in single and mixture treatments at the same concentration were comparatively analyzed with a one-way ANOVA followed by the Duncan test. After exposure for 96 h, the removal efficiency of single E2 treatments was not only obviously less than E2 in mixture, but also significant less than DES in both the single and mixture treatments at 0.1 mg·L−1. In the 0.1 mg·L−1 treatment, the removal efficiency of E2 were all higher than that of DES in both the single and mixture treatments. When the exposure concentration was increased to 1.5 mg·L−1, the removal efficiency of E2 in the mixture was significant higher than the single and mixed DES, and even that of E2 in a single exposure. Moreover, the removal efficiency of the single E2 was also higher than that of DES in the single and mixture treatments. The trend of estrogen biodegradation was general as the removal efficiency except 0.5 mg·L−1. At the 0.5 mg·L−1 concentration treatment, the biodegradation of E2 in mixture was obviously higher than that of E2 in single exposure.The removal efficiency and biodegradation of E2 and DES were calculated to directly show the capacity of R. subcapitata, but that E2 could also cause a significant decline in the growth at 0.8 mg·L−1 after 96 h exposure, indicating that estrogens could cause an adverse effect on R. subcapitata cells. Microalgae can be influenced by contaminants; however, the sensibility and tolerance are related to contaminant compounds and are species-dependent. E2 was slightly toxic to R. subcapitata, it was reported that could even be tolerated at concentrations up to 10 mg·L−1 [50 was 0.87 and 1.01 mg·L−1 for Pseudokirchneriella subcapitata (the same as R. subcapitata) [50 of E2 among the reports, even in the present study. It may cause by some reasons. On one hand, the microalgae cultured by different conditions such as the medium, light intensity, temperature, pH and light-dark cycle could make the different growth situation and adaptive features. These differences may cause the different in accumulation of the intracellular organic matters and extracellular secondary metabolites [R. subcapitata to estrogens [The present study revealed that not only DES could inhibit the growth of 0 mg·L−1 . Howeverapitata) . There wabolites , and it strogens . R. subcapitata in the present study. As an inhibitor of the plasma membrane H+-ATPase, DES has been reported to block proton (H+) transport [Dunaliella [R. subcapitata was exposed to mixed estrogens after 96 h, the toxicology was less than the exposure to a single DES, but more toxic than being exposed to a single E2. That may indicate that E2 could decrease the toxic effects of DES to the microalgae. Additionally, it has been reported that E2 can improve antioxidative enzyme activities and avoid H2O2 generation to reduce oxidative stress injury from heavy metal induced [R. subcapitata, the toxicity concentration was more than 0.5 mg·L−1, indicating the strain was able to remove estrogens with high tolerance.Compared to E2, DES has high toxicity to ransport  and inhiransport , indicatnaliella . When R. induced . Further induced . For mic induced . These r induced ,45. In a induced ,47. The R. subcapitata was assessed. From the results, the removal efficiency of E2 was generally higher than DES in single treatments. The tests showed that about 74.6% and 54.1% of E2 and DES, respectively were removed from culture after 96 h in single treatments. The biodegradation percentage (BDP) was about 72.9% and 52.3% in the single E2 and DES treatments, respectively. As a natural steroidal estrogen, E2 was easier to remove by the microalgae than other synthetic steroidal estrogens such as 17α-ethynylestradiol (EE2) [Chlorella vulgaris, Anabaena cylindrical, Spirulina platensis, and Scenedesmus quadricauda could effectively remove E2 from culture and could accelerate its removal from the wastewater [R. subcapitata was biodegradation. This was consistent with the results from previous reports on the removal of carbamazepine, nonlyphone, and steroid estrogens by the microalgae Chlamydomonas mexicana, Scenedesmus obliquus, Selenastrum capricornutum, and Desmodesmus subspicatus, respectively [The removal of synthetic organic compounds by a variety of microalgae species has been widely demonstrated. In this study, the removal capacity of estrogens E2 and DES in single and mixture by microalgae stewater ,48. Due stewater . Comparestewater ,51. In tstewater . Moreovestewater . In the ectively ,35,56. Tectively . Furtherectively ,48,49. ADesmodesmus sp., Chlorella pyrenoidosa, and Scenedesmus obliquus [Scenedesmus dimorphus [Hydroxylation, glycosylation, and methylation are the most generally accepted transformation processes of organic contaminant degradation by microalgae . Hydroxyobliquus . E2 coulimorphus . Moreoveimorphus . NeverthR. subcapitata demonstrated a high capability for the removal of the E2 and DES at mg·L−1 levels, indicating future prospects for application in the treatment of wastewater and possibility of reducing the risk. Furthermore, microalgae could increase the degradation of contaminants in photodegradation [Previous studies have all shown that estrogens might subsequently cause potential risks to organisms at higher trophic levels with biomagnification along food chains in aquatic ecosystems . The preradation  and alsoradation . It was Raphidocelis subcapitata in this study showed that it could efficiently remove E2 and DES. The removal capacity of E2 was more efficient than that of DES by R. subcapitata. The presence of DES could accelerate the removal of E2 from the mixture treatments in equal concentrations. The removal mechanisms included initial rapid adsorption and absorption, followed by bioaccumulation and biodegradation. In addition, the removal was mainly attributed to the biodegradation or biotransformation process by the microalgae cells rather than simple sorption and accumulation in the cells. The microalgae R. subcapitata demonstrated a high capability for the removal of the E2 and DES at mg·L−1 levels, indicating future prospects its application in the treatment of wastewater. Furthermore, the interactions between estrogen compounds and the mechanisms in the removal, bioadsorption, and transformation in single and mixed estrogens should be further investigated.The investigation of


Ph1 locus promotes accurate synapsis and crossover of homologous chromosomes. Interspecific hybrids between wheat and wild relatives are exploited by breeders to introgress important traits from wild relatives into wheat, although in hybrids between hexaploid wheat and wild relatives, which possess only homoeologues, crossovers do not take place during meiosis at metaphase I. However, in hybrids between Ph1 deletion mutants and wild relatives, crossovers do take place. A single Ph1 deletion (ph1b) mutant has been exploited for the last 40 years for this activity. We show here that chemically induced mutant lines, selected for a mutation in TaZIP4-B2 within the Ph1 locus, exhibit high levels of homoeologous crossovers when crossed with wild relatives. Tazip4-B2 mutant lines may be more stable over multiple generations, as multivalents causing accumulation of chromosome translocations are less frequent. Exploitation of such Tazip4-B2 mutants, rather than mutants with whole Ph1 locus deletions, may therefore improve introgression of wild relative chromosome segments into wheat.Despite possessing related ancestral genomes, hexaploid wheat behaves as a diploid during meiosis. The wheat The online version of this article (doi:10.1007/s11032-017-0700-2) contains supplementary material, which is available to authorized users. Triticum aestivum) is composed of three related ancestral genomes , each containing seven identical (homologous) chromosome pairs (homologue pairs 1–7). Each homologue pair has a corresponding related homoeologous chromosome pair, possessing similar gene order and content, within each of the other two genomes. Despite the similarity between homoeologues, wheat behaves as a diploid at meiosis, with synapsis and crossovers (COs) only occurring between homologous chromosomes, rather than between homoeologous chromosomes . This diploid-like behaviour is predominantly controlled by Ph1, a dominant locus on chromosome 5B  at metaphase I in all meiocytes, while inducing a low level of multivalents and univalents in some meiocytes  produces interspecific hybrids, containing haploid sets of wheat and wild relative homoeologous chromosomes, but exhibiting virtually no COs during meiosis. However, in Ph1 deleted wheat-rye hybrids, an average of 7 COs per cell is observed  has also previously been characterised as a tapetal cell gene  treatment failed to produce mutants exhibiting the full Ph1 locus to the part of the 2.5 MB region containing a duplicated chromosome 3B segment carrying heterochromatin and TaZIP4-B2 , inserted into a cluster of CDK2-like genes interspersed with methyl transferase genes  . Ultimately, the SC is degraded, so that the bivalents are only held together by chiasmata or COs at metaphase I, allowing their correct segregation. Recently, Ph1 has been shown to have a dual effect on synapsis and CO formation in wheat  on synapsed homoeologues from becoming COs. However, the molecular mechanism of how the Ph1 locus affects both synapsis and CO is not currently known.Deletion analysis defines the Ph1 deletion mutant, developed in hexaploid wheat cv. Chinese Spring (CS) (CS ph1b), has been used by breeding programmes worldwide to introgress wild relative chromosome segments into wheat. A recent programme has successfully introgressed hundreds of wild relative chromosome segments into wheat, exploiting CS ph1b  as COs  within the Ph1 locus on chromosome 5B is single copy, compared to the CDK2-like and methyl transferase gene cluster. Moreover, ZIP4 has been shown to have a major effect on homologous COs, but not on synapsis, in both Arabidopsis and rice , but which exhibit homoeologous COs in hybrids with wild-relatives. We describe the identification of two such lines through this approach.Although there are Triticum aestivum cv. Chinese Spring and cv. Cadenza); a Chinese Spring mutant lacking the Ph1 locus (ph1b); two Tazip4-B2 mutant Cadenza lines  and Ae. variabilis (2n = 4× = 28)—using either wild-type Cadenza or Tazip4-B2 mutant lines.Plant material used in this study included the following: wild-type hexaploid wheat (http://www.leica-microsystems.com/). The two remaining anthers were either frozen in liquid nitrogen and stored at −80 °C for RNA-seq analysis or fixed in 100% ethanol/acetic acid 3:1 (v/v) for cytological analysis of meiocytes.Both for the RNA-seq analysis and for meiotic studies, the seedlings were vernalised for 3 weeks at 8 °C and then transferred to a controlled environmental room until meiosis under the following growth conditions: 16 h/8 h, light/dark photoperiod at 20 °C day and 15 °C night, with 70% humidity. Tillers were harvested after 6 to 7 weeks, when the flag leaf was starting to emerge, and anthers collected. For each dissected floret, one of the three synchronised anthers was squashed in 45% acetic acid in water and assigned to each meiotic stage by observation under a LEICA DM2000 microscope  were harvested as described in the “Plant material” section. Anthers at late leptotene-early zygotene stage were later harvested into RNA later . The anthers from three plants of each genotype were pooled in a 1.5-ml Eppendorf until reaching 200 to 400 anthers. Once sufficient anthers had been collected, the material was disrupted using a pestle, centrifuged to eliminate the RNA and then homogenised using QIAshredder spin columns . RNA extraction was performed using a miRNeasy Micro Kit  according to the manufacturer’s instructions. This protocol allows purification of a separate miRNA-enriched fraction  and the total RNA fraction (˃200 nt) used in this study. This process was repeated to obtain three biological samples of each genotype.Anthers from wild-type wheat (WT) and wheat lacking the One microgram of RNA was purified to extract mRNA with a poly-A pull down using biotin beads. A total of six libraries were constructed using the NEXTflex™ Rapid Directional RNA-Seq Kit  with the NEXTflex™ DNA Barcodes–48  diluted to 6 μM. The library preparation involved an initial QC of the RNA using Qubit DNA  and RNA  assays as well as a quality check using the PerkinElmer GX with the RNA assay . The constructed stranded RNA libraries were normalised and equimolar pooled into one final pool of 5.5 nM using elution buffer . The library pool was diluted to 2 nM with NaOH, and 5 μl was transferred into 995 μl HT1 (Illumina) to give a final concentration of 10 pM. Diluted library pool of 120 μl was then transferred into a 200-μl strip tube, spiked with 1% PhiX Control v3 and placed on ice before loading onto the Illumina cBot. The flow cell was clustered using HiSeq PE Cluster Kit v4, utilising the Illumina PE_HiSeq_Cluster_Kit_V4_cBot_recipe_V9.0 method on the Illumina cBot. Following the clustering procedure, the flow cell was loaded onto the Illumina HiSeq2500 instrument following the manufacturer’s instructions. The sequencing chemistry used was HiSeq SBS Kit v4 with HiSeq Control Software 2.2.58 and RTA 1.18.64. The library pool was run in a single lane for 125 cycles of each paired end read. Reads in bcl format were demultiplexed based on the 6 bp Illumina index by CASAVA 1.8, allowing for a one base-pair mismatch per library and converted to FASTQ format by bcl2fastq.http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) to remove adaptor sequences and low-quality reads (-q 20—length 80—stringency 3). A total of 273,739 transcripts (Triticum_aestivum_CS42_TGACv1_scaffold.annotation) were quantified using kallisto v0.43.0 , equipped with a Leica DFC450 camera and controlled by LAS v4.4 system software . Images were processed using Adobe Photoshop CS5  extended version 12.0 × 64.A total of five plants per line were examined in a randomised block design. Generally, 2–3 tillers in each plant were analysed to identify meiocytes at meiotic metaphase I. Anthers from ZIP4 sequence called TRIAE_CS42_5BL_TGACv1_404600_AA1305800 (http://plants.ensembl.org/Triticum_aestivum/Info/Index). The primers used were as follows: forward primer: 5′GCCGCCATGACGATCTCCGAG3′ and reverse primer: 5′GGACGCGAGGGACGCGAG3′ for Cadenza1691 and forward primer: 5′GTGTTCCTAATGCTCACAACTC3′ and reserve primer: 5′ACCAGACATACTTGTGCTTGGT3′ for Cadenza0348. PCR amplification was performed using MyFi Polymerase , according to the manufacturer’s instructions. The primers were amplified as follows: 3 min 95 °C, 35 cycles of 15 s at 95 °C, 15 s at 58 °C and 30 s at 72 °C. PCR products were resolved on 2% agarose gels in 1× TBE and stained with ethidium bromide and visualised under UV light. PCR products were purified using Qiaquick PCR Purification kits  and cloned using a p-GEM T easy vector kit . The ligation mixture was used to transform Escherichia coli DH5a, and transformants were selected on LB agar containing ampicillin (100 mg/ml) , isopropyl β-D-1-thiogalactopyranoside   and 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside  . The PCR fragments were isolated using QIAprep Spin Miniprep kit . All kits were used as described in the manufacturer’s instructions. Sequencing was carried out by the Eurofins Company. Alignment of sequences was carried out using Clustal Omega software .The regions of each mutation in both mutant Cadenza lines were sequenced to confirm the existence of either missense or nonsense mutations in Cadenza1961 and Cadeza0348, respectively. Wheat leaf tissues from wild type Cadenza and mutant Cadenza lines were harvested at growth stages 3–4 . DNA was extracted using the CTAB method . Means were separated using the Dunn’s test with a probability level of 0.05.Statistical analyses were performed using STATISTIX 10.0 software . Both ZIP4 homologues are located within the Ph1 locus on 5B, and also on chromosomes 3A, 3B and 3D. Before undertaking the targeted induced lesion in genomes (TILLING) mutant analysis, we assessed the expression of TaZIP4-B2 to confirm that the TaZIP4-B2 gene within the Ph1 locus is expressed during meiosis; that it has a higher level of expression than the ZIP4 homologues present on chromosome group 3; and finally, that Ph1 deletion significantly reduces overall ZIP4 expression. At the coding DNA sequence and amino acid levels, TaZIP4-B2 (AA1305800.1) showed 95.3 and 89.2% similarity to TaZIP4-B1 (AA0809860.1), 94.1 and 87.5% similarity to TaZIP4-A1 (AA0645950.1) and 94.4 and 87.2% similarity to TaZIP4-D1 (AA0884100.1), respectively  (WT) and the Ph1 deletion mutant (ph1b) at the late leptotene-early zygotene stage, and six libraries prepared for the RNA-seq study. RNA-seq analysis showed that TaZIP4-B2 exhibited a higher level of expression than the ZIP4 homologues on homoeologous group 3 chromosomes  accounted for 97% of the TaZIP4-B2 transcripts. As expected, when the Ph1 locus was deleted, expression of TaZIP4-B2 was also eliminated (p < 0.05), but there was no apparent increase in the transcription of the ZIP4 homologues on homoeologous group 3 chromosomes, to compensate for the absence of ZIP4 on chromosome 5B (p > 0.05)  on chromosome 5B, within the Ph1 locus.In hexaploid wheat, www.wheat-tilling.com database  reveals those plants possessing mutations in the target genes, as well as a list of all mutations possessed by the plant   showed reduced number of COs in cytological analysis, suggesting that their Tazip4-B2 mutations did exhibit a phenotype. Both lines were selected for wide crossing studies with wild relatives to score the effect of their Tazip4-B2 mutations on homoeologous CO frequency in the wheat Tazip4-B2 mutant-wild relative hybrids, as compared to non-mutagenised wheat-wild relative hybrids. Null segregants were not available, so wild-type Cadenza lines were used as controls. Mutations within TaZIP4-B2 were verified by sequencing, and primers were designed to the mutated regions to follow mutated genes during crossing , shown to be important for ZIP4 function   within the coding sequences of 106 other shared genes. Sixteen of these genes, including TaZIP4-B2, were located on chromosome 5B. However, none of the genes apart from TaZIP4-B2 were located within the 2.5 MB Ph1 region defined in our previous study  of Ph1, and the wild relative tetraploid Aegilops kotschyi , to show that homoeologous COs are induced when the Ph1 locus is deleted. Interspecific hybrids of the ph1b mutant and wild relatives have been subsequently used in plant breeding programmes for introgression purposes , were crossed with Ae. variabilis. The frequency of univalents, bivalents, multivalents and total chiasma frequency was scored at meiotic metaphase I in the resulting F1 hybrid  at metaphase I in the wild-type Cadenza. Moreover, the chiasma mean frequency was 1.48 (SE 0.12) in the presence of Ph1 (TaZIP4-B2) and 12.21 (SE 0.19) and 12.23 (SE 0.20) in the Cadenza1691-Ae. Variabilis and Cadenza0348-Ae. variabilis hybrids, respectively. The observed chiasma frequencies at metaphase I, in the two Tazip4-B2 mutant line-Ae. variabilis hybrids, are similar to those previously reported at metaphase I in the Ph1 deletion mutant (ph1b)-Ae. variabilis hybrids. Thus, the data indicate that TaZIP4-B2 within the Ph1 locus is likely to be involved in the suppression of homoeologous COs.In this study, both rid Fig. , with 6.Ph1 deletion mutant (ph1b) have been reported previously  in the wild-type Cadenza, to 3.29 (SE 0.16) in Cadenza1691 and 3.63 (SE 0.18) in Cadenza0348. This indicates a slight reduction in homologous COs in these Tazip4-B2 mutant lines. CO frequency was a mean of 40.50 (SE 0.21) in the wild-type Cadenza, 38.13 (SE 0.20) in Cadenza1691 and 37.30 (SE 0.23) in Cadenza0348. These observed chiasma frequencies at metaphase I in the two mutant Cadenza lines are again similar to those previously reported at metaphase I in wheat in the absence of the Ph1 locus. However, no multivalents were observed, and there was no significant increase in the number of univalents at metaphase I in the Tazip4-B2 mutant lines, as is normally observed in Ph1 deletion mutants , and the probability that they will both be located within the Ph1 region is even lower (2.4 × 10−7). Thus, it is extremely unlikely that the increased homoeologous crossover phenotype found in both Tazip4-B2 mutant lines results from a nonsense mutation in a further gene independently linked with Tazip4-B2 within the Ph1 locus.Thus, seven lines carrying mutations within the Tazip4-B2 lines to clean up background mutations, as well as transferring Tazip4-B2 into the highly crossable hexaploid wheat cv. Chinese Spring. We are currently at BC3. BC lines will be made available once this exercise is complete. We have also complemented the approach of identifying chemically induced Tazip4-B2 mutants, by exploiting CRISPR to generate a large deletion within TaZIP4-B2. Initial analysis reveals that this Tazip4-B2 line has a similar phenotype to the two Tazip4-B2 Cadenza lines described above. We are currently segregating the transgenes away. TaZIP4-B2 is also being over-expressed to assess whether increased ZIP4 levels reduce homologous crossover. Our current hypothesis is that a higher ZIP4 level is optimal for homologous crossover, and a lower ZIP4 level is optimal for homoeologous crossover. Reducing or increasing ZIP4 around the optimal levels reduces the frequency of crossover. Interestingly, increasing the copy number of 5B chromosomes carrying TaZIP4-B2 reduces homologous crossover  both  Feldman . In addi Feldman . Thus, ZTazip4-B2 mutants were identified through a non-GM route, which can be exploited as an alternative to the CS ph1b mutant. Seeds for both mutants have been deposited with the Germplasm Resource Unit at the John Innes Centre (www.jic.ac.uk/research/germplasm-resources-unit). The accession number of seeds for Cad0348 is W10336 and for Cad1691 is W10337. The seeds for both lines are available on request, free of intellectual property restrictions.In summary, two ESM 1(DOCX 38 kb).Supplementary Table 1ph1b deletion) of the Ph1 locus. Data represent mean values ± standard error (SE) from RNA samples collected at late leptotene-early zygotene stage in WT and in ph1b deletion. (XLSX 13 kb).Detailed information of all transcripts obtained by Kallisto and the statistical analysis of transcripts per million (TPM) in wheat chromosomes 3AL, 3B, 3DL and 5BL, both in presence (WT) and in absence (Supplementary Table 2TaZIP4-B2(Traes_5BL_9663AB85C.1). The two mutant lines (Cadenza1691 and Cadenza0348) which showed reduced number of COs in Cadenza mutant lines are indicated in bold. (XLSX 15 kb).Detailed information on the seven EMS mutant lines selected as possessing potentially interesting mutations within


Corynebacterium glutamicum that expresses exogenous GAD gene, gadB2 or gadB1, can synthesize GABA from its own produced Glu. To enhance GABA production in C. glutamicum, ribosomal binding site (RBS) sequence and promoter were searched and optimized for increasing the expression efficiency of gadB2. R4 exhibited the highest strength among RBS sequences tested, with 6 nt the optimal aligned spacing (AS) between RBS and start codon. This combination of RBS sequence and AS contributed to gadB2 expression, increased GAD activity by 156% and GABA production by 82% compared to normal strong RBS and AS combination. Then, a series of native promoters were selected for transcribing gadB2 under optimal RBS and AS combination. PdnaK, PdtsR, PodhI and PclgR expressed gadB2 and produced GABA as effectively as widely applied Ptuf and PcspB promoters and more effectively than Psod promoter. However, each native promoter did not work as well as the synthetic strong promoter PtacM, which produced 20.2 ± 0.3 g/L GABA. Even with prolonged length and bicistronic architecture, the strength of PdnaK did not enhance. Finally, gadB2 and mutant gadB1 were co-expressed under the optimal promoter and RBS combination, thus converted Glu into GABA completely and improved GABA production to more than 25 g/L. This study provides useful promoters and RBS sequences for gene expression in C. glutamicum.Glutamate decarboxylase (GAD) converts  Therefore, optimization of promoters and RBS sequences has been applied for protein expression in C. glutamicum. Through constructing RBS libraries and regulating the pathway genes aroG, aroB, aroD and aroE by RBS of different strengths, 9 genetic modules were built up and shikimic acid synthesis was improved in C. glutamicum  were explored here for expressing a GAD gene, i.e. gadB2 derived from L. brevis Lb85, by tacM promoter. Then, to improve the transcription of gadB2 and yield of GABA, several types of native promoters and various architecture of the strong native promoter were explored. Considering the beneficial effects of gadB1–gadB2 co-expression  medium at 37 °C and 200 rpm. C. glutamicum SH, an l-glutamate-producing strain, was used for expressing gadB2 and gadB1 genes and producing GABA. SH was deposited in the China General Microbiological Culture Collection (CGMCC) center with accession number CGMCC 1.581. C. glutamicum was grown in LBG medium (LB supplemented with 5 g/L glucose) at 200 rpm and 30 °C. When necessary, 30 μg/mL kanamycin was added to the media.Bacterial strains used in this study are listed in Table gadB2. For expressing with normal RBS sequence and AS of 7 nt, gadB2 was amplified from the plasmid pJYW-4-gadB1–gadB2  and AS of 7 nt as well as with RBS of R4 and AS of 6 nt (R4a) or 8 nt (R4b), gadB2 was amplified similarly using the 14 different forward primer Rn-B2-F and the same reverse primer gadB2-R, digested with AflII and BamHI, and ligated into pJYW-5 digested with the same enzymes, resulting in the 14 plasmids of pJYW-5-Rn-gadB2. Finally, these plasmids were transformed into C. glutamicum SH by the method described previously  was amplified via overlap PCR using primers PdnaK-F, PdnaK-2SD-R, PdnaK-2SDB2-F and gadB2-R, and then ligated into pJYW-5, resulting in the plasmid pJYW-5-PdnaK-2SD-gadB2. To express gadB2 with prolonged PdnaK, PdnaK with the downstream 60 bp [PdnaK(+1)], with the upstream 60 bp [PdnaK(−1)], upstream 180 bp [PdnaK(−2)] and upstream 240 bp [PdnaK(−3)] were amplified with corresponding primers and then overlapped with R4a-gadB2 fragment that was amplified with primers PdnaKB2-F and gadB2-R, generating plasmids pJYW-5-PdnaK(+1)-R4a-gadB2, pJYW-5-PdnaK(−1)-R4a-gadB2, pJYW-5-PdnaK(−2)-R4a-gadB2 and pJYW-5-PdnaK(−3)-R4a-gadB2. Finally, the 5 plasmids were transformed into C. glutamicum SH, yielding 5 recombinant strains, PdnaK2SD-B2, PdnaK(+1)-B2, PdnaK(−1)-B2, PdnaK(−2)-B2 and PdnaK(−3)-B2.For expressing gadB1mut gene with the strong RBS (R4a) was amplified from the plasmid pEC-gadB1T17I/D294G/E312S/Q346H  of 1.9 and cultured at 30 °C and 200 rpm for 72 h as previously described  containing 10% glycerol and 0.1% Triton, and applied directly as the crude enzyme of whole cell suspension. The GAD reaction was then performed at 37 °C for 1 h in a reaction mixture (1 mL) consisting of 0.4 M Na2HPO4–citric acid buffer (pH 5.0), 60 mM monosodium glutamate, 0.03 mM pyridoxal 5′-phosphate and appropriate volume of whole cell suspension. GAD activity was determined according to the formation of GABA in this reaction. One unit (U) of GAD activity is defined as 1.0 µmol GABA produced in 1 min in the initial reaction mixture. The specific activity is expressed as U/g of dry cell weight (DCW). The DCW per liter (g/L) was calculated according to an experimentally determined formula: DCW = 0.6495 × OD562 − 2.7925.The cells in the fermentation broth were harvested and washed twice with chilled phosphate buffer saline. However, no GAD activity could be detected after the crude enzyme was extracted from washed cells by method described previously  combined with reverse transcription as described previously  value and was calculated by the The mRNA transcription levels of C. glutamicum SH, the RBS sequences and promoters for expressing GAD gene were explored here. As expression of gadB2 produced more GABA in C. glutamicum than expression of gadB1  were researched.RBS is a pivotal region for controlling translation initiation and protein expression. For expressing target protein(s) in C. glutamicum, the optimal pH for cell growth and Glu biosynthesis is about 7.0, whereas that for GAD activity and conversion of Glu to GABA is 5.0–6.0. For effective production of GABA, the cultivated medium was initially maintained at neutral pH by adding urea during 10–21.5 h of fermentation, and then the pH was let to decline and change spontaneously thereafter. During the fermentation, all the strains that harbor different RBS sequences grew similarly and exhibited similar pH variation. The cell density increased fast before 24 h and nearly maintained thereafter at OD562 of approximately 45–50. Glucose was consumed rapidly before 24 h, slowly thereafter and nearly exhausted at the end of fermentation. The pH value decreased to the lowest level of about 4.9–5.6 at 36 h, partially due to the exhaust of urea and accumulation of acidic Glu  and several native promoters of genes involved in carbon metabolism (dtsR) and Glu biosynthesis (gdh and odhI). Psod and Ptuf have been widely applied to enhance gene expression and optimize metabolic pathways for production of amino acids, such as l-ornithine  was significantly higher than that of strains under other native promoters, it was obviously lower than that of R4a-B2 under PtacM promoter (20.2 ± 0.3 g/L) and more Glu was remained  was significantly lower than that of other strains (3.2–32.0 U/g DCW), especially R4a-B2 under PtacM promoter  and expressing GAD activity (less than 5 U/g DCW), although most of them except PgapA were able to enhance the gadB2 transcription level at GABA conversion stage  was significantly higher than that of strains under other stress response promoters, it was obviously lower than that of R4a-B2 and more Glu was remained  was tested for its strength.Although PgadB2-expressing strains under the prolonged PdnaK promoters, i.e. PdnaK(+1) prolonged to downstream 60 bp, PdnaK(−1) prolonged to upstream 60 bp and PdnaK(−2) prolonged to upstream 180 bp, as well as the strain under the bicistronic PdnaK promoter (PdnaK2SD-B2) grew and consumed glucose in a similar manner with the strain PdnaK-B2  promoter that prolongs to upstream 240 bp grew and consumed glucose much slowly than PdnaK-B2, perhaps due to the reason that tedious fragment might increase the growth burden of bacteria. Meanwhile, its pH value during GABA conversion stage was significantly lower than other strains. The GABA production of PdnaK(+1)-B2 and PdnaK(−1)-B2 (about 16 g/L) was comparable to that of PdnaK-B2 , PdnaK(−1) and PdnaK promoters for producing GABA. But the GABA production of PdnaK(−2)-B2 decreased by 40% and that of PdnaK(−3)-B2 decreased greatly to only 1.3 g/L; meanwhile, more Glu was remained and the total amount of Glu and GABA also decreased, likely due to the further decrease of GAD activity in these two strains, especially in PdnaK(−3)-B2, indicating the repression of PdnaK as it extended to upstream 180 and 240 bp. Therefore, the activity of PdnaK did not increase as PdnaK region extended. Furthermore, although the GAD activity of PdnaK2SD-B2 was always higher than that of PdnaK-B2  and RBS sequence (R4a), expression of gadB2 only was not sufficient for converting all the synthesized Glu into GABA and approximately 5 g/L Glu was remained at the end of fermentation. Therefore, another GAD gene, L. brevis gadB1, was co-expressed with gadB2, both under the robust promoter of PtacM and RBS sequence of R4a. Considering that as the active pH range of GadB1 was broadened to near-neutral pHs after mutagenesis, GABA production increased, especially in gadB1T17I/D294G/E312S/Q346H-expressing strain  was expressed here instead of wild-type gadB1.Even controlled by the robust promoter  and R4a-B2B1mut (127 ± 18 U/g DCW) was 55% and 3.0-fold higher than that of R4a-B2 (32.0 ± 2.5 U/g DCW), respectively. Compared with R4a-B2, more GABA (25.2 ± 3.0 g/L) was produced and nearly no Glu (0.6 ± 0.2 g/L) was remained in R4a-B2B1mut, whereas in R4a-B1mut, less amount of GABA and similar amount of Glu were produced  and the highest amount of both Glu and GABA (290 ± 35 mM) were obtained at 60 h and decreased thereafter, indicating the decomposition and consumption of GABA and Glu after 60 h, likely due to the exhaust of glucose. Therefore, coexpression of gadB1mut and gadB2 under the optimal PtacM promoter and R4a RBS sequence was effective for production of GABA in C. glutamicum.The -B2 Fig. a, b. Theion Fig. c. The GAC. glutamicum. The expression level of heterologous genes has been illustrated to be influenced by multiple factors, including gene dosage, promoter strength, secondary structure of mRNA and RBS sequence  and production of GABA in C. glutamicum at different levels. Two RBS sequences stronger than the frequently used RBS sequence of R, i.e. R4 and R6, were obtained here. Meanwhile, R4 showed the highest strength, whereas R9 and R3 showed the lowest strength. Similarly, the RBS sequence of R4 (GAAAGGAGA) was reported to exhibit higher expression level of NhhBA and specific activity of NHase in C. glutamicum  which exhibited the highest inducibility for expression of a reporter, sfGFP during the transition phase between exponential and stationary phases in C. glutamicum (Kim et al. tacM, partially due to the complex regulation of these native promoters by transcriptional regulators, such as regulation of PgapA by GlxR and SugR, regulation of PdtsR1 by GlxR, repression of PdnaK, PclpB and PclgR by HspR, repression of Pgdh by GlxR, AmtR, FarR and ArgR, repression of PsufR by SufR (Schroder and Tauch H36 was employed for producing single-chain variable fragment of antibody (Yim et al. H30 was proven to be more suitable than PH36 for the production of cadaverine in C. glutamicum (Oh et al. gadB2 expression and GABA production in the future.The strength of promoters tested in this study was also dramatically different as expressing BA Figs. , 4, 5. Tce Figs. , althouggadB2 and gadB1mut, were co-expressed in C. glutamicum, both under the optimal PtacM promoter and R4a RBS sequence. GAD activity increased greatly by 3.0-fold; consequently, GABA production increased to more than 25 g/L and all Glu was converted to GABA (Fig. pknG (Okai et al. C. glutamicum strain harbouring pHGmut (Choi et al. E. coli GAD and much lower than that of GABA6C and GABA6F (Jorge et al. pknG but comparable to that of other recombinant C. glutamicum strains. Considering that more GABA will be produced if the Glu production is high enough, fed-batch fermentation of R4a-B2B1mut will be carried out in the future.Finally, two GAD genes, i.e. ABA Fig. d, e. How


Electron and thermal transport properties, i.e., electrical conductivity, carrier concentration, Hall mobility, Seebeck coefficient, and thermal conductivity, of CuI-Pb co-doped Bi2Te3 were measured in the temperature range from 300 K to 523 K, and compared to corresponding x% of CuI-doped Bi2Te3 and undoped Bi2Te3. The addition of a small amount of Pb significantly decreased the carrier concentration, which could be attributed to the holes from Pb atoms, thus the CuI-Pb co-doped samples show a lower electrical conductivity and a higher Seebeck coefficient when compared to CuI-doped samples with similar x values. The incorporation of Pb into CuI-doped Bi2Te3 rarely changed the power factor because of the trade-off relationship between the electrical conductivity and the Seebeck coefficient. The total thermal conductivity(κtot) of co-doped samples (κtot ~ 1.4 W/m∙K at 300 K) is slightly lower than that of 1% CuI-doped Bi2Te3 (κtot ~ 1.5 W/m∙K at 300 K) and undoped Bi2Te3 (κtot ~ 1.6 W/m∙K at 300 K) due to the alloy scattering. The 1% CuI-Pb co-doped Bi2Te3 sample shows the highest ZT value of 0.96 at 370 K. All data on electrical and thermal transport properties suggest that the thermoelectric properties of Bi2Te3 and its operating temperature can be controlled by co-doping.In order to understand the effect of Pb-CuI co-doping on the thermoelectric performance of Bi A larger ZT leads directly to a higher conversion efficiency. The main challenge lies in the decoupling of the interdependent thermoelectric parameters , which are strongly coupled to the carrier concentration. Commercial TE devices comprise series of p- and n-type semiconductor pairs.Bismuth telluride 0.01Bi2Te3 [0.008Bi2Te2.7Se0.3 have been studied [n-type Cu-intercalated Bi2Te3 changes the electronic band structure, such as band position and band degeneracies, resulting in an increase of the Seebeck coefficient. As a consequence, peak ZT values of 0.88 at 360 K and 0.91 at 320 K were obtained for V-doped and Au-doped Cu0.008Bi1.98Te2.7Se0.3, respectively [n-type Bi2Te3 based materials via compositional tuning approach by adjusting Cu contents or element doping.The ZT values of commercial Bion donor ,7. Howevd alloys ,11,12. Ae Bi2Te3 ,14,15,16e Bi2Te3 . The Cu-at 300 K , which ie Bi2Te3 . Howevere Bi2Te3 . Studiese Bi2Te3 ,20. Cu a studied ,20. The ectively ,20. Ther2Te3 samples were prepared using high temperature solid state reaction method and were consolidated by spark plasma sintering (SPS). The Pb-addition effects on the crystal lattice, the charge transport, and the thermoelectric properties of CuI-doped Bi2Te3 were evaluated.In the present study, CuI-Pb co-doped Bix% CuI-Pb co-doped Bi2Te3  samples are shown in 2Te3 and x% CuI-doped Bi2Te3 were prepared under the same synthetic conditions. All of the diffraction peaks are indexed to rhombohedral Bi2Te3 structure with the space group of R3m  [2−xTe, and CuI were detected in the 10% CuI-Pb co-doped Bi2Te3 samples. This result implies that the solubility limit of CuI and Pb dopants in Bi2Te3 was x < 0.1. While in a previous report, the impurity phase was observed in less than 5% in CuI-doped Bi2Te3 sample [2Te3, the impurity phase was observed only in a 10% CuI-Pb co-doped Bi2Te3 sample. This result indicates that the solubility of CuI in Bi2Te3 is increased by addition of Pb atoms. 2Te3 samples as a function of the dopant fraction. In all of the samples, the in-plane parameter a remains constant, while the unit cell parameter c along the stacking direction expands with an increasing Pb content in the CuI-Bi2Te3 system. The result is presumably a consequence of Cu atoms entering into the interstitial site, which increases the distance between the van der Waals layers [Pb = 0.147 nm) with that of Bi (rBi = 0.146 nm) shows that the size of Pb is very close to that of Bi, and thus the ability of Pb atoms for the substitution of Bi atoms in Bi2Te3 should not be neglected. Halogen atoms such as I (rI = 0.220 nm) are believed to occupy Te (rTe = 0.221 nm) sites in the lattice [Powder X-ray diffraction (PXRD) patterns of 15-0863) , with no3 sample , when Pbs layers . A compaI rI = 0.0 nm are 2Te3 samples with 1% CuI enhanced ZT [2Te3 sample as a reference material to demonstrate the effect of CuI-Pb co-doping on the charge transport properties. The charge transport properties of 1% CuI-Pb co-doping Bi2Te3 at room temperature are investigated by Hall effect analysis and are compared to those of 1% CuI-doped Bi2Te3 and undoped Bi2Te3. Assuming one carrier type and parabolic bands in our analysis, the carrier concentration (n) was calculated from the room temperature  Hall constants using the relationship RH = 1/ne, where RH is the Hall coefficient, n is the carrier concentration, and e is the electronic charge. The Hall coefficients of specimens are negative, indicating n-type conductions. By incorporating Pb in CuI- Bi2Te3 system, the ne value of the bulk samples decreases from ~7.8 × 1019/cm3 (CuI-doped Bi2Te3) to ~3.6 × 1019/cm3 (CuI-Pb co-doped Bi2Te3), and the corresponding mobility value increases from ~164.6 cm2/V∙s to ~216.9 cm2/V∙s at 300 K. In comparison, the undoped Bi2Te3 sample shows the n value of ~1.2 × 1019/cm3and the mobility of 354.9 cm2/V∙s at 300 K. This result verifies that the addition of a small amount of Pb significantly decreases the carrier concentration, which should be attributed to the holes generated by the Pb atoms. This demonstrates that facile control of electron concentration can be easily realized by adding Pb atoms to CuI-doped Bi2Te3 system, yielding an optimal electron concentration of 3−4.5 × 1019/cm3.In our previous work, we demonstrated that doping of Bianced ZT . Thus, w2Te3, 1% CuI-doped Bi2Te3, and 1% CuI-Pb co-doped Bi2Te3. All of the samples exhibit lamellar structures at the micron scale and no obvious large-scale preferred orientation. The microstructures are dense  showing densities of 7.73 g/cm3, 7.77 g/cm3, and 7.82 g/cm3 for undoped Bi2Te3, 1% CuI-doped Bi2Te3, and 1% CuI-Pb co-doped Bi2Te3, respectively. The orientation degree of the (0 0 l) planes, termed as F, was calculated with the Lotgering method [F is expressed as the following equations: F = P − P0/1 − P0, P0 = Il)0(0 0 /∑Ih k l)0(0 0 /∑Ih k l)0(0 0  is the intensity of (0 0 l) peaks and ∑Ih k l)0(0 0  is the (0 0 l) peak intensity and ∑Ih k l)/I(015) of the integrated intensity of (0015) to (015), and represented them in I(0015)/I(015) value for 1% CuI-doped Bi2Te3 and 1% CuI-Pb co-doped Bi2Te3 (17–18%) is slightly higher than those for undoped Bi2Te3 (10%). This indicates that the c-axis of the grains after SPS was preferentially oriented parallel to the pressing direction. This result is consistent with a previous report [2Te3 samples are nearly same.g method . In thiss report , showingS), and power factor of CuI-doped and CuI-Pb co-doped Bi2Te3  system as a function of composition was investigated at room temperature, as shown in 2Te3 (~307 S/cm) is increased by CuI-doping (1% of CuI-doped Bi2Te3 sample gave ~2673 S/cm). The room temperature electrical conductivity of 1% CuI-Pb co-doped Bi2Te3 at 300 K was about ~1462 S/cm. This value is significantly lower than that of 1% CuI-doped Bi2Te3. As shown in x values, confirming the role of Pb as an acceptor [2Te3 are about −115 μV/K and −157 μV/K, respectively, while that for undoped Bi2Te3 is −270 μV/K, which compares well with the previous reported value for n-type Bi2Te3 [2Te3 shows p-type character, however the undoped Bi2Te3 in this study show n-type character. We assume that these differences may arise from the different experimental conditions used for the preparation of undoped Bi2Te3 crystals. The Bi2Te3 prepared by the Bridgman method is a p-type conductor due to the antisite defect of BiTe. However, in this work, the SPS pressed Bi2Te3 samples show n-type characteristics, which arises from the Te vacancy at the interface. This decrease in electrical conductivity and the increase in Seebeck coefficient in co-doped samples can be explained by an increased carrier scattering related to the incorporation of Pb atoms in the CuI-doped lattice and by decreased carrier concentrations caused by Pb atoms, which act as electron acceptors [x values. The power factors decrease with increasing dopant concentrations. The maximum values of the power factors were observed at x = 0.01 for both CuI and CuI-Pb co-doped samples. The benefit of Pb incorporation into CuI-doped Bi2Te3 was not observed in the power factor because of the trade-off relationship between the Seebeck coefficient and the electrical conductivity. . This corresponds to an >80% enhancement over the typical value of undoped Bi2Te3 (22 μW/cm∙K2). tot and open symbols for κlatt) as a function of dopant content. As the dopant concentration increased, the total conductivity of CuI-doped and CuI-Pb co-doped Bi2Te3 increased due to the increase of the electronic contribution. The total thermal conductivity κtot of 1% CuI-Pb co-doped samples (κtot ~ 1.4 W/m∙K at 300 K) is slightly lower than that of 1% CuI-doped Bi2Te3 (κtot ~ 1.5 W/m∙K at 300 K) and undoped Bi2Te3 (κtot ~ 1.6 W/m∙K at 300 K) due to alloy scattering. The lattice part (κlatt) of the thermal conductivity can be estimated by subtracting the electronic component (κelec) from the measured total thermal conductivity, κlatt = κtot − κelec. The electronic component is given by the Wiedemann-Franz relation, κelec = LσT, where L is the Lorenz number. L is taken to be 1.5 × 10−8 V2/K2 for near-degenerate or degenerate semiconductor [2Te3 was 0.66 W/m∙K at 300 K. In contrast to the behavior of κtot upon increasing the dopant concentration, κlatt slightly decreased with increasing dopant concentration. This result demonstrates clearly that the lattice κlatt is reduced by Pb incorporation through the alloy phonon scattering.The thermoelectric properties depend on the dopants , dopant content, and temperature. In order to elucidate the effect of dopants and their contents on the thermoelectric properties, the electrical conductivity (σ), Seebeck coefficient , when compared with 1% CuI-doped Bi2Te3 and undoped Bi2Te3. For all of the samples, a monotonic decrease in electrical conductivity with increasing temperature is observed (n-type). The magnitude of the Seebeck coefficient initially increases and reaches a maximum that is strongly depend on the Pb content x. The onset of intrinsic conduction (the maxima of the curves) in these samples shifts to a higher temperature with an increasing dopant content. While the x = 0% sample has its maxima at ~300 K, the 1% and 3% sample have their maximum at ~425 K, and the x > 5% sample at ~525 K. The maximum value of the Seebeck coefficient (~−176 μV/K) was observed at x = 0.01 CuI-Pb content at 425 K. S2σ) values as a function of temperature. In this system, the power factor values for the 1% CuI-Pb co-doped ranged from 36 μW/cm·K2 at 300 K to 20 μW/cm·K2 at 523 K. The CuI-Pb co-doped sample with x > 0.03 shows a mild temperature dependence. tot of the samples. The κtot of all the doped samples firstly decreases due to the increasing phonon-phonon scattering, and then increases when upon further increase of the testing temperature due to the increase of ambipolar thermal contributions arising from the diffusion of electron-hole pairs with the onset of intrinsic contribution [observed a, which ribution .x) are shown in x. When the temperature is above ~400 K, the ZT values decrease due to the appearance of intrinsic excitation at a higher temperature. In this experiment, the ZTmax of the 1% CuI-Pb co-doped sample was about 0.96 at 370 K, while the highest value ZTmax was about 0.96 at 422 K for the 1% CuI-doped sample. The incorporation of Pb into CuI-doped Bi2Te3 led to a shift of the peak position of ZTmax to lower temperatures. This result shows that the optimization of the operating temperature can be controlled by co-doping. For practical applications of thermoelectric materials, the ZT values at room temperature are also important. 2Te3 sample shows a low ZT of ~0.42 at 300 K due to its very poor electrical properties. The highest ZT of 0.79 and 0.70 at 300 K were achieved for the 1% CuI-Pb doped sample and 1% Cu-doped Bi2Te3 sample, respectively, which are both significantly improved when compared with those of the undoped sample. All of the evidences about electrical and thermal transport properties suggest that the n-type ZT of Bi2Te3 can be enhanced by the incorporation of Pb with CuI dopant. Further improvement in its TE properties can be expected by choosing suitable combination of dopants.The dimensionless figure of merit ZT of the samples with different dopant concentration  were prepared by means of the conventional high-temperature solid-state reaction method, using Bi, Te, CuI and Pb  as starting materials. For convenience, the samples are labeled as dopant contents, such as that Bi2Te3 + x CuI + 1/2x Pb with x is labeled as x% CuI-Pb co-doped Bi2Te3. n-type Bi2Te3 doped with x at % CuI alone was prepared under identical experimental conditions for comparison. The corresponding elements were sealed in appropriate ratio in evacuated fused silica tubes  under a residual pressure of ~10−4 Torr. The sealed tubes were heated to 1000 °C over 12 h, and then held at 1000 °C for 12 h while rocking the liquid to facilitate a complete mixing of the contents. The tubes were slowly cooled to 800 °C over a period of 12 h and then quenched to room temperature. The cast ingot samples were powdered by ball milling in an Ar-filled glove box and the ground powder was passed through a 53 μm-mesh sieve. To obtain dense bulk samples, spark plasma sintering (SPS) was performed under Ar atmosphere by using SPS machine . Typically 12–13 g of the powdered samples were loaded into the graphite die with an inside diameter of 14 mm and heated to 425 °C for 5 min at a heating rate of 100 °C/min and held there for 5 min under an axial pressure of 50 MPa under a vacuum of 1.4 × 10−2 Torr.α radiation (λ = 1.54056 Å). The lattice parameters were obtained by least squares refinement of data in the 2θ range of 10°~70°, with the assistance of a Rietveld refinement program [Powder diffraction pattern was obtained with a Rigaku D/MAX X-ray  diffractometer with CuK program . The cartot, can be obtained from the relationship κtot(T) = D(T)∙pC(T)∙ρ(T), where pC is the specific heat, D(T)is the thermal diffusivity, and ρ(T) is the density of the sample. Thermal diffusivity and specific heat were determined by the flash diffusivity-heat capacity method with a Pyroceram standard using the method described in detail in the literature [In order to investigate the thermoelectric properties, the sample (~13 g) after SPS were cut into rectangular shapes with dimensions of ~3 mm × 3 mm × 10 mm and a disk-shape of about ~14 mm diameter and 2 mm thickness. The former specimens were subjected to the Seebeck coefficient and electrical conductivity measurements , and the latter to thermal diffusivity measurements using a NETZSCH LFA 457 MicroFlash™ instrument . The thermoelectric properties of the samples were measured along the direction perpendicular and parallel to the SPS pressing direction. Only results of perpendicular direction measurements are shown in the manuscript. The thermal conductivity, κterature . Sample n-type Bi2Te3-based compound towards a lower temperature. This demonstrates that facile control of the electron concentration can be realized by adding Pb atoms to the CuI-doped Bi2Te3 system, yielding an optimal electron concentration of 3–4.5 × 1019/cm3. Whereas, the change of room temperature power factor as a consequence of Pb addition was not notable, the thermal conductivity decreased with Pb addition due to the alloying scattering. The maximum ZT of 0.96 was obtained at 370 K for 1% CuI-Pb co-doped Bi2Te3. In comparison with 1% CuI-doped and undoped Bi2Te3, the ZT of 1% CuI-Pb co-doped Bi2Te3 (ZT ~0.79) at room temperature was enhanced by more than 12% and by 88%, respectively.In this work, utilizing second dopant, we successfully shifted the optimum ZT of an